Mora-Boza Ana, Mulero-Russe Adriana, Di Caprio Nikolas, Burdick Jason A, O'Neill Eric, Singh Ankur, García Andrés J
George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA, USA.
Nat Protoc. 2025 Jan;20(1):272-292. doi: 10.1038/s41596-024-01041-8. Epub 2024 Sep 12.
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.
近年来，可灌注水凝胶在微生理系统的制造方面备受关注。然而，目前在水凝胶平台上制造微通道的方法涉及复杂的设备和技术，这阻碍了该领域的发展。在本方案中，我们提出了一种经济高效、简单、通用且超快的方法，用于在可光聚合水凝胶中创建复杂形状的可灌注微通道。我们的方法采用一步紫外光交联和印在廉价透明薄膜上的光掩膜，只需0.8秒就能对合成（聚乙二醇 - 降冰片烯）和天然（透明质酸 - 降冰片烯）水凝胶进行光图案化。此外，这些可灌注水凝胶完全集成到定制的微流控装置中，连接到外部泵系统时可实现连续流体灌注。具有基础实验室技能以及聚合物和材料科学基础知识的专业人员可以轻松重现此方法。在本方案中，我们通过将人内皮细胞接种到微通道中，在动态条件下培养7天，并使其暴露于炎症刺激以引发细胞反应，来展示我们光图案化水凝胶的功能。这突出了我们的平台在制造微生理系统和不同微环境方面的通用性。在水凝胶内制造可灌注通道，包括微流控装置的制造，大约需要3天。构建接种细胞的微生理系统，包括细胞刺激，大约需要7天。总之，我们的方法提供了一种直接且广泛适用的解决方案，可简化并降低使用可灌注三维水凝胶开发功能性体外模型的生物制造技术成本。
