Aspiration-assisted bioprinting of spheroids

Myoung Hwan Kim; Ibrahim T Ozbolat

doi:10.1038/s41596-025-01240-x

. Author manuscript; available in PMC: 2026 Mar 22.

Published in final edited form as: Nat Protoc. 2025 Sep 19;21(4):1325–1373. doi: 10.1038/s41596-025-01240-x

Aspiration-assisted bioprinting of spheroids

Myoung Hwan Kim ^1,², Ibrahim T Ozbolat ^1,^2,^3,^4,^5,^6,^✉

PMCID: PMC13005709 NIHMSID: NIHMS2154587 PMID: 40973814

Abstract

Aspiration-assisted bioprinting (AAB) is a versatile biofabrication technique that enables the precise and selective patterning of biologics, such as tissue spheroids and organoids, addressing limitations of conventional bioprinting techniques. AAB facilitates the fabrication of (1) tissues with physiologically relevant cell densities using spheroids and (2) advanced tissue models that replicate three-dimensional microenvironments essential for studying cellular responses, disease development and drug testing. Here we provide reliable and reproducible guidelines for the precise positioning of abovementioned biologics, incorporating two operational modes: (1) a single-nozzle mode for precise, one-by-one bioprinting and (2) a high-throughput mode using a digitally controllable nozzle array, enabling the rapid and simultaneous placement of multiple spheroids for scalable tissue fabrication. Comprehensive instructions are included for setting up the AAB platform, operating software and key operational procedures, including optimization of bioprinting conditions. This Protocol enables users to build and operate their own AAB platform depending on target applications, achieving fine control over spheroid positioning through successful aspiration and their precise placement under optimized conditions. This Protocol enables the setup of the AAB platform within 1–2 d. Bioprinting time varies depending on the number of spheroids to bioprint: the single-nozzle mode requires ~30 s per spheroid, while the high-throughput mode can print 64 spheroids in 3–4 min. Designed for accessibility and adaptability, this Protocol is suitable for users from a variety of backgrounds, including engineering, biology, pharmacy and medical sciences, who require bioprinting of spheroids for creating microphysiological systems for drug testing and disease modeling and implantable grafts for regenerative medicine.

Introduction

Spheroids, which are three-dimensional (3D) clusters of cells that self-assemble, have become widely used in tissue engineering due to their ability to replicate physiological cell–cell and cell–matrix interactions^1,2. Spheroids provide native-like cell densities, maintain 3D structures and support extracellular matrix deposition—essential features for tissue function and development. However, incorporating spheroids into the fabrication of complex and scalable tissues presents substantial challenges, particularly in achieving precise spatial arrangement.

Aspiration-assisted bioprinting (AAB) has emerged as a promising technique to address these challenges, overcoming limitations faced by conventional spheroid bioprinting techniques. AAB enables the precise spatial positioning of spheroids, accommodating a wide range of spheroid size and types for generation of tissues and tissue microenvironments. AAB supports both scaffold-based and scaffold-free approaches, enabling spheroid bioprinting into functional bioinks as a scaffold³ or sacrificial materials for self-assembly purposes^4,5, respectively. Furthermore, AAB extends the scope of bioprinting by enabling the bioprinting of anisotropic cell aggregates (for example, strands⁶) and single cells (for example, electrocytes⁷, ~400 μm)—capabilities often unachievable with many other bioprinting techniques. In addition, AAB can run in tandem with other bioprinting techniques, such as extrusion-based bioprinting (EBB), to create hybrid approaches that utilize the strengths of both techniques, enabling the fabrication of scalable tissues⁸.

Development of the Protocol

AAB was first introduced in 2020 as a method for the precise placement of biologics⁷. AAB utilizes aspiration forces to lift and transfer biologics, which are then precisely deposited at desired locations upon the release of the applied forces^7,8. This method ensures gentle handling of biologics while maintaining their structural integrity and viability throughout the process. Positive pressure is used to clean the nozzle of any aspirated liquid and mitigate potential clogging issues during bioprinting. Pneumatic channels, which regulate both aspiration and pressure, enable precise pick-and-place operations that are critical for achieving spatially controlled constructs.

Since its development, AAB has been successfully applied to various applications, utilizing diverse bioinks and spheroids^3–15. However, the original AAB platform had limitations in scalability and throughput, which restricted its use in fabricating large-scale tissue constructs with a high number of spheroids. Over the last 5 years, the original AAB system has undergone notable advancements, resulting in improved accuracy, speed and performance with upgraded hardware and software⁸. Key enhancements include the integration of a digitally controllable nozzle array (DCNA), which consists of an expanded array of nozzles that can be selectively activated; and modular printheads for flexibility of bioprinting and an expanded bioprinting workspace. These improvements have focused on addressing the intrinsic drawbacks of the initial AAB platform⁷—low throughput and limited scalability. The improved AAB facilitates the simultaneous placement of numerous spheroids, markedly enhancing throughput and scalability enabling the building of volumetric constructs. These improvements also support hybrid bioprinting strategies, where AAB is integrated with other bioprinting techniques (for example, EBB) to combine the strengths of each bioprinting technique. Moreover, the platform has been adapted for intraoperative bioprinting (IOB), thereby broadening its applications and usability in vitro and in surgical settings⁸.

This Protocol provides a comprehensive, step-by-step guide setting up the hardware and software components of the AAB platform and for performing AAB in various tissue fabrication applications. It highlights the two primary operational modes:

Single-nozzle mode: this mode facilitates precise, one-by-one positioning of biologics to study interactions between multiple cell types, making it ideal for applications that require detailed spatial control, such as microphysiological systems (that is, vascularized tumors used to study tumor invasion and tumor–immune interactions^3,10,16) as well as for building complex 3D structures, such as cylindrical tubular constructs^4,5,7,17.
High-throughput mode: this mode utilizes a DCNA, facilitating the simultaneous deposition of multiple spheroids. Each nozzle in the array operates independently, enabling rapid, precise bioprinting for scalable tissue fabrication⁸.

We present refined instructions based on our extensive experience with AAB across diverse applications. This includes optimization of bioprinting parameters, printability, spheroid preparation and characterization and bioink characteristics to ensure seamless operation and reproducibility. Postbioprinting handling guidelines are also provided to obtain and characterize bioprinted samples properly. To further support its effective use, we provide comprehensive troubleshooting strategies and potential solutions to address common challenges encountered during AAB. To achieve operational reliability and consistency, it is recommended to carefully follow the instructions described in the Protocol and practice procedures thoroughly. These make AAB accessible for researchers seeking a robust, reliable and versatile technique for advanced tissue engineering applications.

This Protocol is designed to be accessible for laboratories equipped with standard aseptic cell culture facilities and can be implemented by users with basic training in both conventional cell culture and engineering skills for setting up and operating the AAB system. The Protocol appeals to a broad audience, including engineers, biologists, bioengineers, pharmaceutical scientists and surgeons.

Applications

AAB enables a versatile and robust technique for precise tissue construction, accommodating a wide range of cell types to support fundamental research and transitional applications. Its capability to create complex, heterogeneous tissue architectures by accurately patterning spheroids (and also organoids) has established its utility across diverse applications, both within^3–15 and beyond our research group^17,18. These include fabrication of bone^4,9,12,13, cartilage^4,11,14 and cardiac¹⁷ tissues and cancer models^3,10,15,16. It has also been employed intraoperatively in surgical settings⁸ and freeform bioprinting^4,5. Furthermore, AAB has been adapted for printing living organisms, such as beetles and zebrafish embryos¹⁸, highlighting its broad applicability. The pick-and-place mechanism of AAB has inspired adaptations in other bioprinting techniques, such as neural organoid bioprinting using magnetic forces¹⁹ and single-cell scale micromasonry²⁰. This wide applicability highlights the system’s capacity to create tissues or tissue models, enabling advancements in tissue engineering, organoid science, organ-on-a-chip devices, in vitro disease modeling and studies of cellular behavior, accommodating diverse experimental needs from small-scale, high-fidelity models to volumetric tissue biofabrication. By utilizing both precision bioprinting and scalable biofabrication, AAB serves as a versatile tool that can be integrated with other bioprinting techniques to utilize complementary strengths and drive innovation in the evolving field of bioprinting.

Comparison with methods

While each spheroid bioprinting strategies offers specific benefits suited to different applications, AAB offers several advantages over existing techniques, including EBB²¹, droplet-based^22,23, Kenzan^24–26, bio-gripper²⁷, magnetic-based¹⁹ and acoustic-based²⁸ approaches. For instance, recent droplet-based technologies—such as the pick-flow-drop system²³—demonstrate high viability, high-throughput and automated spheroid handling with minimal mechanical stress. However, these approaches are limited to two-dimensional spheroid placement and focused on spheroid transfer rather than the fabrication of 3D structures. Kenzan enables the skewering of spheroids onto a needle array to precisely position them in three dimensions. This method facilitates scaffold-free tissue formation through spheroid fusion. While successfully demonstrated for various tissues, this approach is limited by fixed needle spacing, spheroid size compatibility and mechanical damage to spheroids. EBB allows extrusion of spheroids encapsulated within injectable biomaterials, supporting high cell density and enabling the deposition of multiple spheroids simultaneously. However, clogging and breakage due to substantial shear stress are common issues. This approach often results in uneven spheroid densities and limited positional control of spheroids. The magnetic method enables nearly noncontact positioning and spatial patterning of spheroids, yet typically require additional material modifications (for example, magnetic coatings), which limit printing speed and raise concerns about long-term effects of magnetic particles on cellular function. Acoustic spheroid assembly enables noninvasive, high-throughput spheroid patterning but face challenges in accommodating multiple spheroid types with precise spatial arrangements. Biogripper-based techniques can effectively handle large, preformed tissue blocks but are less adaptable to small-scale or high-throughput applications, making them impractical for broader use. By contrast, AAB enables flexible, precise, heterogeneous and scalable spheroid bioprinting across both two-dimensional and 3D configurations. AAB supports both scaffold-free and hybrid bioprinting strategies using two different modes—single-nozzle and high-throughput—offering users fine control over spatial patterning of spheroids along with high-throughput capabilities.

Overview of the procedure

The Protocol is divided into four main parts: (1) AAB system setup (hardware, Steps 1–55; software, Steps 56–63 and hardware–software integration and configuration, Steps 64–98), (2) pre-bioprinting preparation (Steps 99–133), (3) AAB operations (Steps 134–212) and (4) postbioprinting procedures (Steps 213–244). This Protocol provides comprehensive instructions for assembling and operating AAB to perform spheroid patterning for both single-nozzle (Strategy 1, Steps 134–165) and high-throughput mode (Strategy 2, Steps 134–136 and 166–203), with the option to produce a hybrid platform with EBB (Steps 128–133 and 189–192). Moreover, we include protocols for IOB (Steps 204–212), which demonstrates the adaptability of AAB for surgical applications.

Since AAB is designed as a versatile bioprinting platform, users are encouraged to adapt the system and Protocol to their specific needs. Conducting a practice run of AAB using spheroids is strongly recommended before initiating experiments to understand AAB workflow and optimize parameters (for example, nozzle size, aspiration force and so on) for specific spheroids used. The design of parts, bioprinting parameters and strategies described in this Protocol can be adjusted by users to suit their intended applications.

Experimental design

AAB system setup: hardware (Steps 1–55)

AAB utilizes standard components such as screws, an optical breadboard and stainless-steel posts, which are readily available from engineering suppliers. This Protocol provides detailed instructions for assembling the hardware, including camera setup (Steps 16–17), printbed (Steps 32–39), solenoid valves (Steps 41–50) and pneumatic sensors (Step 51). Users should assemble the AAB platform following instructions outlined in Figs. 1–3.

Fig. 1 | — a–j, A CAD schematic illustrating the step-by-step construction of the AAB platform, comprising a three-axis motion stage (a–d), printheads (e–h) and printbed (i and j). The three-axis motion stage was built on an optical breadboard, stainless-steel (SUS) posts (a) and adaptors (b) to mount the motion stage. The assembled motion stage (c) was then mounted on the adaptors (d). The printhead module includes the EBB/single-nozzle AAB holder (e), DCNA holder (f) and isometric camera holder (g), all of which can be mounted on the z axis (h). For the printbed, three separate components were prepared (i), mounted on a lab jack and placed on the breadboard (j). k, A fully assembled AAB platform with the correct directional placement, including keyboard shortcuts for movement control. The printheads and printbed were assembled using 3D-printed parts (.stl files are available at ref. 35).

Fig. 3 | — a–c, Images illustrating the key steps for the assembly of the DCNA, including the recommended nozzle-insertion order (red box) into the stacked acrylic plates (a), the z-axis alignment to level inserted nozzles in the DCNA (.dxf file for acrylic plate laser cutting available via Figshare at https://doi.org/10.6084/m9.figshare.28405133, ref. 35) (b) and the immobilization of leveled nozzles (blue box) using adhesive, followed by a quality check from isometric and side views of the manufactured DCNA (c). d, An example of a manufacturing failure in the DCNA, showing a misaligned nozzle. e, The AAB platform setup for IOB in a surgical setting. f, The assembled printbed equipped with two cameras. g, The assembled DCNA holder mounted on a tilting base for yaw-roll-pitch adjustments. h, The hybridized AAB setup incorporating EBB components for IOB in a rat calvarial defect procedure. Parts a–c and e adapted from ref. 8, Springer Nature Limited.

The motion stage allows precise 3D positioning along x, y and z axes, with built-in drivers and limit switches to prevent exceeding the travel range of 0–203 mm. Modular print heads for single-nozzle (Steps 10–11 and Fig. 1e) and high-throughput mode (Steps 12–15 and Fig. 1f) can be mounted on the z axis (Steps 30–31 and Fig. 1h). The EBB and single-nozzle AAB processes share the same holder, referred to as the EBB/single-nozzle AAB holder (Fig. 1e). If the simultaneous use of EBB and single-nozzle AAB is required, a separate holder should be prepared to enable parallel operation. Hybrid bioprinting using EBB and high-throughput AAB is addressed under Strategy 2. Solenoid valves digitally regulate pneumatic flows, ensuring the gentle manipulation of biologics during bioprinting.

For researchers utilizing the high-throughput mode, detailed guidance is provided on preparing and manufacturing the DCNA in a 4 × 4 array arrangement (Steps 18–29 and Fig. 3a–d), allowing the selective activation of individual nozzles for the simultaneous deposition of multiple spheroids. To augment throughput, users can increase the nozzle array size by modifying the acrylic nozzle holder and adding more solenoid valves. In the current system, nozzle dimensions, ranging from 2.8 to 4.0 mm in width, can be adjusted depending on the application. Based on our experience, 30-gauge nozzles with a 1-inch length are suitable for 200–700-μm spheroids. The fabrication of the DCNA employs stainless-steel nozzles inserted into predefined holes in laser-cut acrylic plates (Step 18 and Fig. 3a). Nozzle alignment calibration ensures that all nozzles are positioned on the same plane (Step 22 and Fig. 3b), and the nozzles are stabilized using glue (Step 23 and Fig. 3c). The assembled nozzle array is treated with Sigmacote to siliconize surfaces of nozzles, minimizing the surface tension at the nozzle–cell medium interface (Step 27). After the silicon treatment, the DCNA is attached to the bioprinting platform via the DCNA holder, enabling precise roll, pitch and yaw adjustments (Figs. 1f and 3g) to achieve alignment parallel to the workspace surface. For 300–350-μm spheroids, bioprinting is standardized at a deposition speed of 10 mm/s, with a slower speed (0.2 mm/s) during lifting to ensure gentle handling.

A solenoid valve control system enables the independent control of nozzles, and a pressure sensor monitors pressure changes in real-time (Fig. 2f). Airflow is digitally regulated via solenoid valves, ensuring gentle manipulation of biologics. To prevent damage to solenoid valves from the aspirated medium, vacuum chambers are installed as liquid traps (Fig. 2c).

Real-time visualization of the process is achieved using cameras with isometric, bottom-view and side-view perspectives. These cameras allow comprehensive monitoring to ensure precision and reproducibility. The side-view camera is used to monitor spheroid detachment during the high-throughput mode operation. Camera specifications can be upgraded to higher resolutions, if needed, with corresponding adjustments to printbed and isometric camera holder dimensions. In the assembled printbed, there are two areas, bed space 1 and bed space 2 (Figs. 1k and 3f), each connected to individual bottom-view cameras. Bed space 1 is designated for spheroid lifting, while bed space 2 is used for bioprinting of aspirated spheroids (Fig. 4). To maintain aseptic conditions for biological applications, the system should be installed inside a biological safety cabinet (BSC), and all components must be disinfected before use. For nonbiological materials, sterility is optional.

Fig. 4 | — a,b, A schematic illustration detailing the step-by-step procedure for the AAB process in single-nozzle AAB (a) or high-throughput AAB mode (b). c, An intraoperative application of the high-throughput AAB mode for calvarial defect regeneration. Part a adapted with permission from ref. 5, IOP Publishing. Parts b and c adapted from ref. 8, Springer Nature Limited.

AAB system setup: software (Steps 56–63)

The AAB system includes a custom-designed software interface that enables the control of the bioprinting process. The software is specifically developed for compatibility with Zaber linear axes and communicates with specific parts for solenoid valves, pneumatic sensor and motion stages. The software operates on Windows operating systems and requires the installation of specific National Instruments (NI) modules. The executable file, HITS-BIO_software_distr.exe, can be downloaded from Supplementary Software.

The software interface consists of several panels (Fig. 5), facilitating control of the pneumatic system and motion stage. For preset positions (Fig. 5, panels 4A–B) and automated motion movement (Fig. 5, panels 9A–B), users can create a comma separated values (.csv) file containing positional data in Cartesian coordinates (x, y and z). Each axis is represented in the .csv file as follows: X (first column), Y (second column) and Z (third column). For the automated motion, the software sequentially reads the .csv file line by line, executing precise nozzle movement, when the ‘play’ button is pressed, after selecting the appropriate moving velocity and acceleration speed. Examples of predefined positions can be found in Supplementary Data 1, while automated movement examples for EBB are provided in Supplementary Data 2. The automatic motion operates in an absolute positioning mode. Once motion begins, the AAB platform sets the initial position as (0, 0, 0), and subsequent movement proceeds according to the designed path defined by absolute coordinates. Users can stop movement instantly by toggling the On/Off switch located in panel 9A (Fig. 5) of the interface. To ensure operational safety, dynamic z-direction limits are implemented, preventing nozzle damage. This dynamic z limit can activate/deactivate by switching on/off using the ‘On/Off’ switch (Fig. 5, panel 6).

The software supports two primary modes: a single-nozzle and high-throughput mode (Fig. 5, panel 10). The single-nozzle mode is optimized for precise one-by-one spheroid placement by activating a channel in solenoid manifolds (Fig. 4a). In the high-throughput mode, selective activation and recognition of individual nozzles is enabled to bioprint multiple spheroids simultaneously (Fig. 4b). The provided software is configured for a 4 × 4 nozzle array but is adaptable for expanded configurations, if needed. Detailed descriptions of software features, panel functions and available shortcuts are summarized in Table 1. These shortcuts enhance operational efficiency of AAB.

Table 1 |.

Descriptions of controls and indicators in the AAB software interface provided in Fig. 5 and their shortcuts

Position	Controls/indicators	Description	Default	Shortcut
Toolbar	Run	Starts executing the software program	–	Ctrl+R
Toolbar	Abort execution	Stops the ongoing software operation	–	Ctrl+
Panel 1	Movement type	Determines whether positions are relative or absolute. Always set to ‘Relative’ for programmed movements	Relative	–
	Units	Specify the unit of motion (for example, mm). Ensure the unit is correct for the experiment	Millimeters	–
	Microstep size	Defines motor specifications. For Zaber X-LSM linear stages, use the default microstep size	0.047625	–
	Motion stage	Choose the correct COM port for connecting the motion stage	COM8	–
	Axis no.	Specify the axis to control: (00: all axes x, y and z; 01: x axis; 02: y axis; 03: z axis)	00	–
Panel 2	Pressure	Displays the current pressure sensor reading	–	–
	Compensat	Shows the corrected pressure value after initialization	–	–
	Received Init	Indicates the pressure value recorded during initialization	–	–
	Pressure sensor	Choose the appropriate COM port to communicate with the pressure sensor	COM20	–
	Initialization	Resets the current pressure reading to zero	–	–
	LED indicator	Displays the current pressure reading	–	–
Panel 3	Pressure SVN#	Assigned SVN no. to control solenoid valve for pressure (Fig. 2c)	48	–
	Min Vac SVN#	Assigned SVN no. to control solenoid valve for the minimum vacuum pressure (Fig. 2c)	21	–
	Aspiration SVN#	Assigned SVN no. to control solenoid valve for aspiration (Fig. 2c)	27	–
	Single SVN#	Assigned SVN no. to control solenoid for EBB/single-nozzle AAB output (Fig. 2c)	0	–
	DCNA starting SVN#	Assigned SVN no. for activating the starting solenoid for DCNA outputs (Fig. 2c)	1	–
	Slave address	IP address for the WAGO Ethernet controller	192.168.1.100	–
	Port	Inputs the Modbus TCP protocol number	502	–
	String indicator	Displays the current connection status with the WAGO Ethernet controller	–	–
	Connect	Button to connect with the WAGO Ethernet controller	–	Ctrl+F1
	Disconnect	Button to disconnect with the WAGO Ethernet controller	–	–
	Initialize controller	Resets all controllers and closes solenoid valves	–	Ctrl+F2
	Switch OFF controller	Opens all valves in the manifold	–	Ctrl+F3
Panel 4A	Reinitialization	Resets all software panels to default settings	–	–
	Read Coordinates from CSV	Opens a file selection dialog for choosing a .csv file from the user’s drive	–	–
	New file path	Displays the file path of the saved .csv file	–	–
	Call table	Transfers assigned positions from Pos 1–10 to panel 4B	–	–
	Save as file	Saves the transferred table data on panel 4B	–	–
	Clear ALL	Removes all values in the table in panel 4B	–	–
	Position Initialization	Sets all values to 0 in the table in panel 4B	–	–
	Pos 1–10	Choose the current motion stage position from panel 5	–	–
	Play buttons	Moves the motion stage to predefined positions	–	–
	SLOW position	Select a Pos no. to move to with SLOW velocity (used for spheroid lifting in the high-throughput mode)	Pos 7	–
	SLOW speed	Sets the velocity for SLOW movement (used for spheroid lifting in the high-throughput mode)	0.25	–
	SLOW play	Moves to the selected SLOW position at the SLOW speed (used for spheroid lifting in the high-throughput mode)	–	Ctrl+F8
Panel 4B	Loaded XYZ Coordinates	Displays position coordinates after using the ‘Call table’ button in panel 4A	–	–
Panel 5	Current X, Y, Z	Displays current X, Y and Z positions of the motion stage as read from the motion driver	–	–
Panel 6	Needle	Always set to ‘Needle’ mode	Needle	–
	Set Maximum Z	Loads the current Z position from the current Z on panel 5. Displays the value after setting maximum Z	–	–
	ON/OFF button	Enables or disables the maximum Z function	OFF	–
Panel 7	X	Move the motion stage along x axis. Input the desired position and press ‘Play’ to execute the movement	37	–
	Y	Move the motion stage along y axis. Input the desired position and press ‘Play’ to execute the movement	153	–
	Z	Move the motion stage along the z axis. Input the desired position and press ‘Play’ to execute the movement	100	–
	Set distance	Displays the currently selected step distance	–	–
	Vel	Defines the velocity of the motion stage. The linear stage has a specified maximum velocity limit. Refer to the manufacturer’s specifications to ensure safe operation	10	–
	Acce	Defines the acceleration of the motion stage in accordance with the selected velocity	10	–
	0.01	Sets step distances to 0.01 mm	–	F1
	0.05	Sets step distances to 0.05 mm	–	F2
	0.1	Sets step distances to 0.1 mm	–	F3
	0.5	Sets step distances to 0.5 mm	–	F4
	1.0	Sets step distances to 1.0 mm	–	F5
	5.0	Sets step distances to 5.0 mm	–	F6
	10.0	Sets step distances to 10.0 mm	–	F7
	20.0	Sets step distances to 20.0 mm	–	F8
	Input	Sets a custom step distance using the numeric input control	0.65	F9
	Move X (+)	Move along +x direction	–	→ key
	Move X (−)	Move along −x direction	–	← key
	Move Y (+)	Move along +y direction	–	↓ key
	Move Y (−)	Move along −y direction	–	↑ key
	Move Z (+)	Move along +z direction	–	PgDn
	Move Z (−)	Move along −z direction	–	PgUp
Panel 8	Home All axes	Move the motion stage to home position (0, 0, 0)	–	–
	Home X	Move x axis to home position	–	–
	Home Y	Move y axis to home position	–	–
	Home Z	Move z axis to home position	–	–
Panel 9A	Automated motion speed	Adjust the velocity for automated motion	4	–
	Acce factor	Defines the acceleration for automated motion: (automated motion speed × acceleration factor = actual acceleration)	10	–
	Absolute	Always set to ‘Absolute’ (relative positioning should not be used for automated motion)	Absolute	–
	Play button	Executes the automated motion based on the imported .csv file from panel 9B	–	F12
	ON/OFF button	Toggles automated motion ON/OFF	OFF	–
Panel 9B	Read coordinates from CSV-automated motion	Opens a file search dialog for .csv file selection in the user’s drive	–	–
Panel 10	1–16 buttons	Individual solenoid valve button assigned in ‘DCNA starting SVN#’ in panel 3	–	–
	Single mode/high-throughput mode buttons	Switch between the single nozzle and high-throughput modes	High-throughput mode
	Pressure	Button to control the solenoid assigned to ‘Pressure SVN#’ in panel 3	–	Ctr1+F5
	Aspiration	Button to control the solenoid assigned to ‘Aspiration SVN#’ in panel 3	–	Ctr1+F6
	Minimum vacuum	Button to control the solenoid assigned to ‘Min. vac SVN#’ in panel 3	–	–
	Single channel ON/OFF button	Button to control the solenoid assigned to ‘Single SVN#’ in panel 3 under the ‘Single’ tab	–	Ctr1+F7
	Aspiration On/OFF	Confirms selected nozzles in the DCNA under the ‘High-throughput’ tab	–	Ctr1+F9
	Aspiration set coils	Activates or deactivates selected nozzles in the DCNA under the ‘High-throughput’ tab	–	Ctr1+F10
	DCNA arrangement set	Selects the DCNA arrangement	On for all nozzles	–
	DCNA recognition array	Configure the arrangement of the DCNA mapping to solenoid valves in a 4 × 4 array. Each number represents the solenoid assigned to a specific nozzle	–	–

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Real-time monitoring is enabled through manufacturer’s software (for example, Digital Viewer, DinoCapture and so on). Cameras configured for isometric, bottom-view and side-view perspectives provide comprehensive visualization of the process. This real-time feedback ensures precise, consistent and gentle handling of biologics.

Hardware and software integration and configuration (Steps 64–98)

After assembling the hardware, establishing and testing the connection between hardware and software is critical to ensure seamless operation of AAB. The Protocol provides detailed instructions for integrating the motion stage (Steps 64–69 and 81–82), solenoid valves (Steps 74–78 and 83–85), a pressure sensor (Steps 70–72 and 86–87) and cameras (Step 73) with the software interface. Proper configuration ensures reliable communication and responsive operation. The solenoid valve control system connects to the software via a transmission control protocol/internet protocol (TCP/IP) connection, enabling the independent regulation of pneumatic channels. Solenoid valves support the simultaneous control of multiple channels and allow the selective activation and recognition of individual nozzles within the DCNA. Real-time pressure monitoring is achieved using an integrated pressure sensor connected through an Arduino microcontroller.

Before performing bioprinting, the system must be validated. Steps 92–98 of the Protocol outline the procedures for configuring motion and pneumatic controls. Users are instructed to conduct a test run to adapt AAB procedures and to validate system function and troubleshoot any potential issues. Adjustments to motion paths and solenoid valve activation timing are critical for optimizing the performance for specific applications.

The Protocol allows flexibility in hardware selection to accommodate various budgets and experimental needs. While the described setup utilizes Zaber linear axes and commercial solenoid valves, equivalent alternatives can be substituted. For example, low-cost motion stages and solenoid valves controlled via Arduino are viable options. Commercially available 3D printers can also be adapted for AAB, provided that proper integration with the software and hardware is achieved, as we reported before^4,7.

Spheroid preparation (Steps 99–102) and characterization (Steps 103–105)

This section outlines prebioprinting steps, including spheroid preparation and characterization. These preparations ensure compatibility with AAB for an optimal performance. Key factors, such as spheroid size and physical properties, must be carefully considered to determine optimal parameters for specific applications. Spheroid size, influenced by cell type and seeding density, must be optimized with respect to the nozzle size to avoid aspiration failure or damage to spheroids. The relationship between spheroid diameter and nozzle size is essential to achieving successful outcomes.

Physical properties, such as spheroid stiffness and deformation under aspiration, greatly affect the success of AAB. Spheroids with low stiffness may deform excessively, increasing the risk of damage or failure. To evaluate spheroid stiffness, commercially available instruments (for example, CellScale) can be used to measure force–displacement curves (Step 104, option A). Alternatively, an accessible method based on the aspiration length-based stiffness measurement can be used with optical microscopy (Step 104, option B). This method measures the deformation length of spheroids subjected to a fixed aspiration force, facilitating approximate stiffness calculations^29,30. This method offers a practical and cost-effective solution without specialized equipment and is directly relevant to AAB while aiding in optimizing aspiration forces for AAB. Aspiration-induced deformation by modulating the aspiration force (gradually increasing from low to high) should be evaluated for various spheroid types to ensure cell viability during bioprinting (Step 105). Testing different aspiration forces is critical in determining the threshold for maintaining structural integrity and minimizing cell damage. If spheroids are damaged even at the lowest aspiration force, such a spheroid type is not deemed suitable for AAB. By ensuring that the spheroid properties (that is, size and stiffness) are optimized and compatible with AAB, users can achieve high reproducibility and minimize bioprinting failures. This preparation step is crucial for creating consistent and viable constructs tailored to specific experimental requirements.

Biomaterial preparation and characterization (Steps 106–115) and EBB preparation and optimization (Steps 116–133)

Biomaterials play a crucial role in supporting spheroids during AAB, serving either as bioink substrates as a scaffold via EBB or as support materials in embedded bioprinting to enable the 3D bioprinting of complex structures. The selection and characterization of biomaterials are essential to ensure compatibility with AAB and achieve reproducible results. Bioink used for EBB or support materials for embedded bioprinting must exhibit specific rheological properties. In EBB, shear-thinning reduces viscosity under high shear rates, facilitating smooth bioink flow while minimizing clogging and excessive pressure in the nozzle. This facilitates uniform filament deposition and prevents cell damage. In embedded bioprinting, shear-thinning provides smooth nozzle movement within the support material by reducing viscosity under applied shear forces, thereby minimizing disruptions to the surrounding matrix. Self-healing allows the bioink to recover its structural integrity postextrusion after a deformation caused by a high shear rate in EBB. In embedded bioprinting, self-healing allows support material to recover its structure after the nozzle passes, maintaining continued mechanical support for printed constructs. Yield-stress enables bioprinted constructs to be stabilized in 3D by holding them in place within the support material, preventing undesired movement or settling. Specifically, low yield-stress (for example, <10 Pa) support materials may result in spheroid sinking and reduced positional accuracy, whereas excessively high yield-stress (for example, >500 Pa) support materials may resist nozzle motion, potentially damaging spheroids. Based on prior testing and practical use in AAB workflows, an intermediate yield stress range of ~20–100 Pa is generally effective for balancing positional accuracy and printability.

These properties should be assessed using rheological analyses, such as frequency sweep, amplitude sweep, flow sweep and recovery tests, to optimize biomaterial characteristics for different needs. The shear-thinning behavior can be evaluated using a flow sweep test across shear rates ranging from 0.1 to 100 s⁻¹. Recovery tests simulate biomaterial deformation and assess its structural recovery under cyclic shear conditions using alternating low (0.1 s⁻¹) and high (100 s⁻¹) shear rates. Yield-stress properties are measured in an oscillatory mode using an amplitude sweep test. This test is performed within the linear viscoelastic region over a strain range of 0.01–100%, while a frequency sweep test evaluates storage (G′) and loss (G″) moduli across angular frequencies of 0.1–100 rad/s. The sol–gel transition point, where G′ and G″ intersect, quantifies yield stress. Parameters for each test can be adjusted on the basis of the experimental requirements and biomaterial properties. Experiments should be repeated under identical conditions to confirm reproducibility and consistent rheological trends across replicates. Particular attention should be paid to irregularities in curves, such as nonmonotonic trends, sudden drops or increases or discontinuities, which may indicate wall slip or other measurement artifacts.

Biocompatibility is another critical factor when selecting biomaterials. Cytocompatibility can be assessed using assays, such as LIVE/DEAD, AlamarBlue or Cell Counting Kit-8 (CCK-8). Biomaterials should support high cell viability. For the biocompatibility test, biomaterials are cultured with cells either seeding cells on them or encapsulating cells (~1 million/mL). For sacrificial support materials, spheroids should be embedded and cultured for 3–5 d to evaluate cytocompatibility. Nonaspirated spheroids or free-standing spheroids in well-plates can serve as controls. All biomaterials should be sterilized using appropriate methods, such as gamma irradiation, ultraviolet (UV) treatment or autoclaving before use. For nonascetical applications, this step can be skipped.

In this Protocol, we also describe how to use EBB for AAB in hybrid bioprinting, while the characterization of support material is detailed in our previous publications^4,5. Detailed instructions for optimizing bioinks are provided in Steps 116–133. Extrudable bioinks can be loaded into a 1-cc nozzle (compatible with the current AAB setup); and the nozzle holder (Fig. 1e) can be modified for larger syringes (for example, 5 or 10 cc), if needed. The extrusion performance for EBB can vary depending on pneumatic pressure, printing speed and nozzle diameter, and users are encouraged to optimize these parameters.

AAB process (Steps 134–136, common preparation; Steps 137–165, (Strategy 1) single-nozzle AAB; Steps 166–203, (Strategy 2) high-throughput AAB; Steps 204–212, intraoperative AAB)

The basic workflow of AAB involves several steps. Spheroids are first placed in a chamber containing a culture medium with an adequate number of spheroids for AAB (Fig. 4). The single-nozzle (see Steps 160–165 and Fig. 4a for single-nozzle mode) or DCNA (see Steps 189–203 and Fig. 4b for high-throughput mode) is then positioned above the chamber. Using the optimized aspiration force, spheroids are gently aspirated and lifted before being transferred to the desired positions. At the designated bioprinting site, the aspiration force is released to place the spheroids. This process can be repeated as needed to build complex and scalable constructs. Factors including humidity, temperature and operator skills can influence bioprinting outcomes. Therefore, their optimization is essential to maximize success rates and minimize cell damage. Regular practice, use of shortcuts (Table 1) and test runs with fixed spheroids (Step 102) following the AAB workflow (Fig. 6) are highly recommended before performing AAB for functional outcomes. Test runs will help optimize parameters (for example, nozzle size, spheroid size corresponding to the nozzle and aspiration forces) and ensure smooth AAB operations. However, after fixation, as the spheroid stiffness can change, the optimal aspiration force may differ from the actual force required for live spheroids.

Fig. 6 | — A step-by-step representation of the control logic and operational flow used in AAB process, including cameras, motion stage, solenoid valves and pneumatic sensor. The diagram adapted from ref. 8, Springer Nature Limited.

The high-throughput AAB mode is more complex and advanced compared to the single-nozzle mode. Users must master single-nozzle AAB first to understand the mechanisms of AAB, improve proficiency and troubleshoot effectively before attempting high-throughput operations with live spheroids. In the high-throughput mode, the DCNA facilitates the simultaneous bioprinting of multiple spheroids. The DCNA is moved into a Petri dish containing spheroids suspended in the medium. While the current DCNA is inherently rectangular, the deposition of spheroids is dynamically controlled through selective nozzle activation, enabling nonrectangular patterns such as circular shapes. Expanding the array size (for example, n × n) not only accelerates the process but also improves the spatial resolution, allowing more flexible deposition of spheroids for irregularly shaped spheroid patterns. A successful aspiration of spheroids in the DCNA is confirmed using a bottom-view camera (Step 197). The DCNA is then gently lifted and positioned over the bioprinting area (Step 197–200). Aspiration pressure is subsequently released; spheroids are deposited at their designated locations (Step 201). For applications requiring bioink substrates, the EBB process is first used to extrude the bioink, forming a substrate, where spheroids are placed (Fig. 4b). If the bioink used for EBB is photocrosslinkable, it can be stabilized by exposing it to an appropriate light source (for example, 405-nm light source), depending on the photoinitiator used.

AAB is also highly adaptable for IOB applications (Fig. 4c and Steps 204–212), such as repairing tissue defects by creating defect-specific constructs tailored to the geometry of the defect. As we demonstrated before⁸, the process begins by extruding a bioink substrate directly into the defect site to serve as a scaffold. Spheroids are then deposited using AAB, which are then overlaid with another layer of the bioink to immobilize spheroids.

Postbioprinting process and downstream analysis (Steps 213–244)

Postbioprinting processes include system cleaning (Steps 213–230) and assessing functionality and quality of constructs (Steps 231–244). This section outlines protocols for handling, culturing and analyzing bioprinted constructs, including optional methods for assessing characteristics, including bioprintability (for positional accuracy of spheroid placement) (Steps 233–238), LIVE/DEAD assays (Steps 239–243) and other analyses (Step 244). These processes vary depending on whether constructs are fabricated on bioink substrates or embedded within support materials. Postbioprinting culture conditions should be optimized on the basis of the cell type and intended application.

After AAB, constructs placed on bioink substrates can be cultured directly by adding the culture medium. Constructs embedded within support materials can be maintained and retrieved after the fusion of bioprinted spheroids. For constructs requiring the removal of support materials, appropriate methods are recommended on the basis of the support material used. Thermally reversible support materials can be liquified by adjusting the temperature^31,32, while chemically crosslinked support materials can be dissolved using appropriate reagents⁵. Mechanically removable support materials can be gently extracted or agitated without damaging the constructs^4,33. Each removal method should be optimized to maintain the integrity and function of constructs. Bioprinted spheroids, especially those designed for spheroid fusion, may lack sufficient structural integrity during the early postbioprinting stage (for example, <2 d, depending on spheroid type and condition). Over time, these constructs undergo spontaneous extracellular matrix deposition, self-assembly through spheroid fusion and tissue remodeling and maturation, which contribute to progressively enhanced mechanical strength. The use of a bioreactor is optional but can be beneficial, especially for accelerating tissue maturation and improving mechanical integrity.

Limitations

Despite its advantages, AAB faces challenges that require further optimization. Both single-nozzle and high-throughput modes are prone to clogging due to aspiration, which can delay bioprinting. Aspiration forces must be carefully adjusted to prevent damage to spheroids, as different spheroid types possess different mechanical properties.

The current AAB setup employs a fixed aspiration force that requires manual adjustments during bioprinting. This can be addressed by replacing the manual valve with a digital one for adaptive control. Several other components also require manual intervention, such as motion and valve control for spheroid lifting and placement. Operators must visually confirm spheroid lifting and placement to prevent damage to spheroids, ensuring gentle and swift handling during lifting to avoid prolonged air exposure and confirming successful placement at desired positions to prevent excessive compression during placement. This is achieved using bottom-view cameras, one for monitoring spheroid lifting from the chamber and another for placement on the printbed. However, an opaque substrate can reduce visibility from the bottom-view cameras, hindering precise spheroid positioning. For such cases, including IOB, side-view cameras have been integrated to monitor the deposition on predefined x and y positions. Integrating computer vision and artificial intelligence could enable fully automated AAB with improved precision, eliminating the need for visual-based control and manual adjustments.

The current DCNA uses a 4 × 4 array of 16 nozzles (30-guage), arranged in a fixed configuration. In addition, a precise alignment along the z axis of the DCNA is critical for an accurate spheroid deposition. Misalignment can lead to uneven deposition or spheroid damage upon substrate contact. While the current DCNA is effective on flat surfaces, bioprinting inside a support material, on irregular or nonplanar surfaces, such as in vivo defects, remains challenging. Creating a bioink substrate on defects for placing spheroids, along with adjustments to the roll, pitch and yaw angles of the DCNA, addresses these issues and helps maintain a consistent deposition on tilted surfaces. Incorporating independent height-adjustable nozzles, such as the adaptive printing developed before³⁴, would further enhance flexibility for AAB.

While the principles of AAB are straightforward, many parameters and bioprinting conditions must be carefully optimized for a reliable and seamless bioprinting procedure. This Protocol provides detailed guidance on these considerations to help users achieve consistent results with minimized spheroid damage and improved success rates across diverse applications.

Materials

Biological materials

Cell line of interest. The following cell types are examples of those used for AAB; however, this Protocol is not limited to those lines
Human adipose-derived stem cells (Lonza, cat. no. PT-5006)
Human bone marrow-derived mesenchymal stem cells (RoosterBio Inc, cat. no. MSC-001)
Human dermal fibroblasts (Lonza, cat. no. CC-2511)
Human umbilical vein endothelial cells (HUVEC, Lonza, cat. no. C2519A)

▲ CRITICAL Cells lacking cadherins do not spontaneously form spheroids and may require additional methods or materials.

▲ CAUTION Cell lines should be regularly checked to ensure that they are contamination-free. The culture medium should be specific; see culture media used in our published studies^3,7,8.
Animal of interest. The animal model listed below is an example used for intraoperative AAB; however, the Protocol can be adapted for other models
Inbred immunodeficient RNU athymic rats, acquired at 5 weeks until the age of 12 weeks (Charles River Laboratories International)

▲ CAUTION Experiments with live animals must be approved by the appropriate institutional animal care and use committee (IACUC). All experiments in this Protocol were approved by the IACUC at the Pennsylvania State University.

Reagents

▲ CAUTION Always wear suitable personal protective equipment when handling chemicals used in this Protocol.

Reagents needed throughout procedure

Dulbecco’s phosphate-buffered saline, 1× without calcium and magnesium (store at room temperature (RT, 20–24 °C)) (DPBS; Corning, cat. no. 21–031-CV)
Sterile deionized water (DI water, obtained from a Milli-Q water system and autoclaved for sterilization)
Ethanol absolute, anhydrous (200 proof) (Decon Labs, cat. no. V1005M)

▲ CAUTION Ethanol is flammable. Do not use near sparks or flames.
0.4% Trypan blue solution (store at RT) (Thermo Scientific, cat. no. 15250061)
Cell culture media appropriate for the specific cells being used

Intraoperative AAB

▲ CAUTION All procedures must be approved by the appropriate animal welfare committee at the user’s institution and adhere to national regulations for animal research.

Isoflurane, United States Pharmacopeia (USP; Dechra, cat. no. Isospire)

▲ CAUTION Isoflurane is an anesthetic gas that has toxicity to the central nervous system. To minimize the risk of inhaling excess isoflurane, ensure adequate ventilation and implement effective gas scavenging systems.
Bupivacaine (0.8 mg/mL, Centralized Biological Laboratory, Penn State University)
Buprenorphine (ZooPharm)
Betadine (povidone-iodine) solution (Covetrus, cat. no. 001618)
Enrofloxacin (Norbrook, Enroflox 2.27%)
Sterile saline solution (Aspen, national drug codes (NDC) no. 46066-807-25)
Ophthalmic eye ointment, USP (Bausch+Lomb, NDC no. 24208-780-55)
Gauze pads (Covidien, cat. no. 9023)
Heating pad (Stryker, cat. no. TP700)
Pulse oximeter (Kent Scientific corporation, MouseSTAT Jr.)

Equipment

Equipment needed throughout the procedure

Class II BSC (NuAire, cat. no. NU-425–400)
3D printer (QIDI TECH, cat. no. X-max)
1.75-mm polylactic acid (PLA⁺) filaments (eSUN, black)
Razor blades (CANOPUS)
35-mm Petri dishes (Celltreat, cat. no. 229638)
0.5-inch blunt 27-gauge stainless steel nozzles (Nordson, cat. no. 7005008)
0.5-inch blunt 30-gauge stainless steel nozzles (Nordson, cat. no. 7018433)
0.5-inch blunt 32-gauge stainless steel nozzles (Nordson, cat. no. 7018462)
22-gauge tapered nozzles (Nordson, cat. no. 7018298)
Sterile pipettors (100–1,000 μL, VWR, cat. no. 89079–974) and P1000 pipette tips (Celltreat, cat. no. 229047)
Sterile pipettors (20–200 μL, VWR, cat. no. 89079–970) and P200 pipette tips (Celltreat, cat. no. 229044)
Sterile pipettors (2–20 μL, VWR, cat. no. 89079–964) and P10 pipette tips (Celltreat, cat. no. 229042)
1-cc sterile disposable slip tip syringes (BH Supplies, cat. no. BH1LS)
0.22-μm sterile syringe filters (EMD Millipore, cat. no. SLGSR33SS)
5% CO₂, 37 °C humidified incubator (Thermo Fisher Scientific, Heracell VIOS 160i, cat. no. 51033553)
Centrifuge (Thermo Scientific, Sorvall X1R Pro-MD, cat. no. 75009261)

▲ CAUTION Ensure the weight and balance of tubes are confirmed before using the centrifuge. Verify the maximum centrifugation force before proceeding.
Water bath set for 37 °C (VWR)
Phase-contrast microscope (Invitrogen, EVOS XL Core)
Confocal microscope (Zeiss, LSM880)
Forceps
Refrigerator for 4 °C (VWR)
Centrifugal mixer (FlackTek, cat. no. DAC 330–100 SE)
Rheometer (Anton Parr, MCR 302)
Autoclave
Allen wrench set (EKLIND)

Cell culture and spheroid preparation

Hemocytometer (Feleolibe, cat. no. JSB348)
96-well cell-repellent U-bottom plates (Greiner Bio-One, cat. no. 650970)
75-cm² cell culture flasks (Corning, cat. no. 353136)
175-cm² cell culture flasks (Corning, cat. no. 353112)
40-μm cell strainer (Corning, cat. no. 352340)
15-mL centrifuge tubes (Celltreat, cat. no. 229418)
50-mL conical tubes (MTC Bio, cat. no. C2602)
LIVE/DEAD viability kit (Invitrogen, cat. no. L3224)

AAB platform

▲ CRITICAL The motion stage should be installed in a BSC for a sterile bioprinting environment, and surfaces should be wiped using 70% (vol/vol) ethanol and UV light before use with cells for sterilization purposes.

EBB/single-nozzle AAB nozzle holder (custom-made). Use computer-aided design (CAD) software, such as Solidworks, to design parts for the platform. See Fig. 1e and provided .stl files (EBB holder_base.stl and EBB holder_cover.stl) for the designs in ref. 35
DCNA holder (custom-made). Use CAD software to design parts for the platform. See Fig. 1f and provided .stl files (DCNA holer_1.stl and DCNA holder_2.stl) for the designs in ref. 35
Isometric camera holder (custom-made). Use CAD software to design parts for the platform. See Fig. 1g and provided .stl files (Isometric camera holder.stl) for the designs in ref. 35
Printbed (custom-made). Use CAD software to design parts for the platform. See Fig. 1i,j and provided .stl files (Print bed_1.stl, Print bed_2.stl and Print bed_3.stl) for the designs in ref. 35
WAGO ECO power supply (WAGO, cat. no. 787–732)
WAGO controller Ethernet (WAGO, cat. no. 750–881)
Eight-channel digital outputs (WAGO, cat. no. 750–1515)

▲ CRITICAL Order as many as necessary.
End module (WAGO, cat. no. 750–600)
FESTO solenoid valve manifold (FESTO, cat. no. MH1)

▲ CRITICAL Order as many as necessary. We used three manifolds, integrated with a total of 52 solenoid valves.
Plug socket with a cable (FESTO, cat. no. 566656)

▲ CRITICAL Order according to the number of solenoid valves used.
Push-in fitting (external thread M3, for 5/32-inch outer diameter (OD) tubing) (FESTO, cat. no. 130592)

▲ CRITICAL Order according to the number of solenoid valves used.
Push-in fitting (External thread M7, for 1/4-inch OD tubing) (FESTO, cat. no. 183740)

▲ CRITICAL Order according to the number of solenoid valves used.
Blanking plug (FESTO, cat. no. B-M7)
Blanking plug (FESTO, cat. no. B-M3)
5/32-inch tee connector (McMaster-carr, cat. no. 5779K32)
Push-to-connect straight tube reducer 1/4–3/8 inch (McMaster-Carr, cat. no. 5779K355)
Push-to-connect straight tube reducer 5/32–1/4 inch (McMaster-Carr, cat. no. 5779K353)
Male luer to 1/16 inch (OD: 1.6 mm) adapter (Nordson, cat. no. MLRLB210)
Male luer integral lock ring to 3/32 inch (inner diameter (ID): 2.4 mm) tubing (Nordson, cat. no. MTLL220)
Tubing extension (ID: 1 mm and OD: 2 mm) (Truecare, cat. no. TCRTCBEXT004EA); cut the female luer to connect tubing (ID: 1.6 mm and OD: 3.2 mm). Retain the male luer to connect with another adapter (female luer: 3/32 inch (ID: 2.4-mm tubing), cat. no. FTLB220)
4 mm × 2.5 mm pneumatic polyurethane tubing (Joywayus)
1/4-inch OD polyvinyl chloride (PVC) clear tubing (EZ-FLO, cat. no. 98615)
5/32-inch OD polyurethane tubing (Utah pneumatic)
3/8-inch OD PVC clear tubing (EZ-FLO, cat. no. 98617)
1/16-inch (1.6 mm) ID 1/8-inch (3.2 mm) OD clear silicone tubing (Uxcell, cat. no. a20103000ux0011)
Pneumatic regulator
12 inch × 12 inch × 1/2 inch solid aluminum optical breadboard (Base Lab Tools, cat. no. SAB1212)
1/4–20 × 3/4 inch stainless steel socket head cap screw, (Base Lab Tools, cat. no. CS25–0750-R)
1/4–20 × 1.25 inch stainless steel socket head cap screw, (Base Lab Tools, cat. no. CS25–1250-R)
Stainless steel set screw—cup point: 1/4–20 × 1/2 inch (Base Lab Tools, cat. no. SS25–0500-R)
Stainless steel set screw—cup point: 1/4–20 × 3/4 inch, (Base Lab Tools, cat. no. SS25–0750-R)
Ø1-inch stainless steel post length 6 inch, no. 8–32 tapped hole and 1/4–20 tapped hole (Base Lab Tools, cat. no. PTA060)
M6–1.0 × 20-mm hex socket head cap screw (McMaster-Carr, cat. no. 91292A137)
M3 × 10-mm hex socket head cap screws (VIGRUE)
M3 × 12-mm hex socket head cap screws (VIGRUE)
M3 × 20-mm hex socket head cap screws (VIGRUE)
M3 × 25-mm hex socket head cap screws (VIGRUE)
M3 nuts (VIGRUE)
Motorized linear axis (Zaber, cat. no. X-LSM200A)

▲ CRITICAL Three linear axes are required for the x, y and z axes.
Miniature angle bracket, 90° (Zaber, cat. no. AB103)
Miniature angle bracket, 90° (Zaber, cat. no. AB103B)
Adaptor plate (Zaber, cat. no. AP101)
Adafruit MPRLS ported pressure sensor (Adafruit MPRLS, cat. no. 3965)
Arduino Uno Rev3 (Arduino, cat. no. A000066)
Breadboard (Deyue, cat. no. 7545924028)
Breadboard jumper cable kit (ELEGOO, cat. no. EL-CP-004)
Tilting base

▲ CRITICAL Obtain the tilting base from the micromanipulator (World Precision Instruments, cat. no. MD4-M3-R).
USB Digital microscope (Dino-Lite, cat. no. AM4113ZT)
USB Digital microscope (Plugable, cat. no. USB2-MICRO-250X)

▲ CRITICAL Two cameras are required.
12MP CMOS 4K microscope camera (Hayear, cat. no. HY-5299)
C-Mount 300× zoom lens (Hayear, cat. no. HY-300XA)
Cover glass 22 mm × 22 mm (VWR, cat. no. 16004–302)
Plain glass slides (Globe Scientific, cat. no. 1301)
Epoxy glue (Gorilla)
Solder and solder wire (Lonove)
4 inch × 4 inch lab jack (Wisamic, cat. no. T060021)
Desktop or laptop running 64-bit Windows 10 or 11
External monitor (any brand will do)

DCNA preparation

Sigmacote (Sigma-Aldrich, cat. no. SL2)

▲ CAUTION Sigmacote is a flammable and corrosive material that can cause severe respiratory and skin irritation/damage. Handle Sigmacote in a fume hood while wearing prospective gloves and glasses. Take safety precautions when handling the material.
2-mm-thick acrylic sheet (SimbaLux)
Glue gun (any brand will do)
Glue gun stick (ELDVAP)
1-inch blunt 30-gauge stainless steel nozzles (CML supply, cat. no. 901-30-100)
Laser cutter (Epilog laser, cat. no. Fusion M2)

Postbioprinting process and spheroid characterization

24-well tissue culture plates (Greiner Bio-One, cat. no. 662160)
Pipette puller (Sutter, cat. no. P-1000)
Capillary glass (Sutter, cat. no. B100-58-10)
Pasteur borosilicate pipettes (VWR, cat. no. 14673-043)
Pressure controller (Nordson EFD, cat. no. Ultimus II)
Paraformaldehyde solution 4% (Santa Cruz Biotechnology, cat. no. sc-281692)

Software

LabVIEW Runtime 2019 SP1 (NI, https://www.ni.com/en/support/downloads/software-products/download.labview-runtime.html?srsltid=AfmBOopJoKR_wvmoOSTB21wtOeq2pVUv2ZcSma6iHagY_sIoZLF0VxZb#348045)
NI-VISA 19.5 (NI, https://www.ni.com/en/support/downloads/drivers/download.ni-visa.html?srsltid=AfmBOoqFJB6k5luHUfmnUXDICwUZFYbP3mIsGpD6Jcj7YAZRvvVlQppM#329456)
Arduino integrated development environment (Arduino IDE, Arduino, https://www.arduino.cc/en/software/)
ImageJ (National Institutes of Health)
Appropriate 3D CAD software (for example, SolidWorks)
Slicing program (for example, Cura)
WAGO Ethernet settings (WAGO, https://downloadcenter.wago.com/wago/software/details/m5mg5kqe77030p67rct)
Zaber Launcher (Zaber, https://software.zaber.com/zaber-launcher/download/)
DinoCapture 2.0 (Dino-Lite, https://www.dino-lite.com/download01.php)
Digital viewer (Plugable Technologies, https://plugable.com/pages/microscope-drivers?srsltid=AfmBOoq60gyshRckQrhpzoFxVXtuEOzYHOqEmZhdPqBohCQd5gb1Fw18)

Reagent setup

LIVE/DEAD viability reagent

To make 2-μM calcein acetoxymethyl ester and 4-μM ethidium homodimer-1 working reagent, add 2.5 μL of calcein acetoxymethyl ester stock solution (4 mM in stock) and 10 μL of ethidium homodimer-1 stock solution (2 mM in stock) in 5 mL of sterile DPBS, followed by vortexing to mix homogeneously.

▲ CRITICAL Use the working reagent immediately. Protect from light.

70% ethanol

Mix 700 mL of 100% ethanol with 300 mL of DI water. Store the solution at RT for up to a month.

Procedure

Hardware setup

● TIMING 1–2 d, depending on handskills and familiarity with assembly

▲ CRITICAL The hardware setup is instructed for Zaber linear axes. The motion stage assembly can be done as same as shown in Fig. 1.

3D motion stage assembly

1
Linear axis connection: connect linear axis for x, y and z axes. Daisy-chain the linear axis using cables provided from the manufacturer. The x axis should be connected first and directly to the computer, followed by the y axis and then z axis. For example, computer → x axis → y axis → z axis. Follow the manufacturer’s manual for detailed instructions.
2
Power connection test: connect the power cable and confirm that the green power indicator is lit on each axis. After confirmation, remove the power.
3
Attach 6 inch SUS pillars to the optical breadboard using cup-point SUS screws (1/4–20 × 3/4 inch) (Fig. 1a).
4
Fix two adaptors (AP101, cyan color in Fig. 1b) onto the SUS pillars using SUS socket head cap screws (1/4–20 × 3/4 inch).
5
Remove a top plate (already built) from the x axis to mount the y axis on top. The removed top plate will be used for DCNA holder (see Step 15).
6
Mount the z axis vertically to the y axis using a 90°-angle bracket (AP103B, blue part in Fig. 1c) and M3 screws provided by the manufacturer.

▲ CRITICAL STEP Follow the assembly sequence (x → y → z). Ensure all axes are aligned at 90° to maintain the positional accuracy in 3D. Misalignment will reduce bioprinting precision. Tighten all connections securely to avoid wobbling during operation.
7
Mount the assembled motion stage (from Steps 5–6) to the AP101 adaptors using four M3 screws on each side (Fig. 1d). Adjust the x axis manually using manual control knob to access and secure screws on the left side.

▲ CAUTION Avoid placing fingers near moving parts during adjustments.
8
Check that all components are parallel and at 90° angles.

▲ CRITICAL STEP Misalignment can introduce substantial positional errors in 3D.
9
Confirm that all screws are tightly fixed to avoid loosening during operation.

▲ CAUTION Overtightening may damage screw threads or deform components.

EBB/single nozzle AAB holder preparation

10
Use the provided .stl files (EBB holder_base.stl and EBB holder_cover.stl)³⁵ to 3D print the EBB/single nozzle AAB holder after slicing them with the slicing software (for example, Cura) or the 3D printer manufacturer’s slicer. Modify slicing settings (for example, filament temperature, printbed temperature, z offset and so on) on the basis of the material, and follow the 3D printer manufacturer’s instructions.
11
Insert a 1-cc syringe between the 3D-printed ‘EBB holder_base’ and ‘EBB holder_cover’³⁵, securing it with M3 × 20-mm screws and nuts. Remove the inserted syringe.

▲ CRITICAL STEP Tighten enough to hold the syringe securely but avoid overtightening, which could make the syringe difficult to remove. This holder can be interchangeable between EBB and the single-nozzle AAB mode (Fig. 1e).

DCNA holder preparation

12
Use provided .stl files (DCNA holer_1.stl and DCNA holder_2.stl)³⁵ to 3D print the DCNA holder (Fig. 1f).
13
Assemble 3D-printed components using M3 × 20 mm screws and nuts. Ensure proper resistance for stable rotation and fixation during tightening screws.
14
Attach the DCNA holder to a tilting base (Fig. 3g) detached from a micromanipulator to adjust yaw, pitch and roll for the alignment of the DCNA surface.
15
Assemble the top plate (detached from Step 5) and tilting base using a M4 screw to mount the assembled DCNA holder on the z axis of motion stage (Fig. 3g). In Fig. 1, the attached tilting base is not included in the design.

Isometric camera holder preparation

16
3D print the isometric camera holder using the provided .stl file (isometric camera holder.stl)³⁵. This holder is specifically designed for a Dino-Lite Premier AM4113ZT digital microscope. If a different camera is used, resize the holder accordingly.
17
Insert the Dino-Lite camera into the printed holder (Figs. 1g and 3h). If the camera slips in the holder, put tape around the camera to stabilize it. This camera will focus on the nozzle tip to monitor EBB or single-nozzle AAB processes. The focus adjustment and alignment will be done after mounting. Misaligned cameras may result in improper monitoring.

DCNA preparation

18
Cut acrylic plates into 10 mm × 10 mm squares with predefined holes using a laser cutter (Fig. 3a). Use the provided design file (Acrylic plate_DCNA.dxf)³⁵ for laser cutting. This design is intended for a 3.4-mm width (nozzle end-to-end) DCNA, compatible with a 30-gauge nozzle. Adjust the hole pattern on the basis of the user-specific DCNA arrangement and the OD of the nozzle to use. Laser-cutting settings may vary for different equipment, and the optimization of the settings may be required.
19
Test the holes with the nozzle (for example, a 30-gauge nozzle) to ensure that they are not clogged.

▲ CRITICAL STEP Puncture clogged holes using a nozzle to open the holes. Clogged holes will damage or block the nozzle during DCNA preparation.

▲ CAUTION Be aware to avoid puncturing your fingers while handing nozzles.
20
Stack nine acrylic plates together and start inserting nozzles in the order shown in Fig. 3a.

▲ CRITICAL STEP Follow the specific insertion order (for example, (1) red, (2) green, (3) blue, (4) yellow) and slightly bend the shafts close to the nozzle hub outward (Fig. 3a) to minimize disturbances between closely packed nozzles and ensure z-positioning accuracy.
21
Adjust the nozzle extrusion lengths during insertion to avoid neighboring nozzle interference (Fig. 3a). After insertion, set the longest extrusion length for the nozzles located in the yellow areas, followed by those in the blue or green areas (these can be equal) and, lastly, those in the red areas (Fig. 3a). The extrusion lengths can be relatively adjusted among the nozzles within each color group.
22
Gently press the stacked acrylic plates against a flat surface to align all nozzle tips with respect to a single plane (Fig. 3b).

▲ CRITICAL STEP Avoid touching the inserted nozzles, as this can result in misalignment on the z position (Fig. 3d).
23
Use a glue gun to immobilize the inserted nozzles and stacked acrylic plates.

▲ CRITICAL STEP Apply glue to individual nozzles but avoid applying glue to two acrylic plates at the nozzle end, as these will be removed in the next step (Step 24). Excess glue may interfere with the proper fitting of the DCNA to the holder.
24
Once the glue dries, gently remove the two acrylic plates at the nozzle end to uncover the nozzle-end within DCNA (Fig. 3c).
25
Use a microscopic camera to inspect the front, side and bottom angles of the assembled DCNA (Fig. 3c).

▲ CRITICAL STEP Misalignment will cause x, y and z positioning errors (Fig. 3d). If misalignment on x axis or y axis is detected, adjust the position of nozzles with tweezers or reassemble DCNA.
26
Rinse the DCNA externally and internally by flushing the nozzles with DI water using a syringe (for example, 10-cc syringe) to remove debris and check for clogging and dry with compressed air.

■ PAUSE POINT Store the manufactured DCNA with the exposed nozzles carefully protected at RT. The fabricated DCNA can be stored indefinitely under this condition.
27
Apply Sigmacote solution dropwise to fully coat the exposed DCNA nozzles.

▲ CRITICAL STEP The coating is applied almost instantaneously. Ensure complete coverage of the surface of nozzle tips to reduce surface tension of the nozzles.
28
After air drying the DCNA in a hood, rinse it with DI water.
29
For biological applications, circulate 70% ethanol through nozzles using a sterile syringe and spray 70% ethanol on the entire DCNA. Rinse with sterile DI water to remove ethanol residues and dry thoroughly. Store the sterilized DCNA in a sterile environment and use promptly.

Mounting bracket and holder

30
On the z axis, attach the 90° bracket (AP103) using M3 screws provided from the manufacturer (Fig. 1h).

▲ CRITICAL STEP Ensure the bracket is tightly secured to prevent any movement during AAB.
31
Mount the required holder (Fig. 1h) onto the 90° bracket using a M6–1.0 × 20-mm screw, depending on the AAB mode and user requirements (see Step 137 for the single-nozzle AAB and Step 168 for the high-throughput AAB modes). Ensure the holder is tightly secured to the bracket.

Printbed assembly

▲ CRITICAL This printbed is designed for a 35-mm Petri dish. For applications involving different cultureware, the printbed and camera views require modifications.

32
3D print the required parts (Print bed_1.stl, Print bed_2.stl and Print bed_3.stl) using the provided .stl files³⁵ (Fig. 1i). These components are designed for use with a 4 inch × 4 inch lab jack.
33
Secure the printed ‘Print bed_1’ part to a 4 inch × 4 inch lab jack using epoxy glue. Allow the glue to fully cure before proceeding.
34
Attach the printed ‘Print bed_2’ to the assembly using M3 × 20-mm screws.
35
Insert two USB cameras into the respective camera holder chambers (Fig. 3f). These cameras are used as the bottom view cameras for bed space 1 and 2. This holder is specifically designed for the Plugable digital USB microscope (USB2-MICRO-250X). When using other cameras, dimensions may need to be adjusted accordingly.
36
Protect bottom-view cameras from accidental liquid exposure by placing a 22 mm × 22 mm glass slide over each lens. Slides protect cameras from potential droplet contamination and can be replaced or cleaned easily.
37
Attach the printed top part ‘Print bed_3’ over the camera holders and secure it with M3 × 12 mm screws.
38
Put M3 × 10 mm screws on the 35-mm Petri dish holder. Different manufacturers may have slight variations in Petri dish dimensions. Adjust the screws to ensure a snug fit.
39
Place the assembled printbed onto the breadboard and secure it in place using socket head cap screws (1/4–20 × 3/4 inch) (Fig. 1j).

Hardware placement

40
Place the motion stage in the BSC. For nonbiological applications, the assembled stage can be installed outside of the BSC.

▲ CRITICAL STEP Ensure the motion stage orientation matches the direction demonstrated in Figs. 1k and 3e to align with the programmed control direction when using the keyboard.

Solenoid valve assembly

▲ CRITICAL Follow the wiring diagram in Fig. 2 for detailed connection instructions.

41
Attach the positive power cable to the ‘L’ slot, the negative power cable to the ‘N’ slot and the ground cable to the ground slot (Fig. 2a).
42
Connect red (positive), black (negative) and yellow (ground) wires to the appropriate terminals of the WAGO Ethernet controller (Fig. 2a).
43
Assemble the eight-channel outputs through a uniform sliding clip design. Ensure you hear a ‘click’, indicating correct positioning (Fig. 2a,b).
44
Attach the end module at the end of the assembly to signal the system’s final point and provide feedback to the Ethernet control section (Fig. 2a,b).
45
Connect the Ethernet cable to the X1 port of the WAGO Ethernet controller, ensuring proper TCP/IP communication with the computer (Fig. 2a,b).
46
Set up the three solenoid manifolds for pressure, vacuum and nozzle pneumatic control as shown in Fig. 2c. To minimize cost, the number of valves on the manifold can be reduced on the basis of the required setup. For a 4 × 4 DCNA arrangement, at least 20 valves are required, including 16 for DCNA, 1 for EBB, 1 for pressure, 1 for vacuum and 1 for minimum vacuum, distributed across three valve manifolds.
47
Connect plug sockets and cables to each solenoid valve and to the WAGO eight-channel digital output modules (Fig. 2a,c).

▲ CRITICAL STEP Ensure that valves are connected in an organized manner. Use the solenoid valve number (SVN no.) described in Fig. 2c for recognition by the AAB software (Fig. 5, panel 3), for example: SVN no. 0: ON/OFF for the single-nozzle AAB; SVN nos. 1–16: ON/OFF for high-throughput AAB; SVN no. 21: ON/OFF for minimum vacuum; SVN no. 27: ON/OFF for vacuum and SVN no. 48: ON/OFF for pressure.
48
Seal unused valve inlets and outlets using M3 blanking plugs.
49
Attach 1/4 inch and 5/32 inch tubing to the push-in tube fittings, chamber, regulator, three-way splitter and pneumatic supply (pressure and vacuum) as shown in Fig. 2c,e. Pressure and vacuum sources are supplied by our building system. If not available at the user’s facility, external pressure and vacuum generators should be used.
50
Arrange electrical wiring and tubing to prevent tangling.

Pneumatic sensor connection

51
Solder pins to the MPRLS pneumatic sensor and connect them to an Arduino board according to the manufacturer’s instructions (Fig. 2f). Briefly, VIN → 5V, GND → GND, SCL → A5 and SDA → A4.

Sterilization for AAB

▲ CRITICAL UV-irradiate for at least 30 min before starting the bioprinting process. This step is optional for nonbiological applications.

52
Decontaminate the BSC and maintain sterile conditions.
53
Disinfect all materials entering the BSC with 70% ethanol.
54
Install syringe filters on the pressure source to purify generated air.
55
Autoclave nozzles under a plastic cycle if they are being used for biologics. See Step 29 for the sterilization of DCNA.

■ PAUSE POINT Autoclaved nozzles can be stored in the BSC until needed.

Software setup

● TIMING ~30 min

▲ CRITICAL The provided software has been verified to work only on PCs running Windows 10 or Windows 11 64-bit operating systems and hardware provided in the Protocol.

56
Download and install LabVIEW Runtime 2019 SP1 from the NI homepage, which is required to run the AAB software.
57
Open the NI Package Manager (available from NI’s website) and install NI-VISA 19.5. This driver enables the communication between the AAB software and hardware via USB connections.
58
Download the AAB software from Supplementary Software and download the ‘HITS-BIO_software_distr.zip’ file on your local computer, followed by extracting the files.
59
Place the extracted folder in a specific directory on your computer for easy access.
60
Double-click on HITS-BIO_software_distr.exe to open and confirm the execution of the AAB software. The AAB software is precompiled and does not require any additional installation steps once extracted.

◆ TROUBLESHOOTING
61
Download Arduino IDE software from the official Arduino website and an example code for the Adafruit MPRLS sensor (provided by Adafruit, https://github.com/adafruit/Adafruit_MPRLS).
62
(Optional) Install DinoCapture 2.0 software if you use the Dino-Lite digital microscope from the Dino-lite website.
63
(Optional) Install Digital Viewer software if you use the Plugable digital microscope from the Plugable website. In addition, alternative camera software can be used (for example, IP Camera Viewer 4).

Hardware and software integration and configuration

● TIMING ~1 h

▲ CRITICAL The provided AAB software and instructions are designed to the specific components demonstrated in this Protocol. If different parts are used (for example, motion stages or solenoid valves from other manufacturers), configuration steps and software may require adjustments. Connect the power and USB to the computer before execution of the AAB software. Any changes in the AAB software settings must be made while the AAB software is stopped by pressing the ‘Abort execution’ button (Fig. 5, toolbar). After changes are made, restart the software by pressing the ‘Run’ button (Fig. 5, toolbar). The default software configurations are shown in Fig. 5. Table 1 provides a detailed list of user interface elements in each panel of the AAB software, along with their functions and corresponding shortcuts.

COM port settings for the motion stage

64
Open the device manager on your computer.
65
Expand the ports (COM (communication) and LPT (line printer terminal)) category and locate the motion stage port.
66
Right-click the port > Properties > Port setting > Advanced.
67
Change the COM port number to an available port (COM8 is recommended as it is the default setting in the AAB software).

▲ CRITICAL STEP Ensure thatno conflicts exist between port numbers for multiple devices.
68
Apply changes.

Motion stage and software connection

69
Open Zaber Launcher software to recognize and assign device numbers to each axis, x axis (device no. 01), y axis (device no. 02) and z axis (device no. 03). Confirm all axes are detected and operational in Zaber Launcher, then exit the software. Before using the AAB software, test the motion stage control using Zaber Launcher to ensure proper function.

Pneumatic sensor connection

70
COM port settings for pneumatic sensor: open the Arduino IDE software and navigate to the Adafruit MPRLS Library. Load the ‘simpletest’ example file provided by Adafruit (https://github.com/adafruit/Adafruit_MPRLS). Change the COM port number (Steps 64–68) to a recommended value (for example, COM20) and apply changes.

▲ CRITICAL STEP Ensure that no conflicts exist between port numbers for multiple devices.
71
Upload the example code to the Arduino board, following the instructions provided in the Adafruit MPRLS Library documentation and monitor the readings in the serial monitor. Ensure the proper wiring and connectivity between the pneumatic sensor and Arduino board.

▲ CRITICAL STEP The default example code outputs readings in hPa and PSI with unit characters. Modify the code to return only numeric values, removing unit characters after converting the readings to the desired unit, as the AAB software processes numeric values only.

◆ TROUBLESHOOTING
72
Cross-check the pressure sensor’s output value with the set value of the pressure controller (Ultimus II, Nordson EFD) at a fixed pressure or vacuum level to ensure accuracy of pressure reading.

Set up cameras

73
Connect all cameras to the computer via USB or other provided interfaces. Confirm all the cameras are functioning properly and displaying the desired field of view in the camera software.

Solenoid valve connection

74
Locate the address selection switch (dip switch) on the top left of the WAGO Ethernet controller (Fig. 2b). Use this switch to configure the IP address of the controller to 192.168.1.100. Each switch corresponds to a binary digit, starting from bit 0 at switch 1 (bottom) to bit 7 at switch 8 (top). The switch toggled left represents binary 1. The switch toggled right represents binary 0. The binary configuration for the WAGO Ethernet controller in this Protocol is 00100110, where the third, sixth and seventh switches are toggled left, while all other switches are toggled right (Fig. 2b). Calculate the decimal equivalent of the binary configuration. For example, binary 00100110 = 2² + 2⁵ + 2⁶ = 100, and the resulting IP address is 192.168.1.100.

▲ CRITICAL STEP To establish the communication between the WAGO Ethernet controller and computer, the IP address in the AAB software (Fig. 5, panel 3) must be adjusted to match the determined IP address by the dip switch.
75
Set the subnet mask to 255.255.255.0 in the WAGO Ethernet Settings software.
76
Navigate to Settings > Network and Internet > Network and Sharing Center > Change Adapter Settings > Local Area Connection > Properties.
77
Locate the Internet Protocol Version 4 (TCP/IPv4) in the list and click Properties.
78
Manually set the IP address to 192.168.1.1 (or any other 192.168.1.x address, except x = 100, to prevent an IP conflict). Set the subnet mask to 255.255.255.0 and leave the default gateway blank.

▲ CRITICAL STEP Ensure that there are no IP conflicts with other devices on the same network. Using the same IP address will prevent communication. Proper alignment between the computer and Ethernet controller’s IP address is crucial for stable communication.

Execute the AAB software

79
Launch the provided AAB software by double-clicking the executable file. Once initiated, the software runs automatically.

▲ CRITICAL STEP Ensure that all connected hardware (motion stage, solenoid valves, pneumatic sensor and cameras) is powered on and connected to the computer before executing the AAB software. If any hardware is not properly connected at launch, press the ‘Abort execution’ button in the software to stop execution, exit the software, reconnect the hardware and then restart the software.
80
(Optional) To modify configurations (related to the hardware) within the software, press the ‘Abort execution’ button, adjust settings as needed and resume operation by pressing the ‘Run’ button.

Connect motion stage to the AAB software

81
Confirm that the AAB software has the correct COM port number assigned for the motion stage (default: COM8) (Fig. 5, panel 1). If needed, reassign the COM port number in the software.

▲ CRITICAL STEP The COM port for the motion stage must match the default or configured port in the software. Avoid connecting the same COM port to multiple programs simultaneously, as this may lead to connection errors.

◆ TROUBLESHOOTING
82
Once connected, verify the connection by checking the ‘current X’, ‘current Y’ and ‘current Z’ values in the software display (Fig. 5, panel 5). These values should read ‘203’ when the motion stage is successfully detected. This value (for example, 203, representing the maximum travel distance) may vary depending on the motion stage specifications.

Connect solenoid valve to AAB software

83
Ensure that the slave address (Fig. 5, panel 3) matches the defined IP address set in Steps 74–78 (default: 192.168.1.100).
84
Press the ‘Connect’ button in the AAB software to establish communication with the WAGO Ethernet controller.
85
Verify a successful connection by observing the green light-emitting diode (LED) indicator on top of the ‘Connect’ button. If the connection fails, an error code will appear in the string indicator. The ‘NS’ LED on the WAGO Ethernet controller (Fig. 2b) will blink if the connection is not established and will remain steady once connected.

◆ TROUBLESHOOTING

Connect pneumatic sensor to the AAB software

86
Confirm the COM port number assigned to the pneumatic sensor in the AAB software (default: COM20) (Fig. 5, panel 2). If needed, reconfigure the port number.

▲ CRITICAL STEP The COM port for the pneumatic sensor must match the default or configured port in the software. Avoid connecting the same COM port to multiple programs simultaneously, as this may lead to connection errors.
87
Confirm the connection. Once successfully connected, a live reading will appear on the LED indicator in the AAB software interface.

◆ TROUBLESHOOTING

Initialization process

88
Initialize motion stage: press the ‘Home All Axes’ button (Fig. 5, panel 8) in the AAB software to set the positions of the x, y and z axes to (0, 0, 0). Observe LED indicators on the driver for each linear stage. A green LED indicates that the motion stage is stable and paused. An orange or other-colored LED indicates that the motion stage is active and in motion.

▲ CRITICAL STEP Ensure that the axes are properly homed to avoid positional inaccuracies during operation. Confirm that the ‘Needle’ button appears on panel 6 in Fig. 5, as it is required for the AAB operation. The other button, ‘Holder’, moves in the opposite direction and is not appropriate for this Protocol.
89
Initialize solenoid valve: after connecting the solenoid valve system (Steps 83–85), press the ‘Initialize controller’ button located in panel 3 (Fig. 5). Confirm that the ‘NS’ LED on the WAGO Ethernet controller is lit (indicating successful connection). Verify that the LED lights in the eight-channel digital output modules are ON, indicating that the solenoid valves are in the OFF state.
90
Initialize pneumatic sensor: press the ‘Initialization’ button (Fig. 5, panel 2) after the pressure reading has stabilized. This action will set the current pneumatic reading to 0.

▲ CRITICAL STEP Perform pressure sensor initialization after the initialization of the solenoid valve. Activating solenoid valves before this step can lead to incorrect pressure readings due to preexisting pressure/vacuum conditions in the pneumatic system. If not properly initialized, subsequent pressure-based operations may yield unreliable results.
91
Configuration: confirm the following parameters in the AAB software and reconfigure if the hardware settings are different.
- Movement type (for example, absolute or relative positioning). We recommend using the default settings
- Units (for example, millimeters for distance). We recommend using the default settings
- Microstep size (to adjust motion precision). This depends on the motion axis specification
- COM port numbers (for motion stage and pneumatic sensor)
- Assigned SVN no. (for solenoid valves as described in Steps 41–50)
- IP address (for the WAGO Ethernet controller)
- Printing velocity (to define the motion speed of the stage during operations)
  
  ▲ CRITICAL STEP Refer to Table 1 for detailed descriptions of each numeric control and button function within the software interface. Double-check all configurations before starting the AAB process to ensure proper function. Misconfigured parameters may lead to errors in hardware control or failed bioprinting operations.

Validation of motion control

▲ CRITICAL Proper validation at this stage ensures smooth and reliable operation during bioprinting.

92
Manually enter coordinates for the x, y and z axes on the AAB software interface (Fig. 5, panel 7).
93
Select predefined movement distances (0.01 to 20 mm or custom values) with shortcut keys for distance adjustments detailed in Table 1.
94
Use keyboard shortcuts (Table 1) to test the motion control in each direction (Fig. 1k).
- x axis: move left using the ← arrow key and right using the → arrow key.
- y axis: move forward using the ↓ arrow key and backward using the ↑ arrow key.
- z axis: move up using the ‘Page Up’ (PgUp) key and down using the ‘Page Down’ (PgDn) key.
  
  ▲ CRITICAL STEP Misplacing the motion stage while setting up can result in reversed or incorrect movements. Ensure the hardware orientation matches Figs. 1k and 3e.
95
Validate motion control accuracy by placing a transparent grid on the printbed. Observe the stage motion to ensure that it moves precisely and aligns with entered coordinates. If this step has been performed previously, skip to the next step.

◆ TROUBLESHOOTING

Validation of solenoid valve control

▲ CRITICAL Proper validation at this stage ensures smooth and reliable operation during bioprinting.

96
After initialization of the WAGO Ethernet Controller (Step 89), activate valves using the corresponding buttons (‘Pressure’, ‘Aspiration’ and ‘Minimum vacuum’) in panel 10 (Fig. 5).

◆ TROUBLESHOOTING
97
Monitor the pressure sensor readings to validate in panel 2 (Fig. 5). When the pressure valve is on, the sensor should display a value above 0. When the aspiration valve is on, the sensor should display a value below 0.

▲ CRITICAL STEP After turning valves on and off, confirm that the pressure sensor reads 0 when all valves are closed. Incorrect readings indicate a need for reinitialization.

◆ TROUBLESHOOTING
98
Test the other pneumatic control buttons for single-nozzle and high-throughput modes to confirm function.

■ PAUSE POINT Once the hardware and software have been validated and are functioning as expected, the setup will remain ready for AAB indefinitely. If the system is not in use, powering it off is recommended. When powered off, repeat Steps 79–98. If any configurations differ from the default settings of the AAB software, the correct configuration must be reapplied each time the software is restarted.

Spheroid preparation

● TIMING 1–2 d, excluding the duration for cell expansion

▲ CRITICAL The final spheroid size varies depending on the cell seeding density and cell type. The following steps provide a general protocol for spheroid preparation using cells expressing cadherins. Cells lacking cadherins do not spontaneously form spheroids and may require additional methods or materials. Modify this Protocol on the basis of the specific requirements of the cell type used.

99
Cell expansion: thaw and culture cells in cell culture-treated flasks using the appropriate culture medium for the cell type. Incubate cells in a humidified incubator (37 °C and 5% CO₂). The initial cell seeding density for passaging varies depending on the cell type and proliferation rate. Ensure that the selected flask size is suitable for the number of cells to be expanded.
100
Spheroid preparation: seed cells in a 96-well cell-repellent U-bottom plate with an optimized cell seeding density based on the desired spheroid size with 100–200 μL of culture medium per well. Incubate the plate in a humidified conditions (37 °C and 5% CO₂) for 24–48 h.

▲ CRITICAL STEP The seeding density directly influences spheroid size. Optimize seeding densities for each cell type to ensure reproducible spheroid dimensions suitable for the target application.
101
Monitoring of spheroids: monitor spheroids daily using an optical microscope. Record the size and morphology of spheroids to confirm that they have the desired size. For most applications, spheroids at 24–48 h postformation are recommended, as spheroids at this stage gain sufficient structural properties, providing consistent performance during bioprinting.

▲ CRITICAL STEP Ensure that spheroids meet the size requirements for your experiment. If using a new cell line for the first time, test a range of seeding densities to identify consistency in spheroid size and compactness.
102
(Optional) Spheroid fixation for practice run: collect spheroids and wash them twice with DPBS, followed by fixing with 4% paraformaldehyde for 30 min at RT. The fixation time may need to be adjusted on the basis of spheroid size; however, 30 min at RT is generally sufficient for most spheroids (~<1,000 μm) used in AAB. After fixation, rinse spheroids three times with DPBS. Fixed spheroids can be stored at 4 °C without drying for 2–4 weeks until used for a practice run.

Spheroid characterization

● TIMING ~1 h

▲ CRITICAL This step is essential to understand the biological and physical properties of spheroids for their effective use in AAB. Key parameters include spheroid size, stiffness, deformation behavior and viability under applied aspiration forces.

103
Spheroid size characterization: measure the size of spheroids using an optical microscope. Ensure the consistency in size across the batch to maintain reproducibility during AAB.
104
Assess stiffness and deformation using commercially available equipment (option A) or aspiration-based measurement (option B).
- (A)
  Commercially available equipment
  1. Use commercially available equipment (for example, CellScale) to measure spheroid stiffness. Follow the manufacturer’s instructions for the setup and measurement protocols.
- (B)
  Aspiration-based stiffness measurement
  ▲ CRITICAL This method is a cost-effective alternative when high-cost mechanical testing instruments are unavailable. However, it relies on image-based analysis and serves as a reference rather than a precise measurement. The calculated stiffness may not fully represent the actual mechanical properties of spheroids. Use this approach to understand spheroid behavior under aspiration pressure and refine experimental parameters accordingly.
  1. Prepare an optical microscope equipped with video-recording capabilities.
  2. Use the pressure controller and micromanipulator (the tilting base is removed at Step 15 and is not required for this step).
  3. Use a micropipette puller to prepare the pipette with an optimized pulling settings depending on the filament type and desired micropipette geometry.
    
    ▲ CRITICAL STEP Optimize the pulling settings for the desired ID on the basis of the filament type. The diameter should be neither too small nor too large compared with the spheroid size. Refer to the Sutter Instrument’s pipette cookbook for guidelines.
  4. Connect a micropipette to the pressure controller using tubing.
  5. Place a spheroid with 1–2 drops of culture medium on a clean glass slide under the microscope and focus on the image.
  6. Mount the micropipette on the micromanipulator and position it gently adjacent to the spheroid.
  7. Connect a micropipette to the pressure controller using tubing.
  8. Start video recording.
  9. Set the aspiration pressure to an appropriate value on the pressure controller and apply it to the micropipette.
  10. Observe the spheroid deformation under the microscope and record the aspirated length of the spheroid until no further changes in deformation are observed.
    
    ▲ CRITICAL STEP Avoid vibrations during this step. Adjust the aspiration pressure on the basis of the spheroid type. For example, use lower pressure for soft (that is, cancer cell) spheroids to prevent overdeformation and use higher pressure for hard (that is, osteogenic) spheroids.
  11. Analyze video to measure the ID (α) and OD (β) of the micropipette and the aspirated length (L) of the spheroid using ImageJ.
  12. Calculate the spheroid stiffness (E) on the basis of the following equations^29,30, where ΔP is the applied aspiration pressure, ∅(η) is the wall function (valid for thin-wall micropipettes, where η ≪ 1).
    $E = \frac{3 α Δ P}{2 π L} \emptyset (η)$ (1)
    
    $\emptyset (η) = \frac{1}{2} \times \{\frac{1 + η}{1 + \frac{η}{2}}\} \times ln (\frac{8}{η})$ (2)
    
    $η = \frac{β - α}{α}$ (3)
105
Cell viability: apply incremental aspiration pressure (for example, 0, 1, 3, 5 and 10 mm Hg) to spheroids using a pressure controller (for example, Ultimus II, Nordson EFD) or the AAB setup and analyze viability using LIVE/DEAD staining to assess the viability of spheroids. A detailed description for viability test is provided in Steps 239–243. Check the viability of spheroids via LIVE/DEAD staining after applying different aspiration forces to understand the effect of aspiration force on viability.

Biomaterial preparation and characterization

● TIMING Depends on the biomaterial types

▲ CRITICAL This section provides a general guideline for preparing biomaterials and their characterization used in AAB. Biomaterial composition, concentration and preparation methods must be optimized on the basis of the user’s specific experimental design. This is the minimum requirement to ensure that biomaterials are compatible with bioprinting.

106
Biomaterial preparation: prepare the biomaterials to be used for bioink substrates via EBB or support materials. Biomaterial composition and concentration will vary depending on the specific experimental design and requirements of the AAB protocol. Ensure that formulations are optimized for the bioprinting performance and compatibility with AAB.
107
Biomaterial sterilization: sterilize the prepared biomaterials using appropriate sterilization methods based on the biomaterial type and stability.

▲ CRITICAL STEP The ideal sterilization method varies depending on the biomaterial. For example, powder or sponge forms can be sterilized before hydrogel formulation using ethylene oxide or gamma irradiation, which are both compatible with a wide range of biomaterials. UV sterilization is suitable for small volumes spread in thin layers, as its limited penetration may not fully sterilize thicker samples. Autoclaving is appropriate for heat-stable materials, as high temperature, moisture and pressure may cause hydrolysis and degradation. Aqueous solutions can typically be sterilized via 0.22-μm filtration, though this is not suitable for highly viscous biomaterials due to filter clogging. Sterilization is optional for nonbiological applications but materials must be sterilized before incorporating cells or biologics to ensure aseptic conditions.

Rheological characterization

▲ CRITICAL This section provides a general overview of rheological characterization. Since optimal settings vary depending on the biomaterial and experimental design, users should optimize parameters and setup (for example, geometry and gap distance) on the basis of their specific system.

108
Prepare and test biomaterials without air bubbles to ensure accurate measurements.
109
Equip the rheometer with suitable geometry (for example, a 25-mm parallel-plate) based on the biomaterial properties and the type of test to be performed. Refer to the rheometer manufacturer’s instructions for operation and setup. Smaller geometry (for example, <25 mm in diameter) is suitable for high-viscosity materials (for example, >10 Pa s), while larger geometry (for example, >40 mm in diameter) suits low-viscosity materials (for example, <0.1 Pa s).
110
For specialized tests, equip the rheometer with additional components (for example, UV-curing accessories for photo-crosslinkable biomaterials).
111
Load the biomaterials on the center of the Peltier plate. Use an appropriate volume to completely fill the measurement gap and avoid air bubbles.
112
Set the initial gap distance slightly higher than the desired measurement gap distance (for example, if the final gap distance is 500 μm, set the initial gap at 550 μm). Adjust the initial gap on the basis of the viscosity: use a smaller additional distance for low-viscosity (for example, <0.1 Pa s) materials and larger ones for high-viscosity (for example, >10 Pa s) materials. If the torque reading is too low after loading, reducing the gap may help stabilize the readings. For particle suspensions, the measurement gap should be at least 10× larger than the maximum particle size. Cone geometries have fixed gaps that should be used as specified.
113
Lower the gap to the final gap distance (for example, 500 μm for the 25-mm parallel-plate geometry). The optimal gap distance depends on the type of geometry and biomaterial.
114
Trim the excessive amount of biomaterials from the edges of the geometry to ensure clean and uniform contact.

▲ CRITICAL STEP Prevent samples drying during extended tests by using a solvent trap or applying oil (for example, silicon oil) around the sample.
115
Perform rheological tests (for example, amplitude sweep, frequency sweep, flow, sweep, recovery sweep tests and so on) to characterize biomaterials properties and ensure that biomaterials are suitable for use as a support material or an extrudable bioink. Adjust test parameters on the basis of the biomaterial type. For frequency sweeps, ensure strain is below the yield point of the biomaterials and within the linear viscoelastic region. Repeat measurements under identical conditions to ensure reproducibility and examine the data for irregularities (for example, sudden drops, phase shifts or nonlinearities). When such behaviors are observed, wall slip or other artifacts may be present in the measurement. If slip is suspected, the use of roughened or serrated geometries may be considered to improve interfacial grip and reduce measurement error.

EBB preparation and optimization (optional for EBB)

● TIMING 1–2 min to create 5-mm diameter of a bioink substrate with 10 mm/s of printing speed, but timing depends on the size of constructs and printing speed

▲ CRITICAL This section is optional and applies to hybrid bioprinting when using both EBB and AAB or EBB-only workflows. The optimization of printing parameters, including pneumatic pressure for extrusion and printing speed, is highly recommended before proceeding with the bioink use to ensure optimal printability.

116
Prepare the bioink for EBB: ensure the bioink is properly mixed, free of air bubbles and sterilized if used for biologics. Bioink properties, such as viscosity and yield stress, should be precharacterized as described in the previous step.
117
Prepare the toolpath (.csv file) for automated EBB motion: design the movement path using absolute coordinates (for example, create a 10 mm × 10 mm square using Fig. 7c as a reference). An example of the automated motion is provided in Supplementary Data 2.
118
Save the sequential motion path into a .csv file with three columns representing x, y and z coordinates (Fig. 7c). Negative (−) value on the third column (for z axis movement) in the .csv file raises the extrusion nozzle, while positive (+) movement lowers it.
119
Import the prepared .csv file for the automated EBB motion by pressing the ‘Read coordinates from CSV – automated motion’ button (Fig. 5, panel 9B). Confirm that the file is imported correctly by verifying the displayed coordinates in the AAB software.
120
Ensure that the EBB/single-nozzle AAB holder and isometric camera holder have been mounted on the z axis using an M6–1.0 × 20-mm mounting screw (Fig. 1h). Mounting the DCNA holder is optional, as this section (Steps 117–121) focuses on EBB optimization.
121
Camera adjustments: adjust the focus and orientation of the cameras utilized (isometric camera and two bottom-view cameras) as needed to monitor the bioprinting process. Confirm that the camera resolution and lighting are appropriate for monitoring fine details during bioprinting.

Fig. 7 | — a, An illustration of the preset positions for AAB. b, A schematic guiding the required positional alignment between EBB and DCNA nozzles for hybrid bioprinting. c, An example of the method used to generate a .csv file for automated motion in Steps 117–118. d, A process for the DCNA recognition and synchronization between connected valves and nozzle arrangement in a 4 × 4 configuration. e, A schematic for the quantitative measurement of positional accuracy of bioprinted spheroids.

Bioink loading and setup

122
Load the bioink into a 1-cc syringe, ensuring that no air bubbles are present.
123
Cut the flange and plunger from the syringe barrel to connect 3/8 inch OD tubing.

▲ CAUTION Use a blade carefully to avoid injury.
124
Mount the syringe barrel onto the EBB/single-nozzle AAB holder (prepared in Steps 10–11).
125
Attach the tubing to a pneumatic controller (for example, Ultimus II, Nordson EFD). To streamline operations, it is recommended to use separate pneumatic controllers for EBB and AAB processes. However, without the separate pneumatic controllers, EBB can be also performed by connecting the EBB tubing to an empty solenoid valve in valve manifold no. 3, followed by assigning this SVN no. to the AAB software setting in panel 3 (Fig. 5).
126
Apply pressure and observe the bioink extruding from the nozzle using the isometric view camera.
127
Adjust the pressure force to determine the optimal extrusion pressure and ensure consistent bioink extrusion.

◆ TROUBLESHOOTING

EBB testing and optimization of printing parameters

128
Move the EBB nozzle to a 35-mm Petri dish placed on the printbed.
129
Align the center of EBB nozzle with the center point of the bottom camera’s field of view as shown in Fig. 7b.
130
Turn the system ‘ON’ using the button located in panel 10 (Fig. 5) in the AAB software.

▲ CRITICAL STEP The automated motion will not work unless the status indicates ‘ON’ in the button.
131
Apply pressure to start bioink extrusion and press the ‘Start’ button (Fig. 5, panel 10). To stop during the automated motion, switch the button to ‘OFF’.
132
Validate that the extrusion nozzle follows the designed motion path (imported .csv file from Step 119) correctly. Confirm ‘absolute’ mode is activated (Fig. 5, panel 9A).
133
Optimize EBB nozzle size, the gap between nozzle-end and substrate, printing velocity (adjust in Fig. 5, panel 9A) and pneumatic pressure by monitoring printability using both bottom view and isometric cameras. Under optimal EBB conditions, the extruded bioink forms a continuous, straight line without interruptions or inconsistencies. Optimal printing parameters will depend on the bioink viscosity, nozzle size and printing speed.

◆ TROUBLESHOOTING

AAB process

● TIMING The time required for bioprinting depends on the number of spheroids and the user’s proficiency

▲ CRITICAL The success rate of spheroid lifting from the chamber is near 100% under optimized conditions. However, user skills and proficiency greatly impact the success of AAB.

134
Prepare spheroids: transfer spheroids into a 35-mm Petri dish filled with an appropriate medium. Place the dish on bed space 1 (Fig. 1k) and swirl gently to collect spheroids at the center. Confirm the spheroid visibility using the bottom camera.

▲ CRITICAL STEP Perform all biological procedures under sterile conditions within a biosafety cabinet.

◆ TROUBLESHOOTING
135
Prepare bioinks for bioprinting using either EBB or support materials.
- For the creation of bioink substrates using EBB, load the bioink into a 1-cc syringe without the entrapment of air bubbles, attach an appropriate nozzle and mount onto the EBB syringe holder. In this Protocol, EBB is integrated with the high-throughput AAB (Strategy 2); however, it can also be used for the single-nozzle AAB (Strategy 1) by preparing separate holders for EBB and the single-nozzle AAB
- For support materials, load the material into a suitable chamber and place it on ‘bed space 2’. If the chamber does not fit the printbed’s camera window, design a suitable adapter for mounting
136
Proceed with AAB using the single-nozzle (Strategy 1 and Fig. 4a) or high-throughput mode (Strategy 2 and Fig. 4b).

Strategy 1: single-nozzle mode AAB

● TIMING ~30 s/spheroids (depends on the size and number of spheroids and the user’s skillset to operate AAB)

137
Printhead setup: mount the EBB/single-nozzle AAB holder and the isometric camera holder on z axis using M6–1.0 × 20-mm screws (Fig. 1h) without the DCNA holder. Organize cables and tubing using cable ties to prevent interference with motion.
138
Switch to the single-nozzle mode: press the ‘Single mode’ button and select the ‘Single’ tab to enable single-channel control (Fig. 5, panel 10).

Single-nozzle AAB preparation

139
Remove the flange from a syringe (1-cc) barrel and connect tubing (3/8 inch OD) to the syringe barrel via a 1/4–3/8 inch reducer (Fig. 2c).

▲ CAUTION Use a blade carefully to avoid injury.
140
Connect the 1/4 inch OD tubing to the 5/32–1/4 inch reducer to connect 5/32 inch OD tubing for single-nozzle AAB assigned solenoid valve (SVN no. 0). Follow Fig. 2c for detailed tubing instructions.
141
Insert the syringe barrel with a mounted nozzle into the single-nozzle AAB holder (Fig. 3h). Ensure that it is the appropriate nozzle size for the spheroid utilized.
142
Adjust the isometric camera focus on the nozzle tip to monitor the process.
143
Place an empty 35-mm Petri dish on ‘bed space 1’.
144
Define maximum Z position: move the nozzle tip to touch the Petri dish surface using fine increments in z axis. Observe nozzle bending or contact with the Petri dish via the bottom camera. Click ‘Set Max Z’ and activate this function (switch to ‘ON’) (Fig. 5, panel 6).

▲ CRITICAL STEP This step is required to protect nozzles and samples from any damages experienced during AAB by setting up the limit switch as a safety setting. If a certain distance step is selected and the sum of the current Z coordinate and the distance step exceeds the defined maximum Z, the motion will not proceed. For example, if the current Z position is 100 mm and the defined maximum Z is 110 mm, the z axis will not move beyond 110 mm. In case of objects with varying Z heights, select the lowest Z height to avoid accidental collisions. Deactivate this function by switching it to ‘OFF’ only when a new maximum Z position needs to be set or when fine adjustments are required.

Preset positions

▲ CRITICAL Detailed illustration is demonstrated in Fig. 7a. To input the current position, press ‘Pos #’ buttons located in panel 4A (Fig. 5). To move to each position, click ‘play button’ located in the same row to move to the position. An example of the preset positions is provided in Supplementary Data 1.

145
Place the nozzle tip above the empty 35-mm Petri dish, where it will be used as a spheroid chamber for bed space 1. Ensure that the nozzle is placed above the surface of the Petri dish. Set this position as position no. 5 (Pos 5).
146
Lower the nozzle tip close to the substrate placed on bed space 1. Once the tip touches the substrate, raise it by ~1 mm and move the tip to the center. Set this position as position no. 6 (Pos 6). Pos 6 will be reconfigured after placing spheroids.
147
Move back to Pos 5 by clicking the ‘Play’ button for Pos 5. Set this position as position no. 7 (Pos 7).
148
Move the nozzle to bed space 2 using x and y axes. Set this position as Position no. 8 (Pos 8). If an object on bed space 2 is taller than the spheroid chamber on bed space 1, set this height as z height on Pos 5, 7, 8 and 10. Update Pos 5 and 7 accordingly, if necessary.
149
Place the nozzle in bed space 2 close to the substrate at the center of the camera window (Fig. 7b). Set this position as position no. 9 (Pos 9).

▲ CRITICAL STEP Avoid placing the nozzle too close.
150
Move back to Pos 8 by clicking the ‘Play’ button for Pos 8. Set this position as position no. 10 (Pos 10).
151
Repeat these movements continuously by pressing each saved position sequentially to confirm that the nozzle moves without contacting any objects.

▲ CRITICAL STEP If the nozzle tip hits any obstacles, reconfigure the positions appropriately to prevent contact. When replacing the nozzle, verify that the saved positions are accurate. If the new nozzle is at a different position, readjust and redefine positions (Steps 145–150).

■ PAUSE POINT At this stage, the printing setup for the single-nozzle AAB is complete and can be paused until spheroids and bioinks are ready to use.
152
(Optional) Save preset positions: press ‘Call table’ (Fig. 5, panel 4A) and confirm displayed positions in the ‘Loaded XYZ Coordinates’ chart (Fig. 5, panel 4B). Next, click ‘Save as file’ (Fig. 5, panel 4A) and save the .csv file containing preset positions for future use. Ignore the next popup window. The saved position can be loaded later if the setup remains unchanged.
153
(Optional) Load preset positions: press the folder button (Fig. 5, panel 4A) to import the saved .csv file from Step 152. Reconfirm the alignment of saved positions by repeating Step 152 if the setup is modified.

Reconfiguration Pos 6

▲ CRITICAL Detailed illustration is demonstrated in Fig. 7a.

154
Spheroid placement: transfer spheroids into a 35-mm Petri dish and add ~3 mL of medium. Ensure that there is sufficient volume of culture medium for proper spheroid aspiration.

▲ CRITICAL STEP A limited volume will affect successful spheroid lifting.
155
Press the ‘Play’ button for Pos 6 to bring the nozzle to the defined position.
156
Adjust the nozzle tip to ~0.2 mm above the top surface of spheroids in the chamber using small step distances (for example, 0.1 mm) and the PgDn key.

▲ CRITICAL STEP Ensure that the nozzle does not fork or damage spheroids. Estimate the distance by gently moving the nozzle side to side (~0.5 mm) while monitoring via the bottom camera.
157
Redefine Pos 6 by setting this position as the new Pos 6.
158
Move back to Pos 5 or 7 before proceeding.

Pneumatic control setting

159
Initialize the WAGO Ethernet controller to close all valves (Step 89). Activate aspiration (press the ‘Aspiration’ button in Fig. 5, panel 3) and set the optimal aspiration force (Step 105) by adjusting the vacuum regulator and monitoring pressure sensor readings.

Single-nozzle AAB process

▲ CRITICAL A detailed illustration and workflow are demonstrated in Figs. 4a and 6. Each step should be monitored carefully via cameras from multiple angles to prevent damages to spheroids or misalignment. The default motion speed and acceleration are set to be 10 mm/s and 10 mm/s², respectively. Adjust these values as needed for optimal performance.

160
Move to Pos 5 and then to Pos 6 to approach the nozzle to spheroids.
161
Align the nozzle tip above a spheroid and apply aspiration by pressing the ‘Single channel’ button in the ‘Single’ tab (enabled after clicking the ‘Single mode’ switch in Fig. 5, panel 10).

▲ CRITICAL STEP Depending on spheroid stiffness and material properties, it may be advisable to deactivate the aspiration force immediately after lifting spheroids from the chamber to minimize deformation and facilitate detachment. Note that even after the aspiration is deactivated, the spheroids remain adhered to the nozzle tip. For example, if the spheroids or the substrate are soft, maintaining the aspiration force during placement may lead to failure. For a rigid support material, maintain the aspiration force to ensure spheroids are placed successfully to their desired positions without detachment.
162
Lift the spheroid to Pos 7 and transfer it to Pos 8 and 9 for placement.

◆ TROUBLESHOOTING
163
Gently place the spheroid at the desired position and cut off the aspiration force immediately.

◆ TROUBLESHOOTING
164
Confirm successful detachment of the spheroid using cameras.
165
Repeat this step until the desired number of spheroids are bioprinted. Keep monitoring the medium volume in the chamber and fill up accordingly.

Strategy 2: high-throughput AAB mode operation

● TIMING 3–4 min to bioprint 64 spheroids, plus ~1 min to create a 5-mm (in diameter) bioink substrate at 10 mm/s printing speed. TIMING depends on the size of constructs and printing speed

166
DCNA mount: mount the DCNA onto the holder and tighten screws to immobilize the DCNA. Ensure the bottom of the DCNA is parallel to the holder roughly.
167
DCNA connection: connect a tubing to the DCNA by securely plugging the male luer connectors into each individual nozzle (Fig. 2c).
168
Printhead setting: mount the DCNA holder, EBB/single-nozzle AAB holder (optional) and isometric camera holder (optional) on the z axis using M6–1.0 × 20-mm screws.

▲ CRITICAL STEP Ensure that all cables and air tubes are organized with cable ties to prevent interference with the motion stage.
169
EBB preparation: follow Steps 116–133 for preparing EBB. Test the extrusion parameters before bioprinting.
170
Side-view camera installation: install a high-resolution, high-magnification side-view camera focused on the print area (Fig. 3h). This camera is useful for monitoring spheroid detachment during the high-throughput mode operation. However, it is optional in the single-nozzle mode as spheroid detachment can be easily monitored using the isometric camera.

▲ CRITICAL STEP The camera should not obstruct the bioprinting process and must have sufficient focal length.
171
High-throughput mode selection: activate the high-throughput mode by pressing the ‘High-throughput mode’ button and enabling the ‘High-throughput’ tab (Fig. 5, panel 10).

DCNA surface leveling for printing alignment

172
Place a glass slide on bed space 1.
173
Lower the DCNA until it gently touches the glass slide.

▲ CRITICAL STEP Avoid damaging the DCNA.
174
Adjust the pitch, roll and yaw using the tilting base and DCNA holder hinge (Fig. 3g) to ensure all nozzles touch the glass slide, verified through bottom and side-view cameras.
175
Once aligned, move the DCNA upward and proceed to nozzle recognition.

DCNA recognition and synchronization

▲ CRITICAL Detailed illustrations are demonstrated in Fig. 7b,d. These steps are optional but highly recommended for identifying the individual pneumatic valve connected to the associated nozzle in the DCNA, especially for selective patterning.

176
Place a 35-mm Petri dish filled with DI water on bed space 1.
177
Lower the DCNA close to the water surface.

▲ CRITICAL STEP Avoid submerging the DCNA in DI water.
178
Turn on pressure using the ‘Pressure’ button and activate nozzles one-by-one by pressing the ‘1’–’16’ buttons (Fig. 5, panel 10) to identify their corresponding channel numbers by observing air bubbles under the bottom-view camera.

◆ TROUBLESHOOTING
179
Observe which nozzle corresponds to which activated valve and record this mapping by labeling the DCNA arrangement (Figs. 5, panel 10 and 7d). This step ensures accurate synchronization between the randomly connected tubing in the DCNA and the actual nozzle arrangement.
180
Halt the AAB software by pressing the ‘Abort execution’ button (Toolbar, Fig. 5).
181
Input the labeled mapping into the array table (Figs. 5, panel 10 and 7d) and restart the AAB software by pressing the ‘Run’ button (Toolbar, Fig. 5) to apply the updated configuration.
182
Confirm that the updated nozzle arrangement in the AAB software matches with that in the camera view.
183
Move the DCNA upward and remove the Petri dish.

Preset positions and spheroid preparation

184
Preset positions: define the positions as described in Steps 145–151 for the single-nozzle AAB.

▲ CRITICAL STEP When Pos 9 is set, locate the center of the DCNA to the center point of the camera view to align between the EBB nozzle and the DCNA (Fig. 7b).
185
(Optional) Preset positions for EBB: define and save positions for EBB nozzle operations (for example, Pos 1—nozzle above Petri dish, Pos 2—nozzle near substrate at the center point (Fig. 7b) and Pos 3—same as Pos 1).

▲ CRITICAL STEP Ensure that the EBB nozzle tip is at approximately the same height, or slightly lower, than the bottom end of the DCNA to prevent collisions when switching between EBB and AAB operations. When the clogged nozzle is replaced during operation, verify that the new nozzle is positioned at the center point (Fig. 7b) in Pos 2 and adjust Pos 2, if necessary.
186
(Optional) Set maximum Z position: set the maximum Z position to protect the nozzles and samples. Activate the function of maximum Z positioning using the ‘ON’ button (Fig. 5, panel 6) and adjust as necessary. The maximum Z position should be set on the basis of the DCNA nozzle-end-height to maintain the safe operation. Refer to Step 144 for detail on setting the Z limit.
187
Confirm saved positions: sequentially test each saved position to ensure nozzles avoid obstacles. Verify both DCNA and EBB nozzles move without interference. Pos 4 is used as a backup position. These positions can be saved as described in Step 152.
188
Spheroid preparation: transfer spheroids into a 35-mm Petri dish placed on bed space 1 and add ~3 mL of medium.

▲ CRITICAL STEP Ensure the medium level in the dish is below the exposed nozzle tip (4 mm) of the DCNA. Submerging the DCNA in medium increases surface tension and forms water droplets, which prevent successful spheroid lifting and placement. Use a 35-mm Petri dish with a lower height to avoid contact with the DCNA holder, if necessary.

High-throughput AAB process

▲ CRITICAL Detailed illustrations and workflows are demonstrated in Figs. 4b and 6. Monitor the entire process using cameras to prevent spheroid damage or misalignment. Adjust parameters as needed on the basis of spheroid stiffness and substrate properties.

▲ CRITICAL If EBB is being used, perform Steps 189–192; otherwise, proceed directly to step 193.

189
Move to Pos 1 to position the EBB nozzle in bed space 2.
190
Lower the EBB nozzle to Pos 2 and find the optimal gap for extrusion (as optimized in Steps 128–133).
191
Apply optimized pressure to extrude bioink and press the ‘Play’ button (Fig. 5, panel 9A) to print the bioink substrate.
192
After extrusion, remove the pressure and return to Pos 3.
193
Move the DCNA to Pos 5 above the spheroid chamber.
194
Activate aspiration with the ‘Aspiration’ button. If required, enable the ‘Minimum vacuum’ (Fig. 5, panel 10).
195
Assign active nozzles using the ‘DCNA arrangement set’ (Fig. 5, panel 10).

▲ CRITICAL STEP To selectively pattern nozzles in the DCNA, Steps 176–183 must be completed before this step.
196
Lower the DCNA to Pos 6 to approach spheroids. Refer to Steps 155–158 for proper positioning of the DCNA above the spheroids.
197
Apply the optimum aspiration force by sequentially pressing ‘Aspiration On/OFF’ and ‘Aspiration set coils’ buttons to activate the selected nozzles in the DCNA (Fig. 5, panel 10) and confirm spheroid capture using bottom-view camera in bed space 1.

◆ TROUBLESHOOTING
198
Lift spheroids gently with the DCNA to Pos 7 under the observation of the bottom camera. (Optional) To slowly lift the spheroids, use SLOW mode (Fig. 5, panel 4A).

▲ CRITICAL STEP For soft spheroids or bioprinting onto bioink substrates, it may be advisable to deactivate the aspiration force immediately after lifting spheroids from the chamber to minimize deformation and facilitate detachment. Note that even after aspiration is deactivated, spheroids remain adhered to the nozzle tip.

◆ TROUBLESHOOTING
199
Move to Pos 8.
200
Lower the DCNA to Pos 9 for spheroid placement.

▲ CRITICAL STEP Avoid harsh contact to prevent damage.
201
Cut off the aspiration force to release spheroids by sequentially pressing ‘Aspiration On/OFF’ and ‘Aspiration Set Coils’ buttons to deactivate the selected nozzles in the DCNA (Fig. 5, panel 10) and confirm spheroid detachment using bottom-view camera in bed space 2 and lift the DCNA to Pos 10.

◆ TROUBLESHOOTING
202
Repeat Steps 193–201 as needed to increase the density of spheroid placement. Continuously monitor nozzle function and medium levels.
203
(Optional) If EBB is being used, return the EBB nozzle to Pos 1 to overlay bioink (if required) and repeat Steps 189–192. If the extruded bioink is photocrosslinkable, use an appropriate light source (for example, 405 nm light for gelatin methacryloyl (GelMA) containing lithium phenyl-2,4,6-trimethylbenzoylphosphinate) to crosslink the bioprinted construct.

▲ CRITICAL STEP The EBB nozzle should not touch bioprinted spheroids.

(Application) Intraoperative AAB

● TIMING ~5 min to create 5-mm constructs containing 64 spheroids into calvarial defects

▲ CAUTION Before performing any animal experiments, ensure the protocol is approved by IACUC or an equivalent ethics committee. Adhere to all institutional and legal regulations regarding animal research.

204
Place all autoclaved surgical tools on a sterile surface.
205
Thoroughly sterilize the workspace using 70% ethanol and disinfect the bioprinting area, including the printbed and surrounding surfaces. Maintain a sterile environment throughout the procedure to minimize the risk of infection or contamination.
206
Wear appropriate personal protective equipment, including sterile gloves, gowns and masks.
207
Anesthesia induction: gently transfer the animal from its home cage into an anesthesia chamber. Use 2–3% (vol/vol) isoflurane in medical-grade oxygen air at a flow rate of 0.5–1 L/min for induction of anesthesia. Administer bupivacaine (0.015 mg/kg) and buprenorphine (0.015 mg/kg) via subcutaneous injection. Continuously monitor the animal’s state of consciousness.

▲ CRITICAL STEP Confirm the animal is fully anesthetized before IOB by performing a toe-pinch test. The animal should not exhibit any response. Be careful not to overdose the animal.
208
Positioning on the printbed: carefully place the anesthetized animal on the printbed, ensuring the bed is covered with a heating pad set to maintain the body temperature at 37 °C. Gently apply ophthalmic eye ointment to both eyes to prevent dryness and irritation during the procedure. Position the animal so that the surgical site is directly aligned with the bioprinting area, ensuring stability and accessibility for precise bioprinting.
209
Surgical and IOB preparation: make an incision and create a defect after cleansing with betadine. Align the defect area as parallel as possible to the bottom end of the DCNA to facilitate precise spheroid placement within the defect.

▲ CRITICAL STEP Ensure the animal’s head is rigidly secured using an appropriate fixation device. Any movement of the animal head or body during IOB may lead to misalignment and failure of accurate spheroid placement.
210
IOB: perform IOB using Strategy 1 (Steps 137–165) or Strategy 2 (Steps 166–203). After bioprinting spheroids, visually confirm that all spheroids are accurately placed within the defect using the side-view camera. Detailed illustrations are demonstrated in Fig. 4c.

▲ CRITICAL STEP A pulse oximeter can be used to monitor oxygen levels and heart rate, with values less than 95% indicating mild hypoxia and below 90% indicating hypoxia. Continuously monitor and regulate oxygen flow and isoflurane concentration to maintain a breathing rate ~60 breaths/min. Assess the toe-pinch response to ensure that the animal remains adequately anesthetized.

◆ TROUBLESHOOTING
211
Defect closure and postsurgical cleaning: suture the skin above the defect area using an appropriate suture material and technique. Gently clean the surgical area with sterile gauze soaked in warm saline to remove any residual blood or debris.
212
Animal recovery: transfer the animal to a recovery area on a heating pad to maintain its body temperature during recovery from anesthesia. Closely monitor the animal for regular breathing and regaining consciousness. Administer analgesics as prescribed to manage pain (for example, buprenorphine at the recommended dose). Check the surgical site daily for signs of infection, proper healing and no disruption of the sutures. Record the animal’s weight, behavior and overall health status.

Cleanup AAB

● TIMING ~15 min

▲ CRITICAL Proper cleanup and sterilization after AAB operations are essential to maintain equipment longevity and ensure sterile conditions for future experiments.

213
Remove the syringe barrel used for EBB or single-nozzle AAB and the DCNA used for high-throughput AAB. Disconnect the tubing.

▲ CRITICAL STEP Do not reuse syringes or nozzles for experiments. Discard them in a biohazard sharps container. However, the acrylic plates from the DCNA can be reused after removing the glue and thoroughly sterilizing them with 70% ethanol.
214
Disconnect the DCNA from the connected tubing:
215
Remove the printheads from the z axis.
216
Move the motion stage to home position (0, 0, 0) by pressing the ‘Home all axes’ button (Fig. 5, panel 8) on the AAB software to reset the motion stage to its starting position for future use.

Sterilization of tubing used in DCNA

▲ CRITICAL Tubing used for the DCNA is reusable but can also be replaced.

217
Prepare a 500-mL container filled with 70% ethanol.
218
Place all tubing into the bottle.
219
Connect the WAGO Ethernet controller and initialize the controller.
220
Apply aspiration to all valves used for the DCNA (SVN no. 1–16).

▲ CRITICAL STEP The aspirated liquid is visible in the tubing. Ensure that it does not flow into the solenoid valve system. Do not overaspirate ethanol as it may damage solenoid valves.
221
Apply positive pressure to flush out aspirated ethanol until air bubbles are visible in the bottle.
222
Repeat Steps 219–221 three to five times to thoroughly clean and sterilize the tubing.
223
Disconnect the WAGO Ethernet controller after switching OFF controller (Fig. 5, panel 3). Keep the pressure regulator open to maintain airflow. Even if solenoid valves are disconnected, residual pressure may persist as long as the main pressure supply remains active.
224
Dry the tubing completely with residual pressure overnight.
225
Remove the tubing from the ethanol container and place it in the BSC.

■ PAUSE POINT The tubing can be stored until further use.
226
Turn off all pneumatic systems and disconnect the WAGO Ethernet controller.
227
Stop the AAB software using the ‘Abort execution’ button (Toolbar).
228
Close the AAB software after confirmation.
229
Turn off the main power switch.
230
Wipe down all exposed surfaces, including the printbed, motion stage and surrounding areas, with 70% ethanol, followed by turning on UV.

■ PAUSE POINT The cabinet can remain closed until further AAB operations.

Postbioprinting process and downstream analysis

● TIMING ~2 h+, varies depending on the specific analytical method used for experiments

▲ CRITICAL At this stage in the procedure, proceed with downstream appropriate characterization. This can be modified to ensure their function and viability.

231
Postbioprinting process: gently add an appropriate culture medium to bioprinted constructs. If required, transfer bioprinted constructs to an appropriate culture plate (for example, 24-well or 6-well plate).

▲ CRITICAL STEP When transferring the bioprinted constructs, handle them gently to prevent breakage or deformation.
232
(Optional) Removal of support material: for constructs bioprinted in a support material, remove the support material using an appropriate method (for example, enzymatic, physical, or chemical and so on) after bioprinted spheroids are fully fused. This fusion process typically takes 2–3 d, depending on cell types and culture conditions. The removal method should be selected on the basis of the properties of the support material used.

Positional accuracy of bioprinted spheroids

● TIMING ~30 min

▲ CRITICAL Detailed illustrations are demonstrated in Fig. 7e. Record a bioprinting video to perform this analysis to validate the positional accuracy of the spheroid placement.

233
Target single or multiple positions on a transparent grid placed under the substrate, where spheroids will be deposited.
234
Begin recording the process using the bottom-view camera.
235
Perform AAB and place spheroids at their designated target positions. Conduct this step using the actual bioinks and parameters intended for the experiment.
236
Repeat the placement process multiple times to ensure statistical significance in the accuracy analysis.
237
Stop recording.
238
Analyze the recorded video by measuring the distance between target positions and the center of bioprinted spheroids (Fig. 7e).

Cell viability assessment via LIVE/DEAD staining

● TIMING ~1 h

▲ CRITICAL Additional cytocompatibility assays, such as CCK-8, CellTiter-Glo or alamarBlue, can also be performed, if required.

239
Retrieve bioprinted constructs gently from the culture environment and wash samples with DPBS.
240
Prepare and add LIVE/DEAD assay reagents (see ‘Reagent setup’ for more details). Adjust the reagent volume and concentration on the basis of the size and thickness of the bioprinted constructs. Ensure the complete coverage of constructs with the staining solution.
241
Incubate the samples with the staining solution for 30 min at RT. Protect from light during incubation.
242
Wash stained constructs once with DPBS for 1–2 min to remove excess dye.
243
Mount samples on a confocal microscope and image the labeled cells at 488 nm (for live cells) and 561 nm (for dead cells) to assess cell viability.

Bioprinted sample analysis

● TIMING varies by method

▲ CRITICAL The specific timing depends on the chosen method and experimental requirements. Standard protocols should be followed accordingly.

244
Carefully collect and process the bioprinted samples using appropriate techniques such as tissue sectioning, immunostaining or real-time quantitative polymerase chain reaction, depending on the required downstream analysis.

Troubleshooting

Troubleshooting advice can be found in Table 2, which provides a reference to the most common errors experienced while following this Protocol.

Table 2 |.

Troubleshooting table

Step	Problem	Possible reason	Solution
60	Fails to open the provided software	Software incompatibility, incorrect driver version or missing required module installation	Ensure that all required modules are installed. Verify that installed modules and drivers are compatible with the existing version of the software
60	Error appears when opening the software	Changed folder name; the folder name does not match the .exe file name	Ensure the folder name remains unchanged as originally provided during extraction. Do not rename the folder or any associated files
71	Unusual or unrealistic numbers appear in the pressure value window	An incorrect configuration input (for example, bits per second) in the COM port setting	Verify and correct the configuration settings (bits per second: 115,200) in the COM port setting
81	Unable to connect motion axes	Disconnected or incorrectly configured serial port, or power is not turned on	Ensure that the motion stage is properly connected to the computer via the correct serial port. Verify that the motion stage is powered. Connect the motion stage again to the computer
	COM port not recognized or incorrect	Incorrect COM port number assigned in the computer or software	Open device manager (Windows) to check the assigned COM port number. Ensure that the assigned COM port in the AAB software matches with the one detected in device manager. Disconnect and connect the motion stage again to the computer
	COM port not recognized or incorrect	The same COM port is being used by another application	Close any other software that may use the same COM port. Then, restart the AAB software and attempt to reconnect. If the issue persists, change the COM port manually in the device manager and update AAB software settings accordingly
85	Unable to connect with the solenoid valve driver. The ‘Connect’ button remains disabled	Disconnected or unstable Ethernet cable	Verify that the Ethernet cable is securely connected to both the computer and WAGO Ethernet controller with power on
85		Incorrect IP address configuration	Open the Ethernet settings in the AAB software and computer and confirm the IP address matches with the one assigned to the WAGO Ethernet controller (Steps 74–78). If the IP address is incorrect, reconfigure it in software settings after pressing the ‘Abort execution’ button. Restart the software after updating by pressing the ‘Run’ button
87	No readings appear or an error occurs in the pressure value window	Wiring connection is incorrect or loose	Confirm that the sensor is securely connected to the appropriate port on the Arduino
		Incorrect COM port settings in the Arduino IDE and the AAB software or the same COM port is being used by another application	Close any other software that may use the same COM port. Then, confirm the correct COM port number in the AAB software and ensure that it matches with the settings in the Arduino IDE and computer. If the issue persists, change the COM port manually in the device manager and update the AAB software settings accordingly. Restart the AAB software and attempt to reconnect
		MPRLS simple test example code is not uploaded to the Arduino board	Reupload the ‘MPRLS simple test code’ to the Arduino board using the Arduino IDE software
	Unusual or unrealistic numbers appear in the pressure value window	An incorrect configuration input (for example, bits per second) in the COM port setting	Verify and correct the configuration settings (bits per second: 115,200) in the COM port setting
		Tubing is not connected to the sensor	Check the tubing and ensure that it is properly connected to the sensor
95	Motion stage moves in an unexpected direction	Axes misalignment	Verify that the motion axes are aligned correctly as per Fig. 1k
	Mismatch between motion distance and grid size	Incorrect motion stage configuration (for example, microstep size)	Confirm the correct microstep size of the linear stage is set in the AAB software
	Mismatch between motion distance and grid size	Wrong unit selection in software	Verify that the selected unit (Fig. 5, panel 1) matches with experiment settings
96	Valve is not working	Incorrect SVN no. configuration for the solenoid valves	Check and update the SVN no. configuration in the AAB software
	Valve is not working	Disconnected or unstable wiring	Ensure that all wiring connections to the solenoid valve controller and valves are secure
	Sudden disconnection and ‘NS’ LED on the WAGO Ethernet controller is blinking	Improper connection or misconfiguration	Confirm that all connections are secure
		Timeout settings in the WAGO Ethernet configuration are too short	As a temporary option, reconnect to the WAGO Ethernet controller by pressing the ‘Connect’ button in the AAB software. However, it is recommended to adjust the timeout setting. Open the WAGO Ethernet setting software, increase the watchdog timeout setting, apply changes and restart the system
97	Incorrect pressure readings or no response	Incorrect configuration input (for example, bits per second) in the COM port setting	Check the troubleshooting steps for Step 87
	Incorrect pressure readings or no response	Pressure value not properly initialized	Reinitialize the pressure value by pressing the ‘Initialization’ button on panel 2 (Fig. 5)
	Pressure sensor readings are unstable, suddenly drop or do not change	Air leakage in the system	Check and tighten all tubing and connections to eliminate leaks
127 and 133	Bioink is not extrudable	Bioink is not suitable for EBB	Ensure the bioink has appropriate properties for EBB
		Insufficient pressure	Adjust the pressure until achieving smooth extrusion
		Nozzle is too narrow	Use a wider nozzle, if needed
		Clogged nozzle	Replace the nozzle
	Leakage during extrusion	Loose connections or damaged components can result in inconsistent pneumatic pressure	Inspect and tighten all tubing and syringe connections. Replace damaged components, if necessary
	Extruded lines are inconsistent (not straight or uniform)	Air bubbles in the syringe	Remove air bubbles before loading the bioink into the barrel
		Extrusion pressure that is too low or too high, or printing velocity that is too slow or too fast relative to the extrusion pressure, can cause printing inconsistencies	Optimize the pressure for smooth extrusion and adjust the printing speed accordingly to maintain consistent extrusion
		Nozzle or printbed misalignment	Adjust nozzle-to-substrate distance for uniform deposition
		Suboptimal bioink rheology	Modify the bioink composition or concentration to improve the flow consistency
		Clogged nozzle	Replace the nozzle
	Nozzle does not follow the .csv-defined motion path	Incorrect or incomplete coordinates in the .csv file	Review and correct errors in the .csv file
	Nozzle does not follow the .csv-defined motion path	Unit mismatch between software and intended settings	Ensure that the software unit matches with the desired unit setting
134	Spheroids move away in the chamber instead of settling at the center	The chamber is not at the same level with the ground	Check and adjust the leveling to ensure that the chamber is parallel to the ground
134		Vibrations are causing displacement	Gently swirl the dish to move spheroids toward the center
162	Failure to lift spheroids	Aspiration force is too low	Adjust the aspiration force for optimal lifting
		Improper balance between spheroid size and nozzle size	Select a nozzle size suitable for spheroid dimensions
		Nozzle clogging due to debris or leakage	Check for clogging by blowing air into a Petri dish filled with DI water or replace the nozzle, if necessary (recommended)
163	Liquid leakage during spheroid placement	Excessive liquid aspiration during spheroid picking	Minimize the spheroid picking time to reduce liquid uptake, or set the minimum vacuum (~4 mm Hg) to retain liquid while allowing spheroid detachment (Step 194). When using the minimum vacuum, keep activate the ‘Minimum vacuum’ (Fig. 5, panel 10) during bioprinting. If necessary, optimize the minimum pressure to balance effective liquid retention and controlled spheroid release
	Liquid leakage during spheroid placement	Loose tubing connections or damaged components in the pneumatic system	Inspect and tighten all tubing and syringe connections. Replace damaged components, if necessary
	Unsuccessful spheroid detachment	Aspiration force is too high	Reduce aspiration force to the lowest effective force
		Nozzle size is too large	Optimize the nozzle size for the corresponding spheroid type
		Prolonged bioprinting process	Perform the overall bioprinting process as quickly as possible
	Software delay during operation	Rapidly clicking multiple buttons simultaneously or low computer performance	Avoid rushing operations, as simultaneous button presses can override commands and cause delays. Allow the software to process each action before proceeding. Consider upgrading the hardware if performance issues persist
	Spheroid damage	Aspiration force is too high	Reduce aspiration force to prevent excessive aspiration
	Spheroid damage	Excessive force applied during placement	Place spheroids gently on the substrate to avoid mechanical damage. When using a support material, ensure its shear modulus is suitable for spheroid handling
178	Unable to see air bubbles or airflow	Insufficient pressure force or DCNA not positioned close enough to the water surface	Increase pressure force to ensure airflow for confirmation or adjust nozzle positioning closer to the medium surface using the side-view camera
		Manufacturer defects (for example, metal obstruction), or bent or clogged nozzles	Inspect for manufacturing defects and replace the DCNA, if necessary
		Loose tubing connections or damaged pneumatic components	Check and tighten all tubing connections; replace damaged components
197	Spheroids not attaching to the DCNA	Nozzle is clogged	Check for nozzle clogging by blowing air into a Petri dish filled with a medium
		The aspiration force is insufficient	Increase the aspiration force as needed, ensuring that it is not excessive to avoid spheroid damage
		The gap between DCNA tip and spheroids is too large or DCNA is not properly aligned (that is, not parallel) with the spheroid chamber	Reconfigure Pos 6 (Step 156) to adjust the distance between the nozzle tip and the top surface of the spheroids. See Step 174 for the DCNA parallelization
		Misaligned or defective DCNA (Fig. 3d)	Replace the DCNA if misalignment or defects are detected
		Loose tubing connections or damaged pneumatic components	Check and tighten all tubing connections; replace damaged components
198	Failure to lift spheroids/detached spheroids during lifting	Improper inter-nozzle distance relative to the spheroid size—too narrow spacing increases barrier energy between the medium and spheroids	Optimize spheroid spacing relative to the prepared spheroids by increasing internozzle distance in the DCNA or alternating nozzle selection (for example, 27-gauge nozzle) in the DCNA arrangement to improve spheroid lifting success
		Size variation between spheroids	If using spheroids of varying sizes and types, prepare separate chambers for each spheroid size or type
		Lifting is realized rapidly	Reduce lifting speed by activating the SLOW speed mode and using the designated Play button on panel 4A (Fig. 5)
		Aspiration force is too low	Increase the aspiration force as needed for effective lifting
		Siliconized coating is no longer effective	Ensure the siliconized coating is still valid; replace with a new DCNA, if needed
201	Liquid leakage during spheroid placement	Check troubleshooting steps for Step 163	Check troubleshooting steps for Step 163
	Unsuccessful spheroid detachment	Check troubleshooting steps for Step 163	Check troubleshooting steps for Step 163. Alternatively, turn off aspiration while keeping the nozzle open by pressing the ‘Aspiration’ button again (OFF). When using the minimum vacuum, keep it active while turning off aspiration to maintain gentle suction, reducing spheroid deformation during the transfer procedure. Avoid closing the DCNA valves to ensure the minimum vacuum remains active. Replace DCNA if necessary
	Spheroids are not simultaneously placed on the bioink substrate	The DCNA is not parallel to the surface	Readjust yaw-pitch-roll alignment using the tilting base on the DCNA holder to ensure that the DCNA surface is parallel to the substrate. Verify alignment by placing a glass slide near the DCNA and checking parallelism from at least two sides (see Steps 172–175)
	Software delay during operation	Check troubleshooting steps for Step 163	Check troubleshooting steps for Step 163
	Spheroid damage	Check troubleshooting steps for Step 163	Check troubleshooting steps for Step 163
210	Bleeding during IOB, leading to inaccurate or failed spheroid placement	Insufficient hemostasis before IOB	Ensure proper hemostasis before starting bioprinting. Use absorbent materials or gentle suction to control bleeding without disturbing spheroid placement

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Timing

Steps 1–55, hardware setup: 1–2 d depending on handskills and familiarity with assembly

Steps 56–63, software setup: ~30 min

Steps 64–98, hardware and software integration and configuration: ~1 h

Steps 99–102, spheroid preparation: 1–2 d, excluding the duration for cell expansion

Steps 103–105, spheroid characterization: ~1 h

Steps 106–115, biomaterial preparation and characterization: depends on the biomaterial types

Steps 116–133, EBB preparation and optimization (optional for EBB): 1–2 min to create a 5-mm diameter of a bioink substrate with 10 mm/s of printing speed, but timing depends on the size of constructs and printing speed

Steps 134–136, AAB process: the time required for bioprinting depends on the number of spheroids and the user’s proficiency

Steps 137–165, (Strategy 1) single-nozzle mode AAB: ~30 s/spheroids (depends on the size and number of spheroids and the user’s skillset to operate AAB)

Steps 166–203, (Strategy 2) high-throughput AAB mode operation: 3–4 min to bioprint 64 spheroids, plus ~1 min to create a 5-mm (in diameter) bioink substrate at 10 mm/s printing speed. Timing depends on the size of constructs and printing speed

Steps 204–212, (application) intraoperative AAB: ~5 min to create 5-mm constructs containing 64 spheroids into calvarial defects

Steps 213–230 cleanup AAB: ~15 min

Steps 231–244, postbioprinting process and downstream analysis: ~2 h+, varies depending on specific analytical method used for the experiments

Anticipated results

The procedures outlined are designed to enable users to build and operate their own AAB platform with hardware (Figs. 1–3) and software (Fig. 5) integration, as well as the preparation of the DCNA (Fig. 3a–d) for the high-throughput mode. By following these steps, users can utilize AAB under varying circumstances to achieve a precise patterning of spheroids for their applications.

The platform setup process itself is efficient, allowing users to build a fully operational platform within a week. Once assembled, the platform requires minimal maintenance. Users are advised to periodically inspect 3D-printed parts for depreciation and ensure fasteners on the motion stage remain tight to maintain optimal performance. Upon successful assembly and operation of the AAB platform, users can bioprint constructs either by individual spheroid placement or through the simultaneous patterning of multiple spheroids for scalable applications. This Protocol provides comprehensive guidance on troubleshooting common challenges experienced during AAB, ensuring high reproducibility and reliable performance. Initial attempts may yield a success rate of 10–30%, particularly for first-time users; however, with practice and optimization of bioprinting parameters and handling of hardware and software, success rates are expected to improve notably (95–100%).

The general mechanism for AAB is the same for both the single-nozzle mode (Strategy 1 and Fig. 4a) and high-throughput mode (Strategy 2 and Fig. 4b). Briefly, the mechanism includes: 1. capturing a spheroid using aspiration, 2. lifting the spheroid, 3. transferring it to the desired position, 4. releasing the aspiration and 5. placing the spheroid at the target position. Due to this common mechanism, the spheroid type or properties typically do not affect the bioprinting outcome—any spheroid that can be handled in the single-nozzle mode can also be used in the high-throughput mode. However, the high-throughput mode involves the simultaneous control of multiple nozzles, which adds operational complexity and can pose additional challenges.

Demonstrations of AAB applications highlight its versatility, utilizing two different modes: single-nozzle (Fig. 8) and high-throughput (Fig. 9), using spheroids and organoids (Fig. 9d). Using the single-nozzle mode (Fig. 4a), spheroids of a variety of cell types and size (as well as nonspherical building blocks, such as tissue strands⁶ (Fig. 8g)) have been precisely bioprinted onto or into a wide range of materials (1) that were generated by other bioprinting techniques (for example, droplet-based bioprinting^11,14 or EBB¹⁵) or (2) yield-stress support materials for freeform bioprinting^4,5 to construct complex geometries in 3D, respectively. Specifically, AAB has been used to fabricate intricate constructs composed of different spheroid size and types including helical structures (Fig. 8a), PSU initials (Fig. 8b), tubular structures (Fig. 8c) and double-helix structures (Fig. 8d) in 1.2% Carbopol yield-stress gel used as a support material⁴. Spheroids were also deposited in a stratified manner for pyramid-shaped constructs on an alginate substrate⁷ (Fig. 8e). In addition, AAB has been applied to generate vascularized cancer microenvironments by placing breast cancer spheroids at defined distances from a vasculature in collagen–fibrin composite gel, enabling investigations into angiogenesis and responses to immunotherapy and chemotherapy³ (Fig. 8f). Furthermore, controlled spacing (400, 800 and 3,000 μm) of HUVEC spheroids has been achieved to study angiogenic sprouting in fibrin hydrogel⁷ (Fig. 8h). These results demonstrate the adaptability, precision and utility of the single-nozzle mode in engineering complex tissue microenvironments.

Using the high-throughput mode (Fig. 4b), multiple spheroids can be bioprinted at an accelerated manner via the DCNA (Fig. 9a,b). The comparison between bioprinted spheroid arrangements (Fig. 9b, right) and manually mixed spheroids (Fig. 9b, left) of varying size demonstrates the precise spatial arrangement achieved through the high-throughput AAB, in contrast to the random distribution observed in manually mixed spheroids. The DCNA also enabled the selective patterning of different spheroid types and sizes into various spatial configurations under the same aspiration setting (Fig. 9c). AAB enabled the fabrication of bone constructs with uniformly positioned, osteogenically committed spheroids at varying densities (16 or 64 spheroids per construct), in combination with EBB to create a gel substrate using a bioink called BONink⁸ (Fig. 9e). The 64-spheroid arrangement (Fig. 9e(iii)) was achieved by iteratively placing four sets of 16 spheroids (Fig. 9e(ii)) at defined positions. The constructs were created in a stratified manner (Fig. 9e(i,iv)), with spheroids deposited between layers of BONink (Fig. 4b). The high-throughput AAB was also applied in IOB (Fig. 4c), enabling the direct bioprinting of bone constructs containing up to 64 spheroids into rat calvarial defects⁸ (Fig. 9f). This IOB procedure, following the same procedure as described in Fig. 9e, was completed in ~5 min and resulted in successful bone regeneration⁸. Scalable bioprinting was demonstrated with the high-throughput mode by layering hundreds of spheroids into complex structures, in a combination with EBB-enabled deposition of CARink⁸ (Fig. 9g). A large number of spheroids were bioprinted through iterative placement cycles of 16 spheroids each, totaling 64 spheroids per layer between deposited CARink layers, resulting in the fabrication of a ~1 cm³ construct containing ~600 chondrogenic spheroids.

The reproducibility of AAB has been validated across independent experiments^3–15, demonstrating consistent outcomes in various applications with a high positional accuracy and establishing AAB as a reliable platform for generating a controlled tissue microenvironment in both experimental and translational research (Figs. 8 and 9). The printing accuracy achieved a positional error of less than 50 μm between the target point and the center of bioprinted spheroids^3,5 (Fig. 7e). However, bioprinting performance may vary depending on the properties of the spheroids, bioink substrate or support material used, as well as the optimization of key parameters. In the high-throughput mode, the success rate of spheroid lifting has been systematically investigated⁸. For instance, a DCNA with a width of 3.4 mm yielded a 100% spheroid lifting rate (for 300–350-μm spheroids), demonstrating the efficiency of the system. This high success rate is critical for accelerating AAB and enabling parallel patterning of multiple spheroids with precision.

Beyond its versatility and precision, this Protocol facilitates customization based on user requirements. Users can reduce costs by substituting lower-specification motion stages or solenoid valves while still maintaining functional integrity. Moreover, the modular design of printheads facilitates hybrid bioprinting strategies, enabling the seamless integration of AAB and EBB. The setup described in this Protocol is not limited to AAB applications; it can also be utilized for general-purpose motion stage control and solenoid valve actuation to regulate pneumatic or liquid flow, using the included hardware and software. This flexibility further expands the range of possible applications, making the platform highly versatile for diverse needs.

In conclusion, when executed according to this Protocol, the AAB platform provides a powerful tool for precision bioprinting of spheroids. Its adaptability supports a wide range of applications including but not limited to scalable tissue fabrication for regenerative medicine and in vitro disease modeling. To achieve consistent and reproducible results, users are encouraged to optimize the parameters, such as the aspiration force, nozzle size and bioink or spheroid properties. With continued practice, the AAB platform will enable the reliable creation of complex tissue architectures, contributing to advancements in bioprinting research and translational applications.

Supplementary Material

Reporting Summary

NIHMS2154587-supplement-Reporting_Summary.pdf^{(155.5KB, pdf)}

Supplementary Data 1

NIHMS2154587-supplement-Supplementary_Data_1.numbers^{(116.9KB, numbers)}

Supplementary Data 2

NIHMS2154587-supplement-Supplementary_Data_2.numbers^{(163.4KB, numbers)}

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41596-025-01240-x.

Key points.

Aspiration-assisted bioprinting (AAB) is a versatile biofabrication technique that enables the precise and selective patterning of biologics, such as tissue spheroids and organoids. This Protocol provides comprehensive instructions for setting up the AAB platform, operating software and key operational procedures, including the optimization of bioprinting conditions.
AAB overcomes the limitations of conventional bioprinting techniques, enabling flexible, precise, heterogeneous and scalable spheroid bioprinting across both two-dimensional and three-dimensional configurations.

Acknowledgements

This work has been supported by the National Institute of Biomedical Imaging and Bioengineering (NIBIB) award no. R01EB034566 (I.T.O.) and National Institute of Dental and Craniofacial Research (NIDCR) award no. R01DE028614 (I.T.O.). We thank J. C. Moses, S. Liu, M. Yeo and D. Gupta at Penn State University for proofreading the Protocol.

Footnotes

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Competing interests

I.T.O. has an equity stake in Biolife4D and is a member of the scientific advisory board for Biolife4D and Healshape. The remaining author declares no competing interests.

Data availability

The main data discussed in this Protocol are available in supporting primary research publications. STL files used in this Protocol are accessible via figureshare.com at https://doi.org/10.6084/m9.figshare.28405133.v1 (ref. 35). Example datasets to demonstrate the software for prepositioning (Steps 145–151) and automated motion movement (Steps 117–118) are provided in the Supplementary data.

Code availability

The AAB software can be accessed in the Supplementary software. Details of the software interface and instructions were included in the manuscript. We will post updated versions of the software via our GitHub repository at https://github.com/MHKim-software/HITS-Bio.git, if necessary.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Reporting Summary

NIHMS2154587-supplement-Reporting_Summary.pdf^{(155.5KB, pdf)}

Supplementary Data 1

NIHMS2154587-supplement-Supplementary_Data_1.numbers^{(116.9KB, numbers)}

Supplementary Data 2

NIHMS2154587-supplement-Supplementary_Data_2.numbers^{(163.4KB, numbers)}

Data Availability Statement

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PERMALINK

Aspiration-assisted bioprinting of spheroids

Myoung Hwan Kim

Ibrahim T Ozbolat

Abstract

Introduction

Development of the Protocol

Applications

Comparison with methods

Overview of the procedure

Experimental design

AAB system setup: hardware (Steps 1–55)

Fig. 1 |. The fabrication and assembly process for the AAB platform.

Fig. 3 |. Key steps in the assembly of the DCNA and full setup of the AAB platform.

Fig. 2 |. The pneumatic system in AAB.

Fig. 4 |. The key workflow of the AAB process.

AAB system setup: software (Steps 56–63)

Fig. 5 |. The AAB software interface.

Table 1 |.

Hardware and software integration and configuration (Steps 64–98)

Spheroid preparation (Steps 99–102) and characterization (Steps 103–105)

Biomaterial preparation and characterization (Steps 106–115) and EBB preparation and optimization (Steps 116–133)

AAB process (Steps 134–136, common preparation; Steps 137–165, (Strategy 1) single-nozzle AAB; Steps 166–203, (Strategy 2) high-throughput AAB; Steps 204–212, intraoperative AAB)

Fig. 6 |. Detailed control diagram for AAB operations.

Postbioprinting process and downstream analysis (Steps 213–244)

Limitations

Materials

Biological materials

Reagents

Reagents needed throughout procedure

Intraoperative AAB

Equipment

Equipment needed throughout the procedure

Cell culture and spheroid preparation

AAB platform

DCNA preparation

Postbioprinting process and spheroid characterization

Software

Reagent setup

LIVE/DEAD viability reagent

70% ethanol

Procedure

Hardware setup

● TIMING 1–2 d, depending on handskills and familiarity with assembly

3D motion stage assembly

EBB/single nozzle AAB holder preparation

DCNA holder preparation

Isometric camera holder preparation

DCNA preparation

Mounting bracket and holder

Printbed assembly

Hardware placement

Solenoid valve assembly

Pneumatic sensor connection

Sterilization for AAB

Software setup

● TIMING ~30 min

Hardware and software integration and configuration

● TIMING ~1 h

COM port settings for the motion stage

Motion stage and software connection

Pneumatic sensor connection

Set up cameras

Solenoid valve connection

Execute the AAB software

Connect motion stage to the AAB software

Connect solenoid valve to AAB software

Connect pneumatic sensor to the AAB software

Initialization process

Validation of motion control

Validation of solenoid valve control

Spheroid preparation

● TIMING 1–2 d, excluding the duration for cell expansion

Spheroid characterization

● TIMING ~1 h

Biomaterial preparation and characterization

● TIMING Depends on the biomaterial types

Rheological characterization

EBB preparation and optimization (optional for EBB)

● TIMING 1–2 min to create 5-mm diameter of a bioink substrate with 10 mm/s of printing speed, but timing depends on the size of constructs and printing speed