Development of a three-dimensional in vitro vascular model

Blood vessels perform essential biological functions in human body, including the transport of oxygen and nutrients and the removal of metabolic waste products. They regulate immune cell trafficking, maintain tissue homeostasis and contribute to processes such as angiogenesis and wound healing. Abnormalities in these structures are associated with different pathologies, including cardiovascular and neurodegenerative diseases, as well as cancer progression. To gain deeper insights into these mechanisms and to develop more physiologically relevant platforms for drug testing and regenerative medicine, it is crucial to integrate vascular-like networks into in vitro models. Nevertheless, engineering three-dimensional (3D) vessel-like architectures able to mimic the complexity of the native microenvironment remains highly challenging. To fill this gap, this study aimed to develop a 3D in vitro vascular model exploiting three different approaches: i) extrusion-based 3D printing, ii) volumetric printing (VP) and iii) electrospinning. Gelatin methacrylate (GelMA), a natural polymer presenting cell-adhesive motifs, served as ink for the first two approaches. For electrospinning, GelMA-coated polycaprolactone (PCL), a mechanically robust synthetic polymer widely used in biomedical applications, was involved. The obtained constructs were characterized in terms of specific parameters, including geometry, consistency/reliability, fabrication time, resolution and scalability. Based on these considerations, VP was assessed as the optimal approach for producing a GelMA-based vascular model. Morphological characterization of the volumetrically printed structures revealed mainly elongated pores with diameters ranging from 25 to 115 μm, a size range which is considered optimal for blood vessel formation. Swelling studies showed the ability of the tubular structure to absorb a large quantity of water (404.9% ± 29.3%) within a few minutes of immersion, thereby providing a moist environment for cell growth. Preliminary biological tests were carried out on volumetrically printed scaffolds using Human Umbilical Vein Endothelial Cells (HUVECs) under static culture conditions. Live/Dead staining confirmed the cytocompatibility of the constructs. Cells were maintained in culture for up to 14 days, during which PrestoBlue assay confirmed their viability but also indicated a predominantly quiescent state. By day 14, a decline in metabolic activity was observed, likely reflecting the need for further optimization of the seeding protocol. Similarly, DAPI/Phalloidin staining demonstrated cell attachment to the lumen over 7 days, with cells exhibiting their elongated morphology aligned with printing planes defined by the VP process and intercellular interactions, followed by a reduction at day 14. Collectively, the reported results demonstrate the suitability of VP for the fabrication of tubular scaffolds for vascular modeling, even though further optimization of the cellularization process is needed to fully develop functional models. Approaches such as direct bioprinting with cells and the integration of perfusion represent promising strategies to achieve this goal. Future directions also include scaling down the structures to replicate microvascular channels, as well as the incorporation of supporting cell types, specifically pericytes, to better mimic the native composition of capillaries. Moreover, exploiting VP for production of multiple structures in parallel paves the way for enhancing process scalability.