Engineered heart tissue for replacement therapy

Abstract

Cardiac tissue engineering could be a new therapeutic strategy for treatment of heart failure, myocardial infarction, and congenital heart defects. We have developed 3D-Engineered Heart Tissue (EHT) by reconstituting freshly isolated cardiac myocytes from neonatal Fischer 344 rats with pH neutralized liquid collagen type I (Fischer 344), matrigel, and serum-containing culture medium (DMEM, 10% horse serum, 2% chick embryo extract). Applicability of EHTs for replacement therapy was tested in a syngenic rat model. After 14 days in culture contractile function of EHTs (n=33) was measured in Tyrode solution under isometric conditions (twitch tension at 1.8 mM Ca2+: 0.60±0.04 mN). Subsequently, implantation was performed in mechanically ventilated adult Fischer rats under isoflurane anesthesia. After left lateral thoracotomy EHTs were sutured around the circumference of the heart (n=16). Graft integration, function, and survival was investigated 1,3,7,14, and 28 days after implantation in the presence of immunosuppression (CyA 5, azathioprine 2, methylprednisolon 2 mg/kg). Apparent contractions of the EHT-grafts were noted upon visual inspection of the implantation site at any of the given times. Light and confocal laser scanning microscopy of hematoxylin&eosin stained and immunolabeled (actin, actinin, myomesin, connexin, cadherin) cryo-sections revealed that EHT-grafts contained intensively interconnected and highly differentiated myocardial structurs. Labeling of endothelial cells (CD31) and fibroblasts (prolylhydroxylase) revealed intensive vascularization and connective tissue proliferation. Electron microscopy demonstrated that implanted cardiac myocytes developed a high degree of differentiation with classical cross-striational pattern including M-bands in situ. These myocyte clusters were mostly located in close vicinity to newly formed capillaries. Taken together, EHTs can be sutured on the heart, retain their contractile function, develop a highly differentiated myocyardial structure, and are heavily vascularised. Thus, EHTs may be useful for cardiac tissue replacement therapy.