Isolation and purification of islet cells from adult pigs.
We used in situ perfusion and a multiple-organ harvesting technique to collect islets from adult pig pancreata. The tissues were digested with collagenase P followed by purification in a lympholyte discontinuous gradient using a COBE2991 cell separator. The yield and purity of isolated islets were evaluated with a light microscope after dithizone (DTZ) staining. Islet function was assessed using an in vitro insulin release assay. The results showed that before purification 275,000 +/- 20,895 islet equivalents (IEQ) were obtained from 1 digested pancreas. After purification with gradient centrifugation, the islet yield was 230,350 +/- 26,679 IEQ/pancreas. Each gram of the purified pancreatic tissues yielded 2710 +/- 229 IEQ with an average purity of 50.2 +/- 2.0%. The purified islet cells responded to stimulation with high glucose concentrations (16.7 mmol/L), namely, 4.74-fold greater than the insulin secretion with exposure to the basal level of glucose (3.3 mmol/L; P < .001). These results suggested that the established isolation method can be applied to large-scale purification of fully functional islets from pig pancreata.
Qiao
A-Y
AY
Department of Hepatobiliary Surgery, Third Affiliated Hospital of Guangzhou Medical College, Guangzhou, Guangdong, China. anyiqiao@yahoo.com.cn
Zhang
W-H
WH
Chen
X-J
XJ
Zhang
J
J
Xiao
G-H
GH
Hu
Y-X
YX
Tang
D-C
DC
D000818:Animals; D002452:Cell Count; D002469:Cell Separation / Q000379:methods; D048429:Cell Size; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000706:statistics & numerical data; D009929:Organ Size; D010179:Pancreas / Q000033:anatomy & histology / Q000166:cytology; D013552:Swine
D007328:Insulin