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liquid enrichment medium, from which mycoplasma cultures are transferred onto
agar after several days. The second is coculture of samples with permissive cell
lines (usually Vero cells) and then staining with fl uorescent DNA-binding dyes
(DAPI or Hoechst). Recently, commercialized kits to detect mycoplasma DNA have
been validated and applied to cell production, such as MycoTOOL™ (Roche
Diagnostics) (a test that amplifi es a region of the 16S rRNA of mycoplasma) that
has been validated with European Pharmacopoeia tests (Chap. 2.6.7) and the
MycoSensor QPCR assay kit developed by Stratagene, which is acceptable in pre-
clinical regulatory validation of amniotic MSC manufacturing protocol.
Another test relating to safety issues is endotoxin testing. Endotoxins are lipo-
polysaccharides from Gram-negative bacteria. They are the most common cause of
toxic reactions resulting in serious health problems, such as diarrhea, septic shock,
and marrow necrosis. Therefore, testing for endotoxins is standard in cellular and
gene therapy products. The acceptable level of endotoxin in these products is usu-
ally 5.0 EU/kg/dose. Endotoxin is generally tested by the Limulus amebocyte lysate
method. Commercialized kits for endotoxin detection and quantitation have been
developed to facilitate endotoxin measurement.
The last safety issue is tumorigenicity of expanded MSCs. This is the most con-
cerning safety issue using expanded MSCs for clinical applications. This risk origi-
nated from observations of spontaneous transformation of human MSCs in culture
(Rosland et al. 2009 ; Rubio et al. 2005 ; Wang et al. 2005 ). However, most studies
have been retracted because of cross contamination with cultures of exogenous
tumor cell lines (de la Fuente et al. 2010 ; Torsvik et al. 2010 ). There are no reports
of tumorigenicity after transplantation of MSCs into patients. Because of these
results, tumorigenicity testing has not been suggested to evaluate MSCs before clin-
ical application.
6.7 Conclusion
MSC production with GMP compliancy appears to be a compelling condition to use
MSCs in clinical application. GMP will maintain the quality and safety of MSCs.
Clinical-grade MSCs are only produced by application of regulations as well as the
requirements or elements of GMP. However, all procedures should originate from
clinical demands. GMP is not a standard but a set of guidelines or rules for produc-
tion procedures with the highest quality and safety.
References
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P.V. Pham and N.B. Vu