Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

Summary

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

Abstract

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Introduction

Three-dimensional (3D) bioprinting has been a promising method to manufacture complex architectural and functional replicas of native tissues in biofabrication and regenerative medicine^1,2,3. The common strategies of bioprinting, including inkjet, extrusion, and stereolithography printing, have pros and cons from different perspectives⁴. Among these techniques, the extrusion procedure is most commonly used due to its cost-effectiveness. Bioink plays a key role in the process stability of extrusion bioprinting. The ideal cell-laden bioink should not only be biocompatible but also be suitable for mechanical properties⁵. Bioinks with low viscosity are typically presented as a liquid-like state. These bioinks can be easily and quickly deposited and avoid cell membrane damage induced by high shear stress during extrusion. However, in complex cases requiring long-term printing periods, low viscosity often gives rise to the inevitable sedimentation of the encapsulated cells in the bioink reservoir, which is usually driven by gravity and leads to an inhomogeneous cell dispersion in the bioink^6,7. Consequently, a bioink with inhomogeneous cell dispersity hampers the in vitro bioprinting of a functional tissue construct.

Several recent studies focusing on bioinks have reported the promotion of homogenous dispersity of encapsulated cells. A modified alginate bioink based on dual-stage crosslinking was used for extrusion bioprinting⁸. An alginate polymer was modified with peptides and proteins in this study. Cells presented a more homogeneous distribution in this modified alginate than in the commonly used alginate due to the attachment sites provided by the peptides and the proteins. Alternatively, blended bioinks have been utilized to solve the sedimentation of cells in bioink. A blended bioink containing polyethylene glycol (PEG) and gelatin or gelatin methacryloyl (GelMA) with improved mechanical robustness was used in another study⁹. The encapsulated cells presented a homogeneous distribution mainly because the viscosity of the blended bioink was improved. In general, there are several factors influencing the dispersity of the encapsulated cells in the bioink, such as the viscosity of the bioink, the gravity of the cells, the density of the cells, and the duration of the working period. Among these factors, the gravity of cells plays a critical role in promoting sedimentation. The buoyancy and friction provided by the viscous bioink have been investigated as the main forces against gravity to date¹⁰.

Herein, we developed a novel strategy to promote homogeneous dispersity of the encapsulated cells in bioink by manipulating multiple liquid interfaces in the bioink reservoir. These liquid interfaces created by the multilayered modification of bioink can not only provide interfacial retention, which retards the sedimentation of cells, but also maintain a suitable biocompatibility and rheological behavior of the bioink. In practice, we modified aqueous GelMA solution (5%, w/v) with silk fibroin (SF) in a multilayered manner to longitudinally produce four interfaces, providing interfacial tensions in the blended bioink. As a result, the gravity loading on the cells was offset by the man-made interfacial tension, and a nearly homogeneous dispersion of the encapsulated cells in the bioink was obtained due to less sedimentation across the adjacent layers of cells. No similar protocol to slow down the sedimentation of encapsulated cells by manipulating interfacial retention in liquid bioinks has been reported to date. We present our protocol here to demonstrate a new way to solve cell sedimentation in bioprinting.

Protocol

1. Preparation of cell-laden SF-GelMA

1. Sterilize all the materials by using 0.22 μm syringe filter units. Perform all the steps in a biological safety cabinet.
2. Warm 1x PBS to 50 °C, and dissolve gelatin in the heated 1x PBS with stirring. The final concentration of gelatin in PBS should be 10% (w/v).
3. Add methacrylic anhydride into the gelatin solution (weight ratio of methacrylic anhydride to gelatin of 0.6 to 1) slowly with stirring, and mix the complex for at least 1 h (50 °C). Typically, prepare 200 mL of 10% gelatin solution with 12 g of methacrylic anhydride; the volume depends on the needs of the study.
4. Transfer the mixed solution containing gelatin and methacrylic anhydride to a 50 mL sterile tube.
5. Centrifuge the mixed solution at 3,500 x g. It usually takes 3-5 min to obtain two layers. Collect the upper layer (GelMA) and discard the bottom layer (unreacted methacrylic anhydride).
6. Dilute the upper layer solution obtained in step 1.5 with two volumes of deionized water (40−50 °C).
7. Dialyze the solution obtained in step 1.6 with a 12-14 kDa molecular weight cutoff dialysis membrane against deionized water for 5−7 days (40−50 °C). Change the water twice every day.
8. Collect and freeze the GelMA solution at −80 °C overnight.
9. Lyophilize the GelMA solution for 3−5 days in a freeze dryer with the temperature set to -45 °C and the pressure set to 0.2 mbar.
10. Dissolve the lyophilized GelMA (degree of substitution of approximately 75%) in 1x PBS containing 10% FBS (fetal bovine serum, v/v), 25 mM HEPES (N-2-hydroxyethylpiperazine-N-ethane-sulfonic acid), and photoinitiator (0.5%, w/v) to obtain the GelMA bioink preparation.
    NOTE: The degree of substitution of GelMA can be calculated by a ninhydrin assay³.
11. Mix the GelMA solution (10%, w/v) with different volumes of initial SF solution (5%, w/v) and different volumes of 1x PBS to obtain SF-GelMA bioinks with different concentrations of SF. The proportion per 1 mL of GelMA and SF solution in the different bioinks is presented in Table 1.
    NOTE: All bioinks must contain a final concentration of 5% (w/v) GelMA, but the concentration of SF varies: 0.5, 0.75, 1.0, 1.25, and 1.5% (w/v) in the different SF-GelMA bioinks. Use SF-M-Layered-GelMA to term the GelMA bioink modified with SF in a layer-by-layer manner. Use SF-X-GelMA to term those GelMA modified with SF in a homogeneous manner (e.g., GelMA with modification of 1% SF was termed SF-1-GelMA).
12. Sonicate all bioinks for 10-20 min.
13. Grow NIH3T3 cells using DMEM (Dulbecco's modified Eagle medium) containing 10% FBS and 1% penicillin-streptomycin in an incubator (37 °C with 5% CO₂). Passage the cells at a ratio of 1:3 when the density reaches 80%.
14. Centrifuge the suspension of NIH3T3 cells at 1500 rpm for 5 min. Remove the supernatant with suction and resuspend the cell pellet with fresh bioink solution in a 15 mL sterile tube.
    NOTE: The concentration of the encapsulated cells in the bioink should be 1 x 10⁶ cells/mL, and the concentration needs to be calculated with a cytometer.
15. Use 2 mL of different SF-GelMA bioinks to suspend the cell pellets to obtain various cell-laden bioinks.

2. Loading, reheating and bioprinting of the SF-M-Layered-GelMA

1. Load 0.4 mL of cell-laden SF-0.5-GelMA into the bottom layer of the syringe.
   NOTE: Use a 2 mL syringe as the bioink reservoir in this study. Load different SF-GelMA bioinks into the syringe in a layer-by-layer manner.
2. Place the syringe in ice water bath (0 °C) for 5 min to cause the bottom-layer bioink to transform into a gel state.
3. Load 0.4 mL of cell-laden SF-0.75-GelMA bioink above the bottom layer.
4. Place the syringe with the two layers of bioinks in an ice water bath (0 °C) for 5 min to cause the two layers of bioinks to transform into a gel state.
5. Cycle the loading and cooling steps another 3 times with the remaining 3 kinds of bioinks (SF-1-GelMA, SF-1.25-GelMA, SF-1.5-GelMA) to obtain a multilayered bioink system with different concentrations of SF in the different layers. The volume used for the loading of all bioinks should be 0.4 mL.
6. Reheat the multilayered bioink by placing the syringe in an incubator (37 °C) for 30 min prior to bioprinting.
7. Use a 2 mL syringe as the bioink reservoir with a 27 G printing nozzle. Set the speed of flow at 50 μL/min, the moving speed of the nozzle at 2 mm/s, and the height of the nozzle at 1 mm. Perform the bioprinting procedure at room temperature (approximately 20 °C).
   1. Print the tissue construct in an extrusion manner using a custom-made bioprinter under the parameters adapted from our previous studies^11,12.
8. Use ultraviolet light (365 nm, 800 mW) for 40 s to crosslink the bioprinted tissue construct.
9. Culture the crosslinked tissue construct in DMEM (Dulbecco's modified Eagle medium) containing 10% FBS and 1% penicillin-streptomycin in an incubator (37 °C with 5% CO₂). The medium was changed every 8 h during the first 2 days and every 2-3 days thereafter.

Results

A schematic of the preparation of cell-laden bioinks is shown in Figure 1. After preparation of the different bioinks, loading, reheating and bioprinting were performed (Figure 2). To evaluate the distribution of the encapsulated cells in the bioink reservoir, a bioprinting procedure was performed using three different cell-laden bioinks in three 96-well plates (Figure 3A). Two control groups (pristine GelMA and SF-1-GelMA bioinks) ...

Discussion

The stability of the multilayered system is a key point to perform this protocol successfully. We theoretically calculated the diffusion of SF molecules in the GelMA solution based on Nauman’s study¹³. It was found that the diffusion of proteins in solution was related to their molecular weight. The average molecular weight (MW) of bovine serum albumin (BSA) is 66.5 kDa, and its diffusion coefficient is 64-72 μm²/s. The average MW of fibrinogen is 339.7 kDa, and its diffusion...

Materials

Name	Company	Catalog Number	Comments
2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (PI2959)	TCI	M64BK-QD
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Gibco	15630080
Dulbecco’s modified Eagle’s medium (DMEM)	Gibco	10569044
fetal bovine serum (FBS)	Gibco	10091
Gelatin	Sigma-Aldrich	V900863MSDS
Methacrylic anhydride (MA)	Sigma-Aldrich	276685MSDS
Penicillin–streptomycin antibiotics	Gibco	15140163
Phosphate-buffered saline (PBS)	Gibco	10010049
Silk fibroin	Advanced BioMatrix	5154