Perfusable cell-laden matrices to guide patterning of vascularization in vivo.

The survival and function of transplanted tissue engineered constructs and organs require a functional vascular network. In the body, blood vessels are organized into distinct patterns that enable optimal nutrient delivery and oxygen exchange. Mimicking these same patterns in engineered tissue matrices is a critical challenge for cell and tissue transplantation. Here, we leverage bioprinting to assemble endothelial cells in to organized networks of large (>100 µm) diameter blood vessel grafts to enable spatial control of vessel formation in vivo. Acellular PEG/GelMA matrices with perfusable channels were bioprinted and laminar flow was confirmed within patterned channels, beneficial for channel endothelialization and consistent wall shear stress for endothelial maturation. Next, human umbilical vein endothelial cells (HUVECs) were seeded within the patterned channel and maintained under perfusion culture for multiple days, leading to cell-cell coordination within the construct in vitro. HUVEC and human mesenchymal stromal cells (hMSCs) were additionally added to bulk matrix to further stimulate anastomosis of our bioprinted vascular grafts in vivo. Among multiple candidate matrix designs, the greatest degree of biomaterial vascularization in vivo was seen within matrices fabricated with HUVECs and hMSCs encapsulated within the bulk matrix and HUVECs lining the walls of the patterned channels, dubbed design M-C_E. For this lead design, vasculature was detected within the endothelialized, perfusable matrix channels as early as two weeks and αSMA+ CD31+ vessels greater than 100 µm in diameter had formed by eight weeks, resulting in durable and mature vasculature. Notably, vascularization occurred within the endothelialized, bioprinted channels of the matrix, demonstrating the ability of bioprinted perfusable structures to guide vascularization patterns in vivo. The ability to influence vascular patterning in vivo can contribute to the future development of vascularized tissues and organs.

Development, characterization, and applications of multi-material stereolithography bioprinting.

As a 3D bioprinting technique, hydrogel stereolithography has historically been limited in its ability to capture the spatial heterogeneity that permeates mammalian tissues and dictates structure-function relationships. This limitation stems directly from the difficulty of preventing unwanted material mixing when switching between different liquid bioinks. Accordingly, we present the development, characterization, and application of a multi-material stereolithography bioprinter that provides controlled material selection, yields precise regional feature alignment, and minimizes bioink mixing. Fluorescent tracers were first used to highlight the broad design freedoms afforded by this fabrication strategy, complemented by morphometric image analysis to validate architectural fidelity. To evaluate the bioactivity of printed gels, 344SQ lung adenocarcinoma cells were printed in a 3D core/shell architecture. These cells exhibited native phenotypic behavior as evidenced by apparent proliferation and formation of spherical multicellular aggregates. Cells were also printed as pre-formed multicellular aggregates, which appropriately developed invasive protrusions in response to hTGF-ß1. Finally, we constructed a simplified model of intratumoral heterogeneity with two separate sub-populations of 344SQ cells, which together grew over 14 days to form a dense regional interface. Together, these studies highlight the potential of multi-material stereolithography to probe heterotypic interactions between distinct cell types in tissue-specific microenvironments.

Projection-based stereolithography for direct 3D printing of heterogeneous ultrasound phantoms.

Modern ultrasound (US) imaging is increasing its clinical impact, particularly with the introduction of US-based quantitative imaging biomarkers. Continued development and validation of such novel imaging approaches requires imaging phantoms that recapitulate the underlying anatomy and pathology of interest. However, current US phantom designs are generally too simplistic to emulate the structure and variability of the human body. Therefore, there is a need to create a platform that is capable of generating well-characterized phantoms that can mimic the basic anatomical, functional, and mechanical properties of native tissues and pathologies. Using a 3D-printing technique based on stereolithography, we fabricated US phantoms using soft materials in a single fabrication session, without the need for material casting or back-filling. With this technique, we induced variable levels of stable US backscatter in our printed materials in anatomically relevant 3D patterns. Additionally, we controlled phantom stiffness from 7 to >120 kPa at the voxel level to generate isotropic and anisotropic phantoms for elasticity imaging. Lastly, we demonstrated the fabrication of channels with diameters as small as 60 micrometers and with complex geometry (e.g., tortuosity) capable of supporting blood-mimicking fluid flow. Collectively, these results show that projection-based stereolithography allows for customizable fabrication of complex US phantoms.

Contextual cues from cancer cells govern cancer-associated fibroblast heterogeneity.

Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.

Disturbed flow disrupts the blood-brain barrier in a 3D bifurcation model.

The effect of disturbed flow profiles on the endothelium have been studied extensively in systemic vasculature, but less is known about the response of the blood-brain barrier (BBB) to these flow regimes. Here we investigate the effect of disturbed flow on the integrity of the BBB using a three-dimensional, perfusable bifurcation model consisting of a co-culture of endothelial cells with mural and glial cells. Experimental flow patterns predicted by computational fluid dynamics mimic in vivo flow regimes, specifically the presence of a recirculation zone immediately downstream of the bifurcation. Dextran permeability assays and immunostaining with markers for tight junctions show that barrier disruption is significantly greater in areas of disturbed flow compared to fully developed regions downstream of the bifurcation. Probing crosstalk between cell types suggests that disturbed flow causes barrier breakdown independent of endothelial-mural and endothelial-glial interaction. Overall, disturbed flow-induced disruption of the blood-brain barrier suggests that flow-mediated mechanisms may contribute to vascular pathologies in the central nervous system.

Generation of model tissues with dendritic vascular networks via sacrificial laser-sintered carbohydrate templates.

Sacrificial templates for patterning perfusable vascular networks in engineered tissues have been constrained in architectural complexity, owing to the limitations of extrusion-based 3D printing techniques. Here, we show that cell-laden hydrogels can be patterned with algorithmically generated dendritic vessel networks and other complex hierarchical networks by using sacrificial templates made from laser-sintered carbohydrate powders. We quantified and modulated gradients of cell proliferation and cell metabolism emerging in response to fluid convection through these networks and to diffusion of oxygen and metabolites out of them. We also show scalable strategies for the fabrication, perfusion culture and volumetric analysis of large tissue-like constructs with complex and heterogeneous internal vascular architectures. Perfusable dendritic networks in cell-laden hydrogels may help sustain thick and densely cellularized engineered tissues, and assist interrogations of the interplay between mass transport and tissue function.

Multivascular networks and functional intravascular topologies within biocompatible hydrogels.

Solid organs transport fluids through distinct vascular networks that are biophysically and biochemically entangled, creating complex three-dimensional (3D) transport regimes that have remained difficult to produce and study. We establish intravascular and multivascular design freedoms with photopolymerizable hydrogels by using food dye additives as biocompatible yet potent photoabsorbers for projection stereolithography. We demonstrate monolithic transparent hydrogels, produced in minutes, comprising efficient intravascular 3D fluid mixers and functional bicuspid valves. We further elaborate entangled vascular networks from space-filling mathematical topologies and explore the oxygenation and flow of human red blood cells during tidal ventilation and distension of a proximate airway. In addition, we deploy structured biodegradable hydrogel carriers in a rodent model of chronic liver injury to highlight the potential translational utility of this materials innovation.

Methods for fabrication and evaluation of a 3D microengineered model of myelinated peripheral nerve.

OBJECTIVE: The cost and low success rates of the neurological drug development pipeline have diverted the pharmaceutical industry to 'nerve-on-a-chip' systems as preclinical models to streamline drug development. We present a novel micro-engineered 3D hydrogel platform for the culture of myelinated embryonic peripheral neural tissue to serve as an effective in vitro model for electrophysiological and histological analysis that could be adopted for preclinical testing. APPROACH: Dorsal root ganglions (DRG) from 15 d old embryonic rats were cultured in 3D hydrogel platforms. The interaction between Schwann cells (SC) and neurons during axonal development and regeneration affects the direction of growth and the synthesis of myelin sheaths. Induction of myelination was performed with two approaches: the addition of exogenous SC and promoting migration of endogenous SC. MAIN RESULTS: Histological analysis of the preparation utilizing exogenous SC showed aligned, highly fasciculated axonal growth with noticeable myelin sheaths around axons. Separately, electrophysiological testing of the preparation utilizing endogenous SC showed increased amplitude of the compound action potential and nerve conduction velocity in the presence of ascorbic acid (AA). SIGNIFICANCE: This platform has immense potential to be a useful and translatable in vitro testing tool for drug discovery and myelination studies.