In situ digestion, in vitro harvest and culture of jugular vein endothelial cells in rabbits

Objective To establish the methodology for in situ digestion,in vitro harvest and culture of jugular vein endothelial cells in rabbits.Methods The jugular vein was dissected and kept in situ unilaterally,with the contralateral jugular vein intact.Both terminals of the dissected vein were occluded for cannulation and were subjected to type Ⅰ collagenase injection for in situ digestion.The operated jugular vein was resectcd for collecting endothelial cells in vitro,followed by culture and passage using EGM-2 culture medium.The jugular vein endothelial cells were observed under inverted light microscopy and transmission electron microscopy for morphological examination.In addition,factor Ⅷ was measured by immunohistochemistry assay.Results Of all jugular vein endothelial cells harvested,80％ underwent fusion after a 7-to-10-day primary passage.These cells appeared in short spindle-like,polygonal or cobblestone-like arrangement under microscope.The characteristic Weible- Palade bodies of endothelial cells were found under transmission electron microscope.Immunohistochemistry assay showed positive evidence of factor Ⅷ-related antigen.The harvested endothelial cells were capable of proliferation after cryopreservation followed by thaw and passages.Conclusion The approach for in situ digestion and in vitro harvest and culture of jugular vein endothelial cells in rabbits is successfully established. Key words: Rabbits; Jugular veins; Endothelial cells; Cell culture techniques; In situ digestion