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Abstract

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

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.

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Keywords Human Pseudoislet

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