Department of Biology, Faculty of Science, Razi University, Kermanshah, 6714414971, Iran.
Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran.
Tissue Eng Regen Med. 2020 Aug;17(4):459-475. doi: 10.1007/s13770-020-00272-6. Epub 2020 Jul 14.
Currently, there is an urgent need for scalable and reliable in vitro models to assess the effects of therapeutic entities on the human liver. Hepatoma cell lines, including Huh-7, show weakly resemblance to human hepatocytes, limiting their significance in toxicity studies. Co-culture of hepatic cells with non-parenchymal cells, and the presence of extracellular matrix have been shown to influence the biological behavior of hepatocytes. The aim of this study was to generate the scalable and functional hepatic micro-tissues (HMTs).
The size-controllable HMTs were generated through co-culturing of Huh-7 cells by mesenchymal stem cells and human umbilical vein endothelial cells in a composite hydrogel of liver-derived extracellular matrix and alginate, using an air-driven droplet generator.
The generated HMTs were functional throughout a culture period of 28 days, as assessed by monitoring glycogen storage, uptake of low-density lipoprotein and indocyanine green. The HMTs also showed increased secretion levels of albumin, alpha-1-antitrypsin, and fibrinogen, and production of urea. Evaluating the expression of genes involved in hepatic-specific and drug metabolism functions indicated a significant improvement in HMTs compared to two-dimensional (2D) culture of Huh-7 cells. Moreover, in drug testing assessments, HMTs showed higher sensitivity to hepatotoxins compared to 2D cultured Huh-7 cells. Furthermore, induction and inhibition potency of cytochrome P450 enzymes confirmed that the HMTs can be used for in vitro drug screening.
Overall, we developed a simple and scalable method for generation of liver micro-tissues, using Huh-7, with improved hepatic-specific functionality, which may represent a biologically relevant platform for drug studies.
目前，迫切需要可扩展且可靠的体外模型来评估治疗实体对人类肝脏的影响。肝癌细胞系，包括 Huh-7，与人类肝细胞的相似性较弱，限制了它们在毒性研究中的重要性。肝细胞与非实质细胞的共培养以及细胞外基质的存在已被证明会影响肝细胞的生物学行为。本研究旨在生成可扩展且功能齐全的肝微组织 (HMT)。
通过间质干细胞和人脐静脉内皮细胞在肝源性细胞外基质和藻酸盐的复合水凝胶中共同培养 Huh-7 细胞，使用气动液滴发生器生成尺寸可控的 HMT。
通过监测糖原储存、低密度脂蛋白摄取和吲哚菁绿，在 28 天的培养期间评估，生成的 HMT 是功能齐全的。HMT 还显示出白蛋白、α-1-抗胰蛋白酶和纤维蛋白原的分泌水平增加，以及尿素的产生。评估参与肝特异性和药物代谢功能的基因表达表明，与二维（2D）培养 Huh-7 细胞相比，HMT 有显著改善。此外，在药物测试评估中，与 2D 培养 Huh-7 细胞相比，HMT 对肝毒素显示出更高的敏感性。此外，细胞色素 P450 酶的诱导和抑制能力证实 HMT 可用于体外药物筛选。
总的来说，我们使用 Huh-7 开发了一种简单且可扩展的生成肝微组织的方法，具有改善的肝特异性功能，这可能代表药物研究的一个具有生物学相关性的平台。
