title: DMSO-Free Programmed Cryopreservation of Fully Dissociated and Adherent Human Induced Pluripotent Stem Cells.
abstract: THREE MODES FOR CRYOPRESERVATION (CP) OF HUMAN IPSC CELLS HAVE BEEN COMPARED: STD: standard CP of small clumps with 10% of CPA in cryovials, ACC: dissociation of the cells with Accutase and freezing in cryovials, and PLT: programmed freezing of adherent cells in plastic multiwell dishes in a programmable freezer using one- and multistep cooling protocols. Four CPAs were tesetd: dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY). The cells in ACC and PLT were frozen and recovered after thawing in the presence of a ROCK inhibitor Y-27632 (RI). EG was less toxic w/o CP cryopreservation than DMSO and allowed much better maintenance of pluripotency after CP than PG or GLY. The cells were cryopreserved very efficiently as adherent cultures (+RI) in plates (5-6-fold higher than STD) using EG and a 6-step freezing protocol. Recovery under these conditions is comparable or even higher than ACC+RI. Conclusions. Maintenance of cell-substratum adherence is a favorable environment that mitigates freezing and thawing stresses (ComfortFreeze(®) concept developed by CELLTRONIX). CP of cells directly in plates in ready-to-go after thawing format for HT/HC screening can be beneficial in many SC-related scientific and commercial applications such as drug discovery and toxicity tests.
authors.affiliation: CELLTRONIX, San Diego, CA 92196, USA.
authors.forename: Igor I
authors.initials: II
authors.lastname: Katkov
authors.forename: Natalia G
authors.initials: NG
authors.lastname: Kan
authors.forename: Flavio
authors.initials: F
authors.lastname: Cimadamore
authors.forename: Brandon
authors.initials: B
authors.lastname: Nelson
authors.forename: Evan Y
authors.initials: EY
authors.lastname: Snyder
authors.forename: Alexey V
authors.initials: AV
authors.lastname: Terskikh