Evaluation of methylcellulose and dimethyl sulfoxide as the cryoprotectants in a serum-free freezing media for cryopreservation of adipose-derived adult stem cells

Authors

Sreedhar Thirumala, Bioengineering Laboratory, Department of Mechanical Engineering, Louisiana State University, Baton Rouge, Louisiana 70803, USA.
Jeffrey M. Gimble
Ram V. Devireddy

Document Type

Article

Publication Date

4-1-2010

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 (10(6) cells/mL) of P1 ASCs were frozen overnight in a -80 degrees C freezer and stored in liquid nitrogen for 2 weeks before being rapidly thawed in a 37 degrees 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 degrees 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, approximately 84% +/- 5% and approximately 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.

Publication Source (Journal or Book title)

Stem cells and development

First Page

513

Last Page

22

Recommended Citation

Thirumala, S., Gimble, J. M., & Devireddy, R. V. (2010). Evaluation of methylcellulose and dimethyl sulfoxide as the cryoprotectants in a serum-free freezing media for cryopreservation of adipose-derived adult stem cells. Stem cells and development, 19 (4), 513-22. https://doi.org/10.1089/scd.2009.0173