[Improved method for optimized isolation and purification of rat islets and identification of function].
OBJECTIVE
To explore good methods for isolation and purification of rat islets.


METHODS
The islets were isolated from male SD rat pancreata by a collagenase perfusion method and purified by a modified method: added 4 kinds of Euro-Ficoll of different densities (F1: D=1.132, F2: D=1.108, F4: D=l.069,F5: D=1.023), discontinuous density gradient centrifuge the tube at 2,000 r/min for 20 minutes at 4 degrees C, then the islets between F1 and F2 were collected. The purity of islets was assessed by dithizone staining with islets counted and scored for size. Islets viabil ity was assessed by fluorescin diacetate / propidium iodide. The function of purified islets was judged by the test of insulin release and islets transplantation.


RESULTS
After an improved method for optimized isolation and purification, (920+/-122) IEQ purified islets were obtained from one rat. Boththe purity and viability of islets were over 90%. The amount of insulin secretion was (18.25+/-0.32) mU/L and (36.70+/-3.57) mU/L at 2.2 mmol/L and 22.2 mmol/L concentration of glucose respectively, there was significant difference between the two phases (P<0.05). The insulin release index was 2.01+/-0.15. Under 1,000 IEQ islets transplantation, the normal glucose level could be remained in diabetic rats.


CONCLUSIONS
Highpurity and highviability islet cells can be got through improved collagenase perfusion and centrifugation on gradients method.
Yuan
Yu
Y
Key Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichuan University, Chengdu Sichuan, 610041, P. R. China.
Cong
Cong
C
Zhang
Jing
J
Wei
Lingling
L
Li
Shengfu
S
Jin
Xi
X
Mai
Gang
G
Li
Youping
Y
Cheng
Jingqiu
J
Lu
Yanrong
Y
D000818:Animals; D001786:Blood Glucose / Q000032:analysis; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000601:surgery*; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation / Q000379:methods*; D008297:Male; D010179:Pancreas / Q000166:cytology* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D046509:Tissue Culture Techniques / Q000379:methods
D001786:Blood Glucose; D007328:Insulin