HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism
HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism
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DOI：
10.1007/s00204-014-1320-9
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发表时间：
2015-08-01
影响因子：
6.1
通讯作者：
Alves, Paula M.
中科院分区：
文献类型：
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作者：
Rebelo, Sofia P.;Costa, Rita;Alves, Paula M.
在药物开发、肝脏疾病研究和生物人工肝支持方面，需要能够概括肝脏生理学的模型。双能细胞系HepaRG是一种有效的肝脏功能替代物，但其分化状态依赖于高浓度的DMSO，这可能影响药物代谢的研究，限制了该肝脏模型的适用性。在此，我们提出了一种基于海藻酸盐微囊化细胞球体和在二甲亚砜（DMSO）无条件下培养HepaRG的三维（3D）分化策略。在3D培养中，肝细胞样细胞与胆道样细胞的比例为2.9:1，与二维（2D）培养相比，肝细胞分化率提高了35.9%。肝脏特性基因HNF4 α和PXR在3D培养物中的表达与2D分化培养物相当，而动态平衡相关基因白蛋白和氨甲酰磷酸合成酶1在3D培养物中的表达更高。此外，球体呈极化组织，显示出相互连接的胆管网络和排泄功能，通过MRP2的特异性活性来评估。重要的是，尽管基础基因表达水平存在差异，但细胞色素P450家族3亚家族A、多肽4和细胞色素P450家族1亚家族A、多肽2在诱导后的I期酶活性与分化后的2D培养物相当，而白蛋白生成和氨解毒在3D培养物中得到增强。该模型适用于毒理学应用，因为它允许在无dmso的环境中对多种化合物进行高通量分析。由于高外源代谢和维持生物合成功能，该模型的适用性可能会扩大到理解肝脏生理学和生物人工肝的应用。
The need for models that recapitulate liver physiology is perceived for drug development, study of liver disease and bioartificial liver support. The bipotent cell line HepaRG constitutes an efficient surrogate of liver function, yet its differentiated status relies on high concentrations of DMSO, which may compromise the study of drug metabolism and limit the applicability of this hepatic model. Herein, we present a three-dimensional (3D) strategy for the differentiation of HepaRG based on alginate microencapsulation of cell spheroids and culture in dimethyl sulfoxide (DMSO)-free conditions. A ratio of 2.9:1 hepatocyte-like to biliary-like cells was obtained in the 3D culture, with an improvement of 35.9 % in the hepatocyte differentiation when compared with two-dimensional (2D) cultures. The expression of the hepatic identity genes HNF4 alpha and PXR in 3D cultures was comparable to 2D differentiated cultures, while the expression of homeostatic-associated genes albumin and carbamoyl phosphate synthase 1 was higher in 3D. Moreover, the spheroids presented a polarized organization, exhibiting an interconnected bile canalicular network and excretory functionality, assessed by specific activity of MRP2. Importantly, despite variability in basal gene expression levels, the activity of the phase I enzymes cytochrome P450 family 3, subfamily A, polypeptide 4 and cytochrome P450 family 1, subfamily A, polypeptide 2 upon induction was comparable to differentiated 2D cultures and albumin production and ammonia detoxification were enhanced in 3D. The presented model is suitable for toxicological applications, as it allows high throughput analysis of multiple compounds in a DMSO-free setting. Due to the high xenobiotic metabolism and maintenance of biosynthetic functions, the applicability of this model might be broadened to understand liver physiology and for bioartificial liver applications.