Raw data_1 from A tumour-spheroid manufacturing and cryopreservation process that yields a highly reproducible product ready for direct use in drug screening assays

If it were possible to purchase tumour-spheroids as a standardized product, ready for direct use in assays, this may contribute to greater research reproducibly, potentially reducing costs. 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, 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 will 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.

若可购置标准化的肿瘤球状体（tumour-spheroids）产品，且可直接用于实验检测，将有助于提升研究的可重复性，同时或可降低研究成本。本研究详述了一套实验流程：借助微孔培养（microwell culture）技术批量制备尺寸均一的肿瘤球状体，经高存活率冷冻保存后，置于中性浮力培养基（neutral buoyancy media）中培养以用于药物检测。C4-2B前列腺癌细胞与MCF-7乳腺癌细胞在培养48小时后，可聚集形成尺寸均一的肿瘤球状体。由每球100个细胞形成的肿瘤球状体，对冷冻保存过程的耐受性略优于由每球200或400个细胞形成的肿瘤球状体。解冻后，肿瘤球状体的代谢活性显著降低，提示存在线粒体损伤。若将肿瘤球状体解冻于添加了10μM N-乙酰-L-半胱氨酸（N-Acetyl-l-cysteine, NAC）的培养基中，其代谢功能可得到恢复。解冻后，使用中性浮力培养基Happy Cell维持肿瘤球状体的离散组织状态，以开展药物检测。新鲜与冷冻保存的C4-2B前列腺癌、MCF-7乳腺癌肿瘤球状体，对多西他赛（Docetaxel）的浓度梯度处理响应相似。本实验方案将助力实现肿瘤球状体作为可靠、可重复的商品化产品的愿景，使实验室可高效复刻已发表研究并在此基础上开展后续工作，在多数场景下可将肿瘤球状体视作常规细胞培养试剂。