Microfluidic Environment for High Density Hepatocyte Culture

by: M. Zhang, P. Lee, P. Hung, T. Johnson, L. Lee, M. Mofrad

When hepatocytes are isolated from tissue and grown in monolayer culture, they experience rapid loss of liver-specific function. Current methods used to improve in vitro hepatocyte culture include the growth of hepatocytes on extracellular matrix treated surfaces or the formation of spheroid hepatocyte aggregates. Although these hepatocyte aggregates maintain long term hepatocyte functions in vitro more effectively than monolayer counterparts, recent microfluidic technologies offer many advantageous features that allow better control of cell culture microenvironments and in turn hepatocyte behavior. This paper describes a bioreactor with a microfluidic environment that mimics physiological liver mass transport to allow hepatocytes to be cultured in high-density arrays and maintained in a tissue-like microarchitecture of extensive cell-cell contact in close contact with nutrient circulation. The microfluidic environment also enables hepatocytes to be cultured at a high density without nutrient limitation for over one week by maintaining a continuous flow of medium that diffuses to the cells across a porous barrier. The microfluidic cell culture array device consists of a cell culture area and a nutrient flow channel separated by a microfluidic perfusion barrier that localizes and concentrates cell within the cell culture area while allowing diffusion of nutrients from the nutrient flow channel to the cells. The cells used in the device were human hepatoma HepG2/C3A cells, which have a protein synthesis profile that closely resembles that of native liver cells and have improved albumin production compared to the HepG2 cell line.

Continuous flow of nutrients such as glucose in the bioreactor was modeled using finite element analysis software and validated by the survival and proliferation of the cells in culture. Using Trypan Blue and Live/Dead fluorescence assay, 80% viability of hepatocytes cultured in the microfluidic device was observed after 1 week of incubation. By day 8, cells exhibited liver tissue morphology of dense packing, cuboidal geometry, and indistinguishable fused membranes. In comparison, cells grown under control conditions in standard tissue culture 12-well plates appeared strongly attached to the surface and more spread with distinct instead of fused cell membranes. Cells grown in the device maintained a cell density that was 3 times higher than that of monolayer culture and results from quantitative dot-blot assays to assess hepatocyte function via albumin secretion revealed that after 4 days of culture HepG2/C3A cells were producing 3 times more albumin per cell compared with cell grown in the 12-well plates. Thus, a microfluidic environment with mass transport conditions that is physiologically similar to that experienced by hepatocytes in vivo can affect cell behavior by inducing hepatocytes to grow in a natural liver configuration with a high density of hepatocytes and maintain liver tissue specific function.

I came across this paper while searching for recent papers involving PDMS and hepatocytes in an attempt to do some background research on my group’s research idea and found the idea of microscale bioreactors to be very interesting. This technology overcomes the limitation of loss of function observed in hepatocytes grown in monolayer culture and allows a more precise control of the microenvironment experienced by the cells in the culture. This opens up many possibilities in research to determine which physiological conditions are most important to maintain differentiation and function of hepatocytes and many other cell types as well. This technology can also be applied in drug metabolism screening in a more efficient manner at the microscale level so would be very useful for testing of many pharmaceutical products.