Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Summary

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Abstract

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Introduction

Hydrogels are thought to be a suitable material in the field of biofabrication^1,2,3,4. Among them, gelatin methacryloyl (GelMA) has become one of the most versatile biomaterials, initially proposed in 2000 by Van Den Bulcke et al.⁵. GelMA is synthesized by the direct reaction of gelatin with methacrylic anhydride (MA). The gelatin, which is hydrolyzed by the mammal collagen, is composed of target motifs of matrix metalloproteinase (MMP). Thus, in vitro three-dimensional (3D) tissue models established by GelMA can idea....

Protocol

1. Cell culturing

1. Prepare Dulbecco's modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, used to culture human breast cancer cell (MDA-MB-231) lines and human umbilical vein endothelial cell (HUVEC) lines.
2. Prepare DMEM with L-glutamine (DMEM/F-12), supplemented with 10% FBS and 1% penicillin/streptomycin, used to culture bone marrow mesenchymal stem cell (BMSC) lines.
3. Set the culturing environment as 37 °C and 5% CO₂. Culture MDA-MB-231, HUVEC, and BMSC, and passage the cells in a 1:2 ratio when 90% confluence is reached.

2. Fab....

Results

During the fabrication of GelMA microspheres, the GelMA droplets were separated by the external electric field force. When the droplets fell into the receiving silicon oil, they remained standard spheroid shape without tails. This is because the GelMA droplets were in an aqueous phase, while the silicon oil was in an oil phase. The surface tension that formed between the two phases caused the GelMA droplets to maintain a standard spheroid shape. In terms of the cell-laden microspheres, cells experienced the high voltage .......

Discussion

This article describes several strategies to fabricate GelMA 3D structures, namely GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. GelMA has promising biocompatibility and formation capability and is widely used in the field of biofabrication. Microsphere structures are suitable for controlled drug release, tissue culturing, and injection into organisms for further therapy^21,22,23<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm filter membrane	Millipore
2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI)	Yeasen Biological Technology Co., Ltd., Shanghai, China
3D bioprinter	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
405nm wavelength light	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
co-axial nozzle	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
confocal fluorescence microscope	OLYMPUS FV3000
digital light processing (DLP) bioprinter	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
DLP printer	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
Dulbecco's Phosphate Buffered Saline (DPBS)	Tangpu Biological Technology Co., Ltd., Hangzhou, China
Dulbecco's Modified Eagle Medium (DMEM)	Tangpu Biological Technology Co., Ltd., Hangzhou, China
Dulbecco's Modified Eagle Medium with L-glutamine (DMEM/F-12)	Tangpu Biological Technology Co., Ltd., Hangzhou, China
EFL Software	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
fetal bovine serum (FBS)	Tangpu Biological Technology Co., Ltd., Hangzhou, China
gelatin	Sigma-Aldrich, Shanghai, China
gelatin methacryloyl (GelMA)	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
high voltage power	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP)	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
paraformaldehyde	Tangpu Biological Technology Co., Ltd., Hangzhou, China
penicillin/streptomycin	Tangpu Biological Technology Co., Ltd., Hangzhou, China
sodium alginate (Na-Alg)	Sigma-Aldrich, Shanghai, China
TRITC phalloidin	Yeasen Biological Technology Co., Ltd., Shanghai, China
Triton X-100	Solarbio Co., Ltd., Shanghai, China