New Islet Model for 3D Study of Endothelial and &#946; Cell Interactions: Relevance for the Screening of Cytoprotective Agents

Amira Saidi; Clémence Tollard; Liyao Liu; Yasmina El Khallouqi; Jean Sévigny; Cyril Auger; Gilles Kauffenstein; Fatiha El Ghazouani; Laurence Kessler; Florence Toti

doi:10.3791/68220

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Method Article

New Islet Model for 3D Study of Endothelial and β Cell Interactions: Relevance for the Screening of Cytoprotective Agents

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DOI:

10.3791/68220

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April 17th, 2026

Amira Saidi¹ , Clémence Tollard¹ , Liyao Liu¹^,² , Yasmina El Khallouqi¹ , Jean Sévigny³^,⁴ , Cyril Auger¹ , Gilles Kauffenstein¹ , Fatiha El Ghazouani¹ , Laurence Kessler¹^,⁵^,⁶ , Florence Toti¹^,⁷

¹Regenerative Nanomedicine (RNM), UMR 1260, INSERM (French National Institute of Health and Medical Research), University of Strasbourg, ²Department of Emergency, The First Affiliated Hospital of Chongqing Medical University, ³Research Center, CHU de Québec -Laval University, ⁴Department of Microbiology-Infectiology and Immunology, Faculty of Medicine, Laval University, ⁵Department of Diabetes and Nutrition Endocrinology, University Hospital of Strasbourg, ⁶Faculty of Medicine, University of Strasbourg, ⁷Faculty of Pharmacy, University of Strasbourg

Summary

This protocol provides a detailed procedure to generate stable spheroids of murine pancreatic islets, and detailed methods for 3D staining, clearing, and mounting, adapted to their small size.

Abstract

This protocol details an optimized method for the production of small stable spheroids, their culture, and 3D imaging, for the study of the endothelial and insulin-producing β cells interactions in a 3D model of pancreatic islets. The 150-200 µm spheroids, mirroring the lowest range of islet sizes, were prepared from a selected ratio combining 1 intra-islet endothelial cells (MS-1 cells) to 20 insulin-secreting cells (β-TC-6). Staining, clearing, and mounting challenges of small spheroids and their tackling by employing low-melting point agarose and the CUBIC clearing technique are detailed, as well as key points for an efficient analysis of the 3D structure with different probes. Data indicate that NTPDASE-ectonucleotidase 3 does not colocalize with insulin in the spheroid model, suggesting varying maturity and functional levels of β-TC6 and that the complete procedure can also be applied to isolated pancreatic islets, with clear probing of intra-islet vessels. These findings underscore the effectiveness of the 3D imaging protocol in revealing complex pancreatic cell organization and interactions within the islet model.

Introduction

Pancreatic islet transplantation is often complicated by instant blood-mediated inflammatory reaction (IBMIR), which significantly damages the intra-islet endothelial cells (IEC) with an impact on graft perfusion and function. In the long run, a reduced mass of functional islets and the loss of insulin independence are described in patients, 4 years post-transplantation¹. While murine or rat models of islet transplantation have been developed, standardized models to investigate pharmacological targets for the pre-conditioning of islets, of particular relevance in the context of cytokine-driven graft damage induced by the IBMIR are lacking