Thériault Brigitte L, Portelance Lise, Mes-Masson Anne-Marie, Nachtigal Mark W
Campbell Family Cancer Research Institute, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada.
Methods Mol Biol. 2013;1049:323-36. doi: 10.1007/978-1-62703-547-7_24.
We have refined the technique for isolating and propagating cultures of primary epithelial ovarian cancer (EOC) cells derived from solid tumors and ascites. Both protocols involve a simple yet rapid method for the growth and propagation of EOC tumor and ascites cells in a basal culture medium without the addition of growth factors. Isolation of tumor EOC cells involves the mechanical disruption of the tumor tissue with the help of a cell scraper, while ascites-derived EOC cells are mixed with growth medium and placed directly into culture with very little manipulation. We further describe a partial trypsinization method to eliminate fibroblast contamination from primary EOC cells derived from solid tumors. These methods allow for the direct application of many molecular, cellular, and functional analyses within a few weeks of initial isolation, with the added potential of retrospective analyses of archived cells and tissues. Thus, we have included steps for long-term cryopreservation of early-passage EOC cells. Initial isolation of EOC cells can be completed within 1 h, and primary cells are further expanded in culture for several weeks.
我们改进了从实体瘤和腹水中分离并培养原发性上皮性卵巢癌（EOC）细胞的技术。这两种方案都涉及一种简单而快速的方法，即在不添加生长因子的基础培养基中培养和扩增EOC肿瘤细胞及腹水细胞。肿瘤EOC细胞的分离需要借助细胞刮勺对肿瘤组织进行机械破碎，而腹水来源的EOC细胞则与生长培养基混合，只需极少的操作即可直接进行培养。我们还描述了一种部分胰蛋白酶消化法，以消除实体瘤来源的原发性EOC细胞中的成纤维细胞污染。这些方法能够在初次分离后的几周内直接进行许多分子、细胞和功能分析，还具有对存档细胞和组织进行回顾性分析的潜在可能性。因此，我们纳入了早期传代EOC细胞长期冷冻保存的步骤。EOC细胞的初次分离可在1小时内完成，原代细胞可在培养中进一步扩增数周。
