脐带间充质干细胞无血清培养体系的筛选

Screening of serum-free culture system for umbilical cord mesenchymal stem cells

目的:筛选培养脐带间充质干细胞的无血清培养基。方法:采集新生儿脐带标本,将脐带剪成组织碎块,随机分为A组、B组、C组,分别加入MesenCult、Ultraculture和AM-V 3种不同的无血清培养基进行培养。观察3组脐带原代细胞游出时间和细胞形态,检测细胞的增殖能力、分化能力和表面抗原的表达。结果:A组、B组、C组原代细胞从组织块长出的平均时间分别是17 d、6 d、7 d。显微镜下观察可见,从组织块游出的细胞散在分布,呈短梭形或多角形,形态饱满,折光性好,生长融合时A组和B组细胞呈旋涡状生长,细胞折光性好,大小均一,C组细胞细长,折光性稍差,无明显的旋涡状生长的特征。3组均表达CD105、CD90、CD73抗原,不表达CD34、CD45、HLA-DR抗原;3组各标志物的表达差异均无统计学意义(P>0.05)。3组间充质干细胞均具有向成脂细胞、成骨细胞分化的能力,C组分化能力弱于A组和B组。结论:B组培养体系用时较短,干细胞特性保持较好,价格适宜,可以作为大规模培养人间充质干细胞的首选培养基。

Objective:To screen serum-free medium for culturing umbilical cord mesenchymal stem cells.Methods:Neonatal umbilical cord specimens were collected and cut into tissue fragments.The samples were randomly divided into group A,group B and group C,and were cultured with three different serum-free media MesenCult,Ultraculture and AM-V,respectively.The migration time and cell morphology of the primary umbilical cord cells in the three groups were observed,and the proliferation ability,differentiation ability and surface antigen expression of the cells were detected.Results:The average time for primary cells to grow out from the tissue blocks in group A,group B and group C was 17 d,6 d and 7 d.Observed under the microscope,the cells growing out of the tissue blocks were scattered distributed,with short spindle or polygonal shape,full shape and good refraction.When growing to fusion,the cells in group A and group B displayed spiral growth,with good refraction and similar size,but cells in group C were slender,slightly worse in refraction,and had no obvious feature of spiral growth.Cells in the three groups all expressed CD105,CD90,CD73 antigens,but did not express CD34,CD45,HLA-DR antigens,and there was no significant difference in the expression of the above markers among the three groups(P>0.05).Mesenchymal stem cells in the three groups all had the ability to differentiate into adipocytes and osteoblasts,and the differentiation ability in group C was weaker than that in group A and group B.Conclusions:The culture system in group B has a shorter time,better stem cell characteristics and reasonable price,which can be used as the first choice medium for large-scale culture of human mesenchymal stem cells.