Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The overall goal of this procedure is to cryopreserve neonatal rat cardiomyocytes, and subsequently thaw the cells for use in in vitro studies. This is accomplished by mincing the neonatal heart into small pieces. The cardiomyocytes are then enzymatically, dissociated from the heart tissue, cryopreserved, and eventually thawed for subsequent cell culture.

Ultimately, the thawed cells can be used for various in vitro assays, including immunofluorescent analysis of the cardiac markers of interest. The main advantage of this technique over existing methods is that it saves time by not requiring a complete cardiomyocyte isolation to be performed when cells are needed for an assay. Visual demonstration of this method is critical as the nuances involved in knowing the correct size and consistency of the samples during the isolation and processing steps are difficult to learn without direct demonstration.

After all of the neonatal hearts have been collected, remove any non heart tissue from the samples and wash the hearts with HBSS and gentle swirling. After a second wash and a new plate, cut the samples into one to three cubic millimeter pieces and transfer the tissue into 40 milliliters of cold tripsin for overnight incubation at four degrees Celsius on a rocker. The next day, confirm that the trypsin is clear and that the tissue appears fluffy.

Allow the tissue to settle at the bottom of the tube and aspirate as much liquid as possible. Next, at 25 milliliters of warm NR cm, medium with 10%FBS to the tube and swirl it by hand. Then place the tube in a rotating 37 degrees Celsius water bath for two minutes.

When the tissue begins to float, aspirate the supernatant from the bottom of the tube, and at 10 milliliters of freshly prepared collagenase, place the tissue back into the water bath for two more minutes until the solution is cloudy. Then quickly aspirate the S supernatant and add 10 milliliters of fresh collagenase for another rotating incubation. After a final two minutes, iterate the tissue slurry a few times and transfer as much of the supernatant as possible into the first of five 15 milliliter tubes containing four milliliters of HBSS on ice.

Repeat the digestion until each of the tubes of HBSS contains supernatant from the sample tube, and then centrifuge all of the tubes. Then aspirate the supernatant from each of the pellets and resuspend the cells in each tube with six milliliters of HBSS. Now pool the cells through a 40 micron strainer into a 50 milliliter conical tube.

Then rinse the side of each 15 milliliter tube with three milliliters of cold HBSS and pull the washes in the 50 milliliter conical tube. After spinning down the cells again, re suspend the pellet in 10 milliliters of prewarm medium. Then transfer the cells to a T 75 flask with five milliliters of wash from the 50 milliliter tube and incubate the cells for one hour at 37 degrees Celsius and 5%carbon dioxide.

At the end of the incubation, transfer the cells to a T 1 75 flask with 15 milliliters of wash from the T 75 flask and incubate the cells for another hour. To cryopreserve the cells aliquot the floating cells from the cell culture into 50 milliliter conical tubes. Reserve 10 microliters for counting by trian blue exclusion and spin down the cells during the centrifugation count the cells.

Then re suspend the pellet in freezing medium at two to four times 10 to the six cells per milliliter. Transfer one milliliter aliquots of the cells into cryogenic vials and place the vials in a controlled cooling rate freezing container, followed by short term storage at minus 80 degrees Celsius within one to two days, transfer the vials into liquid nitrogen. When it is time to thaw the cells.

First coat a dish in enough fibronectin to cover the bottom of the culture plate and incubate the dish at 37 degrees Celsius for at least 30 minutes. When the plate is ready, place the cells on dry ice, followed by warming in a 37 degrees Celsius water bath. When the cell suspension is just thawed, immediately wash the cells by centrifugation four times after the last wash, count the viable cells by triam blue exclusion and resuspend the pellet in fresh medium.

Finally, incubate the cells on the fibronectin coated dishes at the appropriate concentration for the analysis. Refresh the medium daily for one week, at which point the cells should have begun beating. Typically upon thawing, the viability of the cells will be between 40 to 60%Though this is lower than that observed for other cell types.

With proper seeding and culture, the cells will proliferate. Within about three days of culture, the cells should begin to spontaneously contract, which can be confirmed by phase contrast microscopy. In this representative experiment, the cells were plated in four well culture slides at two times 10 to the fifth cells per well labeled with alpha sarcomeric actin, and imaged by fluorescence microscopy.

Numerous alpha sarcomeric actin positive cells, which appear here as green staining were identified confirming the presence of the neonatal rat cardiomyocytes. Other downstream analyses can be performed with these cells as well. For example, in this representative experiment, the neonatal rat cardiomyocytes were cultured with human cardiac stem cell derived exosomes to test the regenerative properties of the human vesicles While performing the thaw portion of this procedure.

It's important to act quickly and accurately to ensure a viable cell yield. After watching this video, you should have a good understanding of how to successfully isolate cryopreserve and then thaw neonatal rat cardiomyocytes for use in in vitro studies.