Investigation of the hepatic respiration and liver zonation on rat hepatocytes using an integrated oxygen biosensor in a microscale device

Abstract

The development of an in vitro functional liver zonation model is a major issue to reproduce physiological liver features. Oxygen concentration is one of the potential explanations of a primary regulating factor of zonation. In this frame, we investigated the oxygen gradient inside a microfluidic device containing rat hepatocyte cultures. The device integrated a platinum (Pt) (II) octaethylporphyrin sensor, allowing a 2D mapping of the oxygen concentration. After 3 hr adhesion of the hepatocytes, the sensor indicated an intense oxygen depletion, leading to an oxygen shortage in the center of the device. After a 30 min perfusion of the culture medium, we monitored the formation of the oxygen gradient along the culture due to cellular respiration. The profile of the oxygen gradient was modulated and controlled by increasing either the perfusion flow rate or the device thickness. In addition, the oxygen gradient was time dependent as far as it decreased with the time of culture. Perivenous and periportal liver patterns were characterized by the immunostaining of the hepatic markers. We put in evidence a spatio temporal hepatic organization. We observed the overexpression since 24 hr of perfusion of the APC and PCK1 proteins upstream in the oxygen‐rich area of the device. The overexpression of GS, GCK, CYP1A, and HIFα proteins were observed downstream in the oxygen‐poor area. Then, CYP3A2 and β‐catenin spatial reorganization was achieved after 48 hr of culture. The results presented a partial zonation‐like pattern that was superimposed with an oxygen gradient profile.

Characterization of liver zonation‐like transcriptomic patterns in HLCs derived from hiPSCs in a microfluidic biochip environment

“…It appears that cells in this region have already sufficiently depleted oxygen levels supplied from the medium, generating perivenous oxygen tension and therefore enabling typical signaling cascades. Indeed, measurement of oxygen levels in a similar setup have shown to rapidly decrease oxygen levels 27 . Hence, it is plausible to consider the central area in our experiment to have low, but physiological oxygen levels.…”

“…Pericellular oxygen levels have been shown to quickly deplete, reaching values that do not promote healthy cell cultures due to the limitation of cellular respiration. 27 Therefore, a device shorter than the one presented might yield a higher spatial resolution regarding cellular patterns when sampled according to our classification and might generally represent a better basis for an efficient maturation of HLCs toward hepatocytes.…”

“…As shown in the literature by both experimental data and numerical models, microfluidic devices can produce oxygen gradients along the cell culture chamber through the combination of the continuous injection and consumption of oxygen by the cells. 14,15,27,49 Nevertheless, the exact geometry of the microfluidic channel requires special attention. Pericellular oxygen levels have been shown to quickly deplete, reaching values that do not promote healthy cell cultures due to the limitation of cellular respiration.…”

“…While it has been demonstrated that the biochip format allows for the formation of an oxygen gradient, the reproduction of physiologically‐relevant values is usually difficult . 27 In the present study, the culture conditions have been optimized as previously described, 17 which allows for the mid to long term culture of the cells in the biochip. In those conditions, we investigated whether liver‐specific patterning can be achieved in the biochip, acting as an acinar model in‐vitro.…”

“…While one of the central hypotheses regarding functional zonation, as it is observed along the porto‐central axis of liver lobules, suggests that the oxygen gradient along the liver sinusoid could be the responsible extracellular stimulus, 47,48 limitation of oxygen availability below a certain threshold can be expected to negatively affect cellular metabolism and thus functionality. As shown in the literature by both experimental data and numerical models, microfluidic devices can produce oxygen gradients along the cell culture chamber through the combination of the continuous injection and consumption of oxygen by the cells 14,15,27,49 . Nevertheless, the exact geometry of the microfluidic channel requires special attention.…”