RESUMO
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.
Assuntos
Movimento Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Modelos Biológicos , Espécies Reativas de Oxigênio/metabolismo , Pele/citologia , Cicatrização , Adulto , Feminino , Resposta Galvânica da Pele , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
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.
Assuntos
Coração/fisiologia , Miocárdio/citologia , Regeneração/fisiologia , Medicina Regenerativa , Alicerces Teciduais/química , Animais , Bioengenharia , Vasos Coronários/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Animais , Miocárdio/imunologia , Técnicas de Cultura de Órgãos/métodos , Células-Tronco/metabolismo , Engenharia Tecidual/métodos , Transplantes/tendênciasRESUMO
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 strategyâmaking 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.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Engenharia Tecidual , HumanosRESUMO
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.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Estimulação Elétrica/instrumentação , Eletrodos , Microfluídica/instrumentação , Miócitos Cardíacos/fisiologia , Animais , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Ratos , Propriedades de SuperfícieRESUMO
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.
Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias/citologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reatores Biológicos , Técnicas de Cultura de Células , Sobrevivência Celular , Estimulação Elétrica , Eletricidade , Eletrodos , Células-Tronco Embrionárias/metabolismo , Fluoresceínas/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Microscopia de Fluorescência , Contração Miocárdica , Sarcômeros/metabolismo , Aço Inoxidável/química , Titânio/química , Troponina T/metabolismoRESUMO
The version of this paper originally published contained an incorrect unit abbreviation in Step 21: "0.20 g/mL" should have been "0.20 mg/mL." In addition, the first sentence in Step 33 should have read "Use a second pipette with a cut-off pipette tip to add Matrigel to the center well," instead of "Use a second pipette to cut off the tip of the pipette and add Matrigel to the center well." These errors have been corrected in the PDF and HTML versions of the protocol.
RESUMO
Live imaging of stem cells and their support cells can be used to visualize cellular dynamics and fluctuations of intracellular signals, proteins, and organelles in order to better understand stem cell behavior in the niche. We describe a simple protocol for imaging stem cells in the Drosophila ovary that improves on alternative protocols in that flies of any age can be used, dissection is simplified because the epithelial sheath that surrounds each ovariole need not be removed, and ovarioles are imaged in a closed chamber with a large volume of medium that buffers oxygen, pH, and temperature. We also describe how to construct the imaging chamber, which can be easily modified and used to image other tissues and non-adherent cells. Imaging is limited by follicle cells moving out of the germarium in culture around the time of egg chamber budding; however, the epithelial sheath delays this abnormal cell migration. This protocol requires an hour to prepare the ovarioles, followed by half an hour on the confocal microscope to locate germaria and set z limits. Successful imaging time depends on germarial morphology at the time of dissection, but we suggest 10-11 h to encompass all specimens.
Assuntos
Drosophila melanogaster/citologia , Desenho de Equipamento , Ovário/citologia , Células-Tronco/citologia , Imagem com Lapso de Tempo/instrumentação , Animais , Divisão Celular , Movimento Celular , Rastreamento de Células/métodos , Colágeno/química , Meios de Cultura/química , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Combinação de Medicamentos , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Laminina/química , Oócitos/citologia , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oogênese/fisiologia , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Proteoglicanas/química , Silício/química , Células-Tronco/metabolismo , Imagem com Lapso de Tempo/métodosRESUMO
Heart disease is a leading cause of death in western society. Despite the success of heart transplantation, a chronic shortage of donor organs, along with the associated immunological complications of this approach, demands that alternative treatments be found. One such option is to repair, rather than replace, the heart with engineered cardiac tissue. Multiple studies have shown that to attain functional tissue, assembly signaling cues must be recapitulated in vitro. In their native environment, cardiomyocytes are directed to beat in synchrony by propagation of pacing current through the tissue. Recently, we have shown that electrical stimulation directs neonatal cardiomyocytes to assemble into native-like tissue in vitro. This chapter provides detailed methods we have employed in taking this "biomimetic" approach. After an initial discussion on how electric field stimulation can influence cell behavior, we examine the practical aspects of cardiac tissue engineering with electrical stimulation, such as electrode selection and cell seeding protocols, and conclude with what we feel are the remaining challenges to be overcome.
Assuntos
Estimulação Elétrica , Coração , Engenharia Tecidual/métodos , Animais , Animais Recém-Nascidos , Órgãos Bioartificiais , Reatores Biológicos , Técnicas de Cultura de Células , Células Cultivadas , Coração/anatomia & histologia , Coração/fisiologia , Humanos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Folículo Ovariano/metabolismo , Nicho de Células-Tronco , Via de Sinalização Wnt , Animais , Animais Geneticamente Modificados , Linhagem da Célula , Movimento Celular , Proliferação de Células , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Genótipo , Folículo Ovariano/citologia , Fenótipo , Fatores de TempoRESUMO
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.
Assuntos
Reatores Biológicos , Engenharia Celular , Nicho de Células-Tronco , Células-Tronco , Animais , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Modelos Biológicos , Medicina RegenerativaRESUMO
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.
Assuntos
Reatores Biológicos , Coração/fisiologia , Perfusão , Engenharia Tecidual/instrumentação , Animais , Colágeno/química , Estimulação Elétrica , Eletrodos , Desenho de Equipamento , Modelos Teóricos , Oxigênio/análise , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Alicerces TeciduaisRESUMO
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.
Assuntos
Materiais Biocompatíveis/química , Estimulação Elétrica , Polímeros/química , Pirróis/química , Retina/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Tamanho Celular , Células Cultivadas , Imunofluorescência , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Células-Tronco/metabolismo , Compostos de Estanho/química , Transativadores/genética , Transativadores/metabolismoRESUMO
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.
Assuntos
Materiais Biomiméticos , Estimulação Elétrica/instrumentação , Retina/citologia , Células-Tronco/fisiologia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Animais , Sinalização do Cálcio , Diferenciação Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
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.
Assuntos
Materiais Biomiméticos , Reatores Biológicos , Contração Miocárdica , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Engenharia Tecidual , Alicerces Teciduais , Animais , Estimulação Elétrica , Proteínas Musculares/biossíntese , Miocárdio/citologia , Miócitos Cardíacos/citologia , Porosidade , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Coração/fisiologia , Engenharia Tecidual/métodos , Animais , Reatores Biológicos , Estimulação Elétrica , Eletrodos , Modelos Biológicos , Miocárdio/ultraestrutura , Ratos , Ratos Sprague-DawleyRESUMO
We investigated the effects of the initial stiffness of a three-dimensional elastomer scaffold--highly porous poly(glycerol sebacate)--on functional assembly of cardiomyocytes cultured with perfusion for 8 days. The polymer elasticity varied with the extent of polymer cross-links, resulting in three different stiffness groups, with compressive modulus of 2.35 ± 0.03 (low), 5.28 ± 0.36 (medium), and 5.99 ± 0.40 (high) kPa. Laminin coating improved the efficiency of cell seeding (from 59 ± 15 to 90 ± 21%), resulting in markedly increased final cell density, construct contractility, and matrix deposition, likely because of enhanced cell interaction and spreading on scaffold surfaces. Compact tissue was formed in the low and medium stiffness groups, but not in the high stiffness group. In particular, the low stiffness group exhibited the greatest contraction amplitude in response to electric field pacing, and had the highest compressive modulus at the end of culture. A mathematical model was developed to establish a correlation between the contractile amplitude and the cell distribution within the scaffold. Taken together, our findings suggest that the contractile function of engineered cardiac constructs positively correlates with low compressive stiffness of the scaffold.
Assuntos
Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Modelos Teóricos , Miócitos Cardíacos/citologia , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Coração/fisiologia , Engenharia Tecidual/métodos , Animais , Estimulação Elétrica/métodos , Transplante de Coração/métodos , Transplante de Coração/fisiologia , Humanos , Modelos Biológicos , Miocárdio/citologia , Técnicas de Cultura de Órgãos , Perfusão/métodos , Engenharia Tecidual/normasRESUMO
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.
Assuntos
Adipócitos/citologia , Adipócitos/fisiologia , Células-Tronco/citologia , Células-Tronco/fisiologia , Adipócitos/efeitos da radiação , Diferenciação Celular/efeitos da radiação , Polaridade Celular/efeitos da radiação , Tamanho Celular/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta à Radiação , Humanos , Doses de Radiação , Células-Tronco/efeitos da radiaçãoRESUMO
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.
Assuntos
Miocárdio/citologia , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Animais , Bovinos , Eletrofisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Miócitos Cardíacos/fisiologia , RatosRESUMO
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.