Written by Nick Trompeter, Ph.D.
Continuing our blog on culturing primary human hepatic cells in vitro (read about culturing hepatic stellate cells here), hepatocytes will be the focus this week.
Hepatocytes play crucial roles in human physiology via their function to metabolize xenobiotics, secrete albumin for the maintenance of oncotic pressure and transport of endogenous and exogenous molecules, provide immune functions, metabolize ammonia, and form bile to name a few functions.(15) Unlike hepatic stellate cells and Liver Sinusoidal Endothelial Cells, hepatocytes have limited proliferative capacity when cultured in vitro. However, like HSCs and LSECs, in vitro culture can notably influence liver-specific function of hepatocytes with extensive efforts taken by scientists to extend the phenotype of hepatocytes for drug discovery, toxicity, drug metabolism, drug-drug interactions, and tissue engineering purposes.
Mosaic Cell Sciences' primary human hepatocytes form bile canalicular structures when cultured on type I collagen coated plates in William's E medium.
After isolation of hepatocytes, plating of the cells on plastic 2D substrates leads to the rapid loss of albumin secretion, cytochrome P450 enzymes (CYP), polarity of the cells, and eventually cell viability. Seeding hepatocytes on culture vessels coated with extracellular matrix proteins native to the liver (Collagen I-IV) can extend the time prior dedifferentiating from hours to days.(3,26) However, long-term culture of hepatocytes remained elusive until Dunn and colleagues sandwiched hepatocytes between type I collagen gels.1 The culture between collagen matrix recreates the morphology of hepatocytes within native tissue while prolonging the production of albumin, bile canalicular formation and production of bile acids.(1–3) Scientists studying drug metabolism and clearance have leveraged sandwich cultures with various substrate compositions (collagen, Matrigel, decellularized ECM from liver) (2–7) to determine which method stabilizes hepatocyte CYP enzyme function and polarity to study basolateral and canalicular expression of integral transporters (BSEP, Mrp2/3, OATP1A4) for xenobiotic metabolism.(8)
In addition to considering the substratum on which to culture hepatocytes, investigation into the role of various media additives and formulations were undertaken to inhibit hepatocyte dedifferntiation . Studies highlight the importance of dexamethasone, insulin, and a subset of amino acids to delay dedifferentiation and increase viability of hepatocyte cultures. The glucocorticoid, dexamethasone, promotes gap junction formation and functional expression of CYP3A enzyme and transport proteins when supplemented in media at concentrations above 100 nM.(8–10) Inclusion of insulin and amino acids into media have shown hepatotrophic effects, stimulating albumin production, promoting cell adhesion to substrates, and maintaining glucose transport of in vitro hepatocytes. (11–13) Multiple formulations of media have been tested to determine which supports hepatocyte function for cytochrome P450 activity on the scale of days to weeks. William’s E medium, DMEM, modified Chee’s medium, and hepatocyte medium are commercially availably formulations that have shown similar efficacy in maintaining CYP activity.(14) However, different media formulations may be better suited when creating increasingly complex in vitro models, such as spheroid models.(14,16,18) Mosaic Cell Sciences’ experts in primary human hepatocyte (PHH) isolation and culture recommend William’s E medium when planning experiments for studies utilizing 2D morphology that require 7 days or less of culture.
While most strategies focus on extending and maintaining hepatocyte morphology and liver-specific function, capturing the zonation is another challenge faced during in vitro culture of hepatocytes. Within the liver sinusoid, hepatocytes display unique expression profiles and functions across the three zones. Periportal hepatocytes (Zone 1) are highly perfused with nutrients and high oxygen concentrations, leading to functions in oxidative metabolism, production of cholesterol, and bile secretion.(15) Zone 3 hepatocytes (perivenous or pericentral) experience hypoxic conditions and are integral for detoxification, metabolism of drugs (requiring CYP enzymes and uptake transporters), lipogenesis, and glutamine formation.(15) Incubating hepatocytes at 5% oxygen during in vitro culture can improve dedifferentiation of hepatocytes, decrease reactive oxygen species formation, and inhibit DNA damage. (17) Furthermore, introduction of flow and creation of an oxygen gradient within a bioreactor appears to recreate the necessary conditions for zonal stratification of hepatocytes.(19,20) Hepatocytes experience fluid flow in vivo and natively form a production line that allows for coordinated function where metabolites from periportal hepatocytes permit additional modification and utilization by pericentral hepatocytes. Recapitulating zonal phenotypes, fluid flow, and oxygen tension offers superior models to study detoxification and drug metabolism using in vitro systems.(21)
Preliminary approaches to improve liver-specific function of hepatocytes recognized the need of endogenous paracrine factors to maintain a physiologically relevant phenotype.(22) Co-culture studies of hepatocytes with fibroblasts, liver sinusoidal endothelial cells, or both cell populations extend the function of of hepatocyte phenotype to 3 weeks when using micropatterned surfaces.(23) Further studies confirm that paracrine factors secreted by hepatocytes and NPCs, but not cell-cell interactions, are sufficient to lengthen liver-specific function of both hepatocytes and NPCs.(22–24)
The development of 3D spheroid models composed of solely of PHHs or PHHs plus NPCs can sustain the functional phenotype PHHs and NPCs during long-term experiments, as well.(18,25–28) Landy and colleagues first discovered the self-aggregating potential of hepatocytes, termed spheroids, in 1985 using cells from rat organs.(29) Leveraging additional strategies by either hanging drop culture, seeding in non-adhesive wells or dishes, rotational culture, microwells, or adding ECM and biomaterials, PHHs form spheroids either as homogenous or heterogenous cultures (with NPCs).(18) Generation of increasingly complex models of the liver allows for various applications that include drug discovery, tissue engineering for liver transplantation or disease modeling, ADME (absorption, distribution, metabolism, excretion), drug-drug interactions, drug-induced liver injury, and multi-organ/tissue systems.(18,25,26,28–30)
To recapitulate in vivo physiology of the liver in vitro, engineers and scientist generate systems with increasingly complex biology, cell composition, and physiological functions. Microphsyiological systems (MPS) allow for scientists to mimic the physiology of fluid flow, cell-cell interactions, and liver-specific functions (such as albumin secretion and xenobiotic metabolism). Microfluidic systems and liver-on-a-chip technologies have been utilized to study novel therapeutics for liver disease, ADME, hepatotoxicity, and disease modeling.(18) While extending liver-specific function to the scale of weeks, these systems have the added benefit of controlling/manipulating the interaction of cellular populations (Hepatocytes, HSCs, LSECs, etc.) and fluid flow to create high-fidelity models. Furthermore, these systems can be run in parallel for high-throughput screening and may be combined into large physiologic systems by integrating other organ and tissues models (intestine, skin, pancreas, heart, etc.).(31,32) Generation of these systems requires extensive knowledge in micromolding, biofabrication, and tissue engineering disciplines, which commercialization has begun to simplify.
Bioprinting of 3D livers provides further promise to create fully functional organs with the hope of avoiding liver transplantation.(33) Organovo, a pioneer in 3D bioprinting,(34) created a 3D bioprinted liver from PHHs and NPCs that can recapitulate native liver function, while providing translational insights into toxicity, drug metabolism, fibrogenesis, and metabolic dysfunction-associated steatohepatitis (MASH). (35–37) Moreover, Mosaic Cell Sciences’ sister company, Viscient Biosciences, harnessed the capability of the 3D bioprinted liver to investigate the role of individual NPC populations (LSECs, HSCs, and KCs) to induce a MASH phenotype. NPCs from MASH donors can induce ECM deposition, and steatosis without requiring the induction of disease with palmitic acid.(38) Harnessing the power of primary human hepatocytes, showed better fidelity capturing the disease phenotype, showing the power of the 3D bioprinted liver.
Learn more about how Mosaic Cell Sciences’ inventory of primary human liver cells (hepatocytes, Kupffer Cells, Hepatic Stellate Cells, and Liver Endothelial Cells) can empower your discoveries by contacting our Technical Sales Specialist, Nick Trompeter, Ph.D. ( ntrompeter@mosaiccellsci.com ). Our inventory of cells from both healthy and MASH donors, plus custom liver cell isolation services can support all of your liver programs and safety testing.
References
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