无血清扩增和分化人骨髓间充质基质细胞。

Animal serum-free expansion and differentiation of human mesenchymal stromal cells.

机构信息

Center for Medical Research (ZMF), University of Tübingen Medical Center (UKT), 72072 Tübingen, Germany.

出版信息

Cytotherapy. 2010 Apr;12(2):143-53. doi: 10.3109/14653240903470647.

DOI:10.3109/14653240903470647

PMID:20141338

Abstract

BACKGROUND AIMS

Mesenchymal stromal cells (MSC) are attracting increasing interest for possible application in cell therapies. Fetal calf serum (FCS) is widely utilized for cell culture, but its use in the context of clinical applications is associated with too many risks. Therefore we tested FCS-free media for the expansion and differentiation of MSC in compliance with the European good manufacturing practice (GMP) regulations for medicinal products.

METHODS

MSC expansion medium was modified by replacing FCS with human plasma and platelet extract. Cells were characterized according to the defined minimal criteria for multipotent MSC. For chondrogenic differentiation, serum-free micromass cultures were employed. For adipogenic and osteogenic differentiation, the FCS was replaced by human plasma. After 28 days of incubation in differentiation media, cells were analyzed by cytochemical and immunohistochemical staining. Furthermore, mRNA expression of chondrogenic, adipogenic and osteogenic markers was investigated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR).

RESULTS

Expansion and differentiation of MSC under FCS-free conditions yielded cells with chondrogenic, adipogenic and osteogenic phenotypes and a characteristic gene expression. Chondrocytes in micromass pellets revealed an accumulation of proteoglycans and type II collagen as well as a significantly increased mRNA expression of chondrogenic marker genes. The adipocytes displayed Oil red O staining and expressed peroxisome proliferator-activated receptor gamma(2) (ppARgamma2) and lipoprotein lipase (LPL) mRNA. The osteoblasts were positive for von Kossa staining and expressed mRNA of osteogenic marker genes. The results did not indicate any spontaneous differentiation.

CONCLUSIONS

Human plasma is a suitable FCS replacement for the expansion and differentiation of MSC, providing a feasible alternative for tissue engineering with GMP-compatible protocols.

摘要

背景目的

间充质基质细胞（MSC）因其在细胞治疗中的潜在应用而受到越来越多的关注。胎牛血清（FCS）广泛用于细胞培养，但在临床应用中使用它存在太多风险。因此，我们按照欧洲药品生产质量管理规范（GMP）的规定，测试了无 FCS 培养基在 MSC 扩增和分化中的应用。

方法

通过用人血浆和血小板提取物替代 FCS，对 MSC 扩增培养基进行了改良。根据多能 MSC 的定义最低标准对细胞进行了特征描述。对于软骨分化，采用无血清微团培养。对于脂肪和成骨分化，用人类血浆代替 FCS。在分化培养基中孵育 28 天后，通过细胞化学和免疫组织化学染色对细胞进行分析。此外，通过定量逆转录聚合酶链反应（qRT-PCR）研究了软骨形成、脂肪形成和成骨标志物的 mRNA 表达。

结果

在无 FCS 条件下，MSC 的扩增和分化产生了具有软骨形成、脂肪形成和成骨表型以及特征性基因表达的细胞。微团培养中的软骨细胞显示出糖胺聚糖和 II 型胶原的积累，以及软骨形成标志物基因的显著增加的 mRNA 表达。脂肪细胞显示出油红 O 染色，并表达过氧化物酶体增殖物激活受体γ 2（ppARγ2）和脂蛋白脂肪酶（LPL）mRNA。成骨细胞对 von Kossa 染色呈阳性，并表达成骨标志物基因的 mRNA。结果表明没有自发分化。