Figure 8 Identification of HepG2 cells and HUVECs by immunostaining. (A) HUVECs (green, stained with VEGF) attached on the surface of channel. (B) HepG2 cells (red, stained with MRP-2) located in Gel-MA hydrogel. Nuclei were stained blue by DAPI. White line was the borderline of microchannel.
To visually depict the cellular organization within the chip, we immunostained MRP-2 and VEGF to identify HepG2 cells and HUVECs, respectively. MRP-2 is specifically expressed in hepatic cells that mediates the drug transportation 36, while VEGF is secreted by HUVECs but not HepG2 cells to regulate the endothelial angiogenesis and permeability 37. As shown in Figure 8A, VEGF-positive HUVECs were located in the channel zone, while the MRP-2 positive HepG2 cells were dispersed inside Gel-MA hydrogel. Zoom-in images showed the specific expression of VEGF (green, Figure 8B) and MRP-2 (red, Figure 8C) by HUVECs and HepG2 cells, respectively. The HepG2 cells formed irregular aggregates in Gel-MA hydrogel (Figure 8C), consistent with previous reports 38.
While the above example did not cover all the possible applications of layer-by-layer adhesion in constructing hydrogel-based microfluidics, it presented a novel and straightforward concept for fabricating complex and precise 3D architectures. Moreover, another example was shown in Figure S5, where a bilayer chip was constructed for coculture of HepG2 cells and fibroblasts. To replicate the physiology of organs, current biofabrication techniques require the spatially-precise organization of cells and extracellular matrix, mimicking physical cues in vivosuch as chemical components, topography, stiffness and shear stress2. Hence, the layer-by-layer adhesion could aid in the rational design of in vivo -like tissues, as multilayered hydrogels facilitate the mimicking of chemical and structural properties of extracellular matrix in vivo . This paper demonstrated the construction of liver-on-a-chip with vascular structure by organizing liver and endothelial cells in the appropriate positions (Figure 7), well mimicking the vasculature and sustaining cellular activity in the liver 39. Similarly, the layer-by-layer adhesion could also benefit the design of other organs-on-chips such as skin consisting of various layers with different extracellular matrix and cells40,41.

4. Conclusion

We have introduced a new method called “layer-by-layer adhesion” for constructing hydrogel-based microfluidic chips. Four types of hydrogels were well stitched together using the adhesive properties of CS-MA, which exhibited adhesion energy of 1.2-140 N/m. The CS-MA diffused into the hydrogels and then crosslinked at the interface of two hydrogels to create a density zone. Such adhesion maintained good stability even after autoclaving, stretching and twisting. This method allowed for the assembly of perfusable hydrogels with snail, spiral, vascular-like and bilayer microchannels with high resolution. As an example of application, we used this method to construct liver-on-a-chip based on Gel-MA/F127-DA layers and coculture of HepG2 cells with HUVECs. Our method of layer-by-layer adhesion offers a new way to design 3D architectures in hydrogels and construct microfluidic organs-on-chipsin vitro .

5. Data availability statement

The data that supports the findings of this study are available in the supplementary material of this article.

6. Conflicts of interest

There are no conflicts of interest to declare.

7. Acknowledgement

We gratefully acknowledge the financial support of this study by NSFC (National Natural Science Foundation of China, No 22078287 and 21978257).

8. References

1. Sun WJ, Luo ZM, Lee J, et al. Organ-on-a-Chip for Cancer and Immune Organs Modeling. Advanced Healthcare Materials. Feb 21 2019;8(4).
2. Sontheimer-Phelps A, Hassell BA, Ingber DE. Modelling cancer in microfluidic human organs-on-chips. Nat Rev Cancer. Feb 2019;19(2):65-81.
3. Rothbauer M, Zirath H, Ertl P. Recent advances in microfluidic technologies for cell-to-cell interaction studies.Lab on a Chip. Jan 21 2018;18(2):249-270.
4. Huang GY, Zhou LH, Zhang QC, et al. Microfluidic hydrogels for tissue engineering. Biofabrication. Mar 2011;3(1):012001.
5. Zhang X, Li L, Luo C. Gel integration for microfluidic applications. Lab Chip. May 21 2016;16(10):1757-1776.
6. Zhao S, Chen Y, Partlow BP, et al. Bio-functionalized silk hydrogel microfluidic systems. Biomaterials. Jul 2016;93:60-70.
7. Paguirigan A, Beebe DJ. Gelatin based microfluidic devices for cell culture. Lab Chip. Mar 2006;6(3):407-413.
8. Gjorevski N, Lutolf MP. Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture. Nat Protoc. Nov 2017;12(11):2263-2274.
9. Feng YM, Lee Y. Microfluidic assembly of food-grade delivery systems: Toward functional delivery structure design. Trends Food Sci Tech. Apr 2019;86:465-478.
10. Kim YH, Kim DJ, Lee S, Kim DH, Park SG, Kim SH. Microfluidic Designing Microgels Containing Highly Concentrated Gold Nanoparticles for SERS Analysis of Complex Fluids. Small. Dec 2019;15(52).
11. Yang JW, Bai RB, Chen BH, Suo ZG. Hydrogel Adhesion: A Supramolecular Synergy of Chemistry, Topology, and Mechanics.Advanced Functional Materials. Jan 2020;30(2).
12. Wang XY, Jin ZH, Gan BW, Lv SW, Xie M, Huang WH. Engineering interconnected 3D vascular networks in hydrogels using molded sodium alginate lattice as the sacrificial templatet. Lab on a Chip. Aug 7 2014;14(15):2709-2716.
13. Abbott RD, Kaplan DL. Strategies for improving the physiological relevance of human engineered tissues. Trends Biotechnol. Jul 2015;33(7):401-407.
14. Lee SH, Sung JH. Organ-on-a-Chip Technology for Reproducing Multiorgan Physiology. Advanced Healthcare Materials. Jan 24 2018;7(2).
15. Knowlton S, Yu CH, Ersoy F, Emadi S, Khademhosseini A, Tasoglu S. 3D-printed microfluidic chips with patterned, cell-laden hydrogel constructs. Biofabrication. Jun 20 2016;8(2):025019.
16. Lutolf MP. Spotlight on hydrogels. Nature Materials.Jun 2009;8(6):451-453.
17. Wu W, Hansen CJ, Aragon AM, Geubelle PH, White SR, Lewis JA. Direct-write assembly of biomimetic microvascular networks for efficient fluid transport. Soft Matter. 2010;6(4):739-742.
18. Thomsen AR, Aldrian C, Bronsert P, et al. A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement. Lab Chip. Dec 19 2018;18(1):179-189.
19. Yuk H, Varela CE, Nabzdyk CS, et al. Dry double-sided tape for adhesion of wet tissues and devices. Nature. Nov 7 2019;575(7781):169-+.
20. Kim IY, Seo SJ, Moon HS, et al. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv.Jan-Feb 2008;26(1):1-21.
21. Li BQ, Wang L, Xu F, et al. Hydrosoluble, UV-crosslinkable and injectable chitosan for patterned cell-laden microgel and rapid transdermal curing hydrogel in vivo. Acta Biomater. Aug 2015;22:59-69.
22. Yin HY, Akasaki T, Sun TL, et al. Double network hydrogels from polyzwitterions: high mechanical strength and excellent anti-biofouling properties. J Mater Chem B. 2013;1(30):3685-3693.
23. Qi Z, Xu J, Wang Z, Nie J, Ma G. Preparation and properties of photo-crosslinkable hydrogel based on photopolymerizable chitosan derivative. International journal of biological macromolecules.2013;53(none):144-149.
24. Monier M, Wei Y, Sarhan AA, Ayad DM. Synthesis and characterization of photo-crosslinkable hydrogel membranes based on modified chitosan. Polymer. 2010;51(5):1002-1009.
25. Galante R, Pinto TJA, Colaco R, Serro AP. Sterilization of hydrogels for biomedical applications: A review. J Biomed Mater Res B Appl Biomater. Aug 2018;106(6):2472-2492.
26. Takei T, Danjo S, Sakoguchi S, et al. Autoclavable physically-crosslinked chitosan cryogel as a wound dressing. J Biosci Bioeng. Apr 2018;125(4):490-495.
27. Yang JW, Bai RB, Suo ZG. Topological Adhesion of Wet Materials. Advanced Materials. Jun 20 2018;30(25).
28. Gao Y, Wu KL, Suo ZG. Photodetachable Adhesion.Advanced Materials. Feb 8 2019;31(6).
29. Zhao X, Lang Q, Yildirimer L, et al. Photocrosslinkable Gelatin Hydrogel for Epidermal Tissue Engineering. Advanced Healthcare Materials. Jan 2016;5(1):108-118.
30. Occhetta P, Visone R, Russo L, Cipolla L, Moretti M, Rasponi M. VA-086 methacrylate gelatine photopolymerizable hydrogels: A parametric study for highly biocompatible 3D cell embedding.Journal of Biomedical Materials Research Part A. Jun 2015;103(6):2109-2117.
31. Wang ZJ, Kumar H, Tian ZL, et al. Visible Light Photoinitiation of Cell-Adhesive Gelatin Methacryloyl Hydrogels for Stereolithography 3D Bioprinting. Acs Appl Mater Inter. Aug 15 2018;10(32):26859-26869.
32. Yajima Y, Yamada M, Yamada E, Iwase M, Seki M. Facile fabrication processes for hydrogel-based microfluidic devices made of natural biopolymers. Biomicrofluidics. Mar 2014;8(2):024115.
33. Shen C, Li Y, Wang Y, Meng Q. Non-swelling hydrogel-based microfluidic chips. Lab Chip. Dec 7 2019;19(23):3962-3973.
34. Baeyens N, Bandyopadhyay C, Coon BG, Yun S, Schwartz MA. Endothelial fluid shear stress sensing in vascular health and disease.J Clin Invest. Mar 1 2016;126(3):821-828.
35. Liu HT, Wang YQ, Cui KL, Guo YQ, Zhang X, Qin JH. Advances in Hydrogels in Organoids and Organs-on-a-Chip. Advanced Materials. Dec 13 2019;31(50).
36. Gissen P, Arias IM. Structural and functional hepatocyte polarity and liver disease. J Hepatol. Oct 2015;63(4):1023-1037.
37. Apte RS, Chen DS, Ferrara N. VEGF in Signaling and Disease: Beyond Discovery and Development. Cell. Mar 7 2019;176(6):1248-1264.
38. Pimentel CR, Ko SK, Caviglia C, et al. Three-dimensional fabrication of thick and densely populated soft constructs with complex and actively perfused channel network. Acta Biomater. Jan 2018;65:174-184.
39. Miller JS, Stevens KR, Yang MT, et al. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat Mater. Sep 2012;11(9):768-774.
40. Kim JJ, Ellett F, Thomas CN, et al. A microscale, full-thickness, human skin on a chip assay simulating neutrophil responses to skin infection and antibiotic treatments. Lab on a Chip. Sep 21 2019;19(18):3094-3103.
41. Mori N, Morimoto Y, Takeuchi S. Skin integrated with perfusable vascular channels on a chip. Biomaterials. Feb 2017;116:48-56.