The establishment of "two-step sequential filtration method" on the yield rate of purified islets in rats / 中国当代儿科杂志

ABSTRACT

<p><b>OBJECTIVE</b>To develop a simple, rapid and reliable method of purifying Sprague-Dawley (SD) rat islets by sequential filtration through two cell strainers of different sizes and to evaluate the efficacy of the method.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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).</p><p><b>CONCLUSIONS</b>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.</p>