Fig. 7
Characterization of MSCs after repeated freeze/thaw/explant cultures. a MSC marker expression in repeated freeze/thaw/explant cultures. Conditioned UC tissue after initial explant culture was subjected to 10 cycles of freeze/thaw/explant culture and cell cultures obtained from each cycle were harvested and analyzed by RT-PCR for the expression of the MSC markers CD73, CD90, and CD105 with GAPDH expression as a control. The cumulative yield of MSC from each freeze/thaw/explant culture cycle was calculated for a 10-day period. b Conditioned UC tissue after initial explant culture was subjected to one, four, seven, and 10 freeze/thaw/explant culture cycles, respectively, and cell surface marker analysis was performed for the presence of the typical MSC markers (CD73, CD90, and CD105) by flow cytometry. c Proliferative capacity of MSC-like cells obtained after reculture of cryopreserved conditioned UC tissues from one, four, seven, and 10 freeze/thaw/explant culture cycles was evaluated by cell counting in a hemocytometer after initial seeding of 104 cells from each MSC population. Relative proliferative capacity was calculated as fold changes after setting the initial cell number 104 cells = 1. Data represent the mean ± SD of four independent experiments. Statistical analysis was performed by Student’s t test. d Conditioned UC tissue after initial explant culture was subjected to one, four, seven, and 10 freeze/thaw/explant culture cycles, respectively, and the SA-β-gal assay was applied to the outgrowing MSC cultures. SA-β-gal-positive cells were quantified as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s t test. bp base pairs, MSC mesenchymal stroma/stem cells, PE phycoerythrin, UC umbilical cord