Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes.

Kengo Nozaki,Tetsuya Nakamura,Tomohiro Mizutani,Yuka Matsumoto,Taichi Matsumoto,Masayoshi Fukuda,Wakana Mochizuki,Mamoru Watanabe

doi:10.1007/s00535-016-1170-8

Kengo Nozaki, Tetsuya Nakamura + Show 6 more

Open Access

https://doi.org/10.1007/s00535-016-1170-8

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Abstract

BackgroundIntraepithelial lymphocytes (IELs) in the intestine play important roles in the regulation of local immune responses. Although their functions have been studied in a variety of animal experiments, in vitro studies on spatiotemporal behaviors of IELs and their interaction with intestinal epithelial cells (IECs) have been hampered due to the lack of a suitable culture system. In this study, we aimed at developing a novel co-culture system of IELs with IECs to investigate dynamic interaction between these two populations of cells in vitro.MethodsWe optimized experimental conditions under which murine IELs can be efficiently maintained with IECs cultured as three-dimensional organoids. We then tested the effect of IL-2, IL-7, and IL-15 on the maintenance of IELs in this co-culture system. By time-lapse imaging, we also examined the dynamic behaviors of IELs.ResultsIELs can be expanded with epithelial organoids in the presence of IL-2, IL-7, and IL-15. IELs were efficiently maintained within and outside of organoids showing a ~four-fold increase in both αβT and γδT IELs for a period of 2 weeks. Four-dimensional fluorescent imaging revealed an active, multi-directional movement of IELs along the basolateral surface of IECs, and also their inward or outward migration relative to organoid structures. Cell tracking analysis showed that αβT and γδT IELs shared indistinguishable features with regard to their dynamics.ConclusionsThis novel co-culture method could serve as a unique tool to investigate the motility dynamics of IELs and their temporal and spatial interaction with IECs in vitro.Electronic supplementary materialThe online version of this article (doi:10.1007/s00535-016-1170-8) contains supplementary material, which is available to authorized users.

Highlights

In the intestine, the epithelial layer covering its inner-most surface functions as the site of substance passage and the active site of immune response [1, 2]
The epithelium consists of different types of intestinal epithelial cells (IECs) that arise from adult stem cells residing at the bottom of the crypt-villus epithelial architecture
We have developed a novel method to culture murine Intraepithelial lymphocytes (IELs) in vitro in which both abT and cdT IELs proliferate efficiently in the presence of epithelial organoids

Summary

Introduction

The epithelial layer covering its inner-most surface functions as the site of substance passage and the active site of immune response [1, 2]. The intestinal epithelial tissue is home to an abundant population of cells of a different lineage, intestinal intraepithelial lymphocytes (IELs). Other abT IELs of the CD4-CD8ab- double negative. Intraepithelial lymphocytes (IELs) in the intestine play important roles in the regulation of local immune responses. Their functions have been studied in a variety of animal experiments, in vitro studies on spatiotemporal behaviors of IELs and their interaction with intestinal epithelial cells (IECs) have been hampered due to the lack of a suitable culture system. We aimed at developing a novel co-culture system of IELs with IECs to investigate dynamic interaction between these two populations of cells in vitro

Methods

Results

Conclusion