Studies On Gelatin/Alginate Hydrogels For Cell Three-Dimensional Cultures

Objective:In vitro cell three-dimensional（3D）cultures are the bases for tissue engineering and organ manufacturing.Among various in vitro cell 3D culture models,hydrogels can better simulate the in vivo cell growth environments.Especailly,natural polymer based hydrogels,such as gelatin and alginate,are super in biocompatibilities with respect to cell accommodation,activity,oxygen/nutrient supply,and metabolite transportation.Nevertheless,the mechanical properties of the natural hydrogels are normally very poor even through physical and chemical crosslinking.Multi-component interpenetrating polymer networks can enhance the structural stability of hydrogels and obtain more suitable matrix materials for in vitro cell 3D cultures.In this thesis,microbial transglutaminase（m TG）,which is commonly used in food engineering,was used for gelatin crosslinking.A double-crosslinked gelatin/alginate polymer network（G-A-IPN）was studied for in vitro cell 3D culture model construction.Methods:A series of G-A-IPN hydrogels were formed by cross-linking gelatin with m TG and alginate with Ca²⁺in the gelatin/alginate solutions.Water holding capacity,hardness,swelling rate and degradation rate of several G-A-IPN hydrogels with different concentrations of sodium alginate were compared with gelatin/alginate hydrogels with single cross-linking of Ca²⁺.Cytotoxicity and cell proliferation rate in the hydrogels with different concentrations of alginate sodium were characterized via CCK-8 kit.A suitable hydrogel for in vitro cell 3D cultures was selected.This hydrogel was used to culture human tongue squamous carcinoma cells（Tca-8113）and rat adipose-derived stem cells（ASCs）,and 3D bioprinting using a pneumatic extrusion 3D printer.The possibility of this hydrogel to construct in vitro cell 3D culture model was analyzed.Results:（1）Compared with the A-semi-IPN hydrogels,the double cross-linked G-A-IPN hydrogels have more stable structural properties,stronger mechanical properties and lower swelling properties,so they are more suitable for in vitro cell 3D culture model construction.（2）In the experimental groups,cells（Tca-8113）in the hydrogel containing 3%（w/v）sodium alginate and 5%（w/v）gelatin grow and proliferate the fastest.（3）The model of adipose stem cell-hydrogel structure in vitro with the shape of blood vessel was established by the method of composite mold and endothelized successfully.（4）A 3D mesh structure was obtained through adjucting the printing parameters of the 3D bioprinter.Conclusion:The structural instability of the double crosslinked G-A-IPN hydrogels is significantly improved.Tca-8113 cells embedded in the selected G-A-IPN hydrogel grow well during the 7 days in vitro cultures.ASCs in the hydrogel can be induced into endothelial cells through adding growth factors in the culture medium.The gelatin/alginate hydrogel can be printed into 3D structures using the pneumatic extrusion 3D bioprinter.