Animal serum-free expansion and differentiation of human mesenchymal stromal cells.
OBJECTIVE
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.
Felka
Tino
T
Center for Medical Research (ZMF), University of Tübingen Medical Center (UKT), 72072 Tübingen, Germany.
Schäfer
Richard
R
De Zwart
Peter
P
Aicher
Wilhelm K
WK
D050156:Adipogenesis / Q000187:drug effects / Q000235:genetics; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects; D020219:Chondrogenesis / Q000187:drug effects / Q000235:genetics; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008648:Mesoderm / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D008875:Middle Aged; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D017154:Stromal Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism
D015415:Biomarkers; D016895:Culture Media, Serum-Free