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Free Content Optimization of Cryopreservation of Stem Cells Cultured as Neurospheres: Comparison Between Vitrification, Slow-Cooling and Rapid Cooling “Freezing” Protocols

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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.

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Keywords: CRYOPRESERVATION; NEUROSPHERES; STEM CELLS; VITRIFICATION; “FREEZING”

Document Type: Research Article

Publication date: November 1, 2007

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  • CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation

    The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.

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