Improved method for isolation of porcine neonatal pancreatic cell clusters

Abstract

Based on the described methods for the isolation of neonatal pancreatic cell clusters (NPCCs), we have developed modifications in order to improve their quality, functionality, and process reproducibility in the isolation technique, for potential use in research. In addition, we indicate techniques for describing yield, functionality, viability and purity of the NPCCs. Methods: Purity of the NPCCs was determined through dithizone staining and subjected to image analysis. Viability and apoptosis was measured through flow cytometry with propidium iodide and annexin, respectively. NPCC functionality was measured through a static glucose stimulation test. Results: We developed a high-yield reproducible technique that had 81 279.55 ± 18 257.05 IEQ/g of pancreas at 4 days of culture, with a 94% viability and an 88 ± 2.73% purity. Stimulation index from the glucose stimulation test was > 10. Conclusion: The technique allowed us to obtain NPCC with optimal viability, functionality, purity, and endurance for use in research. © Blackwell Munksgaard, 2005.