This protocol works with samples from human and animal hearts, even after hours of transportation time. Up to 200 myocytes per milligram can be isolated from vibratome-cut tissue slices. Electrophysiological, structural and biochemical analysis of cardiomyocytes, helps to discover and better understand, mechanisms of cardiac disease.
After filling a 100 millimeter tissue culture dish, with 20 milliliters of cold cutting solution, place the tissue sample in the culture dish. Keep the culture dish on a plate cooled to 4 degrees Celsius. Keep in mind that living tissue samples from animals and especially humans are potentially infectious.
Use scissors or scalpel, to remove excess fibrotic tissue and epicardial fat, from the sample. For optimal vibratome processing of larger tissue samples, use the scalpel to cut eight millimeter cubes from the tissue. For smaller samples from biopsies, this is not necessary.
One can identify the epicardium by the epicardial fat layer. After removing excess fat, place the tissue block, into the dish, with the epicardium facing down. To prepare for tissue embedding, boil 400 milligrams of low melting point agarose in ten milliliters of cutting solution.
Fill a ten milliliter syringe with the hot dissolved agarose gel. Seal the syringe and place it in a 37 degrees Celsius water bath for at least 15 minutes, to allow the agarose to equilibrate. Using forceps, move the trim specimen into a clean 35 millimeter tissue culture dish, with the epicardium facing downward.
Then, remove excess fluid from the specimen, with a sterile swab. Secure the specimen against movement, by holding it with forceps. And empty the syringe of agarose over top of it, making sure to completely immerse the specimen.
Immediately placed the dish on ice and let the agarose solidify for 10 minutes. Mount an unused razor blade to the blade holder of the vibratome. If possible, calibrate the vibratome, by adjusting the Z deflection of the blade.
Use a scalpel to excise an agarose tissue block that fits the specimen holder of the vibratome. To ensure stability, make sure the tissue is still sufficiently immersed in agarose. With agarose margins, of at least eight millimeters.
Apply a thin layer of cyanoacrylate glue to the specimen holder. And gently press the agarose tissue block, onto the holder. Build a vibratome bath, with cutting solution.
Then, fill the outer cooling tank of the vibratome with crushed ice, to maintain a temperature of four to six degrees Celsius, throughout the cutting process. Place the specimen holder, containing the agarose tissue block, into the vibratome bath. Using the vibratome settings described in the manuscript, generate 300 microliter thick slices.
It is crucial to create sections in parallel epicardium because this is how cardiomyocyte fibers are aligned in the ventricular wall. Otherwise, there will be too much damage. Use a standard light microscope, to check the alignment of the cardiomyocytes and the tissue slices.
To avoid damaging the tissue, hold the agarose, instead of the tissue itself. A cardiomyocyte fibers, should be uniformly aligned and the myocytes, should not be contracted. Place a heat plate on the lab shaker and warm it to 37 degrees Celsius.
Start the lab shaker at 65 RPM. Dissolve the proteinase in two milliliters of solution one. And dissolve the collagenase, in two milliliters of solution one.
But do not mix the proteinase and the collagenase. Add calcium chloride to the collagenase solution, for a final concentration, of five micromolar. Incubate both solutions at 37 degrees Celsius.
Use forceps to transfer a tissue slice to a clean 60 millimeter tissue culture dish. With five millimeters of pre-chilled cutting solution. Keeping the dish on ice or a cold plate, carefully remove the agarose from the tissue, using a blade or forceps.
To perform the initial wash of the tissue slices, place a clean 35 millimeter tissue culture dish on the heat blade. And fill it, with two millimeters of pre-warmed solution one. Transfer one or two tissue slices to the prepared dish.
Then use a one millimeter pipette, for the washing steps. After washing the slices, remove solution one from the dish and add two milliliters of the proteinase solution. Incubate the slices for 12 minutes on the heat blade, with the shaker at 65 RPM.
Wash the tissue slices twice more, with two milliliters of pre-warmed solution one. Next, remove solution one from the dish and add two milliliters of collagenase solution. Incubate the slices for at least 30 minutes on the heat blade, with shaking at 65 RPM.
After 15 minutes of incubation, check for free individual myocytes, by examining the dish under a light microscope. Continue checking every five minutes, until individual myocytes are visible. Then, alt digestion by washing the slices twice, with two milliliters of pre-warmed solution two.
And refill the dish, with two milliliters of solution two. Carefully pull the fibers of the tissue slice apart, using fine forceps. Continue to work carefully, to avoid damaging the cardiomyocytes.
And pipette several times, with a single use past your pipette. Then, confirm that the rod-shaped cardiomyocytes have separated, by examining the dish, under a light microscope. Place the dish back on the heat plate at 37 degrees Celsius and agitate by shaking.
By adding 10 millimolar and 100 millimolar calcium chloride stock solutions, slowly increase the calcium concentration of the tissue suspension from five micromolar to 1.5 millimolar. When the calcium increase has complete, use forceps to remove any remaining undigested tissue chunks. First, stop the agitation of the suspension.
Next, using a 1000 microliter pipette, slowly remove 1/3 of the solution, from the top of the suspension. Avoid aspiration of the cardiomyocytes. Then add 700 microliters of solution three, to the cells.
After 10 minutes of agitation, repeat these wash steps. Transfer the suspension to a 15 milliliter centrifuge tube. And allow the cardiomyocytes to sediment at room temperature, for a minimum of 10 minutes and a maximum of 30 minutes.
Remove the supernatant completely. And resuspend the pellet in modified tyrode solution or the desired experimentation buffer. Using a light microscope, verify the cell quality.
30 to 50%of the cardiomyocytes should be rod shaped, smooth without membrane blebs. And display, clear cross striations. To verify isolation efficiency, the protocol was applied to rat myocardium.
And compared with isolation via coronary perfusion and isolation, from small tissue chunks. A lower proportion of rod shaped cells was observed when using the protocol, then after isolation by perfusion. But the total number was still high.
In contrast, isolation from tissue chunks, yielded fewer rod-shaped cells. Flow cytometry, was used to compare the results quantitatively. The isolation of cardiomyocytes from tissue slices, does not reach to the yield obtained by coronary perfusion.
But provides, significantly higher yields than myocyte isolation, from tissue chunks. Next, the protocol was applied to human myocardium. Isolation from myocardial slices, yielded Hyde numbers and a large proportion of rod shaped human myocytes.
And only a small proportion of, rounded cardiomyocytes. Isolation from myocardial slices, resulted in a strikingly higher number of rod-shaped myocytes, than isolation from tissue chunks. Photometric quantification confirmed a substantially higher myocyte viability after isolation from myocardial slices.
Human cells, isolated with the described protocol, can be subjected to structural analysis. Alpha actinin staining, revealed a dense regular Z line pattern and a mean sarcomere length of 1.92 micrometers, in resting cardiomyocytes. LTCC and RyR staining, showed clear cluster is co-localized near the cell membrane and T tubules.
The isolated cardiomyocytes were excitable and could be used for studies on cellular electrophysiology or excitation contraction coupling. Action potential shape and duration, was in the range reported by others, for ventricular cardiomyocytes, from failing human hearts. The calcium transients recorded by Khan vocal line scanning, showed a clear upstroke after stimulation.
And, an acceptable signal to noise ratio. Cardiomyocyte shortening by contraction can be seen from the deflection of the lower cell border. Due to the high number of isolators cardiomyocytes obtained by this protocol, cell culture's possible.
This may allow for gene transfer, pharmacological testing and tissue engineering of human cardiomyocytes. An important application, is the verification of findings, from animal models in human cells. Heart failure is a clinical syndrome with very limited therapeutic options.
Improved cardiomyocyte isolation techniques will help to identify cellular targets for diagnosis and future therapies.
