Study of modified methods of digestion and purification of human islet cells

Abstract

Objective To explore the modified methods of digestion and purification of human islet cells. Methods Donor pancreases from 20 donors after cardiac death (DCD) were provided by the Organ Transplantation Center of the Second Affiliated Hospital of Guangzhou Medical University and the local ethical committee approval was received. Among 20 donors, 13 cases were male and 7 female, aged (45±7) years on average. The pancreas was resected immediately after perfusion of 4℃ UW solution in the abdominal aorta and the pancreatic tissues were stored in 4℃ UW solution. The trimmed pancreatic duct was injected with collagenase V and was digested with collagenase digestion juice at 38℃ for 30 min in a self-designed pancreatic digestion tank. The islet cells were isolated and purified by discontinuous density gradient centrifugation method and were stained with dithizone (DTZ) staining. The islet equivalent quantity was calculated. The survival rate of islet cells was determined by acridine orange (AO) staining. Results A total of 20 samples of adult pancreases were digested with a digestive rate of 92.7%. The amount of collagenase for single digestion was 400 ml. After DTZ staining, the islet cells were observed in scarlet round-like cell clusters. The islet equivalent quantity of each pancreas after digestion was (69±28)×103 IEQ/g, and (53±29)×103 IEQ/g after purification with a purity of 79%. The survival rate of islet was (94.1±2.3)%. Conclusions Excellent digestion and purification effect of human islet cells can be obtained by using the novel self-designed modified pancreatic digestion tank, which can significantly reduce the dosage of collagenase. Key words: Islet cell; Digestion; Purification; Transplantation