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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

Published: November 3rd, 2023

DOI:

10.3791/65259

1Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, 2Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Vanderbilt University Medical Center, 3Department of Biomedical Engineering, University of Miami, 4VA Tennessee Valley Healthcare System
* These authors contributed equally

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

The islets of Langerhans are mini organs scattered throughout the pancreas whose function is crucial for the maintenance of glucose homeostasis. Insulin is secreted from beta cells following the metabolism of glucose, an increase in the ATP/ADP ratio, the closure of ATP-sensitive potassium channels, depolarization of the plasma membrane, and the influx of extracellular calcium1. Glucagon secretion from alpha cells is less understood, but it has been postulated that intracellular and paracrine pathways contribute to glucagon granule exocytosis2,3,4. Bot....

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Human islets (N = 4 preparations) were obtained through partnerships with the Integrated Islet Distribution Program, Human Pancreas Analysis Program, Prodo Laboratories, Inc., and Imagine Pharma. The Vanderbilt University Institutional Review Board does not consider deidentified human pancreatic specimens as human subjects research. This work would not be possible without organ donors, their families, and organ procurement organizations. See Table 1 for donor demographic information. Human islets from pa.......

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Biosensor-expressing human pseudoislets were created via the adenoviral delivery of constructs encoding the cAMP biosensor cADDis (Figure 1A). Figure 1B shows the reaggregation of the transduced human islet cells over time, with fully formed pseudoislets observed after 6 days of culture. The cells began to show visible cADDis fluorescence within 48 h, and there was high biosensor expression in transduced cells by the end of the culture period. Using thi.......

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The integration of a microperifusion system, biosensor-expressing pseudoislets, and laser-scanning confocal microscopy allows for the synchronous assessment of intracellular signaling events and dynamic hormone secretory profiles. The dynamic microperifusion system can deliver a series of well-defined stimuli to the pseudoislets and allows for the collection of the effluent, in which the insulin and glucagon concentrations can be measured by commercially available ELISA. Concurrently, live-cell imaging of the biosensor-e.......

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Organ donors and their families are appreciated for their invaluable donations, and the International Institute for Organ Procurement Organizations, Advancement of Medicine (IIAM) and the National Disease Research Exchange (NDRI) are acknowledged for their partnership in making human pancreatic tissue accessible for research. This work was supported by the Human Islet Research Network (RRID:SCR_014393), the Human Pancreas Analysis Program (RRID:SCR_016202), DK106755, DK123716, DK123743, DK120456, DK104211, DK108120, DK112232, DK117147, DK112217, EY032442, and DK20593 (Vanderbilt Diabetes Research and Training Center), The Leona M. and Harry B. Helmsley Charitable Trus....

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NameCompanyCatalog NumberComments
Ad-CMV-cADDisWelgenNot applicable
 0.01” FEP tubingIDEX1527L
1 M HEPESGibco15630-080Enriched-CMRL Media Component
1.5 mL and conical tubesAnyAny
10 μm PTFE filterCole-ParmerSK-21940-41Change every 8-10 runs
100 mM Sodium PyruvateThermo Scientific11360070Enriched-CMRL Media Component
190 proof EthanolDecon labs2816Acid Ethanol Component
200 mM GlutaMAX-I SupplementGibco35050061Enriched-CMRL Media Component
AscorbateSigmaA5960DMEM Perifusion Buffer Component
Bovine Serum AlbuminSigmaA7888DMEM Perifusion Buffer Component
Bubble trap Omnifit006BT
CellCarrier ULA 96-well MicroplatesPerkin Elmer6055330
cellSens analysis softwareOlympusv3.1Software used for data analysis
CMRL 1066MediaTech 15-110-CVEnriched-CMRL Media Component
Conical adapter (IDEX, P-794)IDEXP-794
D-(+)-GlucoseSigmaG7528Glucose Buffer Component
DMEM SigmaD5030DMEM Perifusion Buffer Component
Environmental chamberokolabIX83
Epinepherine (Epi)SigmaE4250Stimulation Buffer Component
Fetal Bovine Serum (FBS), Heat InactivatedSigma12306CEnriched-CMRL Media Component
Glucagon ELISAMercodia10-1281-01
Glucagon Kit HTRFCisbio62CGLPEH
HCl (12N)AnyAnyAcid Ethanol Component
HEPESSigmaH7523DMEM Perifusion Buffer Component
iCell Endothelial Cells Medium SupplementCell DynamicsM1019iEC Media Component
Idex Derlin nut & ferrule 1/4-24Cole-ParmerEW-00414-LW
Insulin ELISAMercodia10-1113-01
Isobutylmethylonine (IBMX)SigmaI5879Stimulation Buffer Component
Laser scanning confocal microscopeOlympusFV3000
L-GlutamineSigmaG8540DMEM Perifusion Buffer Component
Microchip (University of Miami, FP-3W)University of MiamiFP-3W
Microchip holder Micronit MicrofluidicsFC_PRO_CH4525
Model 2110 Fraction CollectorBiorad7318122
P10, P200, and P1000 pipets and tipsAnyAny
Penicillin/StreptomycinGibco15140-122Enriched-CMRL Media Component
Peristaltic pump InstechP720
Phosphate Buffered SalineGibco14190-144Wash Islets
Sarstedt dishesSarstedtdepends on dish diameter
Sodium BicarbonateSigmaS6014DMEM Perifusion Buffer Component
Sodium PyruvateSigmaP2256 DMEM Perifusion Buffer Component
StereoscopeOlympusSZX12
Steriflip Filter (0.22 μm)MilliporeSCGP00525Filter all buffers twice
VascuLife VEGF Medium Complete KitLifeLine Cell TechnologyLL-0003iEC Media Component

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