Department of Medical Sciences, Section of Cancer Pharmacology, Uppsala University, S-751 85, Uppsala, Sweden.
Exp Cell Res. 2012 Aug 1;318(13):1577-85. doi: 10.1016/j.yexcr.2012.03.026. Epub 2012 Apr 2.
Clinically relevant in vitro methods are needed to identify new cancer drugs for solid tumors. We report on a new 3-D spheroid cell culture system aimed to mimic the properties of solid tumors in vivo. The colon cancer cell lines HCT-116 wt and HCT-116 wt/GFP were grown as monolayers and for 3 or 6 days on 96-well NanoCulture® plates to form spheroids. Expression of surface markers, genes and hypoxia were assessed to characterize the spheroids and drug induced cytotoxicity was evaluated based on fluorescein diacetate (FDA) conversion by viable cells to fluorescent fluorescein or by direct measurement of fluorescence of GFP marked cells after a 72 h drug incubation. The cells reproducibly formed spheroids in the NanoCulture® plates with tight cell-attachment after 6 days. Cells in spheroids showed geno- and phenotypical properties reminiscent of hypoxic stem cells. Monolayer cultured cells were sensitive to standard and investigational drugs, whereas the spheroids gradually turned resistant. Similar results for cytotoxicity were observed using simplified direct measurement of fluorescence of GFP marked cells compared with FDA incubation. In conclusion, this new 3-D spheroid cell culture system provides a convenient and clinically relevant model for the identification and characterization of cancer drugs for solid tumors.
需要临床相关的体外方法来鉴定用于实体瘤的新癌症药物。我们报告了一种新的 3-D 球体细胞培养系统，旨在模拟体内实体瘤的特性。结肠癌细胞系 HCT-116 wt 和 HCT-116 wt/GFP 作为单层细胞培养，并在 96 孔 NanoCulture®板上培养 3 或 6 天以形成球体。评估表面标记物、基因和缺氧的表达以对球体进行特征描述，并根据活细胞将荧光二乙酸酯（FDA）转化为荧光荧光素或在 72 h 药物孵育后直接测量 GFP 标记细胞的荧光来评估药物诱导的细胞毒性。细胞在 6 天后在 NanoCulture®板上可重复地形成紧密附着的球体。球体中的细胞表现出类似于缺氧干细胞的遗传和表型特性。单层培养的细胞对标准药物和研究药物敏感，而球体逐渐产生耐药性。与 FDA 孵育相比，使用简化的 GFP 标记细胞荧光直接测量法观察到类似的细胞毒性结果。总之，这种新的 3-D 球体细胞培养系统为鉴定和表征用于实体瘤的癌症药物提供了一种方便且临床相关的模型。
