ラット心筋細胞の分離と培養

The overall goal of this experiment is to isolate and cultivate adult rat cardiomyocytes, which can be used in various experiments to understand their biological and pathophysiological behavior. This method can help answer key questions in the field of biology and pathophysiology of the heart, such as cardiac dedifferentiation, cardiac hypertension, or heart failure. The main advantage of this technique is that adult cardiomyocytes can be investigated individually without the influence of adjacent cells, such as fibroblasts or endothelial cells.

The implications of this technique extend toward pathophysiological understanding of heart diseases because adult cardiac myocytes can be investigated individually. Though this method can provide insight into the behavior of adult rat cardiomyocytes, it can also be applied to other models, such as ischemia reperfusion models, spontaneously hypertensive rats, or other species. The Langendorff perfusion system consists of a long, double-walled glass tube with a glass funnel at the top and the bottom of the tube.

The apparatus can be heated by water. The top and lower glass funnels are connected with a pump that transports the perfusion medium, which is fumigated with 95%oxygen. To prepare the perfusion system, heat the plating medium and washing medium to 37 degrees Celsius.

Then, defreeze a tube of 500 microliters calcium chloride, and weigh 25 milligrams of collagenase. Next, flush the Langendorff perfusion system with aqua sterile. Afterwards, let Powell medium circulate the system for five minutes.

Next, prepare a tube with 40 milliliters of Powell medium, and cover it with Parafilm. Heat the tube to 37 degrees Celsius, and gas it with 95%oxygen. Remove the circulating Powell medium, and fill the Langendorff perfusion system with 80 milliliters of fresh Powell medium without any air bubbles, and gas the medium with 95%oxygen.

Then, prepare a thread of about 25 centimeters in length for attaching the removed heart to the cannula. Degrease a razor blade with alcohol, and fasten it to the chopper. Finally, clamp a plastic disk into the chopper.

After sacrificing male, Wistar rats, as described in the text protocol, open the abdomen behind the costal arch with an abdominal shear, and with the same pair of scissors, cut through the diaphragm to open the thoracic cavity. Remove the heart together with the lung and thymus by cutting above the thymus highly cranial in the thoracic cavity. Transfer the material to ice-cold saline solution immediately.

Remove the lung and thymus from the heart with a large dissecting scissor. Then, by fixating the material with capsule forceps, transfer the heart to a new saline solution. The following steps must be performed precisely but also quickly to minimize the time between extraction of the heart and fixation to the Langendorff perfusion system.

This is essential for a good result in quality and number of the adult cardiomyocytes. Remove excess tissue, like residues of thymus, trachea, fat, and connective tissue from the heart, using capsule forceps and a dissecting scissor. Uncover the aorta, and sever it with a dissecting scissor between the first and second branchial arch.

Next, start the dripping of the Langendorff perfusion system. Place the heart on the cannula of the Langendorff perfusion system, and fixate it first with a crocodile clamp and later with the prepared thread. Rinse the heart until it is free of blood.

Dissolve 25 milligrams of collagenase in five to six milliliters of warm Powell medium, and add 12.5 microliters of calcium chloride. When the residue volume is 45 milliliters, close circulation by moving a glass funnel, which is connected with the Langendorff perfusion system over the dripping heart, and cover this funnel with Parafilm for protection of the heart. Add the solved collagenase to the perfusion system.

Adjust the perfusion system for 25 minutes with a drop velocity of one drop per second. After 25 minutes of perfusion, the heart appears swollen and waxy. Stop the perfusion after 25 minutes, and remove the heart from the Langendorff perfusion system.

Remove the aorta, atria, and connective tissue from the heart, and open the right and left ventricles. Chop the heart two times at an angle of 90 degrees, repeating this process manually with two scalpels for 10 seconds each side. Next, transfer 12 milliliters of the perfusion medium into a new tube.

Pour the cell slurry into this medium, and digest cells for another five minutes at 37 degrees Celsius. Mix the solution every minute. Filter the solution with the digested heart through a 200 micron nylon mesh into a new tube.

Recalcification is a sensitive process. If other rat models or different species are used, the demonstrated protocol can be too aggressive, which would lead to a high mortality rate of the cells. Centrifuge the filtered solution at 29 g for three minutes.

Discard the supernatant, and add six milliliters of warm Powell medium, including 12.5 microliters of calcium chloride to the cell pellet. Resuspend the pellet through smooth shaking movements. Then, centrifuge again at 29 g for two minutes.

Discard the supernatant, and add six milliliters of warm Powell medium, substituted with 25 microliters of calcium chloride. Dissolve the cell pellet through gentle shaking movements, and add 12 milliliters of warm Powell medium, including 120 microliters of calcium chloride. After centrifuging for a third time at 16 g for one minute, remove the supernatant.

Next, mix the cell pellet with the pre-warmed plating medium. Remove the pre-plating medium from the culture plates. Then, transfer one milliliter of plating medium, including the isolated cardiomyocytes, to each culture plate.

Incubate the fresh isolated cardiomyocytes at 37 degrees Celsius for one hour. Remove the plating medium from the culture plates. Finally, add one milliliter of washing medium to each culture plate.

Store the plates at 37 degrees Celsius up to six days without changing the medium. Freshly isolated ARVC show a typical rod-shaped morphology. ARVC with an irregular surface and no detectable intact cell membrane can be assumed not to be viable.

This morphology changes over the period of cultivation in a medium containing FCS or oncostatin M.ARVCs round down and form pseudopodia-like structures, a process also called spreading. Through fluorescent staining with Phalloidin TRITC, the alteration of the contractile apparatus of ARVC can be investigated during cultivation. Freshly isolated cardiomyocytes display a clearly visible cross striation.

During the first days of cultivation, ARVC lost all their contractile elements. This was followed by a reformation, implicating de novo sarcomerogenesis with actin stress fibers and actin bundles in the perinuclear region. This reformation was preceded by the formation of pseudopodia-like structures.

At the end of the cultivation, a typical cross striation from newly assembled sarcomeres in the spread of ARVC was observed. At the end of the cultivation period, 14.7%plus or minus 1.39%of all counted ARVC showed pseudopodia-like structures. Once mastered, this technique can be performed in one to one and a half hour if it is performed properly.

While attempting the procedure, please keep in mind to work as fast and as precise as possible. After watching this video, you should have a good understanding of how to isolate and cultivate adult cardiomyocytes. Don't forget that working with biological material and chemicals can be hazardous.

After its development, this method paved the way for researchers in the field of cardiology to explore basic physiology of adult cardiac myocytes. Following this procedure, other methods like separation of cardiomyocytes of the left and right ventricle or other model species can be performed in order to answer additional questions, like anatomical or species-related differences.