Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes
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Intraepithelial 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.
We 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.
IELs 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.
This 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.
KeywordsIntraepithelial lymphocytes Intestinal epithelial cells In vitro culture Time-lapse imaging Lymphocyte migration
In the intestine, the epithelial layer covering its inner-most surface functions as not only the site of substance passage but also 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. The intestinal epithelial tissue is also home to an abundant population of cells of a different lineage, intestinal intraepithelial lymphocytes (IELs). For example, IELs are scattered at a density of one IEL per 5–10 IECs in the small intestine , making intimate contact with IECs.
IELs are heterogeneous populations that contain T cell receptor (TCR) αβ T cells (αβT IELs) and TCR γδ T cells (γδT IELs) . Among αβT IELs, CD4 + CD8αβ-CD8αα+, CD4 + CD8αβ-CD8αα−, and CD4-CD8αβ+ subsets are classified as conventional IELs, as they develop through conventional thymic selection and migrate into the intestine . Other αβT IELs of the CD4-CD8αβ− double negative (DN) phenotype (CD4-CD8αβ-CD8αα+ and CD4-CD8αβ-CD8αα- cells) and γδT IELs are classified as unconventional subsets, as they develop and become activated differently from conventional IELs [5, 6]. Although both αβT and γδT IELs are known to play important roles in local immune response [7, 8, 9, 10], functions of IELs in each subset are not fully characterized.
As IELs are highly susceptible to apoptosis when isolated [11, 12, 13], in vitro study of IEL functions has long been hampered. This has been attributed to the lack of some IEC-derived factors important for survival and proliferation of IELs in in vitro culture systems. Indeed, the supply of factors derived from IECs [13, 14] or by direct contact with laboratory-adapted epithelial cell lines [15, 16, 17] was shown to be beneficial for in vitro maintenance of IELs. This supports the idea that IECs that preserve physiological properties would provide a more suitable microenvironment for sustained culture of IELs.
Recent advances have enabled long-term culture of IECs in vitro [18, 19, 20]. For example, with such a technology, IECs of the murine small intestine are now allowed to grow almost in perpetuity as three-dimensional epithelial organoids . IECs expanded by this method are capable of reconstituting normal epithelia when transplanted back into syngeneic mice, indicating that the culture process is not deleterious to maintenance of their original features . Thus, it would be of interest to investigate whether the organoid culture technology could become a novel platform to achieve efficient co-culture systems for IELs. In addition, such co-culture systems could offer the advantages of a physiologically relevant model system to study functional and/or temporal and spatial interaction between these two cell populations.
EGFP transgenic mice , R26-H2B-EGFP mice, and R26-H2B-mCherry mice  of C57BL6 background were bred and maintained in the animal facility of Tokyo Medical and Dental University (TMDU). All animal experiments were performed with the approval of the Institutional Animal Care and Use Committee of TMDU.
Preparation of intestinal organoids
Small intestinal crypts were isolated from 10 to 15-week-old wild-type mice as previously described . The crypts were embedded in Matrigel (BD Biosciences) and Advanced DMEM/F12 containing 500 ng/mL mRspo1 (R&D Systems), 20 ng/mL mEGF (Peprotech), and 100 ng/mL mNoggin (R&D Systems) were added to each well. The medium was changed every 2 days until the following use of cultured organoids.
Isolation of IELs
Intestinal IELs were isolated according to the method described previously . The small intestine of 10–25-week-old mice was incubated in a tube containing 5 mM ethylenediaminetetraacetic acid (EDTA) and 1 mM 1,4-dithio-d-threitol (DTT) in Ca- and Mg-free Hank’s balanced salt solution. After passage of the cell suspension through a nylon mesh filter and a column of glass wool, recovered IELs were collected by Percoll gradient centrifugation. After washing, cells were counted and used immediately for the following assays. When needed, αβT and γδT IELs were sorted using a FACS ARIA II (Becton–Dickinson) by negative selection. Antibodies used were as follows: anti-mouse TCR-β-PE (BioLegend) and anti-mouse TCR-δ-PECy7 (BioLegend). Purity of sorted IEL subsets was analyzed after sorting and was always greater than 99 %.
Co-culture of IELs and IECs
Intestinal organoids were cultured for 2 days prior to the co-culture with IELs. On the day of IEL isolation, cultured organoids were released from Matrigel by incubating whole Matrigel in cell recovery solution (Corning) on ice for 30 min. After washing and counting, organoids and independently isolated IELs were placed together onto a 24-well plate, resulting in 100 organoids and 1.0 × 105 IELs were intermixed with 400 μL of DMEM. After incubation at 37 °C for 30 min, the mixture was gently collected and centrifuged for 1 min at 200g. The pellet was suspended in 30 μL of Matrigel and placed in 24-well plates. After Matrigel polymerization, 500 μL of the same medium as that used for the organoid culture was added to each well. Where indicated, 100 U/mL recombinant human IL-2 (Roche), 10 ng/mL mouse IL-7 (Peprotech) and 10 ng/mL mouse IL-15 (Peprotech) were supplemented to the co-culture. The medium was refreshed every 2 days.
After 7 days of co-culture, IELs could be propagated as follows. Entire cells were released from Matrigel by Cell Recovery Solution as described previously . After centrifugation at 500g for 5 min, supernatants were collected and stored as fraction 1. To facilitate recovery of IELs still incorporated in organoids, the pellet was incubated in 5 mM EDTA/PBS for 10 min on ice. The tube was left for 1 min so that the organoids sedimented under gravity, and the supernatant collected and stored as fraction 2. To recover more IELs, sedimented materials were mechanically disrupted by vigorous pipetting and then passed through a 40 μm nylon mesh filter (fraction 3). Fractions 1–3 were combined and EGFP + IELs were counted by a hemocytometer. In parallel, intestinal crypts were freshly isolated as a counterpart of co-culture. Recovered IELs and isolated organoids were mixed as described previously, and co-cultured.
Immunohistochemistry and fixed cell analysis
For whole-mount analysis, cells were fixed with surrounding Matrigel and permeabilized. Primary antibodies were incubated overnight and secondary antibody reactions were performed for 1.5 h. Nuclei were stained with 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI). Samples were then overlaid with Vectashield Mounting Medium (Vector Laboratories) and microscopically analyzed. Antibodies used were as follows: CD3ε (Becton–Dickinson) and Cdh1 (Santa Cruz Biotechnology). Confocal imaging was performed on a Fluoview FV10i system (Olympus). Images were taken using an Olympus 60× objective (1.35 N.A.) at Z-steps of 0.85 μm. The Z-stacks of images were processed with Adobe Photoshop software when necessary.
IELs were suspended in 0.2 % FCS/PBS. Cells were analyzed on a FACS Canto II (BD Biosciences). Antibodies used for surface marker analysis were as follows: anti-mouse TCR-β-PE (BioLegend) and anti-mouse TCR-δ-PECy7 (BioLegend). For intracellular staining, IELs were fixed, permeabilized with PermI reagents (Becton–Dickinson) and incubated with anti-Ki-67-660eFluor (eBioscience) antibodies.
Time-lapse fluorescent imaging
Time-lapse imaging of the co-culture was performed on a DeltaVision system (Applied Precision) in which a fluorescent microscope IX-71 (Olympus) with an InsightSSI illumination source is incorporated. Before initiation of imaging, cell nuclei were stained with Hoechst 33342 (Nacalai Tesque). Fluorescent images of nuclei, EGFP, and mCherry were acquired through a UplansApo 20x objective (0.75 N.A.) or a UApo340 40x objective (1.34 N.A.) onto a CoolSnap ES2 digital camera (Roper Scientific). Single-plane imaging was performed for 2 h at 20 s intervals. Multi-plane imaging was conducted for 10 min at 20 or 30 s intervals, acquiring Z-stacks at 5 μm steps at a time. SoftWorx software (Applied Precision) was used to obtain maximum intensity projection data of these Z-stacks.
IEL motility analysis
Datasets were imported into Imaris 7.5 (Bitplane) software. Nuclei of IELs were then individually determined by using the spot-tracking feature of Imaris, with a size estimate of 10 µm. The migration of nuclei was analyzed by the “autoregressive motion” tracking algorithm of the software. Tracks were also verified visually to ensure that one track followed the same nucleus for the entire time period. The calculations of the mean speed, maximum speed, track length, and displacement of nuclei were performed using the Imaris software. Data are presented as mean ± S.E.M.
Development of 3D co-culture of IELs and intestinal organoids
As a preliminary experiment to develop a method to maintain IELs with intestinal organoids, we assessed whether the conventional organoid culture allowed concomitantly isolated IELs to remain incorporated. Murine small intestinal crypts were isolated, embedded in Matrigel, and cultured as described previously . The organoids were fixed together with surrounding Matrigel at various time points and immunostained three-dimensionally. This whole-mount analysis revealed that there were no detectable CD3+ cells in any organoids analyzed on day 3 or 7 (data not shown). This showed that the previously reported method of intestinal organoid culture does not allow IELs to be efficiently maintained.
We next assessed whether αβT and γδT IELs were both maintained in culture. Flow cytometry analysis revealed that proportions of these subtypes in the whole population were comparable before and after culture (day 7) (Fig. 2d). αβT and γδT IELs in culture showed higher Ki-67 expression compared to those analyzed before culture, indicating the presence of proliferating cells in both subpopulations (Fig. 2e). Similar data were obtained in IELs propagated for 2 weeks (Suppl. Fig. 1).
Motility analysis of IELs in the co-culture system
Motility parameters of whole IELs, αβΤ IELs, and γδΤ IELs
Mean speed (μm/min)
Max speed (μm/min)
Track length (μm)
Whole IELs (n = 85)
5.07 ± 0.27
9.67 ± 0.60
49.57 ± 2.65
18.74 ± 1.47
0.37 ± 0.02
αβΤ IELs (n = 112)
5.72 ± 0.23
10.69 ± 0.46
56.45 ± 2.33
21.22 ± 1.33
0.37 ± 0.02
γδΤ IELs (n = 154)
5.29 ± 0.19
10.05 ± 0.33
51.96 ± 1.86
17.57 ± 0.95
0.33 ± 0.02
αβΤ IELs (n = 93)
5.32 ± 0.26
10.00 ± 0.49
51.04 ± 2.47
17.05 ± 1.31
0.34 ± 0.02
γδΤ IELs (n = 71)
4.55 ± 0.21
8.96 ± 0.53
43.83 ± 2.02
15.60 ± 1.29
0.34 ± 0.02
We have developed a novel method to culture murine IELs in vitro in which both αβT and γδT IELs proliferate efficiently in the presence of epithelial organoids. Based on the previous observations that IEC-derived factors [13, 14] or direct contact with epithelial cell lines [5, 16, 17] did not allow for efficient expansion of IELs, we assume that non-transformed IEC-derived factors and their three-dimensional presentation to IELs may be important for the maintenance of IELs in our system. Although such niche factors remain to be identified, the culture method presented in this study could serve as a useful tool for the behavioral characterization of IELs.
By combining time-lapse imaging with the culture system, we also showed that IELs move around three-dimensionally with high motility, changing their contact status with epithelial organoids. A previous study assessed motility parameters of γδT IELs using in vivo confocal microscopy and determined the mean and maximum velocities as 3.8 ± 0.1 and 7.7 μm/min, respectively . As quantitative analysis for the motility of both αβT and γδT IELs showed comparable values in our analysis, it was proposed that IELs in this in vitro system are as motile as IELs residing in in vivo settings.
In summary, we have demonstrated a novel co-culture method to maintain and expand murine IELs. Using this in vitro culture system, we also demonstrated the highly dynamic nature of IELs. The method will serve as a unique tool to investigate functions and dynamics of IELs that interact with IECs in vivo.
We thank Lesa Thompson for manuscript editing. This study was supported by MEXT Kakenhi (Grant Number 26112705), JSPS Kakenhi (24390186, 24590936, 26221307), and Health and Labour Sciences Research Grants for research on intractable diseases from Ministry of Health, Labor and Welfare of Japan.
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