Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells

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 modifi ed 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°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-fi eld microscopy and fl ow cytometry. The results suggest that the absence of DMSO (and the presence of MC) signifi cantly 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.

Concise Review: Adipose-Derived Stromal Vascular Fraction Cells and Stem Cells: Let's Not Get Lost in Translation

“…While DMSO is used routinely with blood cell products, it has potential adverse effects on the recipient and may not be optimal for all cells. Alternative CPAs for ASCs and SVF cells include hydroxyethyl starch, trihelose, and polyvinyl and some can be used under serum free conditions [30][31][32]. These alternative options should be explored, validated as reproducible, and considered as future industry standards.…”

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction?

“…The conventional slow-freezing and rapid-thawing procedure using dimethylsulfoxide (DMSO) as a cryoprotectant is the most commonly used method (Grout et al, 1990;Meryman, 2007). While this established technique is effective for somatic cell lines and even murine embryonic stem cells (mESCs), hematopoietic and mesenchymal human stem cells, this is not the case for hPSCs, due to low recovery rates and high levels of differentiation (Berz et al, 2007;Reubinoff et al, 2001;Richards et al, 2004;Thirumala et al, 2010). In contrast, vitrification of hPSCs by the "open pulled straw" method www.intechopen.com Current Frontiers in Cryobiology 140 using high cryoprotectant concentrations together with flash-freezing in liquid nitrogen has reported higher cell survival rates (Li et al, 2010b;Reubinoff et al, 2001;Richards et al, 2004).…”

Inducing Heat Shock Proteins Enhances the Stemness of Frozen–Thawed Adipose Tissue-Derived Stem Cells

“…As a chemically induced apoptotic control, ASCs were incubated in fresh medium enriched with 40 mM etoposide (24 h), as described in earlier publications from our laboratory [6,9,10]. Similarly, for necrotic control, ASCs were incubated for 24 h in fresh medium with 5 mM hydrogen peroxide, as described earlier [6,9,10].…”

“…The postthawed cells that were not adhered (dead) to the flasks and the adhered (healthy) cells were pooled together and washed in PBS and suspended in 1· binding solution supplied by the manufacturer. Approximately, 10 5 cells were stained using 5 mL of each dye, annexin V, and PI, for 15 min in dark and analyzed using a flow cytometer (BD Biosciences) within 1 h. The quadrants were set based on the live and dead control samples as described by Thirumala et al [6,9,10]. In brief, apoptotic analyses for ASCs were performed on a FACS Calibur flow cytometer (BD Biosciences) utilizing 488-nm laser excitation and fluorescence emission at 530 nm (FL1) and >575 nm (FL3).…”