A modified method for isolation of adult rat cardiomyocytes

BACKGROUND: At present, adult rat cardiomyocytes are usually isolated by enzymolysis method with different kinds of perfusion apparatuses, collagenases and methods. It is mainly aimed at emergent isolation of single cardiomyocyte and only appropriate for electrophysiological study. OBJECTIVE: To develop and optimize a stable adult Sprague-Dauley rat cardiomyocytes isolation method for primary culture and research. DESIGN, TIME AND SETTING: The cytology in vitro experiment was performed at the Molecular Cardiology Center, Department of Cardiology, Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology from March to September 2008. MATERIALS: Thirty male Sprague-Dawley rats aged 8 to 10 weeks were obtained from the Laboratory Animal Center, Tongji Medical College. Collagenase type II, protease and bovine serum albumin (BSA) without fatty acid were purchased from Worthington Biochemical Corporation, Sigma Corporation and Merck Corporation, respectively. METHODS: After successful anesthesia, the rat heart was quickly excised out with sufficient length of the aorta and perfused by modified Langendorff system: firstly perfusion with calcium-free Krebs-Henseleit buffer (KHB) in a non-recirculating manner for 4 minutes, and then digestion with 0.06% collagenase (Worthington, type 2) and 0.003% protease for about 15 minutes, thereafter ventricular tissue was minced and incubated in a digestion medium with 10 g/L BSA at 37°C for 10 minutes. After filtered with 200 μm nylon net and centrifuged at 500 g for 1 minute, the isolated cells were performed gradient calcium reintroduction and allowed to natural sedimentation. MAIN OUTCOME MEASURES: Morphology, vitality and yield of isolated rat cardiomyocytes. RESULTS: A large amount of rod-shaped rat cardiomyocytes with clear cross striations and high activity [(73±5.6)%, n=30] were acquired after calcium reintroduction, more than 90% of rod-shaped cardiomyocytes were quiescent with clear transverse striation. The average yield of viable cardiomyocytes was (4.2±0.4)×10⁶ per heart. CONCLUSION: A high yield of cardiomyocytes isolated by aorta retrograde perfusion with digestive enzymes using modified Langendorff system can be acquired stably and applicable for primary culture and cell or molecular biology study.