Isolation and Culture of Neonatal Mouse Cardiomyocytes

The overall goal of this procedure is to isolate and culture neonatal mouse cardiomyocytes for use in subsequent assays. This is accomplished by first dissecting the hearts of neonatal mice, and then manually cutting the heart tissue into small fragments. In the second step, the cardiac cells are dissociated through a series of enzymatic digestions. Next, the isolated cells are plated, to enrich for cardiomyocytes. Finally, the cultured cardiomyocytes can be observed, adhering to the plate, and displaying spontaneous beating behavior. Ultimately, the subcellular localization of proteins within cardiac muscle cells transfected with eukaryotic expression constructs, can be observed by immunofluorescent microscopy.

- The main advantage of this technique over other methods is the robustness and reproducibility in the isolation and culture of neonatal mouse cardiomyocytes.

- This method can help answer key questions in the field of cardiovascular research, and give insights into the unique molecular cell biology of cardiac muscle cells, such as addressing how small molecules can influence cardiac signaling pathways, or how biomechanical stimuli can affect protein localization, transcription, and expression.

- Because of the versatile nature of the resulting cardiomyocyte cells and culture, this technique enables many downstream applications and essays tailored to the needs of the experiment.

- Before beginning the cardiac tissue isolation, dispense 25 milliliters of calcium and magnesium free PBS, freshly supplemented with 20 millimolar of BDM, into each of two sterile bacterial dishes placed on ice. Then, add 10 milliliters of isolation medium to a sterile 50 milliliter conical tube, and place on ice. Now, quickly sterilize the skin of one to three day old neonatal mice with 75% ethanol. After sacrifice, open the chest cavity of each animal, along the sternum, and extract each heart with a pair of sterile curved scissors. Transfer the hearts immediately into one of the bacterial dishes containing PBS, with 20 millimolar BDM. Next, use a pair of forceps to gently squeeze any blood out of the hearts, and then transfer the washed hearts from the first dish into the second. Then transfer the hearts, once again, into a 250 microliter drop of isolation medium in a third bacterial dish. Use a pair of curved scissors to mince the hearts into 0.5 to one millimeter cube pieces, and then transfer the minced pieces into a conical tube containing 10 milliliters of isolation medium. Incubate the heart tissue with gentle agitation at four degrees Celsius overnight. Before digesting the cardiac tissue, coat cell culture plates with collagen solution for a minimum of one hour. Then, remove the collagen solution, and dry the dishes in a sterile, laminar flow cell culture hood. While the dishes are drying, confirm that the tissue fragments are aggregated. Then, let the fragments sink to the bottom of the tube, and remove all but the last one milliliter of the supernatant. Add five milliliters of freshly prepared digestion medium, and five milliliters of L-15 media, supplemented with 20 millimolar BDM, to the tissue fragments, and then oxygenate the suspension for one minute. Seal the tube, and then transfer the tissue to a 37 degree Celsius water bath. After two minutes, incubate the cardiac fragments in a 37 degree Celsius shaker, set to no more than 60 RPM, for 20 to 30 minutes. Then place a sterile cell strainer into a sterile 50 milliliter conical tube. Pre-wet the filter with 5 milliliters of L-15, supplemented with 20 millimolar BDM, and then use a pre-wetted 10 milliliter cell culture pipette to gently triturate the tissue fragments about 10 to 20 times. Let the larger tissue fragments sediment, and then filter the supernatant, containing the suspended cells, into the tube. Resuspend the undigested tissue fragments in five milliliters of digestion medium, and then incubate the fragments for an additional five to 10 minutes, at 37 degrees Celsius, with gentle agitation. After the tissue fragments have fully digested, gently triturate the cell slurry 10 to 20 times, and strain the solution through the filter, into the conical tube containing the cells from the first digestion. Rinse the cell strainer with five milliliters of the L-15 supplemented media, with 20 millimolar BDM, and then spin down the suspended cardiomyocytes for five minutes, at 50 to 100 times G, at room temperature. Remove the supernatant, and then resuspend the pellet in 10 milliliters of plating medium. Then incubate the cells in a 10 centimeter cell culture dish, in a cell culture incubator, to remove any fibroblasts and endothelial cells. After one to three hours, resuspend the non-adherent cardiomyocytes, by repeatedly pipetting the plating medium over the dish, and then transfer the resuspended cells to a new conical tube. After counting the cells, plate them onto the collagen coated cell culture dishes with a density of approximately 1.5 times 10 to the five cells per centimeter squared. Then incubate the plates, undisturbed, for 12 to 18 hours, for the adherence and spreading of the cardiomyocytes. One day after plating, the cardiomyocytes should adhere to the cell culture dish, and start to contract spontaneously. After confirming adherence and contraction, pre-warm freshly prepared maintenance media in a 37 degree Celsius water bath, then replace the plating medium with the warmed maintenance medium, to remove any dead cells from the culture. Finally, culture the cardiomyocytes for an additional one to five days, changing the medium as necessary. This first image shows isolated neonatal mouse cardiomyocytes at the beginning of the pre-plating step. After one to three hours, the cardiac fibroblasts, and endothelial cells, start to adhere to the uncoated cell culture dish. After resuspension of the nonadherent cardiomyocytes, the remaining cardiac fibroblasts, and endothelial cells, can be further cultured if desired. In this image, isolated and purified neonatal mouse cardiomyocytes, plated in the collagen coated cell culture dishes, can be observed. As demonstrated after 18 hours in culture, the cardiomyocytes adhere to the coated dishes. Here, a representative immunofluorescent image of neonatal mouse cardiomyocytes, stained with antibodies against myomesin in red, and beta-catenin in green, is shown. Finally, this immunofluorescent image is of transfected neonatal mouse cardiomyocytes, with the expressed GFP tagged protein in green, and alpha actinin in red.

- Following this procedure, other methods like cardiomyocyte transfection, treatment with small molecule inhibitors or activators, or immunofluorescent biomechanical analyses can be performed, in order to gain insights into the mollecular cell biology of cardiomyocytes, and their biomechanical properties.

- The adaptation of this isolation procedure for neonatal mouse cardiomyocytes paved the way for researchers that worked with the genetically modified knock-in or knock-out mice, to explore the behavior of these cells in an easily controlled cell culture setting, especially when working with model systems that have a postnatal cardiac pathology.