上海交通大学学报(医学版) ›› 2018, Vol. 38 ›› Issue (9): 1033-.doi: 10.3969/j.issn.1674-8115.2018.09.006

• 论著·基础研究 • 上一篇    下一篇

单个细胞来源胎盘间充质干细胞培养方法的比较研究

孙宝航行 1,鲁奕 1,王怡沁 1,周嘉乐 1,林梦璐 1,宋玮 2,姜萌 1,卜军 1   

  1. 1.上海交通大学医学院附属仁济医院心内科,上海 200240;2.上海中医药大学附属曙光医院心血管科,上海 200021
  • 出版日期:2018-09-28 发布日期:2018-10-15
  • 通讯作者: 宋玮,电子信箱:swei222@aliyun.com。姜萌,电子信箱:jiangmeng0919@163.com。卜军,电子信箱:pujun310@hotmail.com。为共同通信作者。
  • 作者简介:孙宝航行(1995—),硕士生;电子信箱: bowensun_0719@126.com。
  • 基金资助:
    国家自然科学基金( 81270206, 81470391);上海市教育委员会高峰高原学科建设计划( 20172014);上海交通大学医工交叉 Med-X基金(YG2014MS49)

Comparative cultivation of single cell derived placental mesenchymal stromal cells in vitro

SUN Bao-hangxing1, LU Yi1, WANG Yi-qing1, ZHOU Jia-le1, LIN Meng-lu1, SONG Wei2, JIANG Meng1, BU Jun1   

  1. 1. Department of Cardiology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200240, China; 2. Department of Cardiology, Shuguang Hospital, Shanghai University of Chinese Medicine, Shanghai 200021, China
  • Online:2018-09-28 Published:2018-10-15
  • Supported by:
    National Natural Science Foundation of China, 81270206, 81470391; Shanghai Municipal Education Commission— Gaofeng Clinical Medicine Support, 20172014; Shanghai Jiao Tong University Medical-Engineering Med-X Foundation, YG2014MS49

摘要: 目的 ·观察胚胎来源间充质干细胞( placental derived mesenchymal stem cells,PMSCs)在人血小板裂解液( human platelet lysate,HPL)、碱性成纤维细胞生长因子( basic fibroblast growth factor,bFGF)及传统的胎牛血清( fetal bovine serum,FBS)不同组合的培养基中的倍增数目及细胞性状,着重探索更合适的 PMSCs培养体系。方法 ·分离单个细胞来源 PMSCs,扩增后分别用 4种培养基孵育,分为 FBS组、FBS+bFGF组、HPL组以及 HPL+bFGF组。观察细胞形态及生长状态、细胞表型及多能分化。在 P1、 P2、P3、P4代进行计数。取 P4代细胞接种,分析集落形成单位的个数。结果 ·经鉴定, PMSCs具有间充质干细胞的生物特性。数量上, FBS+bFGF组在 P4代达到( 1.12×107)个 /cm2,HPL组达到( 1.24×107)个 /cm2(P>0.05),而 FBS组和 HPL+bFGF组则只有(5.58×106)个 /cm2和(8.56×106)个 /cm2。细胞形态上, FBS+bFGF组与 HPL组 P4代的 PMSCs均保持贴壁生长,但 HPL组细胞于 P5代无法贴壁生长。细胞集落数量上, P4代时, FBS+bFGF组为 51个 /孔,HPL为 52个 /孔(P>0.05)。细胞生物学特性上,FBS+bFGF组和 HPL组在 P4代时均能基本保有间充质干细胞的特性和多能分化的能力。结论 ·从细胞数量上来看, HPL组中的 P4代细胞数与目前常用的 FBS+bFGF培养基没有显著差异,且已达到临床治疗剂量需求;从细胞生物学特性上来看, HPL培养基与 FBS+bFGF培养基均能基本保持 PMSCs细胞特性和多能分化的能力。但由于 HPL培养基免疫原性低,因此更适合临床移植应用。然而,由于 HPL培养基无法维持 PMSCs生长至 5代以后,故无法用于大规模细胞扩增。HPL+bFGF在目前实验条件下并不具备优势。

关键词: 胎盘来源间充质干细胞, 细胞扩增, 碱性成纤维生长因子, 人血小板裂解液

Abstract: Objective · To explore optimal placental derived mesenchymal stem cells (PMSCs) culture medium using different combination of human platelet lysate (HPL), basic fibroblast growth factor (bFGF) and traditional fetal bovine serum (FBS) for further basic and clinical study. Methods · Single cell derived PMSCs was harvested and incubated with 4 kinds of culture, i.e, FBS, FBS+bFGF, HPL and HPL+bFGF. The morphology, growth state, cell phenotype and multi-energy differentiation were observed. Cells of P1, P2, P3 and P4 generation were counted respectively. The number of units of colony formation was analyzedinoculating P4 cells. Results · It was identified that PMSCs had the biological properties of MSCs. Quantificationally, cell density reached (1.12×107) cells/cm2 in FBS+bFGF group and (1.24×107) cells /cm2 in HPL group (P>0.05) in P4, while those in FBS group and HPL+bFGF group were (5.58×106) cells /cm2 and (8.56×106) cells /cm2, respectively. For cell morphology, the P4 PMSCs of FBS+bFGF and HPL groups kept adherent growth, but the HPL cells could not be adherent in P5 generation. The number of colony was 51/well in FBS+bFGF group, and it was 52/well in HPL group (P>0.05) in P4. The FBS+bFGF group and the HPL group were able to maintain the characteristics of MSCs and the ability of pluripotent differentiation in the P4 generation. Conclusion · PMSCs in P4 cultured in HPL medium can keep the biological characteristics and meet the clinical transplantation requirements in quality and quantity, which are preferable for their low immunogenicity to clinical applications. In long term, PMSCs cultured in FBS+bFGF medium are preferable for the longer lasting characters of MSCs and larger quantity in basic studies. HPL+bFGF medium has no advantage on quality and quantity.

Key words: placental derived mesenchymal stromal cells, cell proliferation, basic fibroblast growth factor, human platelet lysate

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