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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., Zohar et al. demonstrate a real-time imaging of fluorescently labeled cells embedded in 3D constructs subjected to different flow conditions. This study provides a framework that enables tracking cell activity continuously during tissue development, suggesting its potential utility for drug screens or cytotoxicity assays.

Understanding the mechanosensitivity of tissues is a fundamentally important problem having far-reaching implications for tissue engineering. Here we study vascular networks formed by a coculture of fibroblasts and endothelial cells embedded in three-dimensional biomaterials experiencing external, physiologically relevant forces. We show that cyclic stretching of the biomaterial orients the newly formed network perpendicular to the stretching direction, independent of the geometric aspect ratio of the biomaterial's sample. A two-dimensional theory explains this observation in terms of the network's stored elastic energy if the cell-embedded biomaterial features a vanishing effective Poisson's ratio, which we directly verify. We further show that under a static stretch, vascular networks orient parallel to the stretching direction due to force-induced anisotropy of the biomaterial polymer network. Finally, static stretching followed by cyclic stretching reveals a competition between the two mechanosensitive mechanisms. These results demonstrate tissue-level mechanosensitivity and constitute an important step toward developing enhanced tissue repair capabilities using well-oriented vascular networks.

Endothelial cells form the interior layer of blood vessels and, as such, are constantly exposed to shear stress and mechanical strain. While the impact of shear stress on angiogenesis is widely studied, the role of mechanical strain is less understood. To this end, endothelial cells and fibroblasts are cocultured under oscillatory strain to create a vessel network. The two cell types show distinctly different sensitivities to the mechanical stimulation. The fibroblasts, sense the stress directly, and respond by increased alignment, proliferation, differentiation, and migration, facilitated by YAP translocation into the nucleus. In contrast, the endothelial cells form aligned vessels by tracking fibroblast alignment. YAP inhibition in constructs under mechanical strain results in vessel destruction whereas less damage is observed in the YAP-inhibited static control. Moreover, the mechanical stimulation enhances vessel development and stabilization. Additionally, vessel orientation is preserved upon implantation into a mouse dorsal window chamber and promotes the invading host vessels to orient in the same manner. This study sheds light on the mechanisms by which mechanical strain affects the development of blood vessels within engineered tissues. This can be further utilized to engineer a more organized and stable vasculature suitable for transplantation of engineered grafts.

Today, in vitro vessel network systems frequently serve as models for investigating cellular and functional mechanisms underlying angiogenesis and vasculogenesis. Understanding the cues triggering the observed cell migration, organization, and differentiation, as well as the time frame of these processes, can improve the design of engineered microvasculature. Here, we present first evidence of the migration of endothelial cells into the depths of the scaffold, where they formed blood vessels surrounded by extracellular matrix and supporting cells. The supporting cells presented localization-dependent phenotypes, where cells adjacent to blood vessels displayed a more mature phenotype, with smooth muscle cell characteristics, whereas cells on the scaffold surface showed a pericyte-like phenotype. Yes-associated protein (YAP), a transcription activator of genes involved in cell proliferation and tissue growth, displayed spatially dependent expression, with cells on the surface showing more nuclear YAP than cells situated deeper within the scaffold.

The vascularization of three-dimensional (3D) engineered tissues enhances their in vivo integration with the host vasculature and its ability to serve as sustainable biological substitutes for injured or sick tissues. Engineered vascular networks must comply with the corresponding host’s vasculature structure, which is known to be regulated by mechanical stimuli. The impact of mechanical cues on vascularization of 3D engineered constructs is gaining much attention, as they may allow for control of vascular network structure, directionality, remodeling, and maturation to ensure long-term functionality. Mechanical forces can be generated in vitro either by actively applying an external source of force or following internal induction by cellular contractile forces responding to the mechanical properties of the engineered construct. This chapter provides an overview of recent studies aimed to explore the impact of both internal and external mechanical stimulations on vascularization of 3D engineered tissues and will propose future directions to advance some of the current challenges.

Background & Aim Complete spinal cord injury (SCI) is a debilitating disease which usually leads to permanent functional impairments, with various complications and limited spontaneous recovery or effective treatment. Here, we report that in rats with complete SCI, intranasal administrations of mesenchymal stem cells-derived exosomes (MSC-Exo) could penetrate the blood-brain barrier, home selectively to the spinal cord lesion, and show affinity to neurons within the lesion. When these exosomes were loaded with phosphatase and tensin homolog small interfering RNA, termed ExoPTEN, they migrated from the nose and silenced PTEN expression in the lesion. Furthermore, the loaded exosomes promoted robust axonal regeneration and angiogenesis, accompanied by decreased astrogliosis and microgliosis. Moreover, the intranasal ExoPTEN treatment partially restored electrophysiological and structural integrity, and most importantly, enabled remarkable functional recovery. This rapid, non-invasive approach, using cell-free nano-swimmers carrying molecules to target pathophysiological mechanisms, suggests a novel strategy for clinical translation to SCI and beyond. Methods, Results & Conclusion MSC-exo were extracted from Human bone marrow mesenchymal stem cells. All rats had complete transection of the spinal cord. MSC-exo were loaded with co-incubation together with siRNA for PTEN conjugated to cholesterol. The MSC-exo were given by intranasal administration 1-4 hours post-SCI.

Engineered tissues are a promising tool for addressing the growing need for tissues and organs in surgical reconstructions. Prevascularization of implanted tissues is expected to enhance survival prospects post transplantation and minimize deficiencies and/or hypoxia deeper in the tissue. Here, we fabricate a three-dimensional, prevascularized engineered muscle containing human myoblasts, genetically modified endothelial cells secreting angiopoietin 1 (ANGPT1) and genetically modified smooth muscle cells secreting vascular endothelial growth factor (VEGF). The genetically engineered human muscle shows enhanced host neovascularization and myogenesis following transplantation into a mouse host, compared to the non-secreting control. The vascular, genetically modified cells have been cleared for clinical trials and can be used to construct autologous vascularized tissues. Therefore, the described genetically engineered vascularized muscle has the potential to be fully translated to the clinical setting to overcome autologous tissue shortage and to accelerate host neovascularization and integration of engineered grafts following transplantation., Luba Perry et al. report transplantation of engineered prevascularized human muscle into mice to repair an abdominal muscle defect. They show that genetically engineering smooth muscle cells to secrete VEGF and endothelial cells to secrete ANGPT1 significantly improves host neovascularization and myogenesis.

Spinal cord injury (SCI), involving damaged axons and glial scar tissue, often culminates in irreversible impairments. Achieving substantial recovery following complete spinal cord transection remains an unmet challenge. Here, we report of implantation of an engineered 3D construct embedded with human oral mucosa stem cells (hOMSC) induced to secrete neuroprotective, immunomodulatory, and axonal elongation-associated factors, in a complete spinal cord transection rat model. Rats implanted with induced tissue engineering constructs regained fine motor control, coordination and walking pattern in sharp contrast to the untreated group that remained paralyzed (42 vs. 0%). Immunofluorescence, CLARITY, MRI, and electrophysiological assessments demonstrated a reconnection bridging the injured area, as well as presence of increased number of myelinated axons, neural precursors, and reduced glial scar tissue in recovered animals treated with the induced cell-embedded constructs. Finally, this construct is made of bio-compatible, clinically approved materials and utilizes a safe and easily extractable cell population. The results warrant further research with regards to the effectiveness of this treatment in addressing spinal cord injury.

Antibiotic resistance is a major global health concern that requires action across all sectors of society. In particular, to allow conservative and effective use of antibiotics clinical settings require better diagnostic tools that provide rapid determination of antimicrobial susceptibility. We present a method for rapid and scalable antimicrobial susceptibility testing using stationary nanoliter droplet arrays that is capable of delivering results in approximately half the time of conventional methods, allowing its results to be used the same working day. In addition, we present an algorithm for automated data analysis and a multiplexing system promoting practicality and translatability for clinical settings. We test the efficacy of our approach on numerous clinical isolates and demonstrate a 2-d reduction in diagnostic time when testing bacteria isolated directly from urine samples.

Implanted scaffolds bearing 3D-printed parallel endothelialized channels restore blood perfusion in ischaemic hind limbs and infarcted hearts in rodents.

Sporadic spontaneous network activity emerges during early central nervous system (CNS) development and, as the number of neuronal connections rises, the maturing network displays diverse and complex activity, including various types of synchronized patterns. These activity patterns have major implications on both basic research and the study of neurological disorders, and their interplay with network morphology tightly correlates with developmental events such as neuronal differentiation, migration and establishment of neurotransmitter phenotypes. Although 2D neural cultures models have provided important insights into network activity patterns, these cultures fail to mimic the complex 3D architecture of natural CNS neural networks and its consequences on connectivity and activity. A 3D in-vitro model mimicking early network development while enabling cellular-resolution observations, could thus significantly advance our understanding of the activity characteristics in the developing CNS. Here, we longitudinally studied the spontaneous activity patterns of developing 3D in-vitro neural network "optonets," an optically-accessible bioengineered CNS model with multiple cortex-like characteristics. Optonet activity was observed using the genetically encodable calcium indicator GCaMP6m and a 3D imaging solution based on a standard epi-fluorescence microscope equipped with a piezo-electric z-stage and image processing-based deconvolution. Our results show that activity patterns become more complex as the network matures, gradually exhibiting longer-duration events. This report characterizes the patterns over time, and discusses how environmental changes affect the activity patterns. The relatively high degree of similarity between the network's spontaneously generated activity patterns and the reported characteristics of in-vivo activity, suggests that this is a compelling model system for brain-in-a chip research.

Tissue engineering incorporates wide and multidisciplinary approaches to engineered viable, clinically relevant tissue substitutes for regenerative medicine applications. When engineering substitutes for most functional tissues, the clinical relevance of the engineered tissue relies greatly on the incorporation of a mature and perfusable network of blood vessels within that tissue. The field of vascular tissue engineering is rapidly developing, combining new insights from vascular cell biology together with novel biomaterials engineering approaches and microfabrication techniques. The purpose of this review is to communicate the recent substantial scientific advancements in these fields, with a particular focus on methods which induce neovascular formation in vitro .

Generating transplantable β-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 β-cells. There are several protocols for differentiating hESCs into pancreatic or endocrine precursors. However, so far, production of mature, functional β-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 β-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.

An interdigitated electrode array embedded within a micro-channel with forced flow is shown to enable dielectrophoretic (DEP) characterization of particles and/or cells based on measurements of their trapping percentage over a continuous frequency range. A simplified model of the trapping percentage, using spatial averaging of the convective and DEP force, linearly correlated it to the effective DEP force (in its positive mode). Thus, the Clausius-Mossotti factor was fitted to the experimental data, yielding effective electrical characteristics of the particles and/or cells. Also, the generated trapping percentage curve response over a continuous range of frequencies facilitates sorting and detection based on differences other than just the cross-over frequencies.

Natural polymers share recognition sequences that promote cell adhesion, rendering them attractive candidates for scaffolding in tissue engineering applications. However, challenges remain with regard to the fabrication of robust and porous structures of such raw materials for the design of extracellular matrix (ECM) mimics of living tissues. In this study, we present a fibrous scaffold that solely consists of albumin, the most abundant protein in mammalian blood plasma. The scaffold was fabricated using the electrospinning method, and resulted in microscale fibers that demonstrated mechanical properties which were similar to those of elastin fibers, a common component of connective tissue ECM. Albumin scaffolds proved nontoxic and supported adhesion and the spreading of fibroblasts, muscle cells, and endothelial cells (ECs) in vitro. In vivo studies demonstrated ∼50% biodegradation of the albumin scaffolds within 3 weeks of implantation. In addition, it was found that the fibers were encapsulated by dense fibrosis and evoked a weak inflammatory response, similar to that triggered by poly(L-lactide)/poly(lactic-co-glycolic acid) scaffolds. Albumin tubular structures fabricated to mimic blood vessels successfully guided the formation of blood vessel-like bi-layer structures made of fibroblasts and ECs. Thus, albumin scaffolds featuring biologically relevant characteristics pose a readily applicable alternative to synthetic scaffolding materials.

The robust repair of large wounds and tissue defects relies on blood flow. This vascularization is the major challenge faced by tissue engineering on the path to forming thick, implantable tissue constructs. Without this vasculature, oxygen and nutrients cannot reach the cells located far from host blood vessels. To make viable constructs, tissue engineering takes advantage of the mechanical properties of synthetic materials, while combining them with ECM proteins to create a natural environment for the tissue-specific cells. Tropoelastin, the precursor of the elastin, is the ECM protein responsible for elasticity in diverse tissues, including robust blood vessels. Here, we seeded endothelial cells with supporting cells on PLLA/PLGA scaffolds treated with tropoelastin, and examined the morphology, expansion and maturity of the newly formed vessels. Our results demonstrate that the treated scaffolds elicit a more expanded, complex and developed vascularization in comparison to the untreated group. Implantation of tropoelastin-treated scaffolds into mouse abdominal muscle resulted in enhanced perfusion of the penetrating vasculature and improved integration. This study points to the great potential of these combined materials in promoting the vascularization of implanted engineered constructs, which can be further exploited in the fabrication of clinically relevant engineered tissues.

Spinal cord injury is a devastating medical condition. Recent developments in pre-clinical and clinical research have started to yield neural implants inducing functional recovery after spinal cord transection injury. However, the functional performance of the transplants was assessed using histology and behavioral experiments which are unable to study cell dynamics and the therapeutic response. Here, we use neurophotonic tools and optogenetic probes to investigate cellular level morphology and activity characteristics of neural implants over time at the cellular level. These methods were used in-vitro and in-vivo, in a mouse spinal cord injury implant model. Following previous attempts to induce recovery after spinal cord injury, we engineered a pre-vascularized implant to obtain better functional performance. To image network activity of a construct implanted in a mouse spinal cord, we transfected the implant to express GCaMP6 calcium activity indicators and implanted these constructs under a spinal cord chamber enabling 2-photon chronic in vivo neural activity imaging. Activity and morphology analysis image processing software was developed to automatically quantify the behavior of the neural and vascular networks. Our experimental results and analyses demonstrate that vascularized and non-vascularized constructs exhibit very different morphologic and activity patterns at the cellular level. This work enables further optimization of neural implants and also provides valuable tools for continuous cellular level monitoring and evaluation of transplants designed for various neurodegenerative disease models.

Understanding the forces controlling vascular network properties and morphology can enhance in vitro tissue vascularization and graft integration prospects. This work assessed the effect of uniaxial cell-induced and externally applied tensile forces on the morphology of vascular networks formed within fibroblast and endothelial cell-embedded 3D polymeric constructs. Force intensity correlated with network quality, as verified by inhibition of force and of angiogenesis-related regulators. Tensile forces during vessel formation resulted in parallel vessel orientation under static stretching and diagonal orientation under cyclic stretching, supported by angiogenic factors secreted in response to each stretch protocol. Implantation of scaffolds bearing network orientations matching those of host abdominal muscle tissue improved graft integration and the mechanical properties of the implantation site, a critical factor in repair of defects in this area. This study demonstrates the regulatory role of forces in angiogenesis and their capacities in vessel structure manipulation, which can be exploited to improve scaffolds for tissue repair.

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen supply to the cell microenvironment, as passive diffusion is limited to a very thin layer. Although various materials have been described to restore the integrity of full-thickness defects of the abdominal wall, no material has yet proved to be optimal, due to low graft vascularization, tissue rejection, infection, or inadequate mechanical properties. This protocol describes a means of engineering a fully vascularized flap, with a thickness relevant for muscle tissue reconstruction. Cell-embedded poly L-lactic acid/poly lactic-co-glycolic acid constructs are implanted around the mouse femoral artery and vein and maintained in vivo for a period of one or two weeks. The vascularized graft is then transferred as a flap towards a full thickness defect made in the abdomen. This technique replaces the need for autologous tissue sacrifications and may enable the use of in vitro engineered vascularized flaps in many surgical applications.

Purpose: To identify the factors secreted by lens epithelial cells (LECs) responsible for the altered trabecular meshwork (TM) cells and to compare their effect on monocultured TM cells with that of TM cells co-cultured with LECs. Methods: Such factors were isolated using cytokine antibody array membranes, and their effect on TM cells was assessed by analyzing changes in morphology and gene expression. In addition, inhibition of the isolated factors was performed in the co-culture model by adding specific antibodies to the cell culture media. Results: Transforming growth factor beta-2, interleukin-4 (IL-4), and vascular endothelial growth factor (VEGF) are presented as candidate cytokines responsible for the observed changes in LEC-TM co-cultures. Culturing TM cells in the presence of VEGF and IL-4 triggered alterations closely reflecting those observed in the LEC-TM co-culture model, where their inhibition significantly hindered the alteration of the TM cells. Conclusion: These findings suggest a possible explanation for the development of infantile aphakic glaucoma, based on residual LECs secreting IL-4 and VEGF after removal of congenital cataract, which then alter trabecular meshwork cell morphology and gene expression.

The unlimited proliferative and differentiative capacities of embryonic stem cells (ESCs) are tightly regulated by their microenvironment. Local concentrations of soluble factors, cell-cell interactions and extracellular matrix signaling are just a few variables that influence ESC fate. A common method employed to induce ESC differentiation involves the formation of cell aggregates called embryoid bodies (EBs), which recapitulate early stages of embryonic development. EBs are normally formed in suspension cultures, producing heterogeneously shaped and sized aggregates. The present study demonstrates the usage of a microfluidic traps system which supports prolonged EB culturing. The traps are uniquely designed to facilitate cell capture and aggregation while offering efficient gas/nutrients exchange. A finite element simulation is presented with emphasis on several aspects critical to appropriate design of such bioreactors for ESC culture. Finally, human ESC, mouse Nestin-GFP ESC and OCT4-EGFP ESCs were cultured using this technique and demonstrated extended viability for more than 5 days. In addition, EBs developed and maintained a polarized differentiation pattern, possibly as a result of the nutrient gradients imposed by the traps bioreactor. The novel microbioreactor presented here can enhance future embryogenesis research by offering tight control of culturing conditions.

As droplet-based microfluidic devices evolve, the demand for simple-to-fabricate droplet manipulation modules increases. Of these modules, droplet sorting has drawn much attention due to its ability not only to enrich, but also to selectively isolate droplet subpopulations of interest. In this paper, we present an innovative piezoelectric-driven droplet sorter that is simple to fabricate, reproducible and robust, which provides extensive control over spatio-temporal droplet pattern. This degree of control is demonstrated by sorting droplets of alternating volumes and by grouping defined number of droplets into traveling clusters. The ability to automatically sort droplets is demonstrated by computerized detection and sorting of droplets based on their color. The sorter performance was investigated and found to work on a wide range of sorting parameters. The sorter is created by a single step fabrication process and does not rely on complex electronics or optics. These advantages simplify the adoption of droplet-based microfluidic technology by the scientific community and provide an ideal platform for single cell assays.

Myocardial infarction can lead to irreversible heart failure. In an attempt to restore function in the failing heart, tissue-engineered cardiac constructs can be applied to repopulate scar tissue with a new pool of contractile cells. Effective engineering of viable thick complex tissue-constructs requires intense vascularization. Furthermore, endothelial-cardiomyocyte crosstalk plays a key role in mutually enhancing tissue functionality, which can further improve construct survival. The ability to generate an engineered, vascularized muscle tissue was demonstrated by us using the skeletal and the cardiac muscle models. In the skeletal model, we showed that prevascularization of the construct promoted perfusion of the graft. More recently, we successfully generated a beating human cardiac muscle-construct, containing an endothelial network, by co-culturing human embryonic stem cell-derived-cardiomyocytes, fibroblasts, and endothelial cells within biodegradable scaffolds. Such muscle-constructs could contribute significantly to the emerging discipline of cardiovascular regenerative medicine as well as to the study of the important role of tissue vascularization.

A polyHIPE is a highly porous, emulsion-templated polymer synthesized by polymerizing a monomer and a crosslinking comonomer in the continuous phase of a high-internal phase emulsion (HIPE). The synthesis of degradable polyHIPE could be of interest for biomedical applications such as tissue engineering scaffolds. In this research, a poly(e-caprolactone) (PCL) oligomer with terminal vinyl groups was used as the crosslinking comonomer for a polyHIPE based on t-butyl acrylate (tBA). The porous structure, properties, water absorption, and hydrolytic degradation of the polyHIPE were investigated. The polyHIPE containing 50 wt % PCL exhibited very large voids, 1 to 3 mm in diameter that resulted from the destabilization of the HIPE on addition of PCL, making the polyHIPE more suitable for tissue engineering applications. The relatively flexible PCL enhanced segmental mobility, yielding two glass transition temperatures and a significant reduction in modulus. When exposed to a 3 M aqueous solution of NaOH, the t-butyl groups underwent hydrolysis and the PCL underwent degradation, rapidly leading to the complete disintegration of the macromolecular structure. The tBA-based polyHIPE containing 50 wt % PCL exhibited enhanced cell adhesion, penetration, and growth indicating that it is a suitable candidate for further research and development. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2009

Droplet based microfluidic systems have been shown to be most valuable in biology and chemistry research. However droplet modulation and manipulation requires still further improvement in order to make this technology feasible particularly for biological applications. On demand generation of droplets and droplet synchronization, which is crucial for coalescence, remain largely unanswered. The present study describes a simple and robust droplet generator based on a piezoelectric actuator which is integrated into a microfluidic device. The droplet generator is able to independently control the droplet size, rate of formation and distance between droplets. Moreover, the droplet uniformity is especially high, deviating from the mean value by less than 0.3%. The cross flow and T-junction configurations are tested and show no significant differences, yet the inlet to main channel ratio is found to be important. As this ratio increases, droplets tend to be generated in bursts instead of individually. The physical mechanisms involved are discussed, providing insight into optimized design of such systems.

In this work we propose a new method titled "layered hydrospinning." In this method, nanofibers are being collected on top of a liquid reservoir and assembled layer-by-layer to form a 3D scaffold. The geometrical features of fabricated hydrospun scaffolds show a porosity of 99% and pores of a diameter over 100 microm. Cells were seeded during the hydrospinning process, thus achieving an initial even density of cells in the scaffold. We show that human embryonic stem cells and mouse myoblasts cultured on the scaffolds were able to infiltrate further into the scaffolds and proliferate to a greater extent, compared to conventional electrospun scaffolds collected on a plate.

PolyHIPE are highly porous, open-pore, cross-linked polymers synthesized within high internal phase emulsions (HIPE). Biodegradable polycaprolactone (PCL) oligomers were incorporated into polyHIPE using two approaches. The first approach involved copolymerization with a vinyl-terminated PCL (PCL-VL). This approach yielded a typical polyHIPE structure with voids on the order of tens of microns. The covalent bonds formed by PCL-VL prevented its removal during emulsifier extraction. The second approach involved the formation of semi-interpenetrating polymer networks (semi-IPN) with a PCL diol (PCL-OL). The relatively hydrophilic PCL-OL destabilized the HIPE and produced voids on the order of hundreds of microns, a size more appropriate for tissue engineering applications. The PCL-OL, which is more mobile than the covalently bound PCL-VL, underwent more extensive phase separation. In addition, a significant amount of PCL-OL was removed during emulsifier extraction. Cells were successfully attached to the surface of a semi-IPN polyHIPE, forming a monolayer. Eventually, spontaneous differentiation of the cells and the formation of myotubes were observed.

The present work describes an experimental method and design tools which enable the precise localization of an analyte, a few microns in width, both temporally and spatially using laminar flows and thus improves previous methods in hydrodynamic focusing. The technique is used to adsorb proteins to selected regions within a microfluidic device without any contamination of the surroundings and may serve in applications requiring selective conveying of other reagents. The regions not coated by proteins are modified with poly(ethylene glycol; PEG), known to efficiently resist protein and cell adhesion. Human endothelial and fibroblast cells are later introduced into the device selectively attaching to the protein coated regions and cultured for a few days. A simulation of the convection-diffusion characteristics of the system is presented and compared to the known T-sensor. The results reveal that, by proper design, reagents concentration may be kept nearly constant along the flow direction. This phenomenon is demonstrated here by achieving particularly precise patterning of cells but may be utilized for numerous other applications as well.

Growing interest in using endothelial cells for therapeutic purposes has led to exploring human embryonic stem cells as a potential source for endothelial progenitor cells. Embryonic stem cells are advantageous when compared with other endothelial cell origins, due to their high proliferation capability, pluripotency, and low immunogenity. However, there are many challenges and obstacles to overcome before the vision of using embryonic endothelial progenitor cells in the clinic can be realized. Among these obstacles is the development of a productive method of isolating endothelial cells from human embryonic stem cells and elucidating their differentiation pathway. This review will focus on the endothelial potential of human embryonic stem cells that is described in current studies, with respect to the differentiation of human embryonic stem cells to endothelial cells, their isolation, and their characterization.

Transplantation of a tissue-engineered heart muscle represents a novel experimental therapeutic paradigm for myocardial diseases. However, this strategy has been hampered by the lack of sources for human cardiomyocytes and by the scarce vasculature in the ischemic area limiting the engraftment and survival of the transplanted muscle. Beyond the necessity of endothelial capillaries for the delivery of oxygen and nutrients to the grafted muscle tissue, interactions between endothelial and cardiomyocyte cells may also play a key role in promoting cell survival and proliferation. In the present study, we describe the formation of synchronously contracting engineered human cardiac tissue derived from human embryonic stem cells containing endothelial vessel networks. The 3D muscle consisted of cardiomyocytes, endothelial cells (ECs), and embryonic fibroblasts (EmFs). The formed vessels were further stabilized by the presence of mural cells originating from the EmFs. The presence of EmFs decreased EC death and increased EC proliferation. Moreover, the presence of endothelial capillaries augmented cardiomyocyte proliferation and did not hamper cardiomyocyte orientation and alignment. Immunostaining, ultrastructural analysis (using transmission electron microscopy), RT-PCR, pharmacological, and confocal laser calcium imaging studies demonstrated the presence of cardiac-specific molecular, ultrastructural, and functional properties of the generated tissue constructs with synchronous activity mediated by action potential propagation through gap junctions. In summary, this is the first report of the construction of 3D vascularized human cardiac tissue that may have unique applications for studies of cardiac development, function, and tissue replacement therapy.

(2007). Engineering Human Embryonic Stem Cell Differentiation. Biotechnology and Genetic Engineering Reviews: Vol. 24, No. 1, pp. 243-262.

Background Adipose-derived mesenchymal stem cells (MSCs) have been gaining fame mainly due to their vast clinical potential, simple isolation methods and minimal donor site morbidity. Adipose-derived MSCs and microvascular endothelial cells have been shown to bear angiogenic and vasculogenic capabilities. We hypothesized that co-culture of human adipose-derived MSCs with human adipose-derived microvascular endothelial cells (HAMECs) will serve as an effective cell pair to induce angiogenesis and vessel-like network formation in three-dimensional scaffolds in vitro. Methods HAMECs or human umbilical vein endothelial cells (HUVECs) were co-cultured on scaffolds with either MSCs or human neonatal dermal fibroblasts. Cells were immunofluorescently stained within the scaffolds at different time points post-seeding. Various analyses were performed to determine vessel length, complexity and degree of maturity. Results The HAMEC:MSC combination yielded the most organized and complex vascular elements within scaffolds, and in the shortest period of time, when compared to the other tested cell combinations. These differences were manifested by higher network complexity, more tube alignment and higher α-smooth muscle actin expression. Moreover, these generated microvessels further matured and developed during the 14-day incubation period within the three-dimensional microenvironment. Conclusions These data demonstrate optimal vascular network formation upon co-culture of microvascular endothelial cells and adipose-derived MSCs in vitro and constitute a significant step in appreciation of the potential of microvascular endothelial cells and MSCs in different tissue engineering applications that can also be advantageous in in vivo studies.

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.

Implantable 3D engineered vascular tissue constructs can be formed by co-culturing endothelial and fibroblast cells on macroporous scaffolds. Here we show that these constructs can be used for studying the dynamics of neovascular formation in-vitro by a combination of live confocal imaging and an array of image processing and analysis tools, revealing multiple distinct stages of morphogenesis. We show that this process involves both vasculogenic and angiogenic elements, including an initial endothelial multicellular cluster formation followed by rapid extensive sprouting, ultimately resulting in a stable interconnected endothelial network morphology. This vascular morphogenesis is time-correlated with the deposition and formation of an extensive extra-cellular matrix environment. We further show that endothelial network junctions are formed by two separate morphogenic mechanisms of anastomosis and cluster thinning.

Generation of vessel networks within engineered tissues is critical for integration and perfusion of the implanted tissue in vivo. The effect of mechanical cues in guiding and stabilizing the vessels has begun to attract marked interest. This review surveys the impact of mechanical cues on formation of vascular networks in 2D and 3D gel matrices. We give less emphasis to regulation of endothelial monolayers and single endothelial cells. Several vascularization models have consistently found that the stress generated in the gel, and encountered by embedded cells, control various aspects of vascular network formation, including sprouting, branching, alignment, and vessel maturation. This internal stress is generated by cell contractile forces, and is balanced by gel stiffness and boundary constrains imposed on the gel. Actin and myosin II are key molecular players in controlling initiation of vessel sprouting and branching morphogenesis. Additionally, the impact of external mechanical cues on tissue vascularization, and studies supporting the notion that mechanical forces regulate vascularization in the live animal are reviewed.

Abdominal wall reconstruction following extensive tissue loss is essential and can be achieved using autologous flaps. However, their use is limited due to their inadequate availability and due to post-operative donor site scarification. This work presents a step-by-step technique for fabrication of a vascularized muscle flap, to be applied in full-thickness abdominal wall defect reconstruction. Poly L-lactic acid/poly lactic-co-glycolic acid scaffolds, prepared using a salt leaching technique, were used as the supporting matrix in vitro for simultaneously seeded endothelial cells, fibroblasts and myoblasts. The cell-embedded graft was then implanted around femoral artery and vein vessels, which provided a central blood supply. Vascularization and perfusion were achieved by capillary sprouting from the main host vessel into the graft. A thick and vascularized tissue was formed within one week, and was then transferred as an autologous flap together with its main vessels, to a full-thickness abdominal wall defect. The flap remained viable after transfer and featured sufficient mechanical strength to support the abdominal viscera. Thus, this engineered muscle flap can be used as an alternative source for autologous flaps to reconstruct full-thickness abdominal wall defects.

Endothelial cells organized into blood vessels are critical for the formation and maintenance of most tissues in the body and are involved in regulating physiological processes such as angiogenesis, inflammation and thrombosis. Endothelial cells are of great research interest, because of their potential to treat vascular diseases and to stimulate the growth of ischaemic tissue. They can be used to engineer artificial vessels, repair damaged vessels, and to induce the formation of vessel networks in engineered tissues. For such clinical applications, proliferating human endothelial progenitor cells can be isolated from adult tissues or embryonic stem cells. Recently, these cells were successfully used to engineer single vessels and to stimulate capillary networks, both in vitro and in vivo.

One of the major obstacles in engineering thick, complex tissues such as muscle is the need to vascularize the tissue in vitro. Vascularization in vitro could maintain cell viability during tissue growth, induce structural organization and promote vascularization upon implantation. Here we describe the induction of endothelial vessel networks in engineered skeletal muscle tissue constructs using a three-dimensional multiculture system consisting of myoblasts, embryonic fibroblasts and endothelial cells coseeded on highly porous, biodegradable polymer scaffolds. Analysis of the conditions for induction and stabilization of the vessels in vitro showed that addition of embryonic fibroblasts increased the levels of vascular endothelial growth factor expression in the construct and promoted formation and stabilization of the endothelial vessels. We studied the survival and vascularization of the engineered muscle implants in vivo in three different models. Prevascularization improved the vascularization, blood perfusion and survival of the muscle tissue constructs after transplantation.

A novel method for patterning cellular co-cultures that uses the layer-by-layer deposition of ionic biopolymers is described. Non-biofouling hyaluronic acid (HA) micropatterns were used to immobilize cells and proteins to glass substrates. Subsequent ionic adsorption of poly-L-lysine (PLL) to HA patterns was used to switch the HA surfaces from cell repulsive to adherent thereby facilitating the adhesion of a second cell type. The utility of this approach to pattern co-cultures of hepatocytes or embryonic stem cells with fibroblasts was demonstrated. In addition, the versatility of this approach to generate patterned co-cultures irrespective of the primary cell seeding and relative adhesion of the seeded cells was demonstrated. Thus, the proposed method may be a useful tool for fabricating controlled cellular co-cultures for cell-cell interaction studies and tissue engineering applications.

Human embryonic stem (hES) cells hold promise as an unlimited source of cells for transplantation therapies. However, control of their proliferation and differentiation into complex, viable 3D tissues is challenging. Here we examine the use of biodegradable polymer scaffolds for promoting hES cell growth and differentiation and formation of 3D structures. We show that complex structures with features of various committed embryonic tissues can be generated, in vitro , by using early differentiating hES cells and further inducing their differentiation in a supportive 3D environment such as poly(lactic- co -glycolic acid)/poly( l -lactic acid) polymer scaffolds. We found that hES cell differentiation and organization can be influenced by the scaffold and directed by growth factors such as retinoic acid, transforming growth factor β, activin-A, or insulin-like growth factor. These growth factors induced differentiation into 3D structures with characteristics of developing neural tissues, cartilage, or liver, respectively. In addition, formation of a 3D vessel-like network was observed. When transplanted into severe combined immunodeficient mice, the constructs continue to express specific human proteins in defined differentiated structures and appear to recruit and anastamose with the host vasculature. This approach provides a unique culture system for addressing questions in cell and developmental biology, and provides a potential mechanism for creating viable human tissue structures for therapeutic applications.

Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo , and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo , when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

Poor graft survival is a critical obstacle toward production of clinically relevant engineered tissues. Here we utilize a multicellular culturing approach for induction of vascular networks embedded within cardiac tissue constructs. The construct is composed of human cardiomyocytes, endothelial cells (ECs), and embryonic fibroblast cells co-seeded onto highly porous three-dimensional (3D) scaffolds. The resulting vascularized cardiac constructs showed microstructural details characteristic of cardiomyocytes and nascent vessels and exhibited synchronous beating activity in vitro. Upon implantation, stable grafts were formed presenting intense vascularization, with evidence of anastomosis between the pre-formed endothelial capillaries and host neovessels.

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.

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.

Here, we review the frontier microfluidic techniques for single cell analysis (SCA), which is important for research of many biological systems. Microfluidics provides high-throughput, high-resolution experiments at low cost and reagent use, making it especially useful for single cell analysis. Recent advancements in the field have made SCA more feasible, improving device throughput and resolution, adding capabilities, and combining different functions to bring forth new assays. Developments in incubation have allowed for long-term cell tracking assays to be performed with single cell resolution. The ability of systems to provide chemical isolation or prolonged growth of adherent cells is also discussed.