[An economic and effective method for islet cell isolation from SD rats].
OBJECTIVE
To set up an economic and effective method for islet isolation from rat, and thereby prove a laboratory protocol of animal model for clinical islet transplantation.


METHODS
Twenty-five adult male SD rats weighing 230-380 g were used as organ donor. In each of 5 repeated experiments, pancreatic islets of 5 animals were isolated by intraductal infusion of compound sodium chloride injection (CSCI), and subsequently, digested with low concentration (0.5 mg/mL) of collagenase V solution. Islet purification was performed by using a discontinuous density gradient centrifugation that was prepared with 27.0%, 23.0%, 20.5% and 11.0% of Ficoll 400. Islet yield and purity were determined by dithizon (DTZ) stain, and propidium iodide (PI)/fluorescein diacetate (FDA) double stain was used to check viability of islets. The endocrine secretory function was assessed by insulin secretion in either low (2.8 mmol/L) or high (25.0 mmol/L) glucose incubation after 3 days of culture in RPMI1640 media.


RESULTS
Average islet digestion time of 5 experiments was (13.8 +/- 1.6) min. Before purification, average isolated number was (5,626 +/- 422) islets, and the number was significantly reduced to (2 914 +/- 485) islets after purification (P < 0.01). The average recovery rate was 51.6% +/- 6.0%, and the average yield was (583 +/- 97) islets/pancreas. The average purity and viability of islets were 90.2% +/- 3.4% and 81.6% +/- 7.0%, respectively. After 3 days of culture, insulin secretion of the islets was (116.1 +/- 17.4) EU/L in high glucose incubation, which was significantly higher than that of low glucose environment [(39.7 +/- 7.5) EU/L, P < 0.01)]. The average insulin stimulation index was 3.0 +/- 0.4.


CONCLUSIONS
The islet isolation with the CSCI solution and digestion with low concentration of collagenase V decrease experimental cost and also have a beneficial effect on islet recovery and their function.
Zhang
Jianbo
J
People's Hospital, Chengdu Sichuan, 610072, P.R. China.
Yang
Qian
Q
Chen
Jie
J
Mu
Dezhi
D
Zhang
Lin
L
Mao
Meng
M
Qu
Yi
Y
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D007515:Islets of Langerhans / Q000166:cytology*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley