Cell-based tissue engineering approaches to myocardial repair are 21st century solutions to an intractable problem. Because the adult heart lacks a pool of cells to effect repairs, it is incapable of effective cardiomyocyte regeneration after injury or infarction. Both direct cardiac myocyte cell transplantation into the myocardium and the development of tissue-engineered cardiac tissue constructs show significant promise as potential solutions. However, terminally differentiated adult cardiomyocytes suffer from a limited period of stability in cell culture, whether in a 2-D culture dish setting or in a 3-D scaffold environment. Therefore, the ultimate aim of this project is to develop a marketable process to provide long-term storage methods for tissue-engineered myocardial cells or constructs. Prior published efforts to establish preservation methods for cardiac myocytes and myocardium have encountered fundamental problems. Ice formation within the extracellular matrix of multicellular tissues limits the function of cryopreserved tissues using conventional freezing techniques. This mode of injury can be circumvented, by using a new cryopreservation approach, known as vitreous cryopreservation or amorphous solidification of a supercooled liquid. The working hypothesis that this study seeks to address is that ice-free cryopreservation technology will enhance the survival of myocardial cells and tissue segments to be used in myocardial tissue-engineered constructs compared with conventional cryopreservation involving freezing. The function and structural integrity of vitrified and control specimens will be quantitatively assessed using viability assays (metabolic, apoptotic, contractile, membrane integrity tests) and histology. Following successful completion of the aims of this Phase I SBIR feasibility proposal, several issues will be addressed in a Phase II SBIR submission to fully develop an effective storage protocol for human tissue-engineered myocardial constructs. In the long-term, it is anticipated that cryopreservation by vitrification may permit the storage of tissue-engineered left ventricular assist devices or hearts.
