Flow-enhanced vascularization and maturation of kidney organoids in vitro.

Kidney organoids derived from human pluripotent stem cells have glomerular- and tubular-like compartments that are largely avascular and immature in static culture. Here we report an in vitro method for culturing kidney organoids under flow on millifluidic chips, which expands their endogenous pool of endothelial progenitor cells and generates vascular networks with perfusable lumens surrounded by mural cells. We found that vascularized kidney organoids cultured under flow had more mature podocyte and tubular compartments with enhanced cellular polarity and adult gene expression compared with that in static controls. Glomerular vascular development progressed through intermediate stages akin to those involved in the embryonic mammalian kidney's formation of capillary loops abutting foot processes. The association of vessels with these compartments was reduced after disruption of the endogenous VEGF gradient. The ability to induce substantial vascularization and morphological maturation of kidney organoids in vitro under flow opens new avenues for studies of kidney development, disease, and regeneration.

Renal reabsorption in 3D vascularized proximal tubule models.

Three-dimensional renal tissues that emulate the cellular composition, geometry, and function of native kidney tissue would enable fundamental studies of filtration and reabsorption. Here, we have created 3D vascularized proximal tubule models composed of adjacent conduits that are lined with confluent epithelium and endothelium, embedded in a permeable ECM, and independently addressed using a closed-loop perfusion system to investigate renal reabsorption. Our 3D kidney tissue allows for coculture of proximal tubule epithelium and vascular endothelium that exhibits active reabsorption via tubular-vascular exchange of solutes akin to native kidney tissue. Using this model, both albumin uptake and glucose reabsorption are quantified as a function of time. Epithelium-endothelium cross-talk is further studied by exposing proximal tubule cells to hyperglycemic conditions and monitoring endothelial cell dysfunction. This diseased state can be rescued by administering a glucose transport inhibitor. Our 3D kidney tissue provides a platform for in vitro studies of kidney function, disease modeling, and pharmacology.

Three-dimensional bioprinting of thick vascularized tissues.

The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods. To date, bioprinting methods have yielded thin tissues that only survive for short durations. To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.

In vitro human tissues via multi-material 3-D bioprinting.

This paper highlights the foundational research on multi-material 3-D bioprinting of human tissues, for which the Lewis Bioprinting team at Harvard University was awarded the 2017 Lush Science Prize. The team's bioprinting platform enables the rapid fabrication of 3-D human tissues that contain all of the essential components found in their in vivo counterparts: cells, vasculature (or other tubular features) and extracellular matrix. The printed 3-D tissues are housed within a customised perfusion system and are subjected to controlled microphysiological environments over long durations (days to months). As exemplars, the team created a thick, stem cell-laden vascularised tissue that was controllably differentiated toward an osteogenic lineage in situ, and a 3-D kidney tissue that recapitulated the proximal tubule, a subunit of the nephron responsible for solute reabsorption. This highly versatile platform for manufacturing 3-D human tissue in vitro opens new avenues for replacing animal models used to develop next-generation therapies, test toxicity and study disease pathology.

Bioprinting of 3D Convoluted Renal Proximal Tubules on Perfusable Chips.

Three-dimensional models of kidney tissue that recapitulate human responses are needed for drug screening, disease modeling, and, ultimately, kidney organ engineering. Here, we report a bioprinting method for creating 3D human renal proximal tubules in vitro that are fully embedded within an extracellular matrix and housed in perfusable tissue chips, allowing them to be maintained for greater than two months. Their convoluted tubular architecture is circumscribed by proximal tubule epithelial cells and actively perfused through the open lumen. These engineered 3D proximal tubules on chip exhibit significantly enhanced epithelial morphology and functional properties relative to the same cells grown on 2D controls with or without perfusion. Upon introducing the nephrotoxin, Cyclosporine A, the epithelial barrier is disrupted in a dose-dependent manner. Our bioprinting method provides a new route for programmably fabricating advanced human kidney tissue models on demand.

Controlling Material Reactivity Using Architecture.

3D-printing methods are used to generate reactive material architectures. Several geometric parameters are observed to influence the resultant flame propagation velocity, indicating that the architecture can be utilized to control reactivity. Two different architectures, channels and hurdles, are generated, and thin films of thermite are deposited onto the surface. The architecture offers an additional route to control, at will, the energy release rate in reactive composite materials.

3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs.

A new bioprinting method is reported for fabricating 3D tissue constructs replete with vasculature, multiple types of cells, and extracellular matrix. These intricate, heterogeneous structures are created by precisely co-printing multiple materials, known as bioinks, in three dimensions. These 3D micro-engineered environments open new -avenues for drug screening and fundamental studies of wound healing, angiogenesis, and stem-cell niches.

Embedded 3D printing of strain sensors within highly stretchable elastomers.

A new method, embedded-3D printing (e-3DP), is reported for fabricating strain sensors within highly conformal and extensible elastomeric matrices. e-3DP allows soft sensors to be created in nearly arbitrary planar and 3D motifs in a highly programmable and seamless manner. Several embodiments are demonstrated and sensor performance is characterized.

High-throughput printing via microvascular multinozzle arrays.

Microvascular multinozzle arrays are designed and fabricated for high-throughput printing of functional materials. Ink-flow uniformity within these multigeneration, bifurcating microchannel arrays is characterized by computer modeling and microscopic particle image velocimetry (micro-PIV) measurements. Both single and dual multinozzle printheads are produced to enable rapid printing of multilayered periodic structures over large areas (≈1 m(2)).