Study of the Crosstalk Between Hepatocytes and Endothelial Cells Using a Novel Multicompartmental Bioreactor: A Comparison Between Connected Cultures

Maria Angela Guzzardi, Federico Vozzi, Arti Devi Ahluwalia. Study of the Crosstalk Between Hepatocytes and Endothelial Cells Using a Novel Multicompartmental Bioreactor: A Comparison Between Connected Cultures and Cocultures. Tissue Engineering Part A. November 2009, 15(11): 3635-3644. doi:10.1089/ten.tea.2008.0695.

http://www.liebertonline.com/doi/full/10.1089/ten.tea.2008.0695

Note: All figure captions from text

Introduction

Cell cultures have been widely used to investigate cell behavior in vitro and cocultures are useful for studying the effects of heterotypic cell contact. In fact, using the coculture technique, liver cells have been found to maintain their liver function in vitro for much longer when cocultured together with endothelial cells. However, these techniques can only probe cell-cell interactions within the same tissue and not cell communication across different tissues within the body. To date, there have been very few studies that have explored cell communication via soluble factors released into the bloodstream in vitro. This study develops a novel multicompartmental bioreactor (MCB) that can mimic blood flow and interactions between different tissues. In particular, they utilize their MCB to investigate the crosstalk between hepatocytes (HepG2) and human umbilical vein endothelial cells (HUVEC). Albumin secretion, urea secretion, and glucose consumption were assayed and taken to be measures of liver function. Nitric oxide secretion was also assayed and taken to be a measure of vasodilation of the endothelium.

Summary

A novel MCB, shown in Figure 1, was fabricated. It is composed of separate chambers, each capable of holding a small well of cells, which are connected by tubes either in parallel or series. The entire apparatus is contained within a plexiglass slide and Teflon frame to preserve sterility, and connected to a mixing chamber and peristaltic pump. The MCB culture is a dynamic culture since flow of media is involved.

FIG. 1. (A) Multicompartmental bioreactor (MCB) cell culture chamber closed by a plexiglass slide and Teflon frame, connected to the reservoir chamber and filled with a dye to illustrate the chambers. (B) The connection scheme for the experimental setup described. The gray chambers were empty of cells. Color images available online at www.liebertonline.com/ten.

In all experiments, an alginate coating was used to protect the HepG2 cells from the shear stress of fluid flow. Shear stress had been shown to damage the cells. The justification provided for this is that in the body hepatocytes are not exposed to blood flow and are separated by a thin capillary wall. However, nutrients and oxygen can still be exchanged through the wall. The alginate coating is meant to mimic the capillary wall such that the hepatocytes are protected from shear stress but can still access nutrients in the media.

Traditional static monocultures and cocultures of HepG2 and HUVEC were prepared to set a baseline for activity. The five day results for albumin and urea secretion and glucose consumption are shown in Figures 5-7. Briefly, when compared to monocultures of either HepG2 or HUVEC, the static cocultures secreted more albumin per cell per day and less urea but consumed less glucose.

FIG. 5. Glucose consumption rate per cell in baseline and coculture studies, n=3.

FIG. 6. (A) Total albumin production in static multiwell cultures. HepG2 in monoculture were seeded at a density of 5×104, while HepG2 in coculture were seeded at a density of 8×104. n=3, *p<0.05 n="">

FIG. 7. (A) Total urea production in static multiwell cultures. HepG2 in monoculture were seeded at a density of 5×10⁴, while HepG2 in coculture were seeded at a density of 8×10⁴. n=3, *p<0.05>B) Urea production in HepG2 and coculture studies calculated as urea released per day per cell, n=3.

Dynamic MCB connected cocultures of HepG2 and HUVEC were then compared to dynamic monocultures and static cocultures. For all dynamic experiments, the time point taken was 1 day. The results for all the different assays are shown in Figures 8-10. Briefly, when compared to dynamic monocultures of HepG2 or static cocultures, the dynamic cocultures secreted more albumin per cell per day and more urea but consumed less glucose.

FIG. 8. Glucose consumption per cell after 24h in static mono- and cocultures and in dynamic mono- and connected cultures. The static data are the results obtained from baseline and coculture studies, in which the first point of the curve, on day 1 is considered. n=3, *p<0.05>

FIG. 9. Albumin release per cell in hepatocyte mono- and connected culture in the MCB, and comparison between dynamic and static conditions, n=3, *p<0.05>p<0.005.

FIG. 10. Urea production per cell in HepG2 dynamic mono- and connected culture in the MCB, and comparison between dynamic and static conditions, n=3, *p<0.05>p<0.005.

From these results the authors conclude that the flow of media simulating blood flow increases protein production, nitrogen metabolism, and nitric oxide production. Furthermore, hepatocytes use glucose as an energy source more efficiently when connected in dynamic culture to endothelial cells.

Critiques

While this is a fairly unique paper with interesting results, there are a few areas that the paper could improve on. First, simply choosing a different time point for evaluating the MCB cultures could have changed the results and therefore also the interpretation of the results. For example, albumin secretion was greater in the static coculture at 1 day, but at 2 days, it was greater in the HepG2 culture. Had the MCB cultures been assessed at 2 days, it may have been more difficult to compare them to the static cultures.

Also, their conclusion that dynamic cocultures used up less glucose than static cocultures and therefore are more efficient is not strongly supported because the actual difference was not statistically significant.

In addition, a different cell density was used from monocultures to cocultures, and the reason for this difference in cell density was not explained. The higher cell density in the cocultures could potentially affect the metabolism of the cells. Also, a substantially different ratio of HUVEC to HepG2 cells (1:10 in static coculture and 3:1 in dynamic coculture) introduces another variable that makes it hard to definitively say that the dynamic nature of the culture is what caused the difference in results.

Finally, the allometric scaling method that they use to determine the conditions for cell culture that best mimic physiological conditions isn’t very convincing. They assert that these conditions reproduce physiological conditions quite well but they don’t have adequate evidence for that assertion. So, while their set up is closer to physiological then the static culture, it still has a long way to go. It would also have been nice if there was a way to distinguish how much of each metabolite came from which type of cell. The authors could have investigated the effect of changing the flow rate as well. There is no discussion of what soluble factor may have caused the effects observed in the paper.