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Portable bioreactor for perfusion and electrical stimulation of engineered cardiac tissue

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Abstract

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.

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... Characteristics of the studies. From the selected studies, [30,37] correspond to tissue cultures applying low electrical current stimulation (ii) while the studies [14,[31][32][33][34][35][36]38] correspond to electromechanical strain stimulation (iii). Human mesenchymal stromal cells (hMSCs). ...
... Types of cell tissues. As for the cell types considered in each of the selected references, six studies deal with cardiac tissue engineering [31][32][33][34][35][36]38], one with bone tissue engineering [30], one with muscle/ligament/tendon tissue engineering [14] and two with stem cell tissue engineering, [38]. Regarding the objective of generic cell differentiation, Dodel and his collaborators (see [37]) explicitly show that differentiation into ligament tissue requires electrical stimulation. ...
... The scaffolds employed in the reactors are commonly made out of a mixture of both non-biological and biological cell tissues (synthesized and/or natural). The biological components range from generic components as collagen [30,33], chitosan [37,38], collagen-chitosan hydrogels [32,38], poly(glycerol sebacate) [31] and poly(l-lactic acid) [38] to specific tissues adequate as substrate for each target cell culture. These tissues range from fresh pig hearts [34] (cardiac), human umbilical vein endothelial cells [35] (cardiac), rat hearts [33,36] (cardiac) to adipose-derived stem/stromal cells [14] (ligament differentiation). ...
Article
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We review here the current research status on bioreactors for tissue engineering with cell electrical stimulation. Depending on the cell types, electrical stimulation has distinct objectives, in particular being employed both to mimic and enhance the endogenous electricity measured in the natural regeneration of living organisms as well as to mimic strain working conditions for contractible tissues (for instance muscle and cardiac tissues). Understanding the distinct parameters involved in electrical stimulation is crucial to optimize its application. The results presented in the literature and reviewed here reveal that the application of electrical stimulation can be essential for tissue engineering applications.
... Subsequent studies by Maidhof, Tandon and colleagues [38,39] demonstrated the beneficial effects of combining electrical stimulation with media perfusion for cardiac tissue formation. Protection of cardiomyocytes (localized only in the bulk phase of the porous scaffold) from hydrodynamic shear (associated with the flow that is localized only in perfused channels), and the application of electrical stimulation synergistically enhanced the functionality of tissue constructs. ...
... Protection of cardiomyocytes (localized only in the bulk phase of the porous scaffold) from hydrodynamic shear (associated with the flow that is localized only in perfused channels), and the application of electrical stimulation synergistically enhanced the functionality of tissue constructs. Lower excitation thresholds (indicative of cellular electrical excitability), higher maximum capture rates (indicative of increased intracellular connections), and higher contraction amplitudes (indicative of functional contractile maturation) were measured for young animal cardiomyocytes grown in channeled scaffolds with medium perfusion and electrical stimulation [38,39]. ...
Article
The challenging task of heart regeneration is being pursued in three related directions: derivation of cardiomyocytes from human stem cells, in vitro engineering and maturation of cardiac tissues, and development of methods for controllable cell delivery into the heart. In this review, we focus on tissue engineering methods that recapitulate biophysical signaling found during normal heart development and maturation. We discuss the use of scaffold-bioreactor systems for engineering functional human cardiac tissues, and the methods for delivering stem cells, cardiomyocytes and engineered tissues into the heart. Copyright © 2015. Published by Elsevier B.V.
... Nowadays, the availability of affordable open-source and lowcost electronic solutions for bioprocess monitoring and control purposes and the diffusion of low-cost 3D printing technologies give the opportunity to rethink the design phase as well as to develop highly customizable and flexible bioprocess platforms at limited implementation costs [48][49][50][51][52] . In this perspective, we present here a compact, easy-to-use, tunable stretch bioreactor platform for TE applications. ...
Article
Physical stimuli are crucial for the structural and functional maturation of tissues both in vivo and in vitro. In tissue engineering applications, bioreactors have become fundamental and effective tools for providing biomimetic culture conditions that recapitulate the native physical stimuli. In addition, bioreactors play a key role in assuring strict control, automation, and standardization in the production process of cell-based products for future clinical application. In this study, a compact, easy-to-use, tunable stretch bioreactor is proposed. Based on customizable and low-cost technological solutions, the bioreactor was designed for providing tunable mechanical stretch for biomimetic dynamic culture of different engineered tissues. In-house validation tests demonstrated the accuracy and repeatability of the imposed mechanical stimulation. Proof of concepts biological tests performed on engineered cardiac constructs, based on decellularized human skin scaffolds seeded with human cardiac progenitor cells, confirmed the bioreactor Good Laboratory Practice compliance and ease of use, and the effectiveness of the delivered cyclic stretch stimulation on the cardiac construct maturation.
... Many researchers have studied tissue engineering to fabricate 3D cardiac constructs in vitro [11][12][13] , in which scaffolds such as collagen, fibrin gel, and oriented fibres are generally used. Cardiomyocytes seeded in the scaffold demonstrate heart-specific functions, because the scaffold-based system can provide a 3D culture environment for cells. ...
Article
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Cardiac constructs fabricated using human induced pluripotent stem cells-derived cardiomyocytes (iPSCs-CMs) are useful for evaluating the cardiotoxicity of and cardiac response to new drugs. Previously, we fabricated scaffold-free three-dimensional (3D) tubular cardiac constructs using a bio-3D printer, which can load cardiac spheroids onto a needle array. In this study, we developed a method to measure the contractile force and to evaluate the drug response in cardiac constructs. Specifically, we measured the movement of the needle tip upon contraction of the cardiac constructs on the needle array. The contractile force and beating rate of the cardiac constructs were evaluated by analysing changes in the movement of the needle tip. To evaluate the drug response, contractile properties were measured following treatment with isoproterenol, propranolol, or blebbistatin, in which the movement of the needle tip was increased following isoproterenol treatment, but was decreased following propranolol or blebbistain, treatments. To evaluate cardiotoxicity, contraction and cell viability of the cardiac constructs were measured following doxorubicin treatment. Cell viability was found to decrease with decreasing movement of the needle tip following doxorubicin treatment. Collectively, our results show that this method can aid in evaluating the contractile force of cardiac constructs.
... Moreover, this requires establishing perfusion with multiple blood arteries and veins, and maintenance of co-culture environment, in addition to coupling of electromechanical beating with host tissue [41]. To gain these functions, external electric stimulations and use of electro-conductive scaffold materials are required to improve the involuntary and synchronized beating of synthetic heart tissues [27, 42,43]. Attaining precise control of all of these factors and optimizing them with native host tissue is very challenging and efforts continue in all these fronts to develop fully functional artificial heart constructs. ...
Article
Cardiovascular disease (CVD) is among the leading causes of mortality worldwide. The shortage of donor hearts to treat end-stage heart failure patients is a critical problem. An average of 3500 heart transplant surgeries are performed globally, half of these transplants are performed in the US alone. Stem cell therapy is growing rapidly as an alternative strategy to repair or replace the damaged heart tissue after a myocardial infarction (MI). Nevertheless, the relatively poor survival of the stem cells in the ischemic heart is a major challenge to the therapeutic efficacy of stem-cell transplantation. Recent advancements in tissue engineering offer novel biomaterials and innovative technologies to improve upon the survival of stem cells as well as to repair the damaged heart tissue following a myocardial infarction (MI). However, there are several limitations in tissue engineering technologies to develop a fully functional, beating cardiac tissue. Therefore, the main goal of this review article is to address the current advancements and barriers in cardiac tissue engineering to augment the survival and retention of stem cells in the ischemic heart.
... Thus, perfusion bioreactors play an important role in creating the specific culture conditions necessary for the development of 3D engineered tissues by improving nutrient transportation and waste removal and generate mechanical stimulation in the form of shear stress 6 . Direct perfusion culture has been shown to be beneficial for culturing various types of engineered tissues, such as cardiac [6][7][8] , hepatic 9,10 , cartilage, and bone tissue [11][12][13] , with even very low flow rates inducing widespread changes in gene and protein expression in multiple cell types 14,15 . Flow-induced shear stress has been shown to regulate and enhance angiogenic processes in microfluidic chips [16][17][18][19] and monolayer two-dimensional (2D) flow-over models 20,21 . ...
Article
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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.
... Previous studies have successfully shown that media perfusion through microfluidics has increased the cell survival rate in a few-millimeter-thick cardiac tissues. 118,119 Radisic and her colleagues used this concept and developed a thicker and functional cardiac tissue through microfluidics perfusion system to supply oxygen to seeded cells. They also developed a 3D bioreactor-based scaffold with multiple parallel channels similar to capillary network to enhance perfusion of cells to match the nutrients demand. ...
Article
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Conventional tissue engineering, cell therapy, and current medical approaches were shown to be successful in reducing mortality rate and complications caused by cardiovascular diseases (CVDs). But still they have many limitations to fully manage CVDs due to complex composition of native myocardium and microvascularization. Fabrication of fully functional construct to replace infarcted area or regeneration of progenitor cells is important to address CVDs burden. Three-dimensional (3D) printed scaffolds and 3D bioprinting technique have potential to develop fully functional heart construct that can integrate with native tissues rapidly. In this review, we presented an overview of 3D printed approaches for cardiac tissue engineering, and advances in 3D bioprinting of cardiac construct and models. We also discussed role of immune modulation to promote tissue regeneration.
... Bioreactor systems allowing for a variety of controlled in vitro studies on larger populations of cells have been developed over the past 3 decades. [26][27][28][29][30] Bioreactor designs have been engineered for dynamic nuclear polarization-MRS studies, most typically for MRS on high-field nuclear magnetic resonance systems, 19 and, independently, for optical imaging. 31 However, as of yet, bioreactors for complementary optical and MRS studies of the same 3D cell culture have not been developed, partly because of engineering challenges. ...
Article
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Purpose Fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorescent metabolites permits the measurement of cellular metabolism in cell, tissue and animal models. In parallel, magnetic resonance spectroscopy (MRS) of dynamic nuclear (hyper)polarized (DNP) ¹³C‐pyruvate enables measurement of metabolism at larger in vivo scales. Presented here are the design and initial application of a bioreactor that connects these 2 metabolic imaging modalities in vitro, using 3D cell cultures. Methods The model fitting for FLIM data analysis and the theory behind a model for the diffusion of pyruvate into a collagen gel are detailed. The device is MRI‐compatible, including an optical window, a temperature control system and an injection port for the introduction of contrast agents. Three‐dimensional printing, computer numerical control machining and laser cutting were used to fabricate custom parts. Results Performance of the bioreactor is demonstrated for 4 T1 murine breast cancer cells under glucose deprivation. Mean nicotinamide adenine dinucleotide (NADH) fluorescence lifetimes were 10% longer and hyperpolarized ¹³C lactate:pyruvate (Lac:Pyr) ratios were 60% lower for glucose‐deprived 4 T1 cells compared to 4 T1 cells in normal medium. Looking at the individual components of the NADH fluorescent lifetime, τ1 (free NADH) showed no significant change, while τ2 (bound NADH) showed a significant increase, suggesting that the increase in mean lifetime was due to a change in bound NADH. Conclusion A novel bioreactor that is compatible with, and can exploit the benefits of, both FLIM and ¹³C MRS in 3D cell cultures for studies of cell metabolism has been designed and applied.
... Scaffold-based cardiac tissue engineering is useful as it provides a foundation for the construction of the 3D environment and can help modulate specific heart functions. Several groups have fabricated cardiac constructs from hydrogel mixtures, such as matrigel and collagen, and rat heart cells [13,14]. Further, when electrical stimulation was applied to these constructs for 5 days, the engineered cardiac tissue displayed an enhanced inotropic reserve. ...
Article
Full-text available
A major challenge in cardiac tissue engineering is the host’s immune response to artificial materials. To overcome this problem, we established a scaffold-free system for assembling cell constructs using an automated Bio-3D printer. This printer has previously been used to fabricate other three-dimensional (3D) constructs, including liver, blood vessels, and cartilage. In the present study, we tested the function in vivo of scaffold-free cardiac tubular construct fabricated using this system. Cardiomyocytes derived from induced pluripotent stem cells (iCells), endothelial cells, and fibroblasts were combined to make the spheroids. Subsequently, tubular cardiac constructs were fabricated by Bio-3D printer placing the spheroids on a needle array. Notably, the spheroid fusion and beat rate in the constructs were observed while still on the needle array. After removal from the needle array, electrical stimulation was used to test responsiveness of the constructs. An increased beat rate was observed during stimulation. Importantly, the constructs returned to their initial beat rate after stimulation was stopped. In addition, histological analysis shows cellular reorganization occurring in the cardiac constructs, which may mimic that observed during organ transplantation. Taken together, our results indicate that these engineered cardiac tubular constructs, which address both the limited supply of donor tissues as well as the immune-induced transplant rejection, has potential to be used for both clinical and drug testing applications. To our knowledge, this is the first time that cardiac tubular constructs have been produced using optimized Bio-3D printing technique and subsequently tested for their use as cardiac pumps.
... Lux et al. created a bioreactor that can both provide cyclic mechanical stretch and perfusion of medium to cardiac patches up to 2.5 cm × 4.5 cm in size (106). Tandon et al. developed a portable bioreactor which can both provide perfusion and electrical stimulation to cardiac patches (107). Further engineering is required to scale-up these types of reactors, to design systems able to transmit electrical and mechanical cues in suspension. ...
Article
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Recent advances in the differentiation and production of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) have stimulated development of strategies to use these cells in human cardiac regenerative therapies. A prerequisite for clinical trials and translational implementation of hPSC-derived CMs is the ability to manufacture safe and potent cells on the scale needed to replace cells lost during heart disease. Current differentiation protocols generate fetal-like CMs that exhibit proarrhythmogenic potential. Sufficient maturation of these hPSC-derived CMs has yet to be achieved to allow these cells to be used as a regenerative medicine therapy. Insights into the native cardiac environment during heart development may enable engineering of strategies that guide hPSC-derived CMs to mature. Specifically, considerations must be made in regard to developing methods to incorporate the native intercellular interactions and biomechanical cues into hPSC-derived CM production that are conducive to scale-up.
... A micropump-powered perfusion microbioreactor, providing electrical stimulation necessary to engineer cardiac tissue in vitro, as well, was developed to carry the latter wherever it is needed. This microbioreactor may also function as an external pumping system supplying nutrients and oxygen to the graft post-implantation [12]. In a recent review, Chang and Wu [13] presented the advantageous features of using microreactors for cartilage tissue engineering assays, and described their fabrication, experimental setup, and applications. ...
... In previous studies we have shown that although stimulated whole heart constructs developed less measurable cellular viability in contrast to non-stimulated constructs, they revealed a clearly increased cellular orientation and arrangement [5]. Another approach to tissue stimulation is to subject myocardial constructs isolated to an electrophysiological environment by the application of electric tension or direct current injection [6, 7]. However, only direct current injection has been previously investigated in a setting involving whole heart constructs [8]. ...
Article
Today the concept of Whole-Heart Tissue Engineering represents one of the most promising approaches to the challenge of synthesizing functional myocardial tissue. At the current state of scientific and technological knowledge it is a principal task to transfer findings of several existing and widely investigated models to the process of whole-organ tissue engineering. Hereby, we present the first bioreactor system that allows the integrated 3D biomechanical stimulation of a whole-heart construct while allowing for simultaneous controlled perfusion of the coronary system.
... We are adjusting the molecular size of the PLLA to have its degradation rate match the regeneration of cardiac tissue with the seeded cells and the integration of the constructs with the host tissue. Moreover, electrical [56,57] and mechanical [58,59] stimuli have been reported to facilitate cardiac differentiation and cardiac tissue organization. Thus, both rhythmic electrical and mechanical stimuli will be utilized in the construction of cardiac tissue in our future studies. ...
Article
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Myocardial infarction (MI) is the irreversible necrosis of heart with approximately 1.5 million cases every year in the United States. Tissue engineering offers a promising strategy for cardiac repair after MI. However, the optimal cell source for heart tissue regeneration and the ideal scaffolds to support cell survival, differentiation, and integration, remain to be developed. To address these issues, we developed the technology to induce cardiovascular progenitor cells (CPCs) derived from mouse embryonic stem cells (ESCs) towards desired cardiomyocytes as well as smooth muscle cells and endothelial cells. We fabricated extracellular matrix (ECM)-mimicking nanofibrous poly(l-lactic acid) (PLLA) scaffolds with porous structure of high interconnection for cardiac tissue formation. The CPCs were seeded into the scaffolds to engineer cardiac constructs in vitro. Fluorescence staining and RT-PCR assay showed that the scaffolds facilitated cell attachment, extension, and differentiation. Subcutaneous implantation of the cell/scaffold constructs in a nude mouse model showed that the scaffolds favorably supported survival of the grafted cells and their commitment to the three desired lineages in vivo. Thus, our study suggested that the porous nanofibrous PLLA scaffolds support cardiac tissue formation from CPCs. The integration of CPCs with the nanofibrous PLLA scaffolds represents a promising tissue engineering strategy for cardiac repair. Copyright © 2015. Published by Elsevier Ltd.
... 47 Bioreactors that generate dynamic flow culture systems provide a nutrient-rich environment in which external forces can be applied to cultures, thereby resulting in tissues with increased strength and longevity. 48 Another requirement for creation of truly biomimetic cardiac tissue constructs is the effective mimicking of mechanical and structural properties of native cardiac ECM, composed of aligned collagen fibers with nano-scale diameters, to influence tissue architecture and electromechanical coupling. Therefore, Macadangdang et al. 49 and Kim et al. 50,51 reported the development and analysis of a nano-topographically controlled in vitro model of the myocardium that mimics the structural and functional properties of native myocardial tissue and specifically the underlying hydrogel architecture. ...
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Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately re-create the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when using such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models that accurately mimic the physiological properties of native tissue samples and highlight the advantages of using such 3D microtissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models.
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Since cardiovascular diseases (CVDs) are globally one of the leading causes of death, of which myocardial infarction (MI) can cause irreversible damage and decrease survivors’ quality of life, novel therapeutics are needed. Current approaches such as organ transplantation do not fully restore cardiac function or are limited. As a valuable strategy, tissue engineering seeks to obtain constructs that resemble myocardial tissue, vessels, and heart valves using cells, biomaterials as scaffolds, biochemical and physical stimuli. The latter can be induced using a bioreactor mimicking the heart’s physiological environment. An extensive review of bioreactors providing perfusion, mechanical and electrical stimulation, as well as the combination of them is provided. An analysis of the stimulations’ mechanisms and modes that best suit cardiac construct culture is developed. Finally, we provide insights into bioreactor configuration and culture assessment properties that need to be elucidated for its clinical translation. Graphical abstract
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Cardiovascular diseases are the leading cause of morbidity and mortality in the United States. Cardiac tissue engineering is a direction in regenerative medicine that aims to repair various heart defects with the long-term goal of artificially rebuilding a full-scale organ that matches its native structure and function. Three-dimensional (3D) bioprinting offers promising applications through its layer-by-layer biomaterial deposition using different techniques and bio-inks. In this review, we will introduce cardiac tissue engineering, 3D bioprinting processes, bioprinting techniques, bio-ink materials, areas of limitation, and the latest applications of this technology, alongside its future directions for further innovation.
Chapter
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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.
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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.
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Cardiac tissue engineering has made notable progress in recent years with the advent of an experimental model based on neonatal cardiomyocytes entrapped in collage gels and purified basement membrane extract, known as "engineered heart tissues" (EHTs). EHTs are a formidable display of tissue-level contractile function and cellular-level differentiation, although they suffer greatly from mass transport limitations due to the high density of metabolically active cells and the diffusion-limited nature of the hydrogel. In this report, a mathematical model was developed to predict oxygen levels inside a one-dimensional, diffusion-limited model of EHT. These predictions were then compared to values measured in corresponding experiments with a hypoxia-sensitive stain (pimonidazole). EHTs were cast between two plastic discs, which allowed for mass transfer with the culture medium to occur in only the radial direction. EHTs were cultured for up to 36 h in the presence of pimonidazole, after which time they were snap-frozen, histologically sectioned, and stained for bound pimonidazole. Quantitative image analysis was performed to measure the distance from the culture medium at which hypoxia first occurs under various conditions. As tested by variation of simple design parameters, the trends in oxygen profiles predicted by the model are in reasonable agreement with those obtained experimentally, although a number of ambiguities related to the specific model parameters led to a general overprediction of oxygen concentrations. Based on the sensitivity analysis in the present study, it is concluded that diffusion-reaction models may offer relatively precise predictions of oxygen concentrations in diffusion-limited tissue constructs.
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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.
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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. Copyright © 2011 John Wiley & Sons, Ltd.
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In developing tissues, proteins and signaling molecules present themselves in the form of concentration gradients, which determine the fate specification and behavior of the sensing cells. To mimic these conditions in vitro, we developed a microfluidic device designed to generate stable concentration gradients at low hydrodynamic shear and allowing long term culture of adhering cells. The gradient forms in a culture space between two parallel laminar flow streams of culture medium at two different concentrations of a given morphogen. The exact algorithm for defining the concentration gradients was established with the aid of mathematical modeling of flow and mass transport. Wnt3a regulation of β-catenin signaling was chosen as a case study. The highly conserved Wnt-activated β-catenin pathway plays major roles in embryonic development, stem cell proliferation and differentiation. Wnt3a stimulates the activity of β-catenin pathway, leading to translocation of β-catenin to the nucleus where it activates a series of target genes. We cultured A375 cells stably expressing a Wnt/β-catenin reporter driving the expression of Venus, pBARVS, inside the microfluidic device. The extent to which the β-catenin pathway was activated in response to a gradient of Wnt3a was assessed in real time using the BARVS reporter gene. On a single cell level, the β-catenin signaling was proportionate to the concentration gradient of Wnt3a; we thus propose that the modulation of Wnt3a gradients in real time can provide new insights into the dynamics of β-catenin pathway, under conditions that replicate some aspects of the actual cell-tissue milieu. Our device thus offers a highly controllable platform for exploring the effects of concentration gradients on cultured cells.
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The requirements for engineering clinically sized cardiac constructs include medium perfusion (to maintain cell viability throughout the construct volume) and the protection of cardiac myocytes from hydrodynamic shear. To reconcile these conflicting requirements, we proposed the use of porous elastomeric scaffolds with an array of channels providing conduits for medium perfusion, and sized to provide efficient transport of oxygen to the cells, by a combination of convective flow and molecular diffusion over short distances between the channels. In this study, we investigate the conditions for perfusion seeding of channeled constructs with myocytes and endothelial cells without the gel carrier we previously used to lock the cells within the scaffold pores. We first established the flow parameters for perfusion seeding of porous elastomer scaffolds using the C2C12 myoblast line, and determined that a linear perfusion velocity of 1.0 mm/s resulted in seeding efficiency of 87% +/- 26% within 2 hours. When applied to seeding of channeled scaffolds with neonatal rat cardiac myocytes, these conditions also resulted in high efficiency (77.2% +/- 23.7%) of cell seeding. Uniform spatial cell distributions were obtained when scaffolds were stacked on top of one another in perfusion cartridges, effectively closing off the channels during perfusion seeding. Perfusion seeding of single scaffolds resulted in preferential cell attachment at the channel surfaces, and was employed for seeding scaffolds with rat aortic endothelial cells. We thus propose that these techniques can be utilized to engineer thick and compact cardiac constructs with parallel channels lined with endothelial cells.
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A major challenge of tissue engineering is directing cells to establish the physiological structure and function of the tissue being replaced. Electrical stimulation has been used to induce synchronous contractions of cultured cardiac constructs. The hypothesis adopted for this study is that functional cardiac constructs can be engineered by "mimicking" the conditions present during cardiac development, and in particular, electrical stimulation using supra-threshold signals. For this Master's Thesis research, I have compared the material properties and charge-transfer characteristics at the electrode-electrolyte interface of various biocompatible materials, including carbon, stainless steel, titanium and titanium nitride, for use as electrodes in a biomimetic system for cardiac tissue engineering. I have also designed and implemented an electrical stimulator which is capable of modulating several important parameters of electrical stimulation, including stimulus amplitude and frequency. (cont.) In addition, I have built an experimental setup incorporating this electrical stimulator and used it for experiments with C2C12 mouse myoblast cells and neonatal rat cardiomyocytes. Lastly, I have analyzed cell morphology as well as functional performance of engineered tissue by assessing excitation thresholds and maximum capture rates. Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2006. Includes bibliographical references (leaves 66-69).
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Cell grafting has emerged as a novel approach to treat heart diseases refractory to conventional therapy. We hypothesize that survival and functional and electrical integration of grafts may be improved by engineering cardiac tissue constructs in vitro before grafting. Engineered heart tissue (EHT) was reconstituted by mixing cardiac myocytes from neonatal Fischer 344 rats with liquid collagen type I, matrigel, and serum-containing culture medium. EHTs were designed in circular shape (inner/outer diameter: 8/10 mm; thickness: 1 mm) to fit around the circumference of hearts from syngenic rats. After 12 days in culture and before implantation on uninjured hearts, contractile function of EHT was measured under isometric conditions. Baseline twitch tension amounted to 0.34+/-0.03 mN (n=33) and was stimulated by Ca(2+) and isoprenaline to 200+/-12 and 185+/-10% of baseline values, respectively. Despite utilization of a syngenic model immunosuppression (mg/kg BW: azathioprine 2, cyclosporine A 5, methylprednisolone 2) was necessary for EHT survival in vivo. Echocardiography conducted 7, 14, and 28 days after implantation demonstrated no change in left ventricular function compared with pre-OP values (n=9). Fourteen days after implantation, EHTs were heavily vascularized and retained a well organized heart muscle structure as indicated by immunolabeling of actinin, connexin 43, and cadherins. Ultrastructural analysis demonstrated that implanted EHTs surpassed the degree of differentiation reached before implantation. Contractile function of EHT grafts was preserved in vivo. EHTs can be employed for tissue grafting approaches and might serve as graft material to repair diseased myocardium.
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This protocol describes tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cell populations on porous scaffolds (in some cases with an array of channels) and bioreactors with perfusion of culture medium (in some cases supplemented with an oxygen carrier). The overall approach is 'biomimetic' in nature as it tends to provide in vivo-like oxygen supply to cultured cells and thereby overcome inherent limitations of diffusional transport in conventional culture systems. In order to mimic the capillary network, cells are cultured on channeled elastomer scaffolds that are perfused with culture medium that can contain oxygen carriers. The overall protocol takes 2-4 weeks, including assembly of the perfusion systems, preparation of scaffolds, cell seeding and cultivation, and on-line and end-point assessment methods. This model is well suited for a wide range of cardiac tissue engineering applications, including the use of human stem cells, and high-fidelity models for biological research.
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