Insights on the role of cryoprotectants in enhancing the properties of bioinks required for cryobioprinting of biological constructs

Abstract

Preservation and long-term storage of readily available cell-laden tissue-engineered products are major challenges in expanding their applications in healthcare. In recent years, there has been increasing interest in the development of off-the-shelf tissue-engineered products using the cryobioprinting approach. Here, bioinks are incorporated with cryoprotective agents (CPAs) to allow the fabrication of cryopreservable tissue constructs. Although this method has shown potential in the fabrication of cryopreservable tissue-engineered products, the impact of the CPAs on the viscoelastic behavior and printability of the bioinks at cryo conditions remains unexplored. In this study, we have evaluated the influence of CPAs such as glycerol and dimethyl sulfoxide (DMSO) on the rheological properties of pre-crosslinked alginate bioinks for cryoprinting applications. DMSO-incorporated bioinks showed a reduction in viscosity and yield stress, while the addition of glycerol improved both the properties due to interactions with the calcium chloride used for pre-crosslinking. Further, tube inversion and printability experiments were performed to identify suitable concentrations and cryobioprinting conditions for bioinks containing CPAs & pre-crosslinked with CaCl₂. Finally, based on the printability analysis & cell recovery results, 10% glycerol was used for cryobioprinting and preservation of cell-laden constructs at −80 °C and the viability of cells within the printed structures were evaluated after recovery. Cell viability results indicate that the addition of 10% glycerol to the pre-crosslinked bioink significantly improved cell viability compared to bioinks without CPAs, confirming the suitability of the developed bioink combination to fabricate tissue constructs for on-demand applications.

Graphical abstract

Effect of cryoprotectants on the viscoelastic behavior of bioinks and cell recovery in cryobioprinted tissue constructs.

Introduction

Tissue engineering is an interdisciplinary field that aims to restore or enhance the functions of damaged tissues or organs using cells, biomaterials and bioactive molecules [1]. Recent advancements in regenerative medicine and tissue engineering can fulfil the demands for tissue and organ replacements within the next few years [2]. Globally, researchers are trying to develop various tissues/organs using different scaffold fabrication strategies, including electrospinning, cell sheet engineering, hydrogels, solvent casting, particulate leaching and 3D bioprinting [3, 4]. Among these fabrication techniques, the usage of extrusion-based bioprinting (EBB) to fabricate artificial tissues and organs has significantly increased in the last decade due to its speed, cost-effectiveness, ease of fabrication, versatility in handling multiple materials and capacity to fabricate intricate structures [5]. Bioinks utilized in extrusion bioprinting typically consist of a mixture of cells and hydrogels, which are deposited layer-by-layer through a nozzle attached to the print head, following pre-programmed design instructions [6]. Bioinks typically consist of polysaccharides, proteins and biomolecules like growth factors to replicate the microenvironment of native tissues by supplying the essential components required for cell growth and function [7]. In recent years, bioprinting has made significant advances in successfully fabricating complex tissues such as bone, cornea, skin, heart, kidney, liver, brain, etc., that closely mimic the shape and native features [8].

Although bioprinting process provides structural mimicry of native tissues or organs, preservation of these bioprinted constructs for on-demand applications is still in the development stage [9–11]. On-demand or off-the-shelf cell-laden tissue-engineered products are biological structures (bioprinted constructs/ scaffolds) containing living cells, which are stored in cryo conditions for long term and used when required with minimal or without requiring extensive culturing. To provide quick relief and improve patient quality of life, several researchers are focusing on the preservation and recovery of tissue-engineered constructs using cryoprotective agents (CPAs) to improve the functional efficacy for on-demand applications [10, 12–14]. CPAs are the chemical substances commonly used to preserve biological materials (cells, tissues, and organs) by preventing ice crystal formation and slowing down metabolic processes and cellular degradation. CPAs are majorly divided into two distinct groups, depending on their ability to penetrate biological membranes. Penetrable CPAs work effectively by crossing the cell membranes and forming a protective barrier that decreases the rate of intracellular ice crystal formation [15]. The most commonly used penetrating CPAs are dimethyl sulfoxide (DMSO) and glycerol, which are capable of protecting a wide range of biological materials, including cells, tissues, and organs [16–18].

Non-penetrating cryoprotectants including sugars such as sucrose, lactose, maltose, raffinose, trehalose and melezitose), starches (hydroxyethyl starch) and synthetic polymers (polyvinyl pyrrolidone (PVP) and polyethylene oxide (PEO) are unable to cross cellular membranes but protect cells by reducing osmotic stress during the freezing and thawing processes. Saccharides and fetal bovine serum (FBS) are crucial non-penetrating cryoprotectants that preserve cell viability by reducing shear stress, maintaining osmotic balance and minimizing oxidative stress in the freezing medium [19]. Similarly, natural polymers like dextran, alginate, chondroitin sulfate and chitosan are widely used in the cryopreservation of diverse cell types and tissues, serving as valuable cryoprotective agents [20, 21].

In recent years, researchers have developed various strategies to incorporate CPAs into engineered tissues to preserve their functionality and offer quick therapeutic solutions for individuals suffering from burns, bone defects, muscle loss, etc. [22, 23]. Currently, two fundamental approaches are used for the cryopreservation of biofabricated tissues. The first approach involves fabricating tissues, allowing them to mature, or freezing them in cryoprotective agents containing solutions to utilize them for on-demand applications [16]. In contrast, the second approach integrates CPAs with bioinks, depositing them directly onto a freezing platform, thus eliminating the traditional two-step approach of fabricating and preserving tissues successively. Recently, a new bioprinting method called “cryobioprinting”, has been reported in which low viscous GelMA based bioinks are printed by adding cryoprotective agents to enhance shape fidelity of the bioinks, preserve cell functionalities and provide easy transport of tissues/organs to desired locations under cryopreservation conditions for on-demand applications [10, 12]. Cryobioprinting is a promising approach to overcome current challenges in fabricating ready-to-use tissue constructs that can be cryopreserved for long term [12]. Bioinks used for bioprinting applications need specific viscoelastic properties, including shear thinning, recovery and stability. It is essential to maintain the shape and structure during the printing and post-printing process until the cells have time to grow, self-organize into functional tissue [24]. Further, bioinks should have enough viscosity to dispense through the bioprinter nozzle and maintain the layer thickness of the printed structures [25]. In general, cryoprotectants are added to bioinks or printed structures to minimize freezing-induced damage to the cells within the bioinks. However, the incorporation of cryoprotectants can also change the viscoelastic behavior of the bioinks, affecting their printability and layer stacking ability apart from supporting cell viability. Hence, it is important to carefully select and optimize the use of CPAs within the bioink formulation to achieve a balance between cell viability and printability. However, the changes in the viscoelastic properties of the inks after the addition of DMSO, disaccharides and trisaccharides was not studied [10, 12].

In the present study, the effect of DMSO and glycerol on the viscoelastic properties of the pre-crosslinked alginate bioinks were evaluated. Initially, the viscoelastic properties of the developed cryobioinks were studied to determine the changes in the strength and viscosity of the bioinks. Further, tube inversion and printability experiments were performed to identify suitable concentrations and printing conditions of CPAs containing pre-crosslinked alginate bioinks. Various concentrations of DMSO and glycerol containing storage medium were used to cryopreserve and assess the recovery of adult human dermal fibroblasts. After cryopreservation for 72 h, the cells were recovered and cultured for 72 h to evaluate proliferation and viability. Finally, based on the optimized printing conditions, the ability of cell proliferation while using 10% glycerol was evaluated to use them for on-demand applications.

Materials & methods

Materials

Sodium alginate salt from brown algae (Viscosity ≥2000 cPs, 2% at 2 °C) and calcium chloride were purchased from Sigma – Aldrich, USA. Dulbecco’s phosphate buffer saline (DPBS), Dulbecco’s Modified Eagle Medium (DMEM), penicillin/streptomycin (PS) and Fetal bovine serum (FBS) were purchased from Thermo Fisher Scientific, USA. Dimethyl sulfoxide (DMSO) and glycerol were purchased from Himedia, India.

Preparation of cryopreservable bioinks

Alginate (ADM-20 mM CC) based pre-crosslinked bioinks were prepared according to our previous report with slight modifications [26]. Briefly, ADM-20 mM CC bioink was prepared by dissolving 4% sodium alginate (w/v) in DMEM containing 10% FBS (v/v) and 20 mM CaCl₂. Cryopreservable bioinks were prepared by adding DMSO to maintain the final concentrations such as 0% (ADM–20 mM CC), 5% (ADM–20 mM CC-D5%), 10% (ADM–20 mM CC-D10%) and 15% (ADM–20 mM CC-D15%) DMSO (v/v) in the pre-crosslinked bioinks. Bioinks with glycerol, ADM–20 mM CC-G5%, ADM–20 mM CC-G10% and ADM–20 mM CC-G15% were prepared while maintaining glycerol concentrations at 5%, 10%, and 15% respectively (Table 1). To assess the role of different components in printability and viscoelastic properties, bioinks without pre-crosslinking were prepared by adding various concentrations of CPAs to the DMEM supplemented with 10% FBS (Supplementary Table 1).

Table 1.

List of various alginate based pre-crosslinked cryobioinks used in this study. All combinations contain 10% FBS and 20 mM CaCl₂

Bioinks used	Composition
ADM–20 mM CC	4% alginate dissolved in DMEM
ADM–20 mM CC-D5%	4% alginate dissolved in DMEM with 5% DMSO
ADM–20 mM CC-D10%	4% alginate dissolved in DMEM with 10% DMSO
ADM–20 mM CC-D15%	4% alginate dissolved in DMEM with 15% DMSO
ADM–20 mM CC-G5%	4% alginate dissolved in DMEM with 5% glycerol
ADM–20 mM CC-G10%	4% alginate dissolved in DMEM with 10% glycerol
ADM–20 mM CC-G15%	4% alginate dissolved in DMEM with 15% glycerol

Viscoelastic properties of cryopreservable bioinks

The viscoelastic properties of cryopreservable bioinks were determined using Physica MCR302 rheometer (Anton Paar, Austria). The stage temperature was kept constant at 26 °C throughout the tests and the analysis was carried out on a parallel plate (25 mm in diameter) with a 1 mm gap between plates. Initially, the viscosity of the cryopreservable bioinks was determined by varying the shear rate from 1 s⁻¹ to 100 s⁻¹ to determine the shear-thinning behavior of bioinks. Further, the linear viscoelastic range (LVR) and yield stress of the bioinks were determined by performing an amplitude sweep ranging from 0.01 to 100% strain at a constant frequency of 10 Hz. Furthermore, the strength of the bioinks were determined from the storage and loss modulus values obtained by varying frequency from 0.1 to 100 rad s⁻¹ in the linear viscoelastic range [26].

Tube inversion method

The stability of the pre-crosslinked cryopreservable bioinks were evaluated using the tube inversion method. Various bioinks mentioned in Table 1 were prepared in glass vials and maintained in an inverted position for 30 min at 37 °C. Further, to evaluate the flow characteristics of the bioinks, photographs were taken immediately after inverting the vials and again after 30 min.

Printability & pore factor analysis

Printing experiments were carried out using 3D Bioplotter (EnvisionTEC, Germany) by loading the bioinks (Table 1) into low-temperature cartridges. Printhead temperature was maintained at 26 °C and 0.40 mm flow trap needle was used to dispense the bioinks at pressures ranging from 0.3 to 0.7 bars while varying printing speed (5 mm/s, 10 mm/s and 15 mm/s). The spreading ratio was analyzed by measuring the strand width of the printed constructs using a phase contrast inverted microscope (Olympus CKX53, Japan). The circularity (C) and printability were evaluated by measuring the area (A) and perimeter (L) of the pores in the gird pattern (printing two layers in 0/90^o) using Image J software (Version – 1.54 d) as mentioned in previous reports [27].

$$\text{Spreading}\text{ratio}=\frac{\text{Strand}\text{width}\text{of}\text{the}\text{printed}\text{constructs}}{\text{Needle}\text{inner}\text{diameter}}$$

$$\text{Circularity}=\frac{4\pi A}{L^2}$$

$$\text{Printability}\left(\text{Pr}\right)=\frac{\pi }{4}\text{x}\frac{1}{C}$$

Cell culture

Primary human adult dermal fibroblast (HADF) cells were procured from Himedia, India. Cells were cultured in T-75 flask using DMEM supplemented with 10% FBS and 1% antibiotic–antimycotic (AB-AM) in a CO₂ incubator at 37 °C with 5% CO₂. After reaching cellular confluency of 80–85%, cells were washed with Dulbecco’s phosphate buffered saline (DPBS) and then trypsinized using a 0.05% trypsin/EDTA solution. Cells were collected through centrifugation at 2000 rpm for 5 min at 4 °C and counted using a haemocytometer by trypan blue exclusion method.

Cryopreservation & recovery of cells

To assess the cell recovery post-freezing, solutions containing varying concentrations of cryoprotectants (Supplementary Table 2) were prepared and incubated with the cells. Briefly, trypsinized cells were added to the cryoprotectant solution with a density of 0.02 ×10⁶ cells per mL in a cryovial and gradually cooled at a rate of −1 °C/min to −80 °C. After 72 h of freezing, cells were rapidly thawed by maintaining them at 37 °C and then centrifuged at 2000 rpm for 5 min to remove the cryoprotectant solution. To the cell pellet, 250 µL of fresh DMEM medium supplemented with 10% FBS and 1% AB-AM was added, and the cells were incubated in a CO₂ incubator with 5% CO₂. After 24 h and 72 h of recovery, MTS assay was performed to analyze the cryoprotective and proliferation abilities of the cells frozen in different cryoprotectant solutions [2]. Briefly, after washing the wells with DPBS, 20 μL of MTS reagent was diluted in serum free DMEM (1:10 ratio) and added to the wells. Absorbance was measured at 490 nm after 3 h of incubation using a multimode microplate reader (Infinite 200 M Tecan, Austria).

Cryoprinting of complex shapes

The complex shape printability of the developed ADM–20 mM CC-G10% bioink was evaluated by extruding the bioink using the optimized printing conditions (section 2.5). The print head temperature was initially set at 26 °C, while the printing platform was maintained at −10 °C. Various shapes such as hollow cylindrical tube with 1 cm diameter and 0.5 cm height, 1 cm (L) ×1 cm (W) grid structures with 0.5 cm height, heart outline and our Center name “CeNTAB” were printed to assess the printability.

Cryoprinting of cellular constructs

Based on the results observed in viscoelasticity, printability, and cell recovery experiments, ADM–20 mM CC and ADM–20 mM CC-G10% were printed to further evaluate the suitability of these bioinks to fabricate tissue constructs for on-demand applications. ADM–20 mM CC printed onto the platform maintained at room temperature (25 °C) was used as control. Bioinks consisting of ADM–20 mM CC and ADM–20 mM CC-G10% were 3D printed onto a platform maintained at –10 °C and their properties were compared with constructs printed at room temperature. HADF cells (2 ×10⁶ cells/mL) were mixed with bioinks and cryobioprinted under optimized conditions (0.5 bars at 15 mm/s) (Section 2.5). The platform temperature was maintained at –10 °C during printing and the constructs were immediately stored at –80 °C after printing. During thawing, 0.1 M CaCl₂ was directly added to cryoprinted constructs and incubated for 30 min in CO₂ incubator maintained at 37 °C. After 30 min of crosslinking, constructs were washed twice with DPBS and maintained in DMEM medium supplemented with 10% FBS & 1% AB-AM.

Live-dead assay

At predetermined time points, recovered constructs were analyzed for cell viability using live-dead assay kit (Thermo Fischer, USA). The stained constructs were visualized under confocal laser scanning microscope (CSLM. FV1000, Olympus, Japan) and the percentage of viable cells were counted using the Image J software [28]. The viability of cells was calculated using the below mentioned formula:

$$\text{Cell}\text{viability}\left(\%\right)=\frac{\text{Number}\text{of}\text{live}\text{cells}}{\text{Total}\text{number}\text{of}\text{cells}}\text{X}100$$

Statistical analysis

All data were expressed in terms of mean ± SD with n equal to the number of samples. Statistical difference in the yield stress of different bioinks was calculated using one-way ANOVA. Statistical difference in cell viability was calculated using two–way ANOVA followed by the Tukey post–hoc test. The p-value < 0.05 was considered as statistically significant.

Results

Viscoelastic properties of cryopreservable bioinks

CPAs are extensively used in the cryopreservation of cells & tissues to prevent ice crystal formation and to ensure cell viability during the freezing process. Glycerol and DMSO are widely used penetrative CPAs due to their cytocompatibility and water miscibility, while polymers like alginate, chondroitin sulfate and chitosan are widely employed non-penetrative CPAs [29]. Alginate is an anionic polysaccharide derived from brown seaweed and widely used for various biomedical applications due to its low toxicity, resemblance to mammalian extracellular matrix and crosslinks quickly in the presence of divalent cations like Ca²⁺ and Mg²⁺ [30]. In recent times, alginate has been widely used for cryopreserving & recovery of various scaffolds and tissue constructs, including freeze-dried scaffolds, electrospun scaffolds, hydrogels, and 3D bioprinted structures for on-demand applications [31, 32]. In our previous reports, pre-crosslinking of 4% alginate with 20 mM CaCl₂ (ADM–20 mM CC) improved the shape fidelity and printability of the bioprinted constructs compared to the bioinks without pre-crosslinking [26]. In the present study, ADM–20 mM CC bioink was used to determine the effect of incorporation of cryoprotectants on the viscoelastic properties, printability and their suitability for cryobioprinting applications.

Initially, the viscosity of bioinks was measured and found that all the bioinks (Table 1) showed shear-thinning behavior, which is an ideal property required for printing. Further, the addition of DMSO led to a reduction in the viscosity of the bioinks due to the interactions between DMSO and calcium chloride, which impairs the pre-crosslinking of alginate. Moreover, bioinks with 15% DMSO (ADM–20 mM CC–D15%) showed a significant reduction in the viscosity compared to ADM–20 mM CC, ADM–20 mM CC-D5% and ADM–20 mM CC-D10% bioinks (Fig. 1A). Interestingly, when DMSO was added to bioinks (not pre-crosslinked) showed increased viscosity due to the formation of hydrogen bonding (Supplementary Fig. S1A-a). Pérez-Madrigal et al., observed similar results when DMSO was added to alginate solution and observed changes in viscosity of the alginate solution resulting in the formation of weak gels [33]. The addition of glycerol to both pre-crosslinked and uncrosslinked bioinks increased their viscosity without having any impact on crosslinking (Fig. 2A & Supplementary Fig. S1B-a). The increase in viscosity could be due to the formation of hydrogen bonds between glycerol and alginate chains, as well as an increase in intermolecular cohesion. A recent report by Nie et al. observed that the addition of glycerol significantly improves the viscosity of alginate solutions, leading to the formation of nanofibers using electrospinning [34].

Fig. 1.

Viscoelastic properties of ADM–20 mM CC bioinks containing different concentrations of DMSO. A Viscosity sweep of bioinks with different concentrations of DMSO; B Yield stress of bioinks with DMSO; C Frequency sweep of bioinks with different concentrations of DMSO and D Loss factor of bioinks containing DMSO

Fig. 2.

Viscoelastic properties of ADM–20 mM CC bioinks containing different concentrations of glycerol. A Viscosity sweep of bioinks with different concentrations of glycerol; B Yield stress of bioinks with glycerol; C Frequency sweep of bioinks with different concentrations of glycerol and D Loss factor of bioinks containing glycerol

After determining the LVR range of the bioinks from the amplitude sweep measurements, the changes in the viscoelastic properties of the bioinks with an increase in the frequency was analyzed. Increasing the frequency of bioinks resulted in a corresponding increase in the strength of the bioinks, with gel-like behavior consistently observed across the entire frequency range from 0.1 rad/s to 100 rad/s. This confirms the suitability of these bioinks for 3D bioprinting applications. Further, there was no significant difference in the storage modulus (G′) of the bioinks at 100 rad/s was observed between ADM–20 mM CC (3729.06 ± 154.83 Pa), ADM–20 mM CC-D5% (4029.73 ± 277.17 Pa) and ADM–20 mM CC-D10% (4141.86 ± 479.45 Pa). However, when the concentration of DMSO was increased to 15% (ADM–20 mM CC-D15%), there was a significant reduction in the strength of the bioink (980.22 ± 81.82 Pa), which is not ideal for maintaining the shape fidelity during printing (Fig. 1C). Further, with increase in the concentration of DMSO to ADM bioinks, there was a significant increase in the overall strength compared to control bioink (ADM) (Supplementary Fig. S1A-b). This confirmed that the reduction in the strength of ADM–20 mM CC-D15% bioink is due to interactions with calcium chloride in the pre-crosslinked bioinks. Uncrosslinked and pre-crosslinked bioinks containing various concentrations of glycerol as CPA maintained the gel-like behavior (G′ > G′′) and did not affect the overall storage modulus (G′) of the bioinks (ADM–20 mM CC-G5% - 3889.53 ± 172.24 Pa; ADM–20 mM CC-G10% – 3972.56 ± 305.84 Pa; ADM–20 mM CC-G15% 3716.7 ± 283.53 Pa) compared to bioink without CPAs (Fig. 2C & Supplementary Fig. S1B-b). This suggests that the presence of glycerol does not compromise the stiffness or elasticity of the bioink network.

Loss factor (Tan δ) is the ratio between G′′ and G′ and an indicator of the stability of the bioink over the frequency range from 0.1 rad/s to 100 rad/s. Results of Tan δ measurements showed that all bioinks are in the range from 0.25–0.4, confirming the suitability of these bioinks for 3D bioprinting of tissue constructs [27]. However, ADM–20 mM CC-D15% bioink showed higher Tan δ values, which is an indicator of the fluid-like behavior of bioink and not appropriate for printing applications (Figs. 1D and 2D). Similar to our previous reports, uncrosslinked bioinks containing both DMSO and glycerol showed poor stability with higher Tan δ values, indicating poor viscoelastic behavior and hence not suitable for 3D printing (Supplementary Fig. S1C).

The yield stress of the bioinks was measured to understand the changes in the stress required to initiate the bioink flow upon the addition of DMSO and glycerol. Addition of glycerol significantly increased the yield stress of bioinks proportional to its concentration, whereas the addition of DMSO leads to a significant reduction in the yield stress (Figs. 1B and 2B) similar to the results observed for the viscosity of bioinks. Apart from ADM–20 mM CC-D15% bioink, all other combinations possess the ideal yield stress for successful extrusion of bioinks [35, 36]. Although the incorporation of glycerol and DMSO enhanced the yield stress of non-crosslinked bioinks, inadequate viscosity and poor gel strength make them unsuitable for printing (Supplementary Fig. S1D). From rheological assessments, it may be concluded that the incorporation of DMSO as CPA alters the viscoelastic behavior of alginate-based pre-crosslinked bioinks, and 15% of DMSO-containing bioinks showed poor viscoelastic behavior and not suitable for printing. Bioinks with glycerol have no significant effect on the overall strength of the bioinks, but increasing glycerol concentrations leads to an increase in yield stress.

Printability assessment

The flowability of the bioinks were studied using tube inversion method after loading them into the glass vials [2]. After maintaining the glass vials in the inverted position for 30 min, all bioinks except ADM–20 mM CC-D15%, maintained their position in the glass vials, confirming the stability of these bioinks (Supplementary Fig. S2). Further, the printability of the bioinks and suitable printing conditions were identified by varying the printing parameters such as print head temperature, printing pressure, speed, nozzle diameter, etc. The parameters were set to smoothly extrude the bioink through a nozzle, with precise structure formation, strong interlayer adhesion, high resolution, and cytocompatibility [37]. Based on the results of rheological parameters, bioinks containing 15% DMSO were not used for printability assessment due to their poor viscosity and yield stress. In corroboration with the viscosity results, the pressure required for extruding DMSO-containing bioinks was less (0.3 bars to 0.6 bars) as the concentration of DMSO increased (Supplementary Fig. S3) and for glycerol based bioinks 0.5 bars to 0.7 bars were applied to extrude (Supplementary Fig. S4). Optimal printing conditions were identified for all the bioinks by extruding them with a 0.4 mm diameter needle at different speeds (5 mm/s, 10 mm/s, and 15 mm/s) (Fig. 3). The strand width measured under different printing conditions ranged from a maximum of 1288 µm to a minimum of 499 µm. Also, as the printing pressure remained less than 0.7 bars, it is considered safe for bioprinting applications.

Fig. 3.

Printability analysis of bioinks A without CPAs; CPAs containing B 5% DMSO; C 10% DMSO; D 5% glycerol; E 10% glycerol and F 15% glycerol

Spreading ratio and pore factor values for different bioinks printed at various conditions were analyzed to identify the printability [38]. The spreading ratio was measured by comparing the width of printed strands to the needle’s inner diameter. ADM–20 mM CC-G10% bioink showed lowest spreading ratio (1.24 ± 0.09) when printed at 0.5 bars and 15 mm/s speed compared to all other bioinks and printing conditions (varied from 1.34 ± 0.34 to 1.88 ± 0.17) (Fig. 4A). The same conditions were used to analyze the pore factor and found to be in the range of 0.91 ± 0.01 to 0.97 ± 0.03 (Fig. 4B). A pore factor or printability index (Pr) close to 1 indicates a perfect square, while less than or greater than one indicates poor or over-gelation [39]. The Pr values observed in the present study, ranged from 0.9 to 1, which correlate with the findings reported in earlier [40, 41].

Fig. 4.

A Spreading ratio and B pore factor analysis of bioinks extruded at different speeds and pressures

Cryopreservation & recovery of cells

Cryopreservation is the process of storing biological materials such as cells, tissues and organs at very low temperatures to preserve their viability and maintain their functional characteristics for in vitro and in vivo applications. In the present study, we initially assessed different concentrations of DMSO and glycerol to determine their effectiveness as cryoprotectants for cryobioprinting. After freezing for a short term (72 h) at –80 °C, cells were recovered and seeded on 96 well plates and MTS assay was performed to assess the recovery percentage compared to the control (without cryopreservation). DMEM medium supplemented with 10% FBS was used as a negative control to understand the role of penetrable CPAs in cell preservation. Interestingly, 10% DMSO, 15% DMSO and 5% glycerol showed significantly lower recovery compared to 5% DMSO, 5% and 10% glycerol (Supplementary Fig. S5). Similarly, cells preserved with 5% DMSO displayed significantly higher proliferation efficiency compared to those preserved with 10% and 15% DMSO, while no notable difference in cell proliferation was observed among the cells preserved using glycerol (Supplementary Fig. S5). The results demonstrate that 5% DMSO maintains the functionality of various cell types, such as fibroblasts, keratinocytes and T-cells without impacting proliferation and correlates with published results [42, 43]. In contrast, dermal fibroblasts preserved using 5% glycerol showed significantly lower cell recovery compared to 10% and 15% glycerol. Interestingly, this study found no significant differences in cell proliferation between HDF cells stored in 5% DMSO, 10% glycerol, and 15% glycerol. Moreover, several studies reported that cryopreservation of sensitive cells such as primary cells and stem cells (embryonic stem cells, mesenchymal stem cells, induced pluripotent stem cells) using DMSO has been associated with toxicity [18, 44, 45]. To overcome the problems associated with DMSO, 10% glycerol containing bioink (ADM–20 mM CC-G10%) was used for cryoprinting application to determine its ability for cryopreservation and on-demand tissue engineering applications.

Complex shape printability

For further experiments, ADM–20 mM CC-G10% bioink was used due to good viscoelastic properties, printability and cryoprotective abilities compared with other concentrations of glycerol and DMSO. Initially, the complex shape printability of ADM–20 mM CC-G10% bioink was determined by extruding it using optimized printing conditions (0.5 bars, 15 mm/s, and 0.4 mm NID) by maintaining the platform temperature at –10 °C. After printing each layer, 2 s of waiting time was given to freeze the deposited constructs quickly. However, instantaneous freezing of the deposited bioink was observed only up to 2 mm height (tubular and grid structures) (Fig. 5). This is similar to previous reports on cryobioprinting [9, 12], which is mainly due to the differences in homogenous distribution of temperature (freezing) across different layers. Further, the outline of the heart model and our Centre name were printed up to 2 mm in height, which showed quick freezing and maintained shape fidelity. Although pre-crosslinked alginate-based bioinks show good printability, due to the limitations in the static cryoprinting process, freezing printed constructs after a certain height is not possible [46]. However, these complex shape printability experiments revealed that constructs with 1 mm height can be rapidly frozen within 5 s, and thus the constructs with similar size were printed in our further experiments.

Fig. 5.

Complex shape printability analysis of ADM–20 mM CC-G10% bioink. A Tubular construct ((a) Top view, (b) side view); B Square construct with 2 mm height ((a) Top view, (b) side view); C Square construct with 5 mm height ((a) Top view, (b) side view) (Yellow arrow – frozen region; Black arrow – unfrozen area); D Heart outline and E Our Center name – CeNTAB

Cell viability in cryoprinted constructs

This study aims to produce tissue constructs for on-demand applications with high cell viability in the printed structures after recovery. Previous studies have demonstrated that the use of high printing pressures (>1.5 bars), small diameter needles and temperature changes has a significant impact on the viability of cells [47, 48]. To avoid these complications, we have used a printing pressure of 0.5 bars, which is in the cytocompatible range, flow trap needle (0.4 mm NID) to minimize the complications reported in previous reports [49]. Further, ADM–20 mM CC bioink was printed at two different conditions by maintaining the printing platform at room temperature (25 °C) and –10 °C. Also, ADM–20 mM CC-G10% bioink was printed onto the freezing platform to evaluate the cytocompatibility of the cryoprinting process.

Bioinks were prepared by mixing HADF cells at the final density of 2 × 10⁶ cells/mL and the constructs (1 cm (L) × 1 cm (W) × 0.1 cm (H)) were printed. Constructs printed using ADM–20 mM CC bioink at room temperature were immediately crosslinked with 0.1 M CaCl₂ and more than 85% of the cells were viable after culturing for 24 h (Fig. 6). The results observed in this study are similar to our previous reports, where the incorporation of human ventricular cardiomyocytes and neonatal ventricular rat myocytes into these pre-crosslinked bioinks did not affect the viability of the cells in the printed constructs [26]. Moreover, HADF cells in the printed constructs maintained greater than 84% viability for upto 7 days. In contrast, when ADM–20 mM CC constructs were printed onto the freezing printing platform, maintained at –10 °C and subsequently frozen at –80 °C for 3 days, a significant reduction in the viability of HADF cells (66.83 ± 7.81%) was observed after 24 h of recovery. This could be due to thermal stress on the cells during the freezing process, which compromise the integrity of the cell membranes and adversely affect the viability [15, 50]. Interestingly, we observed greater than 60% of cell viability on day 3 (69.09 ± 4.79%) and 62.58 ± 6.78% viability at day 7 due to the cryoprotective behavior of alginate as a non-penetrating CPA. However, this is not sufficient to achieve the main objective of this study of developing tissue-engineered constructs for on-demand applications [12, 32].

Fig. 6.

Live-dead staining of 3D constructs printed with HADF cells and recovered after 72 h of cryopreservation. Cells after maintaining in culture for A 1 day; B 3 days and C 7 days in [a] ADM–20 mM CC printed at normal conditions; [b] ADM–20 mM CC bioink printed at –10 °C and cryopreserved for 72 h at –80 °C; [c] ADM–20 mM CC-G10% bioink printed at –10 °C and cryopreserved for 72 h at –80 °C (Green - live cells & red - dead cells) and D Quantitative analysis of HADF cell viability using CLSM images

ADM–20 mM CC-G10% bioink was used as bioink and printed onto the printing platform maintained at cryo conditions of –10 °C. Here, greater than 80% of cells were viable and the viability of the cells were maintained upto day 7, which confirmed the capability of glycerol incorporated pre-crosslinked bioink to fabricate tissue constructs for on-demand applications (Fig. 6). Since ADM–20 mM CC-G10% bioink contains 10% glycerol as penetrable CPA and alginate as non-penetrating CPA, the printed constructs were protected from cryoinjuries both by reducing the ice crystal formation, osmotic shock and helped in maintaining cell viability (day 1: 81.07 ± 4.62%, day 3: 83.15 ± 7.04% and day 7: 79.54 ± 6.99%).

Conclusions

Cryobioprinting offers a better solution to overcome the current challenges related to the limited shelf life and long-term storage of bioprinted constructs by combining bioprinting with cryopreservation strategies. Researchers have recently utilized GelMA–based bioinks supplemented with DMSO and various di– and trisaccharides as cryoprotectants for cryobioprinting of various tissues. Although cryobioprinting showed promising outcomes in terms of the printing process and tissue preservation post-printing, there is a notable lack of focus on the impact of these cryoprotective agents on the viscoelastic properties of the bioinks employed in cryobioprinting. In the present study, we have studied the addition of CPAs such as DMSO and glycerol on the viscoelastic behavior of alginate-based bioinks for cryoprinting of tissue-engineered products for on-demand applications. Pre-crosslinked alginate was used as bioink due to printability, quick crosslinking, shape fidelity, cytocompatibility and cryoprotective abilities. Increasing DMSO concentration in pre-crosslinked bioinks lowered the viscosity and yield stress, as DMSO reacted with calcium chloride, which impaired crosslinked alginate chains. Interestingly, the addition of glycerol to the bioinks resulted in increased viscosity, as it formed hydrogen bonds with alginate. Further, printability analysis confirmed that DMSO-containing bioinks required lower printing pressure to extrude the bioinks compared to bioinks without CPAs and glycerol-containing bioinks. Bioink containing 10% glycerol was further assessed for complex shape printability by extruding it onto the freezing platform, which confirmed the ability of the developed bioinks to freeze instantaneously. Finally, the cryobioprinting capabilities of the developed method were assessed by printing HDF cell-laden ADM–20 mM CC-G10% bioink onto the freezing platform at –10 °C. Following preservation and recovery, constructs with cryoprotective agents demonstrated good cell viability compared to controls, confirming the potential of these constructs for on-demand applications. In future, addition of CPAs to different bioinks may become essential for comprehending their interaction with bioink components, allowing the development of tissues with enhanced functionality for on-demand applications. Additionally, further investigations into the long-term viability and functionality of these cryopreserved tissue constructs will be crucial for regenerative medicine applications.

Author contributions

Harshavardhan Budharaju: Methodology, Investigation, Data curation, Formal analysis, Writing – original draft. Dhakshinamoorthy Sundaramurthi: Methodology, Investigation, Data curation, Writing – original draft, Writing – review & editing, Project administration. Swaminathan Sethuraman: Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Supervision, Writing – review & editing.

Data availability

The data supporting this article have been included as part of the Supplementary Information.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1007/s10856-024-06855-2.