Abstract
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.
Highlights
Studies on primary beta cells are required in almost all fields of islet research
We evaluated the effects of glucose and amino acids on beta-cell purity separately; dispersed islets were incubated in unsupplemented MEM medium (5.5 mmol/L D-glucose and 553 mg/L amino acids), or MEM medium supplemented to either 10, 15, or 20 mmol/L D-Glucose, or 1000, 1500, or 2000 mg/L amino acids for one hour at 37◦C prior to flow cytometry
Nonbeta cells responded to incubations with increasing amino acid concentration by increasing FAD fluorescence and decreasing NAD(P)H fluorescence. We show that both beta-cell and nonbetacell flavin adenine dinucleotide (FAD) and nicotinamideadenine dinucleotide (NAD(P)H) fluorescence can be manipulated by preincubation of dispersed islet cells in culture medium containing graded loads of D-glucose and amino acids
Summary
Studies on primary beta cells are required in almost all fields of islet research. it has been far from simple to obtain high numbers of pure and viable betacells. Published methods for purification of beta cells from other endocrine islet cells are based on the sedimentation velocity [1], labelling of beta-cell surface antigens [2], or the endogenous (auto) fluorescence of beta cells [3,4,5,6]. Most of these methods have a limited applicability, as they are associated with low yields of betacells, or permanent deterioration of cellular function caused by mandatory labelling of the cellular membrane. The principle applicability of isolation of beta cells by autofluorescence was shown by Rabinovitch et al [4] and Van de Winkel et al [10], but resulted in a preparation with considerable amounts of alpha-cells
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