Cryopreservation of organotypic brain spheroid cultures.
The cryopreservation of hen and rat brain spheroids was investigated. Brain spheroid cultures were prepared from 7-day-old hen embryos or 16-day-old rat embryos, by using a rotation-mediated culture system. The spheroids were cryopreserved in medium containing 5-15% dimethyl sulphoxide (DMSO) and stored in liquid nitrogen, by using a two-stage cooling procedure. The results show that the viability, as indicated by the total protein content of hen embryo brain spheroids at 24 hours, and at 3, 7 and 28 days after thawing, ranged from 45.5% to 64.2% of control values. It took 3 days for the post-thaw brain spheroids to stabilise, as indicated by their morphology and selected neural markers of functionality. These functions were maintained over a 28-day observation period. Spheroids cultured for 12-15 days in vitro before cryopreservation survived better than those that were cryopreserved after 5-7 days in vitro. The viability and biochemical functionality of spheroids after long-term (up to 6 months) storage were similar to those following short-term storage. The viability of rat brain spheroids cryopreserved in 15% DMSO, as indicated by total protein content, at 24 hours, and at 3 or 7 days after thawing, ranged from 23.1% to 32.1% of control values. This study shows for the first time that brain spheroids prepared from primary tissue can be successfully cryopreserved.
Purcell
Wendy M
WM
Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK.
Atterwill
Christopher K
CK
Xu
Jinsheng
J
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000196:embryology* / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002642:Chick Embryo; D015925:Cryopreservation / Q000379:methods*; D005260:Female; D005947:Glucose / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D011247:Pregnancy; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018874:Spheroids, Cellular
D011506:Proteins; D007770:L-Lactate Dehydrogenase; D000110:Acetylcholinesterase; D005978:Glutathione; D005947:Glucose