Improved automated method for isolation of canine pancreatic islets / 中国普通外科杂志

ABSTRACT

Objective To study an improved automated method for isolation and purification of large amounts of human pancreatic islets from large mammals,and try to create conditions for preparation and isolation of large amounts of human islets.Methods An improved automated system was used to isolate and purify panreatic islets of sogs.Under the general anaesthesia(GA) condition,the pancreas of the dogs was via in situ vascular perfused using cold HC-A solution and then removed en bloc with duodenum and spleen.Then they were placed in cold UW aolutian.Intraductal collagenase-Ⅴ and pefabloc(4 ℃) was delivered with controlled perfusion.lslets were dissociated in system of Ricordi Chamber and purified islets separated with Ficoll density gradient centrifugation under controlled temperature.Digestion time,islets remaining trapped in exocrine tissue,final islet purity,insulin and C-pep secretory activity,and islet recovery were observed.The purified islets were observed under light microscope and electronic microscope after 24 h culture.Results The digestion time rate was(25.0?6.0) min,islets remaining trapped in exocrine tissue was(9.4?2.4)%,final islet purify rate was(89.7?3.5)%,islet recovery after digestion was(17.2?3.6)?104 IEQ/pancreas.Islet recovery after purification was(8.3?2.0)?104IEQ/pancreas.Insulin and C-pep secretory activity of the purified islets and their ultrastructure after 24 h culture were ideal.Conclusions Our improved automated method and facilities for isolation of canine pancreatic islets are reliable,The morphology and function of the procured islets are excellent and they can foreseeably be used for large-scale preparation of huma islets for clinical use.