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Many academic researchers are familiar with the technical challenges inherent in creating patient-specific, tissue-engineered therapeutics for clinical applications. However, for the potential of these products to mature into clinical practice and the profound possibilities to be realized, developers must apply not only technical expertise but also a comprehensive translational strategy─making a product marketable, scalable, manufacturable, and approvable by regulators, while providing genuine advantages to patients. Here, we provide a brief overview of three crucial steps for successful translation: adoption of a "translational mindset," consideration of three categories of core challenges (economic, regulatory, and manufacturing), and detailed planning at the earliest stages of development.

Adult stem cells provide a renewable source of differentiated cells for a wide variety of tissues and generally give rise to multiple cell types. Basic principles of stem cell organization and regulation underlying this behaviour are emerging. Local niche signals maintain stem cells, while different sets of signals act outside the niche to diversify initially equivalent stem cell progeny. Here we show that Drosophila ovarian follicle stem cells (FSCs) produced two distinct cell types directly. This cell fate choice was determined by the anterior-posterior position of an FSC and by the magnitude of spatially graded Wnt pathway activity. These findings reveal a paradigm of immediate diversification of stem cell derivatives according to stem cell position within a larger population, guided by a graded niche signal. We also found that FSCs strongly resemble mammalian intestinal stem cells in many aspects of their organization, including population asymmetry and dynamic heterogeneity.

Electrical signals have been implied in many biological mechanisms, including wound healing, which has been associated with transient electrical currents not present in intact skin. One method to generate electrical signals similar to those naturally occurring in wounds is by supplementation of galvanic particles dispersed in a cream or gel. We constructed a three-layered model of skin consisting of human dermal fibroblasts in hydrogel (mimic of dermis), a hydrogel barrier layer (mimic of epidermis) and galvanic microparticles in hydrogel (mimic of a cream containing galvanic particles applied to skin). Using this model, we investigated the effects of the properties and amounts of Cu/Zn galvanic particles on adult human dermal fibroblasts in terms of the speed of wound closing and gene expression. The collected data suggest that the effects on wound closing are due to the ROS-mediated enhancement of fibroblast migration, which is in turn mediated by the BMP/SMAD signaling pathway. These results imply that topical low-grade electric currents via microparticles could enhance wound healing.

Stem cells hold promise to revolutionize modern medicine by the development of new therapies, disease models and drug screening systems. Standard cell culture systems have limited biological relevance because they do not recapitulate the complex 3-dimensional interactions and biophysical cues that characterize the in vivo environment. In this review, we discuss the current advances in engineering stem cell environments using novel biomaterials and bioreactor technologies. We also reflect on the challenges the field is currently facing with regard to the translation of stem cell based therapies into the clinic.

The idea of extending the lifetime of our organs is as old as humankind, fueled by major advances in organ transplantation, novel drugs, and medical devices. However, true regeneration of human tissue has become increasingly plausible only in recent years. The human heart has always been a focus of such efforts, given its notorious inability to repair itself following injury or disease. We discuss here the emerging bioengineering approaches to regeneration of heart muscle as a paradigm for regenerative medicine. Our focus is on biologically inspired strategies for heart regeneration, knowledge gained thus far about how to make a “perfect” heart graft, and the challenges that remain to be addressed for tissue-engineered heart regeneration to become a clinical reality. We emphasize the need for interdisciplinary research and training, as recent progress in the field is largely being made at the interfaces between cardiology, stem cell science, and bioengineering.

In vitro application of pulsatile electrical stimulation to neonatal rat cardiomyocytes cultured on polymer scaffolds has been shown to improve the functional assembly of cells into contractile engineered cardiac tissues. However, to date, the conditions of electrical stimulation have not been optimized. We have systematically varied the electrode material, amplitude and frequency of stimulation to determine the conditions that are optimal for cardiac tissue engineering. Carbon electrodes, exhibiting the highest charge-injection capacity and producing cardiac tissues with the best structural and contractile properties, were thus used in tissue engineering studies. Engineered cardiac tissues stimulated at 3 V/cm amplitude and 3 Hz frequency had the highest tissue density, the highest concentrations of cardiac troponin-I and connexin-43 and the best-developed contractile behaviour. These findings contribute to defining bioreactor design specifications and electrical stimulation regime for cardiac tissue engineering.

Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Engineered constructs can also serve as high-fidelity models for studies of cardiac development and disease. In a general case, the biological potential of the cell—the actual “tissue engineer”—is mobilized by providing highly controllable three-dimensional environments that can mediate cell differentiation and functional assembly. For cardiac regeneration, some of the key requirements that need to be met are the selection of a human cell source, establishment of cardiac tissue matrix, electromechanical cell coupling, robust and stable contractile function, and functional vascularization. We review here the potential and challenges of cardiac tissue engineering for developing therapies that could prevent or reverse heart failure.

We describe a protocol for tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cells with the application of pulsatile electrical fields designed to mimic those present in the native heart. Tissue culture is conducted in a customized chamber built to allow for cultivation of (i) engineered three-dimensional (3D) cardiac tissue constructs, (ii) cell monolayers on flat substrates or (iii) cells on patterned substrates. This also allows for analysis of the individual and interactive effects of pulsatile electrical field stimulation and substrate topography on cell differentiation and assembly. The protocol is designed to allow for delivery of predictable electrical field stimuli to cells, monitoring environmental parameters, and assessment of cell and tissue responses. The duration of the protocol is 5 d for two-dimensional cultures and 10 d for 3D cultures.

Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Although bioreactors have facilitated the engineering of cardiac patches of clinically relevant size in vitro, a major drawback remains the transportation of the engineered tissues from a production facility to a medical operation facility while maintaining tissue viability and preventing contamination. Furthermore, after implantation, most of the cells are endangered by hypoxic conditions that exist before vascular flow is established. We developed a portable device that provides the perfusion and electrical stimulation necessary to engineer cardiac tissue in vitro, and to transport it to the site where it will be implantated. The micropump-powered perfusion apparatus may additionally function as an extracorporeal active pumping system providing nutrients and oxygen supply to the graft post-implantation. Such a system, through perfusion of oxygenated media and bioactive molecules (e.g. growth factors), could transiently support the tissue construct until it connects to the host vasculature and heart muscle, after which it could be taken away or let biodegrade.

The goal of this study was to simulate in vitro the spontaneous electrical wave activity associated with retinal development and investigate if such biometrically designed signals can enhance differentiation of mouse retinal progenitor cells (mRPC). To this end, we cultured cells on an electroconductive transplantable polymer, polypyrrole (PPy) and measured gene expression and morphology of the cells. Custom-made 8-well cell culture chambers were designed to accommodate PPy deposited onto indium tin oxide-coated (ITO) glass slides, with precise control of the PPy film thickness. mRPCs were isolated from post-natal day 1 (P1) green fluorescent protein positive (GFP+) mice, expanded, seeded onto PPY films, allowed to adhere for 24 hours, and then subjected to electrical stimulation (100 μA pulse trains, 5 s in duration, once per minute) for 4 days. Cultured cells and non-stimulated controls were processed for immunostaining and confocal analysis, and for RNA extraction and quantitative PCR. Stimulated cells expressed significantly higher levels of the early photoreceptor marker cone-rod homebox (CRX, the earliest known marker of photoreceptor identity), and protein kinase-C (PKC), and significantly lower levels of the glial fibrillary acidic protein (GFAP). Consistently, stimulated cells developed pronounced neuronal morphologies with significantly longer dendritic processes and larger cell bodies than non-stimulated controls. Taken together, the experimental evidence shows that the application of an electrical stimulation designed based on retinal development can be implemented to direct and enhance retinal differentiation of mRPCs, suggesting a role for biomimetic electrical stimulation in directing progenitor cells toward neural fates.

Electrical activity is abundant in early retinal development, and electrical stimulation has been shown to modulate embryonic stem cell differentiation towards a neuronal fate. The goal of this study was to simulate in vitro retinal developmental electrical activity to drive changes in mouse retinal progenitor cell (mRPC) gene expression and morphology. We designed a biomimetic electrical stimulation protocol based on spontaneous waves present during retinal development, and applied it to retinal progenitor cells (RPCs) over 3 days of culture. Analysis of protein localization and calcium dynamics, indicate that mRPCs undergo functional neuronal maturation. Our findings suggest that this type of electrical stimulation may be utilized for application in neural tissue engineering and open possibilities for understanding mechanisms guiding active electric membrane development and functional organization during early retinogenesis.

An increasing number of protocols are used to create engineered heart tissue (EHT) constructs, using different progenitors of cardiac cells with various scaffolds and bioreactors. Different laboratories develop their own EHT protocols, which are in most cases highly specialized and involve methods that do not transfer easily from one lab to another. Likewise, the imaging protocols used to evaluate the functional, structural and molecular properties of EHTs are also highly specialized and can involve equipment and methods that do not transfer well between the labs. As a result, it is often necessary to move EHTs between the location where they are engineered to locations where they are used for physiological testing or advanced imaging. In this work, we establish the feasibility of long-distance overnight shipping of EHTs grown in at Columbia University with electrical stimulation to George Washington University for high resolution optical mapping. The EHTs survived overnight shipping in sealed culture media flasks, as evidenced by robust synchronized contractions upon arrival at the destination institution. Calcium transients were readily recordable upon loading of the constructs with Fluo-4. Waves of electrical activity propagating throughout the constructs were imaged using high quantum efficiency back illuminated CCD camera. Due to perfusion limitations, cardiomyocyte-containing layers of EHT are usually restricted to 100-300 microns and the cell density is significantly lower than in intact myocardium. Therefore it was critical to show that the intensity and fidelity of the signals from EHTs stained with the potentiometric dye RH237 are sufficient to record wave propagation throughout the entire 7 mm-wide EHT with 50 micron spatial resolution. The data confirm the feasibility of collaborative arrangements between tissue engineering centers and other laboratories for high resolution fluorescence imaging.

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction, driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue but, due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation and unconstrained (i.e. not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate these three key factors in concert. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modelling studies. We then cultured cardiac cells obtained from neonatal rats in porous, channelled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After 8 days of culture, constructs grown with simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23 ± 0.10% vs 0.14 ± 0.05%, 0.13 ± 0.08% or 0.09 ± 0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization compared to control groups. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs.

Exogenous electric fields have been implied in cardiac differentiation of mouse embryonic stem cells and the generation of reactive oxygen species (ROS). In this work, we explored the effects of electrical field stimulation on ROS generation and cardiogenesis in embryoid bodies (EBs) derived from human embryonic stem cells (hESC, line H13), using a custom-built electrical stimulation bioreactor. Electrical properties of the bioreactor system were characterized by electrochemical impedance spectroscopy (EIS) and analysis of electrical currents. The effects of the electrode material (stainless steel, titanium-nitride-coated titanium, titanium), length of stimulus (1 and 90 s) and age of EBs at the onset of electrical stimulation (4 and 8 days) were investigated with respect to ROS generation. The amplitude of the applied electrical field was 1 V/mm. The highest rate of ROS generation was observed for stainless steel electrodes, for signal duration of 90 s and for 4-day-old EBs. Notably, comparable ROS generation was achieved by incubation of EBs with 1 nM H(2)O(2). Cardiac differentiation in these EBs was evidenced by spontaneous contractions, expression of troponin T and its sarcomeric organization. These results imply that electrical stimulation plays a role in cardiac differentiation of hESCs, through mechanisms associated with the intracellular generation of ROS.

Electrical stimulation has been shown to improve functional assembly of cardiomyocytes in vitro for cardiac tissue engineering. The goal of this study was to assess the conditions of electrical stimulation with respect to the electrode geometry, material properties and charge-transfer characteristics at the electrode-electrolyte interface. We compared various biocompatible materials, including nanoporous carbon, stainless steel, titanium and titanium nitride, for use in cardiac tissue engineering bioreactors. The faradaic and non-faradaic charge transfer mechanisms were assessed by electrochemical impedance spectroscopy (EIS), studying current injection characteristics, and examining surface properties of electrodes with scanning electron microscopy. Carbon electrodes were found to have the best current injection characteristics. However, these electrodes require careful handling because of their limited mechanical strength. The efficacy of various electrodes for use in 2-D and 3-D cardiac tissue engineering systems with neonatal rat cardiomyocytes is being determined by assessing cell viability, amplitude of contractions, excitation thresholds, maximum capture rate, and tissue morphology.

We present a microscale cell culture system with an interdigitated microarray of excimer-laser-ablated indium tin oxide electrodes for electrical stimulation of cultured cells. The system has been characterized in a range of geometeries and stimulation regimes via electrochemical impedance spectroscopy and used to culture primary cardiomyocytes and human adipose derived stem cells. Over 6 days of culture with electrical stimulation (2 ms duration, 1 Hz, 180 microm wide electrodes with 200 microm spacing), both cell types exhibited enhanced proliferation, elongation and alignment, and adipose derived stem cells exhibited higher numbers of Connexin-43-composed gap junctions.

Overcoming the limitations of diffusional transport in conventional culture systems remains an open issue for successfully generating thick, compact and functional cardiac tissues. Previously, it was shown that perfusion systems enhance the yield and uniformity of cell seeding and cell survival in thick cardiac constructs. The aim of our study was to form highly functional cardiac constructs starting from spatially uniform, high density cell seeded constructs. Disk-shaped elastomeric poly(glycerol sebacate) scaffolds were seeded with neonatal rat cardiomyocytes and cultured for eight days with direct perfusion of culture medium or statically in a six-well plate. In the perfusion experimental group, the integrity of some disks was well maintained, whereas in others a central hole was formed, resulting in ring-shaped constructs. This allowed us to also study the effects of construct geometry and of interstitial flow versus channel perfusion. The ring-shaped constructs appeared to have a denser and more uniform deposition of extracellular matrix. In response to electrical stimulation, the fractional area change of the ring-shaped constructs was 7.3 and 2.7 times higher than for disk-shaped tissues cultured in perfusion or statically, respectively. These findings suggest that a combination of many factors, including scaffold elasticity and geometry and the type of perfusion system applied, need to be considered in order to engineer a cardiac construct with high contractile activity.

Electrical stimulation has been shown to improve functional assembly of cardiomyocytes in vitro for cardiac tissue engineering. Carbon electrodes were found in past studies to have the best current injection characteristics. The goal of this study was to develop rational experimental design principles for the electrodes and stimulation regime, in particular electrode configuration, electrode ageing, and stimulation amplitude. Carbon rod electrodes were compared via electrochemical impedance spectroscopy (EIS) and we identified a safety range of 0 to 8 V/cm by comparing excitation thresholds and maximum capture rates for neonatal rat cardiomyocytes cultured with electrical stimulation. We conclude with recommendations for studies involving carbon electrodes for cardiac tissue engineering.

In vivo, direct current electric fields are present during embryonic development and wound healing. In vitro, direct current (DC) electric fields induce directional cell migration and elongation. For the first time, we demonstrate that cultured human adipose tissue-derived stem cells (hASCs) respond to the presence of direct-current electric fields. Cells were stimulated for 2–4 hours with DC electric fields of 6 V/cm that were similar to those encountered in vivo post-injury. Upon stimulation, hASCs were observed to elongate and align perpendicularly to the applied electric field, disassemble gap junctions, and upregulate the expression of genes for connexin-43, thrombomodulin, vascular endothelial growth factor, and fibroblast growth factor. In separate related studies, human epicardial fat-derived stem cells (heASCs) were also observed to align and elongate. It is interesting that the morphological and phenotypic characteristics of mesenchymal stem cells derived both from liposuction aspirates and from cardiac fat can be modulated by direct current electric fields. In further studies, we will quantify the effects of the electrical fields in the context of wound healing.