This method can help answer key questions about generating vascularized scaffolds, which are really the Holy Grail of the tissue engineering field. The main advantage of this technique is that a pressurized pouch in an inverted orientation improves the decellularization of non-transplantable human organs and lets us do that in a sterile fashion over an appropriate time frame. To prepare the heart for decellularization, first perform an internal inspection for possible defects.
If a septal defect is present, correct the defect with the appropriate sutures. Next, ligate the superior and inferior vena cavas with 2-0 silk suture. Suture the right atrium wall with 5-0 PROLENE, and dissect the aorta away from the main pulmonary artery for subsequent cannulation.
Insert the connectors, according to the diameter of the vessel, into the aorta and the pulmonary artery, and secure the connectors with 2-0 silk sutures. Using one of the pulmonary vein orifices, insert a tubing line through the left atrium toward the left ventricle, and connect an infusion line to the connector in the aorta and an outflow line to the connector in the pulmonary artery. Place the prepared heart into a polyester pouch in the inverted orientation, and place the pouch into a perfusion container.
Connect each of the lines to the respective ports in the rubber stopper, according to the diameter of container, and insert the stopper into the lid of the perfusion container to seal the polyester pouch. Then, perfuse PBS via the infusion port of the rubber stopper to verify the outflow from the pulmonary artery and from the line inserted into the left ventricle, using this flow to clean the organ of any residual traces of blood within the vasculature. When the heart is ready, place the assembled bioreactor in an upright orientation, and connect the infusion line, pressure-head line, pulmonary artery-outflow line, and bioreactor draining line to the rubber cap surface ports on top of the perfusion bioreactor.
Then, decellularize the heart for four hours with hypertonic solution, two hours with hypotonic solution, 120 hours with sodium dodecyl sulfate, or SDS, and a final wash with 120 liters of PBS, all under constant 120 millimeters of mercury pressure as measured at the aortic root. During the last 10 liters of the PBS wash, add 500 milliliters of sterile 2.1%peracetic acid solution neutralized with 10 normal sodium hydroxide to the perfusion solution to sterilize the scaffold. Typically, the flow rate into the aorta during the decellularization process gradually decreases as the perfusion solution changes from hypertonic to hypotonic.
In contrast, the flow rate increases when the perfusion solution is changed from a hypotonic solution to SDS, at which point the infusion flow rate demonstrates fluctuations. The outflow rate from the pulmonary artery demonstrates a similar trend with the hyper-and hypotonic solutions. However, the outflow rate during the SDS perfusion exhibits an overall decrease.
The coronary perfusion efficiency also decreases over time as the different reagents are perfused through the vasculature. As the outflow perfusate from the pulmonary artery and left ventricle are simultaneously collected, their debris content can be compared by spectroscopy. The turbidity of the effluent from both the vessels decreases over time during the perfusions, although the turbidity from the pulmonary artery exhibits a more abrupt color change compared to that observed from the left ventricle during the initial perfusion period.
Evaluation of the correlation between the outflow turbidity and the cell debris by a bicinchoninic acid protein assay from six decellularized human hearts reveals a linear correlation between the protein concentration and the effluent turbidity. While attempting this procedure, it's important to monitor the flow rate and to collect the perfusate periodically to actually monitor the perfusion process. Following this procedure, other techniques like mechanical evaluation or sterility testing can be used to evaluate the fidelity of the scaffold and its potential use for recellularization to generate a functional cardiac tissue.
After its development, this technique paved the way for researchers in the regenerative medicine and tissue engineering field to explore the generation of whole vascularized organs, literally changing the solid organ transplant field. Don't forget that working with human organs and tissues and chemicals such as SDS can be extremely hazardous, and always wear appropriate personal protection equipment when undertaking this procedure.
