Progressive hypoxia‐on‐a‐chip: An in vitro oxygen gradient model for capturing the effects of hypoxia on primary hepatocytes in health and disease

Abstract

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo.Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3-6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply.The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia-inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient.This progressive hypoxia-on-a-chip platform can be used to study the role of oxygen and hypoxia-associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen-limited conditions. K E Y W O R D S hepatocytes, HIF, hypoxia, microfluidics, oxygen gradient, ROS

A microfluidic patterned model of non-alcoholic fatty liver disease: applications to disease progression and zonation

“…We have previously performed hypoxia studies, where we generated chemically induced oxygen gradients of 6.9-0.3%. 20 Here, we generated a more physiologically relevant oxygen gradient of 11.2-6.9% in a similar fashion (Fig. 4).…”

“…An oxygen gradient was generated using sodium sulfite and cobalt nitrate, a commonly employed approach for oxygen removal and depletion. 20,52 The oxygen scavenger solution was prepared with 0.13% (w/v) sodium sulfite and 13 μM cobalt nitrate in basal experimental medium. 20-24 hours after cell seeding, media with and without oxygen scavengers were loaded into 3 mL syringes individually and connected to the gradient generator of the microfluidic device.…”

“…19 We have previously used this platform to recapitulate metabolic liver zonation in both rat and human primary hepatocytes using hormones, inducers and oxygen gradients. 19,20 This approach can be used to study either a) the progression of NAFLD spectrum or b) the spatial physiological heterogeneity, i.e. zonal lipid accumulation 21 and other heterogeneity 22 observed during the disease, on a single integrated microfluidic chip.…”

Tissue engineering of the biliary tract and modelling of cholestatic disorders

“…79 For tissues with different compartments, such as a lumen or a vascular bed, each compartment can be maintained in different conditions; luminal flow can be modelled through perfusion with media at different rates, 80 while diffusion of nutrients or oxygen can be captured using chemical gradients. 81 Consequently, tissue engineering enables the transition from single cell, static culture platforms to dynamic model systems, providing the complexity required to reproduce the salient aspects of disease in vitro. Conversely, tissue engineered platforms are simpler than in vivo organs and animal models.…”

Metabolic Switching of Tumor Cells under Hypoxic Conditions in a Tumor-on-a-chip Model

“…In a different approach, and by taking advantage of the gas permeability of PDMS or similar silicone materials, dedicated channels perfused with pre-conditioned fluids were placed on the top [15,[32][33][34] or next to culture chambers [35,36], allowing a quick switch of the oxygen tension in a 3D tumor model [37], to create oxygen gradients in cellular models [38,39] or to simply supply enough oxygen to hepatocytes [15]. Along a similar line, solutions supplemented with an oxygen-scavenging chemical were utilized to create hypoxic conditions [40] or oxygen gradients [41][42][43][44][45], in combination in one report with a thick oxygen-impermeable material (PMMA or polymethylmethacrylate) sheet placed on the top of the device [46].…”