Isolation and culture of mesenchymal stem cells: From laboratory research to clinical application

doi:10.3969/j.issn.2095-4344.2013.14.026

Abstract

Abstract:

BACKGROUND: Mesenchymal stem cells are multipotent adult stem cells. The source, isolation and culture of mesenchymal stem cells differ greatly in different studies in terms of concepts, research methods and outcomes.
OBJECTIVE: To investigate the progress in isolation and culture of mesenchymal stem cells.
METHODS: The PubMed database (http://www.ncbi.nim.nih.gov/PubMed) was retrieved for articles regarding application of stem cells from 2007 to 2011 by computer. The English key words are “mesenchymal stem cells” and “source isolation or culture”. The papers published recently or in authoritative journals were selected. Forty-three papers were suitable for final analysis.
RESULTS AND CONCLUSION: A standardized culture system should be established to achieve isolation and expansion in vitro of mesenchymal stem cells. Therefore, basic research of mesenchymal stem cells should be further developed, and the system of in vitro expansion and oriented differentiation of mesenchymal stem cells should be established, and study on the effects of transplantation of gene-transfected mesenchymal stem cells is of great significance.

Key words: stem cells, stem cell academic discussion, mesenchymal stem cells, source, isolation, non-differentiation expansion, serum-free, microcarrier suspending culture, phenotype, differentiation, umbilical cord, provincial grants-supported paper

CLC Number:

R318

Li Bing-yao, Wu Xiao-yun, Wu Yan1. Isolation and culture of mesenchymal stem cells: From laboratory research to clinical application [J]. Chinese Journal of Tissue Engineering Research, 2013, 17(14): 2649-2655.

Figures/Tables 1

2.1 间充质干细胞的来源和生物学特性间充质干细胞最早在骨髓中发现，因培养后细胞表型为成纤维细胞样，而被称为成纤维细胞集落形成单位。由于对骨髓血系细胞起支持诱导作用，可促进造血细胞克隆的形成，因而推测这种细胞可能是间质细胞的前体细胞，又因其来自于骨髓基质，将其称为“骨髓基质细胞”。早期实验研究用得最多的是骨髓来源的骨髓间充质干细胞。骨髓间充质干细胞占骨髓有核细胞的0.01%-0.000 1%。目前，越来越多的研究表明还能够从脂肪、滑膜、骨骼、肌肉、肺、肝、胰腺、羊水以及脐血等组织中分离和制备间充质干细胞[1]。但实验室和临床上用得最多的仍然还是骨髓来源的间充质干细胞。骨髓来源的间充质干细胞存在以下问题：随着年龄的老化，干细胞数目显著降低，增殖分化能力大幅度衰退；制备过程不容易质控；移植给异体可能引起免疫反应；取材时对患者有损伤，患者有骨髓疾病时不能采集。即使是健康供者，亦不能抽取太多的骨髓。这都限制了骨髓间充质干细胞临床应用，使得寻找骨髓以外其他可替代的间充质干细胞来源成为一个重要的问题。临床上作为脐血来源的间充质干细胞培养成功率很低，一般只有20%-30%的脐血样本培养后能分离出间充质干细胞。为了有效地提高脐血间充质干细胞原代培养成功率，应用正交实验设计法在间充质干细胞传统培养方法的基础上，通过添加细胞因子白细胞介素3和粒细胞-集落刺激因子的方法可以使脐血间充质干细胞的原代培养成功率由传统方法的25%左右提高到90%以上[2]。脐带是孕妇与胎儿之间的物质交通支，也是分娩后的废弃物，收集脐带对产妇和胎儿均不造成伤害，取材也不受伦理学的困扰。另外从胚胎发育而言，脐带的免疫原性低，可以降低或避免免疫排斥。一些研究者比较了脐带间充质干细胞和骨髓间充质干细胞，发现前者比后者有更快的增殖能力和更好的成骨分化能力，因此脐带是临床上间充质干细胞的主要来源。也有一些研究者认为脂肪组织是间充质干细胞最理想的来源[3]，因为其来源的间充质干细胞具有和骨髓源间充质干细胞相似的表型、增殖能力和分化能力，而且脂肪非常丰富且取材简单，捐献者可以避免痛苦[3]。目前，国际干细胞治疗学会提出了鉴定人来源间充质干细胞的3条最低标准[4]：①在标准培养条件下，间充质干细胞具备对塑料底物的贴附性。②间充质干细胞群体表达CD105、CD73及CD90，而不表达造血标志物CD45和CD34。③经体外诱导，间充质干细胞能向成骨细胞、脂肪细胞及软骨细胞分化。这仅仅是间充质干细胞的体外鉴定，一些研究者一直寻求间充质干细胞的体内鉴定[5]。随着对间充质干细胞的深入研究，发现间充质干细胞可以分化成血细胞和肌细胞，还具有跨胚层分化的可塑性。间充质干细胞具有向外胚层神经细胞分化和内胚层肝细胞、胰岛细胞分化的能力[6]。而且间充质干细胞具有非常低得免疫原性。间充质干细胞分泌多种细胞因子发挥造血支持和免疫调节等功能[7-8]。间充质干细胞的这些生物学特性，显著推动了间充质干细胞的应用范围和应用价值。间充质干细胞的标准定义："

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