Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells
Long-Term Coculture Strategies for Primary Hepatocytes and Liver Sinusoidal Endothelial Cells
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DOI：
10.1089/ten.tec.2014.0152
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发表时间：
2015-04-01
影响因子：
3
通讯作者：
Yarmush, Martin L.
中科院分区：
文献类型：
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作者：
Bale, Shyam Sundhar;Golberg, Inna;Yarmush, Martin L.
肝细胞及其体外模型是影响肝脏的药物临床前筛选研究的重要工具。目前大多数模型主要集中在肝细胞单独和缺乏非实质细胞（NPC）的贡献，这是显着的通过分子和NPC本身的响应。将NPC与肝细胞合并的模型能够更真实地再现和阐明细胞相互作用和累积药物反应。肝细胞和肝窦内皮细胞（LSEC）占肝脏质量的80%，其中LSEC排列在血管壁上，并作为肝细胞和血液之间的屏障。用肝细胞培养LSEC以产生多细胞生理相关的体外肝脏模型一直是主要障碍，因为LSEC在分离后迅速丧失其表型。为此，我们描述了胶原凝胶的应用（1）在三明治和（2）作为一个干预细胞外基质层与LSEC的共培养肝细胞延长的时间。这些共培养配置提供了肝细胞和LSEC通过细胞-细胞接触和/或分泌因子导致共培养物的功能和稳定性增强的环境。我们的研究结果表明，在这些配置中，肝细胞和LSEC保持其表型时，一起培养的混合物，并显示稳定的分泌和代谢活动长达4周。窦内皮1（SE-1）抗体的免疫染色证明在培养期间保留LSEC表型。此外，单独培养的LSEC在胶原夹层结构中培养长达4周时保持高活力和SE-1表达。当LSEC作为底层（中间有胶原层）和作为夹心结构的混合物培养时，共培养物的白蛋白产量高10-15倍，并且天然的α 1A 1/2活性比单培养对照高至少20倍。总之，这些数据表明，基于胶原凝胶的肝细胞-LSEC共培养物是两种细胞类型的稳定化和长期培养的高度合适的模型。总之，这些结果表明基于胶原凝胶的肝细胞-LSEC共培养模型对于体外毒性测试和肝脏模型开发研究是有希望的。
Hepatocytes and their in vitro models are essential tools for preclinical screening studies for drugs that affect the liver. Most of the current models primarily focus on hepatocytes alone and lack the contribution of non-parenchymal cells (NPCs), which are significant through both molecular and the response of the NPCs themselves. Models that incorporate NPCs alongside hepatocytes hold the power to enable more realistic recapitulation and elucidation of cell interactions and cumulative drug response. Hepatocytes and liver sinusoidal endothelial cells (LSECs) account for similar to 80% of the liver mass where the LSECs line the walls of blood vessels, and act as a barrier between hepatocytes and blood. Culturing LSECs with hepatocytes to generate multicellular physiologically relevant in vitro liver models has been a major hurdle since LSECs lose their phenotype rapidly after isolation. To this end, we describe the application of collagen gel (1) in a sandwich and (2) as an intervening extracellular matrix layer to coculture hepatocytes with LSECs for extended periods. These coculture configurations provide environments wherein hepatocyte and LSECs, through cell-cell contacts and/or secretion factors, lead to enhanced function and stability of the cocultures. Our results show that in these configurations, hepatocytes and LSECs maintained their phenotypes when cultured together as a mixture, and showed stable secretion and metabolic activity for up to 4 weeks. Immunostaining for sinusoidal endothelial 1 (SE-1) antibody demonstrated retention of LSEC phenotype during the culture period. In addition, LSECs cultured alone maintained high viability and SE-1 expression when cultured within a collagen sandwich configuration up to 4 weeks. Albumin production of the cocultures was 10-15 times higher when LSECs were cultured as a bottom layer (with an intervening collagen layer) and as a mixture in a sandwich configuration, and native CYP 1A1/2 activity was at least 20 times higher than monoculture controls. Together, these data suggest that collagen gel-based hepatocyte-LSEC cocultures are highly suitable models for stabilization and long-term culture of both cell types. In summary, these results indicate that collagen gel-based hepatocyte-LSEC coculture models are promising for in vitro toxicity testing, and liver model development studies.