Study On Decellularized Testicular Matrix Hydrogel And Its Effect On Proliferation And Differentiation Of Spermatogonial Stem Cell

Objective:The aim of this study is to further develop and optimize the culture system of spermatogonial stem cells in vitro.Since tissue-specific extracellular matrix hydrogels have special structures and bioactive substances,which can regulate the proliferation and differentiation of cells.Our study intends to study the effect of testicular tissue-derived decellularized matrix hydrogels on the proliferation and differentiation of spermatogonial stem cells.The culture system of spermatogonial stem cells based on DTM hydrogel was established to study the mechanism of spermatogonial stem cells proliferation and differentiation in vitro.Methods:Firstly,we prepared decellularized testicular matrix(DTM)hydrogel by chemical decellularized abstergent and pepsin dissolution method,and optimized the conditions of decellularized and hydrogel preparation through a series of characterization experiments.Secondly,we compared the effects of Laminin,Matrigel and DTM hydrogels on proliferation and stemness maintenance of spermatogonial stem cells by staining of living dead cells,alkaline phosphatase staining,RT-PCR,flow analysis and BSP sequencing.Finally,we explored the feasibility of DTM hydrogel in promoting spermatogonial stem cell differentiation into sperm in vitro by RT-qPCR,immunofluorescence and flow analysis.Results:Results showed that the combination of freeze-thaw and 1% SDS treatment for 18 hours could effectively remove cell components from testicular tissue and retain the fine structure and bioactive compounds of testicular tissue.After freeze-drying,shearing and crushing,enzymatic hydrolysis,centrifugation and polymerization,decellularized testicular matrix formed hydrogel in different concentrations(2.5 mg/mL,5 mg/mL and 10 mg/mL).DTM hydrogel had good biocompatibility and could promote the proliferation and maintain the stemness and function of spermatogonial stem cells in vitro.In addition,compared with laminin and Matrigel culture system,DTM hydrogel 3D culture system could significantly promote spermatogonial stem cells differentiating into haploid round sperm.Conclusion:The extracellular matrix of testis can be self-assembled into a bioactive DTM hydrogel by changing the pH and PBS.DTM hydrogel can promote the proliferation and differentiation of spermatogonial stem cells.