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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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Name Company Catalog Number Comments
Ad-CMV-cADDis Welgen Not applicable
 0.01” FEP tubing IDEX 1527L
1 M HEPES Gibco 15630-080 Enriched-CMRL Media Component
1.5 mL and conical tubes Any Any
10 μm PTFE filter Cole-Parmer SK-21940-41 Change every 8-10 runs
100 mM Sodium Pyruvate Thermo Scientific 11360070 Enriched-CMRL Media Component
190 proof Ethanol Decon labs 2816 Acid Ethanol Component
200 mM GlutaMAX-I Supplement Gibco 35050061 Enriched-CMRL Media Component
Ascorbate Sigma A5960 DMEM Perifusion Buffer Component
Bovine Serum Albumin Sigma A7888 DMEM Perifusion Buffer Component
Bubble trap  Omnifit 006BT
CellCarrier ULA 96-well Microplates Perkin Elmer 6055330
cellSens analysis software Olympus v3.1 Software used for data analysis
CMRL 1066 MediaTech  15-110-CV Enriched-CMRL Media Component
Conical adapter (IDEX, P-794) IDEX P-794
D-(+)-Glucose Sigma G7528 Glucose Buffer Component
DMEM  Sigma D5030 DMEM Perifusion Buffer Component
Environmental chamber okolab IX83
Epinepherine (Epi) Sigma E4250 Stimulation Buffer Component
Fetal Bovine Serum (FBS), Heat Inactivated Sigma 12306C Enriched-CMRL Media Component
Glucagon ELISA Mercodia 10-1281-01
Glucagon Kit HTRF Cisbio 62CGLPEH
HCl (12N) Any Any Acid Ethanol Component
HEPES Sigma H7523 DMEM Perifusion Buffer Component
iCell Endothelial Cells Medium Supplement Cell Dynamics M1019 iEC Media Component
Idex Derlin nut & ferrule 1/4-24 Cole-Parmer EW-00414-LW
Insulin ELISA Mercodia 10-1113-01
Isobutylmethylonine (IBMX) Sigma I5879 Stimulation Buffer Component
Laser scanning confocal microscope Olympus FV3000
L-Glutamine Sigma G8540 DMEM Perifusion Buffer Component
Microchip (University of Miami, FP-3W) University of Miami FP-3W
Microchip holder  Micronit Microfluidics FC_PRO_CH4525
Model 2110 Fraction Collector Biorad 7318122
P10, P200, and P1000 pipets and tips Any Any
Penicillin/Streptomycin Gibco 15140-122 Enriched-CMRL Media Component
Peristaltic pump  Instech P720
Phosphate Buffered Saline Gibco 14190-144 Wash Islets
Sarstedt dishes Sarstedt depends on dish diameter
Sodium Bicarbonate Sigma S6014 DMEM Perifusion Buffer Component
Sodium Pyruvate Sigma P2256  DMEM Perifusion Buffer Component
Stereoscope Olympus SZX12
Steriflip Filter (0.22 μm) Millipore SCGP00525 Filter all buffers twice
VascuLife VEGF Medium Complete Kit LifeLine Cell Technology LL-0003 iEC Media Component

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