Figure 6 Construction of perfusable microfluidic chips vialayer-by-layer adhesion. (A) Perfusable microfluidic chip with spiral pattern. (B) Perfusable microfluidic chip with snail pattern. (C) Bilayer chip for coculture of cells. Scale bar=1 cm.
3.5 Application of layer-by-layer adhered microfluidic as liver-on-a-chip
As a common practice with microfluidics, liver-on-a-chip was constructed to mimic the hepatic structures. The hydrogel microfluidic chip is typically constructed using Gel-MA, a photo-crosslinkable polymer29. However, Gel-MA is mechanically weak and cannot withstand perfusing-induced shear stress. To address this issue, we encapsulated the HepG2 cells in Gel-MA and stitched it onto a tough F127-DA sheet to reinforce the Gel-MA layer (Figure 7A). HUVECs were seeded on the surface of the channel for perfusion culture (Figure 7A).
The key step in the construction was the adhesion of the cell-laden Gel-MA hydrogel to the F127-DA sheet. The neutral CS-MA adhesive used was well-tolerated by HepG2 cells (Figure S4). To minimize the injury during stitching, a short UV exposure of 30 s with a flashlight was applied for the CS-MA photo-crosslinking in the presence of VA-086, since long-wave UV light (395 nm) triggered low toxicity to cells30. After 2 days of perfusing culture, cell viability was assessed using Calcein AM/PI staining, which detects living and dead cells 29,31. Based on fluorescent images, most of cells (both HepG2 and HUVECs) were alive (stained green, Figure 7B), with only a very small amount of dead cells (stained red, Figure 7C). The spread cells in the channel zone (shown by the white line in Figure 7B to 7D) were likely HUVECs, while the round cells and aggregates located in the entire chip would be HepG2 cells, as the encapsulated HepG2 cells possibly grew or migrated to aggregates within 4 days of culture. Quantitative analysis showed that encapsulation decreased the viability of HepG2 cells to 75%, but it increased to over 80% after 1 day’s incubation due to the proliferation of living cells (Figure 7E). Moreover, the seeded HUVECs were mostly alive within 2 days of perfusion culture (Figure 7E).