title: Cryopreservation by slow cooling with DMSO diminished production of Oct-4 pluripotency marker in human embryonic stem cells.
abstract: We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.
authors.affiliation: UCSD Cancer Center, San Diego, CA 92122, USA; Stem Cell Center Core, Burnham Institute for Medical Research (BIMR), La Jolla, CA 92037, USA.
authors.forename: Igor I
authors.initials: II
authors.lastname: Katkov
authors.forename: Min S
authors.initials: MS
authors.lastname: Kim
authors.forename: Ruchi
authors.initials: R
authors.lastname: Bajpai
authors.forename: Yoav S
authors.initials: YS
authors.lastname: Altman
authors.forename: Marc
authors.initials: M
authors.lastname: Mercola
authors.forename: Jeanne F
authors.initials: JF
authors.lastname: Loring
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Terskikh
authors.forename: Evan Y
authors.initials: EY
authors.lastname: Snyder
authors.forename: Fred
authors.initials: F
authors.lastname: Levine
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002470:Cell Survival; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004622:Embryo, Mammalian / Q000166:cytology*; D016166:Free Radical Scavengers / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D051379:Mice; D008856:Microscopy, Fluorescence; D050814:Octamer Transcription Factor-3 / Q000378:metabolism*; D013234:Stem Cells / Q000166:cytology*
chemical_list: D003451:Cryoprotective Agents; D016166:Free Radical Scavengers; D050814:Octamer Transcription Factor-3; D049452:Green Fluorescent Proteins; D004121:Dimethyl Sulfoxide