Detailed protocol for evaluation of dynamic perifusion of human islets to assess β-cell function

Abstract

The definitive measure of β-cell quality in an islet is the measurement of β-cell function, i.e., the ability of the islets to release insulin in a controlled manner in response to minute changes in ambient glucose levels. Continuous flow or dynamic perifusion of the solution containing glucose and secretagogues through the islets is the most accurate assessment of regulated insulin release in vitro. Here, we describe in detail a low cost, mini-perifusion system that can be adapted to any laboratory to assess islet function by examining dynamic insulin release in response to elevated glucose concentrations and addition of secretagogues. Human islets with purity > 80% and viability > 90% were perifused with low glucose (1 mM) and subsequently challenged with high glucose (16.8 mM ± KCl, 25 mM). A prototypical biphasic response to elevated glucose concentrations was observed with an average 8-fold (above basal) increase in insulin concentration at peak values. Similarly, perifusion with carbachol or exendin-4 (Byetta) with glucose (6 mM) resulted in 1.32- and 1.35-fold increase in insulin secretion above basal. Islets could be maintained in the perifusion apparatus and continued to respond to glucose for up to 3 h. At minimal financial cost and technical expertise, this apparatus can be set-up in any biological laboratory to evaluate regulated hormone release from many cell types in less than 6 h. This will allow other laboratories to measure insulin responses to their drug or modifier of interest in vitro, in a manner that better approximates islet function in vivo.

Figure 1

Perifusion system. (A) A simplified diagram of the perifusion system showing the basic elements of the four column apparatus. (B) Photograph of the perifusion system, consisting of a water bath, ismatec digital pump, cooling tray for 96-well plate, tubing and columns. (C) Blue print of the 0.73 cm³ volume islet perifusion column with technical specifications. (D) Blue print of the islet perifusion column shown with rack that is fabricated from polycarbonate plastic.

Figure 2

Validation of perifusion assay to assess human islet functional quality. 200 IEQ/column each from the same cadaveric donor were perifused for 1 h with 1 mM glucose prior to fraction collection. They were then stimulated with 16.8 mM glucose for a period of 10 min. This was followed by a 17 min perifusion with 1 mM glucose. Subsequently, the islets were then stimulated with 16.8 mM glucose + 25 mM KCl for an additional 10 min. Collected fractions were measured for insulin by ELISA. Insulin measurements were divided by basal release to obtain normalized values (stimulation index, SI). (A) Values for insulin measured from the fractions collected from each of the four columns run in parallel using the same islet preparation. Normalized values of insulin release are consistent across replicate columns. (B) Retrospective analysis of perifusion data from 12 different donor islet preparations showing the mean ± SEM of each point.

Figure 3

Effect of second glucose stimulation at different time points on the same islets. Human islets (200 IEQ/column) were perifused for 1 h with 1 mM glucose prior to collection of samples for insulin measurement. Islets were then stimulated twice with 16.8 mM glucose for 10 min each. The time between the two stimulations was (A) 17 min (first two columns) and (B) 60 min (last two columns). (C) The second stimulation with data from all four columns superimposed on another. Although lower than the first, the second stimulations clearly overlap with each other indicating that islets from the same donor have similar physiological response when challenged twice with glucose at different time points. Insulin measurements were divided by basal release to obtain normalized values of insulin release.

Figure 4

Effect of carbachol and exendin-4 on islet insulin release.