Visikol announces introduction of assay for screening islet cell proliferation using InSphero microtissues and advanced 3Screen™ imaging and analysis.
Understanding pancreatic β-cell function in the regulation of glucose homeostasis and metabolism is of paramount importance in the research of the most common human metabolic disease – diabetes. Although isolated primary islets are considered the gold standard tool of diabetes research, their experimental use is limited due to their inherent heterogeneity in size, cellular composition and purity, as well as their rapid decline in functionality and viability ex vivo.
The ability of the small molecule harmine to activate β-cell proliferation in vivo is a well-established phenomenon and, for this reason, has initiated interest developing analog compounds to treat diabetes. This in turn has given rise to interest in the development of high-throughput screening (HTS) assays to survey the efficacy of these drugs in in vitro models of pancreatic islets.
In this study, we combine the 3D InSight™ Human Islet Microtissues provided by InSphero and the Visikol HISTO-M tissue clearing reagent to illustrate the power of tissue clearing on high content screening (HCS) of novel drugs targeting β-cell proliferation.
Materials and Methods
Reagents and cell culture
3D InSight™ Human islet microtissues were obtained from InSphero Inc. Antibodies and fluorescent dyes and antibodies were obtained from Invitrogen (Thermo Scientific). Harmine was obtained from Sigma Aldrich.
Treatment of microtissues with antiproliferative cisplatin
3D InSight™ Islet Microtissues were prepared from human islets (Age 29, Male, Hispanic, BMI 22.17, HbA1c 5.5%) and delivered in an Akura™ 96 well plate. Medium exchanges were performed every 2 days to maintain the microtissues in culture. On day 19, a 4.5-day treatment with 10 µM Harmine was started, with re-dosing after 2.5 days. Harmine was dissolved in DMSO to make a 10 mM stock solution, and a vehicle control was treated alongside for comparison.
Fixation and immunolabeling
On day 23, microtissues were fixed using 4% PFA, followed by washing in PBS to remove fixative. Microtissues were treated with methanol, followed by 20% DMSO/methanol, followed by 0.2% Triton X-100 in PBS to improve penetration of antibodies. Microtissues were blocked with 10% donkey serum, then incubated with guinea pig anti-PDX1 (1:150 dilution, Abcam Cat# ab47308), rabbit anti-Ki67 antibody (1:200 dilution, Abcam Cat# ab15580), and with goat anti-rabbit AlexaFluor568 and goat anti-guinea pig AlexaFluor488 conjugated secondaries (1:250 dilution, available from Invitrogen) to label β-cells and proliferating cells. Nuclei were counterstained with DAPI (Molecular Probes).
Clearing and high throughput imaging of microtissues
Microtissues were dehydrated with methanol, then cleared with Visikol HISTO-M. Imaging of spheroids was conducted in InSphero Akura™ 96 well plates using a CX7-LZR High Content Screening platform confocal imager (ThermoFisher). Multi-channel z-stacks were collected for each tissue, using 5 µm steps at 20X magnification. Images were processed using automated ImageJ macros, and cells were counted using CellProfiler.
Results and Discussion
Drug-induced β-Cell Proliferation
The islet microtissues were treated with harmine at a concentration known to induce significant proliferation in dispersed islets cells (10 µM). After intervention with harmine, the effects on islet function were studied. Harmine did not alter the basal levels of secretion at 2.8 mM glucose. Similarly, stimulated secretion levels were maintained in harmine treated islets, reflecting unaltered β-cell function. However, harmine induced a significant reduction in the levels of total content (Figure 1).