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 onl....

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. Tra....

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