Fig 1.
The post-thaw viability (A) and plating efficiency (B) of hASCs cryopreserved using undefined and defined cryopreservation media. The conventional undefined cryopreservation medium consisted of 10% DMSO+10% DMEM/F12+80% FBS while defined 1 freezing medium was composed of 3.5% DMSO+3.5% EG in DMEM/F-12 containing 0.25 M trehalose, 2% PVA, 5% ficoll, and 0.1 mM EGTA. Defined 2 freezing medium was similar to defined 1 medium but additionally contained two antioxidants (i.e., 3 mM reduced glutathione+5 mM ascorbic acid 2-phosphate). Data shown are mean±SEM. Columns with different letters in each thawing category are significantly different (p < 0.05).
Fig 2.
The post-thaw viability (A) and plating efficiency (B) of hASCs after further optimization of the defined cryopreservation medium. The undefined cryopreservation medium consisted of 10% DMSO+10% DMEM/F12+80% FBS while defined 2 medium was made up of 3.5% DMSO+3.5% EG+3 mM reduced glutathione+5 mM ascorbic acid 2-phosphate in DMEM/F-12 containing 0.25 M trehalose, 2% PVA, 5% ficoll, and 0.1 mM EGTA. Defined 3 cryopreservation medium had a slightly increased concentration (5%) of DMSO and EG; otherwise it was similar to defined 2 medium). Data shown are mean±SEM. Columns with different letters in each thawing category are significantly different (p < 0.05).
Fig 3.
Representative images of ASCs in culture prior to differentiation (A) and after both cryopreservation and differentiation into adipogenic (B), osteogenic (C), and chondrogenic (D) lineages as shown by Oil Red-O, Alizarin Red, and Alcian Blue staining, respectively. In this set of experiments, ASCs were cryopreserved using defined 3 medium (5% DMSO+5% EG+3 mM reduced glutathione+5 mM ascorbic acid 2-phosphate in DMEM/F-12 containing 0.25 M trehalose, 2% PVA, 5% ficoll, and 0.1 mM EGTA). Scale bar = 50 μm.
Fig 4.
Normal human female karyotype of ASCs cryopreserved under defined conditions at passage 6 using defined 3 cryopreservation medium (5% DMSO+5% EG+3 mM reduced glutathione+5 mM ascorbic acid 2-phosphate in DMEM/F-12 containing 0.25 M trehalose, 2% PVA, 5% ficoll, and 0.1 mM EGTA).