| CPC A61L 27/3633 (2013.01) [A61L 27/26 (2013.01); A61L 27/52 (2013.01); C08L 5/08 (2013.01); C08L 89/06 (2013.01); C12N 5/0018 (2013.01); C12N 5/0663 (2013.01); A61L 2300/64 (2013.01); A61L 2430/02 (2013.01); A61L 2430/40 (2013.01); C12N 2500/38 (2013.01)] | 6 Claims |
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1. A method for preparing extracellular matrix hydrogel microspheres, the method comprising:
a. preparing a neonatal rat bone marrow mesenchymal stem cell-derived extracellular matrix by a method comprising the following steps:
S1, inoculating neonatal rat-derived bone marrow mesenchymal stem cells into a cell culture dish, and culturing the inoculated neonatal rat-derived bone marrow mesenchymal stem cells using a complete medium until a cell density reaches 90%;
S2, adding ascorbic acid at a concentration of 80-200 UM to the complete medium for induction for 3-10 d;
S3, removing the complete medium and adding a decellularization solution for incubation;
S4, removing the decellularization solution, and adding a deoxyribonuclease for further incubation;
S5, removing the deoxyribonuclease;
S6, washing with a phosphate buffer solution (PBS); and
S7, using a cell scraper to collect the extracellular matrix;
b. preparing hydrogel microspheres by a microfluidic technique comprising the following steps:
S8, preparing a hydrogel precursor solution comprising deionized water and a photoinitiator as an aqueous phase, the hydrogel precursor solution comprising hyaluronic acid methacrylate with a concentration of 1%-5%, gelatin methacryloyl with a concentration of 5%-20%, silk fibroin with a concentration of 7.5%-20%, and collagen methacrylate with a concentration of 1%-20%;
S9, uniformly mixing Span 80 with isopropyl myristate to serve as an oil phase;
S10, filling the aqueous phase and the oil phase into respective injectors, and adjusting and controlling different flow rates by using syringe pumps to push out the aqueous phase and the oil phase at constant speeds, and curing the aqueous phase into stable spheres by exposure to ultraviolet radiation;
S11, washing the surfaces of the hydrogel microspheres by using ethanol; and
S12, collecting and lyophilizing the washed hydrogel microspheres;
C. activating carboxyl groups on the surfaces of the hydrogel microspheres by a method comprising the following steps:
S13, preparing a solution of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) for use as a condensing agent;
S14, immersing the hydrogel microspheres in the DMTMM solution; and
S15, stirring and centrifuging the solution with hydrogel microspheres at room temperature to form activated hydrogel microspheres; and
d. reacting the activated hydrogel microspheres with amino groups on the neonatal rat bone marrow mesenchymal stem cell-derived extracellular matrix to obtain the extracellular matrix hydrogel microspheres, by a method comprising the following steps:
S16, performing lyophilization on the extracellular matrix;
S17, thoroughly grinding the lyophilized extracellular matrix; and
S18, adding the ground extracellular matrix to sterile deionized water to obtain an extracellular matrix suspension; and
S19, adding the activated hydrogel microspheres to the extracellular matrix suspension for reaction on a shaker to form the extracellular matrix hydrogel microspheres.
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