Feeder Preparation Of Several Cells

Objective:Sepretae Sertoli cell, BMSC, MEF, Pancreatic fibroblast and establish cell lines, constructed different feeder layers for a source as a coculture system for stem cells.Methods:1.Separate Sertoli cell with collagenase and Tris-HCl, Optimize the condition of BMSC with several nutrient solution, Separate MEF or PF by the way of tissue digestion.2.Measure MEFs and PFs viability and proliferation forms with MTT, Preserve cells with DMSO in liquid nitrogen, Treat MEFs and PFs with Mitomycir C(10μg/ml) to make feeder layers.Results:Procure pure Sertoli cell by hypotonic treatment, BMSC cultured with DMEM(L) nutrient solution include 15% FBS grows better, Separate PF from connective tissue of pancreas, PF and MEF have the same viability and proliferation forms, Cells Preserved with DMSO in liquid nitrogen, have the capability of serial subcultivation after revival, made MEF feeder layer and PF feeder layer.Conclusion:1.Hypotonic treatment can Purify Sertoli cell.2.DMEM(L) nutrient solution include 15% FBS ptomote BMSC culture. 3.Separate PF from connective tissue of pancreas,PF and MEF have the same viability and proliferation forms.4.Establish a cell repository of MEF and another of PF.5.Prepare MEF and PF feeder layers.