Amniotic fluid-derived stem cells (AFSCs) are a potential cell source for therapeutic applications. They can be easily mass produced, cryopreserved and shipped to clinics for immediate use. However, one major obstacle to the manufacturing of clinical grade stem cells is the need for current good manufacturing practices for cryopreservation, storage, and distribution of these cells. Most current cryopreservation methods used for stem cells include the potentially toxic cryoprotectant (CPA) dimethylsulfoxide (Me2SO) in the presence of animal serum proteins that prevent direct use of these cells in human therapeutic applications. To avoid any potential cryoprotectant related complications, it will be essential to develop non-toxic CPAs or reduce CPA concentration in the freezing media used. In this study, we assessed the use of disaccharides, antioxidants and caspase inhibitors for cryopreservation of AFSCs in combination with a reduced concentration of Me2SO. The thawed cells were tested for viability with MTT assays and a growth curve was created to measure population doubling time. In addition, we performed flow cytometry analysis for cell surface antigens, RT-PCR for mRNA expression of stem cell markers, and assays to determine the myogenic differentiation potential of the cells. A statistically significant (p<0.05) increase in post-thawed cell viability in solutions containing trehalose, catalase and ZVAD-fmk with 5% Me2SO was observed. The solutions containing trehalose and catalase with 5% or 2.5% (v/v) Me2SO produced results similar to those for the control (10% (v/v) Me2SO and 30% FBS) in terms of culture growth, expression of cell surface antigens and mRNA expression of stem cell markers in AFSCs cryopreserved for a minimum of 3weeks. Thus, AFSCs can be cryopreserved with 1/4 the standard Me2SO concentration with the addition of disaccharides, antioxidants and caspase inhibitors. The use of Me2SO at low concentrations in cell freezing solutions may support the development of clinical trials of AFSCs.
- Caspase inhibitor
- Stem cell
ASJC Scopus subject areas
- Agricultural and Biological Sciences(all)
- Biochemistry, Genetics and Molecular Biology(all)