Luda Khait

Cell-based cardiac pumps and tissue-engineered ventricles

Mortalities resulting from cardiovascular disorders remain high, with an urgent need to develop novel treatment modalities. Tissue-engineering therapies aim to provide cell-based alternatives to conventional options. Significant technological advancements have occurred during the last decade towards the fabrication of functional 3D heart muscle in vitro. More recent research has focused on the development of cell-based cardiac pumps and tissue-engineered ventricles. The global objective of this collective work is to simulate the functional performance of the left ventricle, utilizing completely cell-based options. Current prototypes have shown several physiological performance metrics, including the ability of these devices to generate intraluminal pressure upon electrical stimulation. This review will highlight the transition from tissue engineering 3D heart muscle to cell-based cardiac pumps/ventricles.

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.

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.

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.

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).

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.

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, α-myosin heavy chain, and β-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.

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.

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 μN on stimulation with phenylephrine. Bioengineered VSM strips exhibited Ca2+‐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.

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.