title: Optimization of cryopreservation of stem cells cultured as neurospheres: comparison between vitrification, slow-cooling and rapid cooling freezing protocols.
abstract: We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 0.6 M sucrose) or that do not (37% v/v) EG, 0.6 M sucrose and 30% (v/v) EG, 0.6 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs. To protect against contamination a straw-in-straw technique was employed. Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (>96%), multipotency and karyotype. Rapid cooling in 37% (v/v) EG, 0.6 M sucrose afforded good viability but did not preserve structural integrity. Rapid cooling in 30% (v/v) EG, 0.6 M sucrose additionally reduced cell viability to 77%. Slow-cooling reduced cell viability to 65% and damaged the neurospheres. This study suggests that, in contrast to freezing, vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures.
authors.affiliation: Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
authors.forename: Francis Chee Kuan
authors.initials: FC
authors.lastname: Tan
authors.forename: Kong Heng
authors.initials: KH
authors.lastname: Lee
authors.forename: Sok Siam
authors.initials: SS
authors.lastname: Gouk
authors.forename: Raquel
authors.initials: R
authors.lastname: Magalhaes
authors.forename: Anuradha
authors.initials: A
authors.lastname: Poonepalli
authors.forename: Manoor Prakash
authors.initials: MP
authors.lastname: Hande
authors.forename: Gavin S
authors.initials: GS
authors.lastname: Dawe
authors.forename: Lilia L
authors.initials: LL
authors.lastname: Kuleshova
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods / Q000592:standards*; D005615:Freezing; D007621:Karyotyping; D051379:Mice; D039902:Multipotent Stem Cells; D009584:Nitrogen; D013997:Time Factors
chemical_list: D009584:Nitrogen