Neonatal Rat Ventricular Myocyte Isolation & Culture
Primary NRVMs were isolated from postnatal day 1–2 (P1–P2) Sprague-Dawley rat pups using serial enzymatic digestion. Hearts were excised, ventricles dissected, and tissue minced into ~1 mm pieces. Sequential digestion with collagenase type II (0.5 mg/mL) and pancreatin (0.6 mg/mL) in HBSS was performed in 5–7 rounds (15 min each, 37°C with gentle agitation). Supernatants were pooled, filtered (70 μm), and subjected to differential pre-plating (90 min) to deplete fibroblasts.
Culture Conditions
FIBRONECTIN DMEM/M199 5% HORSE SERUM
Purified myocytes were plated on fibronectin-coated (25 μg/mL) dishes at 1,500–2,500 cells/mm² in DMEM/M199 (4:1) supplemented with 5% horse serum, 100 μM BrdU (anti-fibroblast), and penicillin/streptomycin. Serum was reduced to 1% after 24 h to minimize fibroblast overgrowth. Spontaneous beating was typically observed by 48–72 h post-plating, with synchronous monolayer contractility established by day 4–5.
Isolation Yield & Quality Metrics
| Parameter | Value | Method |
|---|---|---|
| Yield per litter (10–12 pups) | 18.5 ± 3.2 × 106 cells | Hemocytometer |
| Post-plating viability | 92.8 ± 2.4% | Trypan blue |
| Cardiomyocyte purity (day 3) | 89.4 ± 3.2% | cTnT+ / DAPI |
| Fibroblast contamination | 8.2 ± 2.6% | Vimentin+ / DAPI |
| Onset of beating | 58 ± 12 h | Visual inspection |
| Synchronous monolayer (day) | 4.2 ± 0.8 | Video analysis |
18.5M
Cells per litter
89%
Cardiomyocyte purity (cTnT+)
~58 h
Onset of spontaneous beating