The development of physiologically relevant in vitro tissue models has long been constrained by the absence of a perfusable vasculature. Native organs and tumors are sustained not only by their cellular components but also by vascular networks that deliver oxygen and nutrients, remove waste, and mediate immune and pharmacological interactions. To date, most engineered tissues have been generated at low densities (LDs; 10 6 –10 7 cells/mL) at the cellular level, where diffusion alone sustains viability. These LD constructs are experimentally tractable but physiologically simplistic because they systematically overestimate drug penetration and fail to capture metabolic gradients. By contrast, high-density (HD; 10 8 –10 9 cells/mL) tissues replicate the diffusion-limited transport, heterogeneous drug exposure, and hypoxia-driven phenotypes observed in vivo. However, HD constructs rapidly undergo necrosis in the absence of vascularization, which highlights the fact that perfusable vasculature is not an enhancement but an enabling factor. This review summarizes methodological innovations that address this challenge. First, we discuss endothelial cell-based strategies including self-assembly, angiogenic stimulation, and co-culture with stromal or pericyte cells, which recapitulate biological morphogenesis but often lack hierarchical fidelity. Then, we survey microfluidic platforms that provide patterned reproducible conduits under flow and enable systematic investigation of angiogenesis, barrier function, and drug transport. Bioprinting and sacrificial templating approaches are highlighted for their scalability and ability to create branching perfusable networks within millimeter-scale tissues. Synthetic microvascular scaffolds generated by two-photon polymerization and related methods offer notable precision at capillary resolution, although biocompatibility and scalability remain challenging. Moreover, hybrid and emerging approaches integrate organoids, bioprinting, and microfluidics into unified platforms to yield physiologically adaptable yet structurally controlled vascularized constructs. Finally, we consider the functional consequences of vascularization in HD tissues. Perfusable networks sustain metabolic activity, preserve tissue-specific phenotypes, and enable physiologically relevant drug penetration and metabolism. In oncology, vascularized HD tumor models reproduce drug gradients, angiogenesis, and vessel-guided invasion, which provide platforms for evaluating therapeutic resistance. In regenerative contexts, vascularization of liver or cardiac tissues preserves cytochrome P450 activity, electrophysiology, and contractility, which makes these systems suitable for toxicity prediction and pharmacokinetics. Furthermore, a perfusable vasculature transforms cell delivery into a dynamic process that enables immune, stromal, and progenitor cells to infiltrate tissues under flow. These advances enable the modeling of immune infiltration, immunotherapy response, and metastatic spread in manners not achievable with static LD models. Thus, immune-integrated vascularized systems constitute a frontier with future directions that include modeling physiological immune trafficking, probing endothelial contributions to therapy-associated toxicities, and developing standardized readouts of perfusion stability and vascular function. By combining HD tissue architecture with perfusable vasculature, these emerging systems narrow the gap between in vitro constructs and in vivo physiology. We propose that vascularized HD tissues constitute the next generation of translational models with the capacity to improve drug safety prediction, advance immunotherapy testing, and accelerate effective treatment development. • High-density (HD) tissue constructs replicate diffusion-limited transport. • However, HD constructs rapidly undergo necrosis in absence of vascularization. • Methodological innovations that address these challenge are reviewed here. • Vascularized HD tissues are proposed as next-generation translational models.
Singh et al. (Fri,) studied this question.