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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 medicine1,2,3. 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 °.......

Representative 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.......

Acknowledgements

The authors acknowledge grants from the National Natural Science Foundation of China (81771971, 81970442, 81703470 and 81570422), National Key R&D Program of China (2018YFC1005002), Science and Technology Commission of Shanghai Municipality (17JC1400200), Shanghai Municipal Science and Technology Major Project (Grant No. 2017SHZDZX01), and Shanghai Municipal Education Commission (Innovation Program 2017-01-07-00-07-E00027).

....

Materials

NameCompanyCatalog NumberComments
2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (PI2959)TCIM64BK-QD
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)Gibco15630080
Dulbecco’s modified Eagle’s medium (DMEM)Gibco10569044
fetal bovine serum (FBS)Gibco10091
GelatinSigma-AldrichV900863MSDS
Methacrylic anhydride (MA)Sigma-Aldrich276685MSDS
Penicillin–streptomycin antibioticsGibco15140163
Phosphate-buffered saline (PBS)Gibco10010049
Silk fibroinAdvanced BioMatrix5154

References

  1. Khademhosseini, A., Langer, R. A decade of progress in tissue engineering. Nature Protocol. 11 (10), 1775-1781 (2016).
  2. Heinrich, M. A., et al. 3D bioprinting: from benches to translational applications. Small. ,....

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