Customizable Microfluidic Origami Liver-on-a-Chip (oLOC)

Abstract

The design and manufacture of an origami-based liver-on-a-chip device are presented, together with demonstrations of the chip's effectiveness at recapitulating some of the liver's key in vivo architecture, physical microenvironment, and functions. Laser-cut layers of polyimide tape are folded together with polycarbonate nanoporous membranes to create a stack of three adjacent flow chambers separated by the membranes. Endothelial cells are seeded in the upper and lower flow chambers to simulate sinusoids, and hepatocytes are seeded in the middle flow chamber. Nutrients and metabolites flow through the simulated sinusoids and diffuse between the vascular pathways and the hepatocyte layers, mimicking physiological microcirculation. Studies of cell viability, metabolic functions, and hepatotoxicity of pharmaceutical compounds show that the endothelialized liver-on-a-chip model is conducive to maintaining hepatocyte functions and evaluation of the hepatotoxicity of drugs. Our unique origami approach speeds chip development and optimization, effectively simplifying the laboratory-scale fabrication of on-chip models of human tissues without necessarily reducing their structural and functional sophistication.

Keywords: biofabrication; organ-on-a-chip; tissue modeling; vascularization.

Figure 1.

(a) 3D architecture of liver, liver lobule, and sinusoid structure; (b) conceptual schematic cross-section of an oLOC, showing a semipermeable membrane that has been folded, seeded on opposite sides with hepatocyte and endothelial cell monolayers, and capped with top and bottom plates to define upper and lower flow channels (vascular chambers) that mimic sinusoids and a central static chamber that contains hepatocyte monolayers on the upper and lower membrane surfaces; (c) schematic diagram of the implemented oLOC, showing membrane layers (black and white speckle), flowing medium pathways (red), static medium pathway (blue), hepatocytes (purple), endothelial cells (maroon), and how it simulates liver structure with functions of flow (red arrows) and transport via diffusion (white arrows).

Figure 2.

(a) Schematic diagram of the laser-patterned polyimide tape and nanoporous membrane oriented for cross-folding; (b) exploded schematic diagram of the folded structure of the oLOC (gray, red, dark blue, and yellow) and its top and bottom acrylic caps (light blue); (c) schematic and (d) cross-sectional cutaway diagrams of the completed oLOC; (e - j) photographs showing the fabrication process for the Generation 3 devices, including (e) hole punching and trimming to pattern the nanoporous membrane, (f) folding the nanoporous membrane between chamber panels to form origami sub-units, (g) layering flow panels to form additional sub-units, (h) stacking the origami sub-units on top of the solid acrylic base plate to form the device’s flow structure, (i) capping the stack with an acrylic top plate (laser-cut with four inlet/outlet ports) and adhering PVC tubing into ports, and (j) final Generation 3 device structure after assembly; (k) flow pattern of Generation 3 device shown by flowing yellow dye through the simulated sinusoids and blue dye through the hepatocyte chambers; (l) plot of concentration of trypan blue in the flow exiting the middle flow path as a function of inverse flow rate when a 0.1% solution of trypan blue in deionized water is flowed through a chip’s upper and lower flow paths and deionized water is flowed through its middle flow path with no pressure difference between flow paths.

Figure 3.

(a) Viability of primary hepatocytes and HUVECs assessed using calcein AM (green) and EthD-1 (red) staining on day 7 of culture; (b) quantified metabolic activities of primary hepatocytes and HUVECs at different periods of culture; (c) F-actin and nuclei staining of primary hepatocytes and HUVECs at day 7 of culture; (d) immunostaining of primary hepatocytes for CYP3A and HUVECs for CD31 and VE-cadherin at day 7 of culture.

Figure 4.

(a) Cytotoxicity of acetaminophen on primary hepatocytes shown via cell viability assay using calcein AM (green) and EthD-1 (red) staining after 48 h of acetaminophen treatment; (b) quantified cell metabolic activity of hepatocytes at different concentrations of acetaminophen; (c) quantified 48-h urea production by hepatocytes at different concentrations of acetaminophen; and (d) quantified 48-h albumin production by hepatocytes at different concentrations of acetaminophen. Asterisk represents significant difference between the dynamic culture group and the static culture group using two-way ANOVA (p≤0.05).

Figure 5.

(a) Cytotoxicity of tamoxifen on HepG2 cells shown via cell viability assay using Calcein AM (green) and EthD-1 (red) staining after 48 h of tamoxifen treatment; (b) quantified cell metabolic activity of HepG2 cells at different concentrations of tamoxifen; (c) quantified 48-h urea production by HepG2 cells at different concentrations of tamoxifen; and (d) quantified 48-h albumin production by HepG2 cells at different concentrations of tamoxifen. Asterisk represents a significant difference between the dynamic culture group and the static culture group using two-way ANOVA (p≤0.05).