A Cell Culture Platform to Maintain Long-term Phenotype of Primary Human Hepatocytes and Endothelial Cells

Abstract

Background and AimsModeling interactions between primary human hepatocytes (PHHs) and primary human liver sinusoidal endothelial cells (LSECs) in vitro 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.MethodsPrimary 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.ResultsLSECs, 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.ConclusionsPHH/fibroblast/endothelial tricultures constitute a robust platform to elucidate reciprocal interactions between PHHs and endothelial cells in physiology, disease, and after drug exposure.

PNPLA2 influences secretion of triglyceride-rich lipoproteins by human hepatoma cells

“…Recent advances in maintaining the human liver morphology and specific functions of isolated primary human hepatocytes during standard cell culture include the co-culture with other cell-types, such as with endothelial cells or 3T3-J2 fibroblasts 93 . Additionally, formation of 3D-like structures, such as 3D-spheroid cultures, has demonstrated the potential to closely mimic human liver function in vitro discovery 94 .…”

Liver Tissue Engineering as an Emerging Alternative for Liver Disease Treatment

“…20,30,31 An approach that had promising results to overcome these limitations is coculturing hPHs with other nonparenchymal cells (NPCs) such as sinusoidal endothelial and stellate cells and fibroblasts to mimic their microenvironment. 17,32,33 Animal primary hepatocytes. Two kinds of animal primary hepatocytes have been used in liver regenerative medicine: rat and porcine hepatocytes.…”

Primary Human Hepatocytes Maintain Long-term Functions in Porous Silk Scaffolds Containing Extracellular Matrix Proteins

“…Such interactions have been long replicated in vitro by co-culturing primary hepatocytes with various liver-and non-liver-derived NPCs, including C3H/10T1/2 mouse embryo cells [38], 3T3-J2 murine embryonic fibroblasts [39], and human fibroblasts [40]. The 3T3-J2 fibroblasts, in particular, have been shown to express key liver-like molecules, such as decorin [41] and Tcadherin [42], which help these fibroblasts induce higher levels of functions in PHHs in 2D cocultures than primary human dermal fibroblasts (not shown), human liver-derived hepatic stellate cells [43], liver sinusoidal endothelial cells [44], and Kupffer cells [45]. The positive effects of 3T3-J2 on PHH functions have also been shown in 3D PEGDA-RGDS hydrogels [17].…”