Modified method of isolating and purifying bovine chromaffin cells

Aim: To establish a more stable, economic and convenient method of in vitro isolation and purification bovine chromaffin cells (BCCs), and provide a plenteous and healthy source of viable cells for clinical transplantation. Methods: The experiment was carried out in the Biomedical Material Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences between January 2004 and December 2005. BCCs of new-born calves were isolated by collagenase digestion combined with mechanical isolation and then purified by Percoll discontinuous density gradient centrifugation. Phase contrast microscope was used to determine the morphology of BCCs. The functional activity and purification of BCCs were identified by immunocytochemical/histochemical staining, respectively. Trypan blue assay were used to detect the viability of BCCs. Results: 1 Healthy BCCs concentrated between the two separating medium, and erythrocytes settled on the bottom of centrifuge tube. Cortex cells and cellular debris scattered on the separating medium of specific density. 2 On average, (0.7-3.1)×10⁸ cells could be isolated from per adrenal gland with 99% viability. 3 The isolated BCC had the functional activity of catecholamine-secretion in culture. Conclusion: Collagenase perfusion digestion combined with mechanical dissociation and discontinuous density gradient centrifugation can be used to isolate and purify BCCs. The purity of the cells depends on the amount of isolated cells and has general biological characteristics of catecholamine.