An important number of healthy and diseased tissues shows spatial variations in their metabolic capacities across the tissue. The liver is a prime example of such heterogeneity where the gradual changes in various metabolic activities across the liver sinusoid is termed as “zonation” of the liver. Here, we introduce the Metabolic Patterning on a Chip (MPOC) platform capable of dynamically creating metabolic patterns across the length of a microchamber of liver tissue via actively enforced gradients of various metabolic modulators such as hormones and inducers. Using this platform, we were able to create continuous liver tissues of both rat and human origin with gradually changing metabolic activities. The gradients we have created in nitrogen, carbohydrate and xenobiotic metabolisms recapitulated an in vivo like zonation and zonal toxic response. Beyond its application in recapitulation of liver zonation in vitro as we demonstrate here, the MPOC platform can be used and expanded for a variety of purposes including better understanding of heterogeneity in many different tissues during developmental and adult stages.

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