Improved islet isolation by three-cannula method for collagenase infusion / 中华器官移植杂志

ABSTRACT

Objective Pancreas perfusion is an essential step in human islet isolation.To develop the new methods for introductal canulation,collagenase infusion and to observe their effects on islets isolation.Methods A total of 17 pancreases were digested from March 2005 to April 2010.The pancreases were distended by three methods:the standard method (n =3),the one-cannula method (n =11) and three-cannula method (n =3).In the standard group,the pancreases were completely cut into half at the mid-body.Two catheters were inserted into the main ductone directed toward the tail and the other to the head.In the one-cannula method group,a long tube was inserted into the duct at the head,advancing to the tail In the three-cannula method group,pancreatic parenchyma was then minimally cut at the mid-body and three catheters were inserted into the main pancreatic ductone at the head (the first catheter) and two at the mid-body,one toward the tail (the second catheter) and the other toward the head (the third catheter).The pancreases were digested by improved Ricordi technique.Ficoll continuous density/grads centrifuge method was performed to purify the islets.DTZ staining was adopted to identify islets and count islet equivalent (IEQ). AO/EB fluorescence examination was used to count active islet percentage.Static glucose stimulating test (SGS) in vitro was designed to estimate islet function and calculate SI.Results The distension volume of the threecannula method group was 1.24 rnl/g pancreas,and higher than the other groups (for the standard group0.71 ml/g pancreas; for one-cannula method group0.96 ml/g pancreas,P＜0.05).The yield of islet in the three-cannula method group and the one-cannula method group was 2514 and 2270 IEQ/g,which was significantly more than that in the standard group (1914 IEQ/g pancreas,P＜0.05).The purity and viability of the islets were 74 ％/79.3 ％,75.6 ％/79.4 ％ and 78.3 ％/84.0 ％ respectively in the three groups with the difference being not significant among the groups.SI in the one cannula method group (4.74) and the three-cannula method group (5.27) was significantly higher than that in the standard group (3.46).ConclusionThe three-cannula method improved collagenase infusion and the islet yields.