Oxygen Gradient Induced in Microfluidic Chips Can Be Used as a Model for Liver Zonation

Abstract

Availability of oxygen plays an important role in tissue organization and cell-type specific metabolism. It is, however, difficult to analyze hypoxia-related adaptations in vitro because of inherent limitations of experimental model systems. In this study, we establish a microfluidic tissue culture protocol to generate hypoxic gradients in vitro, mimicking the conditions found in the liver acinus. To accomplish this, four microfluidic chips, each containing two chambers, were serially connected to obtain eight interconnected chambers. HepG2 hepatocytes were uniformly seeded in each chamber and cultivated under a constant media flow of 50 µL/h for 72 h. HepG2 oxygen consumption under flowing media conditions established a normoxia to hypoxia gradient within the chambers, which was confirmed by oxygen sensors located at the inlet and outlet of the connected microfluidic chips. Expression of Hif1α mRNA and protein was used to indicate hypoxic conditions in the cells and albumin mRNA and protein expression served as a marker for liver acinus-like zonation. Oxygen measurements performed over 72 h showed a change from 17.5% to 15.9% of atmospheric oxygen, which corresponded with a 9.2% oxygen reduction in the medium between chamber1 (inlet) and 8 (outlet) in the connected microfluidic chips after 72 h. Analysis of Hif1α expression and nuclear translocation in HepG2 cells additionally confirmed the hypoxic gradient from chamber1 to chamber8. Moreover, albumin mRNA and protein levels were significantly reduced from chamber1 to chamber8, indicating liver acinus zonation along the oxygen gradient. Taken together, microfluidic cultivation in interconnected chambers provides a new model for analyzing cells in a normoxic to hypoxic gradient in vitro. By using a well-characterized cancer cell line as a homogenous hepatocyte population, we also demonstrate that an approximate 10% reduction in oxygen triggers translocation of Hif1α to the nucleus and reduces albumin production.

Keywords: CoCl2; DFX; HepG2; Hif1α; albumin; hypoxic condition; in vitro; liver; microfluidic chips; oxygen.

Figure 1

Serial connection of MCs and oxygen measurement. Schematic drawing of MCs connected by tubes with oxygen-sealing fitted adapters to obtain eight interconnected microfluidic tissue culture chambers (A). Oxygen sensors (Presens OXY-1 ST Fiber optic trace oxygen meter connected to O₂ SensorPlug and placed in a sensor MC) were inserted before the first inlet and after the final outlet of the connected MCs; (B). HepG2 cells were seeded in one MC and treated with 250 µM DFX or 100 µM CoCl₂ (C).

Figure 2

Oxygen levels in the medium before and after passage through 4 serially connected MCs. Oxygen percentage measured in the media before chamber1 (inlet) and after chamber2 (1 MC), chamber4 (2 MC), chamber6 (3 MC), and chamber8 (4 MC, outlet) of connected MCs during 72 h of cultivation with constant media flow of 50 µL/h (error bars are not visible for some of the points due to a small standard deviation).

Figure 3

Albumin and Hif1α immunofluorescence co-staining. IF staining of HepG2 cells was completed in all eight chambers of connected MCs after 72 h of cultivation with a media flow of 50 µL/h. Albumin is visualized in green and Hif1α is displayed in red. Scale bar 50 µm (A). Higher magnification of images for comparison of albumin (in green) and Hif1α (in red) expressed in HepG2 cells in chamber1 and 8 (as in the experiment above). Scale bar 20 µm (B).

Figure 3

Albumin and Hif1α immunofluorescence co-staining. IF staining of HepG2 cells was completed in all eight chambers of connected MCs after 72 h of cultivation with a media flow of 50 µL/h. Albumin is visualized in green and Hif1α is displayed in red. Scale bar 50 µm (A). Higher magnification of images for comparison of albumin (in green) and Hif1α (in red) expressed in HepG2 cells in chamber1 and 8 (as in the experiment above). Scale bar 20 µm (B).

Figure 4

Quantification of albumin and Hif1α immunofluorescence staining and nuclear translocation. Albumin and Hif1α fluorescence in HepG2 cells in the connected MCs were quantified in immunofluorescence images from three independent experiments using ImageJ. Graphs were generated using GraphPad Prism. Staining intensities of Hif1α in the nucleus and cytoplasm show increased translocation of Hif1α from cytoplasm to the nucleus when comparing chamber1 and chamber8 with oxygen depletion (p = 0.02). Hif1α fluorescence intensity is normalized to 100% total cellular fluorescence (nucleus and cytoplasm) (A). The percentage fluorescence intensity of albumin in HepG2 cells in chamber1 and chamber8 shows downregulation of albumin expression from chamber1 to 8 (p = 0.02) (B).

Figure 5

Fluorescence in situ hybridization (FISH) for ALBUMIN and HIF1A mRNA. ALBUMIN and HIF1A mRNA expressed in HepG2 cells in chamber1 and chamber8 of connected MCs. Scale bar 50 µm.

Figure 6

Quantification of ALBUMIN and HIF1A mRNA fluorescence signals of FISH images. Relative fluorescence intensity of HIF1A (A) and ALBUMIN (B) in situ hybridization signals in chamber1 and chamber8 along the oxygen gradient in the connected MCs. The levels of HIF1A mRNA increase from chamber1 to 8 (p = 0.02) (A); the levels of ALBUMIN mRNA decrease from chamber1 to 8 (p = 0.04) (B).