Fabrication of functional cardiac, skeletal, and smooth muscle pumps in vitro.

Cardiovascular disease is one of the leading causes of death in the United States, and new treatments need to be developed in order to provide novel therapies. Tissue engineering aims to develop biologic substitutes that restore tissue function. The purpose of the current study was to construct cell-based pumps, which can be viewed as biologic left ventricular assist devices. The pumps were fabricated by culturing cardiac, skeletal, and smooth muscle cells within a fibrin gel and then each 3-D tissue construct was wrapped around a decellularized rodent aorta. We described the methodology for pump fabrication along with functional performance metric, determined by the intra-luminal pressure. In addition, histologic evaluation showed a concentric organization of components, with the muscle cells positioned on the outermost surface, followed by the fibrin gel and the decellularized aorta formed the innermost layer. Though early in development, cell-based muscle pumps have tremendous potential to be used for basic and applied research, and with further development, can be used clinically as cell-based left ventricular assist devices.

Human thymus mesenchymal stromal cells augment force production in self-organized cardiac tissue.

BACKGROUND: Mesenchymal stromal cells have been recently isolated from thymus gland tissue discarded after surgical procedures. The role of this novel cell type in heart regeneration has yet to be defined. The purpose of this study was to evaluate the therapeutic potential of human thymus-derived mesenchymal stromal cells using self-organized cardiac tissue as an in vitro platform for quantitative assessment. METHODS: Mesenchymal stromal cells were isolated from discarded thymus tissue from neonates undergoing heart surgery and were incubated in differentiation media to demonstrate multipotency. Neonatal rat cardiomyocytes self-organized into cardiac tissue fibers in a custom culture dish either alone or in combination with varying numbers of mesenchymal stromal cells. A transducer measured force generated by spontaneously contracting self-organized cardiac tissue fibers. Work and power outputs were calculated from force tracings. Immunofluorescence was performed to determine the fate of the thymus-derived mesenchymal stromal cells. RESULTS: Mesenchymal stromal cells were successfully isolated from discarded thymus tissue. After incubation in differentiation media, mesenchymal stromal cells attained the expected phenotypes. Although mesenchymal stromal cells did not differentiate into mature cardiomyocytes, addition of these cells increased the rate of fiber formation, force production, and work and power outputs. Self-organized cardiac tissue containing mesenchymal stromal cells acquired a defined microscopic architecture. CONCLUSIONS: Discarded thymus tissue contains mesenchymal stromal cells, which can augment force production and work and power outputs of self-organized cardiac tissue fibers by several-fold. These findings indicate the potential utility of mesenchymal stromal cells in treating heart failure.

Variable optimization for the formation of three-dimensional self-organized heart muscle.

Cardiac tissue-engineering research is focused on the development of functional three-dimensional (3D) heart muscle in vitro. These models allow the detailed study of critical events in organogenesis, such as the establishment of cell-cell communication and construction and modification of the extracellular matrix. We have previously described a model for 3D heart muscle, termed cardioids, formed by the spontaneous delamination of a cohesive monolayer of primary cells in the absence of any synthetic scaffolding material. In an earlier publication, we have shown that, upon electrical stimulation, cardioids generate a twitch force in the range of 200-300 microN, generate a specific force (twitch force normalized to total cross-sectional area) of 2-4 kN/m(2), and can be electrically paced at frequencies of up to 10 Hz without any notable fatigue. We have two objectives for the current study: model development and model optimization. Our model development efforts are focused on providing additional characterization of the cardioid model. In this study, we show for the first time that cardioids show a pattern of gene expression comparable to that of cells cultured in two dimensions on tissue culture plastic and normal mammalian heart muscle. Compared with primary cardiac cells cultured on tissue culture plastic, the expression of alpha-myosin heavy chain (MHC), beta-MHC, SERCA2, and phospholamban was significantly higher in cardioids. Our second objective, model optimization, is focused on evaluating the effect of several cell culture variables on cardioid formation and function. Specifically, we looked at the effect of plating density (1.0-4.0 x 10(6) cells per cardioid), concentration of two adhesion proteins (laminin at 0.2-2.0 microg/cm(2) and fibronectin at 1-10 microg/cm(2)), myocyte purity (using preplating times of 15 and 60 min), and ascorbic acid stimulation (1-100 microl/ml). For our optimization studies, we utilized twitch force in response to electrical stimulation as our endpoint metric. Based on these studies, we found that cardioids formed with a plating density in the range 3-4 x 10(6) cells per cardioid generated the maximum twitch force, whereas increasing the surface adhesion protein (using either laminin or fibronectin) and increasing the myocyte purity both resulted in a decrease in twitch force. In addition, increasing the ascorbic acid concentration resulted in an increase in the baseline force of cardioids, which was recorded in the absence of electrical stimulation. Based on the model development studies, we have shown that cardioids do indeed exhibit a gene expression pattern similar to normal mammalian heart muscle. This provides further validity for the cardioid model. Based on the model optimization studies, we have identified specific cell culture regimes which support cardioid formation and function. These results are specific to the cardioid model; however, they may be translated and applied to other tissue-engineering models. Collectively, the work described in this study provides insight into the formation of functional 3D heart muscle and the effect of several cell culture variables on tissue formation and function.

Novel bench-top perfusion system improves functional performance of bioengineered heart muscle.

Research in the area of cardiac tissue engineering is focused on the development of functional 3-dimensional cardiac muscle tissue in vitro, which includes bioengineered cardiac patches, pumps and ventricles. One of the major challenges in the field of cardiovascular tissue engineering is determining how to support the increased metabolic demands of 3-dimensional tissue constructs, due to the increase in both cellular mass and density compared to monolayer cultures. Traditional culture systems rely on passive diffusion for the delivery of oxygen and soluble factors. However, perfusion systems can provide continuous delivery of cell culture media to 3D tissue constructs, which promotes more active delivery of oxygen, soluble factors, and shear stress, which can be utilized to guide tissue maturation and functional remodeling of bioengineered tissues. We have previously described a perfusion system and demonstrated compatibility over short time periods (approximately hours) with 2-dimensional monolayer cell culture and 3-dimensional tissue constructs. The objectives of our current study were to: introduce CO2 buffering to stabilize media pH in order to achieve long term culture within the system, incorporate sensors capable of recording high media oxygen concentrations, and to increase the culture time of bioengineered heart muscle within the perfusion system in order to increase their functional performance. We showed that exposure of bioengineered heart muscle to perfusion for a period of 24 h increased their functional performance, as measured by cellular viability, total protein, total RNA, spontaneous contractility, twitch force, and specific force.

Changes in gene expression during the formation of bioengineered heart muscle.

A three-dimensional bioengineered heart muscle (BEHM) construct model had been previously developed, exhibiting contractile forces up to 800 microN. The interest of this study was to determine gene expression levels of biologic markers involved in calcium-handling between BEHM, cell monolayer, and neonatal heart. Cardiac cells were isolated from one litter of F344 rats and organized into groups (n = 5): 4-, 7-, 10-day BEHM and cell monolayer; BEHM was evaluated for cell viability and contractility. Groups were then analyzed for mRNA expression of calcium-handling proteins: myosin heavy chain (MHC) alpha and beta, Sarcoplasmic reticulum Ca++ ATPase (SERCA) 2, phospholamban (PBL), and ryanodine receptor. BEHM exhibited electrically stimulated active force (208 +/- 12 microN day 4, 361 +/- 22 microN day 7, and 344 +/- 29 microN day 10) and no decrease in cell number. Real-time polymerase chain reaction (PCR) showed an increase in gene expression of all calcium-handling proteins in BEHM at 7 and 10 days compared with monolayers, for example, comparing BEHM to monolayer (7 and 10 days, respectively), MHC-alpha: 2600-fold increase and a 100-fold increase; MHC-beta: 70-fold increase at 10 days; ryanodine receptor: 74-fold increase at 10 days; SERCA: 19-fold increase and sixfold increase; PBL: 158-fold increase and 24-fold increase. It was concluded that a three-dimensional environment is a better culturing condition of cardiac cells than a monolayer. Also, BEHM constructs demonstrated a high similarity to a native myocardium, and is, thus, a good starting foundation for engineered heart muscle.

Effect of thyroid hormone on the contractility of self-organized heart muscle.

Tissue-engineered heart muscle may provide an alternative treatment modality for end-stage congestive heart failure. We have previously described a method to engineer contractile heart muscle in vitro (termed cardioids). This study describes a method to improve the contractile properties of cardioids utilizing thyroid hormone (T3) stimulation. Cardioids were engineered by promoting the self-organization of primary neonatal cardiac cells into a contractile tissue construct. Cardioids were maintained in standard cell culture media supplemented with varying concentrations of T3 in the range 1-5ng/ml. The contractile properties of the cardioids were evaluated 48h after formation. Stimulation with T3 resulted in an increase in the specific force of cardioids from an average value of 0.52 +/- 0.16kPa (N = 6) for control cardioids to 2.42 +/- 0.29kPa (N = 6) for cardioids stimulated with 3ng/ml T3. In addition, there was also an increase in the rate of contraction and relaxation in response to T3 stimulation. Cardioids that were stimulation with T3 exhibited improved pacing characteristics in response to electrical pacing at 1-5Hz and an increase in the degree of spontaneous contractility. Changes in the gene expression of SERCA2, phospholamban, alpha-myosin heavy chain, and beta-myosin heavy chain correlated with the changes in contractile properties. This study demonstrates the modulation of the contractile properties of tissue-engineered heart muscle using T3 stimulation.

Modulating the functional performance of bioengineered heart muscle using growth factor stimulation.

Congestive heart failure (CHF) is a major medical challenge in developed countries. The field of cardiac tissue engineering may provide alternative treatment methods of CHF to current surgical and pharmacological therapies. We previously described a model for the formation of Bioengineered Heart Muscle (BEHM), using fibrin gel as a support matrix for primary cardiac cells during 3D construct formation. In the current study, we describe modulating the contractile properties of BEHMs utilizing clenbuterol, insulin like growth factor-1 (IGF-1), and thyroid hormone (T3), as additive factors to primary cardiac cells. We found significant changes in force production with the addition of clenbuterol (control: 95.0 +/- 32.9 microN, clenbuterol: 201.0 +/- 15.8 microN) and IGF-1 (control: 202.9 +/- 18 microN, IGF-1: 272.6 +/- 19.5 microN) was seen. Selecting IGF-1-treated BEHMs for further analysis, we found an increase in force production during extended culturing at 7, 14 days. Also, at 25 ng/mL, myosin heavy chain alpha and SERCA2 expression increased by 1.3 +/- 0.188 and 1.1 +/- 0.04 fold, respectively. Our findings provide preliminary data that can be used to produce BEHMs with higher force of contraction. Exposing BEHMs to these factors would condition the engineered muscle for possible implantation onto injured hearts without cell shock.

Methodology for the formation of functional, cell-based cardiac pressure generation constructs in vitro.

We have previously described a model to engineer three-dimensional (3-D) heart muscle in vitro. In the current study, we extend our model of 3-D heart muscle to engineer a functional cell-based cardiac pressure generating construct (CPGC). Tubular constructs were fabricated utilizing a phase separation method with chitosan as the scaffolding material. Primary cardiac cells isolated from rat hearts were plated on the surface of fibrin gels cast in 35 mm tissue culture dishes. CPGCs (N = 8) were formed by anchoring the tubular constructs to the center of the plate with primary cardiac cells seeded in fibrin gels wrapped around the tubular constructs. Intraluminal pressure measurements were evaluated with and without external electrical stimulation and histological evaluation performed. The fibrin gel spontaneously compacted due to the traction force of the cardiac cells. By 14 d after original cell plating, the cardiac cells had completely formed a monolayer around the tubular construct resulting in the formation of a cell-based CPGC. The spontaneous contractility of the CPGC was macroscopically visible and resulted in intraluminal pressure spikes of 0.08 mmHg. Upon electrical stimulation, the CPGCs generated twitch pressures of up to 0.05 mmHg. In addition, the CPGC constructs were electrically paced at frequencies of up to 3 Hz. Histological evaluation showed the presence of a continuous cell monolayer around the surface of the tubular construct. In this study, we describe a novel in vitro method to engineer functional cell-based CPGCs and demonstrate several physiological metrics of functional performance.

Development of a microperfusion system for the culture of bioengineered heart muscle.

Tissue engineering strategies are being used to develop functional 3D heart muscle in vitro. Work within our own group has been focused on the development of bioengineered heart muscle using fibrin gel as a support matrix. As tissue engineering models of heart muscle are developed in the laboratory, a critical technologic challenge remains the ability to delivery nutrients to the entire tissue construct. To address this specific need, we have developed a novel perfusion system for cardiac tissue engineering applications. The system consists of a custom microincubator, designed to house ten 35-mm tissue culture plates on independent platforms for controlled fluid delivery and aspiration. Temperature, pH, and media flow rate and oxygenation are all regulated. In the current study, we describe the compatibility of this microperfusion system with bioengineered heart muscles. We demonstrate that the perfusion system is capable of supporting construct viability (mitochondrial activity, total protein, and total RNA) and maintaining contractile properties (twitch force, specific force, and electrical pacing).

Micro-perfusion for cardiac tissue engineering: development of a bench-top system for the culture of primary cardiac cells.

Tissue-engineered constructs have high metabolic requirements during in vitro culture necessitating the development of micro-perfusion systems to maintain high functional performance. In this study, we describe the design, fabrication, and testing of a novel micro-perfusion system to support the culture of primary cardiac cells. Our system consists of a micro-incubator with independent stages for 35-mm tissue culture plates with inflow/outflow manifolds for fluid delivery and aspiration. A peristaltic pump is utilized for fluid delivery and vacuum for fluid aspiration. Oxygen saturation, pH, and temperature are regulated for the media while temperature is regulated within the micro-incubator, fluid reservoir, and oxygenation chamber. Validation of the perfusion system was carried out using primary cardiac myocytes, isolated from 2- to 3-day-old neonatal rat hearts, plated on collagen-coated tissue culture plates. Two million cells/plate were used and the perfusion system was run for 1 h (without the need for a cell culture incubator) while controls were maintained in a standard cell culture incubator. We evaluated the cell viability, cell adhesion, total protein, total RNA, and changes in the expression of SERCA2 and phospholamban using RT-PCR, with N = 6 for each group. We found that there was no significant change in any variable during the 1-h run in the perfusion system. These studies served to demonstrate the compatibility of the perfusion system to support short-term culture of primary cardiac cells.

Getting to the heart of tissue engineering.

Cardiovascular disease affects 80 million people in the USA and is the leading cause of death. Significant limitations of current treatments necessitate the development of novel strategies. Cardiovascular tissue engineering is an emerging field focused on the development of biological substitutes to restore, maintain, or improve tissue function. In this article, we present an overview of trends in the field and scientific milestones achieved during the last decade. Various 3D bioengineered models of functional cardiovascular structures, including cell-based cardiac pumps, ventricles, patches, vessels, and valves, are described. We discuss critical technological hurdles that must be addressed for continued progress and an outlook for the future of cardiovascular tissue engineering.

Bioengineering functional human aortic vascular smooth-muscle strips in vitro.

The contraction and relaxation of VSM (vascular smooth muscle) are responsible for the maintenance of vascular tone, which is a major determinant of blood pressure. However, the molecular events leading to the contraction and relaxation of VSM are poorly understood. The development of three-dimensional bioengineered tissues provides an opportunity to investigate the molecular events controlling vascular tone in vitro. In the present study we used fibrin-gel casting to bioengineer functional VSM strips from primary human aortic VSM cells. Our bioengineered VSM strips are functionally similar to VSM in vivo and remained viable in culture for up to 5 weeks. VSM strips demonstrate spontaneous basal tone and can generate an active force (contraction) of up to 85.2 microN on stimulation with phenylephrine. Bioengineered VSM strips exhibited Ca(2+)-dependent contraction and calcium-independent relaxation. The development of functional bioengineered VSM tissue provides a new in vitro model system that can be used to investigate the molecular events controlling vascular tone.

Functional evaluation of isolated zebrafish hearts.

Traditional working heart preparations, based on the original Langendorff setup, are widely used experimental models that have tremendously advanced the cardiovascular field. However, these systems can be deceivingly complex, requiring the maintenance of pH with CO(2), the delivery of oxygenated perfusate, and the need for extensive laboratory equipment. We have examined the feasibility of using isolated zebrafish (Danio rerio) hearts as an experimental model system, in which experimental procedures can be performed in the absence of the traditional requirements and sophisticated setup equipment. Isolated zebrafish hearts exhibited spontaneous contractile activity, could be electrically paced, and were responsive to pharmacologic stimulation with isoproterenol for 1.5 h after in vivo removal. Isolated zebrafish hearts offer a time- and cost-effective alternative to traditional Langendorff/working heart preparation models, and could be used to investigate cardiac function and repair.

Contractile three-dimensional bioengineered heart muscle for myocardial regeneration.

Tissue engineered heart muscle may be able to provide a treatment modality for early stage congestive heart failure. In this study, we describe a new method to engineer functional 3-dimensional heart muscle utilizing a biodegradable fibrin gel. Primary cardiac myocytes were isolated from hearts of 2- to 3-day-old rats and processed in one of the two ways. For the first method (layering approach), the cells were plated directly on the surface of a fibrin gel-coated on polydimethylsiloxane (PDMS) surfaces. The cells were cultured in growth media and the contractile performance evaluated after formation of 3-dimensional tissue constructs. For the second method (embedding approach), the cells were suspended with thrombin and plated on 35 mm tissue culture surfaces coated with PDMS. Fibrinogen was then added to the surface. Within 7 days after initial cell plating, a 3-dimensional tissue construct of cells derived from primary heart tissue (termed bioengineered heart muscle, BEHM) resulted for both approaches. Histological evaluation showed the presence of uniformly distributed cardiac cells throughout the BEHM, both in longitudinal and cross sections. The stimulated active force of BEHMs formed using the layering approach was 835.5 +/- 57.2 muN (N = 6) and 145.3 +/- 44.9 muN (N = 6) using the embedding approach. The stimulated active force was dependent on the initial plating density. It was possible to maintain the contractile function of BEHM in culture for up to 2 months with daily medium changes. The BEHMs exhibited inotropy in response to external calcium and isoproterenol and could be electrically paced at frequencies of 1-7 Hz. We describe a novel method to engineer contractile 3-dimensional cardiac tissue construct with a fourfold increase specific force compared to our previous model.