Purcell Wendy M, Atterwill Christopher K, Xu Jinsheng
Centre for Research in Biomedicine, Faculty of Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK.
Altern Lab Anim. 2003 Dec;31(6):563-73. doi: 10.1177/026119290303100605.
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.
对鸡和大鼠脑类器官的冷冻保存进行了研究。通过旋转介导的培养系统，从7日龄鸡胚胎或16日龄大鼠胚胎制备脑类器官培养物。使用两阶段冷却程序，将类器官保存在含有5 - 15%二甲基亚砜（DMSO）的培养基中，并储存在液氮中。结果表明，以解冻后24小时、3天、7天和28天鸡胚胎脑类器官的总蛋白含量表示的活力，为对照值的45.5%至64.2%。解冻后的脑类器官需要3天才能稳定下来，这从它们的形态和选定的功能性神经标志物可以看出。这些功能在28天的观察期内得以维持。在冷冻保存前体外培养12 - 15天的类器官比体外培养5 - 7天后冷冻保存的类器官存活得更好。长期（长达6个月）储存后类器官的活力和生化功能与短期储存后的相似。以总蛋白含量表示，在15% DMSO中冷冻保存的大鼠脑类器官在解冻后24小时、3天或7天的活力为对照值的23.1%至32.1%。这项研究首次表明，从原代组织制备的脑类器官可以成功冷冻保存。
