The overall goal of this methodology is to generate novel scaffolds from neonatal mirian cardiac tissue for their use in regenerative studies. This method can help answer key questions in regenerative cardiology. Provides a novel model for the study of cell-matrix interactions, as the extracellular matrix is derived from proliferation-capable cardiac tissue.
The main advantage of this technique is that it allows for the discovery of the novelties of the neonatal extracellular matrix. Demonstrating the procedure will be Stefan Kren, a technician in my laboratory. Before beginning the procedure, draw a four centimeter section of PE 50 tubing over a small alcohol flame to create a smaller, thinner catheter.
Trim the ends to the appropriate dimensions to provide two symmetrical catheters from each side of the PO and briefly place a cut end of the tubing into the flame to melt one phalange onto the opening at the thin end. Next, fill a 12 milliliter syringe with PBS and place a three-way stopcock onto the syringe. Attach a 22 gauge needle to the stopcock and drive the needle through the septum.
Then, slide the PE tubing catheter onto the needle and irrigate the assembly with PBS. When the catheter assembly is ready, swab the thorax of the neonate with 70%ethanol. Then use standard scissors to cut the skin away from the chest, while pulling laterally on the tissue with a number five forceps.
Next, perforate the abdominal wall just inferior to the sternum and grasp the xyphoid process with the forceps. Retract the sternum rostrally from the body while cutting through the ribs on either side of the chest, and reflect the rib cage superiorly. When the heart is visible, pull laterally on the thymus to dissect the two main lobes, exposing the arch of the aorta and the caval and pulmonary veins.
Using 10 centimeter spring scissors, transect the major arteries from the aortic arch, as well as the aorta itself. Grasp the ends of the severed vessels to reflect the heart forward, separating it from the trachea and esophagus, and cut between the lungs and heart and on both sides of the heart to transect the pulmonary vasculature and other major veins. Then, grasp the severed ends of the major vessels and remove the heart from the mediastinum, placing the heart in a 60 millimeter culture dish containing sterile PBS.
To decellularize the heart, place the culture dish under a dissecting microscope and insert the catheter assembly needle into the aorta up to, but not extending past, the aortic valve. Secure the needle with one tie of a 7-0 suture, resting the phalange of the catheter proximally against the tie and making a tight seal, and gently perfuse the heart with PBS. If there are no leaks, the tissue will homogeneously blanch as the latent blood is removed.
Place the septum into the neck of the inlet adaptor and fold the sides of the septum over the glass. Then, attach a reservoir filled with 60 milliliters of 1%Sodium dodecyl sulfate in distilled water via a line of sufficient length to produce a column that generates 20 millimeters of mercury of pressure, and perfuse the heart with SDS for 14 hours. At the end of the perfusion, the heart will be translucent with no observable remaining tissue.
Flush the system up to the stopcock with the remaining SDS solution and fill the reservoir with 10 milliliters of deionized water. Then, flush the heart with consecutive perfusions of 10 milliliters of 1%Triton X-100, 10 milliliters of deionized water, and 60 milliliters of PBS containing penicillin and streptomycin. After the last perfusion, store the heart in PBS and antibiotics at four degrees Celsius.
To recellularize the tissue, transfer the catheterized heart matrix into a 60 millimeter culture dish under a dissecting microscope and connect a one milliliter syringe equipped with a 22 gauge needle loaded with the appropriate cells of interest into the catheter. Confirm that the assembly is free of bubbles and gently perfuse 20 microliters of the cell suspension per minute into the heart matrix via the coronary arteries. When 100 microliters of the cells have been delivered, detach the syringe and push the catheter onto a 22 gauge blunt stub secured to the lure fitting on the underside of a bioreactor heart jar lid.
Now, start the peristaltic pump and confirm that the flow proceeds from the reservoir via the pump through the bubble trap to the catheter and that the cardiac circulation flows out of the veins and drips from the apex, recirculating to the media reservoir. In these images, the hematoxylin and eosin staining of intact and decellularized hearts is shown. Note the absence of hematoxylin positive nuclei and the diminution of eosinophilic structures in the decellularized heart.
To evaluate the content of the neonatal extracellular matrix in intact and decellularized neonatal hearts, immunostaining demonstrates that collagen IV is robustly expressed in both the intact and decellularized heart and that the localization of this protein is maintained following cell removal, while the DAPI positive nuclei are effectively removed. The DNA content is also decreased in neonatal hearts following detergent-based decellularization, consistant with reports using detergent-based decellularization in other tissues. In these images, recellularized cardiomyocytes that have migrated into the wall of the left ventricle can be observed.
Up to 23 days after perfusion, these migrated cells may be positive for cardiac progenitor cell markers, mCherry, differentiated cardiomyocyte markers, or DAPI, with the majority of cells expressing all four markers. Once mastered, this technique can be completed in 34 hours if it is performed properly. While attempting this procedure, it's important to remember to ensure not to embolize the heart by introducing air via the perfusion apparatus.
Following this procedure, gel production and 3D printing can be used to create proliferative grafts or even 3D hearts in vitro. This technique could pave the way for researchers to promote cardiac regeneration. After watching this video, you should have a good understanding of how to decellularize and recellularize a neonatal mouse heart.
Don't forget that working with Sodium dodecyl sulfate can be hazardous, and precautions such as an N-95 mask or a fume hood should always be used while handling the dry powdered reagent.
