Liver zonation is a fundamental characteristic of hepatocyte spatial heterogeneity, which is challenging to recapitulate in traditional cell cultures. This study presents a novel microfluidic device designed to induce zonation in liver cell cultures by establishing an oxygen gradient using standard laboratory gases. The device consists of two layers; a bottom layer containing a gas channel network that delivers high (cell incubator air, 19% oxygen) and low oxygenated (nitrogen) gases to create three distinct zones within the cell culture chamber in the layer above. Computational simulations and ratiometric oxygen sensing were employed to validate the oxygen gradient, demonstrating that stable oxygen levels were achieved within two hours. Liver zonation was confirmed using immunofluorescence staining, which showed zonated albumin production in HepG2 cells directly correlating with oxygen levels and mimicking in-vivo zonation behavior. This user-friendly device supports studies on liver zonation and related metabolic disease mechanisms in vitro. It can also be utilized for experiments that necessitate precise gas concentration gradients, such as hypoxia-related research areas focused on angiogenesis and cancer development.
The liver is composed of a set of highly dedicated cell types which organize into functional units known as liver lobules. The cells in the liver lobules specialize in essential tasks such as detoxification, bile production, protein synthesis, glucose regulation, and vitamin storage (Arias et al. 2020). Each liver lobule has six subunits (acinuses) where blood capillaries called sinusoids, are arranged radially and culminates at the central vein (Juza & Pauli, 2014), as depicted in Fig. 1. At the periportal end of the liver lobule, the hepatic artery and portal vein converge to supply the cells with blood rich in oxygen and nutrients. As these resources are consumed by the cells, concentration gradients will form along the sinusoids, with the highest concentration near the periportal end and the lowest near the central vein at the pericentral end. Consequently, cells encounter varying concentrations of nutrients, oxygen, and waste products based on their location, driving the phenomena of metabolic zonation within the liver lobule (Tomlinson et al. 2019). Metabolic zonation is a critical physiological feature that allows liver cells to exhibit diverse activities and characteristics depending on their position along the sinusoidal space. This zonation is often overlooked in conventional cell cultures, raising concerns about the accuracy of in-vitro models (Scheidecker et al. 2020)
For modeling the oxygen distribution, the oxygen diffusion coefficient in PDMS was set to 3.25 × 10−9 m2/s (Markov, Lillie, Garbett, & McCawley, 2014), with saturated oxygen concentration applied at the boundaries exposed to the atmosphere, measuring 1.8 mol/m3. The interface between the cell culture medium and PDMS was characterized using a partition coefficient of 10…
