JP6662518B2 - Method for maintaining and expanding intestinal interepithelial lymphocytes in vitro - Google Patents

Description

  The present invention relates to a method for maintaining and expanding intestinal interepithelial lymphocytes in vitro, a method for producing intestinal crypt organoids in which intestinal interepithelial lymphocytes are increased, and an intestinal interepithelial lymphocyte produced by the production method. Related to increased intestinal crypt organoids and the like.

  In the gut, the epithelial layer covering the innermost surface functions not only as a site for substance passage, but also as an active site for the immune response that constitutes the forefront of host defense. The epithelium is composed of different types of intestinal epithelial cells (IEC) that arise from adult stem cells located at the bottom of the crypt epithelial structure. Intestinal epithelial tissue also has an intestinal interepithelial lymphocyte (IEL), a rich population of cells of different lineages. IELs are dispersed in the small intestine at a density of one IEL per 5 to 10 IECs, for example, and are in intimate contact with the basal surface of the IECs. IELs are heterogeneous T cells and contain more γd + T cell receptor (TCR) T cells than other peripheral T cell compartments. IELs are classified into subsets of type a and type b based on their surface marker expression, and γδT IELs constitute the major fraction of type b IELs. γδT IEL is known to have an important role in epithelial barrier function (Non-patent Documents 1 and 2) and mucosal homeostasis (Non-patent Document 3). Although αβT IEL still represents a heterogeneous population, it also contributes to host defense mechanisms (Non-Patent Document 4).

  When isolated, IEL is very susceptible to apoptosis (Non-Patent Document 5), and it was only possible to maintain it for about two to three days by the conventional in vitro culture method. In vitro studies have been hindered for a long time. Therefore, development of a method capable of maintaining and growing the IEL in vitro has been attempted. For example, in Non-Patent Document 5 described above, IL-2 or IL-15 was added to a culture solution for culturing the isolated γδ IEL in vitro, and IL-15 was added. It is described that a larger amount of γδ IEL was obtained in the case of performing the above. Further, Non-Patent Document 5 describes that when the isolated γδ IEL was cultured in a culture solution to which IL-15 was added, the γδ IEL grew until day 6 (Non-Patent Document 5). FIG. 5A). However, after 6 days from the culture, the number of living γδ IELs decreased (FIG. 5A of Non-Patent Document 5) and the apoptosis rate (%) further increased (FIG. 5C of Non-Patent Document 5). Thereafter, the number of living γδ IELs is expected to decrease rapidly, so that it was not sufficiently practical. In the method of Non-Patent Document 5, although γδ IEL can be proliferated, αβ IEL cannot be proliferated (FIG. 1B of Non-Patent Document 5). In addition, in an in vivo analysis of IEL migration, it is known that IEL actively migrates between the intestinal epithelium and the like (Non-Patent Document 6). The γδ IEL is not expected to show such active migration. On the other hand, for example, Non-Patent Document 7 discloses a method for maintaining an IEL derived from a mouse by co-culturing the isolated IEL with a mouse keratinocyte cell line. However, according to the method of Non-Patent Document 7, the IEL can be maintained at most up to Day 6, and the IEL cannot be proliferated (see FIG. 4 of Non-Patent Document 7). In Non-Patent Document 7, mouse-derived isolated IEL is co-cultured with a mouse intestinal epithelial cell line, or mouse-derived IEL is cultured in an epithelial cell culture solution containing IL-2, IL-7 and IL-15. However, none of the cultures maintained the IEL for more than 7 days. Thus, the method of Non-Patent Document 7 could not maintain and proliferate the IEL at a practical level.

  On the other hand, recent advances in technology have made possible long-term culture of small and large intestinal epithelial stem cells (Non-Patent Documents 8 to 10 and Patent Documents 1 and 2). For example, with such a technique, the IEC of the mouse small intestine can be grown almost forever as a three-dimensional intestinal organoid that includes a crypt-like ridge area surrounding a central lumen lined by villous compartments ( Non-Patent Document 8). IEC grown by this method was shown to be able to reconstitute normal small intestinal epithelium when transplanted back into syngeneic mice, indicating that such a culture step is detrimental to maintaining the intrinsic characteristics of small intestinal epithelial cells (Non-Patent Document 11).

  As described above, small intestinal crypt organoids and large intestinal crypt organoids were known.However, these intestinal crypt organoids are used for maintaining and proliferating IEL, and for intestinal crypt organoids having increased IEL, , Had not been reported before.

JP 2012-254081 A WO 2013/061608 pamphlet

Boismenu, R., and W.L.Havran. 1994.Modulation of epithelial cell growth by intraepithelial gamma delta T cells.Science 266: 1253-1255. Chen, Y., K. Chou, E. Fuchs, W.L.Havran, and R. Boismenu. 2002.Protection of the intestinal mucosa by intraepithelial gamma delta T cells.Proc Natl Acad Sci USA 99: 14338-14343 Ismail, A.S., C.L.Behrendt, and L.V.Hooper. 2009.Reciprocal interactions between commensal bacteria and gamma delta intraepithelial lymphocytes during mucosal injury.J Immunol 182: 3047-3054 Muller, S., M. Buhler-Jungo, and C. Mueller. 2000.Intestinal intraepithelial lymphocytes exerts potent protective cytotoxic activity during an acute virus infection.J Immunol 164: 1986-1994 Chu, C.L., S.S. Chen, T.S.Wu, S.C. Kuo, and N.S. Liao. 1999.Differential effects of IL-2 and IL-15 on the death and survival of activated TCR gamma delta + intestinal intraepithelial lymphocytes.J Immunol 162: 1896-1903 Edelblum, KL, L. Shen, CR Weber, AM Marchiando, BS Clay, Y. Wang, I. Prinz, B. Malissen, AI Sperling, and JR Turner. 2012. Dynamic migration of γδ intraepithelial lymphocytes requires occludin. Proc Natl Acad Sci USA 109: 7097-7102 Chennupati, Y., T. Worbs, X. Liu, FH Malinarich, S. Schmitz, JD Haas, B. Malissen, R. Forster, and I. Prinz. 2010. Intra- and intercompartmental movement of gammadelta T cells: intestinal intraepithelial and peripheral gammadelta T cells represent exclusive nonoverlapping populations with distinct migration characteristics.J Immunol 185: 5160-5168 Sato, T., RG Vries, HJ Snippert, M. van de Wetering, N. Barker, DE Stange, JH van Es, A. Abo, P. Kujala, PJ Peters, and H. Clevers. 2009. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.Nature 459: 262-265 Sato, T., DE Stange, M. Ferrante, RG Vries, JH Van Es, S. Van den Brink, WJ Van Houdt, A. Pronk, J. Van Gorp, PD Siersema, and H. Clevers. 2011. Long- term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.Gastroenterology 141: 1762-1772 Yui, S., T. Nakamura, T. Sato, Y. Nemoto, T. Mizutani, X. Zheng, S. Ichinose, T. Nagaishi, R. Okamoto, K. Tsuchiya, H. Clevers, and M. Watanabe. 2012.Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5 + stem cell.Nat Med 18: 618-623 Fukuda, M., T. Mizutani, W. Mochizuki, T. Matsumoto, K. Nozaki, Y. Sakamaki, S. Ichinose, Y. Okada, T. Tanaka, M. Watanabe, and T. Nakamura. 2014. Small intestinal stem cell identity is maintained with functional Paneth cells in heterotopically grafted epithelium onto the colon.Gelles Dev 28: 1752-1757

  The present invention relates to a method for maintaining and expanding intestinal interepithelial lymphocytes in vitro, a method for producing intestinal crypt organoids in which intestinal interepithelial lymphocytes are increased, and an intestinal interepithelial lymphocyte produced by the production method. It is intended to provide an increased intestinal crypt organoid and the like.

As described in the background art, the method of maintaining and growing (maintaining and / or growing) an isolated IEL in vitro has been insufficient. The present inventors have conducted intensive research in the background art described above, and as a result, have found the following findings (H) to (L) and completed the present invention.
(H) The isolated IEL is embedded in an extracellular matrix together with the small intestinal crypt organoid, and then co-cultured in a culture medium capable of growing the small intestinal crypt organoid, to obtain IL-2, IL-7, IL-7 IEL can be maintained for a longer period of time as compared with the conventional method not using -15. In such a co-culture, when either of IL-7 and IL-15 is added to the culture solution, IEL is maintained for a longer period of time as compared with the conventional method not using IL-2, IL-7 or IL-15. Be able to maintain or grow the IEL more. In particular, in such a co-culture, when IL-2 (preferably all of IL-2, IL-7 and IL-15) is added to the culture solution, the conventional method using IL-2, IL-7 or IL-15 is used. The ability to maintain the IEL for a longer period of time or to grow the IEL more than in the method.
(I) The co-culturing method of (H) allows the IEL to be maintained and / or expanded while generally maintaining the same diversity (such as the ratio between γδ and αβ) as the IEL used at the beginning of the co-culture. What you can do.
(J) The isolated IEL is embedded in an extracellular matrix together with the colon crypt organoid, and then a culture solution capable of growing the colon crypt organoid (provided that IL-2, IL-7 and IL-15 are ), The degree of increase in IEL is slightly superior to that in the case of co-culture with small intestinal crypt organoids, but it is still an excellent method for culturing and maintaining IEL.
(K) When an imaging analysis was performed on the IEL obtained by the co-culturing method of (H) above, it showed active migration as reported in Non-Patent Document 6 concerning in vivo analysis of IEL migration. . That is, according to the co-culturing method of (H), an IEL exhibiting a kinetics closer to an IEL in vivo than an IEL obtained by a conventional method can be maintained and proliferated.
(L) According to the co-culture method of (H) or (J), an intestinal crypt organoid or an intestinal crypt organoid having an increased IEL can be produced.

That is, the present invention
(1) (A) Step A of embedding the isolated intestinal interepithelial lymphocytes together with intestinal crypt organoid in an extracellular matrix: and (B) in a culture solution capable of growing the intestinal crypt organoid A step of co-culturing intestinal interepithelial lymphocytes and intestinal crypt organoids embedded in the extracellular matrix, wherein the method comprises the steps of:
(2) (A) Embedding the isolated intestinal interepithelial lymphocytes together with the intestinal crypt organoid in an extracellular matrix: A: and (B) in a culture solution capable of proliferating the intestinal crypt organoid And co-culturing the intestinal interepithelial lymphocytes and the intestinal crypt organoid embedded in the extracellular matrix, comprising the steps of: And
(3) The above-mentioned (1) or (2), wherein the culture solution capable of growing intestinal crypt organoids comprises one or more selected from IL-2, IL-7 and IL-15. How and
(4) When the intestinal crypt organoid is a small intestinal crypt organoid, the culture solution capable of growing the small intestinal crypt organoid is a culture solution containing a mitogenic growth factor, a Wnt agonist, and a BMP inhibitor; When the organoid is a colon crypt organoid, it is determined that the culture solution capable of growing the colon crypt organoid is a culture solution containing serum albumin, Wnt3a, and R-spondin 1 and not containing serum. The method according to any one of (1) to (3) above,
(5) mitogenic growth factor is EGF, a Wnt agonist R- spondinl and / or Wnt-3a, Ya method described above SL (4) to BMP inhibitor characterized in that it is a Noggin ,
(6) The culture solution capable of growing colonic crypt organoids further contains one or more selected from the group consisting of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and Noggin The method according to the above ( 4 ) or ( 5) ,
(7) When the intestinal crypt organoid is a small intestinal crypt organoid, the extracellular matrix is Matrigel, and when the intestinal crypt organoid is a large intestinal crypt organoid, the extracellular matrix is collagen. The method according to any one of the above (1) to (6),
(8) An intestinal crypt organoid produced by the production method according to any one of (2) to (7) and having increased intestinal interepithelial lymphocytes.

  According to the present invention, intestinal interepithelial lymphocytes can be maintained and expanded in vitro. The method of the present invention has a longer IEL compared to the conventional method using no L-2, IL-7 or IL-15 or the conventional method using L-2, IL-7 or IL-15. It can be maintained or the IEL can grow more. Further, according to the present invention, not only γδ IEL but also αβ IEL can be grown. Further, according to the present invention, an intestinal crypt organoid having an increased IEL can be produced. Such intestinal crypt organoids can be used in a method for evaluating the effect on intestinal interepithelial lymphocyte motility. According to the present invention, it is possible to maintain and proliferate an IEL exhibiting a kinetics closer to that of an IEL in vivo than maintaining and proliferating by a conventional in vitro culture method.

FIG. 1A shows three z-axis slice fluorescence images of Cdh1 (cadherin) and CD3 (interepithelial lymphocytes [IEL; Intraepithelial Lymphocytes]) on day 1 after culture of the small intestinal crypt organoid. The scale bar indicates 50 μm. The upper part of FIG. 1B shows three z-axis slice fluorescence images of CD3 (IEL) and DAPI (cell nucleus) on day 3 after co-culture of the IEL with the intestinal crypt organoid, and the lower part of FIG. 3 shows three z-axis slice fluorescence images of EGFP (IEL) and DAPI (cell nuclei) on day 3 after co-culture with the small intestinal crypt organoid. Note that the upper panel in FIG. 1B and the lower panel immediately below it differ only in the fluorescent signal to be acquired in the same z-axis slice image of the same sample. The arrowheads in the fluorescent images in FIGS. 1A and 1B indicate IELs incorporated in the small intestinal crypt organoids, and the arrows in the fluorescent images in FIG. 1B indicate IELs present outside the small intestinal crypt organoids. The scale bar indicates 50 μm. In the upper part of FIG. 2A, the IEL was compared with the intestinal crypt organoid in the presence of IL-2, IL-7, and IL-15, and then EGFP (IEL) on days 1, 3, and 7 after co-culture. 2A shows an image obtained by superimposing the fluorescence image in the upper part of FIG. 2A and the corresponding phase difference image (PC) in the middle part of FIG. 2A, and the lower part of FIG. 7 shows an enlarged image of a region surrounded by a dotted line in the middle image. 2B, z-axis slice fluorescence of CD3 (IEL) and DAPI (cell nuclei) on day 7 after co-culture of IEL with intestinal crypt organoid in the presence of IL-2, IL-7, and IL-15 Images are shown, and the lower part of FIG. 2B shows that EEL (EEL) and DAPI (cell nuclei) on day 7 after co-culture with intestinal crypt organoid in the presence of IL-2, IL-7, and IL-15. 3 shows three z-axis sliced fluorescent images. Note that the upper panel in FIG. 2B and the lower panel immediately below it differ only in the fluorescent signal to be acquired in the same z-axis slice image of the same sample. In addition, the upper left of FIG. 2C shows a fluorescence image of EGFP (IEL) 14 days after co-culture of IEL with intestinal crypt organoid in the presence of IL-2, IL-7, and IL-15. 2 shows an enlarged image of the area surrounded by the dotted line in the upper left diagram, the lower left of FIG. 2C shows an image obtained by superimposing the phase difference image (PC) on the upper left diagram, and the lower right of FIG. 3 shows an enlarged image of a region surrounded by a dotted line in the lower left diagram. FIG. 3A shows the results of measuring the number of IELs on the 1st, 7th, and 14th days after co-culture of IEL with small intestinal crypt organoid in the presence of IL-2, IL-7, and IL-15. FIG. Bar graphs are expressed as mean ± standard deviation (SEM). Figure 3B were measured ratio of TCRarufabeta ⁺ and TCRganmaderuta ⁺ IEL population in 1 day after intestinal crypts organoid cocultured and 7 days in IL-2, IL-7, and IL-15 the presence of IEL It is a figure showing a result. Figure 3C, for TCRarufabeta ⁺ and TCRγδ ⁺ IEL population of the IL-2, IL-7, and 1 days after intestinal crypts organoid cocultured with IL-15 the presence and 7 days IEL, Ki-67 FIG. 4 shows the results of analyzing the expression level of. Figure 3D is a diagram showing a result of measuring the ratio of TCRarufabeta ⁺ and TCRganmaderuta ⁺ IEL population in 14 days after intestinal crypts organoid cocultured with IL-2, IL-7, and IL-15 the presence of IEL It is. Figure 3E, IL-2 and IEL, IL-7, and the TCRarufabeta ⁺ and TCRγδ ⁺ IEL population in 14 days after intestinal crypts organoid cocultured with IL-15 in the presence, expression levels of Ki-67 It is a figure showing the result of analysis. Figure 4A shows IEL counts on day 1 and day 7 after co-culture with small intestinal crypt organoids in the presence of IL-2, IL-7 or IL-15, or in the presence of these three ILs. It is a figure which shows the result of having measured. Bar graphs are expressed as mean ± standard deviation (SEM). FIG. 4B is a diagram showing the results of measuring the number of IELs on day 1 and day 7 after co-culture with colonic crypt organoids in the presence of IL-2, IL-7, and IL-15. Bar graphs are expressed as mean ± standard deviation (SEM). FIG. 4 shows the results of a motion analysis of IEL co-cultured with small intestinal crypt organoids. FIG. 5A shows the results of analysis of IEL derived from an EGFP-tg mouse and small intestinal crypt organoid derived from a wild-type mouse by time-lapse fluorescent imaging on the third day after co-culture. Representative IEL images showing active movement along the basal side of epithelial cell monolayers (FIG. 5A, top) and IEL images showing migration into and out of small intestinal crypt organoids (FIG. 5A, bottom) . The scale bar indicates 10 μm. FIG. 5B shows the results of analysis by time-lapse fluorescence imaging on day 3 after co-culture of IEL derived from R26-H2B-EGFP mouse and small intestinal crypt organoid derived from wild-type mouse. An image of the IEL migrating along the epithelial cell monolayer (FIG. 5B, top) and an image of the IEL showing migration into and out of the small intestinal crypt organoid (FIG. 5B, bottom). The scale bar indicates 10 μm. The numerical display at the lower right of each fluorescent image in FIGS. 5A and 5B indicates the elapsed time from the start of imaging. FIG. 5C shows the results of IEL motility analysis on day 3 after co-culture of R26-H2B-EGFP mouse-derived IEL and wild-type mouse-derived small intestinal crypt organoid. In the initial (left upper figure in FIG. 5C) and last (upper right figure in FIG. 5C) still images of a representative data set, the cell nuclei of IEL (small white spheres) and their trajectories are shown as 4D images. The enlarged image of the area surrounded by the dotted line is shown in the lower part. The scale bar indicates 50 μm. FIG. 5D shows that TCRαβ ⁺ and TCRγδ ⁺ IEL populations were independently isolated from H2B-EGFP reporter mice and pretreated with 5 μM PD184352 3 days after co-culture with small intestinal crypt organoids from wild-type mice. The results of IEL kinetic analysis are shown (Fig. 5D upper row) or without pretreatment (Fig. 5D lower row). The scale bar indicates 10 μm. FIG. 5E shows that TCRαβ ⁺ and TCRγδ ⁺ IEL populations were independently isolated from H2B-EGFP reporter mice and phosphorylated in the IEL on day 3 after co-culture with intestinal crypt organoids from wild-type mice. The result of having analyzed the expression of ERK protein (phospho-ERK) by flow cytometry is shown.

<1. Method for maintaining and expanding intestinal interepithelial lymphocytes in vitro>
The “method of maintaining and expanding intestinal interepithelial lymphocytes in vitro” of the present invention (hereinafter, also simply referred to as “maintenance / proliferation method of the present invention”) includes:
(A) Embedding the isolated intestinal interepithelial lymphocytes together with intestinal crypt organoids in an extracellular matrix A:
(B) Step B of co-culturing intestinal interepithelial lymphocytes and intestinal crypt organoid embedded in the extracellular matrix in a culture solution capable of growing the intestinal crypt organoid:
Is not particularly limited as long as it includes.

(Isolated intestinal interepithelial lymphocytes)
The “isolated intestinal interepithelial lymphocyte” in the above step A is not particularly limited as long as it is a mammalian intestinal interepithelial lymphocyte and is an isolated intestinal interepithelial lymphocyte. It may be intestinal interepithelial lymphocytes directly isolated from the intestinal epithelium of an animal, or may be intestinal interepithelial lymphocytes obtained by maintaining or expanding such intestinal interepithelial lymphocytes. Further, the term "isolated intestinal interepithelial lymphocyte" in the present invention means that it is not an intact living tissue containing intestinal interepithelial lymphocyte, and is composed of only intestinal interepithelial lymphocyte. And a cell mixture in which intestinal interepithelial lymphocytes are more concentrated than in a living tissue. As such a cell mixture, the content ratio of intestinal interepithelial lymphocytes (the ratio of the number of cells) is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 80% or more. Things can be exemplified.

  The “intestinal interepithelial lymphocytes” in the present specification include small intestinal interepithelial lymphocytes and large intestinal interepithelial lymphocytes, and preferably small intestinal interepithelial lymphocytes. In the present specification, "mammals" preferably include humans, monkeys, mice, rats, hamsters, guinea pigs, cows, pigs, horses, rabbits, sheep, goats, cats, dogs, and the like, with humans being more preferable. It is listed.

Intestinal interepithelial lymphocytes can be isolated from a large intestine tissue or a small intestine tissue of a mammal by a known method (for example, Kohyama et al., Proc Natl Acad Sci USA 1999, 96: 7451-7455). More specifically, the following isolation method can be mentioned.
The intestinal tissue collected from the mammal is washed away with a buffer such as PBS, the Peyer's patch is removed, and cut into thin (eg, about 5 mm) sections. Thereafter, 2-8 mM (preferably 5 mM) ethylene-dinitrilotetraacetic acid (EDTA) and 0.3-2 mM (preferably 1 mM) 1,4-dithio-D- are added to a Hanks balanced salt solution containing no Ca or Mg. Incubate in a container containing threitol (DTT) at 20-40 ° C (preferably 37 ° C) for 10 minutes to 1 hour (preferably 30 minutes) at 50-250 rpm (preferably 150 rpm) with shaking. Next, the contents in the container are passed through a nylon mesh filter having an aperture of 100 to 500 μm (preferably 300 μm) and a glass wool column, and the collected cells are suspended in 20 to 40% (preferably 30%) Percoll. It becomes turbid and centrifuged at 600-1000 g (preferably 800 g) for 10-30 minutes (preferably 20 minutes). IEL can be isolated by centrifugation at 800 g, 40/70% Percoll gradient for 30 minutes. The isolated IEL may be used for assays after washing and cell counting.

The number of “isolated intestinal interepithelial lymphocytes” and the number of “intestinal crypt organoids” used in the above-mentioned step A include the “maintenance / proliferation method of the present invention” and the “production method of the present invention” described later. The number of the above-mentioned “isolated intestinal interepithelial lymphocytes” is, for example, in the range of 1.0 × 10 ² to 1.0 × 10 ⁸ , The number is preferably in the range of × 10 ³ to 1.0 × 10 ⁷ , in the range of 1.0 × 10 ⁴ to 1.0 × 10 ⁶ , and as the number of “intestinal crypt organoids” described above. Is preferably in the range of 10 to 10000, in the range of 50 to 200, or in the range of 70 to 150. The ratio of the number of “isolated intestinal interepithelial lymphocytes” to the number of “intestinal crypt organoids” used in the above step A is defined as “the maintenance / proliferation method of the present invention” or “ The IEL is not particularly limited as long as it can be used in the “production method of the present invention”, but the IEL is in the range of 100 to 100,000, 100 to 10,000, and 500 to 2000 per intestinal crypt organoid. Within the range.

(Intestinal crypt organoid)
The “intestinal crypt organoid” in the above step A is not particularly limited as long as it is a mammalian intestinal crypt organoid. As used herein, the term “intestinal crypt organoid” refers to a tissue structure having a central lumen whose inner surface is covered by intestinal epithelium, and a plurality of crypt-like sites projecting to the outer surface. Specific examples of the “intestinal crypt organoid” in the present specification include small intestinal crypt organoids and large intestinal crypt organoids, and preferably include small intestinal crypt organoids.

  The intestinal crypt organoid can be prepared by a known method. A method for producing a small intestinal crypt organoid is described in detail, for example, in Patent Document 1 (Japanese Patent Application Laid-Open No. 2012-254081), and a method for producing a large intestinal crypt organoid is described, for example, in Patent Document 2 (International Publication No. WO 2013/061608 pamphlet). ) Is described in detail.

  As described above, the method for producing the small intestinal crypt organoid is described in detail in, for example, Patent Document 1, but will be briefly described below. Small intestinal tissue including crypts is collected from the small intestine of the mammal using forceps or the like. Crypts can be isolated from such small intestinal tissue according to known methods. For example, it is possible to isolate crypts by incubating chelators (which release cells from their calcium and magnesium dependent interactions with basement membrane and stromal cell types) and harvested small intestinal tissue. it can. After washing the small intestine tissue, the epithelial cell layer is peeled off from the submucosal layer with a glass slide and cut into small pieces. This is followed by incubation in trypsin or, more preferably, EDTA and / or EGTA, and the undigested small intestinal tissue fragments and single cells from the crypts, for example using a filtration and / or centrifugation step. To separate. Instead of trypsin, other proteolytic enzymes such as collagenase and / or dispase I can be used.

  The cell population containing multiple types of single cells derived from the small intestine crypt obtained by separation contains small intestinal epithelial stem cells. By culturing such a cell population, a small intestinal crypt organoid can be produced.From the viewpoint of more efficiently producing a small intestinal crypt organoid, it is possible to culture the small intestinal epithelial stem cells isolated from the aforementioned cell population. preferable. Such small intestinal epithelial stem cells can be isolated using Lgr5 and / Lgr6, which are markers of adult intestinal stem cells, as indices.

  The small intestinal epithelial stem cells or a cell population containing the small intestinal epithelial stem cells is preferably embedded in an extracellular matrix and cultured three-dimensionally. As the extracellular matrix, the same as the extracellular matrix used in the case of using intestinal interepithelial lymphocytes as intestinal interepithelial lymphocytes and intestinal crypt organoids as intestinal crypt organoids in step A described below. Preferred are mentioned. In addition, as a method for embedding in an extracellular matrix, a method similar to the embedding method in step A described below can be used.

  As the culture solution for producing the small intestinal crypt organoid, a “culture solution capable of proliferating the small intestinal crypt organoid” among the culture solutions used in step B described below is preferable. However, the culture solution for producing the small intestinal crypt organoid may contain the “further optional component” described below, but may not contain it, and is preferably not contained from the viewpoint of production cost. .

  Culture conditions for producing the small intestinal crypt organoid are not particularly limited as long as the small intestinal crypt organoid can be produced, but the culture temperature is, for example, in the range of 30 to 40 ° C, preferably in the range of 36 to 38 ° C. , More preferably 37 ° C. The culture period is, for example, 4 to 12 days, preferably 5 to 10 days, and more preferably 6 to 7 days. The exchange of the culture solution is preferably performed at an appropriate time. The frequency of exchanging the culture solution is not particularly limited as long as the small intestinal crypt organoid can be prepared, and may be, for example, every 1 to 5 days, preferably every 2 to 3 days.

  On the other hand, as described above, a method for producing a colonic crypt organoid is described in detail in, for example, Patent Document 2, but will be briefly described below. From the large intestine of a mammal, large intestine tissue including crypts is collected using forceps or the like. Crypts can be isolated from such large intestine tissue according to known methods. For example, it is possible to isolate crypts by incubating the harvested colon tissue with chelators (releasing cells from their calcium and magnesium dependent interactions with basement membrane and stromal cell types). it can. After washing the colon tissue, the epithelial cell layer is detached from the submucosal layer with a glass slide and cut into small pieces. This is followed by incubation in trypsin or, more preferably, EDTA and / or EGTA to separate undigested colon tissue fragments and crypt-derived single cells using, for example, a filtration and / or centrifugation step. To separate. Instead of trypsin, other proteolytic enzymes such as collagenase and / or dispase I can be used.

  The cell population containing multiple single cells derived from colon crypts obtained by separation contains colon epithelial stem cells. By culturing such a cell population, colonic crypt organoids can be produced, but from the viewpoint of more efficiently producing colonic crypt organoids, it is possible to culture colonic epithelial stem cells isolated from the aforementioned cell population. preferable. Such colonic epithelial stem cells can be isolated using Lgr5 and / Lgr6, which are markers for intestinal adult stem cells, as an indicator.

  The colonic epithelial stem cells or a cell population containing the colonic epithelial stem cells is preferably embedded in an extracellular matrix and cultured three-dimensionally. Examples of such extracellular matrix include those similar to the extracellular matrix used in the case of using colonic epithelial lymphocytes as intestinal interepithelial lymphocytes and colonic crypt organoids as intestinal crypt organoids in step A described below. Preferred are mentioned. In addition, as a method for embedding in an extracellular matrix, a method similar to the embedding method in step A described below can be used.

  As the culture solution for preparing the colonic crypt organoid, a “culture solution capable of proliferating the colonic crypt organoid” out of the culture solution used in Step B described below is preferable. However, the culture solution for preparing the colonic crypt organoid may contain one, two, or three selected from IL-2, IL-7, and IL-15 described below. However, from the viewpoint of manufacturing cost, it is preferable not to include it.

  The culture conditions for preparing the colonic crypt organoid are not particularly limited as long as the colonic crypt organoid can be prepared, but the culture temperature is, for example, in the range of 30 to 40 ° C, preferably in the range of 36 to 38 ° C. , More preferably 37 ° C. The culture period is, for example, 4 to 12 days, preferably 5 to 10 days, and more preferably 6 to 7 days. The exchange of the culture solution is preferably performed at an appropriate time. The exchange frequency of the culture solution is not particularly limited as long as the colon crypt organoid can be produced, and may be, for example, every 1 to 5 days, preferably every 2 to 3 days.

  The intestinal crypt organoid used in the present invention may be an intestinal crypt organoid obtained by primary culture, or obtained by subculturing cells collected from a primary culture (eg, intestinal crypt organoid). It may be an intestinal crypt organoid, or may be an intestinal crypt organoid obtained by subculturing cells collected from a subcultured culture (eg, intestinal crypt organoid). The passage time and frequency are not particularly limited as long as intestinal crypt organoids can be produced, but can be appropriately performed at a timing when the population of intestinal epithelial stem cells or the like (preferably, intestinal crypt organoids) has increased, For example, after 4 to 12 days or every time, preferably after 6 to 10 days or every time. The passaging method is not particularly limited as long as the intestinal epithelial stem cells and the like can be maintained or expanded, and a method of coexisting intestinal epithelial stem cells and the like (for example, intestinal crypts) separated from the extracellular matrix with a new extracellular matrix Intestinal epithelium obtained by separating a semi-solidified extracellular matrix (preferably Matrigel or collagen) by decomposing it with a degrading enzyme (preferably collagenase or the like) or depolymerizing it by cooling to separate it A preferred example is a method of embedding stem cells and the like (for example, intestinal crypts) in a new extracellular matrix.

(Step A in the present invention)
The step A in the present invention is not particularly limited as long as it is a step of embedding the isolated intestinal interepithelial lymphocytes together with the intestinal crypt organoid in an extracellular matrix, and for example, in a culture vessel such as a well. Preferred is a method of polymerizing extracellular matrix while coexisting with isolated intestinal interepithelial lymphocytes, intestinal crypt organoid and extracellular matrix. A more preferred method is to put a mixture of fossil organoids into a culture vessel such as a well, and then add an extracellular matrix to the culture vessel to polymerize the extracellular matrix.

  In a preferred embodiment, before the above step A, the isolated intestinal interepithelial lymphocytes and the intestinal crypt organoid are cultured in a culture solution capable of growing the intestinal crypt organoid for 10 minutes (preferably, a DMEM culture solution). It is preferable to culture for ２2 hours, preferably for 20 minutes to 1 hour.

(Step B in the present invention)
Step B in the present invention is not particularly limited as long as it is a step of co-culturing “intestinal interepithelial lymphocytes and intestinal crypt organoid embedded in extracellular matrix” in a culture solution capable of growing intestinal crypt organoids. Not done.

  The culturing conditions for the co-culture in step B of the present invention are not particularly limited as long as they can be used for the “maintenance / proliferation method of the present invention” and the “production method of the present invention” described below. A range of 40 ° C, preferably a range of 36 to 38 ° C, and more preferably 37 ° C can be suitably exemplified. The culture period is not particularly limited, but may be, for example, 4 to 14 days. In the case of growing intestinal interepithelial lymphocytes, from the viewpoint of obtaining more intestinal interepithelial lymphocytes, 7 to 14 days are preferable. And more preferably 8 to 13 days. In addition, as described below, when producing an intestinal crypt organoid having increased intestinal interepithelial lymphocytes, the culture period of step B is preferably 3 to 7 days, more preferably 5 to 7 days.

  In the co-culture in the step B of the present invention, it is preferable that the culture solution is changed at an appropriate time. The frequency of exchanging the culture solution is not particularly limited as long as it can be used for the “maintenance / proliferation method of the present invention” or the “production method of the present invention” described below. For example, every 1 to 5 days, preferably 2 to 3 days It can be every day.

  In the co-culture in the step B of the present invention, the intestinal crypt organoid may not be replaced, but from the viewpoint of maintaining or increasing the intestinal interepithelial lymphocytes for a longer period, From the viewpoint of obtaining a more increased intestinal crypt organoid, it is preferable to replace at an appropriate time. The exchange frequency of the intestinal crypt organoid is not particularly limited as long as it can be used in the “maintenance / proliferation method of the present invention” or the “production method of the present invention” described below. For example, every 5 to 8 days, preferably 6 to It can be every 7 days.

  The method of exchanging the intestinal crypt organoid is not particularly limited.For example, after isolating the IEL in the co-culture, the IEL is embedded in an extracellular matrix together with the separately cultured intestinal crypt organoid, A method of continuing co-culture in a culture solution capable of growing intestinal crypt organoids may be mentioned. In addition, as a method of isolating the IEL in the co-culture, the following method can be mentioned. After removing `` intestinal interepithelial lymphocytes and intestinal crypt organoids embedded in extracellular matrix '' from the culture solution during co-culture, the extracellular matrix is cooled and depolymerized or decomposed with collagenase, etc. Separate interepithelial lymphocytes and intestinal crypt organoids. After centrifugation, the supernatant fraction (fraction 1) is collected. The precipitate is incubated in a buffer solution on ice, allowed to stand for a while, and then the supernatant fraction (fraction 2) is collected. Further, the precipitate is destroyed by vigorous pipetting or the like, and the fraction passed through a mesh filter (for example, openings of 40 μm) is referred to as fraction 3. Fractions 1 to 3 are all suspensions containing IEL. Any of fractions 1 to 3 may be used, but by combining fractions 1 to 3, IEL can be more efficiently isolated from the co-culture.

(Culture solution of step B)
The culture solution in the step B of the present invention (hereinafter, also simply referred to as “the culture solution in the step B”) is not particularly limited as long as it is a culture solution capable of growing intestinal crypt organoids. The culture solution in step B may or may not contain serum, but preferably does not contain serum. According to the present invention, IEL can be maintained for a long period of time or IEL can be proliferated more without using serum.

  The culture solution of step B contains the following essential components in addition to the basic culture solution components (carbon sources such as sugars, nitrogen sources such as amino acids, and inorganic salts). When the intestinal crypt organoid is a small intestinal crypt organoid, such essential components are mitogenic growth factors, Wnt agonists, and BMP inhibitors (hereinafter, these three components are collectively referred to simply as “small intestinal essential three components”). ), And when the intestinal crypt organoid is a colonic crypt organoid, serum albumin, Wnt3a, and R-spondin 1 (hereinafter, these three components are collectively referred to as simply “the three essential components of the large intestine”) ).

  Examples of the basic culture solution component include carbon sources such as saccharides that can be assimilated by intestinal interepithelial lymphocytes and intestinal crypt organoids and amino acids that can be digested by such intestinal interepithelial lymphocytes and intestinal crypt organoids. Preferable examples include a nitrogen source and an inorganic salt. As the basic culture solution components, those known to those skilled in the art and commercially available ones (for example, those manufactured by Invitrogen) can be used. Specifically, MEM (Minimum Essential Medium), BME (Basal Medium Eagle) ), DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's minimal essential medium), IMDM (Iscove's Modified Dulbecco's Medium), GMEM (Glass-gow's MEM), F12 (Ham's F12 Medium), DMEM / F12, RPMI-3640, BMOC-3 (Brinster's BMOC-3 Medium), CMRL-1066, L-15 (Leibovitz's L-15 medium), McCoy's 5A, Media 199, MEM αMedia, MCDB105, MCDB131, MCDB153, MCDB201, Williams' medium E, Advanced MEM, Advanced DMEM, Advanced DMEM / F- 2, Advanced RPMI1640, it is possible to use the culture solution, such as. In the present invention, Advanced DMEM / F-12 (manufactured by Invitrogen) and the like can be preferably exemplified. Glutamine is a preferred example of the amino acid.

(3 essential components of small intestine)
When the intestinal interepithelial lymphocytes (ie, small intestinal interepithelial lymphocytes or large intestinal interepithelial lymphocytes, preferably small intestinal interepithelial lymphocytes) and small intestinal crypt organoids are co-cultured, the culture solution of step B comprises small intestinal crypt organoids Is a culture solution that can proliferate. The culture solution capable of growing the small intestinal crypt organoid contains three components of the small intestine essential (mitogenic growth factor, Wnt agonist and BMP inhibitor).

  The mitogenic growth factor, which is one of the three essential components of the small intestine, includes epidermal growth factor (EGF), transforming growth factor-α (TGF-α), fibroblast growth factor (FGF), and One or more mitogenic growth factors selected from the group consisting of brain-derived neurotrophic factor (BDNF) are preferred, among which EGF and FGF are more preferred, and EGF is most preferred. . In addition, as the FGF, one or more selected from FGF2 (bFGF), FGF7 (also called keratinocyte growth factor (KGF)) and FGF10 are preferable, and FGF7 and / or FGF10 are more preferable. Can be Preferred combinations of mitogenic growth factors include “EGF alone”, “EGF and FGF7”, and “EGF and FGF10”.

  The concentration of the mitogenic growth factor in the culture solution of step B in the case of co-culturing intestinal interepithelial lymphocytes and small intestinal crypt organoids includes the "method of maintaining and growing the present invention" and the "method of producing the present invention" There is no particular limitation as long as it can be used for EGF, but in the case of EGF, for example, within the range of 5 to 500 ng / mL, preferably within the range of 10 to 250 ng / mL, more preferably within the range of 20 to 125 ng / mL, and still more preferably. Is in the range of 30 to 70 ng / mL. Similar concentrations can be applied to FGF (preferably FGF10 or FGF7). When a plurality of types of FGF are used, the aforementioned concentration range of FGF indicates the total concentration of FGF to be used. During the culture in the step B, it is preferable to add a mitogen to the culture solution every 1 to 3 days (preferably every 2 days).

  The Wnt agonist, one of the three essential components of the small intestine, refers to a substance that activates TCF / LEF-mediated transcription in cells. The Wnt signaling pathway begins with the Wnt protein binding to a cell surface receptor of the Frizzled receptor family, causing the receptor to release a Dishevelled family protein. Dishevelled family proteins usually suppress a complex of molecules including Axin, which promotes β-catenin signaling molecule, glycogen synthase kinase 3 (GSK-3) and APC (Adenomatous polyposis coli) proteins. When this complex is suppressed, β-catenin is stabilized and can enter the nucleus, and the β-catenin that has entered the nucleus promotes transcription by TCF / LEF family transcription factors. Therefore, as the Wnt agonist, a substance having an action of binding to and activating a Frizzled receptor family member including all kinds of Wnt family proteins (a Wnt agonist in a narrow sense), an inhibitor of intracellular β-catenin degradation, And one or more Wnt agonists selected from the group consisting of TCF / LEF activators. The extent to which the Wnt agonist enhances Wnt activity in the cell is not particularly limited, but the level of Wnt activity in the presence of the Wnt agonist is proportional to the level of Wnt activity in the absence of the Wnt agonist. 10% or more, preferably 20% or more, more preferably 30% or more, further preferably 50% or more, more preferably 70% or more, still more preferably 90% or more, and most preferably 100% or more. Are preferred. The Wnt activity can be measured by a known method, for example, by measuring the transcriptional activity of Wnt using a pTOPFLASH and pFOPFLASH Tcf luciferase reporter construct (Korinek et al., 1997. Science 275: 1784- 1787).

  Wnt-1 / Int-1; Wnt-2 / Irp (Int-1 related protein); Wnt-2b / 13, Wnt-3 / Int-4; Wnt-3a (for example, R & D systems) Wnt-4; Wnt-5a; Wnt-5b; Wnt-6 (Kirikoshi H et al. 2001. Biochem Biophys Res Com 283: 798-805); Wnt-7a (R & D systems); Wnt-7b Wnt-8a / 8d; Wnt-8b; Wnt-9a / 14; Wnt-9b / 14b / 15; Wnt-10a; Wnt-10b / 12; 1 selected from the group consisting of Wnt-11 and Wnt-16. Species or two or more Wnt proteins. Wnt proteins are secreted glycoproteins. An overview of human Wnt proteins is provided in "THE WNT FAMILY OF SECRETED PROTEINS", R & D Systems Catalog, 2004). In addition, Wnt agonists are members of the R-Spondin family of secreted proteins (which are involved in the activation and regulation of the Wnt signaling pathway and four members (R-spondin 1 (NU206, Nuvelo, San Carlos). , CA), R-spondin2 (consisting of (R & D systems), R-spondin3 and R-spondin-4)) and binds with high affinity to the Frizzled-4 receptor and binds to the Wnt signaling pathway. Including Norin (also called Norrin, Norrie Disease Protein or NDP) (R & D systems), a secretory regulatory protein that functions like the Wnt protein in inducing activation (Kestutis Planutis et al.) al. (2007) BMC Cell Biol. 8:12) A small molecule agonist of the Wnt signaling pathway, an aminopyrimidine derivative, has recently been identified and is also Expressly included in the agonist (Liu et al. (2005) Angew Chem Int Ed Engl.44,1987-90).

  Known substances having GSK-3 inhibitory activity include small interfering RNA (siRNA, Cell Signaling), lithium (manufactured by Sigma), kenpaulon (Biomol International; Leost, M et al. (2000) Eur J Biochem. 267). , 5983-5994), 6-bromoindirubin-3′-acetoxime (Meijer, L et al. (2003) Chem Biol. 10, 1255-1266), SB216763 and SB415286 (manufactured by Sigma-Aldrich), and “ FRAT-derived peptides that block the interaction of GSK-3 with FRAT-family members and axin. A summary is provided by Meijer et al. (2004) Trends in Pharmacological Sciences 25,471-480. Methods and assays for determining the level of GSK-3 inhibitory activity are known to those of skill in the art and include, for example, those described in Liao et al. 2004, Endocrinology, 145 (6): 2941-9). No.

  In a preferred embodiment, the Wnt agonist includes one or more selected from the group consisting of Wnt family proteins, R-spondins 1-4, norin and GSK-3 inhibitors. In a further preferred embodiment, the Wnt agonist comprises or consists of R-spondin1. The concentration of R-spondin 1 in the culture solution of step B in the case of co-culturing intestinal interepithelial lymphocytes and small intestinal crypt organoids includes the “maintaining / proliferating method of the present invention” and the “production method of the present invention” described later. There is no particular limitation as long as it can be used for, for example, within the range of 50 to 5000 ng / mL, preferably within the range of 100 to 2500 ng / mL, more preferably within the range of 200 to 1250 ng / mL, and still more preferably 300 to 850 ng. / Ml, more preferably from 400 to 625 ng / mL, still more preferably from 450 to 550 ng / mL, most preferably 500 ng / mL. During the culture in the step B, the Wnt family protein is preferably added to the culture medium every two days, and the culture medium is preferably replaced with fresh one every four days.

  In a preferred embodiment, the Wnt agonist is one or more selected from the group consisting of R-spondin (preferably R-spondin 1), Wnt-3a and Wnt-6, and more preferably R-spondin ( Preferably, both R-spondin 1) and Wnt-3a are used. This combination is particularly preferred because it surprisingly has a synergistic effect on organoid formation. Preferred concentrations of R-spondin (preferably R-spondin 1) in the culture solution of step B when co-culturing intestinal interepithelial lymphocytes and small intestinal crypt organoids include the “maintenance / proliferation method of the present invention”, There is no particular limitation as long as it can be used in the “production method of the present invention” described below, but for example, in the range of 50 to 5000 ng / mL, preferably in the range of 100 to 2500 ng / mL, and more preferably in the range of 200 to 1250 ng / mL. Of these, more preferably within the range of 300 to 850 ng / mL, more preferably within the range of 400 to 625 ng / mL, even more preferably within the range of 450 to 550 ng / mL, and most preferably 500 ng / mL. The concentration of Wnt-3a in the culture solution of step B is preferably in the range of 10 to 1000 ng / mL, preferably in the range of 20 to 500 ng / mL, more preferably in the range of 40 to 250 ng / mL, and furthermore It is preferably in the range of 60 to 170 ng / mL, more preferably in the range of 80 to 125 ng / mL, still more preferably in the range of 90 to 110 ng / mL, and most preferably 100 ng / mL.

  The BMP inhibitor, one of the three essential components of the small intestine, binds as a dimeric ligand to a receptor complex consisting of two different receptor serine / threonine kinases, type I and type II receptors. . The type II receptor phosphorylates the type I receptor, which activates this receptor kinase. This type I receptor then phosphorylates a specific receptor substrate (SMAD), which leads to transcriptional activity through signaling pathways.

  BMP inhibitors include substances that block or inhibit the binding of a BMP molecule to a BMP receptor by binding to a BMP molecule to form a complex, and BMPs of a BMP molecule by binding to a BMP receptor. Substances that block or inhibit binding to the receptor are included. The latter inhibitors include BMP receptor antagonists and inverse agonists, and more specifically, antibodies that block or inhibit the binding of BMP molecules to the BMP receptor by binding to the BMP receptor. No.

  The degree to which the BMP inhibitor inhibits BMP activity in a cell is not particularly limited, but the level of BMP activity in the presence of the BMP inhibitor is the level of BMP activity in the absence of the BMP inhibitor. To 90% or less, preferably 80% or less, more preferably 70% or less, further preferably 50% or less, more preferably 30% or less, still more preferably 10% or less, and most preferably 0% or less. Is preferred. The BMP activity can be measured by a known method. For example, as described in Zilberberg et al., 2007. BMC Cell Biol. 8:41, the BMP activity can be measured. You can find out.

  BMP inhibitors include Noggin (Peprotech), chordin, chordin-like protein containing chordin domain (R & D systems), follistatin, and follistatin-related protein containing follistatin domain (R & D). systems), DAN protein, DAN-like protein containing DAN cysteine-knot domain (manufactured by R & D systems), sclerostin / SOST (manufactured by R & D systems), decorin (R & D systems), and One or more selected from the group consisting of α-2 macroglobulin (manufactured by R & D systems), among which are Noggin, DAN protein, and DAN-like protein (Cerberus and Gremlin) (Including R & D systems)) or one or more selected from the group consisting of Gerare, noggin may be mentioned more preferably. These BMP inhibitors can inhibit the binding of the BMP ligand to the signal transducer receptor by binding to the BMP ligand.

  As the concentration of the BMP inhibitor in the culture solution of step B when co-culturing intestinal interepithelial lymphocytes and small intestinal crypt organoids, the “maintenance / proliferation method of the present invention” and the “production method of the present invention” described later There is no particular limitation as long as it can be used, but when the BMP inhibitor is noggin, for example, in the range of 1 ng / mL to 10 μg / mL, preferably in the range of 10 to 1000 ng / mL, more preferably 30 to 300 ng / ML, more preferably in the range of 50 to 200 ng / mL, more preferably in the range of 70 to 130 ng / mL. During the culture in the step B, a BMP inhibitor (preferably noggin) is added to the culture solution preferably every two days, and the culture solution is preferably replaced with fresh one every four days.

(3 essential components of large intestine)
When co-culturing intestinal interepithelial lymphocytes (ie, small intestinal interepithelial lymphocytes or colon intestinal interepithelial lymphocytes, and preferably intestinal interepithelial lymphocytes), the culture solution of step B comprises colonic crypt organoids Is a culture solution that can proliferate. The culture solution capable of growing colonic crypt organoids contains three essential components of the large intestine (serum albumin, Wnt3a, and R-spondin 1).

  The above-mentioned serum albumin, which is one of the three essential components of the large intestine, is not particularly limited as long as it is albumin contained in the serum of mammals. Examples of such serum albumin include those purified from mammalian serum and those chemically purified. Alternatively, any of biochemically synthesized products and commercially available products (for example, BSA manufactured by Sigma) may be used. The origin of serum albumin is not particularly limited as long as it is a mammal, but human serum albumin (human serum albumin; HSA) and bovine serum albumin (BSA) can be preferably exemplified, and BSA is more preferably used. Examples can be given. It is also preferable to use serum albumin derived from a mammal of the same species as the mammalian species from which the intestinal interepithelial lymphocytes and colon crypt organoids to be cultured are derived.

  The concentration of serum albumin in the culture solution of step B in the case of co-culturing intestinal interepithelial lymphocytes and colon crypt organoids includes the "maintaining / proliferating method of the present invention" and the "production method of the present invention" described later. There is no particular limitation as long as it can be used. For example, in the culture solution of the above-described step B, the upper limit is 30% by weight or less (eg, 20% by weight or less, 10% by weight or less, 5% by weight or less), and the lower limit. Is 0.01% by weight or more (for example, 0.05% by weight or more, 0.1% by weight or more, 0.3% or more). As a more preferable concentration, a range of 0.3% or more and 3% or less can be mentioned.

  The above-mentioned Wnt3a, which is one of the three essential components of the large intestine, is not particularly limited as long as it is a protein encoded by the mammalian WNT3A gene, and such Wnt3a is produced by Wnt3-producing cells such as Paneth cells. May be used, those produced by the gene-expressing cells prepared by genetic engineering, those synthesized chemically or biochemically, and those commercially available (for example, manufactured by R & D Systems, Inc.) Wnt3a) may be used. The origin of Wnt3a is not particularly limited as long as it is a mammal, but human Wnt3a (hWnt3a) and mouse Wnt3a (mWnt3a) can be preferably exemplified, and among them, mWnt3a can be more preferably exemplified. It is also preferable to use Wnt3a, which is the same as the mammalian species from which the intestinal interepithelial lymphocytes or colonic crypt organoids are derived.

  The concentration of Wnt3a in the culture solution of step B when co-culturing intestinal interepithelial lymphocytes and colon crypt organoids is used in the “maintenance / proliferation method of the present invention” or the “production method of the present invention” described later. Although not particularly limited as long as it can be obtained, for example, in the culture solution of the above-mentioned step B, the upper limit is 3 mg / mL or less (for example, 500 μg / mL or less, 100 μg / mL or less, 30 μg / mL or less, 3 μg / mL or less, 1 μg / mL or less). mL or less), and the lower limit is 300 pg / mL or more (eg, 1 ng / mL or more, 3 ng / mL or more, 10 ng / mL or more, 30 ng / mL or more). A more preferred final concentration is 30 ng / mL or more.

  The above-mentioned R-spondin 1, which is one of the three essential components of the large intestine, is not particularly limited as long as it is a protein encoded by the mammalian RSPO1 gene. Cells produced by Rspo1-producing cells such as expression cells may be used, or those chemically or biochemically synthesized or commercially available (for example, R-Spondin1 manufactured by R & D Systems) may be used. Good. The origin of Rspo1 is not particularly limited as long as it is a mammal, but human Rspo1 (hRspo1) and mouse Rspo1 (mRspo1) can be preferably exemplified, and among them, mRspo1 can be more preferably exemplified. It is also preferable to use R-spondin 1 derived from a mammal of the same species as the mammalian species from which the intestinal interepithelial lymphocytes or colon crypt organoids to be cultured are derived.

  The concentration of R-spondin 1 in the culture solution of step B in the case of co-culturing intestinal interepithelial lymphocytes and colon crypt organoids includes the “maintaining / proliferating method of the present invention” and the “production method of the present invention” described later. Is not particularly limited as long as it can be used, for example, in the culture solution of the above-mentioned step B, as an upper limit, 5 mg / mL or less (for example, 1 mg / mL or less, 200 μg / mL or less, 40 μg / mL or less, 5 μg / mL or less) , 1 μg / mL or less), and the lower limit is 500 pg / mL or more (eg, 5 ng / mL or more, 50 ng / mL or more, 250 ng / mL or more).

(3 components of large intestine)
When co-culturing intestinal interepithelial lymphocytes (ie, small intestinal interepithelial lymphocytes or large intestinal interepithelial lymphocytes, preferably small intestinal interepithelial lymphocytes) and colonic crypt organoids, the culture solution of step B comprises a basic culture solution component And three essential components of the large intestine, but in addition to those components, selected from the group consisting of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and noggin (Noggin) It can be suitably exemplified that the composition further contains one, preferably two, and more preferably three components (hereinafter, these three components are also referred to as “any three components of the large intestine”).

  The above-mentioned EGF, which is one of the three components of the large intestine, is not particularly limited as long as it is a protein encoded by a mammalian EGF gene. Any of those produced by EGF-producing cells, such as those produced chemically or biochemically, and those commercially available (for example, EGF manufactured by Peprotech) may be used. Although the origin of EGF is not particularly limited as long as it is a mammal, human EGF (hEGF) and mouse EGF (mEGF) can be preferably exemplified, and among them, mEGF can be more preferably exemplified. It is also preferable to use EGF derived from a mammal of the same species as that of the mammal from which the intestinal interepithelial lymphocytes or colon crypt organoid to be cultured are derived.

  The concentration of EGF in the culture solution of step B in the case of co-culturing intestinal interepithelial lymphocytes and colon crypt organoids is used in the “maintenance / proliferation method of the present invention” and the “production method of the present invention” described below. There is no particular limitation as long as it is obtained. For example, in the culture solution of the above-mentioned step B, as an upper limit, 200 μg / mL or less (for example, 40 μg / mL or less, 8 μg / mL or less, 2 μg / mL or less, 200 ng / mL or less, 40 ng / mL or less) mL or less), and the lower limit is 1 pg / mL or more (for example, 8 pg / mL or more, 100 pg / mL or more, 1 ng / mL or more, 10 ng / mL or more). Further, as a more preferable concentration of EGF, a range of 1 ng / mL to 40 ng / mL is exemplified.

  The above-mentioned HGF, which is one of the three components of the large intestine, is not particularly limited as long as it is a protein encoded by a mammalian HGF gene. Any of those produced by HGF-producing cells, such as those synthesized chemically or biochemically, and those commercially available (for example, HGF manufactured by R & D Systems) may be used. Although the origin of HGF is not particularly limited as long as it is a mammal, human HGF (hHGF) and mouse HGF (mHGF) can be preferably exemplified, and among them, mHGF can be more preferably exemplified. It is also preferable to use HGF derived from a mammal of the same species as the mammal from which the intestinal interepithelial lymphocytes or colon crypt organoids to be cultured are derived.

  The concentration of HGF in the culture solution of step B when co-culturing intestinal interepithelial lymphocytes and colon crypt organoids is used in the “maintenance / proliferation method of the present invention” or the “production method of the present invention” described later. Although not particularly limited as long as it can be obtained, for example, in the culture solution of the above-mentioned step B, the upper limit is 500 μg / mL or less (for example, 100 μg / mL or less, 20 μg / mL or less, 4 μg / mL or less, 500 ng / mL or less, 100 ng / mL or less). mL or less), and the lower limit is 50 pg / mL or more (for example, 500 pg / mL or more, 5 ng / mL or more, 25 ng / mL or more). A more preferred concentration of HGF is in the range of 5 ng / mL to 100 ng / mL.

  The above-mentioned noggin, which is one of the three components of the large intestine, is not particularly limited as long as it is a protein encoded by a mammalian NOG gene. Any of those produced by Noggin-producing cells, such as those produced chemically or biochemically, and those commercially available (for example, Noggin manufactured by R & D Systems) may be used. The origin of Noggin is not particularly limited as long as it is a mammal, but human Noggin (hNoggin) and mouse Noggin (mNoggin) can be preferably exemplified, and mouse Noggin can be more preferably exemplified. It is also preferable to use Noggin derived from a mammal of the same species as the mammalian species from which the intestinal interepithelial lymphocytes or colonic crypt organoids to be cultured are derived.

  The concentration of noggin in the culture solution of step B in the case of co-culturing intestinal interepithelial lymphocytes and colon crypt organoids is used in the “maintenance / proliferation method of the present invention” and the “production method of the present invention” described later. Although not particularly limited as long as it can be obtained, for example, in the culture solution of the above-mentioned step B, the upper limit is 500 μg / mL or less (for example, 100 μg / mL or less, 20 μg / mL or less, 4 μg / mL or less, 500 ng / mL or less, 100 ng / mL or less). mL or less), and the lower limit is 50 pg / mL or more (for example, 500 pg / mL or more, 5 ng / mL or more, 25 ng / mL or more). A more preferable concentration of Noggin is in the range of 5 ng / mL to 100 ng / mL.

(Further optional ingredients)
The culture solution of step B may be used for co-culturing intestinal interepithelial lymphocytes (ie, small intestinal interepithelial lymphocytes or large intestinal interepithelial lymphocytes, preferably small intestinal interepithelial lymphocytes) and small intestinal crypt organoids. Even when co-culturing intestinal interepithelial lymphocytes (ie, colonic epithelial lymphocytes or small intestinal interepithelial lymphocytes, preferably colonic epithelial lymphocytes) and colonic crypt organoids, no additional optional components are included. However, from the viewpoint of maintaining or increasing intestinal interepithelial lymphocytes for a longer period of time, or from the viewpoint of obtaining intestinal crypt organoids in which intestinal interepithelial lymphocytes are more increased, an additional optional component may be included. Is preferred. Further optional components include one or more selected from IL-2, IL-7, IL-15, a Rho kinase inhibitor, a Notch agonist, and an antibiotic, among which IL-2, IL- 7 or IL-15, preferably one or more (preferably three), among which "IL-2", "IL-2 and IL-7", "IL-2 and IL" Any one selected from "-15" and "IL-2, IL-7 and IL-15" is more preferable, and "IL-2, IL-7 and IL-15" is most preferable. In particular, IL-2 is extremely superior in maintaining or increasing intestinal interepithelial lymphocytes for a longer period of time and in increasing intestinal interepithelial lymphocytes in intestinal crypt organoids. It is particularly preferable that the culture solution of B contains at least IL-2 as an optional component. In addition, the above-mentioned Rho kinase inhibitor and the above-mentioned Notch agonist are preferable in terms of improving cell survival rate and proliferation efficiency.

  The above-mentioned IL-2 (interleukin 2) is not particularly limited as long as it is mammalian IL-2, and IL-similar to the mammalian species from which intestinal interepithelial lymphocytes and intestinal crypt organoids are derived are used. 2 is preferred. As the mammalian IL-2, commercially available IL-2 (for example, manufactured by Roche) may be used, or the nucleotide sequence or amino acid of the IL-2 gene disclosed in a sequence database such as GenBank. IL-2 chemically synthesized based on the sequence may be used, or IL-2 produced based on the above-described sequence by using a genetic recombination technique may be used.

  The concentration of IL-2 in the culture solution in step B is not particularly limited as long as it can be used in the “maintenance / proliferation method of the present invention” or the “production method of the present invention” described below. / U, preferably in the range of 10-1000 U / mL, more preferably in the range of 30-300 U / mL, even more preferably in the range of 50-200 U / mL, more preferably 70-130 U / mL. Within the range.

  The above-mentioned IL-7 (interleukin 7) is not particularly limited as long as it is mammalian IL-7, and IL-similar to the mammalian species from which intestinal interepithelial lymphocytes and intestinal crypt organoids are derived 7 is preferred. As the mammalian IL-7, commercially available IL-7 (for example, manufactured by Peprotech) may be used, or the nucleotide sequence or amino acid of the IL-7 gene disclosed in a sequence database such as GenBank. IL-7 chemically synthesized based on the sequence may be used, or IL-7 produced based on the above-described sequence using a gene recombination technique may be used.

  The concentration of IL-7 in the culture solution of step B is not particularly limited as long as it can be used in the “maintenance / proliferation method of the present invention” or the “production method of the present invention” described below. For example, 0.1 to 1000 ng / ML, preferably in the range of 1 to 100 ng / mL, more preferably in the range of 3 to 30 ng / mL, still more preferably in the range of 5 to 20 ng / mL, more preferably in the range of 7 to 13 ng / mL. Within the range.

  The above-mentioned IL-15 (interleukin 15) is not particularly limited as long as it is mammalian IL-15. However, IL-15 of the same species as the mammalian species from which intestinal interepithelial lymphocytes and intestinal crypt organoids are derived is used. 15 is preferred. As the mammalian IL-15, commercially available IL-15 (for example, manufactured by Peprotech) may be used, or the nucleotide sequence or amino acid of the IL-15 gene disclosed in a sequence database such as GenBank. IL-15 chemically synthesized on the basis of the sequence may be used, or IL-15 produced on the basis of the above-mentioned sequence using a gene recombination technique may be used.

  The concentration of IL-15 in the culture solution of step B is not particularly limited as long as it can be used in the “maintenance / proliferation method of the present invention” or the “production method of the present invention” described below. For example, 0.1 to 1000 ng / ML, preferably in the range of 1 to 100 ng / mL, more preferably in the range of 3 to 30 ng / mL, still more preferably in the range of 5 to 20 ng / mL, more preferably in the range of 7 to 13 ng / mL. Within the range.

  When a culture solution containing one or more (preferably three) selected from IL-2, IL-7, and IL-15 is used as the culture solution in step B, the interleukin may be 1 to 3 It is preferable to add to the culture solution every day (preferably every two days).

  The Rho kinase inhibitor, one of the further optional components, includes Y-27632 ((R)-(+)-trans-4- (1-aminoethyl) -N- (4 -Pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate) and H 1152 dihydrochloride ((S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl) sulfonyl]) manufactured by Tocris Bioschience -hexahydro-1H-1,4-diazepine dihydrochloride), among which Y-27632 is preferable. The concentration of the Rho kinase inhibitor in the culture solution of step B is not particularly limited as long as it can be used in the “maintenance / proliferation method of the present invention” and the “production method of the present invention” described below. In the culture solution of 2 to 50 μM, preferably in the range of 5 to 20 μM.

  Examples of the above Notch agonist, which is one of the further optional components, include Jagged 1 protein, Delta 1 protein, DSL peptide (Dontu et al., 2004, Breast Cancer Res 6: R605-R615), and among others, DSL Peptides are preferred. The concentration of the Notch agonist in the culture solution of step B is not particularly limited as long as it can be used in the “maintenance / proliferation method of the present invention” or the “production method of the present invention” described below. For the solution, the range is 10 to 100 μM.

  The above-mentioned antibiotics, which are one of the further optional components, include penicillin, streptomycin, gentamicin and the like.

  When a culture solution containing one or more selected from Rho kinase inhibitors, Notch agonists, and antibiotics is used as the culture solution in step B, the culture solution is used every 1 to 3 days (preferably 2 days). Is preferably added to the culture solution.

  As a method for preparing the culture solution in the step B, in addition to the basic culture solution components and the essential three components (small intestine essential components or large intestine essential three components), if necessary, the large intestine optional three components and / or further optional components Ingredients may be mixed with a liquid solvent such as water or PBS, or a culture solution containing a basic culture solution component may contain, in addition to the above-mentioned essential three components (small intestine essential component or large intestine essential three components), if necessary, A method of blending three optional components of the large intestine and / or further optional components can be exemplified.

(Isolation of IEL from co-culture)
The maintenance / proliferation method of the present invention may not further include a step of isolating IEL from the co-culture obtained in step B, but preferably includes this step. Methods for isolating IEL from co-culture include the following methods. After removing `` intestinal interepithelial lymphocytes and intestinal crypt organoids embedded in extracellular matrix '' from the culture solution during co-culture, the extracellular matrix is cooled and depolymerized or decomposed with collagenase, etc. Separate interepithelial lymphocytes and intestinal crypt organoids. After centrifugation, the supernatant fraction (fraction 1) is collected. The precipitate is incubated in a buffer solution on ice, allowed to stand for a while, and then the supernatant fraction (fraction 2) is collected. Further, the precipitate is destroyed by vigorous pipetting or the like, and the fraction passed through a mesh filter (for example, openings of 40 μm) is referred to as fraction 3. Fractions 1 to 3 are all suspensions containing IEL. Any of fractions 1 to 3 may be used, but by combining fractions 1 to 3, IEL can be more efficiently isolated from the co-culture.

(Intestinal interepithelial lymphocytes obtained by the maintenance / proliferation method of the present invention)
“Intestinal interepithelial lymphocytes” (small intestinal interepithelial lymphocytes or large intestinal interepithelial lymphocytes) obtained by the maintenance / proliferation method of the present invention can be used in the epithelium of the small intestine or large intestine of mammals or in the epithelium and the lamina propria It is considered that the administration in the middle of the intestine can improve the barrier function of the intestinal epithelium, the homeostasis of the intestinal mucosa, and the host (mammalian) defense mechanism in the intestine. The improvement of the barrier function of the intestinal epithelium and the improvement of homeostasis of the intestinal mucosa can be achieved mainly by administration of γδT IEL among the intestinal interepithelial lymphocytes. This can be achieved by administration of αβT IEL.

<2. Method for producing intestinal crypt organoid with increased intestinal interepithelial lymphocytes>
The “method for producing intestinal crypt organoid with increased intestinal interepithelial lymphocytes” (hereinafter also simply referred to as “the production method of the present invention”) of the present invention includes:
(A) Embedding the isolated intestinal interepithelial lymphocytes together with intestinal crypt organoids in an extracellular matrix A:
(B) Step B of co-culturing intestinal interepithelial lymphocytes and intestinal crypt organoid embedded in the extracellular matrix in a culture solution capable of growing the intestinal crypt organoid:
Is not particularly limited as long as it includes.

  The steps A and B in the production method of the present invention are the same as the above steps A and B in the above-described method of maintaining and growing the present invention, respectively, unless otherwise specified. According to the production method of the present invention, “intestinal crypt organoid with increased intestinal interepithelial lymphocytes” can be produced in vitro.

  The production method of the present invention may not further include a step of isolating “intestinal crypt organoid with increased intestinal interepithelial lymphocytes” from the co-culture obtained in step B. It is preferable to include. The method for isolating “intestinal crypt organoids with increased intestinal interepithelial lymphocytes” from the co-culture is not particularly limited, but the “intestinal interepithelial lymphocytes embedded in the extracellular matrix and After removing the “fossa organoid” from the culture solution, the extracellular matrix may be cooled, depolymerized or decomposed with collagenase or the like, and the interepithelial lymphocytes and the intestinal crypt organoid may be fractionated.

<3. Intestinal crypt organoid with increased interepithelial lymphocytes>
The “intestinal crypt organoid with increased interepithelial lymphocytes” (hereinafter, also simply referred to as “intestinal crypt organoid of the present invention”) of the present invention includes “intestinal epithelium produced by the production method of the present invention”. It is not particularly limited as long as it is an intestinal crypt organoid with increased interlymphocytes. As used herein, "intestinal crypt organoid with increased intestinal interepithelial lymphocytes" refers to intestinal crypt produced by the same method as the production method of the present invention except that isolated intestinal interepithelial lymphocytes are not used. It means that intestinal interepithelial lymphocytes are increased intestinal crypt organoids as compared to crypt organoids. Specifically, as the “intestinal crypt organoid with increased intestinal interepithelial lymphocytes”, the number of intestinal interepithelial lymphocytes per intestinal crypt organoid on the third day from the start of the culture in step B is defined as The number of intestinal interepithelial lymphocytes per intestinal crypt organoid when not co-cultured with interepithelial lymphocytes is preferably 8 times or more, more preferably 16 times or more, and still more preferably 32 times or more in proportion to the number of intestinal interepithelial lymphocytes per one intestinal crypt organoid. Intestinal crypt organoids, which are increasing in number, can be suitably mentioned. The upper limit of these ratios is not particularly limited, but may be, for example, 200 times or less, 100 times or less. The term “intestinal crypt organoid in which intestinal interepithelial lymphocytes are increased” in the present specification includes the number of intestinal interepithelial lymphocytes per intestinal crypt organoid on the third day from the start of the culture in step B. Preferably, the intestinal crypt organoid is preferably 2 or more, more preferably 4 or more, and still more preferably 6 or more. In addition, the upper limit of the number of intestinal interepithelial lymphocytes per intestinal crypt organoid is not particularly limited, and may be, for example, 36 or less, 18 or less.

  The number of intestinal interepithelial lymphocytes in the intestinal crypt organoids can be measured, for example, by immunohistochemical staining using a fluorescently labeled anti-lymphocyte antibody. Detection of the labeled fluorescence can be performed using a commercially available fluorescence imaging device.

  In addition, the “intestinal crypt organoid with increased intestinal interepithelial lymphocytes” produced by the production method of the present invention is used to prevent intestinal diseases used in transplantation into the intestine (small intestine or large intestine) of mammals (preferably humans). -It can be used as a therapeutic agent (prophylactic and / or therapeutic agent). The “prophylactic / therapeutic agent for intestinal disease” of the present invention (hereinafter, also simply referred to as “the prophylactic / therapeutic agent of the present invention”) includes “intestinal interepithelial lymphocyte increased by the production method of the present invention”. The intestinal crypt organoid is not particularly limited.

  Specific examples of such intestinal diseases include inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, intestinal injury due to chemotherapy, radiation enteritis, and the like. When such a prophylactic / therapeutic agent of the present invention is administered, “intestinal (small or large intestine) crypt organoids with increased interepithelial lymphocytes” engraft in the intestinal (small or large intestine) epithelium, (Small or large intestine) Epithelial injury can be prevented and treated.

  As used herein, “preventive / therapeutic effect of intestinal disease” refers to a preventive effect of an intestinal (small or large intestine) disease, a therapeutic effect of an intestinal (small intestine or large intestine) disease, or Including preventive and therapeutic effects, preventive effects on intestinal (small or large intestine) diseases include the effects of preventing the onset of intestinal (small or large intestine) diseases and the effects of delaying the onset of intestinal (small or large intestine) diseases The therapeutic effect of intestinal (small or large intestine) disease includes an effect of improving symptoms of intestinal (small or large intestine) disease and an effect of preventing or delaying deterioration of intestinal (small or large intestine) disease. . The “intestinal crypt organoid with increased interepithelial lymphocytes” in the “prophylactic / therapeutic agent for intestinal disease” of the present invention is different from a normal intestinal crypt organoid in that intestinal interepithelial lymphocytes are increased. Therefore, as compared with the case of using a normal intestinal crypt organoid (for example, a normal colon crypt organoid in Patent Document 2), the intestinal epithelial barrier function, intestinal mucosal homeostasis, and intestinal host ( (Mammals) It is considered that one or more (preferably three) selected from defense mechanisms can be improved.

  The subject to which the prophylactic / therapeutic agent of the present invention is administered is not particularly limited as long as it is a mammal, and humans can be more preferably exemplified. The mammalian species from which the “intestinal crypt organoid with increased intestinal interepithelial lymphocytes” contained in the prophylactic / therapeutic agent of the present invention is preferably the same as the mammalian species to be administered.

The administration method of the prophylactic / therapeutic agent of the present invention is not particularly limited as long as it can prevent and treat intestinal diseases, and examples thereof include enteral administration, intravenous administration, and the like. Anal enema administration can be suitably exemplified. The dose of the prophylactic / therapeutic agent of the present invention depends on the type of intestinal disease, degree of symptoms, body weight, age of the administration subject, etc., but 1 × 10 to 1 × 10 ¹⁰ / kg cells (1 to ¹⁰ May be administered in divided doses).

  The method for preparing the prophylactic / therapeutic agent of the present invention is not particularly limited, and a pharmaceutically acceptable normal to the "intestinal crypt organoid with increased intestinal interepithelial lymphocytes" produced by the production method of the present invention. Carriers, excipients, diluents, pH buffers, water-soluble solvents such as physiological saline, isotonic agents such as sodium chloride, glycerin, D-mannitol, stabilizers such as human serum albumin, methylparaben, etc. And a method of blending various compounding ingredients such as preservatives and local narcotics such as benzyl alcohol. In addition, the prophylactic / therapeutic agent of the present invention contains an extracellular matrix component such as various types I to VIII collagen and Matrigel from the viewpoint of improving the engraftment efficiency to the intestinal (small or large intestine) epithelium to be administered. More preferably, Matrigel is blended. In addition, in addition to the “intestinal crypt organoid with increased intestinal interepithelial lymphocytes” obtained by the production method of the present invention, a prophylactic / therapeutic agent of the present invention is used in combination with other preventive / therapeutic agents for intestinal diseases. You can also.

<4. Method for assessing effect on intestinal interepithelial lymphocyte motility>
The “method of evaluating the effect of intestinal interepithelial lymphocytes on motility” (hereinafter, also simply referred to as “evaluation method of the present invention”) of the present invention includes:
(P) a step P of bringing a test substance into contact with “intestinal crypt organoid with increased interepithelial lymphocytes” (preferably small intestinal crypt organoid) produced by the production method of the present invention:
(Q) analyzing the motility of the intestinal interepithelial lymphocytes in the intestinal crypt organoid after step P:
(R) comparing the motility obtained in the analysis of step Q with the motility of intestinal interepithelial lymphocytes in intestinal crypt organoids without contact with the test substance:
(S) As a result of the comparison in Step R, if the motility obtained in the analysis in Step Q is higher, the test substance is a substance having an activity of improving the motility of intestinal interepithelial lymphocytes. If the motility obtained in the analysis of step Q is lower, the test substance is evaluated as a substance having an activity of reducing motility of intestinal interepithelial lymphocytes S:
Is not particularly limited as long as it includes.

  The step P is not particularly limited as long as it is a step of bringing a test substance into contact with the “intestinal crypt organoid having increased intestinal interepithelial lymphocytes” produced by the production method of the present invention. There is a method of adding a test substance to a culture solution for culturing the fossa organoid.

  The step Q is not particularly limited as long as it is a step of analyzing the motility of the intestinal interepithelial lymphocytes in the intestinal crypt organoid after the step P. There is a method of analyzing the motility of a sphere.

  The step R is particularly limited as long as the motility obtained in the analysis of the step Q is compared with the motility of intestinal interepithelial lymphocytes in the intestinal crypt organoid in the absence of the test substance. Not done.

  In the step S, as a result of the comparison in the step R, when the motility obtained in the analysis in the step Q is higher, the test substance has an activity of improving the motility of intestinal interepithelial lymphocytes. If the motility obtained in the analysis in step Q is lower, the test substance is evaluated as a substance having an activity to reduce motility of intestinal interepithelial lymphocytes. There is no particular limitation as long as it is a step.

  A substance having an activity to improve the motility of intestinal interepithelial lymphocytes can be evaluated as, for example, a substance that may activate a host defense mechanism in the intestine. A substance having an activity of reducing the activity can be evaluated, for example, as a substance that may inactivate the host defense mechanism in the intestine. In addition, the evaluation method of the present invention relates to a dynamic interaction between intestinal epithelium and lymphocytes between intestinal epithelia (interaction between intestinal epithelium and lymphocytes between intestinal epithelia, and It can also be used to analyze interactions that affect motility.

(Other aspects of the present invention)
The present invention provides "a method for preventing and / or treating intestinal disease, comprising administering to a mammal an intestinal crypt organoid having increased intestinal interepithelial lymphocytes", "preventing and / or treating intestinal disease." Includes "intestinal crypt organoids with increased intestinal interepithelial lymphocytes for treatment", "use of intestinal crypt organoids with increased intestinal interepithelial lymphocytes in the manufacture of a prophylactic and / or therapeutic agent for intestinal disease" It is.

  Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. In the following examples, the day when the culture was started in vitro is defined as the first day after the culture.

1. Materials and Methods 1-1 Experimental mice EGFP recombinant C57BL6 mice (EGFP-tg mice) in which EGFP is constantly expressed under the control of the chicken β-actin promoter and cytomegalovirus enhancer (Okabe et al., FEBS Lett) 1997, 407: 313-319. ") Was provided by Osaka University. In addition, an R26-H2B-EGFP mouse into which a gene encoding an EGFP protein fused to the C-terminal side of histone H2B in the ROSA26 locus has been inserted (see "Abe et al., Genesis 2011, 49: 579-590.") Was provided by RIKEN (Accession No. CDB0203K). C57BL6 background mice and wild type C57BL6 mice were bred and maintained at an animal facility at Tokyo Medical and Dental University (TMDU). All animal experiments were performed with the approval of the Animal Experiment Committee of Tokyo Medical and Dental University.

1-2 Preparation of Small Intestinal Crypt Organoid Small intestinal crypts were isolated from wild-type C57BL6 mice aged 10 to 15 weeks according to the method described in Non-Patent Document 9, and R-spo1 (R-spondin 1), EGF and Noggin were used. Cultured in the presence of That is, about 300 small intestinal crypts were suspended in 30 μL Matrigel (manufactured by BD Biosciences), placed in a 24-well plate, and polymerized at room temperature, and then 500 ng / mL mRspo1 (manufactured by R & D Systems), 20 ng / ML mEGF (manufactured by Peprotech) and 500 μL Advanced DMEM / F12 culture medium (manufactured by Life Technologies) containing 100 ng / mL mNoggin (manufactured by R & D Systems) (hereinafter referred to as “culture medium for small intestinal crypt organoid culture”) .) Was added. The culture solution was replaced with a new culture solution every two days until immediately before use in the following experiments.

1-3 Isolation of IEL from small intestine IEL from small intestine was isolated according to the method described in the literature (Kohyama et al., Proc Natl Acad Sci USA 1999, 96: 7451-7455). That is, the small intestine of an EGFP-tg mouse or an R26-H2B-EGFP mouse of 10 to 15 weeks of age was washed away with PBS, the Peyer's patch was removed, cut into 5 mm sections, and then Hanks equilibrium containing no Ca or Mg. Incubate in a 50 mL conical tube containing 5 mM ethylene-dinitrilotetraacetic acid (EDTA) and 1 mM 1,4-dithio-D-threitol (DTT) in the saline at 37 ° C. for 30 minutes with shaking at 150 rpm; After passing through a 300 μm nylon mesh filter and a glass wool column, the recovered cells were suspended in 30% Percoll and centrifuged at 800 g for 20 minutes. IELs were isolated by centrifugation at 800 g, 40/70% Percoll gradient for 30 minutes. The isolated IEL was used for the assay after washing and cell counting. In addition, 5 to 10 × 10 ⁶ surviving IELs were obtained per mouse. Further, the IEL population of TCRαβ ^+, TCRγδ ⁺ IEL population is (manufactured BioLegend Co.) isolated total IEL anti-mouse TCR-beta-PE antibody, anti-mouse TCR-δ-PECy7 antibody (BioLegend Co.) and It was prepared by staining with an anti-mouse CD3ε-APC antibody (Becton Dickinson) and sorting by FACS ARIAII (Becton Dickinson) (purity> 99%).

1-4 Co-culture of small intestinal crypt organoid and IEL The small intestinal crypt organoid prepared according to the method described in the section “1-2 Preparation of small intestinal crypt organoid” above was cultured for 2 days, and then Cell Recovery Solution (Corning Was added, and the whole matrigel was depolymerized by incubating on ice for 30 minutes. The small intestinal crypt organoid was recovered from Matrigel, washed, and then 100 small intestinal crypt organoids and the above “1-3 small intestine 1.0 × 10 ⁵ IELs isolated according to the method described in “Isolation of IELs of Origin” were mixed in a 400 μL DMEM culture solution on a 24-well plate. After incubating at 37 ° C. for 30 minutes, the mixture was collected, centrifuged at 200 g for 1 minute, and the precipitate (pellet) was suspended in 30 μL of Matrigel and transferred to a 24-well plate. After polymerizing Matrigel at room temperature, 500 μL of a culture solution for intestinal crypt organoid culture, or 100 U / mL recombinant human IL-2 (Roche), 10 ng / mL mouse IL-7 (Peprotech) and 10 ng / mL Small intestinal crypt organoid culture medium containing mL mouse IL-15 (manufactured by Peprotech) (hereinafter, referred to as “+ IL-2 / IL-7 / IL-15 small intestinal crypt organoid culture medium”) 500 μL was added to the wells, and the cells were cultured at 37 ° C. under 5% CO ₂ for 7 days. The culture solution was replaced with a new culture solution every two days.

1-5 Isolation of IEL from co-culture of small intestinal crypt organoid and IEL According to the method described in the section “1-4 Co-culture of small intestinal crypt organoid and IEL” above, After co-culture, Cell Recovery Solution (manufactured by Corning) was added, and the whole matrigel was depolymerized by incubating on ice for 30 minutes. After recovering small intestinal crypt organoids from Matrigel, the cells were collected at 500 g for 5 minutes. After centrifugation, the supernatant fraction (fraction 1) was collected. Further, in order to recover the IEL contained in the precipitate fraction, the precipitate was incubated in 5 mM EDTA / PBS on ice for 10 minutes, and allowed to stand for 1 minute to deposit the precipitate. The fraction (fraction 2) was collected. Further, in order to collect the IEL contained in the precipitate fraction obtained thereafter, the precipitate was mechanically broken by vigorous pipetting, and then the precipitate passed through a 40 μm nylon mesh filter was fractionated. It was set to 3. The combination of fractions 1 to 3 was used as an IEL suspension. The culture of the IEL after the 7th day was performed by counting the number of EGFP-positive (+) IELs contained in the IEL suspension on the 7th day using a hemocytometer, and then counting the above "1-4 small intestinal crypt organoids". Co-culture with the small intestinal crypt organoid according to the method described in the section of “Co-culture of IEL”.

1-6 Immunohistochemical staining and three-dimensional imaging analysis of small intestinal crypt organoid and small intestinal crypt organoid and IEL obtained according to the method described in the section “1-4 Co-culture of small intestinal crypt organoid and IEL” and the like The co-culture was fixed in a 4% paraformaldehyde solution for 40 minutes, permeabilized in a PBS solution containing 0.2% Triton-X for 30 minutes, and then containing 2% BSA and 0.2% Triton-X. A blocking treatment was performed in a PBS solution for 40 minutes (all were treated at room temperature). A primary antibody (mouse anti-CD3ε antibody [Becton Dickinson] and mouse anti-Cdh1 antibody [Santa Cruz Biotechnology]) was incubated overnight at 4 ° C., and a secondary antibody labeled with a fluorescent substance recognizing the primary antibody ( Antibody reaction treatment with an anti-mouse Alexa Fluor 488 antibody and an anti-mouse Alexa Fluor 594 antibody (all manufactured by Life Technologies) was performed for 1.5 hours at room temperature. Cell nuclei were stained with 4 ', 6-diamidino-2-phenylindole dihydrochloride (DAPI), and immunostained samples were then mounted with Vectashield Mounting Medium (Vector Laboratories) and observed under a microscope. Three-dimensional imaging analysis of small intestinal crypt organoids was performed at room temperature with a Fluoview FV10i system (Olympus), and fluorescence images were taken at 0.85 μm z steps using an Olympus 60x objective (1.35 NA). I got it. Note that the maximum depth that could be observed was 250 μm, which was sufficient to cover the entire organoid cultured for 2 days after the start of the culture or co-culture. The z-stack image was processed by Adobe Photoshop software as needed.

1-7 Flow cytometry According to the method described in the above section "1-3 Isolation of IEL derived from small intestine" and "1-5 Isolation of IEL from co-culture of small intestinal crypt organoid and IEL". The isolated IEL was suspended at a concentration of 5 × 10 ⁵ cells in a PBS solution containing 0.2% FCS and diluted with anti-CD16 / 100 diluted 1: 100 in a PBS solution containing 0.2% FCS for 10 minutes at room temperature. After performing FcR blocking treatment in a CD32 antibody (manufactured by BioLegend), flow cytometry analysis using FACS CantoII (manufactured by BD Biosciences) was performed. Separation of lymphocytes (IEL) and intestinal epithelial cells (IEC) based on size and particle size was performed using forward scatter (FSC) and side scatter (SSC) settings. Further, the TCRarufabeta ⁺ of IEL population (subset), to define the IEL subset of TCRganmaderuta ^+, anti-TCR-beta-PE antibody and anti-TCR-δ-PECy7 antibodies were used at optimal concentration.

  Intracellular staining is performed by fixing cells in a 4% paraformaldehyde solution, permeating with Perm II reagent (Becton Dickinson), and then using anti-Ki-67-660eFluor antibody (eBioscience) or anti-phospho Antibody reaction treatment was performed at room temperature for 1 hour in the presence of -ERK-Alexa647 antibody (manufactured by Cell Signaling). In the case of cell surface staining, the antibody reaction treatment was performed at 4 ° C. for 20 minutes.

1-8 Time-lapse fluorescence imaging Time-lapse fluorescence imaging was performed using a DeltaVision system (Applied Precision) equipped with a fluorescence microscope IX-71 (Olympus) having an Insight SSI light source. That is, in the time-lapse fluorescence imaging, a glass-bottom culture dish containing cells is placed on a stage of the microscope covered with a chamber, and a humidified mixed gas containing 5% CO ₂ and 95% air is placed. Was injected and temperature-controlled at 37 ° C. Prior to starting imaging, cell nuclei were stained with 1 μg / mL Hoechst 33342 (Nacalai Tesque) for 30 minutes. In addition, 5 μM PD184352 was added to the culture solution one hour before the start of imaging. Fluorescence images of nuclei and EGFP were acquired with an UplansApo 20x objective lens (0.75 NA) on a CoolSnap ES2 digital camera (Roper Scientific). Single plane imaging was performed at 20 second intervals for 2 hours. Multi-sided imaging was performed for 10 minutes at 30 second intervals, and z-stacks were acquired in 5 μm steps at a time. SoftWorx software (Applied Precision) was used to obtain the maximum intensity projection data for these z-stacks.

1-9 IEL Motion Analysis Imaging data was imported into Imaris 7.5 (Bitplane) software. IEL nuclei were then individually determined using the Imaris spot tracking feature with a size estimate of 10 μm. Nuclear migration was analyzed by software "autoregressive motion" tracking algorithm. The tracks were visually checked to ensure that one track (track) followed the same nucleus throughout the period. The data set consists of information obtained from the 3D Matrigel area (data acquisition space), but the determination of the spot (nucleus) is inaccurate if close enough to reach the surface of this data acquisition space. There were many things. In order to avoid such peripheral effects, Imaris' filtering function was used to define a smaller cuboid (inner cuboid) with edges along the xyz direction 10 μm shorter than the outer data acquisition space. Only nuclei that remained in this inner cuboid for more than 80% of the observation period were used for statistical analysis. Calculations of Mean Speed, Max Speed, track length, and Displacement of Nuclei were performed using Imaris software.

1-10 Preparation of Colonic Crypt Organoid The colonic crypt was isolated from a 7-9 week old wild type EGFP-tg mouse according to the method described in Patent Document 2 (WO 2013/061608 pamphlet), and serum The cells were cultured in the presence of albumin (BSA), Wnt3a, R-spondin 1, epidermal growth factor (EGF), hepatocyte growth factor (HGF), and Noggin. That is, 2000 colon crypts were suspended in 200 μl of type I collagen solution (Nitta Gelatin Inc.) and placed on a 48-well plate. After collagen polymerization, 1% BSA (Sigma), 30 ng / ml mWnt3a (R & D Systems), 500 ng / ml mouse (m) Rspo1 (R & D Systems), 20 ng / ml 500 μl of Advanced DMEM / F12 containing mEGF (Peprotech), 50 ng / ml mHGF (R & D Systems) and 50 ng / ml mNoggin (R & D Systems) was added (hereinafter referred to as “TMDU culture”). Also displayed.) The culture was changed every two days. For passage, the whole gel was digested in DMEM containing collagenase type XI at 37 ° C. for 5 minutes, and the recovered colonic crypt organoid was washed with PBS containing BSA. The colonic crypt organoid precipitate was suspended in PBS containing 2 mM EDTA and 0.5% BSA and shaken vigorously. The disaggregated colonic crypt organoid lumps were mixed with a type I collagen solution and used for subculture. The size of the cell mass used for subculture from a single cell to a cell mass consisting of a plurality of cells was adjusted for the EDTA treatment time while observing under a microscope. For the first two days after cell growth, the Rho kinase inhibitor Y-27632 was added to the culture at 10 μM. In order to induce goblet cell differentiation, colon organoids were treated with the γ-secretase inhibitor LY-411,575 (100 nM) for a predetermined period.

1-11 Co-Culture of Colon Crypt Organoid and IEL After culturing the colon crypt organoid prepared according to the method described in the section “1-10 Preparation of Colon Crypt Organoid” above for 2 days, XI type collagenase is cultured When added to the solution, the whole gel was digested at 37 ° C. for 5 minutes, and the colonic crypt organoid was recovered from collagen. After washing the colonic crypt organoid with PBS containing BSA, 100 such colonic crypt organoids and IEL 1.0 isolated according to the method described in the above section “1-3 Isolation of IEL derived from small intestine”. × 10 ⁵ were mixed in a 400 μL DMEM culture on a 24-well plate. After incubating at 37 ° C. for 30 minutes, the mixture was collected, centrifuged at 200 g for 1 minute, and the precipitate (pellet) was suspended in 200 μl of type I collagen solution (Nitta Gelatin Inc.), and the mixture was suspended in a 24-well plate. It was transferred to the top and allowed to stand until the collagen polymerized. IL-2, IL-7 and IL-15 were added to the above-mentioned TMDU culture, and 100 U / mL recombinant human IL-2 (Roche), 10 ng / mL mouse IL-7 (Peprotech) and A TMDU culture solution containing 10 ng / mL mouse IL-15 (manufactured by Peprotech) (hereinafter, also referred to as “+ IL-2 / IL-7 / IL-15TMDU culture solution”) was prepared. After the above-mentioned collagen is polymerized, “+ IL-2 / IL-7 / IL-15TMDU culture solution” is added to each well of the 24-well plate in a volume of 500 μL and cultured at 37 ° C. under 5% CO ₂ for 7 days. Was done. The culture solution was replaced with a new culture solution every two days.

1-12 Isolation of IEL from co-culture of colon crypt organoid and IEL Co-culture of colon crypt organoid and IEL according to the method described in the section “1-11 Co-culture of colon crypt organoid and IEL” above After culturing, type XI collagenase was added to the culture solution, and the entire gel was digested at 37 ° C. for 5 minutes, and the colonic crypt organoid was recovered from collagen. Subsequent IEL isolation and counting were performed according to the method described in the section “1-5 Isolation of IEL from co-culture of small intestinal crypt organoid and IEL”. In this way, IEL was isolated from a colonic crypt organoid / IEL co-culture and IEL was counted.

2. Results and Discussion 2-1 In Vitro Cultivation of IEL Existing in Small Intestinal Crypt Organoid In order to develop a method for culturing and maintaining IEL in vitro in the presence of a small intestinal crypt organoid, the present inventors first used conventional methods. It was confirmed whether or not the IEL present in the small intestinal crypt organoid can be cultured and maintained by the small intestinal crypt organoid culture method described above. The small intestinal crypt organoid is isolated and cultured according to the method described in the section “1-2 Preparation of Small Intestinal Crypt Organoid”, and the above “1-6 Immunohistochemical staining and three-dimensional imaging analysis of small intestinal crypt organoid” is performed. When immunohistochemical staining was performed according to the method described in the section, CD3 ⁺ T cells (IEL) were detected in the small intestinal crypt organoids on day 1 after the culture (see the arrowhead in FIG. 1A). When the number of IELs in the small intestinal crypt organoids was measured, the number of IELs per small intestinal crypt organoid was 0.21 ± 0.06 (n = 90), and the proportion of small intestinal crypt organoids in which IEL was detected was It was 16% (n = 90). On the other hand, the same analysis was performed on the third and seventh days after the culture, but no CD3 ⁺ T cells (IEL) were detected in the small intestinal crypt organoid. These results indicate that the conventional method for culturing small intestinal crypt organoids cannot culture and maintain the IEL present in small intestinal crypt organoids in the medium to long term. Therefore, a person skilled in the art could not have an idea of using the small intestinal crypt organoid for culturing and maintaining IEL. However, the inventors of the present invention have conducted free studies without being bound by the idea of a person skilled in the art, and as a result, have isolated IEL from small intestine tissue and co-cultured the IEL with small intestinal crypt organoid. For example, I thought that IEL could be cultured and maintained over the medium to long term.

2-2 Co-culture of small intestinal crypt organoid and IEL The IEL was isolated from the small intestine tissue, and it was examined whether the IEL could be cultured and maintained when co-cultured with the small intestinal crypt organoid. According to the method described in the section of “1-4 Co-culture of small intestinal crypt organoid and IEL”, co-culture of small intestinal crypt organoid and IEL was performed in a small intestinal crypt organoid culture medium, and 3 days after the culture. In the above, immunohistochemical staining was performed according to the method described in the item of “1-6 Immunohistochemical staining and three-dimensional imaging analysis of small intestinal crypt organoid”, and EGFP ⁺ CD3 ⁺ T cells (IEL) were detected ( (See arrowheads and arrows in FIG. 1B). The number of IELs per small intestinal crypt organoid was 6.9 ± 0.8 (n = 24), and the ratio of small intestinal crypt organoids in which IEL was detected was 100% (n = 24). . This result indicates that co-culture of the isolated IEL with the small intestinal crypt organoid allows efficient culture and maintenance of the IEL at least up to three days after the culture. Interestingly, EGFP ⁺ CD3 ⁺ T cells (IEL) were present not only in the small intestinal crypt organoid but also outside the small intestinal crypt organoid without contact with the small intestinal crypt organoid (arrow in FIG. 1B). reference). On the other hand, it has been reported that the in vitro survival time of the isolated IEL is limited to less than 48 hours under conditions without cytokine supplementation or TCR stimulation (see Brunner et al., Cell Death Differ 2001, 8: 706-714 "and" Lai et al., J Immunol 1999, 163: 5843-5850 "). Considering such a report and the results of the above Example (the IEL was able to be efficiently cultured and maintained at least up to 3 days after the culture), the isolated IEL was co-organized with the small intestinal crypt organoid. It was shown that the method of culturing can maintain the IEL for a longer period of time as compared with the conventional method not using IL-2, IL-7 or IL-15. In addition, since the IEL can survive and maintain outside the small intestinal crypt organoids, these IELs, which seem to be invisible at first glance, may also be affected by survival and maintenance activities from the small intestinal crypt organoids coexisting in the same culture. Sex is considered.

Although co-culture of small intestinal crypt organoids with IEL was performed until day 7 thereafter, the percentage of IEL detected was reduced. On the other hand, various cytokines have been reported to be involved in the survival and proliferation of IEL, in particular, the presence of TCRarufabeta ⁺ of IEL population, and TCRganmaderuta ⁺ of IL-2 the IEL population, IL-7 and IL-15 It has been reported that culturing in IEL prolongs the survival time of these IEL populations through various mechanisms (Non-Patent Document 5, "Inagaki-Ohara et al., Eur J Immunol 1997, 27: 2885-2891", See Lai et al., J Immunol 1999, 163: 5843-5850, Yada et al., Cell Immunol 2001, 208: 88-95). Therefore, it was investigated whether the co-culture of the intestinal crypt organoid with the IEL in the presence of IL-2, IL-7 and IL-15 would increase the survival time of the IEL. The method described in the above section “1-4 Co-culture of small intestinal crypt organoid and IEL” and the above section “1-5 Isolation of IEL from co-culture of small intestinal crypt organoid and IEL” According to the method described, co-culture of small intestinal crypt organoid and IEL was performed in “+ IL-2 / IL-7 / IL-15 small intestinal crypt organoid culture medium” until day 14, and 1 to 14 days after culture. In the above, when immunohistochemical staining was performed according to the method described in the item of “1-6 Immunohistochemical staining and three-dimensional imaging analysis of small intestinal crypt organoid”, EGFP ⁺ CD3 ⁺ T was obtained at least until day 7 or 14. The percentage of the cell (IEL) population was shown to have increased (see FIGS. 2A-C). For example, FIG. 2A shows that EGFP fluorescence of IEL (white small whitish spots in FIG. 2A) increased as days of culture elapsed, on days 1, 3, and 7. FIG. 2B shows that, on day 7, the positions of the fluorescence of CD3 (upper part of FIG. 2B) and the fluorescence of EGFP (lower part of FIG. 2B) coincide with each other, and IEL is present between the cells of the organoid epithelium. Is shown. FIG. 2C shows that the EGFP fluorescence of the IEL (white small whitish point in FIG. 2C) increased on day 14 after the culture. In addition, since the small intestinal crypt organoid on the 7th day was too large for three-dimensional imaging analysis, the item of “1-5 Isolation of IEL from co-culture of small intestinal crypt organoid and IEL” was used. The IEL was isolated according to the method described, and the number of EGFP ⁺ T cells (IEL) was measured according to the method described in the above section “1-7 Flow Cytometry”. It became clear that the number was doubled (see FIG. 3A). The results indicate that co-culture of IEL with small intestinal crypt organoids in the presence of IL-2, IL-7, and IL-15 results in the proliferation and maintenance of IEL for at least 14 days, as well as small intestinal crypts. This shows that the number of IELs can be increased up to about 4 times on the 14th day after the start of the co-culture with the organoid.

Next, by co-culture with small intestinal crypts organoid, rather than a specific cell populations contained in the IEL population, to ensure that the entire IEL population grows, the IEL population of TCRαβ ^+, TCRγδ ⁺ IEL population Was analyzed for growth. According to the method described in item "1-7 Flow Cytometry", the measured ratio of TCRarufabeta ⁺ and TCRγδ ⁺ IEL population in co day 1 after culture and day 7 of the small intestine crypt organoid, On day 1, and on days 7 and 14, no significant change was observed in the proportions of these IEL populations (see FIGS. 3B and D). In addition, when the expression of Ki-67, which is a cell proliferation marker, was analyzed in the TCRαβ ⁺ and TCRγδ ⁺ IEL populations, most of the IEL populations were Ki-67 positive (see FIGS. 3C and E). ). These results indicate that co-culture with small intestinal crypt organoids proliferates the entire IEL population, not specific cell populations included in the IEL population.

  The above results indicate that a new culture system capable of growing and maintaining the IEL population in vitro without affecting the diversity of the IEL population has been developed.

Next, the isolated IEL is co-cultured with a small intestinal crypt organoid, and any of IL-2, IL-7 and IL-15 used for culturing and maintaining the IEL is essential for culturing and maintaining the IEL. Was considered. According to the method described in the above section "1-4 Co-culture of small intestinal crypt organoid and IEL", co-culture of small intestinal crypt organoid and IEL was performed in a culture solution for small intestinal crypt organoid culture. However, an experiment using any one of the three ILs and an experiment not using any of the ILs were also performed. The concentration of IL in these experiments was in accordance with the method described in the section of “1-4 Co-culture of small intestinal crypt organoid and IEL” above. On the first day and the seventh day after the culture, the number of EGFP ⁺ T cells (IEL) was measured according to the method described in the item of “1-7 Flow Cytometry” above. The result is shown in FIG. 4A.

  As can be seen from the results of FIG. 4A, when IL-7 or IL-15 was used alone among the three ILs, the number of IELs decreased from Day 1 on day 7 after the culturing (Day 7). An improvement in the number of surviving IELs was seen compared to the inability of the IELs to survive without the addition of any of the species ILs. In addition, when IL-2 was used alone among the three ILs, the number of IELs on day 7 after culture increased to about 1.8 times the number of IELs on day 1 after culture. did. From these results, it was shown that all three types of ILs had an effect of promoting the survival and proliferation of IEL, and that IL-2 was particularly strong in this effect (FIG. 4A). In fact, when three kinds of ILs (IL-2, IL-7 and IL-15) are used in combination, the number of IELs on the 7th day after culture is about 3 times less than the number of IELs on the 1st day after culture. It increased by a factor of two (FIG. 4A). That is, when three kinds of ILs (IL-2, IL-7 and IL-15) are used in combination, the number of IELs on day 7 after culturing is about 1 compared to the case where IL-2 is used alone. 0.7-fold (FIG. 4A). From these results, it is particularly preferable to use at least IL-2 of the three types of IL in order to co-culture the isolated IEL with the small intestinal crypt organoid and to culture and maintain the IEL. It was shown that when IL-7 and IL-15 were used in addition to IL-2 and IL-2, a remarkable effect was obtained in terms of IEL growth efficiency.

2-3 Co-culture of colonic crypt organoid with IEL IEL isolated from small intestinal tissue (i.e., small intestinal interepithelial lymphocytes) was not only co-cultured with small intestinal crypt organoid, but also co-cultured with large intestinal crypt organoid. It was examined whether the IEL can be cultured and maintained even in the case of culturing.

According to the method described in the above section “1-11 Co-culture of colonic crypt organoid and IEL”, co-culture of colonic crypt organoid and IEL was performed in “+ IL-2 / IL-7 / IL-15TMDU culture solution”. went. On the first day and the seventh day after the culture, respectively, the IEL was isolated according to the method described in the section of “1-12 Isolation of IEL from co-culture of colon crypt organoid and IEL”, and the above “1. The number of EGFP ⁺ T cells (IEL) was measured according to a method similar to the method described in the section “-7 Flow Cytometry”. The result is shown in FIG. 4B. Surprisingly, it was shown that small intestinal interepithelial lymphocytes can be cultured and maintained even when co-cultured with colonic crypt organoids. It became clear that the number of IELs increased about 1.86 times in 6 to 7 days (FIG. 4B). Although the extent of this increase in IEL numbers is somewhat inferior to that when co-cultured with small intestinal crypt organoids (day 7 in FIG. 3A), such a method is an excellent method for culturing and maintaining IELs. It was shown that.

2-4 Analysis of IEL motility in small intestinal crypt organoids Next, after co-culturing according to the method described in the items of “1-8 Time-lapse fluorescence imaging” and “1-9 IEL motion analysis” above Analysis of the dynamic interaction between the IEL and the small intestinal crypt organoids on day 3 showed that most EGFP ⁺ T cells (IELs) had high motility (see Figure 5A). Although this single-plane imaging did not allow accurate three-dimensional information to be obtained, it showed movement in both in-focus and out-of-focus images, and thus many EGFP ⁺ T cells (IEL) Migrate along the z-axis. It was also shown that IELs in contact with the small intestinal crypt organoids migrated randomly and along the basal side of the epithelial monolayer, changing their cell shape continuously and on a large scale (FIG. 5A). (See upper section). These IELs inserted their cytoplasmic processes or entire structures between epithelial cells and allowed contact with the outer surface of the epithelial cells (see top row of FIG. 5A). It was also shown that IELs that stayed outside the small intestinal crypt organoid also had motility. Some of these IELs were observed migrating towards the small intestinal crypt organoids, making direct contact with the small intestinal crypt organoids, temporarily staying therein, and then exiting (see lower part of FIG. 5A). ). These results indicate that in the co-culture system developed by the present inventors, IEL has high motility and contacts many IECs. In addition, IELs have been shown to be able to enter and exit small intestinal crypt organoids in this co-culture system, altering IEC contact status. This is thought to be due to the fact that the contact between the IEL and the IEC is involved in leading the overall proliferation of the IEL by extending the survival time of the IEL and stimulating the proliferation of the IEL.

Next, the migration activity of IEL was quantitatively analyzed. When EGFP ⁺ T cells (IEL) derived from EGFP-tg mice were used, the asymmetrically deformed shape and large amount of such EGFP ⁺ T cells were particularly close to two EGFP ⁺ T cells. Sometimes it was not possible to properly define individual cell centers. For this reason, using EGFP ⁺ T cells (IEL) derived from R26-H2B-EGFP mice in which only the nucleus where the fusion partner histone H2B is localized is labeled with EGFP, the movement between the IEL and the small intestinal crypt organoids An analysis was made on the dynamic interaction (see FIG. 5B). As a result, H2B-EGFP ⁺ T cells (IEL) were co-cultured with wild-type organoids, imaging was performed at 30-second intervals for 10 minutes, and about 20 z-stack images were acquired at a time. Maximum intensity projection showed that individual cell nuclei could be clearly detected by this method (see FIG. 5C). As a result, IEL migrating along the epithelial layer of the small intestinal crypt organoid was observed, and IEL dynamically migrating into and out of the small intestinal crypt organoid was also observed.

  The three-dimensional data was reconstructed and the migration parameters of the IEL were measured using image processing software. In practice, to avoid inaccurate tracking of nuclei that reached the surface of the space, a smaller space was defined, and only cells that remained within more than 80% of the observation period were analyzed. The result is shown in FIG. 5B. The average velocity of IEL migration in FIG. 5B was 5.07 ± 0.27 μm / min and the maximum velocity was 9.67 ± 0.60 μm / min (see FIG. 5C and Table 1).

On the other hand, when the motility parameter of TCRγδ ⁺ IEL was evaluated in vivo using a confocal microscope, the average velocity and the maximum velocity were 3.8 ± 0.1 μm / min and 7.7 μm / min, respectively. (Non-Patent Document 6). The results obtained in this example were shown to support the above in vitro results. Further, the TCRarufabeta ⁺ of IEL, was analyzed by comparing both the migration of TCRganmaderuta ⁺ of IEL, significant differences in both was observed (upper FIG. 5D, and see Table 1). This result, TCRarufabeta ⁺ of IEL have essentially shown to have the same mobility and TCRganmaderuta ⁺ of IEL.

The factors and factors that influence the interaction between the IEC and the IEC remain largely unclear. In an in vivo experiment, it has been reported that administration of TNF-α alters the migration of TCRγδ ⁺ in IEL and retention in epithelial tissue (Non-Patent Document 6). Pretreatment with TNF-α (10 ng / ml) and IEL kinetic analysis revealed no change in IEL kinetics. On the other hand, when IEL motility analysis was performed after pretreatment with PD184352, which is an inhibitor of MEK1 / MEK2, TCRαβ ⁺ IEL migration was reduced by about 20%, while TCRγδ ⁺ IEL migration No change was observed (see the lower part of FIG. 5D and Table 1). Interestingly, the analysis by flow cytometry, ERK phosphorylation, and TCRarufabeta ⁺ of IEL, to occur at the same level in a subset of both TCRganmaderuta ⁺ of IEL shown (see FIG. 5E). These results indicate that, even though MEK1 / MEK2 is similarly activated in the entire IEL population, whether the MEK1 / MEK2-ERK cascade is involved in cell-to-cell migration activity depends on cell type. Suggests. Thus, the co-culture system developed by the present inventors is expected to be a useful tool for analyzing the mechanism controlling the in vitro interaction between IEL and IEC.

  The present invention relates to a method for maintaining and expanding intestinal interepithelial lymphocytes in vitro, a method for producing intestinal crypt organoids having increased intestinal interepithelial lymphocytes, and an increase in intestinal interepithelial lymphocytes produced by the production method. Intestinal crypt organoids can be provided.

Claims (6)

(A) Embedding the isolated intestinal interepithelial lymphocytes together with intestinal crypt organoids in an extracellular matrix A:
(B) Step B of co-culturing intestinal interepithelial lymphocytes and intestinal crypt organoid embedded in the extracellular matrix in a culture solution capable of growing the intestinal crypt organoid:
It is characterized by including
A method for maintaining and growing intestinal interepithelial lymphocytes in vitro, wherein the culture medium capable of growing the intestinal crypt organoid contains IL-2, IL-7 and IL-15. (A) Embedding the isolated intestinal interepithelial lymphocytes together with intestinal crypt organoids in an extracellular matrix A:
(B) Step B of co-culturing intestinal interepithelial lymphocytes and intestinal crypt organoid embedded in the extracellular matrix in a culture solution capable of growing the intestinal crypt organoid:
It is characterized by including
A method for producing an intestinal crypt organoid having increased intestinal interepithelial lymphocytes, wherein the culture solution capable of growing the intestinal crypt organoid contains IL-2, IL-7, and IL-15.   When the intestinal crypt organoid is a small intestinal crypt organoid, the culture solution capable of growing the small intestinal crypt organoid is a culture solution containing a mitogenic growth factor, a Wnt agonist and a BMP inhibitor; In the case of a crypt organoid, the culture solution capable of growing the colon crypt organoid is a culture solution containing serum albumin, Wnt3a, and R-spondin 1, and not containing serum. The method according to claim 1.   The method according to claim 3, wherein the mitogenic growth factor is EGF, the Wnt agonist is R-spondin1 and / or Wnt-3a, and the BMP inhibitor is Noggin.   The culture medium capable of growing the colonic crypt organoid further comprises one or more selected from the group consisting of epidermal growth factor (EGF), hepatocyte growth factor (HGF), and Noggin. The method according to claim 3 or 4, wherein the method is characterized in that:   When the intestinal crypt organoid is a small intestinal crypt organoid, the extracellular matrix is Matrigel, and when the intestinal crypt organoid is a large intestinal crypt organoid, the extracellular matrix is collagen. Item 6. The method according to any one of Items 1 to 5.