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Although MSCs exhibit self-renewal, they also undergo senescence after long-term
culture. MSCs typically proliferate for 20–50 doublings, depending on the cell source
and culture conditions (Cholewa et al. 2011 ; Izadpanah et al. 2006 ; Suchanek et al.
2007 ). Senescent cells display aneuploidy without transformation and exhibit muta-
tions in certain genes, such as the p53 gene (Tarte et al. 2010 ); upregulation of hyal-
uronan and proteoglycan link protein 1, keratin 18, brain-derived neurotrophic factor,
or renal tumor antigen; and downregulation of pleiotrophin (Schallmoser et al. 2010 ).
To date, senescence is easy to evaluate by a β-galactosidase staining assay.
Differentiation is also an important characteristic of MSCs. According to
Dominici et al., MSCs must be able to differentiate into three kinds of mesodermal
cells, namely, osteoblasts, adipocytes, and chondroblasts ( 2006 ). This suggestion
has been used as a guideline to evaluate MSCs. Some reports show that senescent
MSCs have a reduced differentiation potential for only osteoblasts. Differentiation
assays are easy to perform with commercial differentiation kits. When cultured in
inducing medium for 14–21 days, MSCs differentiate into adipocytes, osteoblasts,
or chondroblasts depending on the media. Similar to self-renewal testing, differen-
tiation potential tests are also performed for 2–3 weeks. Therefore, this test is usu-
ally applied during evaluation of the production procedure.
To evaluate MSC quality before transplantation, there are two popular indica-
tors, surface marker expression and cell viability. Assessment of both can be carried
out by fl ow cytometry. For cell viability, collected MSCs are stained with
7- aminoactinomycin D (7-AAD), and dead cells are identifi ed based on the signal
of 7-AAD. Although there is no guideline or regulation concerning the percentage
of live MSCs for clinical grafts, most studies only use MSC samples with more than
95 % live cells. In terms of surface markers for MSCs, according to Dominici et al.
( 2006 ), there are two groups of markers used to confi rm MSCs: positive markers
(CD13, CD44, CD73, CD90, and CD105) and negative markers (CD14, CD34,
CD45, and HLA-DR). Profi le marker kits for these have been commercialized to
confi rm MSC phenotypes (Dominici et al. 2006 ) (Fig. 6.6 ).
6.6.5.2 MSC Safety
The fi rst safety issue relates to contamination by viruses, including hepatitis B and C,
HIV, human T-cell leukemia virus type 1, and syphilis. This issue must be addressed
at donor or sample collection. All donors must be carefully checked according to
blood bank guidelines. Only viral negative samples are used in further processes.
The second safety issue relates to bacterial and fungal contamination. Both bac-
terial and fungal contamination of classical pharmaceutical products are excluded
by standardized tests, as set, for example, in Europe [Pharmacopoeia (EP), Chap.
2.6.1] or in the USA [Pharmacopoeia (USP), Chap. 71]. These growth promotion
tests involve two cultivation media, fl uid thioglycolate and soybean casein digest
media, and two temperatures, 22.5 and 32.5 °C, for growth of each tested sample.
However, this test requires 14 days and is clearly unsuitable for products with a
short shelf-life. There are instructions on validation of rapid microbiological tests in
P.V. Pham and N.B. Vu