Makena Ford and Professor Alonzo Cook, Mechanical Engineering
Introduction
Over 120,000 people in the United States alone are currently on the waiting list to receive a lifesaving organ transplanti. Even if these patients receive an invaluable transplant, they will have to take immunosuppressants for the rest of their life, to prevent the body from attacking foreign heart cells. As heart disease prevails as the leading cause of death in America, an alternative solution to heart transplantation begs to be found. Our research team has been investigating the decellularization and recellularization of porcine hearts. We hope to reintroduce human cells onto the remaining protein structure, creating a fully functioning heart that is tailored to the patient in need. If successful, we could eliminate the need for immunosuppressants and dramatically reduce the waiting time and cost of heart transplants.
Methodology
Harvesting
Our research starts with the harvesting of porcine hearts from female pigs around 6 months of age. The sternum is cut and the rib cage is spread open to allow for the extraction of the porcine heart. The pulmonary veins, aorta and main pulmonary artery are cut to free the heart. The heart is then rinsed with phosphate saline and heparin to clean the heart of blood and to prevent clotting of arteries. The heart is then stored in heparinized water and returned to the lab.
Decellularization
Once harvested, we will begin the decellularization process. Excessive fat is stripped from the heart. We then connect the heart to our bioreactor through the aorta. The pump will begin at a normal flow and gradually progress to a pressure of 5 PSI. The porcine heart is then decellularized via our protocol shown in Table 1.
Analysis of the Extracellular matrix (ECM)
Decellularized tissue is fixed in buffered glutaldehyde overnight. These strips of tissue are plated in platinum for image enhancing. Histology samples are sliced thinly and stained with H&E and Masson’s trichrome and then analyzed.
Recellularization
Recellularization experiments were performed on the decellularized tissue. Murine endothelial (MS-1) cells were cultured in 100 U/mL penicillin and 100 µg/mL streptomycin and tagged with green fluorescent proteins. MS-1 cells were incubated at 70% confluency in 5 mL of cell media and 25 µL of labeling solution from DiO lipophilic tracers cell labeling kits. Cells were detached from their containers using Trypsin and then cell solution was dispensed onto 4mm diameter round ECM samples, suspended in cell medium. Fluorescent imaging was acquired 1 week later and fixed for histology testing.
Cytotoxicity
The viability of the ECM was also measured via cytotoxicity assessment. 1X1 cm samples were cut from decellularized porcine hearts. ECM samples were immersed in 70% ethyl alcohol overnight, and then exposed to 1X PBS and cell growth medium for 48 hours. Samples were placed in well plates, with wells without ECM samples serving as controls. MS-1 cells were labeled with 2 µM calcein and 4 µM EthD and then placed onto the ECM and into the vacant control wells. Samples were observed and tested after 1 and 3 days of cell exposure.
Results
Decellularization
Multiple decellularization of porcine hearts following our standard protocol shows that the heart’s structure and shape are consistently preserved. Red and purple coloration is completely removed from the ECM, leaving a pure protein skeleton.
Histology
Histology imaging shows the cytoplasm stained pink and cell nuclei stained blue have been completely removed from the recellularized structure (see Figure 1).
Recellularization
Tagged ECM samples were visualized at 549 and 565 nm. Figure 2 indicates, via abundant green fluorescence, that labeled MS-1 cells adhered to the ECM. Histology sampling further proves the maintained viability of the ECM following recellularization.
Cytoxicity Test
MS-1 cells reached full confluency in both positive and negative controls. Abundance of
growth on all samples indicates no cytotoxicity on the ECM.
Discussion
While previous studies have been conducted to decellularize porcine hearts, our protocol minimizes exposure to chemical detergent, preserving the integrity of the protein structure. In 2008, Ott et al published a study that succeeded in recellularizing rat hearts with endothelial cellsii. No research has successfully produced full recellularization of a porcine heart. Our study has successfully attached MC-1 cells to the ECM, demonstrating the possibility of further recellularization. Continued testing has also proven the effectiveness of our specific decellularization process and the immunogenic potential of the ECM.
Conclusion
While our study has made great steps forward in the area of tissue engineering, a lot of work is yet to be done. I hope that further research in the lab will produce a fully recellularized aorta and eventually a full functioning, recellularized porcine heart.
References
- American Transplant Foundation. (n.d.). Retrieved December 29, 2014, from http://www.americantransplantfoundation.org/about-transplant/facts-and-myths/
- Ledford, H. (2008, January 13). Ghost heart has a tiny beat. Retrieved December 29, 2014, from http://www.nature.com/news/2008/080113/full/news.2008.435.html