380247 Porcine Heart Decellularization Improvement and Optimization
Heart failure is the number one cause of death in the United States and for the patients with end stage heart disease, the only option is to receive a heart transplant; however, the demand for heart transplants is higher than the supply of suitable hearts. Artificial hearts also have not been able to provide an appropriate solution because of inherent thrombogenicity and blood incompatibility problems that increase the risk of stroke and hemolytic anemia in patients receiving these hearts. Tissue engineered hearts that are created by decellularization and then recellularization with the patient’s own cells exhibit promising advantages such as creating an unlimited source of donor hearts. Also tissue engineered hearts are not immunogenic, meaning they will not be rejected by the recipient’s body because the natural scaffold is not immunogenic, and the cells are sourced from the same patient. Advances made in research of stem cells such as the discovery of IPS (Induced pluripotent stem cell) and STAP (Stimulus-triggered acquisition of pluripotency) cells have brought a lot of hope to create the ideal tissue engineered heart.
DNA and some membrane proteins of the cells, if enter the body will initiate an immune response; therefore, decellularization is an important step in creating tissue engineered hearts. Ideal decellularization can be achieved by completely removing all cells and cell debris from the heart, while keeping the heart scaffold’s vasculature intact and also preserving the important growth factors, glycosaminoglycans (GAGs) and proteins in the extracellular matrix (ECM).
In this work, we optimized the decellularization process of porcine hearts in terms of time and cost by using a custom-designed bioreactor. In the bioreactor, a peristaltic pump that is capable of creating pulsatile, high pressure flow was used and the hearts were connected to the bioreactor by the aorta and the solutions were pumped through the coronary arteries. A combination of type-1 distilled water, phosphate buffered saline, sodium dodecyl sulfate and Triton X-100 with minimum detergent exposure to the ECM were used for the decellularization process. Decellularized samples were characterized by histology and immunohistochemistry staining and tested for residual DNA, GAGs and collagen. Biocompatibility of the decellularized ECM was demonstrated with a hemolysis assay which measured the rupture of erythrocytes in contact with the scaffolds. An immunogenicity test was performed by measuring the stimulation of macrophage cells when they were in contact with the decellularized ECM compared to a fresh heart. Samples of the decellularized hearts were then recellularized with porcine aortic endothelial cells and murine MS-1 endothelial cells, and the proliferation and function of the cells on the scaffolds were studied.
This study introduced and validated a decellularization method that is optimized for porcine hearts which are similar to human hearts in terms of size and anatomy. This method may be very beneficial in aiding the development of tissue engineered human hearts. We showed that the decellularized ECM has minimal DNA residue and stimulation of macrophages which means that the ECM should not be immunogenic when transplanted into the body. Also the hemolysis assay that was developed showed minimal erythrocyte cell rupture which demonstrates cell compatibility of the decellularized ECM. Furthermore, repopulation of the endothelial cells in the ECM demonstrated the recellularization capability of the decellularized hearts. Next steps will be towards recellularization with stem cells and improving the function of tissue engineered hearts.
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