Improved method for isolation of porcine neonatal pancreatic cell clusters.
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.


CONCLUSIONS
The technique allowed us to obtain NPCC with optimal viability, functionality, purity, and endurance for use in research.
Valdés-González
Rafael A
RA
Xenotransplant Laboratory of the Hospital Infantil de México "Federico Gómez", México, D.F., Mexico. rvaldes@xenomexico.org
Silva-Torres
María L
ML
Ramírez-González
Briceyda
B
Terán
Luis
L
Ormsby
Christopher E
CE
Ayala-Sumuano
Jorge T
JT
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D005434:Flow Cytometry; D005947:Glucose / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D008297:Male; D008856:Microscopy, Fluorescence; D015203:Reproducibility of Results; D013552:Swine
D007328:Insulin; D005947:Glucose