New Automatized Method of 3D Multiculture Viability Analysis Based on Confocal Imagery: Application to Islets and Mesenchymal Stem Cells Co-Encapsulation.

Clovis Chabert,Florence Rivera,Arnold Fertin,Pierre-Yves Benhamou,Anaick Moisan,Cécile Cottet-Rousselle,Camille Laporte,Eric Fontaine,Emily Tubbs,Magali Orhant-Prioux,Sandrine Lablanche

doi:10.3389/fendo.2018.00272

Abstract

Co-encapsulation of pancreatic islets with mesenchymal stem cells in a three-dimensional biomaterial’s structure is a promising technique to improve transplantation efficacy and to decrease immunosuppressant therapy. Currently, evaluation of graft quality after co-encapsulation is only based on insulin secretion. Viability measurement in a 3D conformation structure involving two different cell types is complex, mainly performed manually, highly time consuming and examiner dependent. Standardization of encapsulated graft viability analysis before transplantation is a key point for the translation of the method from the bench side to clinical practice. In this study, we developed an automated analysis of islet viability based on confocal pictures processing of cells stained with three probes (Hoechst, propidium iodide, and PKH67). When compared with results obtained manually by different examiners, viability results show a high degree of similarity (under 3% of difference) and a tight correlation (r = 0.894; p < 0.001) between these two techniques. The automated technique offers the advantage of reducing the analysis time by 6 and avoids the examiner’s dependent variability factor. Thus, we developed a new efficient tool to standardize the analysis of islet viability in 3D structure involving several cell types, which is a key element for encapsulated graft analysis in clinical practice.

Highlights

Islet transplantation is a promising therapy for millions of patients with type 1 diabetes as it offers a perspective of an efficient metabolic control with a prevention of severe hypoglycemia without insulin injections [1,2,3]
The aim of this study was to develop and validate a standardized method to substitute the current manual viability analysis of islets co-encapsulated with mesenchymal stem cells (MSCs), in agreement with the clinical constraints
Once the blue channel is transformed in a binary signal, the picture is merged with the regions of interest (ROIs) previously detected

Summary

Introduction

Islet transplantation is a promising therapy for millions of patients with type 1 diabetes as it offers a perspective of an efficient metabolic control with a prevention of severe hypoglycemia without insulin injections [1,2,3]. To prevent graft rejection, an Automatized Viability Analysis of Co-Encapsulated Islets immunosuppressive therapy is required, which can lead to severe adverse events such as infection or neoplasic disease. The capsule allows the diffusion of insulin, oxygen, and nutriments while protecting islets from the immune system [11]. This technique has already shown interesting results in different species [12,13,14] and paves the way for free-immunosuppressive islets transplant therapy [15]. Co-encapsulation of islets with MSCs appears to be a promising approach to improve treatment strategies for diabetic patients [21]

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