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

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.

A novel intestinal organoid-based in vitro co-culture system to dissect out the initial host defense system

“…IELs do not recirculate and that intestinal epithelial space provides the & Atsushi Mizoguchi mizoguchi_atsushi@med.kurume-u.ac.jp final destination of T cells [3,4]. In addition, the authors demonstrate another novel finding that, during the movement, IELs can continuously change their cellular shapes [8]. As compared to the in vivo system, the organoid-based in vitro co-culture system therefore may provide a more useful tool to closely examine the interaction of epithelial cells and IELs at a cellular level.…”

“…IELs are found to migrate into, and then come out from, the organoids [8]. This finding is certainly of interest because it challenges current concepts that TCRcd ?…”

“…Alternatively, the nature of non-transformed epithelial cells that rapidly enter into programmed cell death, a phenomenon known as ''anoikis'' [7], has hampered investigators to utilize the freshly isolated intestinal epithelial cells in a physiological setting for a long-term co-culture with IELs. In this issue of Journal of Gastroenterology, Nozaki et al introduce an organoid-based in vitro co-culture system with murine IELs to overcome the fundamental problems [8]. This novel co-culture system employs three-dimensional organoids in Matrigel composed of stem cell-derived crypt-villus structures without mesenchymal niche [9].…”

Translational applications of adult stem cell-derived organoids

“…Co-culture of organoids with intraepithelial lymphocytes (IELs) (Nozaki et al, 2016;Zumwalde et al, 2016) has opened up the possibility of exploiting organoids to expand immune cells in vitro, which could be used for immunotherapy purposes, for instance by predicting cytotoxicity when cultured together with patient-derived tumor organoids. IELs play important roles in local immune responses in the tissue in which they reside and could potentially be triggered to specifically attack tumor cells.…”

“…IELs play important roles in local immune responses in the tissue in which they reside and could potentially be triggered to specifically attack tumor cells. Nozaki et al established a novel strategy to maintain and expand IELs in a co-culture system with murine small intestinal organoids (Nozaki et al, 2016). Importantly, the authors showed that IEL migration dynamics are comparable to those of IELs residing in an in vivo setting, suggesting that IELs do not lose functional activity in vitro.…”