Ware Brenton R, Durham Mitchell J, Monckton Chase P, Khetani Salman R
School of Biomedical Engineering, Colorado State University, Fort Collins, Colorado.
Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois.
Cell Mol Gastroenterol Hepatol. 2017 Nov 24;5(3):187-207. doi: 10.1016/j.jcmgh.2017.11.007. eCollection 2018 Mar.
Modeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) can help elucidate human-specific mechanisms underlying liver physiology/disease and drug responses; however, existing hepatocyte/endothelial coculture models are suboptimal because of their use of rodent cells, cancerous cell lines, and/or nonliver endothelial cells. Hence, we sought to develop a platform that could maintain the long-term phenotype of PHHs and primary human LSECs.
Primary human LSECs or human umbilical vein endothelial cells as the nonliver control were cocultivated with micropatterned PHH colonies (to control homotypic interactions) followed by an assessment of PHH morphology and functions (albumin and urea secretion, and cytochrome P-450 2A6 and 3A4 enzyme activities) over 3 weeks. Endothelial phenotype was assessed via gene expression patterns and scanning electron microscopy to visualize fenestrations. Hepatic responses in PHH/endothelial cocultures were benchmarked against responses in previously developed PHH/3T3-J2 fibroblast cocultures. Finally, PHH/fibroblast/endothelial cell tricultures were created and characterized as described previously.
LSECs, but not human umbilical vein endothelial cells, induced PHH albumin secretion for ∼11 days; however, neither endothelial cell type could maintain PHH morphology and functions to the same magnitude/longevity as the fibroblasts. In contrast, both PHHs and endothelial cells displayed stable phenotype for 3 weeks in PHH/fibroblast/endothelial cell tricultures; furthermore, layered tricultures in which PHHs and endothelial cells were separated by a protein gel to mimic the space of Disse displayed similar functional levels as the coplanar tricultures.
PHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.
对原代人肝细胞（PHH）和原代人肝窦内皮细胞（LSEC）之间的相互作用进行建模，有助于阐明肝脏生理/疾病及药物反应背后的人类特异性机制；然而，现有的肝细胞/内皮细胞共培养模型并不理想，因为它们使用的是啮齿动物细胞、癌细胞系和/或非肝脏内皮细胞。因此，我们试图开发一个能够维持PHH和原代人LSEC长期表型的平台。
将原代人LSEC或作为非肝脏对照的人脐静脉内皮细胞与微图案化的PHH集落共培养（以控制同型相互作用），然后在3周内评估PHH的形态和功能（白蛋白和尿素分泌以及细胞色素P-450 2A6和3A4酶活性）。通过基因表达模式和扫描电子显微镜评估内皮细胞表型，以观察窗孔。将PHH/内皮细胞共培养中的肝脏反应与先前开发 的PHH/3T3-J2成纤维细胞共培养中的反应进行比较。最后，按照先前描述的方法创建并表征PHH/成纤维细胞/内皮细胞三培养物。
LSEC可诱导PHH白蛋白分泌约11天，而人脐静脉内皮细胞则不能；然而，两种内皮细胞类型均无法将PHH的形态和功能维持到与成纤维细胞相同的程度/寿命。相比之下，在PHH/成纤维细胞/内皮细胞三培养物中，PHH和内皮细胞在3周内均表现出稳定的表型；此外，用蛋白质凝胶将PHH和内皮细胞分隔开以模拟狄氏间隙的分层三培养物显示出与共面三培养物相似的功能水平。
PHH/成纤维细胞/内皮细胞三培养物构成了一个强大的平台，可用于阐明PHH与内皮细胞在生理、疾病及药物暴露后的相互作用。
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