Pancreatic beta-cell purification by altering FAD and NAD(P)H metabolism.
Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD) and nicotinamide-adenine dinucleotide phosphate (NAD(P)H) autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(P)H fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(P)H contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(P)H fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.
Smelt
M J
MJ
Transplantation Biology and Immunoendocrinology, Section of Medical Biology, Department of Pathology and Laboratory Medicine, University Medical Centre Groningen, 9700 RB Groningen, The Netherlands. m.j.smelt@med.umcg.nl
Faas
M M
MM
de Haan
B J
BJ
de Vos
P
P
D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D003470:Culture Media; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D050416:Glucagon-Secreting Cells / Q000166:cytology; D007150:Immunohistochemistry; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology; D008297:Male; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew
D003470:Culture Media; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP