结合人血小板裂解液培养人脐带间充质干细胞的基础培养基选择  

Selection of basal medium for culture of human umbilical cord mesenchymal stem cells in combination with human platelet lysate

作　　者：王灵娟 刘春香 蔡光辉 芦慧颖 周慧敏[3] 张怡 WANG Lingjuan;LIU Chunxiang;CAI Guanghui;LU Huiying;ZHOU Huimin;ZHANG Yi(Heilongjiang Tian Qing Stem Cell Co.,Ltd.,Harbin 150028,Heilongjiang Province,China;不详)

机构地区：[1]天晴干细胞股份有限公司,黑龙江哈尔滨150028 [2]老年性疾病干细胞技术国家地方联合工程研究中心,黑龙江哈尔滨150028 [3]河北医科大学附属第一医院内分泌科,河北石家庄050031

出　　处：《中国生物制品学杂志》2024年第9期1062-1069,共8页Chinese Journal of Biologicals

基　　金：黑龙江省“百千万”工程科技重大专项项目(2019ZX12C01)。

摘　　要：目的结合人血小板裂解液(platelet lysate,PL)选取一种安全性高、成本低、适用于人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUC-MSCs)生长,且在培养过程中不影响细胞各项功能的基础培养基,用于大量高代次hUC-MSC的培养。方法将MSCBM、α-MEM、IMDM 3种基础培养基分别与PL UltraGROG Advanced进行配比,培养P0~P8代hUC-MSCs,对比不同代次的细胞形态、扩增数量、增殖功能、分化功能及免疫表型,确定适用于HUC-MSCs培养的最佳基础培养基。结果3种培养基培养的P1~P5代细胞,形态均一,呈放射状生长,与标准的细胞形态相比差异无统计学意义(t_(IMDM VSα-MEM)=0.159,t_(MSCBM VSα-MEM)=0.147,t_(IMDM VS MSCBM)=0.161;P均>0.05),培养至P6~P8代时,MSCBM、α-MEM培养的细胞与P0~P5代细胞形态相比,差异无统计学意义(t_(IMDM VS MSCBM)=0.132,t_(IMDM VSα-MEM)=0.128;P均>0.05);MSCBM、α-MEM、IMDM培养的P1~P8代细胞阳性表达CD73、CD90、CD105,表达率均约达99%,阴性表达CD34、CD45,表达率均低于2%,不同培养基间差异无统计学意义(t_(IMDM VSα-MEM)=0.102,t_(MSCBM VSα-MEM)=0.106,t_(IMDM VS MSCBM)=0.113;P均>0.05);3种培养基培养的P8代细胞克隆形成性不同,MSCBM培养的细胞形成的单个克隆团和克隆团内细胞数量均高于α-MEM、IMDM培养的同代次细胞(t分别为0.023、0.049,P均<0.05);3种培养基培养的细胞增殖功能由高到低分别为MSCBM、α-MEM、IMDM,其中MSCBM显著高于IMDM(t=0.041,P<0.05),而与α-MEM差异无统计学意义(t=0.211,P>0.05),倍增时间(DT)与增殖功能结果一致;3种培养基培养的同代次细胞均具有较强分化功能,培养的P8代细胞分化成骨和成软骨的细胞数量无显著差异(t_(IMDM VSα-MEM=0.119,t_(MSCBM VSα-MEM)=0.112,t_(IMDM VS MSCBM)=0.111;P均>0.05),MSCBM培养的P8代细胞分化的成脂细胞数量高于α-MEM培养的P8代细胞(t=0.036,P<0.05),α-MEM培养的P8代细胞分化的成脂细胞数量高于IMDM培养的P8代细�Objective To select a safe,low-cost basal medium in combination with platelet lysate(PL),suitable for the growth of human umbilical cord mesenchymal stem cells(hUC-MSCs)with no influence on the functions of cells during the cultivation process,and to use it for the culture of a large number of high-generation hUC-MSCs.Methods Three different basal media,MSCBM,α-MEM and IMDM,were mixed with PL UltraGROG Advanced respectively to culture hUC-MSCs from P0 to P8.The cell morphology,cell expansion quantity,proliferation function,differentiation function and cell surface markers of different generations were compared to determine the optimal basal medium suitable for hUC-MSCs cultivation.Results The morphology of P1-P5 cells cultured in the three media was uniform and radial,with no significant difference between them and the standard cells(t_(IMDM VSα-MEM)=0.159,t_(MSCBM VSα-MEM)=0.147,t_(IMDM VS MSCBM)=0.161;each P>0.05).The morphology of P6-P8 cells cultured in MSCBM andα-MEM showed no significant difference compared with that of P0-P5cells(t_(IMDM VS MSCBM)=0.132,t_(IMDM VSα-MEM)=0.128;each P>0.05).The positive expression rate of CD73,CD90 and CD105all reached about 99%in P1-P8 cells cultured in MSCBM,α-MEM and IMDM,while the expression of CD34 and CD45 was negative with the expression rate of lower than 2%,and there was no significant difference among different media(t_(IMDM VSα-MEM)=0.102,t_(MSCBM VSα-MEM)=0.106,t_(IMDM VS MSCBM)=0.113;each P>0.05).The clonal formation of P8 cells cultured in the three media was different,the numbers of single clonal cluster and MSCBM cells in clonal cluster were higher than those of the same generation cells cultured inα-MEM and IMDM(t=0.023 and 0.049,respectively,each P<0.05).The proliferative function of cells cultured in the three media from high to low was MSCBM,α-MEM and IMDM,among which,the proliferation function of cells cultured in MSCBM was significantly higher than that in IMDM(t=0.041,P<0.05),while there was no significant difference between MSCBM andα-MEM(t

关 键 词：细胞培养 培养基 血小板裂解液 脐带间充质干细胞 

分 类 号：Q253[生物学—细胞生物学]