Recellularization of Whole Decellularized Porcine Hearts
with Human Cardiac Fibroblasts and Endothelial Cells
Nima Momtahan, Beverly L Roeder, Alonzo D Cook
Cardiovascular diseases in the United States account for approximately 30% of all deaths and 60% on a global scale . During myocardial infarction, a portion of the heart does not receive a supply of nutrients and oxygen which leads to irreversible cell damage. For patients who progress to 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. Advances in technology (e.g. total artificial heart, ventricular assist devices, improved diagnostic techniques, etc.) have slowed the accelerating demand for end-stage organ transplantation, yet are not sufficient to satisfy the need. Clinical efforts to restore lost function after an infarction through cell delivery to the injured areas of the heart have not been completely effective . Tissue engineered hearts that are produced by decellularization and then recellularization with the patient’s own cells hold promising advantages such as creating an unlimited source of donor hearts . Also tissue engineered hearts are non-immunogenic, meaning they will not be rejected by the recipient’s body because the natural scaffold is non-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) cells as well as improved decellularization techniques that produce naturally vascularized scaffolds with preserved components, have brought a lot of hope to create the ideal tissue engineered heart.
DNA and some membrane proteins of the cells can initiate an immune response leading to the rejection of the tissue. Ideal decellularization will 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 order to replicate the normal heart’s function, the ECM should be reseeded with the necessary cell types and be able to provide support for the cells from preserved structural and signaling molecules. The heart consists of many cell types, including cardiomyocytes, endothelial cells, vascular smooth muscle cells, pericytes, and cardiac fibroblasts in which fibroblasts outnumber all other cell types.
In this work, we improved the decellularization process of porcine hearts in terms of time and cost by using a custom-designed perfusion apparatus. Automation systems were implemented in the system to control pressure and temperature during the process, as well as programmed gradual pressure increase. 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.
After decellularization, the right ventricles of the hearts were dissected, cannulated through the main right coronary artery and any large open blood vessels were closed using a cyanoacrylate tissue adhesive (Vetbond®). Sections of the hearts were placed in sealed, agitated and oxygenated bioreactors and sterilized with a protocol optimized for being less destructive and enhancing cell attachment to the ECM. Sections were then perfused with growth medium for 24 hours and reseeded with 1×108 fluorescence-labeled human umbilical vein endothelial cells (HUVECs) and human cardiac fibroblasts (HCFs). The HUVECs were introduced through vasculature perfusion and HCFs by needle injection into the ECM at multiple time points. After at least 7 days of growth, histological images showed HUVECs dispersed throughout the right ventricle, covering the vasculature and HCFs present in the myocardium.
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, perfusion recellularization of the right ventricles with HUVECs and HCFs demonstrated the feasibility of recellularizing whole decellularized porcine heart tissues. The next steps will be towards repopulating the ECM with cardiomyocytes in addition to supportive cells, in order to obtain a functional heart tissue that can be transplanted into an infarcted heart to help restore its lost function.
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2. Robertson, M.J., et al., Optimizing recellularization of whole decellularized heart extracellular matrix. PLoS One, 2014. 9(2): p. e90406.
3. Momtahan, N., et al., Strategies and Processes to Decellularize and Recellularize Hearts to Generate Functional Organs and Reduce the Risk of Thrombosis. Tissue Eng Part B Rev, 2015. 21(1): p. 115-132.
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