A Simple Method for the Isolation, Cultivation, and Characterization of Endothelial Cells from Rabbit Coronary Circulation

Abstract

During the last few years, endothelial cell 1.1. Endothelial Cell Harvesting and Culture functions have been intensively studied in several in vitro and in vivo preparations New Zealand white rabbits of either sex (1.5–1.8 [1–3]. However, there are many methodological kg body weight) were anesthetized with a mixture problems that hinder or exclude biochemical analyof xylazine (5 mg/kg) and ketamine (35 mg/kg). ses of intact endothelial cells under in situ condiAnimals received heparin i.v. and 10 minutes later tions. For this reason, a system of cultured coronary were sacrificed with a sodium pentobarbital overendothelial cells grown under physiologically dedose. Then, under careful sterile conditions, the fined and carefully controlled conditions needs to hearts were excised and promptly placed in a sterile be established. Successful cultivation has been rePetri dish. All subsequent work was performed in ported for endothelial cells derived from human a sterile hood under laminar flow conditions. umbilical vein [4], bovine vena cava [5], rabbit pulThe aortas were cannulated with a sterile polymonary artery [6], bovine pulmonary artery [7], ethylene cannula secured with a surgical suture and and from other tissues [8]. Very few studies have the hearts were perfused with 500 mL of sterile described the isolation of endothelial cells from Krebs solution (117 mM NaCl, 6 mM KCl, 3 mM the heart [9,10]; however, the methods described CaCl2, 1 mM MgSO4, 0.5 mM EDTA, 16.7 mM in these reports had limitations represented by a low glucose, 24 mM NaHCO3, pH 7.4) at room temperacellular yield and the need for complex equipment. ture; the Krebs solution was injected manually with Thus, the aim of the present study was to develop a sterile 20-mL syringe to remove all blood compoa method to obtain endothelial cell cultures from nents trapped within the coronary circulation. After the coronary circulation of rabbit hearts, with the this washout, hearts were perfused with 5 mL of a main objectives being a good reproducibility and harvesting solution composed of collagenase type a high cellular yield of pure endothelial cells. IV (Sigma Chemical, St. Louis, MO, USA, cat. no. C-5138, 0.2 U/mL) and trypsin (Sigma Chemical, St. Louis, MO, USA, cat. no. T-8003, 0.3 U/mL) Abbreviations: DMEM, Dulbecco’s modified eagle medium; FCS, dissolved in Krebs solution. After injection of the fetal calf serum; PBS, phosphate-buffered solution; BSA, bovine initial 5 mL, the perfusion was stopped and, beserum albumine; cGMP, guanosine 39:59-cyclic monophospate; ACE, angiotensin converting enzyme. cause the hearts were not beating, the enzyme soluCorresponding author: Paolo Golino, M.D., Ph.D., Division of tion was not washed away, but remained in contact Cardiology, University of Naples “Federico II”, via Sergio Pansini with the coronary vasculature. After 10 minutes of 5, 80131 Naples, Italy. Tel: 139 (81) 746 2216; Fax: 139 (81) 746 2229; E-mail: ,golino@unina.it.. incubation at room temperature, the hearts were