The overall goal of this procedure is to isolate culture and visualize contraction and calcium handling in adult mirroring cardiomyocytes. This is accomplished by first removing the muren heart and placing it in room temperature. PBS, the heart can be cannulated in the dish as shown in the cartoon or directly on the needle attached to the perfusion system, as in the movie.
The third step is to wash and digest the heart by pumping perfusion, buffer, and then enzyme buffer through the system. The final step is to cut the ventricles into a dish of transfer buffer and gently mince the tissue with forceps. Ultimately, results can show individual cardiomyocyte cell contraction and handling of calcium through whole cell and or sarcomere length measurements and ratio metric.
Calcium indicators like RO 2:00 AM with the ion optic System. Generally individuals new to this method will struggle because it can be difficult to quickly hang the heart through the aorta without damaging the vessel or the heart itself. Inject adult mice at about 12 weeks of age with 150 USP of heparin sodium into the abdominal space.
If the mouse is dehydrated, inject with one cc of saline solution. Anesthetize the mouse with an injection of ketamine xylazine mixture At a weight dependent dose, mice are checked to ensure that they are deeply anesthetized via loss of hind limb toe pinch reflex, and slowing of respiratory rate. Secure the sedated mouse to the operating pad, then excise the heart and place it in a dish of physiologic saline.
Using opti vision dissecting goggles, identify and expose the aorta. Removing the tissue around the aorta is not required for cell isolation. As long as the aortic root is clearly visible, prime the pump with perfusion buffer.
Begin pumping at a fast speed to fill the lines and check for the absence of bubbles. Then pump at a slow speed in order to set the flow rate for heart perfusion. Use a circulating water bath to maintain the temperature of the perfusion buffer at 37 degrees Celsius.
Use micro dissecting forceps to place the aorta on the needle like a sock. Be sure that the end of the needle rests in the ascending aorta, ideally in the aortic root distal to the right innominate. Next, secure the heart to the needle by tying a 10 centimeter length of suturing silk around the top of the aorta.
Ensure that the tie is at the level of the ascending aorta below the right innominate artery, but it is also above the end of the needle and above the aortic valve. Now lower the heart into the interior of the conical. This helps maintain the ambient temperature.
Perfuse the heart with perfusion buffer for five minutes at a rate of one milliliter per minute, and cla the glass chamber outflow to allow the PERFU eight to collect in the conical glass and envelop the heart. At this point, you should see the volume of the heart increase. Additionally, blood in the cardiac tissue will be replaced by PERFU eight causing tissue.Palor.
Stop the pump to move the tubing from the perfusion buffer container to the enzyme buffer container. Release the outflow clamp to drain the PERFU eight. Restart the pump to begin to perfuse the heart.
With enzyme buffer at one milliliter per minute, clamp the outflow again to allow the enzyme buffer to envelop the heart.Here. As the enzyme is perfusing the heart and digesting the connective tissue, the heart will become palor and the structural integrity will diminish. For the novice, it's helpful to connect the perfusion line with a manometer.
The manometer can also be used to ensure the position of the needle is above the aortic valve and not in the ventricle. Low pressure indicates that the needle is in the ventricular chamber, and high pressure indicates that the needle touches the valve. When the heart is getting digested, the pressure will rapidly decline as the connective tissue does not hold the resistance.
To determine when to cut the ventricles from the digested heart, lift the heart out of the conical glass, gently squeeze the heart with forceps and collect a few drops of PERFU eight in a small Petri dish to check. If single cells are dropping in eight to 10 minutes, the ventricular pressure will have dropped and you should be able to see single cells enter the Perfu. Eight when either is first seen, cut the ventricles from the heart into a dish full of transfer, buffer A for more uniform preparation.
Cut out and remove the right ventricle, leaving the left ventricle behind to mince and study. If interested in the right ventricle, it can be minced after separation. Using micros dissecting forceps mince each ventricle separately, A successful digestion will leave almost no solid chunks of tissue after mincing with most of the tissue becoming amorphous upon dissociation.
To further dissociate the tissue, flow it through a plastic paster pipette from which the tip has been cut. At a 45 degree angle, secure a piece of squared mesh to a small funnel using clamps and place this filtering apparatus into a 15 milliliter tube. Use the modified pasture pipette to remove the cell solution from the dish and filter the solution into the 15 milliliter tube.
Allow the live cells to settle to the bottom of the tube, which will take a couple of minutes. Splitting the volume into two tubes yields faster. Pelleting after Ali.
Quoting 2.5 milliliters of each of four calcium solutions into four separate 15 milliliter tubes. Use the standard pipette to carefully transfer the settled cell pellet to the first of the calcium solutions. Allow the cells to settle to the bottom of the tube and repeat the process.
Transferring the settled cells through each of the calcium solutions. When the cells are transferred to the fourth calcium solution, cap the tube and turn it on its side. Power the MM SIS system ensuring that the arc lamp is initiated first.
Then prime the system with calcium buffer B.Connect the tubes from the pump to the appropriate inlet and outlets of the MM cis chamber. Fasten down an appropriately sized glass cover slip, typically 20 by 25 millimeters in the chamber. Transfer 500 microliters of the cell solution to a small eend orph tube.
Add 0.5 microliters of PHE 2:00 AM to the cells and allow them to incubate in the dark at room temperature for five to seven minutes after darkening the room. Now carefully begin to flow calcium buffer B through the chamber. Once flow has been initiated and no sooner, begin increasing the chamber temperature to 37 degrees Celsius.
Use a modified pipette to load one or two drops of cell solution onto the cover slip, depending on the cell concentration, and allow the cells to settle. The density of the cells in the chamber. Should be such that single non-overlapping cells can be easily in the sys data acquisition platform.
Make sure that the chamber is connected to the myo Pacer via the connection wires and begin to set the voltage and frequency for the experiment. Choose a cell that is beating at the correct frequency and move it into the framing aperture. Adjust the camera and framing aperture dimensions so that the entire cell is in the center of the window directed horizontally with the sarco mirrors apparent.
Next, place the box in an area of the cell containing well-defined sarcomeres and adjust the focus to optimize the peak of the power spectrum. Also adjust the brightness of the microscope to optimize the blue smoothing window and black contrast information. Turn on the photo multiplier and begin recording.
After enough data has been recorded, click pause to temporarily stop recording. Do not click stop. This will terminate the recording and no background will be able to be recorded for that cell.
Now without altering the focus nor dimensions of the viewing window, move the microscope stage to an area of the cover slip in which no cells or cellular material can be seen. Then click resume to record a background measurement. After enough background has been recorded, click stop to terminate the recording.
Then click file save to save all the traces for that cell In a single ZPT file, the isolation of adult cardiomyocytes results in rod-shaped striated and quiescent cells that do not spontaneously beat the arrowhead shows rounded dead cells with no striations. Quiescent cells can be cultured and transfected with adenovirus to manipulate gene expression. After 24 hours of culture, the morphology of the live cells does not change.
They're still calcium tolerant and they can be paced by field stimulation. A yield of 85 to 90%viable cells is feasible. The MM SIS system was used to measure contractility and calcium transient of myocytes taken from a wild type C 57 black six mouse.Simultaneously.
Measurement of contractility and calcium handling was made by measuring the ratio of the bound to the unbound forms of fira 2:00 AM ratio measurements may vary slightly between setups, but healthy cells usually score between 1.5 and 2.5. In order to analyze the data from the contractility and calcium traces, the traces were averaged to create a characteristic trace for each cell. Using a mono transient data analysis algorithm found in the sys software parameters for systolic and diastolic function were generated from the characteristic traces.
After watching this video, you should have a good understanding of how to isolate individual adult marine cardiomyocytes.
