Development of Liver-on-Chip Integrating a Hydroscaffold Mimicking the Liver's Extracellular Matrix

Abstract

The 3Rs guidelines recommend replacing animal testing with alternative models. One of the solutions proposed is organ-on-chip technology in which liver-on-chip is one of the most promising alternatives for drug screening and toxicological assays. The main challenge is to achieve the relevant in vivo-like functionalities of the liver tissue in an optimized cellular microenvironment. Here, we investigated the development of hepatic cells under dynamic conditions inside a 3D hydroscaffold embedded in a microfluidic device. The hydroscaffold is made of hyaluronic acid and composed of liver extracellular matrix components (galactosamine, collagen I/IV) with RGDS (Arg-Gly-Asp-Ser) sites for cell adhesion. The HepG2/C3A cell line was cultured under a flow rate of 10 µL/min for 21 days. After seeding, the cells formed aggregates and proliferated, forming 3D spheroids. The cell viability, functionality, and spheroid integrity were investigated and compared to static cultures. The results showed a 3D aggregate organization of the cells up to large spheroid formations, high viability and albumin production, and an enhancement of HepG2 cell functionalities. Overall, these results highlighted the role of the liver-on-chip model coupled with a hydroscaffold in the enhancement of cell functions and its potential for engineering a relevant liver model for drug screening and disease study.

Keywords: extracellular matrix; hydroscaffold; liver; organ-on-chip; spheroid.

Figure 1

Experimental procedures used for HepG2/C3A cell culture in the biochip and Petri containing the HA-hydroscaffold.

Figure 2

Microfluidic devices and hydroscaffold characterization. (a) microfluidic biochip; (b) microscopic observation of biochip microstructures; (c) biochip containing the hydroscaffold; (d) microscopic observation of dehydrated hydroscaffold inside biochip; (e) microscopic observation of hydrated hydroscaffold inside biochip; (f) SEM observation of the hydroscaffold; and (g) characterization of the pressure variation in the biochip with and without hydroscaffold.

Figure 3

Morphology of HepG2/C3A cells cultivated in (A) biochip containing the hydroscaffold and (B) biochip coated with collagen.

Figure 4

Cell viability for different seeding densities after 96h of culture in the biochip containing the hydroscaffold: DAPI (nuclei, blue), calcein (living cells, green), and ethidium (dead cells, red).

Figure 5

Albumin secretion by HepG2/C3A cultivated in a dynamic biochip and static Petri containing the hydroscaffold. Starting cell density 20,000 (a), 125,000 (b), and 250,000 cells/cm² (c); * p < 0.05 The insert in panel (a) is a close-up on the vertical axis.

Figure 6

Long-term (21 days) culture of HepG2/C3A cells in a biochip with a hydroscaffold (Starting cell density of 20,000 cells/cm²). (a) evolution in the morphology of HepG2/C3A spheroids throughout the 21 days of culture; (b) pressure evolution during the first 14 days of culture; (c,d) pressure measured inside the biochip on day 14 and 21, respectively.

Figure 7

Characterization of HepG2/C3A spheroids after 21 days of dynamic culture in a biochip containing a hydroscaffold. (a) cell viability: DAPI (nuclei, blue), calcein (living cells, green) and ethidium (dead cells, red); (b) F-actin staining: DAPI (nuclei, blue) and phalloidin (F-actin, green); (c) E-cadherin staining: DAPI (nuclei, blue) and E-cadherin (purple); (d) SEM observation (TV: top view and CS: cross section). The immunostaining images (b,c) correspond to z-stack projections.

Figure 8

Characterization of HepG2/C3A spheroids after 21 days of dynamic culture in a biochip containing the hydroscaffold. (a) F-actin and MRP2 staining showing the formation of bile canalicular-like structures: DAPI (nuclei, blue), phalloidin (F-actin, green); MRP2 (red) and biliary-like network (co-localization MRP2 and F-actin signals, yellow overlay signal, the two pictures in the bottom correspond to an enlargement from the merge picture); (b) BSEP staining: DAPI (nuclei, blue) and BSEP (yellow); (c,d) albumin and urea production in the dynamic biochip and static Petri conditions throughout 21 days of culture (both biochip and Petri contained the hydroscaffold). The immunostaining images (a,b) correspond to z-stack projections. * p < 0.05.