A programmable, open-source robot that scratches cultured tissues to investigate cell migration, healing, and tissue sculpting

Summary

Despite the widespread popularity of the “scratch assay,” where a pipette is dragged manually through cultured tissue to create a gap to study cell migration and healing, it carries significant drawbacks. Its heavy reliance on manual technique can complicate quantification, reduce throughput, and limit the versatility and reproducibility. We present an open-source, low-cost, accessible, robotic scratching platform that addresses all of the core issues. Compatible with nearly all standard cell culture dishes and usable directly in a sterile culture hood without specialized training, our robot makes highly reproducible scratches in a variety of complex cultured tissues with high throughput. Moreover, the robot demonstrates precise removal of tissues for sculpting arbitrary tissue and wound shapes, enabling complex co-culture experiments. This system significantly improves the usefulness of the conventional scratch assay and opens up new possibilities in complex tissue engineering for realistic wound healing and migration research.

Graphical abstract

Highlights

• •Traditional scratch assay is a manual process that is prone to user variability
• •We developed a low-cost and programmable scratching system
• •SCRATCH demonstrates up to 4$\times$ improvement in scratching uniformity
• •The platform also creates complex patterns and enables tissue co-culture

Motivation

The “scratch assay” is one of the most popular approaches for studying cell migration and healing. However, the manual nature of the method leads to low reproducibility and high variability, and current automated solutions lack flexibility and are prohibitively expensive. We address these issues by developing a low-cost and open-source platform that allows programmable scratching across virtually all types of dish and plate culture vessels, demonstrating new approaches in tissue co-cultures for complex tissue engineering.

Lin et al. develop a low-cost and automated robot platform that performs the widely used scratch assay for wound healing and cell migration studies. This system shows improved scratch width uniformity across different cell lines, shapes tissue into arbitrary geometries, and enables complex tissue co-culture.

Introduction

The “scratch assay” (Figure 1A)—dragging a pipette tip or sharp object through a cultured tissue and monitoring the cellular healing response in the resulting gap—is among the most common approaches to study cell migration and healing in vitro^1,2 but also, perhaps, among the least reproducible and scalable due to the manual nature of the process.^2,3,4 While a popular protocol paper on the manual method has nearly 5,000 citations at this point,¹ and the method is largely free, the traditional scratch assay relies on pressure, tool orientation and brand, speed, and manual stability and is inherently limited in precision, throughput, and scalability (e.g., it is more difficult in a 96-well plate than in a 6-well plate). Moreover, there is a missed opportunity to use “scratching” as a form of subtractive manufacturing to produce much more complex tissue geometries and easily prepare unique systems-level co-cultures. Given the ubiquity and importance of scratch assays, new approaches improving the reproducibility, throughput, and versatility can benefit a broad range of research fields.

Figure 1.

System mechanism and capability

(A) The scratch assay is performed by a pipette tip moving across the cell monolayer, leaving a cell-depleted region.

(B) System overview. The lateral movement of the pipette tip is actuated by a stepper motor-driven belt system. Red and green arrows represent the x and y direction, respectively. The vertical movement of the tip is actuated by a servo, indicated with a magenta arrow. The 35-mm dish is placed on a custom-designed fixture.

(C) A close-up photo of the device operating on a 60-mm dish.

(D) Phase-contrast image of the dish-scale scratch pattern demonstration. Scale bar: 2 mm.

(E) Cytoplasmic staining of the scratch pattern in a 96-well dish. Scale bar: 1 mm.

(F) Close-up photo of the device operating on a 96-well plate. An arbitrary pattern and well location can be selected.

While alternative solutions to generate gaps in tissues are well represented in the literature, none of them address all of the challenges.² One popular approach is the “barrier removal assay,” where cells are seeded on either side of a rubber stencil, and then the stencil is removed to generated a “gap.”^5,6,7,8 While versatile, the approach requires precision pipetting⁹ and simply does not scale to small culture vessels. Commercial rubber inserts are available but are limited in geometry and configuration and are costly consumables. Further, there is a concern that barrier removal may not properly damage the surrounding tissue consistent with actual injury.¹⁰ Similarly, DIY (do-it-yourself) parallel scratchers based on machined or molded tips have been used effectively in multi-well plate studies,^3,11,12 but the approach relies on sophisticated machine-shop CNC (computer numerical control) capabilities, still requires user-applied pressure and speed, can only make straight lines, and is intrinsically limited to a single specific substrate (e.g., 96-well plates only). While commercial scratch systems exist,¹³ they are also limited to only a few well plate options (e.g., 24/96 well) and straight lines, and the cost is prohibitively high, relatively speaking (∼$10,000—$20,000 at the time of writing). A variety of alternative approaches to remove cells and tissue also exist that are based on the use of microfluidics.^{2,4,14,15,16,17} These approaches typically rely either on spatially dosing enzymes such as trypsin for controlling cell removal^18,19 or physically removing cells based on pneumatic membrane compression removal.²⁰ While precise and reproducible, these methods require expensive microfluidics manufacturing capabilities, are not programmable without creating new devices, and are relatively inaccessible to the broader community that performs scratch assays due to the required expertise and infrastructure. Hence, there is an exciting opportunity to redevelop the common, mechanical form of the scratch assay around flexible, programmable, open-source hardware that can be adopted by any laboratory.

All of the key variables and challenges discussed here are the things at which a robot excels: precision, reproducibility, throughput/repetition, and programmability. Inspired by these advantages, we modified a low-cost robotic platform originally intended for art generation. We call this device SCRATCH (scalable cellular resection apparatus to characterize healing). SCRATCH allows (1) complete programmability to produce almost any pattern, (2) the use of any scratching tip (e.g., pipette tips, needles, wires, etc.), (3) compatibility with nearly all standard culture vessels (3.5-cm dishes to 96-well plates), (4) direct use in a sterile culture hood, and (5) a low net cost of <$500 at the time of writing. The remainder of this report summarizes how SCRATCH works and demonstrates its capabilities.

Results

SCRATCH device working principles and system architecture

SCRATCH is a fully automated scratch assay system, and its key advantages stem from computer control of a robotic gantry (Figure 1B). The core of the SCRATCH device is a writing/drawing robot that provides programmable lateral (xy) and vertical (z) movement of the scratching apparatus (Figure 1C, color-coded arrows). While SCRATCH can be built using off-the-shelf components from the 3D printing community, for simplicity, here we modified a hobby “art bot” (AxiDraw V3 but many others exist) originally intended to hold pens and markers, as this saves considerable time for a minimal cost (∼$500). This chassis consists of an xy stepper motor-belt system to position the pipette tip tool over a tissue culture region and a servo motor to precisely and gently bring the tool into contact with the tissue in preparation for scratching. Instead of a pen or marker, we 3D-printed a customized pipette tip holder for 10 μL pipette tips (this can be tuned for any pipette tip style) (Figure 1C). To ensure stability of the tip during scratching, we applied a thin layer of reusable adhesive putty (e.g., FunTak) between the tip and the holder. This tip carrier can then be attached to the xyz gantry as if it were a pen (see CAD [computer-aided design] file access instructions under Data and code availability). At this point, SCRATCH is ready for use (see Video S1 for its operation).

Video S1. The SCRATCH system operates on a 96-well plate, related to Figure 1

(A) A recording of SCRATCH in operation, accessing arbitrary wells and scratching a “cross” shape in a 96-well plate.

A key design goal was to make SCRATCH as user friendly and reproducible as possible to enable rapid adoption in cell biology labs, so a key feature of our design is our modular sample holder directly attached to the frame of SCRATCH that allows most standard culture vessels—from 3.5-cm Petri dishes to 96-well plates (Figures 1D–1F)—to be positioned precisely and reproducibly relative to the pipette tool (see CAD file access instructions under Data and code availability; Video S1). This sample holder also incorporates an alignment ring to calibrate the tip position at the beginning of the scratch (STAR Methods). The use of this fixture allows SCRATCH to be controlled using pre-made template files in open-source drawing software (Inkscape already has plug-in support for many drawing bots) (Figure S1; see also our shared template files). The user then loads an appropriate template for a given culture vessel, draws the desired patterns in each well, and “prints” the scratch pattern on SCRATCH via a USB connection.

We demonstrated the versatility of SCRATCH by creating unique patterns in different types of Petri dishes and culture plates. First, we scratched a large-scale “star” pattern across a layer of primary mouse skin keratinocytes in a 35 mm dish (Figure 1D) to demonstrate the ability to generate complex, precise patterns (Figure 1D, right).

We then tested SCRATCH on a more challenging culture vessel: a 96-well plate. Here, the small well diameter prevents reproducible or precise manual scratching, and the throughput required to scratch 96 wells is not feasible using the traditional manual approach. However, SCRATCH was able to reliably pattern standard scratches in all 96 wells in <4 min; more complex geometries will take longer. Figure 1F shows a fluorescence image of “+” patterns scratched into the wells of a 96-well plate. Once calibrated, SCRATCH can automatically and reproducibly scratch arbitrary patterns in most standard culture dishes or plates at high throughput.

Reproducibility and dynamics characterization

We first assessed how reproducible SCRATCH patterns were relative to manual patterns using linear scratches made in primary mouse skin keratinocyte layers cultured in 60 mm plates (STAR Methods); representative results are shown in Figure 2A. We used the standard deviation of the width of each scratch as the metric for evaluating uniformity. As shown in Figure 2B, SCRATCH exhibited significantly improved uniformity vs. manual scratching (nearly 4$\times$ reduction in standard deviation and on the order of a single cell) while maintaining an average width of ∼700 μm (approximate diameter of the 10 μL pipette tip). The observed variations we do see with SCRATCH likely reflect both biological variability in cell orientations and minor vibrations from the motor-belt system (see Figure S2 for high-resolution data on the tip trajectory and Figure S3 for a demonstration of the effective resolution limit).

Figure 2.

Linear scratch quantification and comparison

(A) 12 scratches performed by SCRATCH. Scale bar: 2 mm.

(B) Scratch uniformity on a keratinocyte monolayer. The edge outline is highlighted in yellow. The device scratch demonstrates lower variation than a manual scratch. Scale bar: 1 mm.

(C) Wound-healing assay on 8 scratches, showing uniform wound closure. Time-lapse photos 0, 12, and 24 h after scratch are shown. Scale bar: 1 mm. Error bars represent standard deviation.

(D) Fast and consistent pipette movement from SCRATCH allows low scratch variation on high-viscoelasticity tissues. An MDCK monolayer is scratched without calcium chelation. Scale bar: 1 mm.

Therefore, SCRATCH demonstrates superior uniformity to manual scratches in basic tissues, which improves reproducibility of scratch assays and allows higher throughput. As a demonstration of these benefits, we rapidly produced an array of 15 linear gaps into a primary mouse skin monolayer and quantified the wound closure rate to validate the uniformity (Figure 2B). Phase-contrast images of 0 h, 12 h, and 24 h after scratching are shown alongside the quantification (Figure 2C), and the closure curves indicate relatively uniform and tight healing dynamics.

We next investigated the importance of scratching speed (how quickly the tool is translated through the tissue). This is something impossible to control manually, whereas SCRATCH allows scratch speed to be programmed up to 380 mm/s. Tissues are viscoelastic materials, meaning that their mechanical properties, adhesion to the substrate, and mechanobiological responses depend on the rate at which they are mechanically deformed, not just how much they are deformed, so being able to regulate the scratching rate should provide unique advantages and a new dimension to consider. In particular, we hypothesized that the high-speed, precise motion of SCRATCH would be particularly useful when working with more challenging tissues possessing strong cell-cell adhesion and relatively weaker cell-substrate adhesion where slow or irregular manual scratching can cause the tissues to delaminate rather than being “cut.”²¹

Here, we used the widespread MDCK (Madin-Darby canine kidney) kidney epithelial model, commonly used in all manner of collective migration experiments and screens and known to exhibit strong cell-cell adhesion and develop collective cell behaviors as a result.^{8,19,22,23,24} We first established a baseline by manually scratching engineered, mature MDCK layers (STAR Methods) as best as we could (Figure 2D), which resulted in massive, irregular gaps and widespread delamination due to inherent irregularities in the manual process. We observed similar results when we set SCRATCH to a slow speed (38 mm/s) and repeated the experiment (Figure 2D). By contrast, when we repeated the experiment with SCRATCH set to the fastest translation speed (380 mm/s), we were able to produce highly uniform and more regular scratch patterns in comparison to slower mechanical or manual scratching (Figure 2D). Overall, SCRATCH was able to deliver more precision, reproducibility, and throughput than manual scratching.

Subtractive tissue manufacturing: Designing complex tissue patterns

Only laboratory wounds are perfect straight lines, and many studies have emphasized the importance of tissue and wound shape in governing cellular migration and growth.^{9,25,26,27,28,29,30} We explored this concept by adapting SCRATCH for subtractive manufacturing of living tissues—gradually removing existing regions of tissue to produce complex patterns (returning to the primary mouse skin monolayer model). SCRATCH enables this by “raster cutting,” where it can gradually move the pipette tip tool back and forth while ensuring an overlap in the pattern to fully clear a given region of cells (Figures 3A and 3B). Here, we chose an approximate overlap of 75%. “Positive” or “negative” patterns can be achieved by selectively scratching the “center” or “edge” of a monolayer, either leaving a solid tissue (positive) or cleared region (negative) (Figure 3C). This subtractive manufacturing method extends the application of SCRATCH beyond pure scratch assays to complex assays evaluating the role of wound size and shape, for example. Moreover, this process is also fully automated within the free software used to control SCRATCH, allowing arbitrarily complex patterns, as shown in Figure 3D.

Figure 3.

Raster mode capabilities and demonstration

(A) Demonstration of area clearance from tip overlap. The programmed path diameters are 3, 2, 1, and 0.5 mm. Scale bar: 2 mm.

(B) Raster mechanism cartoon and calculation of raster overlap.

(C) Demonstration of positive and negative area clearance. Scale bar: 2 mm.

(D) Complex shape achieved through rastering. Scale bar: 2 mm.

SCRATCH for complex co-cultures

The “empty space” created by SCRATCH offers new potential for tissue co-culture because additional cell types can be back-filled into the newly created empty regions (Figure 4A). As a demonstration of this, we created a complex co-culture using a dermal/epidermal model of fibroblasts (NIH 3T3 fibroblasts) and keratinocytes (primary mouse keratinocytes). The resulting spiral pattern is shown in Figures 4B and 4C and was produced by first scratching a layer of keratinocytes (pre-stained with a membrane dye) and then washing with PBS and backfilling fibroblasts (pre-stained with a different membrane dye) as described in STAR Methods. The initial population of keratinocytes is shown in cyan and NIH 3T3 cells in magenta. We also used a nuclear dye (Hoechst 33342) to stain all cell types. The spiral is clearly visible, and the expanded view shows good spatial separation between keratinocytes and fibroblasts. Note that the quality of the backfilling method relies on the confluency of the first monolayer, since the seeded cells will also attach to the area that is outside of the intended region. In the case shown here, we did not attempt to optimize the first tissue layer (e.g., by increasing cell density, allowing more culture time, or increase cell-cell junction strength with additional calcium), so some of the mesenchymal NIH 3T3 fibroblasts were able to “infiltrate” the keratinocyte layer (up to around 20% in the image shown). In practice, the co-culture conditions should be tuned for a desired application or question. Similar to planar lithography, this process can be repeated multiple times for additional “layers” of cells as long as a co-culture medium exists that can support each cell type. These data further emphasize the versatility offered by the SCRATCH system to enable not only scratch assays but more complex tissue engineering and cell-cell communication assays.

Figure 4.

Using SCRATCH for arbitrary geometry tissue co-culture

(A) Tissue co-culture through backfilling. A scratch is made on the cell A monolayer. After washing with PBS, the desired secondary cell suspension is added. After attachment, the dish is washed multiple times with PBS to remove any unattached cells. Co-culture medium is then added, and the dish is ready for the experiment.

(B) Fluorescence image of a spiral scratch on keratinocytes backfilled with NIH 3T3 fibroblasts. Keratinocytes are stained with Cellbrite Green, and NIH 3T3 fibroblasts are stained with Cellbrite Red; both cells are stained with NucBlue. Scale bar: 2 mm.

(C) Magnification of the center of the spiral backfill. Scale bar: 500 μm.

Discussion

SCRATCH demonstrates a low cost, fully programmable, and high-throughput tool for the popular scratch assay that brings many significant advantages to the method, including improved reproducibility, throughput, and versatility with compatibility for nearly all standard culture plates and dishes. In particular, we showed improved precision, throughput, and reproducibility over manual scratches as well as the ability to use scratching to produce unique tissue shapes and co-cultures without the need for microfabrication or manual stenciling.

The open-source and open hardware nature of SCRATCH, combined with its low cost, should substantially aid its adoption, as it can be incorporated cheaply and easily into most cell biology laboratories and used in or outside of tissue culture hoods. A key aspect of SCRATCH is that it is easy to modify as a platform, allowing nearly any tip to be incorporated, and allowing for custom programming in Python if unique features are required that the standard graphics software does not allow (for instance, the tip can be programmed to go through a “wash” step where it is agitated in a buffer or ethanol well in between scratching different wells in a multiwell plate to avoid cross-contamination). Due to the potential for tip wear and cross-contamination, we strongly recommend replacing tips between different samples, but the user should evaluate the need for this in their own assays. Similarly, the SCRATCH platform can easily be modified with a more precise Z-drive to regulate scratching pressure or enable tip changes, and even the tips themselves can be adjusted or custom machined (e.g., from PTFE—Teflon) to reduce the risk of wear. Scratching inevitably leaves cell debris on the substrate, which may affect migration. However, compared to manual scratching, SCRATCH’s programmable path allows the robot to make multiple passes, reducing cell debris attaching to the substrate (Figure S4).

Moreover, SCRATCH is not dependent on one specific piece of hardware, as any traditional “maker” tools, such as a diode laser cutter or 3D printer, can be modified to do something similar, and this concept is active area of research. Overall, this type of versatility can substantially improve the types of applications where scratch-style assays are useful and further aid in their adoption and accessibility to the broader community.

Limitations of the study

Despite demonstrating improved uniformity and flexibility in the scratch assay or tissue shaping, SCRATCH is highly dependent on cell-cell adhesion due to its physical contact with the cells. A highly connective monolayer would result in greater width variation but, at the same time, would reduce “infiltration” of the second cell type for tissue co-culture. Carefully modulating tissue connectivity before and after scratching balances this tradeoff. Alternatively, non-contacting methods, such as laser ablation, can be used.^31,32,33 Additionally, due to the conical shape of the pipette tips, areas close to the edge of the culture vessel cannot be reached (Figure S1). However, custom manufacturing of the scratching tips can alleviate the problem.

Resource availability

Lead contact

Requests for further information, resources, and reagents should be directed to and will be fulfilled by the lead contact, Daniel Cohen (danielcohen@princeton.edu).

Materials availability

This study did not generate new materials.

Data and code availability

Given large file sizes, the data that support the findings of this study are available from the lead contact upon request.

All CAD files for 3D printing and code necessary to perform the work shown here are available at our laboratory GitHub repository (https://github.com/CohenLabPrinceton/SCRATCH), and we are happy to provide support as needed. The DOI is listed in the key resources table.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Acknowledgments

Support for this work was provided in part by NIH award R35 GM133574-06. We also thank members of the Cohen lab for advice and support.

Author contributions

Conceptualization, D.J.C. and Y.L.; methodology, D.J.C. and Y.L.; investigation, Y.L., A.S.-D., and M.B.; visualization, Y.L., M.B., and D.J.C.; writing – original draft, Y.L. and D.J.C.; writing – review & editing, Y.L. and D.J.C.; funding acquisition, D.J.C.; resources, D.J.C.; supervision, D.J.C.

Declaration of interests

D.J.C. and Y.L. have filed patent applications based on the method developed in this work.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Cellbrite Green	Biotium	Cat #: 30021
Cellbrite Red	Biotium	Cat #: 30023
NucBlue (Hoechst 33342)	Thermo Fisher	Cat #: R37605
Chemicals, peptides, and recombinant proteins
Low glucose DMEM	Sigma-Aldrich	Cat #: D5523-10L
Sodium bicarbonate	Sigma	Cat #: S5761-500G
Fetal bovine serum	Atlanta Biologicals	Cat #: S11550
Penicillin-streptomycin	Gibco	Cat #: 15140-122
Phosphate buffered saline	Gibco	Cat #: 14-190-250
Primary keratinocyte media DMEM/F-12 (3:1)	Fibco Invitrogen Life Technologies	Nowak and Fuchs, 2009
Paraformaldehyde	Electron Microscopy Science	Cat #: 15710
Experimental models: Cell lines
Wild-type MDCK II	Nelson Laboratory, Stanford University	N/A
Primary mouse Keratinocytes	Devenport Laboratory, Princeton University	N/A
NIH 3T3 fibroblasts	Schwarzbauer Laboratory, Princeton University	N/A
Software and algorithms
Inkscape with Axidraw driver	Inkscape Project	V1.3.2
ImageJ/FIJI	https://imagej.net/software/fiji	V2.14.0
Image stitching	Preibisch, Saalfeld and Tomancak (2009)	N/A
MRI wound healing tool	Montpellier Ressources Imagerie	N/A
Slidebook	3i intelligent imaging innovations	V5
MicroManager	https://micro-manager.org/	V2.0
Prism	GraphPad Software	V10
Customized template	https://doi.org/10.5281/zenodo.13994473	V0.1
Other
XY gantry	Evil Mad Scientist, Inc.	Model: Axidraw V3
3D printer	Prusa Research a.s.	Model: MK3S+
PLA filament	Prusa Research a.s.	Model: PLA Jet black
M4 x 16 screw	McMaster-Carr	Cat #: 94500A282
M4 Hex nut	McMaster-Carr	Cat #: 90592A090
Adhesive putty	Loctite	Cat #: 10079340647432
Pipette tips (10μL)	Alkali Scientific	Cat #: ST1011-CS
Inverted microscope (phase contrast imaging)	Leica	DMi8
Inverted microscope (fluorescence imaging)	Zeiss	Axio Observer Z1

Experimental model and study participant details

Primary mouse keratinocytes were provided by the Devenport Laboratory at Princeton University and cultured in E-medium (Nowak and Fuchs, 2009) supplemented with 15% serum (S11550, Atlanta Biologicals) and 50 μM calcium. Wild-type MDCK-II cells (courtesy of the Nelson Laboratory, Stanford University) were cultured in Dulbecco’s Modified Eagle’s Medium (D5523-10L, Sigma-Aldrich) with 1 g/L sodium bicarbonate, 10% fetal bovine serum (S11550, Atlanta Biologicals), and 1% penicillin–streptomycin (15140-122, Gibco). NIH 3T3 fibroblasts were provided by the Schwarzbauer Laboratory at Princeton University. 3T3 cells were cultured in Dulbecco’s Modified Eagle’s Medium with phenol red (D5523-10L, Sigma-Aldrich), 10% fetal bovine serum (S11550, Atlanta Biologicals), and 1% streptomycin/penicillin (15140-122, Gibco). Tissue co-culture media consists of 50% Keratinocyte media and 50% 3T3 fibroblast media.³⁴ All cells were maintained at 37°C under 5% CO2 and 95% relative humidity. Cells were split before reaching 70% of confluence for maintenance culture, but all the dishes used for scratching had over 90% confluence to ensure even monolayers.

Method details

SCRATCH hardware setup

Here, we used the Axidraw v3 drawing robot (Evil Mad Scientist, Inc.) to provide XYZ control of our scratching tip. All of the CAD files for the customized attachments and templates we describe here are available at our github repository (See data and code availability section). Critical note: As of the time of publication, Axidraw is now only available through Bantam Tools and the cost has gone up to ∼$700. While this is still reasonable (similar to a single primary antibody), a variety of similar systems also exist through numerous suppliers that enable a variety of options and software approaches.

We designed and 3D printed a custom, modular plate holder that we attached to the Axidraw chassis using two M4 16mm long screws (94500A282, McMaster-Carr) and two M4 nuts (90592A090, McMaster-Carr), and this allows us to mount standard cultureware from 3.5 cm dishes to 96-well plates. We then designed and 3D printed a custom pipette holder with a thin layer of reusable adhesive (10079340647432, Loctite) (Video S1). The pipette holder assembly was then gently clamped to the vertical stage of the Axidraw using the built-in clamping screw. We calibrated SCRATCH using an alignment ring around the target dish, and press-fit the dish into the modular plate holder. If needed, reusable adhesive can be added to improve stability. With the gantry in pen-up position and powered down (or its motors disengaged), we moved the gantry arm across the dish to ensure vertical clearance through the dish walls, and then aligned the pipette tip with the mark on the alignment ring, this establishes the “origin” of the drawing and the starting point.

Upon completion, the pipette tip holder assembly was removed from the vertical stage of Axidraw. Then the dish was removed from the holder and washed with PBS three times to remove cell debris.

Scratch assay configuration

The Axidraw V3 is programmed using its official plugin in Inkscape (The Inkscape Team). The “Pen-up” and “Pen-down” range is set to 100% and 0% to ensure vertical clearance between the wells. Drawing speed is set to 10% (38 mm/s) and pen-up movement speed is set to 75% (285 mm/s). For contiguous tissues that have high cell-cell adhesions, drawing speed is set to 100% (380 mm/s). Dialogue box “Use constant speed when pen is down” is selected to ensure consistency. Pen raising speed and pen lowering speed is set to “Dead slow” to minimize pipette tip bouncing upon contact. Motor resolution is set to “∼2780DPI” for smooth operation and plot optimization set to least to avoid random starting point on a path. For all scratch assays, the programmed path is set to 0.01mm thick and is copied 4 times to the same place for repeated scratches. This ensures good area clearance and avoids uneven scratching due to non-conformal contact.

For raster mode, we use hatch fills options in Inkscape. Hatch spacing is set to a conservative value 0.1mm, which ensures each region is passed by the pipette tip at least 6 times to avoid any missed scratch zones due to non-conformal contact between the tip and the surface. Hatch angle is set to 45° but can be modified based on the tip. Inset fill from edges option is selected to compensate for the finite tip width, and inset distance is set to 0.187mm (a 75% overlap to ensure path clearance) but should be determined experimentally.

Tissue co-culture

A 35mm dish with confluent keratinocytes was scratched with the steps shown previously. Then the dish was washed with PBS three times and stained with Cellbrite Green (30021, Biotium) at 5μL/mL for 30 min. A dish of 3T3 fibroblasts was also stained with Cellbrite Red (30023, Biotium) at 5μL/mL in suspension for 30 min. The stained dish is washed with PBS and 2mL co-culture media is added. Stained 3T3 suspension is washed with co-culture media 3 times using a centrifuge (5702, Eppendorf). 3T3 suspension is then added to the keratinocyte dish with a density of 1000 cells/mmˆ2. The dish is then incubated for 30 min for 3T3 attachment. Then the dish is fixed using 4% paraformaldehyde and stained with Hoechst 33342 (Thermo Fisher) for nucleus.

Microscopy

Phase-contrast images were captured with an automated inverted microscope (Leica DMI8) with a 5X objective. For wound healing assays, time-lapse images were captured every 20 min. Fluorescence images were captured using an inverted microscope (Zeiss Axio Observer Z1) with a 5x objective, controlled using Slidebook (3I Intelligent Imaging Innovations) with Cy5, FITC, and DAPI filter sets. In both experiment setups, the microscopes were equipped with custom-built incubators maintaining 37°C and 5% CO2.

Quantification and statistical analysis

Image and data analysis

FIJI (https://imagej.net/software/fiji) is used to process all images, including stitching³⁵ and wound area calculation for wound healing assay (MRI Wound Healing Tool, Montpellier Ressources Imagerie). Stitched phase images are processed through FFT bandpass filter (40px max, 2px min) to minimize flat fielding. A custom script is developed to analyze scratch width uniformity. Each scratch is thresholded and segmented to calculate the distance between the edges. Data visualization is performed using GraphPad Prism 10 (GraphPad Software).

Published: December 9, 2024