![]() To demonstrate that our primary cardiomyocyte cultures can serve as good physiological model systems, we looked at the activation of two cell signaling pathways. However, once the Cardiomyocyte Growth Supplement, a reagent supplied in our kit that reduces fibroblast contamination, was included in the growth media for all of the cultures the overall culture purity at Day 7 increased by 20-30% except for the culture prepared with the Roche blend (8%)(Figure 4B). Cultures prepared using trypsin or Roche enzyme blends were significantly less pure (51% and 45%, respectively). At Day 1, cultures prepared by our method were 61% pure cardiomyocytes, similar to cultures prepared with the Worthington kit (Figure 4B). Cardiomyocyte purity was calculated as the ratio of total troponin T cardiac isoform stained cells to total cells (determined by nuclear staining). To assess the purity of the cardiomyocyte cultures, we immunostained cells with an antibody specific to the cardiomyocyte marker protein troponin T cardiac isoform (green in Figure 3A, lower panel). Despite the presence of some PI-labeled cells at Day 1 in cultures prepared by each isolation method, the ratio of PI-labeled cells versus total cells in cultures prepared by our method (Figure 4A) was similar to that obtained from cultures prepared using the Worthington kit (19% at Day 1 and 8% at Day 7) but much lower than the ratio obtained in cultures prepared by the trypsin homebrew method (34% at Day 1 and 16% at Day 7) and using the Roche enzyme blends (55% at Day 1, and over 50% at Day 7). Propidium Iodide (PI, red), a nuclear fluorescent dye normally excluded from viable cells, was used to reveal dead cells in cultures (Figure 3A, lower panel). To assess the health of the cultures over time, we evaluated the viability and purity of mouse cardiomyocytes at Day 1 and Day 7 in culture. For the enzyme blends of Liberase™ DH and TM (Roche Diagnostics), we performed one 35-minute incubation at 37☌ to match our method. As described in Table 1, the do-it-yourself (DIY) trypsin method uses 5 to 8 repeated incubations, and the Neonatal Cardiomyocyte Isolation System (#LK003300 Worthington Biochemical Corporation) uses an overnight digestion followed by a secondary 35- to 40-minute digestion the following day. We selected our best enzyme combination (papain plus thermolysin at certain concentrations) and protocol (Figure 1, Pierce Kit) and then compared it to a standard literature protocol using trypsin, a commercially-available kit, and a selection of commercially-available enzyme blends recommended for cardiomyocyte isolation. We screened several different proteases and collagenases individually and in combination to determine the most efficient digestion strategy (data not shown). The choice of enzyme(s), concentration, and timing of the digestions has a profound effect on cardiomyocyte yield and viability. Isolating primary cardiomyocytes is a delicate process involving the controlled use of enzymes to disrupt complex protein and intercellular matrix interactions found in heart tissue. Current methods to isolate neonatal mouse/rat cardiomyocytes use either a series of trypsin digests (typically five to eight incubations for 10 to 20 minutes each), or a single, long digestion, usually 16 hours to overnight (Table 1). A common problem during cardiomyocyte isolation is that the heart is a solid organ with strong intercellular attachments, so the dissociation process is more difficult and time consuming. Isolating primary cardiomyocytes from neonatal mouse and rat hearts has typically been a time-consuming, labor-intensive task. In addition, preparations of heart cells isolated from small mammals like mouse and rat enable a large number of quick, relatively low-cost experiments compared to studies conducted in whole animals. Compared with the whole heart, cardiomyocyte cultures are relatively pure, with limited contaminating cell types such as endothelial cells. Cardiomyocyte cultures provide a homogeneous population of single cells, which are easy to visualize and manipulate. Cultured primary cardiomyocytes are widely used to study and understand the mechanisms underlying normal cardiovascular function, cardioprotection, and cardiovascular diseases. ![]()
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