Abstract
The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-β1. Hepatocytes are not generally thought of as significant producers of GFs–this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function.
Highlights
To harness cell secreted signals, we fabricated a simple microsystem comprised of a cell culture microchamber 3 mm in length, 5 mm in width and 75 μm in height connected by transport channels (600 μm in width and 75 μm in height) to reservoirs at the inlet and outlet holding 500 μlof media (Fig. 1a,b)
Cultivation of primary hepatocytes began over four decades ago[54,55]
Liver biologists identified a number of strategies that recapitulate aspects of the in vivo microenvironment to rescue hepatic phenotype in culture
Summary
In microscale cultures hepatocytes expressed high levels of E-cadherin, remained polarized, and synthesized albumin at elevated levels over the course of three weeks Such phenotype maintenance in hepatocytes requires collagen gel (or matrigel) sandwich cultures[6,8,10], co-cultures with stromal cells[5,11,12,13] or spheroid cultures[9,14] and has not, to the best of our knowledge, been observed previously in hepatocytes cultured as a monolayer on simple collagen-coated surfaces. In a series of experiments, we demonstrated that phenotype enhancement is the result of endogenous hepato-inductive growth factors (GFs) accumulating inside the microchambers These observations are interesting because hepatocytes are epithelial cells and are not known for secretion of GFs in vitro. Efforts are under way in our lab to develop low-volume multi-well plates that mimic traditional biology cultureware while capturing endogenous signals produced by the hepatocytes
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