Experimental Study Of The Biological Characteristics Of Platelet Lysate On Human Mesenchymal Stem Cells

Objective:This study aims to get the biological characteristics of BMMSC and ATMSC under the effect of PL-supplemented culture conditions, providing some experimental data for clinical cell therapy and regenerative medicine.Methods:1.PL was prepared by repeated freezing and thawing from fresh blood.2.Collect healthy adult bone marrow and adipose tissue, then obtain primary BMMSC and ATMSC by density gradient centrifugation and type I collagenase digestion, combined cell adherence method.3.PL cultured primary BMMSC and ATMSC.4.Use inverted phase contrast microscope to observate morphology of BMMSC and ATMSC.5.Use flow cytometry detection to determine CD13,CD29,CD90,CD105,CD14, CD19, CD45,CD34,HLA-DR and HLA-DR expression changes of BMMSC and ATMSC cultured by PL.6.Use cell counting method to calculate each generation group multiples of ATMSC and BMMSC.7.Using colony forming experiment to test colony-forming unit-fibroblast(CFU-F) of BMMSC and ATMSC.8.To assess the differentiation potential of BMMSC and ATMSC,both types of cells were differentiated into chondrocyte,osteoblast and adipocyte phenotypes with Alcian Blue staining,Alizarin Red staining and Oil Red-O staining.9.To investigate the production of cytokines IL-6,TNF-?,IL-8 and VEGF of BMMSC and ATMSC with ELISA.Results:1.On PL-supplemented culture conditions,BMMSC and ATMSC exhibited similar fibroblastlike morphology,and grew in parallel or vortex-like patterns.2.The results of flow cytometry detection show that both BMMSC and ATMSC have similar cell phenotype( p>0.05),CD13,CD29,CD90,CD105 and HLA-ABC are all positive expression,CD14,CD19,CD45,CD34 and HLA-DR are all present negative expression.3.Cell proliferation capacity showed that ATMSC which through the cultivation of P1 ~ P5 have a higher cumulative cell population multiples than BMMSC(p<0.05).4.Colony forming experiment analyses showed that there are no statistical difference between BMMSC and ATMSC(p > 0.05).5.Differentiation in vitro experiments show that BMMSC and ATMSC have the capacity of going into chondrogenic,osteogenic and into adipogenic.6.There are no statistical differences of both BMMSC and ATMSC are all secretion IL-6,TNF-?,IL-8 and VEGF(p > 0.05).Conclusions: Application of PL succeeded in cultivating amplification in vitro BMMSC and ATMSC.BMMSC and ATMSC which training by PL have the capacity of going into chondrogenic,osteogenic and into adipogenic.Both BMMSC and ATMSC have no significant differences of biological characteristics on cell morphology,cell phenotype,colony forming ability and the level of cytokine secretion.ATMSC have a higher cumulative cell population multiples than BMMSC, ATMSC in vitro cultivation system of PL is more suitable for large-scale expansion.PL training system established in this experiment provide a safe and effective method for ATMSC and BMMSC amplification in vitro.