The objective of this video is to demonstrate a standardized protocol for adult cardiomyocytes isolation, long-term culture and transfection. Isolating and culturing cardiomyocytes from mouse or rat continue to be an essential method to investigate the cell autonomous biology of cardiomyocytes within the healthy and disease heart. There's several questions we can answer using isolated cardiomyocytes.
Such as the proliferative potential. There are gene expression changes or specific cytokine expressions upon injury response. Unfortunately, mouse cardiomyocytes do not survive past one week in normal culture conditions.
Therefore, in this video, we describe an optimized method to isolate adult mouse cardiomyocytes, transfixed them and cultured them well beyond 21 days, this technique is initially hard to master. But with practice one can achieve about 80 to 90%survive in cardiomyocytes after isolation and about a 100 percent transfection efficiency. And most of these cardiomyocytes in correct conditions can survive past 20 days.
Clean and sterilize your surgical instruments by soaking them in 70%alcohol for 15 minutes and subsequently washing them with double distilled water. After the water, leave the tools in the air to dry next, clean the profusion apparatus by running 70%alcohol twice for five minutes each. Increasing the flow rate will help to clean the tubing after alcohol rinse out any remaining alcohol by running double distilled water for 10 minutes.
set up three dishes to clean the heart after excision. Fill each of the dishes with 20 milliliters of myocyte buffer. Add two to three drops of heparin to each dish with the Pasteur pipette and mix Well by pipetting up and down multiple times.
Take a 10 milliliter syringe and fill it with myocyte buffer. Remove any of the air bubbles from the syringe and place it in the third dish in an angled position, securing it with tape. Prepare a loose Knot with surgical suture and place it around the needle.
Inject the mouse with heparin through IP injection 20 minutes prior to anesthesia. Circulate the myocyte profusion buffer through the profusion apparatus at a flow rate of three milliliters per minute. After five minutes, replace the profusion buffer with enzyme solution, saturate the enzyme solution with oxygen during the process, again, set the enzyme solution to a flow rate of three mLs per minute.
Confirm anesthesia by toe pinching and place the mouse on the surgical platform. Sterilize the skin with 70%alcohol. Carefully open the chest, exercise the heart and put the heart in the first dish.
Clear the blood from the heart by gentle squeezing, then transfer the heart to the second dish to further clean the blood from the heart and to remove any non-cardiac tissues. Subsequently transfer the heart to the third dish. Find the aorta and use your forceps to keep it Peyton more placing it around the cannulating needle.
Secure it by pulling down your pre tight knot tightening and then making a second knot. For additional stability fix the order and place with the help of a clip. Trim any excess suture thread, and finally start the heart profusion using the myocyte buffer in the syringe to clear any of the blood remaining in the heart.
Carefully remove the calculating needle from the syringe and hook it to the profusion apparatus. Attempt to prevent any air bubbles from going into the heart. As this may affect the flow of the enzyme solution and digestion.
Move the water jacket up, to provide a homogeneous environment to the heart during profusion. Allow the enzyme solution to flow through the heart with a speed of two to three mLs per minute. Let the enzyme solution flow through the heart for two minutes.
At two minutes, mix in 40 microliters of a hundred micromolar calcium chloride solution to the enzyme solution. Let the enzyme solution pass through the heart for another 10 to 15 minutes. Once the flow becomes smooth and the heart starts to look brown and soft, you know that you have an even distribution of the collagenase enzyme to achieve proper digestion of the heart.
When you think your digestion is complete, take the heart down from the Langendorff perfusion system and put it into a 16 millimeter Petri dish filled with five milliliters of enzyme solution and take it to a biosafety foot. Carefully remove the atria and extra fat tissue. Mince the heart in defined pieces with the help of forceps.
Take a sterile Pasteur pipette and cut its tip at 45 degrees. Use the Pasteur pipette to break up the heart tissue by gentle pipetting up and down. Optimal digestion provides a suspension of single cell cardiomyocytes after that add five milliliters of stop solution to stop the enzyme activity to avoid over digestion, Take a first 50 milliliter conical tube and place a sterile a hundred micron cell strainer on it.
Pass the cardiomyocyte suspension through the cell strainer to remove any large chunks of tissue. Finally wash the strainer with stop solution to collect any attached cardiomyocyte remains. Centrifuge the cell suspension at 20 GS for three minutes and discard the supernatant.
Re suspend the cardiomyocytes and 10 milliliters of stop solution, at three minute intervals at 10 microliters of 100 millimolar calcium chloride solution four times and mix. After the fourth edition centrifuge the cardiomyocytes suspension at 20 GS for three minutes and discard the supernatant. Re suspend the cardiomyocytes into culture media Pre plate the cells into a 60 millimeter dish and put into CO2 incubator for two to three hours.
In two to three hours. The main contaminant cell types like fiber blasts will be adhered to the service of the dish, allowing for pure isolation of cardiomyocytes. After that, take the dish out of the incubator and collect the floating cardiomyocytes in a conical tube and replayed the cardiomyocytes in a laminin coated 24 well culture plate.
Four to six hours is sufficient for the cardiomyocytes to adhere to the surface. So transfection could be performed at six hours after plating. RNA IMAX transfection reagent was used to transplant cardiomyocytes with siRNAs.
The culture media contains 25 micromolar blood Staten supplemented with 10%FBS, which we have found is better suited to perform longterm cardiomyocyte culture and induce proliferation analysis. After transfection, you can view the cardiomyocytes by microscopy utilizing a micro incubator to how's the cells for time-lapse imaging. These are brightfield images of transfected rod shaped cardiomyocytes at low and high magnification.
Here, you can see day by day imaging, which showcases changes in adult mouse cardiomyocyte morphology. We confirm that the cardiomyocytes are healthy and contractile at early time points, as well as after D differentiation and longterm culture. Here's an example of Ki67 immunofluorescent staining demonstrating induced proliferation of adult cardiomyocytes in culture after siRNAs, a transfection.
As you can see from the results using this technique, one can obtain healthy adult cardiomyocytes from mouse and rat and culture them for longterm study. In conclusion, once mastered this technique will allow us to study cardiomyocytes well beyond the current culturing protocols.
