Multi-cellular 3D human primary liver cell culture elevates metabolic activity under fluidic flow

Abstract

We have developed a low-cost liver cell culture device that creates fluidic flow over a 3D primary liver cell culture that consists of multiple liver cell types, including hepatocytes and non-parenchymal cells (fibroblasts, stellate cells, and Kupffer cells). We tested the performance of the cell culture under fluidic flow for 14 days, finding that hepatocytes produced albumin and urea at elevated levels compared to static cultures. Hepatocytes also responded with induction of P450 (CYP1A1 and CYP3A4) enzyme activity when challenged with P450 inducers, although we did not find significant differences between static and fluidic cultures. Non-parenchymal cells were similarly responsive, producing interleukin 8 (IL-8) when challenged with 10 μM bacterial lipoprotein (LPS). To create the fluidic flow in an inexpensive manner, we used a rocking platform that tilts the cell culture devices at angles between ±12°, resulting in a periodically changing hydrostatic pressure drop between reservoirs and the accompanying periodically changing fluidic flow (average flow rate of 650 μL min(-1), and a maximum shear stress of 0.64 dyne cm(-2)). The increase in metabolic activity is consistent with the hypothesis that, similar to unidirectional fluidic flow, primary liver cell cultures increase their metabolic activity in response to fluidic flow periodically changes direction. Since fluidic flow that changes direction periodically drastically changes the behavior of other cells types that are shear sensitive, our findings support the theory that the increase in hepatic metabolic activity associated with fluidic flow is either activated by mechanisms other than shear sensing (for example increased opportunities for gas and metabolite exchange), or that it follows a shear sensing mechanism that does not depend on the direction of shear. Our mode of device operation allows us to evaluate drugs under fluidic cell culture conditions and at low device manufacturing and operation costs.

Figure 1

schematic of device assembly and operation. the system consists of two silicone gaskets with 6 mm holes that serve as cell culture chambers (a). the silicone gaskets are sandwiched between two 3d printed plastic pieces that both provide fluidic channels. medium that flows through the channels reach the cell culture from the top and the bottom (b–c). the device is placed on a rocker platform to create gravity-driven, periodically changing bidirectional medium flow.

Figure 2

fluorescence microscopy images of 3d cell cultures with scaffold and liver cells that were recovered from the cell culture device after 14 days of culture. hepatocytes were immunostained against albumin (yellow) and are shown at magnifications of 4x (a), and 10x (b). the 3d scaffold is visible as a grid in blue. the cell cultures appear tissue-like with a high cell density. stellate cells and kupffer cells were stained for cd38 (green, shown in c) and cd68 (green, shown in d) using separate samples. nuclei were stained with dapi.

Figure 3

rates of albumin (a) and urea (b) production throughout the 14-day culture period. values are means ± standard deviations, n = 3 with each separate experiment consisting of six technical replicates. significant differences (p>0.05) between static and dynamic cell culture conditions are indicated with an asterisk.

Figure 4

activity of cyp 1a1 (a) and cyp 3a4 enzymes (b) throughout the 14-day cell culture period. values are shown as percent increase compared to uninduced cultures. values are means ± standard deviations, n = 3 with each separate experiment consisting of two technical replicates. significant differences (p>0.05) between static and dynamic cell culture conditions are indicated with an asterisk.

Figure 5

concentrations of il-8 in response to a 24-hour challenge with bacterial lipopolysaccharide (lps). values are means ± standard deviations, n = 3 with each separate experiment consisting of two technical replicates. significant differences (p>0.05) between static and dynamic cell culture conditions are indicated with an asterisk.

Figure 6

results of theoretical simulation of the fluidic flow in the cell culture device. the flow velocity depends on the tilting angle (a) and oscillates (b). shear stress is highest in the middle of the cell culture chamber (c). the letters a–h in (c) refer to tilting times and the corresponding flow rates marked in (b).