如果可以购买肿瘤球体作为标准化产品,准备直接用于检测,这可能有助于提高研究的可重复性,有可能降低成本和加速成果。在这里,我们描述了一种工作流程,其中使用微孔培养大量生产大小均匀的癌症肿瘤球体,冷冻保存具有高活力,然后在中性浮力培养基中培养以进行药物测试。培养48小时后,C4-2B前列腺癌或MCF-7乳腺癌细胞合并为均匀的肿瘤球体。由100个细胞形成的肿瘤球体的耐受性比由200或400个细胞形成的肿瘤球体略好。解冻后,肿瘤球体代谢活性显著降低,提示线粒体损伤.通过将肿瘤球体解冻到补充有10µMN-乙酰-1-半胱氨酸(NAC)的培养基中,可以挽救代谢功能。解冻后,中性浮力介质,快乐细胞ASM,用于在药物测试期间将肿瘤球体保持为离散组织。新鲜和冷冻保存的C4-2B或MCF-7肿瘤球体对多西他赛的滴定反应相似。该协议将有助于未来的肿瘤球体可以作为可靠和可重复的产品购买,允许实验室有效地复制和建立已发表的研究,在许多情况下,使肿瘤球体只是另一种细胞培养试剂。
If it were possible to purchase tumour-spheroids as a standardised product, ready for direct use in assays, this may contribute to greater research reproducibility, potentially reducing costs and accelerating outcomes. Herein, we describe a workflow where uniformly sized cancer tumour-spheroids are mass-produced using microwell culture, cryopreserved with high viability, and then cultured in neutral
buoyancy media for drug testing. C4-2B prostate cancer or MCF-7 breast cancer cells amalgamated into uniform tumour-spheroids after 48 h of culture. Tumour-spheroids formed from 100 cells each tolerated the cryopreservation process marginally better than tumour-spheroids formed from 200 or 400 cells. Post-thaw, tumour-spheroid metabolic activity was significantly reduced, suggesting mitochondrial damage. Metabolic function was rescued by thawing the tumour-spheroids into medium supplemented with 10 µM N-Acetyl-l-cysteine (NAC). Following thaw, the neutral
buoyancy media, Happy Cell ASM, was used to maintain tumour-spheroids as discrete tissues during drug testing. Fresh and cryopreserved C4-2B or MCF-7 tumour-spheroids responded similarly to titrations of Docetaxel. This protocol will contribute to a future where tumour-spheroids may be available for purchase as reliable and reproducible products, allowing laboratories to efficiently replicate and build on published research, in many cases, making tumour-spheroids simply another cell culture reagent.