Convergence of Organ‐on‐a‐Chip and Freeform Printing of Sacrificial Poly(2‐cyclopropyl‐2‐oxazoline) Enables the Generation of Perfusable Endothelialized Channels in Hydrogels

ABSTRACT The fabrication of physiologically relevant microvascular networks remains a major challenge in Organ‐on‐a‐chip (OoC) technologies, largely due the dependence on plastic materials and the generation of channels with non‐physiological, rectangular cross‐sections. Here, we present a novel approach for the in‐situ generation of perfusable micro‐vessels within OoC platforms using freeform printing (FFP) of the thermo‐responsive poly(2‐cyclopropyl‐2‐oxazoline) (PcycloPrOx). We integrated FFP with a fluidic custom‐designed OoC device to directly print suspended sacrificial vascular templates, enabling the creation of circular cross‐section channels with resolutions down to 200 µm, without post‐processing. Following hydrogel casting and template dissolution, green fluorescent protein human umbilical vein endothelial cells (GFP‐HUVECs) were seeded into the channels and cultured under continuous perfusion (unidirectional and bidirectional) for 7 days. Confocal fluorescence microscopy revealed rapid endothelialization, with a confluent monolayer established by day 3. By day seven, immunostaining confirmed expression of endothelial markers CD31 and VE‐cadherin, indicating proper endothelialization. This work demonstrates the first functional application of PcycloPrOx‐based FFP for OoC vascularization and provides a scalable, automatable strategy for engineering perfusable, endothelialized microvascular 3D tissue models. Our platform offers new opportunities for vascularized organ‐on‐chip models in drug screening and disease modelling.