Abstract OBJECTIVE: To develop a simple, rapid and reliable method of purifying SpragueDawley (SD) rat islets by sequential filtration through two cell strainers of different sizes and to evaluate the efficacy of the method. METHODS: Islets were isolated from 8 to 12-week-old clean grade Sprague-Dawley rat pancreases using the standard collagenase digestion procedure and purified with either the generally used Ficoll density gradient method or the innovative two-step sequential filtration method. The purity and vitality of the isolated islets were visualized and assessed with DTZ and AO/PI staining. Glucose stimulating tests were performed to assay cell activity, and immunohistochemical staining was used to evaluate the synthesis function of islet cells. RESULTS: The yield of islets in the two-step filtration method group was 782±115 IEQ per rat, which was significantly higher than in the conventional Ficoll density gradient method group (598±135 IEQ per rat, P<0.01). Purity of the isolated islets in the two-step filtration method group was 90%-100% and vitality was over 95%. In the conventional Ficoll density gradient method group, islet purity was 65%-85% and vitality was 85%-95%. With regard to the high-sugar stimulation test in the two-step filtration method group, insulin concentrations in islets cultured for 24 hours were significantly higher than in those that were freshly purified (76.9 ± 6.1 μg/L vs 49.4 ± 3.9 μg/L; P<0.01). CONCLUSIONS: A two-step sequential filtration method for rat islet purification was developed and the method was simple and reliable, with high islet vitality, purity and yield.
LU Zhong,SHEN Shui-Xian,ZHI Di-Jing et al. The establishment of “two-step sequential filtration method” on the yield rate of purified islets in rats[J]. CJCP, 2013, 15(7): 572-576.
LU Zhong,SHEN Shui-Xian,ZHI Di-Jing et al. The establishment of “two-step sequential filtration method” on the yield rate of purified islets in rats[J]. CJCP, 2013, 15(7): 572-576.
［1］Patterson CC, Dahlquist GG, Gyürüs E, Green A, Soltész G; EURODIAB Study Group. Incidence trends for childhood type 1 diabetes in Europe during 1989-2003 and predicted new cases 2005-20: a multicentre prospective registration study［J］. Lancet, 2009, 373(9680): 2027-2033.
［2］McCall M, James Shapiro AM. Update on islet transplantation. Cold Spring Harb Perspect Med［J/OL］. 2012, 2(7): a007823.
［3］Li DS, Yuan YH, Tu HJ, Liang QL, Dai LJ. A protocol for islet isolation from mouse pancreas［J］. Nat Protoc, 2009, 4(11): 1649-1652.
［4］de Groot M, de Haan BJ, Keizer PP, Schuurs TA, van Schilfgaarde R, Leuvenink HG. Rat islet isolation yield and function are donor strain dependent［J］. Lab Anim, 2004, 38(2): 200-206.
［7］Shimoda M, Noguchi H, Fujita Y, Takita M, Ikemoto T, Chujo D, et al. Islet purification method using large bottles effectively achieves high islet yield from pig pancreas［J］. Cell Transplant, 2012, 21(2-3): 501-508.
［9］Liao YT, Jiang JX, Ye J, Qi H, Li FR. A novel protocol for mouse islet isolation［J］. Pancreas, 2012, 41(7): 1134-1135.
［10］Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas［J］. Science, 1980, 21: 908-910.
［11］Carter JD, Dula SB, Corbin KL, Wu R, Nunemaker CS. A practical guide to rodent islet isolation and assessment［J/OL］. Biol Proced Online, 2009, 11: 3-31.
［12］Gürol AO, Yillar G, Kur?瘙塂un AO, Kü-ük M, Deniz G, Aktas E, et al. A modified automated method for isolation of viable pancreatic islets in laboratory animals［J］. Transplant Proc, 2004, 36(5): 1526-1527.
［13］Morrison CP, Wemyss-Holden SA, Dennison AR, Maddern GJ. Islet yield remains a problem in islet autotransplantation［J］. Arch Surg, 2002, 137(1): 80-83.
［14］Salvalaggio PR, Deng S, Ariyan CE, Millet I, Zawalich WS, Basadonna GP, et al. Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves islet mass and function［J］. Transplantation, 2002, 74(6): 877-879.
［15］Jo J, Choi MY, Koh DS. Size distribution of mouse Langerhans islets［J］. Biophys J, 2007, 93 (8):2655-2666.
Stull ND, Breite A, McCarthy R, Tersey SA, Mirmira RG. Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease[J/OL]. J Vis Exp, 2012. doi: 10.3791/4137.
［17］Cavallari G, Zuellig RA, Lehmann R, Weber M, Moritz W. Rat pancreatic islet size standardization by the "hanging drop" technique［J］. Transplant Proc, 2007, 39 (6): 2018-2020.
［18］Clark SA, Borland KM, Sherman SD, Rusack TC, Chick WL. Staining and in vitro toxicity of dithizone with canine, porcine, and bovine islets［J］. Cell Transplant, 1994, 3(4): 299-306.
［19］Bank HL. Rapid assessment of islet viability with acridine orange and propidium iodide［J］. In Vitro Cell Dev Biol, 1988, 24(4): 266-273.
