The procedure begins with excising the heart from an adult rat and hanging it on the perfusion system. Enzyme solutions are allowed to perfuse for about 30 minutes after which the heart is removed. Minced and enzymatic digestion allowed to continue in a small beaker.
At the end of digestion, cells are dissociated into a single cell suspension by mechanical agitation and filtered into a sterile tube. Primary heart cells are washed three times in buffers with increasing calcium concentration, resuspended in culture, medium and plated on laminate coated tissue culture dishes. Hi, I'm Shahi from the laboratory of Henry Cowa in the Department of Physiology and the Cellular by Physics at Columbia University.
Today we'll show you a procedure for isolating and culturing other rat heart cells. We use this procedure in our lab to study calcium channels in heart. So let's get started.
The day before the rat surgery make certain that all solutions are made, but do not add any enzymes yet. Buffers a B and the wash buffer should all be filtered for sterility prior to storage at four degrees Celsius. Six, well tissue culture plates must be plated with laminate solution the day before as well and stored at 37 degrees Celsius in a CO2 incubator.
On the day of the surgery. Prepare the surgical instruments by sanitizing the clamp scissors and forceps with 70%ethanol and let them dry on a paper towel. A one milliliter syringe filled with half a milliliter of heparin solution is made ready.
Now it is good practice to wash the perfusion apparatus running through 70%ethanol with the peristaltic pump in reverse. Once finished, rinsing with ethanol, rinse the apparatus with double distilled water and finally rinse with buffer A.The flow through is collected in a beaker and placed in a 37 degree Celsius water bath. To prepare the cleaned up profusion apparatus, filled the right syringe tube with 40 milliliters of buffer A and the left syringe tube with 40 milliliters of buffer B.Prime the perfusion tubing to the tip of the catheter by allowing buffer A to flow through the apparatus.
Refill the buffer a syringe to the 40 milliliter level. Make sure that there are no air bubbles trapped in the tubing after this step, now shut off the buffer, a syringe with the stop cock valve and repeat the process such that the profusion tubing is now primed with buffer B.The profusion apparatus is finally ready and is left with the buffer. B stop cock valve open, but the flow is stopped.
Using the regulator on the IV line, discard the buffer flow through in the collecting beaker. The last thing to do before the surgery is to add the required enzymes to your stock solution and then filter the enzyme solution just as the other solutions were filtered the day before. Once filtered, this solution can be left at room temperature following standard protocol.
Euthanize the rat with iso fluorine in a gas chamber. Sanitize the incision area on the thorax with 70%ethanol. Open the chest using scissors and expose the heart.
Inject the left ventricle with 0.5 milliliters heparin solution. Remove the heart with a large portion of the aorta intact. Immediately insert the catheter into the aorta.
A typical rat heart should yield a high fraction of healthy rod shaped myocytes and few dead rounded cells. If the following procedure is properly adhered to, to begin clamp the aorta to the catheter. The tip of the catheter should not be pushed too far into the heart to ensure good perfusion of the heart through the coronary artery.
Once clamped, tied the aorta to the catheter with the suture. Next, open the IV line regulator to allow a fast drip rate and allow buffer bee to wash out the heart during the buffer bee wash. Use the small scissors and forceps to remove the atria and any fatty or lung tissue clinging to the heart.
After buffer B is finished, switch the flow to buffer a while. Buffer A is allowed to flow for five minutes. Warm the enzyme solution in a 37 degree Celsius water bath.
When buffer A is depleted from the syringe but still present in the tubing load 50 milliliters of the enzyme solution in syringe A of the profusion apparatus. It is now necessary to discard all the flow through from the collection beaker in the water bath until the enzyme solution peruses the heart. As soon as the enzyme solution peruses the heart, activate the peristaltic pump, which is set up to transfer the enzyme solution from the collecting beaker to the replenished syringe.
A allow the enzyme solution to flow through the heart for 10 minutes. As the heart digests, it begins to look bloated at 37.5 microliters of 0.1 molar calcium chloride to the enzyme solution in syringe A to give an effective concentration of 0.1 millimolar calcium. This is the first of several calcium chloride additions used to increase the concentration after 10 minutes, increase the concentration of calcium to 0.2 millimolar by adding an additional 50 microliters of 0.1 molar calcium chloride to syringe A and let the profusion proceed for a further 10 minutes.
After 10 minutes have elapsed, cut off the ventricles and transfer the heart to a small sterile beaker containing 20 milliliters of enzyme solution in the beaker. Increase the calcium concentration to 0.4 millimolar by adding 40 more microliters of 0.1 molar calcium chloride gently mince the heart into 10 or more pieces with a pair of small sterile scissors. Now incubate the beaker at 37 degrees Celsius with gentle rocking.
After five minutes of incubation, add 40 microliters of 0.1 molar calcium chloride to give an effective concentration of 0.6 millimolar calcium and gently iterate the heart pieces with a plastic transfer pipette three to five times after incubating for an additional five minutes at 37 degrees Celsius, add 40 microliters of 0.1 molar calcium chloride and gently iterate as before. Use a sterile 500 micrometer filter to separate the digested single myocytes from indigested connective tissue. Allow the cells to settle in a 50 milliliter tube for 10 minutes at room temperature jerk discard the supernatant with a transfer VI Gently resuspend the cells in wash buffer number one, and allow the cells to settle for 10 to 20 minutes at room temperature while the cells are settling.
Take a small aliquot and use this time as an opportunity to assess the quality and viability of the cells in suspension under an inverted microscope. A good preparation will have a high proportion of rod like cells with crisp striations. Once the cells have settled, discard the snat and gently resuspend the cells in wash buffer.
Number two, let the cells settle at room temperature, which should again take 10 to 20 minutes and discard the supernatant. Gently resuspend the cells in 5%serum media before transferring the cells to tissue culture plates. Wash the plates with one XSFM.
Transfer the cells at a desired density and incubate in a CO2 tissue culture incubator. For three to five hours, switch the media to one XSFM Cells can be cultured for up to four days and used as required for experiments. There should be more than 70%live heart myocytes under inverted microscope when the protocol is done correctly.
These are isolated myocytes transfected with YFP REM after being cultured for 48 hours. Cells of this quality are typically cultured from this procedure. We'll just show you how to isolate and the culture are that hot cells.
When doing this procedure, it's important to remember to be as quick and as clean as possible. So that's it. Thanks for watching and good luck with your experiments.
