Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells
- 1 April 2010
- journal article
- research article
- Published by Mary Ann Liebert Inc in Stem Cells and Development
- Vol. 19 (4), 513-522
- https://doi.org/10.1089/scd.2009.0173
Abstract
Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells (ASCs) could increase the usefulness of these cells in tissue engineering and regenerative medicine. To this end, we investigated the post-freeze/thaw viability and apoptotic behavior of Passage 1 (P1) adult stem cells (ASCs) in 11 different media: (i) the traditional media containing Dulbecco’s modified Eagle’s medium (DMEM) with 80% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO), (ii) DMEM with 80% human serum (HS) and 10% DMSO, (iii) DMEM with 1% methyl cellulose (MC) and 10% of either HS or FCS or DMSO, and (iv) DMEM with 0%, 2%, 4%, 6%, 8%, or 10% DMSO. Approximately 1 mL (106 cells/mL) of P1 ASCs were frozen overnight in a −80°C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37°C water bath (1–2 min of agitation), resuspended in culture media, and seeded in separate wells of a 6-well plate for a 24-h incubation period at 37°C. After 24 h, the thawed samples were analyzed by bright-field microscopy and flow cytometry. The results suggest that the absence of DMSO (and the presence of MC) significantly increases the fraction of apoptotic and/or necrotic ASCs. However, the percentage of viable cells obtained with 2% DMSO and DMEM was comparable with that obtained in freezing media with 10% DMSO and 80% serum (HS or FCS), that is, ∼84% ± 5% and ∼84% ± 8%, respectively. Adipogenic and osteogenic differentiation behavior of the frozen thawed cells was also assessed using histochemical staining. Our results suggest that post-thaw ASC viability, adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum but with a minimal concentration of 2% DMSO in DMEM.Keywords
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