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

Summary

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.

Abstract

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.

Introduction

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 exocytosis^2,3,4. Bot....

Protocol

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.......

Discussion

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.......

Materials

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