Large-scale perfused tissues via synthetic 3D soft microfluidics

The creation of larger, more functional engineered tissues and organoids is a critical goal in regenerative medicine and drug discovery. Current limitations in generating these tissues include poorly defined structural organization, small size, and slow maturation. In vivo, tissue development relies on a complex vascular network supplying oxygen and nutrients, removing waste, and mediating paracrine signaling. The size of this microvasculature is crucial for efficient diffusion; most cells are within 200 µm of a capillary. Without vascular support, maintaining physiological conditions in vitro is restricted to this narrow range, resulting in necrosis in the core of larger organoids. Existing approaches to vascularize in vitro tissues, such as extrinsic angiogenesis induction, genetic manipulation, or grafting, have limitations in controlling flow rates and vascular network topology. Various templating approaches using artificial vessels have also been explored, but these have been limited in the minimum vessel diameter (150 µm) and the size of the perfused tissues.

Numerous strategies have been developed to address the challenge of vascularizing engineered tissues and organoids. Extrinsic angiogenesis, involving the infiltration of vessel sprouts from co-cultured endothelial cells or pre-established microvascular beds, has shown promise but resulted in limited organoid size. Other approaches include grafting organoids into host animals, overexpression of the transcription factor hETV2, and enhancement of pro-endothelial differentiation by ambient flow. However, these methods lack precise control over flow rates and vascular network architecture. Artificial vessel creation using templating methods such as layer-by-layer deposition, droplet-based techniques, and stereolithography have been investigated. Bioprinting techniques employing bioinks with embedded vascular templates have also been explored, but minimum vessel diameters remained a limitation. The Kenzan method, utilizing micro-needles as temporary structural supports, produces perfusable channels, but these are limited in diameter (150-200 µm). Laser photo-ablation of hydrogels has been used to form artificial vessels but faces similar size constraints. Despite these advances, the integration of artificial microvessels with diameters below 150 µm into a complete perfusion system and creation of large perfused tissues has remained elusive.

To overcome the limitations of existing technologies, the researchers developed a novel 3D soft microfluidic strategy using two-photon laser scanning photo-polymerization. A critical advancement was the formulation of a custom hydrophilic photo-polymer based on polyethylene glycol diacrylate (PEGDA) to prevent the swelling observed with other materials. This non-swelling hydrogel, incorporating pentaerythritol triacrylate (PETA) for cell binding and Triton-X 100 for porosity, enabled the precise printing of capillary-scale vessels (10 µm to >70 µm diameter) within a three-dimensional grid structure. These grids were integrated into a perfusion chip connected to a peristaltic pump. Human pluripotent stem cells (hPSCs) were aggregated into organoids and seeded into the grids within a Matrigel matrix, creating a 'gel-in-gel' construct. Vessel permeability was confirmed using fluorescein. For neural differentiation experiments, hPSC aggregates were cultured in the grids and subjected to neural differentiation protocols. For liver tissue experiments, hPSCs were first differentiated into liver progenitors, then aggregated into spheroids and seeded into the perfused grids. Single-cell RNA sequencing (scRNAseq), immunohistochemistry, flow cytometry, and functional assays were performed to assess tissue viability, differentiation, and functionality. Drug clearance experiments were conducted with perfused liver constructs using quinidine, metoclopramide, and theophylline to assess CYP450 enzyme activity.

The developed 3D soft microfluidic platform enabled the creation of densely packed capillary networks with high precision and control, circumventing the limitations of previous approaches. The non-swelling hydrogel ensured a tight seal between the microfluidic grid and the perfusion system, allowing for reliable perfusion over extended periods. scRNAseq analysis revealed significant transcriptomic differences between perfused and non-perfused tissues. Perfusion significantly reduced hypoxia and apoptosis, as evidenced by decreased HIF1α and cleaved Caspase 3 expression, respectively. Neural differentiation was markedly accelerated in perfused tissues, as indicated by increased expression of neuroepithelial markers (PAX6, PAX7, PAX3, CDH2) and decreased expression of pluripotency markers (NANOG, POU5F1). In long-term cultures (2 months), perfused neural organoids exhibited extensive neuronal and glial arborization, including myelination (MBP expression), which was absent in non-perfused controls. Radial glia networks in perfused organoids demonstrated significantly greater arborization compared to controls, suggesting a role in neuronal guidance. The perfused hepatic constructs displayed improved hepatocyte differentiation, with increased expression of hepatocyte-specific genes (HNF6, NTCP, ALB, AAT, CYP2C9, CYP3A4) and functional activity, including albumin and urea production. Drug clearance studies using quinidine and theophylline showed good in vitro-in vivo correlation (IVIVC), demonstrating the potential of this platform for drug metabolism studies. The platform allows generation of functional and viable tissues significantly larger ( >15 mm³) than those previously reported.

This study presents a significant advance in generating large-scale, perfused engineered tissues. The ability to create capillary-scale vessels with unprecedented accuracy and integrate them into a robust perfusion system addresses a critical limitation in tissue engineering. The findings highlight the importance of perfusion in promoting tissue viability, preventing apoptosis, and accelerating differentiation. The observed metabolic shift from glycolysis to oxidative phosphorylation in perfused tissues further underscores the physiological relevance of this approach. The successful prediction of human in vivo drug clearance using perfused hepatic constructs demonstrates the platform's potential for drug development and personalized medicine applications. The uniform distribution of nutrients and oxygen provided by the densely spaced capillary network likely contributes to the lack of tissue zonation and layering observed in some constructs; future modifications could potentially address this by manipulating inter-capillary spacing or introducing spatially-engineered cues.

The 3D soft microfluidic technology provides a powerful platform for creating large, functional, perfused in vitro tissues. This platform enables the study of tissue development, disease modeling, and drug discovery at an unprecedented scale. Future research could explore the incorporation of vascular cells into the microfluidic network, the use of alternative extracellular matrices, and the optimization of perfusion parameters for specific tissue types. The ability to generate and maintain large, viable tissues will undoubtedly accelerate advancements in regenerative medicine and drug development.

While this platform provides a significant improvement in tissue perfusion, it does not fully replicate the complexity of the in vivo vasculature. Adaptive vascular remodeling and features such as the blood-brain barrier are not represented in this model. The use of Matrigel as the extracellular matrix, while widely used, might not fully recapitulate the complexity of native tissue environments. Further optimization of the hydrogel formulation and cell-seeding techniques may be needed to fully replicate the in vivo environment.