Recent progress in cell transplantation therapy to repair impaired hearts has encouraged further attempts to bioengineer 3-dimensional (3-D) heart tissue from cultured cardiomyocytes. Cardiac tissue engineering is currently pursued utilizing conventional technology to fabricate 3-D biodegradable scaffolds as a temporary extracellular matrix. By contrast, new methods are now described to fabricate pulsatile cardiac grafts using new technology that layers cell sheets 3-dimensionally. We apply novel cell culture surfaces grafted with temperature-responsive polymer, poly(N-isopropylacrylamide) (PIPAAm), from which confluent cells detach as a cell sheet simply by reducing temperature without any enzymatic treatments. Neonatal rat cardiomyocyte sheets detached from PIPAAm-grafted surfaces were overlaid to construct cardiac grafts. Layered cell sheets began to pulse simultaneously and morphological communication via connexin43 was established between the sheets. When 4 sheets were layered, engineered constructs were macroscopically observed to pulse spontaneously. In vivo, layered cardiomyocyte sheets were transplanted into subcutaneous tissues of nude rats. Three weeks after transplantation, surface electrograms originating from transplanted grafts were detected and spontaneous beating was macroscopically observed. Histological studies showed characteristic structures of heart tissue and multiple neovascularization within contractile tissues. Constructs transplanted into 3-week-old rats exhibited more cardiomyocyte hypertrophy and less connective tissue than those placed into 8-week-old rats. Long-term survival of pulsatile cardiac grafts was confirmed up to 12 weeks. These results demonstrate that electrically communicative pulsatile 3-D cardiac constructs were achieved both in vitro and in vivo by layering cardiomyocyte sheets. Cardiac tissue engineering based on this technology may prove useful for heart model fabrication and cardiovascular tissue repair. The full text of this article is available at http://www.circresaha.org.

Fabrication of Pulsatile Cardiac Tissue Grafts Using a Novel 3-Dimensional Cell Sheet Manipulation Technique and Temperature-Responsive Cell Culture Surfaces

The concept of regenerating diseased myocardium by implantation of tissue-engineered heart muscle is intriguing, but convincing evidence is lacking that heart tissues can be generated at a size and with contractile properties that would lend considerable support to failing hearts. Here we created large (thickness/diameter, 1-4 mm/15 mm), force-generating engineered heart tissue from neonatal rat heart cells. Engineered heart tissue formed thick cardiac muscle layers when implanted on myocardial infarcts in immune-suppressed rats. When evaluated 28 d later, engineered heart tissue showed undelayed electrical coupling to the native myocardium without evidence of arrhythmia induction. Moreover, engineered heart tissue prevented further dilation, induced systolic wall thickening of infarcted myocardial segments and improved fractional area shortening of infarcted hearts compared to controls (sham operation and noncontractile constructs). Thus, our study provides evidence that large contractile cardiac tissue grafts can be constructed in vitro, can survive after implantation and can support contractile function of infarcted hearts.

Engineered heart tissue grafts improve systolic and diastolic function in infarcted rat hearts

Cardiac tissue engineering is an emerging field. The suitability of engineered heart tissue (EHT) for both in vitro and in vivo applications will depend on the degree of syncytoid tissue formation and cardiac myocyte differentiation in vitro, contractile function, and electrophysiological properties. Here, we demonstrate that cardiac myocytes from neonatal rats, when mixed with collagen I and matrix factors, cast in circular molds, and subjected to phasic mechanical stretch, reconstitute ring-shaped EHTs that display important hallmarks of differentiated myocardium. Comparative histological analysis of EHTs with native heart tissue from newborn, 6-day-old, and adult rats revealed that cardiac cells in EHTs reconstitute intensively interconnected, longitudinally oriented, cardiac muscle bundles with morphological features resembling adult rather than immature native tissue. Confocal and electron microscopy demonstrated characteristic features of native differentiated myocardium; some of these features are absent in myocytes from newborn rats: (1) highly organized sarcomeres in registry; (2) adherens junctions, gap junctions, and desmosomes; (3) a well-developed T-tubular system and dyad formation with the sarcoplasmic reticulum; and (4) a basement membrane surrounding cardiac myocytes. Accordingly, EHTs displayed contractile characteristics of native myocardium with a high ratio of twitch (0.4 to 0.8 mN) to resting tension (0.1 to 0.3 mN) and a strong β-adrenergic inotropic response. Action potential recordings demonstrated stable resting membrane potentials of −66 to −78 mV, fast upstroke kinetics, and a prominent plateau phase. The data indicate that EHTs represent highly differentiated cardiac tissue constructs, making EHTs a promising material for in vitro studies of cardiac function and tissue replacement therapy.

Tissue Engineering of a Differentiated Cardiac Muscle Construct

The major challenge of tissue engineering is directing the cells to establish the physiological structure and function of the tissue being replaced across different hierarchical scales. To engineer myocardium, biophysical regulation of the cells needs to recapitulate multiple signals present in the native heart. We hypothesized that excitation–contraction coupling, critical for the development and function of a normal heart, determines the development and function of engineered myocardium. To induce synchronous contractions of cultured cardiac constructs, we applied electrical signals designed to mimic those in the native heart. Over only 8 days in vitro, electrical field stimulation induced cell alignment and coupling, increased the amplitude of synchronous construct contractions by a factor of 7, and resulted in a remarkable level of ultrastructural organization. Development of conductive and contractile properties of cardiac constructs was concurrent, with strong dependence on the initiation and duration of electrical stimulation.

https://www.pnas.org/content/pnas/101/52/18129.full.pdf

Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds.

Cell sheet engineering for myocardial tissue reconstruction

A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3-dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro-coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte-populated matrices (CMPMs) anchored at opposite ends to the Velcro-covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue-like structure of alpha-actin and alpha-tropomyosin-positive cardiac myocytes exhibiting typical cross-striation, sarcomeric myofilaments, intercalated discs, desmosomes, and tight junctions. Force measurements of paced or unpaced CMPMs were performed in organ baths after 6-11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive "staircase"), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant beta-galactosidase-carrying adenovirus. Transduction efficiency increased from about 5% (MOI 0.1) to about 50% (MOI 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgenic animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions.

Hans H. Dohmen

Papers

Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system.