The overall goal of this procedure is to isolate viable cardiac melanocyte like cells from the mouse heart. This is accomplished by first harvesting hearts from euthanized mice. The second step is to mince the hearts and dissociate the cells using enzymatic digestion and mechanical agitation.
Next, the slurry of cells is centrifuged and the pellet containing the cardiomyocytes and melanocyte like cells is resus suspended. The final step is to plate and culture. The cells ultimately isolated cardiac melanocyte like cells can be used for patch studies or microarray analysis in response to pathophysiological stressor to assess cellular excitability or the status of the transcriptome.
The main advantage of this technique over existing method designed to isolate and culture the cardiomyocytes, is that it results in the isolation of many wearable cardiac melanocytes like cells. Well, we first had the idea for this method when we discovered a new cell population, the mouse heart, and we wanted to investigate its physiology. To begin, prepare two sets of sterilized surgical instruments, one for dissecting out the hearts and another for mincing up the tissue.
Next, prepare the media supplements, but wait to add them to the culture media until just before plating the cells. After preparing the culture media filter to sterilize the solution while working in a tissue culture hood, store the media at four degrees Celsius. Prepare the DNA's one solution by adding 0.05 grams of DNA's, one to 100 milliliters of DPBS with 10%FBS plus antibiotics.
Next, prepare a 0.5%tryin solution by adding 0.5 grams of trypsin to 100 milliliters of DPBS plus antibiotics. Lastly, gather together other equipment needed to complete the procedure, including Petri dishes, 50 milliliter conical tubes, 40 micron cell strainers, DPBS plus antibiotics, 60 millimeter and 35 millimeter culture dishes. After euthanizing P zero to P two neonatal mouse pups, wipe their chest with alcohol and place them in a 10 centimeter P two dish With DPBS working under a stereo microscope, carefully open the chest cavity and remove the entire heart with the atria attached.
Wash the hearts in DPBS and transfer them to a new 10 centimeter Petri dish. Move the Petri dish with the excised hearts into a tissue culture hood and follow sterile technique for the following steps. First, wash the hearts three times in sterile DPBS plus antibiotics.
Place the hearts in a 60 millimeter culture dish and add six to eight milliliters of 0.5%trips in solution. Next, cut the hearts into small pieces and incubate them at 37 degrees Celsius for 30 minutes. After the incubation is complete, pull the resulting solution from the dish into a sterile 10 milliliter conical tube.
Using a 10 milliliter sterile pipette quickly but gently rerate the solution 30 to 50 times, filter the resulting suspension through a 40 micron cell strainer into a new clean 50 milliliter conical tube. After centrifugation, gently aspirate off the supinate and resuspend the remaining pellet in six to eight milliliters of sterile DPBS. Repeat this step two more times.
Following the third rinse after centrifugation, carefully drain or aspirate off the wash solution and resuspend the pellet in six milliliters of supplemented culture media. Next, draw the resuspended solution into a sterile transfer pipette and place the cells in a 35 millimeter dish. Pull the isolated cardiac cells from three neonatal mice after an overnight incubation, aspirate off the culture media along with any unattached cells, and then rinse the plates once with DPBS, add fresh media to the plates and replace the media.
Every two days, cells isolated from neonatal or embryonic hearts can be maintained in culture for up to three weeks, whereas cells isolated from adult hearts can be maintained in culture for up to 10 days. To isolate melanocyte like cells from an adult mouse heart, first, wash the excised hearts in sterile DPBS containing antibiotics in a tissue culture hood. Squeeze the heart slightly with curved dissecting forceps to remove any remaining blood, and then rinse the heart in DPBS one more time.
Next, remove the atria and atrial ventricular annulus from the hearts and place the excise tissue specimens in a 35 millimeter dish. Cut the hearts into small pieces using curved scissors and forceps. Add six to eight milliliters of 0.5%trips in solution to the dish and incubate a 37 degrees Celsius for 45 to 60 minutes.
After the incubation, repeat the steps demonstrated earlier to obtain a suspension of adult cardiac cells and platers before. In this example, the white arrow identifies the cardiac melanocyte in both panels. Note that without the aid of the fluorescent marker, the cardiac melanocyte is difficult to identify and distinguish from the atrial myocytes attached to the dish, the light blue arrowhead point to atrial cells that are not well attached to the dish shown here, a healthy cardiac melanocytes isolated from a neonatal mouse heart.
They have developed extensive dendritic processes and closely resemble cutaneous melanocytes. Once mastered, this technique can be done in a few hours if it's performed properly. After watching this video, you should have a good understanding of how to isolate viable melanocyte cells from the heart.
That'll be suitable for physiologic and molecular analysis.
