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.

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.