Abstract
Perfusable hydrogels have garnered substantial attention in recent years for the fabrication of microphysiological systems. However, current methodologies to fabricate microchannels in hydrogel platforms involve sophisticated equipment and techniques, which hinder progress of the field. In this protocol, we present a cost-effective, simple, versatile and ultrafast method to create perfusable microchannels of complex shapes in photopolymerizable hydrogels. Our method uses one-step UV photocross-linking and a photomask printed on inexpensive transparent films, to photopattern both synthetic (PEG-norbornene) and natural (hyaluronic acid-norbornene) hydrogels in just 0.8 s. Moreover, these perfusable hydrogels are fully integrated into a custom-made microfluidic device that allows continuous fluid perfusion when connected to an external pump system. This methodology can be easily reproduced by professionals with basic laboratory skills and a fundamental knowledge of polymers and materials science. In this protocol, we demonstrate the functionality of our photopatterned hydrogels by seeding human endothelial cells into the microchannels, culturing them under dynamic conditions for 7 d, and exposing them to inflammatory stimuli to elicit cellular responses. This highlights the versatility of our platform in fabricating microphysiological systems and different microenvironments. The fabrication of perfusable channels within the hydrogels, including the fabrication of the microfluidic devices, requires ~3 d. The development of the cell-seeded microphysiological system, including the stimulation of cells, takes ~7 d. In conclusion, our approach provides a straightforward and widely applicable solution to simplify and reduce the cost of biofabrication techniques for developing functional in vitro models using perfusable three-dimensional hydrogels.
Key points
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This protocol describes the fabrication of perfusable microchannels of complex shapes by photopatterning hydrogels. It uses photomask transparency films to photopattern hydrogels and includes representative examples to recreate different physiological microenvironments.
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This is performed by a one-step UV light-triggered cross-linking using a photomask printed on inexpensive transparent films. Compared with existing methods for fabricating perfusable hydrogels, this makes it a cost-effective, facile, versatile and ultrafast approach.
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Data availability
The main data discussed in this protocol are available in the supporting primary research paper4.
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Acknowledgements
The authors acknowledge financial support from the Wellcome Leap HOPE program awarded to A.S. and A.J.G. A.M.-B. acknowledges support from the European Research Executive Agency under Individual GLOBAL Marie Skłodowska-Curie Actions Fellowship (project no. 101028216 — SYNMAT FOR ORGANOIDS). A.M.-R. acknowledges support from the National Institute of Diabetes and Digestive and Kidney Diseases (F31 DK130581). N.D.C. and J.A.B. acknowledge the National Science Foundation through the Center for Engineering Mechanobiology STC (CMMI 15-48571). A.S. acknowledges the financial support from NIH/NCI (5R01CA238745-04).
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Contributions
A.M.-B. designed the study, developed and optimized the protocol, performed the main experiments, interpreted the data and wrote the main manuscript. A.M.-R. performed the immunostaining experiments, imaged the samples and edited the manuscript. N.D.C. and J.A.B. provided the NorHA reagents and protocol, and edited the manuscript. E.O. contributed to the preparation of reagents, provided equipment information and edited the manuscript. A.S. and A.J.G. jointly conceived the study and edited the manuscript.
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A.M.-B., A.S. and A.J.G. are inventors on a patent application related to this work and owned by the Georgia Tech Research Corp. The remaining authors declare no competing interests.
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Key reference using this protocol
Mora-Boza, A. et al. Adv. Mater. 35, 2306765 (2023): https://doi.org/10.1002/adma.202306765
Supporting information
Supplementary Data 1
Examples of perfusable channel designs.
Supplementary Data 2
Design of features for maskless instruments.
Supplementary Video 1
Fabrication steps.
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Mora-Boza, A., Mulero-Russe, A., Di Caprio, N. et al. Facile photopatterning of perfusable microchannels in hydrogels for microphysiological systems. Nat Protoc 20, 272–292 (2025). https://doi.org/10.1038/s41596-024-01041-8
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DOI: https://doi.org/10.1038/s41596-024-01041-8
