Microfluidic environment for high density hepatocyte culture

Introduction:

Upon isolation, monolayer hepatocytes lost liver-specific function rapidly; this limits their application in drug screening and as a dependable cell source for bioartificial liver support. Hepatocytes function is influenced by the microenvironment such as flow rate, cell density, media composition, geometry configuration, extracellular matrix, and cell-cell contacts. Microfludic technology has recently offered the advantages in controlling fluid flows and in improving cell culture microenvironments.

Summary:

The objective of the study is to “controllably maintain cells in a “tissue-like” state by using a microfluidics mass transport design that mimics the tissue microvasculature.” The fluidic device, in short, was a PDMS based device containing one inlet, one outlet, and one cell loading area (Figure 1).

Fig. 1 (a) Microfluidic culture unit design. The microfluidic unit consisted of three parts: a 150 μm wide by 440 μm long cell culture area (blue), a microfluidic perfusion barrier (gray), and a medium flow channel (red). Cells were introduced from the top port, and localized into the cell culture area. The perfusion barrier consisted of a grid of channels 5 μm wide and 2 μm tall, serving to prevent cells from passing through, while enabling nutrient exchange from the flow channel on the opposite side.Inset shows SEM micrograph of perfusion channels. Scale bar represents 5 μm. (b) Glucose concentration at the center of cell culture mass was determined using finite element analysis software and plotted against medium flow rate. (c) Surface plot of glucose concentration throughout one culture unit with an inlet glucose concentration of 25 μmM. At a flow rate of 10μl/day, glucose concentration at the center of the cell mass after consumption was theoretically determined to be 22 mM.

The hepatocytes (HepG2/G3A) cells were seeded in the device (incubated on a 45 degree slope rack to maintain continous gravity driven flow) and in the standards tissue culture 12-well plates (control). The flow rate for the device was measured by collecting the outlet media (10.3±1.6 μl/day/well).

Characterization of different cell cultures:

Cell viability and proliferation were determined daily using Trypan Blue and Live/Dead fluoresence assay. The cell viability in the device after one week is 80%. The cells in the control appeared strongly attached and more spread, with distinct membrane The cells in the device exhibited a “tissue-like” morphology (i.e. dense packing, cuboidal geomery, and indistinguishable fused membrane (Figure 3).

Fig. 2 HepG2/C3A human hepatoma cell growth inside the microfluidic cell culture array. Scale bar represents 100 μm

Fig. 3 High density cell culture. Phase contrast micrographs of confluent cell morphology on a standard 12-well tissue culture plate (a) and inside one sinusoid of the microfluidic cell culture array (b) after 8 days of culture. Images are at equal magnification, with scale bar representing 50 μm. (c) Cells were seeded at 10^{5 cells/well for controls (filled circles) and about 500 cells/well for devices (filled triangles) and measured for cell density over 1 week.}

Liver-specific synthetic function of albumin was examined via a quantitative dot-blot assay (a method using immunostaining on nitrocellulose membrane). The result (Figure 4) shows that the hepatocytes on the device were able to produce three times albumin on per-cell basis compared to the control culture.

Fig. 4 Albumin secretion was characterized in TC dish and the microfluidic device after 4 days of culture. Albumin secretion from cells cultured in the microfluidic device increased threefold compared with on a dish.

Comment:

Overall, this work is impressive because the design took into account the physiological relevant mass transport condition; therefore, sustaining a high cell density (>2000 cells/mm2) without nutrient depletion over one week is possible. Additionally, the design enables the cells to assembly in close proximity thus might enhance cell-cell contacts normally found in native liver tissue. Furthermore, this work also shows how the mass transport can affect maintenance of differentiated phenotype and biological function of hepatocytes. Finally, the configuration (i.e. flow rate, geometry) also enhanced the production of albumin in the cells by threefold comparing to a 12-well plate.

The researchers suggest that the close proximity assembly of hepatocytes cell in the device might enhance cell-cell contacts such as tight junctions and desmosome, which normally found in native liver tissue. However, no chemical assay (i.e. Cadherin immunostaining assay or Cadherin gene expression assay) was used to test their suggestion. Meanwhile the end goal is to use the device in drug screening, drug metabolism, fundamental physiology studies; the detoxification activity mediated by cytochrome p450 (via CYP2B6, CYP2E1, CYP1A2 gene expression) of the hepatocytes cells was not examined.

Reference:
Mimi Y. Zhang & Philip J. Lee & Paul J. Hung &Terry Johnson & Luke P. Lee & Mohammed R. K. Mofrad, “Microfluidic Environment for High Density Hepatocyte Culture”, Biomed Microdevices (2008) 10:117–121

Link: http://biomechanics.berkeley.edu/assets/papers/LiverChip_Mimi2007.pdf