Influence Of Mixed Human Umbilical Cord Serum On The Proliferation Ability Of Human Bone Marrow Mesenchymal Stem Cells

Background and purpose:Mesenchymal stem cells (MSCs) are adult stem cells from mesoblastema with ability to self-renew and differentiate, and they are becoming ideal seed cells with good clinical application prospect in tissue engineering and regenerative medicine.There are several methods in MSCs cultivation by now, However,routine use of fetal bovine sera (FBS) in vitro expansion is notoptimal and may bring health risk for patients, because FBS may carry Viruses, bacteria and may cause anaphylaxis. So, In recent years,"Humanized" substitute of FBS is becoming attractive.Human Umbilical cord blood serum(UCBS) is a rich source of growth factors that support the growth, proliferation, and differentiation of MSCs, and it has other advantages such as:i. low cost, ii. easy availability.ⅲ. harmless to the mother and fetus, iv. Low infective rate of CMV, EB virus, v. Low immunity, et al.therefor, These evidences strongly support substitution of UCBS in the place of FBS as a growth supplement for MSCs growth and expansion.Research work is divided into two parts:Part1Identification of MSCs cultured with Mixed UCBS, autologous serum and FBS, and comparison of MSCs proliferation ability between three groups. Methods:Ten bone marrow samples were obtained under aseptic conditions.The mononuclear cells were extracted by density gradient centrifugation method, and MSCs were cultured with10%Mixed UCBS,10%autologous serum and10%FBS+DMEM/F12culture medium respectively. The morphology of MSCs was observed and the third passage MSCs were used to detect their surface antigens by flow cytometry. Observe the differentiation capacity of MSCs into osteoblasts and adipocytes in conditioned medium.and Compare the proliferation ability of MSCs between Mixed UCBS, autologous serum and FBS medium.Results:There were no difference on the morphology of MSCs between three groups. The positive rates of CD73, CD105of MSCs cultured in Mixed UCBS medium were higher than those in FBS and autologous serum (p<0.05), whereas the positive rate of CD34、CD45was lower than2%in three groups.there was no statistically significant difference in the differentiation capacity of MSCs into osteoblasts and adipocytes between three groups (p>0.05). The ability of proliferation and total population of MSCs in Mixed UCBS was better than FBS and autologous serum (p<0.05).Conclusion:The fiber-like cell cultured with Mixed UCBS, FBS and autologous serum were identificated to be MSCs. the ability of proliferation in Mixed UCBS is superior to the FBS and autologous serum group.Part2Influence of different volume fractions of mixed human umbilical cord serum on the proliferation ability of human bone marrow mesenchymal stem ellsMethods:Ten bone marrow samples were obtained under aseptic conditions.The mononuclear cells were extracted by density gradient centrifugation method, and cultured with the volume fraction of5%、7.5%、10%、15%、20%of the umbilical cord serum+DMEM/F12culture medium respectively. count the cell number at the end of original generation and third generation, the third passage MSCs were used in this study, MTT was used to investigate the effect of different volume fractions of Umbilical cord serum on the MSCs proliferation.Results:Compared with the5%、15%、20%volume fraction of umbilical serum groups,The MSCs proliferation ability and Cell number were better in7.5%、10%volume fraction of umbilical cord serum groups, there was statistically significant difference (P<0.05)Conclusion:The MSCs proliferation are more active at7.5%,10%volume fraction of human umbilical cord serum culture system.