Selection of cryopreservation medium for slow-freezing.

<p>(A) Recovery frequencies (rate, %) of iPSC (201B7) colonies treated with Pronase/EDTA dissociation followed by cryopreservation with 5 different media (Formulas A–E). Recovery frequencies (rate, %) were determined by the percentage of ALP+ colonies 5 days after thawing compared with those at day 5 after passaging with Pronase/EDTA without cryopreservation. Recovery frequencies (rate, %) are shown as bars with S.D. Formula A: [6% HES, 5% DMSO, 4% BSA, and 50% D-MEM/F12 in saline]; B: [6% HES, 5% DMSO, and 50% D-MEM/F12 in saline]; C: [6% HES, 5% DMSO, and 4% BSA in saline]; D: [6% HES and 5% DMSO in saline]; E: [6% HES, 5% DMSO, and 5% ethylene glycol (EG) in saline]. Results of 3 independent experiments are shown. Differences between E and the others are significant. *; <i>P</i><0.05. (B) The effects of EG addition on cryopreservation efficacy of freezing media. Various concentrations (1, 2, 3, 4, 5, 7.5, 10, 12.5, or 15% v/v) of EG were added to cryopreservation Formula D (6% HES, 5% DMSO in saline). Recovery frequencies (rate, %) were determined by scoring the post-thaw number of ALP+ colonies and those without cryopreservation. Results of 3 independent experiments are shown. *; <i>P</i><0.05 (C) ALP staining of colonies of iPSC 201B7 maintained for 5 days after passage (left photo: post-plating, non-frozen control) and those 5 days after thaw (right photo: post-thawing, dissociation with Pronase/EDTA and cryopreservation with CP-5E). Magnified photos are attached. Scale bars indicate 500 µm. (D) Cell colonies of hiPSC cell lines (201B7, 253G1) or hESC cell lines (KhES-1, H1) were dissociated with Pronase/EDTA, followed by cryopreservation with CP-5E (Formula E: 6% HES, 5% DMSO, and 5% EG in saline). Recovery frequencies (%) were determined by scoring the number of ALP+ colonies after thawing for comparison with nonfrozen cells. Results of 3 independent experiments are shown.