title: Cryopreservation of stromal vascular fraction of adipose tissue in a serum-free freezing medium.
abstract: Developing effective techniques for the cryopreservation of human adipose-derived adult stem cells could increase the usefulness of these cells in tissue engineering and regenerative medicine. Unfortunately, the use of serum and a commonly used cryoprotectant chemical, dimethyl sulphoxide (DMSO), during cryopreservation storage restricts the direct translation of adult stem cells to in vivo applications. The objective of this study was to test the hypothesis that the stromal vascular fraction (SVF) of adipose tissue can be effectively cryopreserved and stored in liquid nitrogen, using a freezing medium containing high molecular weight polymers, such as methylcellulose (MC) and/or polyvinylpyrollidone (PVP), as the cryoprotective agent (CPA) instead of DMSO. To this end, we investigated the post-freeze/thaw viability and apoptotic behaviour of SVF of adipose tissue frozen in 16 different media: (a) the traditional medium containing Dulbecco's modified Eagle's medium (DMEM) with 80% fetal calf serum (FCS) and 10% DMSO; (b) DMEM with 80% human serum (HS) and 10% DMSO; (c) DMEM with 0%, 2%, 4%, 6%, 8% or 10% DMSO; (d) DMEM with 1% MC and 10% of either HS or FCS or DMSO; (e) DMEM with 10% PVP and varying concentrations of FCS (0%, 10%, 40% or 80%); (f) DMEM with 10% PVP and 10% HS. Approximately 1 ml (10(6) cells/ml) of SVF cells 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 agitation), resuspended in culture medium and seeded in separate wells of a six-well plate for a 24 h incubation period at 37 degrees C. After 24 h, the thawed samples were analysed by brightfield 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 SVF cells. However, the percentage of viable cells obtained with 10% PVP and DMEM was comparable with that obtained in freezing medium with DMSO and serum (HS or FCS), i.e. approximately 54 +/- 14% and approximately 63 +/- 10%, respectively. Adipogenic and osteogenic differentiation behaviour of the frozen thawed cells was also assessed, using histochemical staining. Our results suggest that post-thaw SVF cell viability and adipogenic and osteogenic differentiability can be maintained even when they are frozen in the absence of serum and DMSO but with 10% PVP in DMEM.
authors.affiliation: Department of Mechanical Engineering, Louisiana State University, Baton Rouge, LA, USA.
authors.forename: Sreedhar
authors.initials: S
authors.lastname: Thirumala
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Gimble
authors.forename: Ram V
authors.initials: RV
authors.lastname: Devireddy
mesh_terms: D000273:Adipose Tissue / Q000098:blood supply / Q000166:cytology*; D002478:Cells, Cultured; D015925:Cryopreservation; D016895:Culture Media, Serum-Free; D005615:Freezing; D006801:Humans; D010012:Osteogenesis; D017154:Stromal Cells / Q000166:cytology*
chemical_list: D016895:Culture Media, Serum-Free