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As engineered tissues progress toward therapeutically relevant length scales and cell densities, it is critical to deliver oxygen and nutrients throughout the tissue volume via perfusion through vascular networks. Furthermore, seeding of endothelial cells within these networks can recapitulate the barrier function and vascular physiology of native blood vessels. In this protocol, we describe how to fabricate and assemble customizable open-source tissue perfusion chambers and catheterize tissue constructs inside them. Human endothelial cells are seeded along the lumenal surfaces of the tissue constructs, which are subsequently connected to fluid pumping equipment. The protocol is agnostic with respect to biofabrication methodology as well as cell and material composition, and thus can enable a wide variety of experimental designs. It takes ~14 h over the course of 3 d to prepare perfusion chambers and begin a perfusion experiment. We envision that this protocol will facilitate the adoption and standardization of perfusion tissue culture methods across the fields of biomaterials and tissue engineering. Customizable tissue perfusion chambers facilitate seeding and perfusion culture of human endothelial cells within vascularized tissue constructs.

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-β1. 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.

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. Cell-laden hydrogels can be patterned with algorithmically generated sacrificial dendritic vessel networks made of laser-sintered carbohydrate powders.

Recently developed biofabrication technologies are enabling the production of three-dimensional engineered tissues containing vascular networks which can deliver oxygen and nutrients across large tissue volumes. Tissues at this scale show promise for eventual regenerative medicine applications; however, the implantation and integration of these constructs in vivo remains poorly studied. Here, we introduce a surgical model for implantation and direct in-line vascular connection of 3D printed hydrogels in a porcine arteriovenous shunt configuration. Utilizing perfusable poly(ethylene glycol) diacrylate (PEGDA) hydrogels fabricated through projection stereolithography, we first optimized the implantation procedure in deceased piglets. Subsequently, we utilized the arteriovenous shunt model to evaluate blood flow through implanted PEGDA hydrogels in non-survivable studies. Connections between the host femoral artery and vein were robust and the patterned vascular channels withstood arterial pressure, permitting blood flow for 6 h. Our study demonstrates rapid prototyping of a biocompatible and perfusable hydrogel that can be implanted in vivo as a porcine arteriovenous shunt, suggesting a viable surgical approach for in-line implantation of bioprinted tissues, along with design considerations for future in vivo studies. We further envision that this surgical model may be broadly applicable for assessing whether biomaterials optimized for 3D printing and cell function can also withstand vascular cannulation and arterial blood pressure. This provides a crucial step toward generated transplantable engineered organs, demonstrating successful implantation of engineered tissues within host vasculature.

Thermofluidics actuates genetic patterning in artificial tissue., Spatial patterns of gene expression in living organisms orchestrate cell decisions in development, homeostasis, and disease. However, most methods for reconstructing gene patterning in 3D cell culture and artificial tissues are restricted by patterning depth and scale. We introduce a depth- and scale-flexible method to direct volumetric gene expression patterning in 3D artificial tissues, which we call “heat exchangers for actuation of transcription” (HEAT). This approach leverages fluid-based heat transfer from printed networks in the tissues to activate heat-inducible transgenes expressed by embedded cells. We show that gene expression patterning can be tuned both spatially and dynamically by varying channel network architecture, fluid temperature, fluid flow direction, and stimulation timing in a user-defined manner and maintained in vivo. We apply this approach to activate the 3D positional expression of Wnt ligands and Wnt/β-catenin pathway regulators, which are major regulators of development, homeostasis, regeneration, and cancer throughout the animal kingdom.

Cancer cells function as primary architects of the tumor microenvironment. Yet, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblasts (CAFs) are distinguishable on the basis of gene expression signatures they acquire in co-culture with epithelial- or mesenchymal-like lung adenocarcinoma (LUAD) cells. High expression of the EMT activator ZEB1 endows LUAD cells with the capacity to activate a soluble factor exchange that leads to CAF reprogramming, to generate CAF-led invasive projections in multicellular aggregates, and to respond to pro-metastatic signals from CAFs in mice. Thus, ZEB1-expressing LUAD cells are positioned at the apex of a signaling hierarchy in the tumor microenvironment.

Manufacturing has historically followed a mass production approach due to economies of scale and the engineering challenges of large-scale customization, leading to a one-size-fits-all paradigm. This manufacturing-centric approach has forced consumers and patients to adapt to medical devices in terms of anatomical fit and biological performance, often significantly decreasing their quality of life. In order to improve the biological interface with the human body, the materials science and bioengineering communities are rapidly adopting three-dimensional (3D) printing, which promises high precision, automation, and a customized fit. However, numerous design and engineering constraints, many posed by the fragile nature of living cells and soft gels, suggest exciting opportunities for further research in materials synthesis, characterization, and integration. Specifically, materials innovations in bioinks and support materials, coupled with improved 3D bioprinting processes for multiple materials, have the potential to empower the next generation of biology by enabling precision engineered tissues, organoids, and eventually whole organs.

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.

There is a growing clinical need to address high failure rates of small diameter (

Lung cancer is the foremost cause of cancer related deaths in the U.S. It is a heterogeneous disease composed of genetically and phenotypically distinct tumor cells surrounded by heterotypic cells and extracellular matrix dynamically interacting with the tumor cells. Research in lung cancer is often restricted to patient-derived tumor specimens, in vitro cell cultures and limited animal models, which fail to capture the cellular or microenvironment heterogeneity of the tumor. Therefore, our knowledge is primarily focused on cancer-cell autonomous aberrations. For a fundamental understanding of lung cancer progression and an exploration of therapeutic options, we focused our efforts to develop an Ex Vivo Tumor platform to culture tumors in 3D matrices, which retains tumor cell heterogeneity arising due to in vivo selection pressure and environmental influences and recapitulate responses of tumor cells to external manipulations. To establish this model, implanted syngeneic murine tumors from a mutant KRAS/p53 model were harvested to yield multicellular tumor aggregates followed by culture in 3D extracellular matrices. Using this system, we identified Src signaling as an important driver of invasion and metastasis in lung cancer and demonstrate that EVTs are a robust experimental tool bridging the gap between conventional in vitro and in vivo models.

Traditional three-dimensional printing technologies, originally meant for synthetic materials, have been adapted for biotechnology and regenerative medicine. The technology works but there is high variability in the resins needed for successful integration with biological components. We define these print resins as bioinks, which encompass printable materials that interface with biocomponents during or after the actual print or are involved in the structural construction of scaffolds that will interface with biocomponents. Although synthetic materials are traditionally accepted as mechanically robust, are finely tunable, and are processed using commercially available methods, natural materials have inherent biochemical cues that induce cell attachment and differentiation. The balance of the strong properties of these materials has led to combined technologies such as co-printing and hybrid bioinks, and the development of specialized resins such cell-laden, sacrificial, and support bioinks. Here, we review the evolution of bioinks from traditional methods and materials to highly complex and specialized resins that have been used successfully for in vitro and in vivo bioengineering applications.

SUMMARY 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., In brief Bota-Rabassedas et al. show that EMT in lung adenocarcinoma cells activates a secretory process that governs CAF heterogeneity and, in turn, sensitizes lung adenocarcinoma cells to pro-metastatic signals from CAFs. Thus, EMT positions lung adenocarcinoma cells at the apex of a signaling hierarchy in the tumor microenvironment., Graphical Abstract

The field of tissue engineering has advanced the development of increasingly biocompatible materials to mimic the extracellular matrix of vascularized tissue. However, a majority of studies instead rely on a multiday inosculation between engineered vessels and host vasculature rather than the direct connection of engineered microvascular networks with host vasculature. We have previously demonstrated that the rapid casting of three-dimensionally-printed (3D) sacrificial carbohydrate glass is an expeditious and a reliable method of creating scaffolds with 3D microvessel networks. Here, we describe a new surgical technique to directly connect host femoral arteries to patterned microvessel networks. Vessel networks were connected in vivo in a rat femoral artery graft model. We utilized laser Doppler imaging to monitor hind limb ischemia for several hours after implantation and thus measured the vascular patency of implants that were anastomosed to the femoral artery. This study may provide a method to overcome the challenge of rapid oxygen and nutrient delivery to engineered vascularized tissues implanted in vivo.

Background: Though in vivo models provide the most physiologically relevant environment for studying tissue function, in vitro studies provide researchers with explicit control over experimental conditions and the potential to develop high throughput testing methods. In recent years, advancements in developmental biology research and imaging techniques have significantly improved our understanding of the processes involved in vascular development. However, the task of recreating the complex, multi-scale vasculature seen in in vivo systems remains elusive. Results: 3D bioprinting offers a potential method to generate controlled vascular networks with hierarchical structure approaching that of in vivo networks. Bioprinting is an interdisciplinary field that relies on advances in 3D printing technology along with advances in imaging and computational modeling, which allow researchers to monitor cellular function and to better understand cellular environment within the printed tissue. Conclusions: As bioprinting technologies improve with regards to resolution, printing speed, available materials, and automation, 3D printing could be used to generate highly controlled vascularized tissues in a high throughput manner for use in regenerative medicine and the development of in vitro tissue models for research in developmental biology and vascular diseases. Developmental Dynamics 244:629–640, 2015. © 2015 Wiley Periodicals, Inc.

Engineered tissue constructs have the potential to augment or replace whole organ transplantation for the treatment of liver failure. Poly(ethylene glycol) (PEG)-based systems are particularly promising for the construction of engineered liver tissue due to their biocompatibility and amenability to modular addition of bioactive factors. To date, primary hepatocytes have been successfully encapsulated in non-degradable hydrogels based on PEG-diacrylate (PEGDA). In this study, we describe a hydrogel system based on PEG-diacrylamide (PEGDAAm) containing matrix-metalloproteinase sensitive (MMP-sensitive) peptide in the hydrogel backbone that is suitable for hepatocyte culture both in vitro and after implantation. By replacing hydrolytically unstable esters in PEGDA with amides in PEGDAAm, resultant hydrogels resisted non-specific hydrolysis, while still allowing for MMP-mediated hydrogel degradation. Optimization of polymerization conditions, hepatocellular density, and multicellular tissue composition modulated both the magnitude and longevity of hepatic function in vitro. Importantly, hepatic PEGDAAm-based tissues survived and functioned for over 3 weeks after implantation ectopically in the intraperitoneal (IP) space of nude mice. Together, these studies suggest that MMP-sensitive PEGDAAm-based hydrogels may be a useful material system for applications in tissue engineering and regenerative medicine., National Institutes of Health (U.S.) (R01EB008396), National Institutes of Health (U.S.) (R01DK85713), National Institutes of Health (U.S.) (EB00262)

While tissue-mimicking phantoms have been useful in the validation of ultrasonic equipment and image processing techniques, they are often restricted to simple vascular geometries due to limitations in fabrication techniques. To address this need, a novel technique has been employed to fabricate 3D-printed photo-curable poly(ethylene glycol) (PEG) hydrogel constructs containing complex, small-scale (e.g., 100s of μm in diameter) vascular channels that can be imaged with ultrasound. In this study, we use color Doppler ultrasound to obtain 3D velocity vector fields through vascular geometries within hydrogel phantoms and compare these results to optical flow assessment and numerical modeling results.

Here, we investigate the tubulogenic potential of commercially-sourced iPS-ECs with and without supporting commercially-sourced hMSCs within 3D natural fibrin or semi-synthetic gelatin methacrylate (GelMA) hydrogels. We developed a selectable dual color third generation lentiviral reporter (hEF1α-H2B-mOrange2-IRES-EGFP PGK-Puro) to differentially label the nucleus and cytoplasm of iPS-ECs which allowed real-time tracking of key steps of vascular morphogenesis such as vacuole formation and coalescence to form shared multicellular lumens. We implement 3D quantification of the network character and validate that transduced and untransduced iPS-ECs can form tubules in fibrin with or without supporting hMSCs. In addition to natural fibrin gels, we also investigated tubulogenesis in GelMA, a semi-synthetic material that has received increased interest due to its ability to be photopatterned and 3D printed, and which may thus boost development of complex 3D models for regenerative medicine studies. We find that iPS-ECs alone have a muted tubulogenic response within GelMA, but that their tubulogenic response is enhanced when they are co-cultured with a small fraction of hMSCs (2% of total cells). Our work bolsters previous findings by validating established tubulogenic mechanisms with commercially available iPS-ECs, and we expect our findings will benefit biologic studies of vasculogenesis and will have applications in tissue engineering to pre-vascularize tissue constructs which are fabricated with advanced photopatterning and three-dimensional printing.

Tissue vascularization and integration with host circulation remains a key barrier to the translation of engineered tissues into clinically relevant therapies. Here, we used a microtissue molding approach to demonstrate that constructs containing highly aligned “cords” of endothelial cells triggered the formation of new capillaries along the length of the patterned cords. These vessels became perfused with host blood as early as 3 d post implantation and became progressively more mature through 28 d. Immunohistochemical analysis showed that the neovessels were composed of human and mouse endothelial cells and exhibited a mature phenotype, as indicated by the presence of alpha-smooth muscle actin–positive pericytes. Implantation of cords with a prescribed geometry demonstrated that they provided a template that defined the neovascular architecture in vivo. To explore the utility of this geometric control, we implanted primary rat and human hepatocyte constructs containing randomly organized endothelial networks vs. ordered cords. We found substantially enhanced hepatic survival and function in the constructs containing ordered cords following transplantation in mice. These findings demonstrate the importance of multicellular architecture in tissue integration and function, and our approach provides a unique strategy to engineer vascular architecture.

Fabrication of vascular networks within engineered tissue remains one of the greatest challenges facing the fields of biomaterials and tissue engineering. Historically, the structural complexity of vascular networks has limited their fabrication in tissues engineered in vitro. Recently, however, key advances have been made in constructing fluidic networks within biomaterials, suggesting a strategy for fabricating the architecture of the vasculature. These techniques build on emerging technologies within the microfluidics community as well as on 3D printing. The freeform fabrication capabilities of 3D printing are allowing investigators to fabricate fluidic networks with complex architecture inside biomaterial matrices. In this review, we examine the most exciting 3D printing-based techniques in this area. We also discuss opportunities for using these techniques to address open questions in vascular biology and biophysics, as well as for engineering therapeutic tissue substitutes in vitro.

Aggregates of cells, also known as multicellular aggregates (MCAs), have been used as microscale tissues in the fields of cancer biology, regenerative medicine, and developmental biology for many decades. However, small MCAs (fewer than 100 cells per aggregate) have remained challenging to manufacture in large quantities at high uniformity. Forced aggregation into microwells offers a promising solution for forming consistent aggregates, but commercial sources of microwells are expensive, complicated to manufacture, or lack the surface packing densities that would significantly improve MCA production. To address these concerns, we custom-modified a commercial laser cutter to provide complete control over laser ablation and directly generate microwells in a poly(dimethylsiloxane) (PDMS) substrate. We achieved ultra rapid microwell production speeds (>50 000 microwells per h) at high areal packing densities (1800 microwells per cm2) and over large surface areas for cell culture (60 cm2). Variation of the PDMS substrate distance from the laser focal plane during ablation allowed for the generation of microwells with a variety of sizes, contours, and aspect ratios. Casting of high-fidelity microneedle masters in polyurethane allowed for non-ablative microwell reproduction through replica molding. MCAs of human bone marrow derived mesenchymal stem cells (hMSCs), murine 344SQ metastatic adenocarcinoma cells, and human C4-2 prostate cancer cells were generated in our system with high uniformity within 24 hours, and computer vision software aided in the ultra-high-throughput analysis of harvested aggregates. Moreover, MCAs maintained invasive capabilities in 3D migration assays. In particular, 344SQ MCAs demonstrated epithelial lumen formation on Matrigel, and underwent EMT and invasion in the presence of TGF-β. We expect this technique to find broad utility in the generation and cultivation of cancer cell aggregates, primary cell aggregates, and embryoid bodies.

Selective Laser Sintering (SLS) is an additive manufacturing process that uses a laser to fuse powdered starting materials into solid 3D structures. Despite the potential for fabrication of complex, high-resolution structures with SLS using diverse starting materials (including biomaterials), prohibitive costs of commercial SLS systems have hindered the wide adoption of this technology in the scientific community. Here, we developed a low-cost, open-source SLS system (OpenSLS) and demonstrated its capacity to fabricate structures in nylon with sub-millimeter features and overhanging regions. Subsequently, we demonstrated fabrication of polycaprolactone (PCL) into macroporous structures such as a diamond lattice. Widespread interest in using PCL for bone tissue engineering suggests that PCL lattices are relevant model scaffold geometries for engineering bone. SLS of materials with large powder grain size (~500 μm) leads to part surfaces with high roughness, so we further introduced a simple vapor-smoothing technique to reduce the surface roughness of sintered PCL structures which further improves their elastic modulus and yield stress. Vapor-smoothed PCL can also be used for sacrificial templating of perfusable fluidic networks within orthogonal materials such as poly(dimethylsiloxane) silicone. Finally, we demonstrated that human mesenchymal stem cells were able to adhere, survive, and differentiate down an osteogenic lineage on sintered and smoothed PCL surfaces, suggesting that OpenSLS has the potential to produce PCL scaffolds useful for cell studies. OpenSLS provides the scientific community with an accessible platform for the study of laser sintering and the fabrication of complex geometries in diverse materials.

Recent methods have revealed that cells on planar substrates exert both shear (in-plane) and normal (out-of-plane) tractions against the extracellular matrix (ECM). However, the location and origin of the normal tractions with respect to the adhesive and cytoskeletal elements of cells have not been elucidated. We developed a high-spatiotemporal-resolution, multidimensional (2.5D) traction force microscopy to measure and model the full 3D nature of cellular forces on planar 2D surfaces. We show that shear tractions are centered under elongated focal adhesions whereas upward and downward normal tractions are detected on distal (toward the cell edge) and proximal (toward the cell body) ends of adhesions, respectively. Together, these forces produce significant rotational moments about focal adhesions in both protruding and retracting peripheral regions. Temporal 2.5D traction force microscopy analysis of migrating and spreading cells shows that these rotational moments are highly dynamic, propagating outward with the leading edge of the cell. Finally, we developed a finite element model to examine how rotational moments could be generated about focal adhesions in a thin lamella. Our model suggests that rotational moments can be generated largely via shear lag transfer to the underlying ECM from actomyosin contractility applied at the intracellular surface of a rigid adhesion of finite thickness. Together, these data demonstrate and probe the origin of a previously unappreciated multidimensional stress profile associated with adhesions and highlight the importance of new approaches to characterize cellular forces.

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core [1]. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture [2–4]. Here, we 3D printed rigid filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks which could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization, and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices (ECMs), and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.

The implementation of engineered surfaces presenting micrometer-sized patterns of cell adhesive ligands against a biologically inert background has led to numerous discoveries in fundamental cell biology. While existing surface patterning strategies allow patterning of a single ligand, it is still challenging to fabricate surfaces displaying multiple patterned ligands. To address this issue we implemented laser scanning lithography (LSL), a laser-based thermal desorption technique, to fabricate multifaceted, micropatterned surfaces that display independent arrays of subcellular-sized patterns of multiple adhesive ligands with each ligand confi ned to its own array. We demonstrate that LSL is a highly versatile “maskless” surface patterning strategy that provides the ability to create patterns with features ranging from 460 nm to 100 μ m, topography ranging from − 1 to 17 nm, and to fabricate both stepwise and smooth ligand surface density gradients. As validation for their use in cell studies, surfaces presenting orthogonally interwoven arrays of 1 μ m × 8 μ m elliptical patterns of Gly-Arg-Gly-Asp-terminated alkanethiol self-assembled monolayers and human plasma fi bronectin are produced. Human umbilical vein endothelial cells cultured on these multifaceted surfaces form adhesion sites to both ligands simultaneously and utilize both ligands for lamella formation during migration. The ability to create multifaceted, patterned surfaces with tight control over pattern size, spacing, and topography provides a platform to simultaneously investigate the complex interactions of extracellular matrix geometry, biochemistry, and topography on cell adhesion and downstream cell behavior.

Regenerative medicine offers the potential to develop patient-specific three-dimensional (3D) tissue constructs for treatment of human pathologies. However, our field generally lacks the ability to recreate, in vitro , the heterogeneous patterns of cells and matrix which are hallmarks of native tissue. 3D printing and patterning techniques now offer the ability to control the shape and architecture of constructs with high precision, and have transitioned to include vasculature to boost mass transport. The controlled exploration of the wide parameter space available with biocompatible manufacturing technologies will allow us to engineer 3D matrices for future studies assessing cell behavior in heterogeneous and physiologically relevant settings.

Techniques that control the spatial presentation of moieties such as cell adhesion peptides on surfaces are important for the advancement of tissue engineering, the elucidation of fundamental tissue structure-function relationships, and the formation of immobilized cell and protein arrays for biotechnology. Cell and protein patterns have previously been created using a range of methods, including photolithography 3] and soft lithographic approaches such as microcontact printing, microfluidic patterning, 6] and micromolding. 8] The patterned substrates have generally been modified silicon or glass, 10] whereas surface patterning of deformable, solvated substrates has not received a similar degree of attention. High fidelity patterning of deformable, solvated substrates is challenging, particularly since many commonly used patterning techniques cannot be readily applied. For example, microcontact printing is generally not applicable to such substrates due to lateral diffusion of the patterned molecule when the inked stamp comes into contact with the solvated substrate. Given the importance of deformable, solvated substrates, such as hydrogels, as scaffolds for soft tissue engineering applications, development of methods for direct, high fidelity surface patterning of these substrates is desirable.

We have developed proteolytically degradable hydrogels with covalently immobilized fluorogenic protease substrates to visualize extracellular proteolytic activity and cell migration in three dimensions. Dye quenched-bovine serum albumin (DQ-BSA), a quenched, proteolytically activated fluorogenic substrate, was conjugated to poly(ethylene glycol) (PEG)-monoacrylate, and the product (DQ-BSA-PEG) was then covalently incorporated into proteolytically degradable and cell adhesive PEG hydrogels via photopolymerization. The DQ-BSA-PEG substrate in solution and incorporated into hydrogels exhibited significantly enhanced fluorescence after exposure to enzymes. Fibroblasts seeded within this hydrogel spread in three dimensions and extended lamellipodia. Cell migration and proteolytic activity were visualized using confocal microscopy. Proteolytic activity was concentrated near cell surfaces and remained present in the tracks where cell migration had occurred.

The fabrication of scaffolds for tissue engineering requires elements of customization depending on the application and is often limited due to the flexibility of the processing technique. This investigation seeks to address this obstacle by utilizing an open-source three-dimensional printing (3DP) system that allows vast customizability and facilitates reproduction of experiments. The effects of processing parameters on printed poly(ε-caprolactone) scaffolds with uniform and gradient pore architectures have been characterized with respect to fiber and pore morphology and mechanical properties. The results demonstrate the ability to tailor the fiber diameter, pore size, and porosity through modification of pressure, printing speed, and programmed fiber spacing. A model was also used to predict the compressive mechanical properties of uniform and gradient scaffolds, and it was found that modulus and yield strength declined with increasing porosity. The use of open-source 3DP technologies for printing tissue engineering scaffolds provides a flexible system that can be readily modified at a low cost and is supported by community documentation. In this manner, the 3DP system is more accessible to the scientific community, which further facilitates the translation of these technologies toward successful tissue engineering strategies.

Effective utilization of three-dimensional printing for tissue and organ engineering remains nontrivial. Here, Jordan Miller identifies key challenges and discusses conceptual targets on the horizon., How structure relates to function—across spatial scales, from the single molecule to the whole organism—is a central theme in biology. Bioengineers, however, wrestle with the converse question: will function follow form? That is, we struggle to approximate the architecture of living tissues experimentally, hoping that the structure we create will lead to the function we desire. A new means to explore the relationship between form and function in living tissue has arrived with three-dimensional printing, but the technology is not without limitations.

The fabrication of scaffolds for tissue engineering requires elements of customization depending on the application and is often limited due to the flexibility of the processing technique. This investigation seeks to address this obstacle by utilizing an open-source three-dimensional printing (3DP) system that allows vast customizability and facilitates reproduction of experiments. The effects of processing parameters on printed poly(e-caprolactone) scaffolds with uniform and gradient pore architectures have been characterized with respect to fiber and pore morphology and mechanical properties. The results demonstrate the ability to tailor the fiber diameter, pore size, and porosity through modification of pressure, printing speed, and programmed fiber spacing. A model was also used to predict the compressive mechanical properties of uniform and gradient scaffolds, and it was found that modulus and yield strength declined with increasing porosity. The use of open-source 3DP technologies for printing tissue-engineering scaffolds provides a flexible system that can be readily modified at a low cost and is supported by community documentation. In this manner, the 3DP system is more accessible to the scientific community, which further facilitates the translation of these technologies toward successful tissue-engineering strategies.

By delivering payloads in response to specific exogenous stimuli, smart bacterial therapeutics have the potential to overcome many limitations of conventional therapies, including poor targeting specificity and dosage control in current cancer treatments. Although not yet explored as a trigger for bacterial drug delivery, light is an ideal induction mechanism because it offers fine spatiotemporal control and is easily and safely administered. Using recent advances in optogenetics, we have engineered two strains of Escherichia coli to secrete a potent mammalian cytotoxin in response to blue or red light. The tools in this study demonstrate the initial feasibility of light-activated bacterial therapeutics for applications such as tumor cytolysis, and their modular nature should enable simple substitution of other payloads of interest.

The ability to self-regulate can become impaired when people are required to engage in successive acts of effortful self-control, even when self-control occurs in different domains. Here, we used functional neuroimaging to test whether engaging in effortful inhibition in the cognitive domain would lead to putative dysfunction in the emotional domain. Forty-eight participants viewed images of emotional scenes during functional magnetic resonance imaging in two sessions that were separated by a challenging attention control task that required effortful inhibition (depletion group) or not (control group). Compared to the control group, depleted participants showed increased activity in the left amygdala to negative but not to positive or neutral scenes. Moreover, whereas the control group showed reduced amygdala activity to all scene types (i.e. habituation), the depletion group showed increased amygdala activity relative to their pre-depletion baseline; however this was only significant for negative scenes. Finally, depleted participants showed reduced functional connectivity between the left amygdala and ventromedial prefrontal cortex during negative scene processing. These findings demonstrate that consuming self-regulatory resources leads to an exaggerated neural response to emotional material that appears specific to negatively valenced stimuli and further suggests a failure to recruit top-down prefrontal regions involved in emotion regulation.

Quantitative measurements of cell-generated forces have heretofore required that cells be cultured on two-dimensional substrates. We describe a technique to quantitatively measure three-dimensional traction forces exerted by cells fully encapsulated in well-defined elastic hydrogel matrices. Using this approach we measured traction forces for several cell types in various contexts and revealed patterns of force generation attributable to morphologically distinct regions of cells as they extend into the surrounding matrix.

Synthetic hydrogels based on poly(ethylene glycol) (PEG) have been used as biomaterials for cell biology and tissue engineering investigations. Bioactive PEG-based gels have largely relied on heterobifunctional or multi-arm PEG precursors that can be difficult to synthesize and characterize or expensive to obtain. Here, we report an alternative strategy, which instead uses inexpensive and readily available PEG precursors to simplify reactant sourcing. This new approach provides a robust system in which to probe cellular interactions with the microenvironment. We used the step-growth polymerization of PEG diacrylate (PEGDA, 3400Da) with bis-cysteine matrix metalloproteinase (MMP)-sensitive peptides via Michael-type addition to form biodegradable photoactive macromers of the form acrylate-PEG-(peptide-PEG)(m)-acrylate. The molecular weight (MW) of these macromers is controlled by the stoichiometry of the reaction, with a high proportion of resultant macromer species greater than 500kDa. In addition, the polydispersity of these materials was nearly identical for three different MMP-sensitive peptide sequences subjected to the same reaction conditions. When photopolymerized into hydrogels, these high MW materials exhibit increased swelling and sensitivity to collagenase-mediated degradation as compared to previously published PEG hydrogel systems. Cell-adhesive acrylate-PEG-CGRGDS was synthesized similarly and its immobilization and stability in solid hydrogels was characterized with a modified Lowry assay. To illustrate the functional utility of this approach in a biological setting, we applied this system to develop materials that promote angiogenesis in an ex vivo aortic arch explant assay. We demonstrate the formation and invasion of new sprouts mediated by endothelial cells into the hydrogels from embedded embryonic chick aortic arches. Furthermore, we show that this capillary sprouting and three-dimensional migration of endothelial cells can be tuned by engineering the MMP-susceptibility of the hydrogels and the presence of functional immobilized adhesive ligands (CGRGDS vs. CGRGES peptide). The facile chemistry described and significant cellular responses observed suggest the usefulness of these materials in a variety of in vitro and ex vivo biologic investigations, and may aid in the design or refinement of material systems for a range of tissue engineering approaches.

Development of robust 3D tissue analogs in vitro is limited by passive, diffusional mass transport. Perfused microfluidic tissue engineering scaffolds hold the promise to improve mass transport limitations and promote the development of complex, metabolically dense, and clinically relevant tissues. We report a simple and robust multilayer replica molding technique in which poly(dimethylsiloxane) (PDMS) and poly(ethylene glycol) diacrylate (PEGDA) are serially replica molded to develop microfluidic PEGDA hydrogel networks embedded within independently fabricated PDMS housings. We demonstrate the ability to control solute-scaffold effective diffusivity as a function of solute molecular weight and hydrogel concentration. Within cell laden microfluidic hydrogels, we demonstrate increased cellular viability in perfused hydrogel systems compared to static controls. We observed a significant increase in cell viability at all time points greater than zero at distances up to 1 mm from the perfused channel. Knowledge of spatiotemporal mass transport and cell viability gradients provides useful engineering design parameters necessary to maximize overall scaffold viability and metabolic density. This work has applications in the development of hydrogels as in vitro diagnostics and ultimately as regenerative medicine based therapeutics.