Department of Biological Sciences and Bioengineering & Centre for Environmental Sciences and Engineering, Indian Institute of Technology Kanpur, Kanpur 208016, India.
Department of Chemical Engineering, Kyushu University, Fukuoka, Japan.
Biotechnol J. 2017 Oct;12(10). doi: 10.1002/biot.201700274. Epub 2017 Sep 6.
While hepatic cell lines are mainly used for in vitro drug induced toxicity studies, they exhibit limited functionalities. To overcome this, the authors have employed genetically engineered mouse hepatoma cells, Hepa/8F5, wherein expression of liver enriched transcription factors is induced by doxycycline leading to increased functionality. Further enhancement in functionality is achieved by spheroid culture in a previously developed 3D cell culture platform. Cells are seeded in presence of temperature-responsive poly(N-isopropylacrylamide) on poly(N-isopropylacrylamide--co-gelatin) cryogel scaffold based high throughput platform. Cells seeded in presence of poly(N-isopropylacrylamide) and induced with doxycycline exhibited highest functionalities. There is an increase of ≈26, 36, and 39% in albumin secretion, ammonia removal, and CYP3A4 activity, respectively. Morphological analysis showed arrest in cell proliferation and enlarged nucleus in presence of doxycyline and spheroid formation in presence of poly(N-isopropylacrylamide). Drug induced liver toxicity studies revealed that cells induced with doxycycline are resistive to tamoxifen but sensitive to acetaminophen whereas, cultures initiated in presence of poly(N-isopropylacrylamide) are resistive to both the drugs which is indicative of diffusional barrier of the spheroids. The authors conclude that Hepa/8F5 cells show enhanced functionality in cryogel based spheroid culture platform which can be successfully used for high throughput screening of hepatic toxicity in vitro.
虽然肝细胞系主要用于体外药物诱导毒性研究，但它们表现出有限的功能。为了克服这一问题，作者采用了经基因工程改造的小鼠肝癌细胞 Hepa/8F5，其中肝富集转录因子的表达受强力霉素诱导，从而增强了功能。通过在先前开发的 3D 细胞培养平台中进行球体培养进一步增强了功能。在温度响应性聚（N-异丙基丙烯酰胺）存在下将细胞接种在基于高通量平台的聚（N-异丙基丙烯酰胺-co-明胶）冷冻凝胶支架上。在存在聚（N-异丙基丙烯酰胺）和强力霉素诱导的情况下接种的细胞表现出最高的功能。白蛋白分泌、氨去除和 CYP3A4 活性分别增加了≈26%、36%和 39%。形态分析显示，在强力霉素存在下细胞增殖停滞且细胞核增大，在聚（N-异丙基丙烯酰胺）存在下形成球体。药物诱导的肝毒性研究表明，经强力霉素诱导的细胞对他莫昔芬具有抗性，但对乙酰氨基酚敏感，而在聚（N-异丙基丙烯酰胺）存在下起始的培养物对两种药物均具有抗性，这表明球体的扩散屏障。作者得出结论，Hepa/8F5 细胞在基于冷冻凝胶的球体培养平台中表现出增强的功能，可成功用于体外肝毒性的高通量筛选。
