Optimization of cryopreservation of stem cells cultured as neurospheres: comparison between vitrification, slow-cooling and rapid cooling freezing protocols.
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.
Tan
Francis Chee Kuan
FC
Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Lee
Kong Heng
KH
Gouk
Sok Siam
SS
Magalhaes
Raquel
R
Poonepalli
Anuradha
A
Hande
Manoor Prakash
MP
Dawe
Gavin S
GS
Kuleshova
Lilia L
LL
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
D009584:Nitrogen