KR20220043010A - Method for three dimensional co-culture of organoids from human tonsil and immune cells - Google Patents

Abstract

The present invention relates to a method for three-dimensional (3D) co-culture of organoids from human tonsil and immune cells. More specifically, culture conditions optimized for culturing tonsil organoids are combined with culture conditions optimized for culturing tonsil immune cells, so that it is proved that co-culture of tonsillar organoids and immune cells is possible and in vitro implementation of tonsillar tissue was possible by applying same. The present invention includes a composition of specific culture conditions capable of long-term co-cultivation of organoids and immune cells of tonsillar tissue, a culture technique using the same, and a technique for measuring immune cell activity using a corresponding platform. A tonsillar tissue organoid-immune cell co-culture platform can be widely applied to proliferation of antigen-specific T cells, formation of antibodies in vitro, and screening of therapeutic agents related to tonsillar and immune diseases.

Description

Method for three dimensional co-culture of organoids from human tonsil and immune cells

The present invention relates to a three-dimensional co-culturing method of human tonsil-derived organoids and immune cells.

"Organoid" means that cells derived from tissues or embryonic stem cells can be cultured in 3D form to form an artificial organ. Organoid is a suffix that has the same meaning as ‘organ’ of an organ, and has the word ‘organ-like’. Organoids have a more well-arranged cell and cell function through a three-dimensional culture method, and have a functional organ-like form and function. Organoids are receiving attention along with research on optimization of growth and differentiation factors that can differentiate into various tissues, with stem cell research and 3D cell culture being developed.

The tonsil is a type of lymphoid follicle composed of B cells and T cells, which is responsible for oral immune functions, and a non-keratinized epithelium that surrounds lymphoid follicles in addition to immune cells. epithelium). Existing organoid culture technology has the disadvantage of culturing mainly stem cells and epithelial cells, and in particular, it has not succeeded in optimizing and maintaining culture conditions for stromal cells such as immune cells. . Accordingly, there is a need to establish culture conditions optimized for culturing tonsil organoids and culturing immune cells.

Korean Patent Registration No. 10-1953977 (Registered on February 25, 2019)

It is an object of the present invention to provide a culture medium composition for co-culture of tonsil stem cell-derived tonsil organoids and tonsil immune cells comprising IGF-1, IL-2 and anti-CD28 antibody as active ingredients. In addition, it is to provide a culture solution composition further comprising additional prostaglandin E2 (Prostaglandin E2; PGE2).

Another object of the present invention is to provide a method of co-culturing tonsil stem cell-derived tonsil organoids and tonsil immune cells using the composition.

In order to achieve the above object, the present invention provides a culture medium composition for co-culture of tonsil stem cell-derived tonsil organoids and tonsil immune cells comprising IGF-1, IL-2 and anti-CD28 antibody as active ingredients. In addition, it provides a culture solution composition further comprising additional prostaglandin E2 (Prostaglandin E2; PGE2).

In addition, the present invention comprises the steps of (1) isolating tonsil stem cells from tonsil tissue; (2) contacting the isolated tonsil stem cells with an extracellular matrix (ECM); and (3) culturing the tonsil stem cells in contact with the ECM in the culture solution composition, and culturing tonsil organoids and tonsil immune cells. to provide.

The present invention relates to a three-dimensional co-culturing method of human tonsil-derived organoids and immune cells. It was revealed that the co-culture of nooids and immune cells was possible, and by applying this, it was possible to implement the tonsil tissue in vitro. The present invention includes a composition of specific culture conditions capable of long-term co-culture of organoids and immune cells of tonsil tissue, a culture technique using the same, and a technique for measuring immune cell activity using the corresponding platform. The tonsil tissue organoid-immune cell co-culture platform according to the present invention can be widely applied to antigen-specific T cell proliferation, in vitro antibody formation, and screening for therapeutic agents related to tonsils and immune diseases.

1 shows the cell distribution results after 2 weeks of culture of tonsil organoids.
Figure 2 shows the decrease in immune cells according to the culture of tonsil organoids.
Figure 3 shows the organoid culture results through the composition of the culture medium containing the active ingredient of the present invention in order to solve the immune cell reduction phenomenon.
4 shows the results of microscopic findings (a) and immune cell detection through flow cytometry (b) according to tonsil organoid culture. Figure 4a shows the growth of tonsil organoids and the formation of immune cell clusters in passage 1, and Figure 4b shows the results of confirming the proportion of immune cells according to passage culture.
5 shows the results of comparing the expression of markers in organoids with actual tonsil tissue.
6 is a result of analyzing the degree of organoid generation according to the concentration of PGE2 through an optical microscope image in an organoid-immunocyte co-culture environment.
7 shows the results of analyzing the distribution of immune cells according to the concentration of PGE2 by flow cytometry in an organoid-immunocyte co-culture environment.

The present invention provides a culture medium composition for co-culture of tonsil stem cell-derived tonsil organoids and tonsil immune cells comprising IGF-1, IL-2 and anti-CD28 antibody as active ingredients. Preferably, the culture medium composition may further include prostaglandin E2 (PGE2).

Preferably, the culture solution composition comprises IGF-1 at a final concentration of 10 to 200 ng/ml, IL-2 at a final concentration of 1 to 20 ng/ml, an anti-CD28 antibody at a final concentration of 1 to 10 μg/ml and a final concentration 0.1 to 0.5 μM of PGE2 may be included, but is not limited thereto.

Preferably, the culture solution composition may consist of the components shown in Table 1, but is not limited thereto.

In addition, the present invention comprises the steps of (1) isolating tonsil stem cells from tonsil tissue; (2) contacting the isolated tonsil stem cells with an extracellular matrix (ECM); and (3) culturing the tonsil stem cells in contact with the ECM in the culture solution composition according to the above, and culturing tonsil organoids and tonsil immune cells. provide a way

Preferably, the cultured tonsil organoids may express p75, CD44, CK19, CK14, MUC1, CK13 and CK8, but is not limited thereto.

Preferably, PGE2 may be added in the step of culturing the tonsil organoids and tonsil immune cells.

In the present invention, the method of culturing tonsil stem cells includes culturing the tonsil stem cells by contacting them with an extracellular matrix (ECM). Any suitable extracellular matrix may be used. The isolated tonsil stem cells are preferably cultured in an environment that partially mimics the cellular microenvironment in which the tonsil stem cells naturally exist. This cell niche can be mimicked by culturing the stem cells in the presence of a biomaterial such as an extracellular matrix that provides key regulatory signals that control stem cell fate. The cell niche is determined in part by the extracellular matrix produced by stem cells and surrounding cells and cells in the niche. In a preferred method of the present invention, the tonsil stem cells are cultured in contact with an extracellular matrix. "Contact" means physical or mechanical or chemical contact, meaning that force needs to be used to separate the resulting population of tonsil organoids or tonsil stem cells from the extracellular matrix. Preferably, the tonsil stem cells are embedded in the ECM. The culture medium of the present invention can be diffused into the extracellular matrix.

In the present invention, "organoid" means that it can be made in the form of an artificial organ by culturing it in a 3D form using cells derived from tissues or embryonic stem cells. Organoid is a suffix that has the same meaning as ‘organ’ of an organ, and has the word ‘organ-like’. Organoids have a more well-arranged cell and cell function through a three-dimensional culture method, and have a functional organ-like form and function. Organoids are receiving attention along with research on optimization of growth and differentiation factors that can differentiate into various tissues, with stem cell research and 3D cell culture being developed.

Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited by these examples.

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Reagents

The following reagents were used in the present invention.

Bovine Serum Albumin (BSA), 1×HBSS solution (# 14025092, Gibco), Collagenase, type II (# 4176, Worthington), Hyaluronidase (# H3506, Sigma), Y-27632 dihydrochloride (# 1254, Tocris) ), 48 well plate for suspension culture (# 677102, Greiner Bio-one), Reduced Growth Factor Basement Membrane Matrix, Type 2 (BME 2) (# 3533-001-02, Trevigen), X-VIVO 15 media (# 04 -418Q, Lonza), Primocin (#anti-pm, Invivogen), HEPES (#15630-080, Gibco), GlutaMAX (#35050-061, Gibco), B27 (#12587, Gibco), NAC (N-acetyl- cysteine;#A9165, Sigma), Nicotinamide (#N0636, Sigma), A83-01 (#2939, Tocris), Noggin (#6057-NG, R&D), IGF-1 (#100-11, Peprotech), FGF2 ( # 100-18B, Peprotech), FGF10 (# 100-26, Peprotech), IL-2 (# 200-02, Peprotech), PGE2 (# P0409, Sigma), Purified anti-human CD3 antibody (#566685, BD) , Purified anti-human CD28 antibody (#555725, BD), DMEM, High Glucose, w/ stable glutamine w/ sodium pyruvate (# L0103, Biowest), Fetal Bovine Serum (FBS), 1× Phosphate buffered saline (PBS), Zeoci n (# R250-01, Life technologies), G-418 solution (# 4727878001, Roche), Rspo1 cells (3710-001-K, Cultrex), L-WNT3A cells (CRL-2647, ATCC), TrypLE express (# 12605-010, Life technologies), CellBanker 1 (Zenoaq)

2. Derived from human tonsil stem cells organoid produce

(1) After collecting a human tonsil sample, put it in a preservative solution (1× HBSS with 1% BSA) and store it in a refrigerator at 4°C until the next day or the next day before the experiment.

(2) Collagenase type II (Collagenase type II) 0.5 mg/ml, hyaluronidase 0.5 mg/ml, Y-27632 10 mM was added to the preservation solution with tonsil samples to obtain a digestion solution After making about 20 ml for each sample, it is stored in a stirred incubator at 37°C at 200 rpm for 30 minutes.

(3) Filter the reacted tonsil sample with a 70 μm strainer to remove undissolved tissue, and then centrifuge the filtered solution at 300 g for 5 minutes to obtain only cells.

(4) After counting the number of cells, count the number of cells to contain 1.0 × 10 ⁴ cells based on a 48-well plate, and centrifuge again under the same conditions.

(5) Take out the 48-well plate heated at 37° C. for at least 1 hour in advance, mix 20 ul of BME 2 matrix per well with the cell pellet, and then dispense into each well to make a dome shape. Thereafter, after storage at 37° C. for 20 minutes, the culture medium is put into each well of 250 ul and cultured.

(6) At this time, the composition of the culture medium is shown in Table 1.

(7) After putting the existing culture solution into a conical tube every 2-3 days during culture, centrifuge at 300 g for 5 minutes. After that, the cells (mainly immune cells) contained in the pellet are dissolved in a new culture medium and put into the well.

(8) Perform subculture after 2 weeks.

final concentration X-VIVO 15 media Primocin 1 × HEPES 1 × Glutamax 1 × B27 1 × NAC 1 mM Nicotinamide 10 mM A83-01 0.5 uM Wnt3a CM 5% Rspo1CM 5% Noggin 100 ng/ml IGF-1 50 ng/ml FGF10 20 ng/ml FGF2 10 ng/ml Y-27632 5 uM** IL-2 10 ng/ml anti-CD28 antibody 2 μg/ml**

** Y-27632, anti-CD28 antibody is added only for 3-4 days after incubation.

3. organoid Preparation of conditioned media for culture medium

(1) Take out the cell stock (1.0 × 10 ⁶ cells/vial) from the nitrogen tank and react until it is half-dissolved in a water bath. Then, add 5 ml of a growth medium to the dissolved cell line and incubate it in a T25 flask. (Growth medium: DMEM with 10% FBS)

(2) After one day, add the selection reagent to the T25 flask as follows.

i) For L-WNT3A cells, add 8 ul/ml G-418 solution (50 mg/ml) to a final concentration of 0.4 mg/ml

ii) For Rspo1 cells, add 3 ul/ml Zeocin (100 mg/ml) to a final concentration of 0.3 mg/ml

(3) Start subculture because the T25 flask is full after one day.

(4) Aspirate the medium and wash once with 1×PBS pre-warmed.

(5) Remove the supernatant, add 2 ml of pre-warmed TrypLE, and put it in the incubator for 3 minutes.

(6) Take out the T25 flask, add 2 ml of growth medium, harvest the cells, and transfer to a 15 ml tube.

(7) Centrifuge at 300 g for 3 minutes.

(8) Remove the supernatant and add 2 ml of the growth medium, dissolve the pellet, aliquot 1 ml and put it in the T75 flask, and then add the rest of the medium (final 12 ml) and the selection reagent according to the composition. After that, it is stored in an incubator.

< WNT3A CM >

(9-1) After 2-3 days, passaging once more to prepare a total of 10 T75 flasks. In this case, a split ratio of about 1:10 is suitable. At this time, after splitting, 10 ml of a harvest medium, not a selection medium, is added. (harvest medium: 10 ml of DMEM with 10% FBS)

(9-2) As L-WNT3A cells grow, they secrete WNT3A. After 4 days, harvest the first medium, refrigerate (1 ^st Batch), and add 10 ml of harvest medium.

(9-3) After 3 days, the medium is harvested (2 ^nd Batch), mixed with 1 ^st Batch medium, and centrifuged at 3,000 rpm for 10 minutes (4 ℃).

(9-4) After pouring the supernatant onto the 0.22 μm vacuum filter flask, apply vacuum to filter.

< Rspo1 CM >

(10-1) After 2-3 days, passaging once more to prepare a total of 10 T75 flasks. In this case, a split ratio of about 1:10 is suitable.

(10-2) After 2-3 days, when the T75 flask is about 70-80% full, add the next 15ml of harvesting medium. (Harvest medium: 15 ml of AddDMEM/F12 with 1×GlutaMAX)

(10-3) Incubate in an incubator for 1 week.

(10-4) After one week, the supernatant is harvested in a 50 ml tube, and centrifuged at 3,000 rpm, 4° C. for 10 minutes.

(10-5) After pouring the supernatant onto a 22 μm vacuum filter flask, apply vacuum to filter.

(11) Aliquot the harvested conditioned medium and store it frozen in a 50ml tube. If necessary, take out one at a time and keep it refrigerated for use.

5. Human Tonsil Stem Cell Derived organoid passage

(1) After putting the culture solution in a new conical tube, centrifuge at 300 g for 5 minutes. After removing the supernatant, 500 ul of TrypLE Express (10 mM Y-27632 added) per well of a 48-well plate was added and pipetting was performed. react for 10 minutes.

(2) After adding 1 ml of PBS, pipetting vigorously more than 20 times, and then centrifuging at 300 g for 5 minutes.

(3) Filter the cell pellet with a 40 μm strainer to filter out unremoved organoids.

(4) The filtered cell suspension is counted and the above organoid preparation method is repeated.

< Example >

Organoid culture was attempted by extracting cells from tonsil samples. At this time, the following strategies were tried to promote the growth of immune cells; 1) Make an organoid culture medium based on X-VIVO 15 medium optimized for immune cell culture instead of Advanced DMEM/F12, which is the basic medium for organoid culture. 2) For T cell activation, anti-CD3 antibody (1 ug/ml) and anti-CD28 antibody (2 ug/ml) were added to the medium in soluble form. 3) IL-2 was added to the medium for additional T cell growth. After screening for 2 weeks (passage 0) the conditions that can promote the growth of immune cells compared to the conventional medium with the above three strategies, optimal conditions are selected and cultured of immune cells and organoids even after subculture And it was confirmed whether growth is maintained.

1. X- VIVO 15 Immune cell growth according to the use of media and T cell stimulating antibody

The maintenance level of organoids and immune cells cultured in X-VIVO 15 medium-based one-way organoid culture medium (abbreviated as TS-media, see Table 1 for composition) was analyzed by flow cytometry after 2 weeks of culture. It was confirmed through the method. At this time, as a control, a condition using only X-VIVO 15 medium was added, and T cell activation was promoted through stimulation of anti-CD3, anti-CD28, or anti-CD3 and anti-CD28, respectively (Table 2).

( - ) Anti-CD3 Anti-CD28 Anti-CD3/28 X-VIVO 15 # One # 2 #3 # 4 TS-media #5 #6 #7 # 8

Experimental results according to each condition are shown in FIG. 1 .

The production of epithelial cell organoids was unsuccessful only with the X-VIVO 15 culture medium. However, an increase in T cells according to CD3/CD28 stimulation was observed in the X-VIVO 15 culture medium (FIG. 1, #4). In TS-media suitable for culturing epithelial cell organoids, epithelial cells, which are cells derived from organoids, are increased, and immune cells are also partially maintained. However, after performing one passage, when culture was maintained for about a month, it was confirmed that the growth of organoids was dominant, and as a result, the ratio of immune cells was reduced to an extent not suitable for the experiment (FIG. 2).

To build a model system that maintains immune cells even after passage and studies the interaction with organoids, the above experiment was performed in a culture medium in which IL-2, which promotes T cell growth, is added to the relevant conditions. In order to maintain the growth of immune cells even after passage, the experiment was performed based on the culture medium condition in which IL-2, which promotes the growth of T cells, is added. As a result, the formation of relatively large organoids under the conditions in which IL-2 and anti-CD28 antibody were added was confirmed at passage 0. At this time, it was confirmed that the growth of the organoid was rather inhibited when the anti-CD3 antibody was added (Fig. 3a), which is thought to have occurred because the epithelial cells constituting the organoid were attacked by the immune cells due to an excessive immune response. (Fig. 3b).

Therefore, it was confirmed whether immune cells were maintained under the condition of anti-CD28 antibody + IL-2 in passage 1, and as a result, tonsil organoids were maintained with the composition of the corresponding combination (black arrow), It was confirmed that clusters were formed (red arrow) (Fig. 4a).

In fact, when the cells were analyzed by flow cytometry using the corresponding cells, it was confirmed that a similar level of immune cell ratio was maintained when compared to passage 0. It was confirmed in passage 1 (after culture for one month) that it was maintained to some extent (FIG. 4b).

2. One way organoid my marker Expression confirmation

Tonsil organoids were generated normally, and to confirm whether the markers expressed in the original tissue were maintained in the organoids, the presence or absence of the marker expression of the organoids was checked through the IF staining technique after culturing the organoids (FIG. 5).

As a result, it was confirmed that all of the markers expressed in existing tissues were also expressed in organoids. However, markers expressed in the basal part of the tissue (p75, CD44, CK19, CK14) are expressed outside the organoid, and the markers expressed in the surface part (MUC1, CK13, CK8) are organoids was observed to have an orientation opposite to that of the tissue, which is a common phenomenon observed in other organoids such as intestinal organoids.

3. In the culture medium PGE2 according to the addition organoid Check the level of production

Additionally, the change in the degree of organoid generation according to the concentration of PGE2 in the culture medium was confirmed through optical microscopy image analysis. While the number of organoids was significantly reduced in the culture medium without PGE2, it was confirmed that the number of organoids and the size of the organoids significantly increased as the concentration of PGE2 was increased in a concentration-dependent manner (FIG. 6). This result was also demonstrated by single cell analysis through flow cytometry (Fig. 7, left), and when the concentration of PGE2 was 5 μM, the ratio of epithelial cells constituting organoids significantly increased compared to the group not treated with PGE2. confirmed that. On the other hand, it was confirmed that the ratio of immune cells was significantly decreased (FIG. 7, right), and through this, it was proved that PGE2 is an important factor regulating the ratio of epithelial cells constituting immune cells and organoids in co-culture conditions. As a result of the experiment, it was concluded that it is appropriate to set the concentration of PGE2 to 0.1 ~ 0.5 μM in order to maintain the optimal ratio between immune cells and epithelial cells under the relevant conditions.

Claims (6)

A culture medium composition for co-culture of tonsil stem cell-derived tonsil organoids and tonsil immune cells comprising IGF-1, IL-2 and anti-CD28 antibody as an active ingredient. According to claim 1, wherein the culture composition is a culture medium composition characterized in that it further comprises prostaglandin E2 (Prostaglandin E2; PGE2). The method according to claim 2, wherein the culture composition comprises IGF-1 at a final concentration of 10 to 200 ng/ml, IL-2 at a final concentration of 1 to 20 ng/ml, an anti-CD28 antibody at a final concentration of 1 to 10 μg/ml, and A culture solution composition comprising PGE2 at a final concentration of 0.1 to 0.5 μM. The culture solution composition according to any one of claims 1 to 3, wherein the culture solution composition consists of the components shown in Table 1. (1) isolating tonsil stem cells from tonsil tissue;
(2) contacting the isolated tonsil stem cells with an extracellular matrix (ECM); and
(3) Tonsil stem cell-derived tonsils comprising the step of culturing tonsil stem cells in contact with the ECM in the culture solution composition according to any one of claims 1 to 4, and culturing tonsil organoids and tonsil immune cells Methods of co-culturing organoids and amygdala immune cells. According to claim 5, wherein the cultured tonsil organoids p75, CD44, CK19, CK14, MUC1, CK13 and CK8 is characterized in that the expression of tonsil stem cell-derived tonsil organoids and tonsil immune cells co-culture method.

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