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Vascularization of 3D engineered tissues poses a great challenge in the field of tissue engineering. One promising approach for vascularizing engineered tissue is cocultivation with endothelial cells (ECs), which spontaneously self-assemble into a natural capillary network in the presence of supportive cells. However, the ECs do not self-assemble according to physiological hierarchy which is required to support blood supply. This work describes the design and fabrication of an AngioTube, a biodegradable engineered macro-vessel surrounded by cylindrical micro-channel array, which is designed to support physiological flow distribution and enable the integration with living capillaries. The well-defined geometry of the engineered micro-channels guides endothelial cells to form patent micro-vessels which sprouted in accordance with the channel orientation. Three different in-vitro models were used to demonstrate anastomosis of these engineered micro-vessels with self-assembled vascular networks. Finally, in-vivo functionality was demonstrated by direct anastomosis with the femoral artery in a rat hindlimb model. This unique approach proposes a new micro-fabrication strategy which introduces uncompromised micro-fluidic device geometrical accuracy at the tissue-scale level. STATEMENT OF SIGNIFICANCE: This study proposes a micro-fabrication strategy suitable for processing real-scale cylindrical implants with very high accuracy, which will enable translation of the high-resolution geometry of micro-fluidic devices to clinically relevant implants containing functional multi-scale vascular networks. Moreover, this approach promises to advance the field of tissue engineering by opening new opportunities to explore the impact of well controlled and uncompromised 3D micro-geometry on cellular behavior.

A growing number of technologies are being developed to promote vascularization and innervation in engineered tissues. Organ-on-a-chip, organoid and 3D printing technologies, as well as pre-vascularized and oriented scaffolds, have been employed for vascularization and innervation of engineered tissues both in vivo and in vitro. Both vascularization and innervation are critical for neural tissue engineering, as these complex tissues require provision of both blood and nerves. As such, this review will have particular focus on neural tissue engineering. We examine state-of-the-art approaches for tissue vascularization and innervation and identify promising methods for developing vascularized and innervated engineered neural constructs.

Progress in bottom-up synthetic biology has stimulated the development of synthetic cells (SCs), autonomous protein-manufacturing particles, as dynamic biomimetics for replacing diseased natural cells and addressing medical needs. Here, we report that SCs genetically encoded to produce proangiogenic factors triggered the physiological process of neovascularization in mice. The SCs were constructed of giant lipid vesicles and were optimized to facilitate enhanced protein production. When introduced with the appropriate genetic code, the SCs synthesized a recombinant human basic fibroblast growth factor (bFGF), reaching expression levels of up to 9⋅106protein copies per SC. In culture, the SCs induced endothelial cell proliferation, migration, tube formation, and angiogenesis-related intracellular signaling, confirming their proangiogenic activity. Integrating the SCs with bioengineered constructs bearing endothelial cells promoted the remodeling of mature vascular networks, supported by a collagen-IV basement membrane–like matrix. In vivo, prolonged local administration of the SCs in mice triggered the infiltration of blood vessels into implanted Matrigel plugs without recorded systemic immunogenicity. These findings emphasize the potential of SCs as therapeutic platforms for activating physiological processes by autonomously producing biological drugs inside the body.

Engineering implantable, functional, thick tissues requires designing a hierarchical vascular network. 3D bioprinting is a technology used to create tissues by adding layer upon layer of printable biomaterials, termed bioinks, and cells in an orderly and automatic manner, which allows for creating highly intricate structures that traditional tissue engineering techniques cannot achieve. Thus, 3D bioprinting is an appealing in vitro approach to mimic the native vasculature complex structure, ranging from millimetric vessels to microvascular networks. Advances in 3D bioprinting in granular hydrogels enabled the high-resolution extrusion of low-viscosity extracellular matrix-based bioinks. This work presents a combined 3D bioprinting and sacrificial mold-based 3D printing approach for fabricating engineered vascularized tissue flaps. 3D bioprinting of endothelial and support cells using recombinant collagen-methacrylate bioink within a gelatin support bath is utilized for the fabrication of a self-assembled capillary network. This printed microvasculature is assembled around a mesoscale vessel-like porous scaffold, fabricated using a sacrificial 3D printed mold, and is seeded with endothelial cells. This assembly induces the endothelium of the mesoscale vessel to anastomose with the surrounding capillary network, establishing a hierarchical vascular network within an engineered tissue flap. The engineered flap is then directly implanted by surgical anastomosis to a rat femoral artery using a cuff technique. The described methods can be expanded for the fabrication of various vascularized tissue flaps for use in reconstruction surgery and vascularization studies.

PolyHIPEs, macroporous polymers templated within high internal phase emulsions (HIPEs), emulsions with over 74% internal phase, are almost always crosslinked to prevent collapse during drying. Here, elastomeric poly(urethane urea) (PUU) polyHIPEs with highly interconnected open-cell structures were synthesized in water-in-oil (w/o) HIPEs. The urethane reactions between a diisocyanate and an oligomeric polyol (a poly(e-caprolactone) (PCL) diol, a PCL triol, or an aliphatic polycarbonate (PC) diol) occurred in the external phase, while the water-diisocyanate urea reactions occurred at the oil–water interface. The resulting linear macromolecular structures produced unusually soluble polyHIPEs whose thermal transitions and mechanical properties could be fine-tuned through the polyol macromolecular structure and molecular weight and through the urea content (the hydroxyl to isocyanate ratio). The polyHIPEs underwent almost complete degradation in 3 M NaOH, with the PCL-based PUUs undergoing a significantly more rapid degradation. Cells growing in the polyHIPEs adhered to the walls, spread, and penetrated into the porous structures. This work demonstrates that elastomeric, degradable polyHIPEs with potential for tissue engineering applications can be synthesized through the emulsion templating of PUUs with linear macromolecular structures.

Generating comprehensive image maps, while preserving spatial three-dimensional (3D) context, is essential in order to locate and assess quantitatively specific cellular features and cell-cell interactions during organ development. Despite recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on two-dimensional histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in three dimensions and map tissue interactions at key time points in the mouse embryo. We demonstrate the utility of the approach by providing volumetric data, 3D distribution of three main cellular components (epithelial, mesenchymal and endothelial cells) within the developing pancreas, and quantification of their relative cellular abundance within the tissue. Interestingly, our 3D images show that endocrine cells are constantly and increasingly in contact with endothelial cells forming small vessels, whereas the interactions with mesenchymal cells decrease over time. These findings suggest distinct cell-cell interaction requirements for early endocrine cell specification and late differentiation. Lastly, we combine our image data in an open-source online repository (referred to as the Pancreas Embryonic Cell Atlas)., Development, 149 (3), ISSN:0950-1991, ISSN:1477-9129

Cell-based meat (CBM) production is a promising technology that could generate meat without the need of animal agriculture. The generation of tissue requires a three-dimensional (3D) scaffold to provide support to the cells and mimic the extracellular matrix (ECM). For CBM, the scaffold needs to be edible and have suitable nutritional value and texture. Here, we demonstrate the use of textured soy protein—an edible porous protein-based biomaterial—as a novel CBM scaffold that can support cell attachment and proliferation to create a 3D engineered bovine muscle tissue. The media composition was optimized for 3D bovine satellite cell (BSC) proliferation and differentiation by adding myogenic-related growth factors. Myogenesis of several cell combinations was compared, and elevated myogenesis and ECM deposition were shown in co-culture of BSCs with bovine smooth muscle cells and tri-cultures of BSCs, bovine smooth muscle cells and bovine endothelial cells. The expression of proteins associated with ECM gene sets was increased in the co-culture compared with BSC monoculture. Volunteers tasted the product after cooking and noted its meaty flavour and sensorial attributes, achieving the goal of replicating the sensation and texture of a meat bite. This approach represents a step forward for the applied production of CBM as a food product. Animal skeletal muscle cell culture has the potential to provide new protein sources without the need for conventional animal agriculture. Ben-Ayre et. al. address one of the challenges of growing cell-based meat in vitro by using textured soy protein as an extracellular matrix scaffold. This approach allowed three-dimensional skeletal muscle cell development to produce a meat-like product for human consumption.

The past decade has witnessed tremendous endeavors to deliver novel preclinical

In recent years, extracellular vesicles (EVs), specifically exosomes, have emerged as a promising strategy for treating a wide spectrum of pathologies, such as cancer and COVID-19, as well as promoting tissue regeneration in various conditions, including cardiomyopathies and spinal cord injuries. Despite the great potential of EV-based therapies, poor yield and unscalable production of EVs remain big challenges to overcome to translate these types of treatment to clinical practices. Here, we review different strategies for enhancing EV yield by physical, biological or chemical means. Some of these novel approaches can lead to about 100-fold increase in EV production yield, thus bringing closer the clinical translation with regard to scalability and efficiency.

Description, GLUT4-overexpressing engineered muscle constructs improve mice diabetic state and show a potential as a therapeutic tool., Skeletal muscle insulin resistance is a main defect in type 2 diabetes (T2D), which is associated with impaired function and content of glucose transporter type 4 (GLUT4). GLUT4 overexpression in skeletal muscle tissue can improve glucose homeostasis. Therefore, we created an engineered muscle construct (EMC) composed of GLUT4-overexpressing (OEG4) cells. The ability of the engineered implants to reduce fasting glucose levels was tested in diet-induced obesity mice. Decrease and stabilization of basal glucose levels were apparent up to 4 months after implantation. Analysis of the retrieved constructs showed elevated expression of myokines and proteins related to metabolic processes. In addition, we validated the efficiency of OEG4-EMCs in insulin-resistant mice. Following high glucose load administration, mice showed improved glucose tolerance. Our data indicate that OEG4-EMC implant is an efficient mode for restoring insulin sensitivity and improving glucose homeostasis in diabetic mice. Such procedure is a potential innovative modality for T2D therapy.

Cultivated meat harnesses tissue engineering (TE) concepts to create sustainable, edible muscle tissues, for addressing the rising meat product demands and their global consequences. As 3D-printing is a promising method for creating thick and complex structures, two plant-protein-enriched scaffolding compositions were primarily assessed in our work as 3D-printable platforms for bovine satellite cells (BSC) maturation. Mixtures of pea protein isolate (PPI) and soy protein isolate (SPI) with RGD-modified alginate (Alginate(RGD)) were evaluated as prefabricated mold-based and 3D-printed scaffolds for BSC cultivation, and ultimately, as potential bioinks for cellular printing. Mold-based protein enriched scaffolds exhibited elevated stability and stiffness compared to ones made of Alginate(RGD) alone, while allowing unhindered BSC spreading and maturation. Extrusion based 3D-printing with the two compositions was then developed, while using an edible, removable agar support bath. Successfully fabricated constructs with well-defined geometries supported BSC attachment and differentiation. Finally, cellular bioprinting was demonstrated with PPI-enriched bioinks. Cell recovery post-printing was observed in two cultivation configurations, reaching ∼80-90% viability over time. Moreover, cells could mature within 3D-printed cellular constructs. As animal-derived materials were avoided in our scaffold fabrication process, and pea-protein is known for its low allergic risk, these findings have great promise for further cultivated meat research.

Individuals with spinal cord injury (SCI) usually suffer from permanent neurological deficits, while spontaneous recovery and therapeutic efficacy are limited. Here, we demonstrate that when given intranasally, exosomes derived from mesenchymal stem cells (MSC-Exo) could pass the blood brain barrier and migrate to the injured spinal cord area. Furthermore, MSC-Exo loaded with phosphatase and tensin homolog small interfering RNA (ExoPTEN) could attenuate the expression of PTEN in the injured spinal cord region following intranasal administrations. In addition, the loaded MSC-Exo considerably enhanced axonal growth and neovascularization, while reducing microgliosis and astrogliosis. The intranasal ExoPTEN therapy could also partly improve structural and electrophysiological function and, most importantly, significantly elicited functional recovery in rats with complete SCI. The results imply that intranasal ExoPTEN may be used clinically to promote recovery for SCI individuals.

Graft vascularization remains one of the most critical challenges facing tissue-engineering experts in their attempt to create thick transplantable tissues and organs. In vitro prevascularization of engineered tissues has been suggested to promote rapid anastomosis between the graft and host vasculatures; however, thrombotic events have been reported upon graft implantation. Here, we aimed to determine whether in vitro vessel maturation in transplantable grafts can accelerate vascular integration and graft perfusion and prevent thrombotic events in the grafts. To this end, endothelial cells and fibroblasts were cocultured on 3D scaffolds for 1, 7, or 14 d to form vasculature with different maturation degrees. Monitoring graft–host interactions postimplantation demonstrated that the 14-d in vitro-cultured grafts, bearing more mature and complex vessel networks as indicated by elongated and branched vessel structures, had increased graft–host vessel anastomosis; host vessel penetration into the graft increased approximately eightfold, and graft perfusion increased sixfold. The presence of developed vessel networks prevented clot accumulation in the grafts. Conversely, short-term cultured constructs demonstrated poor vascularization and increased thrombus formation. Elevated expression levels of coagulation factors, von Willebrand factor (vWF), and tissue factor (TF), were demonstrated in constructs bearing less mature vasculature. To conclude, these findings demonstrate the importance of establishing mature and complex vessel networks in engineered tissues before implantation to promote anastomosis with the host and accelerate graft perfusion.

Autologous muscle flaps are commonly used to reconstruct defects that involve muscle impairment. To maintain viability and functionality of these flaps, they must be properly vascularized and innervated. Tissue-engineered muscles could potentially replace autologous muscle tissue, but still require establishment of sufficient innervation to ensure functionality. In this study, we explored the possibility of innervating engineered muscle grafts transplanted to an abdominal wall defect in mice, by transferring the native femoral nerve to the graft. Six weeks posttransplantation, nerve conduction studies and electromyography demonstrated increased innervation in engineered grafts neurotized with the femoral nerve, as compared to non-neurotized grafts. Histologic assessments revealed axonal penetration and formation of neuromuscular junctions within the grafts. The innervation process described here may advance the fabrication of a fully functional engineered muscle graft that will be of utility in clinical settings.

Extracellular vesicles (EVs) have emerged as a promising strategy to promote tissue regeneration. However, overcoming the low EV production yield remains a big challenge in translating EV-based therapies to the clinical practice. Current EV production relies heavily on 2D cell culture, which is not only less physiologically relevant to cells but also requires substantial medium and space. In this study, we engineered tissues seeded with stem cells from dental pulp or adipose tissues, or skeletal muscle cells, and significantly enhanced the EV production yield by applying mechanical stimuli, including flow and stretching, in bioreactors. Further mechanistic investigation revealed that this process was mediated by yes-associated protein (YAP) mechanosensitivity. EVs from mechanically stimulated dental pulp stem cells on 3D scaffolds displayed superior capability in inducing axonal sprouting than the 2D counterparts. Our results demonstrate the promise of this strategy to boost EV production and optimize their functional performance toward clinical translation.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineering hierarchical vasculatures is critical for creating implantable functional thick tissues. Current approaches focus on fabricating mesoscale vessels for implantation or hierarchical microvascular in vitro models, but a combined approach is yet to be achieved to create engineered tissue flaps. Here, millimetric vessel-like scaffolds and 3D bioprinted vascularized tissues interconnect, creating fully engineered hierarchical vascular constructs for implantation. Endothelial and support cells spontaneously form microvascular networks in bioprinted tissues using a human collagen bioink. Sacrificial molds are used to create polymeric vessel-like scaffolds and endothelial cells seeded in their lumen form native-like endothelia. Assembling endothelialized scaffolds within vascularizing hydrogels incites the bioprinted vasculature and endothelium to cooperatively create vessels, enabling tissue perfusion through the scaffold lumen. Using a cuffing microsurgery approach, the engineered tissue is directly anastomosed with a rat femoral artery, promoting a rich host vasculature within the implanted tissue. After two weeks in vivo, contrast microcomputer tomography imaging and lectin perfusion of explanted engineered tissues verify the host ingrowth vasculature's functionality. Furthermore, the hierarchical vessel network (VesselNet) supports in vitro functionality of cardiomyocytes. Finally, the proposed approach is expanded to mimic complex structures with native-like millimetric vessels. This work presents a novel strategy aiming to create fully-engineered patient-specific thick tissue flaps.

Generating comprehensive image maps, while preserving spatial 3D context, is essential to quantitatively assess and locate specific cellular features and cell-cell interactions during organ development. Despite the recent advances in 3D imaging approaches, our current knowledge of the spatial organization of distinct cell types in the embryonic pancreatic tissue is still largely based on 2D histological sections. Here, we present a light-sheet fluorescence microscopy approach to image the pancreas in 3D and map tissue interactions at key development time points in the mouse embryo. We used transgenic mouse models and antibodies to visualize the three main cellular components within the developing pancreas, including epithelial, mesenchymal and endothelial cell populations. We demonstrated the utility of the approach by providing volumetric data, 3D distribution of distinct progenitor populations and quantification of relative cellular abundance within the tissue. Lastly, our image data were combined in an open source online repository (referred to as Pancreas Embryonic Cell Atlas). This image dataset will serve the scientific community by enabling further investigation on pancreas organogenesis but also for devising strategies for the in vitro generation of transplantable pancreatic tissue for regenerative therapies.

Producing meat without the drawbacks of conventional animal agriculture would greatly contribute to future food and nutrition security. This Review Article covers biological, technological, regulatory and consumer acceptance challenges in this developing field of biotechnology. Cellular agriculture is an emerging branch of biotechnology that aims to address issues associated with the environmental impact, animal welfare and sustainability challenges of conventional animal farming for meat production. Cultured meat can be produced by applying current cell culture practices and biomanufacturing methods and utilizing mammalian cell lines and cell and gene therapy products to generate tissue or nutritional proteins for human consumption. However, significant improvements and modifications are needed for the process to be cost efficient and robust enough to be brought to production at scale for food supply. Here, we review the scientific and social challenges in transforming cultured meat into a viable commercial option, covering aspects from cell selection and medium optimization to biomaterials, tissue engineering, regulation and consumer acceptance.

Fabrication of a functional hierarchical vascular network remains an unmet need for cultivation and transplantation of 3D engineered tissues. In this work, an effective approach was developed to fabricate a functional, perfusable and biocompatible, multi-scale vascular network (MSVT) within thick, implantable engineered tissues. Using a templating technique, macro-vessels were patterned in a 3D biodegradable polymeric scaffold seeded with endothelial and support cells within a collagen gel. The lumen of the macro-vessel was lined with endothelial cells, which further sprouted and anastomosed with the surrounding self-assembled capillaries. Anastomoses between the two-scaled vascular systems displayed tightly bonded cell junctions, as indicated by vascular endothelial cadherin expression. Moreover, MSVT functionality and patency were demonstrated by dextran passage through the interconnected hierarchical vasculature. Additionally, physiological flow conditions were applied with home-designed flow bioreactors, to achieve a MSVT with a natural endothelium structure. Finally, implantation of a multi-scale-vascularized graft in a mouse model resulted in a clear beneficial effect, as reflected by extensive host vessel penetration into the graft and an increase in blood perfusion via the engineered vessels as compared to control microscale-vascularized graft. Designing and fabricating such multi-scale vascular architectures within 3D engineered tissues is essential, both for in-vitro models and for therapeutic translation research.

The regeneration of injured spinal cord is hampered by the lack of vascular supply and neurotrophic support. Transplanting tissue-engineered constructs with developed vascular networks and neurotrophic factors, and further understanding the pattern of vessel growth in the remodeled spinal cord tissue are greatly desired. To this end, highly vascularized scaffolds embedded with human dental pulp stem cells (DPSCs) are fabricated, which possess paracrine-mediated angiogenic and neuroregenerative potentials. The potent pro-angiogenic effect of the prevascularized scaffolds is first demonstrated in a rat femoral bundle model, showing robust vessel growth and blood perfusion induced within these scaffolds postimplantation, as evidenced by laser speckle contrast imaging and 3D microCT dual imaging modalities. More importantly, in a rat complete spinal cord transection model, the implantation of these scaffolds to the injured spinal cords can also promote revascularization, as well as axon regeneration, myelin deposition, and sensory recovery. Furthermore, 3D microCT imaging and novel morphometric analysis on the remodeled spinal cord tissue demonstrate substantial regenerated vessels, more significantly in the sensory tract regions, which correlates with behavioral recovery following prevascularization treatment. Taken together, prevascularized DPSC-embedded constructs bear angiogenic and neurotrophic potentials, capable of augmenting and modulating SCI repair.

Exosomes, a subtype of extracellular vesicles, are nanovesicles of endocytic origin. Exosomes contain a plethora of proteins, lipids, and genetic materials of parent cells to facilitate intercellular communications. Tracking exosomes in vivo is fundamentally important to understand their biodistribution pattern and the mechanism of biological actions in experimental models. Until now, a number of tracking protocols have been developed, including fluorescence labeling, bioluminescence imaging, magnetic resonance imaging, and computed tomography (CT) tracking of exosomes. Recently, we have shown the tracking and quantification of exosomes in a spinal cord injury model, by using two tracking approaches. More specifically, following intranasal administration of gold nanoparticle-encapsulated exosomes to rats bearing complete spinal cord injury, exosomes in the whole central nervous system were tracked by using microCT, and quantified by using inductively coupled plasma and flame atomic absorption spectroscopy. In addition, optical imaging of fluorescently labeled exosomes was performed to understand the abundance of migrating exosomes in the spinal cord lesion, as compared to the healthy controls, and to further examine their affinity to different cell types in the lesion. Thus, the protocol presented here aids in the study of exosome biodistribution at both cellular and organ levels, in the context of spinal cord injury. This protocol will also enable researchers to better elucidate the fate of administered exosomes in other models of interest.

Engineered neural implants have a myriad of potential basic science and clinical neural repair applications. Although there are implants that are currently undergoing their first clinical investigations, optimizing their long-term viability and efficacy remain an open challenge. Functional implants with pre-vascularization of various engineered tissues have proven to enhance post-implantation host integration, and well-known synergistic neural-vascular interplays suggest that this strategy could also be promising for neural tissue engineering . Here, we report the development of a novel bio-engineered neuro-vascular co-culture construct, and demonstrate that it exhibits enhanced neurotrophic factor expression , and more complex neuronal morphology. Crucially, by introducing genetically encoded calcium indicators (GECIs) into the co-culture, we are able to monitor functional activity of the neural network , and demonstrate greater activity levels and complexity as a result of the introduction of endothelial cells in the construct. The presence of this enhanced activity could putatively lead to superior integration outcomes. Indeed, leveraging on the ability to monitor the construct's development post-implantation with GECIs, we observe improved integration phenotypes in the spinal cord of mice relative to non-vascularized controls. Our approach provides a new experimental system with functional neural feedback for studying the interplay between vascular and neural development while advancing the optimization of neural implants towards potential clinical applications.

Engineering of functional tissue, by combining either autologous or allogeneic cells with biomaterials, holds promise for the treatment of various diseases and injuries. Prevascularization of the engineered tissue was shown to enhance and improve graft integration and neovascularization post-implantation in immunocompromised mice. However, the neovascularization and integration processes of transplanted engineered tissues have not been widely studied in immunocompetent models. Here, we fabricated a three-dimensional (3D) vascularized murine muscle construct that was transplanted into immunocompetent and immunocompromised mice. Intravital imaging demonstrated enhanced neovascularization in immunocompetent mice compared to immunocompromised mice, 18 days post-implantation, indicating the advantageous effect of an intact immune system on neovascularization. Moreover, construct prevascularization enhanced neovascularization, integration, and myogenesis in both animal models. These findings demonstrate the superiority of implantation into immunocompetent over immunocompromised mice and, therefore, suggest that using autologous cells might be beneficial compared to allogeneic cells and subsequent immunosuppression. Taken together, these observations have the potential to advance the field of regenerative medicine and tissue engineering, ultimately reducing the need for donor organs and tissues.

A multitude of cell screening assays for diagnostic and research applications rely on quantitative measurements of a sample in the presence of different reagent concentrations. Standard methods rely on microtiter plates of varying well density, which provide simple and standardized sample addressability. However, testing hundreds of chemical dilutions requires complex automation, and typical well volumes of microtiter plates are incompatible with the analysis of a small number of cells. Here, we present a microfluidic device for creating a high-resolution chemical gradient spanning 200 nanoliter wells. Using air-based shearing, we show that the individual wells can be compartmentalized without altering the concentration gradient, resulting in a large set of isolated nanoliter cell culture wells. We provide an analytical and numerical model for predicting the concentration within each culture chamber and validate it against experimental results. We apply our system for the investigation of yeast cell metabolic gene regulation in the presence of different ratios of galactose/glucose concentrations and successfully resolve the nutrient threshold at which the cells activate the galactose pathway.

Microtia is a small, malformed external ear, which occurs at an incidence of 1–10 per 10 000 births. Autologous reconstruction using costal cartilage is the most widely accepted surgical microtia repair technique. Yet, the method involves donor-site pain and discomfort and relies on the artistic skill of the surgeon to create an aesthetic ear. This study employed novel tissue engineering techniques to overcome these limitations by developing a clinical-grade, 3D-printed biodegradable auricle scaffold that formed stable, custom-made neocartilage implants. The unique scaffold design combined strategically reinforced areas to maintain the complex topography of the outer ear and micropores to allow cell adhesion for the effective production of stable cartilage. The auricle construct was computed tomography (CT) scan-based composed of a 3D-printed clinical-grade polycaprolactone scaffold loaded with patient‐derived chondrocytes produced from either auricular cartilage or costal cartilage biopsies combined with adipose-derived mesenchymal stem cells. Cartilage formation was measured within the construct in vitro, and cartilage maturation and stabilization were observed 12 weeks after its subcutaneous implantation into a murine model. The proposed technology is simple and effective and is expected to improve aesthetic outcomes and reduce patient discomfort.

Spinal cord injury (SCI) is a debilitating condition, often leading to severe motor, sensory, or autonomic nervous dysfunction. As the holy grail of regenerative medicine, promoting spinal cord tissue regeneration and functional recovery are the fundamental goals. Yet, effective regeneration of injured spinal cord tissues and promotion of functional recovery remain unmet clinical challenges, largely due to the complex pathophysiology of the condition. The transplantation of various cells, either alone or in combination with three-dimensional matrices, has been intensively investigated in preclinical SCI models and clinical trials, holding translational promise. More recently, a new paradigm shift has emerged from cell therapy towards extracellular vesicles as an exciting “cell-free” therapeutic modality. The current review recapitulates recent advances, challenges, and future perspectives of cell-based spinal cord tissue engineering and regeneration strategies.

Human adipose-derived microvascular endothelial cells (HAMEC) and mesenchymal stem cells (MSC) have been shown to bear angiogenic and vasculogenic capabilities. We hypothesize that co-culturing HAMEC:MSC on a porous biodegradable scaffold in vitro, later implanted as a graft around femoral blood vessels in a rat, will result in its vascularization by host vessels, creating a functional vascular flap that can effectively treat a range of large full-thickness soft tissue defects. HAMEC were co-cultured with MSC on polymeric three-dimensional porous constructs. Grafts were then implanted around the femoral vessels of a rat. To ensure vessel sprouting from the main femoral vessels, grafts were pre-isolated from the surrounding tissue. Graft vascularization was monitored to confirm full vascularization before flap transfer. Flaps were then transferred to treat both abdominal wall and exposed bone and tendon of an ankle defects. Flaps were analysed to determine vascular properties in terms of maturity, functionality and survival of implanted cells. Findings show that pre-isolated grafts bearing the HAMEC:MSC combination promoted formation of highly vascularized flaps, which were better integrated in both defect models. The results of this study show the essentiality of a specific adipose-derived cell combination in successful graft vascularization and integration, two processes crucial for flap survival. Copyright © 2017 John Wiley & Sons, Ltd.

Engineered three-dimensional (3D) constructs have received much attention as in vitro tools for the study of cell–cell and cell–matrix interactions, and have been explored for potential use as experimental models or therapeutic human tissue substitutes. Yet, due to diffusion limitations, the lack of stable and perfusable blood vessel networks jeopardizes cell viability once the tissue dimensions extend beyond several hundred microns. Direct perfusion of 3D scaffold cultures has been shown to enhance oxygen and nutrient availability. Additionally, flow-induced shear stress at physiologically relevant levels, positively impacted endothelial cell migration and alignment in various two-dimensional (2D) culture models and promoted angiogenic sprouting in microfluidic systems. However, little is known about the effect of flow on vascularization in implantable 3D engineered tissue models. The present study investigated the effect of direct flow-induced shear stress on vascularization in implantable 3D tissue. Th...

Biodegradable polyesters have been extensively used for preparation of nerve guidance scaffolds, due to their high biocompatibility and defined degradation periods. However, conventional methods for fabrication of porous polyester scaffolds provide limited control over shape and micro-architecture. Here, a fabrication procedure based on 3D printing was developed to generate highly ordered and anatomically personalized, polyester scaffolds for soft tissue regeneration. Scaffolds composed of Poly-lactic-glycolic acid (PLGA) and poly-L-lactic acid (PLLA) were specifically customized for nerve injuries. This was obtained by using an oriented multi-layer printing pattern which established a linear structure in the fabricated scaffolds to match the aligned topography of nerve tissues. The oriented scaffold was shown to guide regenerating axons to linear conformations and support growth of induced pluripotent stem cell-derived neurons in vitro and in vivo in a model of spinal cord injury. The described scaffolds may advance the field of nerve regeneration. Furthermore, modifications could be integrated to generate soft implants for various types of tissues.

In spite of current therapy protocols many T2DM patients fail to achieve blood glucose homeostasis goals. Insulin resistance in T2DM is associated with impaired GLUT4 function and cellular content. Thus, our aim was to improve overall glucose homeostasis by constructing engineered muscle tissue, using genetically modified skeletal muscle cells that over-express the GLUT4 transporter (OEG4). The OEG4 cells were grafted on biodegradable scaffolds and cultured to allow the formation of engineered muscle tissue constructs (EMC). Using an in-vitro 2-deoxyglucos (DOG) uptake assay, the OEG4-EMCs showed a 1.8-fold increase in DOG uptake with insulin stimulation, compared to unmodified controls. For in-vivo efficacy evaluation OEG4-EMCs and empty constructs, not seeded with cells, as control were implanted in the abdominal wall of immunodeficient recombination-activating gene 1 (Rag1)/MKR mice model of T2DM. Following i.p glucose tolerance test mice implanted with OEG4-EMCs had significantly (p Disclosure M. Beckerman: None. E. Karnieli: None. C. Harel: None. A. Klip: None. P.J. Bilan: None. E. Gallagher: None. D. LeRoith: Advisory Panel; Self; AstraZeneca, MannKind Corporation. S. Levenberg: None. Funding Rina and Avner Schneur Charitable Giving Fund