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.

To develop stem cell therapy for small intestinal (SI) diseases, it is essential to determine whether SI stem cells in culture retain their tissue regeneration capabilities. By using a heterotopic transplantation approach, we show that cultured murine SI epithelial organoids are able to reconstitute self-renewing epithelia in the colon. When stably integrated, the SI-derived grafts show many features unique only to the SI but distinct from the colonic epithelium. Our study provides evidence that cultured adult SI stem cells could be a source for cell therapy of intestinal diseases, maintaining their identity along the gastrointestinal tract through an epithelium-intrinsic mechanism.

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N(10-) methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7-OH-MTX, DAMPA and 2,4-diaminopteroic acid (DAPA) as an internal standard were separated on a reversed-phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7-OH-MTX and DAMPA from plasma at 100 pmol/mL were 81.5±5.4, 82.5±5.3 and 56.2±7.0%, respectively. The limits of quantification for MTX, 7-OH-MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within- and between-day variations for MTX, 7-OH-MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy.

No abstract

No abstract