A Perfusable Multi‐Hydrogel Vasculature On‐Chip Engineered by 2‐Photon 3D Printing and Scaffold Molding to Improve Microfabrication Fidelity in Hydrogels

Abstract Engineering vasculature networks in physiologically relevant hydrogels represents a challenge in terms of both fabrication, due to the cell–bioink interactions, as well as the subsequent hydrogel‐device interfacing. Here, a new cell‐friendly fabrication strategy is presented to realize perfusable multi‐hydrogel vasculature models supporting co‐culture integrated in a microfluidic chip. The system comprises two different hydrogels to specifically support the growth and proliferation of two different cell types selected for the vessel model. First, the channels are printed in a gelatin‐based ink by two‐photon polymerization (2PP) inside the microfluidic device. Then, a human lung fibroblast‐laden fibrin hydrogel is injected to surround the printed network. Finally, human endothelial cells are seeded inside the printed channels. The printing parameters and fibrin composition are optimized to reduce hydrogel swelling and ensure a stable model that can be perfused with cell media. Fabricating the hydrogel structure in two steps ensures that no cells are exposed to cytotoxic fabrication processes, while still obtaining high fidelity printing. In this work, the possibility to guide the endothelial cell invasion through the 3D printed scaffold and perfusion of the co‐culture model for 10 days is successfully demonstrated on a custom‐made perfusion system.