Effects of Interleukin-4 (IL-4)-releasing microparticles and adoptive transfer of macrophages on immunomodulation and angiogenesis.

Macrophages are major regulators of angiogenesis in response to injury, but the mechanisms behind their diverse and phenotypically specific functions are still poorly understood. In particular, the effects of interleukin-4 (IL-4) on macrophage behavior have been well studied in vitro, but it remains unclear whether the release of IL-4 from biomaterials can be used to control macrophage phenotype and subsequent effects on angiogenesis in vivo. We used the murine hindlimb ischemia model to investigate the effects of IL-4-releasing poly(lactic-co-glycolic acid) microparticles co-delivered with IL-4-polarized macrophages on host macrophage phenotype and angiogenesis in vivo. We established a minimum dose of IL-4 required to modulate macrophage phenotype in vivo and evaluated effects on macrophage subpopulation diversity using multidimensional flow cytometry and pseudotime analysis. The delivery of IL-4-releasing microparticles did not affect the density or size of blood vessels as measured by immunohistochemical (IHC) analysis, but it did increase perfused tissue volume as measured by 3D microcomputed tomography (microCT). In contrast, the co-delivery of IL-4-releasing microparticles and exogenously IL-4-polarized macrophages increased the size of blood vessels as measured by IHC, but without effects on perfused tissue volume via microCT. These effects occurred in spite of low recovery of adoptively transferred macrophages at 4 days after administration. Spatial analysis of macrophage-blood vessel interactions via IHC showed that macrophages closely interacted with blood vessels, which was slightly influenced by treatment, and that blood vessel size was positively correlated with number of macrophages in close proximity. Altogether, these findings indicate that delivery of IL-4-releasing microparticles and exogenously IL-4-polarized macrophages can be beneficial for angiogenesis, but further mechanistic investigations are required.

Engineered marble-like bovine fat tissue for cultured meat.

Cultured meat can provide a sustainable and more ethical alternative to conventional meat. Most of the research in this field has been focused on developing muscle tissue, as it is the main component of meat products, while very few studies address cultured fat tissue, an essential component in the human diet and determinant of meat quality, flavor, juiciness, and tenderness. Here, we engineered bovine fat tissue for cultured meat and incorporated it within engineered bovine muscle tissue. Mesenchymal stem cells (MSCs) were derived from bovine adipose tissue and exhibited the typical phenotypic profile of adipose-derived MSCs. MSC adipogenic differentiation and maturation within alginate-based three-dimensional constructs were optimized to yield a fat-rich edible engineered tissue. Subsequently, a marble-like construct, composed of engineered bovine adipose and muscle tissues, was fabricated, mimicking inter- and intra-muscular fat structures.

Fabrication of Engineered Vascular Flaps Using 3D Printing Technologies.

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.

Boosting extracellular vesicle secretion.

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.

Development of a 3D atlas of the embryonic pancreas for topological and quantitative analysis of heterologous cell interactions.

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).

Microcomputed Tomography-Based Analysis of Neovascularization within Bioengineered Vascularized Tissues.

In the field of tissue engineering, evaluating newly formed vascular networks is considered a fundamental step in deciphering the processes underlying tissue development. Several common modalities exist to study vessel network formation and function. However, a proper methodology that allows through three-dimensional visualization of neovessels in a reproducible manner is required. Here, we describe in-depth exploration, visualization, and analysis of vessels within newly formed tissues by utilizing a contrast agent perfusion protocol and high-resolution microcomputed tomography. Bioengineered constructs consisting of porous, biocompatible, and biodegradable scaffolds are loaded with cocultures of adipose-derived microvascular endothelial cells (HAMECs) and dental pulp stem cells (DPSCs) and implanted in a rat femoral bundle model. After 14 days of in vivo maturation, we performed the optimized perfusion protocol to allow host penetrating vascular visualization and assessment within neotissues. Following high-resolution microCT scanning of DPSC:HAMEC explants, we performed the volumetric and spatial analysis of neovasculature. Eventually, the process was repeated with a previously published coculture system for prevascularization based on adipose-derived mesenchymal stromal cells (MSCs) and HAMECs. Overall, our approach allows a comprehensive understanding of vessel organization during engraftment and development of neotissues.

Human-engineered auricular reconstruction (hEAR) by 3D-printed molding with human-derived auricular and costal chondrocytes and adipose-derived mesenchymal stem cells.

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 constructin 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 Repair: From Cells and Tissue Engineering to Extracellular Vesicles.

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.

Investigating lymphangiogenesis in vitro and in vivo using engineered human lymphatic vessel networks.

The lymphatic system is involved in various biological processes, including fluid transport from the interstitium into the venous circulation, lipid absorption, and immune cell trafficking. Despite its critical role in homeostasis, lymphangiogenesis (lymphatic vessel formation) is less widely studied than its counterpart, angiogenesis (blood vessel formation). Although the incorporation of lymphatic vasculature in engineered tissues or organoids would enable more precise mimicry of native tissue, few studies have focused on creating engineered tissues containing lymphatic vessels. Here, we populated thick collagen sheets with human lymphatic endothelial cells, combined with supporting cells and blood endothelial cells, and examined lymphangiogenesis within the resulting constructs. Our model required just a few days to develop a functional lymphatic vessel network, in contrast to other reported models requiring several weeks. Coculture of lymphatic endothelial cells with the appropriate supporting cells and intact PDGFR-ß signaling proved essential for the lymphangiogenesis process. Additionally, subjecting the constructs to cyclic stretch enabled the creation of complex muscle tissue aligned with the lymphatic and blood vessel networks, more precisely biomimicking native tissue. Interestingly, the response of developing lymphatic vessels to tensile forces was different from that of blood vessels; while blood vessels oriented perpendicularly to the stretch direction, lymphatic vessels mostly oriented in parallel to the stretch direction. Implantation of the engineered lymphatic constructs into a mouse abdominal wall muscle resulted in anastomosis between host and implant lymphatic vasculatures, demonstrating the engineered construct's potential functionality in vivo. Overall, this model provides a potential platform for investigating lymphangiogenesis and lymphatic disease mechanisms.

Use of Simvastatin, Fibrin Clots, and Their Combination to Improve Human Ovarian Tissue Grafting for Fertility Restoration After Anti-Cancer Therapy.

Anticancer treatments, particularly chemotherapy, induce ovarian damage and loss of ovarian follicles. There are limited options for fertility restoration, one of which is pre-chemotherapy cryopreservation of ovarian tissue. Transplantation of frozen-thawed human ovarian tissue from cancer survivors has resulted in live-births. There is extensive follicular loss immediately after grafting, probably due to too slow graft revascularization. To avoid this problem, it is important to develop methods to improve ovarian tissue neovascularization. The study's purpose was to investigate if treatment of murine hosts with simvastatin or/and embedding human ovarian tissue within fibrin clots can improve human ovarian tissue grafting (simvastatin and fibrin clots promote vascularization). There was a significantly higher number of follicles in group A (ungrafted control) than in group B (untreated tissue). Group C (simvastatin-treated hosts) had the highest levels of follicle atresia. Group C had significantly more proliferating follicles (Ki67-stained) than groups B and E (simvastatin-treated hosts and tissue embedded within fibrin clots), group D (tissue embedded within fibrin clots) had significantly more proliferating follicles (Ki67-stained) than group B. On immunofluorescence study, only groups D and E showed vascular structures that expressed both human and murine markers (mouse-specific platelet endothelial cell adhesion molecule, PECAM, and human-specific von Willebrand factor, vWF). Peripheral human vWF expression was significantly higher in group E than group B. Diffuse human vWF expression was significantly higher in groups A and E than groups B and C. When grafts were not embedded in fibrin, there was a significant loss of human vWF expression compared to groups A and E. This protocol may be tested to improve ovarian implantation in cancer survivors.

Intranasal Delivery of Mesenchymal Stem Cell Derived Exosomes Loaded with Phosphatase and Tensin Homolog siRNA Repairs Complete Spinal Cord Injury.

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.

Multi-flow channel bioreactor enables real-time monitoring of cellular dynamics in 3D engineered tissue.

The key to understanding, harnessing, and manipulating natural biological processes for the benefit of tissue engineering lies in providing a controllable dynamic environment for tissue development in vitro while being able to track cell activity in real time. This work presents a multi-channel bioreactor specifically designed to enable on-line imaging of fluorescently labeled cells embedded in replicated 3D engineered constructs subjected to different flow conditions. The images are acquired in 3D using a standard upright confocal microscope and further analyzed and quantified by computer vision. The platform is used to characterize and quantify the pace and directionality of angiogenic processes induced by flow. The presented apparatus bears considerable potential to advance scientific research, from basic research pursuing the effect of flow versus static conditions on 3D scaffolds and cell types, to clinically oriented modeling in drug screening and cytotoxicity assays.

Engineering vascularized flaps using adipose-derived microvascular endothelial cells and mesenchymal stem cells.

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.

Dielectrophoretic characterization of cells in a stationary nanoliter droplet array with generated chemical gradients.

A novel design of reusable microfluidic platform that generates a stationary nanoliter droplet array (SNDA) for cell incubation and analysis, equipped with a complementary array of individually addressable electrodes for each microwell is studied. Various solute concentration gradients were generated between the wells where dielectrophoresis (DEP) was used to characterize the effect of the gradients on the cell's response. The feasibility of generating concentration gradients and observation of DEP responses was demonstrated using a gradient of salts in combination with microparticles and viable cells. L1210 Lymphoma cells were used as the model cells in these experiments. Lymphoma cells' cross-over frequency (COF) decreased with increasing stress conditions. Specifically, a linear decrease in the cell COF was measured as a function of solution tonicity and blebbistatin dose. Lymphoma cells were incubated under a gradient of the chemotherapeutic agent doxorubicin (DOX), which led to saturation in the cell-COF response at 30 nM DOX, demonstrating the potential of the platform in screening of label-free drugs.

Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis.

Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required.

An engineered muscle flap for reconstruction of large soft tissue defects.

Large soft tissue defects involve significant tissue loss, requiring surgical reconstruction. Autologous flaps are occasionally scant, demand prolonged transfer surgery, and induce donor site morbidity. The present work set out to fabricate an engineered muscle flap bearing its own functional vascular pedicle for repair of a large soft tissue defect in mice. Full-thickness abdominal wall defect was reconstructed using this engineered vascular muscle flap. A 3D engineered tissue constructed of a porous, biodegradable polymer scaffold embedded with endothelial cells, fibroblasts, and/or myoblasts was cultured in vitro and then implanted around the femoral artery and veins before being transferred, as an axial flap, with its vascular pedicle to reconstruct a full-thickness abdominal wall defect in the same mouse. Within 1 wk of implantation, scaffolds showed extensive functional vascular density and perfusion and anastomosis with host vessels. At 1 wk posttransfer, the engineered muscle flaps were highly vascularized, were well-integrated within the surrounding tissue, and featured sufficient mechanical strength to support the abdominal viscera. Thus, the described engineered muscle flap, equipped with an autologous vascular pedicle, constitutes an effective tool for reconstruction of large defects, thereby circumventing the need for both harvesting autologous flaps and postoperative scarification.

The effect of endothelial cells on hESC-derived pancreatic progenitors in a 3D environment.

Generating transplantable ß-like-cells from human embryonic stem cells (hESC) could serve as an ideal cell-based therapy for treatment of type 1 diabetes, which is characterized by the destruction of insulin-secreting pancreatic ß-cells. There are several protocols for differentiating hESCs into pancreatic or endocrine precursors. However, so far, production of mature, functional ß-like-cells has been achieved mainly by transplanting hESC derived pancreatic progenitors (PPs) and allowing several months for maturation to occur in vivo. One approach, believed to have potential in promoting differentiation into ß-like-cells prior to transplantation, is culturing PPs alongside blood vessels. Endothelium and blood vessels have been shown to direct pancreatic development during embryogenesis and also induce endocrine differentiation in vitro. Here we designed a three-dimensional (3D) construct utilizing highly porous polymeric scaffolds that mimic natural conditions and provide cells with mechanical support, and used it in the differentiation protocol. Clusters of hESC derived pancreatic precursor cells were embedded within the scaffolds along with human endothelial cells (ECs) and fibroblasts forming vessel-like networks. Culturing these clusters with ECs for one week significantly increased the population of PPs, characterized by co-expression of the pancreatic markers Pdx1 and Nkx6.1 and also highly induced Ngn3 expression which indicates commitment to endocrine fate. The presence of fibroblasts, however, reduced this cell population. Three months upon implantation of constructs containing clusters and ECs or clusters alone, implanted mice retained normal blood glucose levels after treatment with STZ, while un-implanted mice became diabetic. These findings may lay the foundation for creating an optimal tissue-construct that will support PPs' maturation in vitro and enhance graft function upon implantation.

Olfactory bulb-derived cells seeded on 3D scaffolds exhibit neurotrophic factor expression and pro-angiogenic properties.

Olfactory bulb (OB)-derived cells include fibroblasts, astrocytes, and olfactory ensheathing cells (OECs). OECs are a distinctive type of glia that secrete neurotrophic factors and form myelin sheaths around axons projecting from the olfactory mucosa into the OB of the central nervous system. Their unique properties make them candidates for cell therapy of spinal cord injury (SCI). Current SCI cellular repair techniques suffer from massive cell loss post implantation. To overcome this difficulty, we propose to seed and propagate OB-derived cells on biodegradable scaffolds to form a stable tissue construct. Upon implantation, scaffolds may serve as carriers to introduce the tissue into the lesion site. In this study, we characterized OB-derived cells cultured on biodegradable poly-l-lactic acid/polylactic-co-glycolic acid scaffolds in vitro. We showed that cells remained viable and proliferative for up to 2 weeks on the scaffolds. We have shown that OB-derived cells induce neuronal differentiation of pheochromocytoma cells (PC12) on scaffolds, and that a purified population of OECs is sufficient for the differentiation. Selective inhibition of nerve growth factor (NGF) on PC12 cells blocks the differentiation. We have shown that the expression of BDNF and NGF genes in OB-derived cells grown on 3D scaffolds compared to 2D monolayer cultures was significantly upregulated. In addition, OB-derived cells stimulated network formation of endothelial cells grown on the same scaffolds. Taken together, these results clearly demonstrate the vast potential of 3D scaffolds in maintaining and strengthening the unique therapeutic properties of embedded OB-derived cells. This strategy will enable more efficient therapeutic usage of OB-derived cells for treatment of SCI.

Fibroblasts derived from human pluripotent stem cells activate angiogenic responses in vitro and in vivo.

Human embryonic and induced pluripotent stem cells (hESC/hiPSC) are promising cell sources for the derivation of large numbers of specific cell types for tissue engineering and cell therapy applications. We have describe a directed differentiation protocol that generates fibroblasts from both hESC and hiPSC (EDK/iPDK) that support the repair and regeneration of epithelial tissue in engineered, 3D skin equivalents. In the current study, we analyzed the secretory profiles of EDK and iPDK cells to investigate the production of factors that activate and promote angiogenesis. Analysis of in vitro secretion profiles from EDK and iPDK cells demonstrated the elevated secretion of pro-angiogenic soluble mediators, including VEGF, HGF, IL-8, PDGF-AA, and Ang-1, that stimulated endothelial cell sprouting in a 3D model of angiogenesis in vitro. Phenotypic analysis of EDK and iPDK cells during the course of differentiation from hESCs and iPSCs revealed that both cell types progressively acquired pericyte lineage markers NG2, PDGFRß, CD105, and CD73 and demonstrated transient induction of pericyte progenitor markers CD31, CD34, and Flk1/VEGFR2. Furthermore, when co-cultured with endothelial cells in 3D fibrin-based constructs, EDK and iPDK cells promoted self-assembly of vascular networks and vascular basement membrane deposition. Finally, transplantation of EDK cells into mice with hindlimb ischemia significantly reduced tissue necrosis and improved blood perfusion, demonstrating the potential of these cells to stimulate angiogenic responses in vivo. These findings demonstrate that stable populations of pericyte-like angiogenic cells can be generated with high efficiency from hESC and hiPSC using a directed differentiation approach. This provides new cell sources and opportunities for vascular tissue engineering and for the development of novel strategies in regenerative medicine.