COMPOSITION AND METHOD FOR CULTURING ORGANOIDS

Abstract

The present invention relates to a composition for culturing organoids and a method for culturing organoids by using the same. Since the present invention includes a compound, which can replace an essential protein component in a conventional medium, consistent stability during organoid culturing can be maintained and culturing can be performed at low cost.

Claims

1. A composition for culturing an organoid comprising a compound of the following chemical formula 1: ##STR00007##

2. The composition according to claim 1, wherein the organoid is derived from an adult stem cell.

3. The composition according to claim 1, wherein the organoid is a small intestinal organoid.

4. The composition according to claim 1, wherein a concentration of the compound in the composition is between 25 and 50 M.

5. The composition according to claim 1, wherein the composition further comprises one or more selected from the group consisting of epidermal growth factors (EGF), noggin, thiazovivin, CHIR99021 and a pharmaceutically acceptable salt of CHIR99021.

6. A method for culturing an organoid, comprising: culturing cells in a composition comprising a compound of the following chemical formula 1: ##STR00008##

7. The method according to claim 6, wherein the cell is a stem cell, a population of stem cells, or an isolated tissue fragment.

8. The method according to claim 7, wherein the stem cell is an adult stem cell.

9. The method according to claim 6, wherein a concentration of the compound in the composition is between 25 and 50 M.

10. The method according to claim 6, wherein the composition further comprises one or more selected from the group consisting of epidermal growth factors (EGF), noggin, thiazovivin, CHIR99021 and a pharmaceutically acceptable salt of CHIR99021.

Description

BRIEF DESCRIPTION OF FIGURES

[0024] FIG. 1A is a photograph of one of 105 96-well plates used for compound screening. The six wells in the leftmost column were used as a positive control group, the six wells in the rightmost column were used as a negative control group, and the entire ten columns in the middle were used as an experimental group.

[0025] FIG. 1B shows the result of selecting a second candidate material using the total small intestinal organoid number (viability factor) and the budding small intestinal organoid number (budding factor).

[0026] FIG. 1C is an optical micrograph of small intestinal organoid grown in a culture solution comprising a compound determined to be significant as a result of screening.

[0027] FIG. 2A is an optical micrograph of mouse intestinal crypt treated with RS-246204 and cultured for 4 days, where ENR is a positive control group (including R-spondin), EN is a negative control group, and EN+RS-246204 is an experimental group.

[0028] FIG. 2B is a graph showing the result of WST analysis for confirming the survival rate of small intestinal organoids according to RS-246204 concentration. The Y axis is a value obtained by converting the WST activity value into percentage, and a negative control group is set to 100%.

[0029] FIG. 2C is a graph showing the number of different forms of organoids after growth of small intestinal organoids according to RS-246204 concentration. The Y axis is the number of organoids present in the well. Budding organoids refer to budded organoids, and non-budding organoids refer to living organoids which have not been budded. Total viable organoids refer to a sum of the number of budding organoids and non-budding organoids.

[0030] FIG. 2D is a graph measuring the average length of organoid circumference by date. In the graph, DIV refers to day in vitro, the Y axis is the percentage value of the average organoid circumference, and DIV-1 is set to 100%.

[0031] FIG. 2E is an optical micrograph of subculturing after primarily culturing small intestinal organoids under ENR and EN+RS246204 conditions, respectively.

[0032] FIG. 3A is a photograph showing the result of electrophoresis after RT-PCR using RNA extracted from small intestinal organoids cultured under ENR and EN+RS246204 conditions, respectively.

[0033] FIG. 3B is a graph showing qRT-PCR results using RNA extracted from small intestinal organoids cultured under ENR and EN+RS246204 conditions, respectively.

[0034] FIG. 3C shows an immunofluorescence staining results of small intestinal organoids cultured for 4 days under ENR and EN+RS246204 conditions. Hoechst (blue) represents the nucleus, and Alexa594 (red) represents the fluorescence for each antibody.

[0035] FIG. 4 is an optical micrograph of inducing forskolin stimulation after culturing small intestinal organoids for 4 days under ENR and EN+RS246204 conditions, respectively.

EXAMPLES

[0036] In the following, exemplary embodiments of the inventive concept will be explained in further detail with reference to examples. However, the following examples are meant to exemplify the present invention, and the scope of the invention is not restricted by these examples.

Example 1. Isolation of Mouse Small Intestinal Crypt

[0037] Small intestinal crypts were isolated from a mouse to be used in experiments for preparing and culturing small intestinal organoids. Specifically, the small intestine was isolated after killing a 5-7 weeks old C57BL/6 mouse weighing 20-25 g by cervical vertebrae dislocation. The small intestine was cut longitudinally from a proximal end to a distal end, and also laterally cut into pieces of about 5 mm length. The piece of small intestine obtained was washed with ice-cooled Dulbecco's phosphate-buffered saline (DPBS) until the supernatant liquid was sufficiently clear. Then, the crypts were isolated by treating with a Gentle Cell Dissociation Reagent (StemCell Technologies, Cambridge, Mass.), and filtering with a cell strainer.

Example 2. Screening of Compound Library Using Organoid Proliferation Measurement

[0038] Compounds which can replace R-spondin in organoid cultures were screened for 8,364 types of compounds included in a representative library of the Korea Chemical Bank.

[0039] The small intestinal crypts derived from the small intestine of a 7-week-old C57BL/6 mouse isolated in Example 1 were mixed in Matrigel, and placed in each well of a 96-well plate. An experimental group of eighty wells was used per 96-well plate, three wells were used for each of the positive control group and the negative control group, and three wells were used for each of the positive control group and the negative control group including 0.5% of DMSO. The negative control group used an EN culture solution which does not contain R-spondin nor compounds (composition: advanced DMEM/F-12, Hepes buffer solution, GLUTAMAX-I SUPPLEMENT, penicillin-streptomycin solution, N-acetyl-L-cysteine, B-27 serum-free supplement, N-2 supplement, animal-free recombinant murine EGF, recombinant murine noggin, CHIR99021, thiazovivin). The positive control group used the EN culture solution in which 10% of R-spondin is contained. Hereinafter, the EN culture solution containing 10% of R-spondin is referred to as ENR culture solution. As the experimental group, 8,364 different types of compounds were added to the EN culture solution at a concentration of 50 M, respectively. The compounds used for screening were treated immediately after completing the polymerization of matrigel and crypt, and was applied without replacement for 4 days immediately after isolating crypts. After keeping in 37 C. humidification incubator (5% CO.sub.2) for 4 days, the cultured organoids were observed and optical microscope photos were taken for analysis (see FIG. 1A).

[0040] The number of living organoids, the number of budding organoids, and the circumference of each organoid were measured using the optical microscope photos. For counting, a cell counter plugin of Image J Software was used, and for measuring the circumference, a free curve tool of Dixi eXcope software was used. The compounds were ranked based on the measured values, and then 295 candidate compounds were selected to be used in a second screening. The 295 candidate compounds for second screening were re-examined in the same manner as first screening. After that, 21 candidates were selected by collecting and ranking the results of the first and second screening. After performing a third screening for the 21 candidate materials in the same manner, all three results were collected to finally select seven candidate compounds (see FIG. 1B).

[0041] Among the final seven candidate compounds, the compound of the following chemical formula 1 (Compound Library No.: STK611777) showed the highest growth of small intestinal organoids among the candidate materials, and it was observed from numerical data and visual confirmation that said compound could grow and maintain organoids most similar to R-spondin (see FIG. 1C):

##STR00006##

[0042] The compound is referred to as RS-246204.

Example 3: Small Intestinal Organoid Culture Effect of RS-246204

3.1. Culture Effect According to Concentration of Compound

[0043] An optimal concentration on the small intestinal organoids culture effect of RS-246204 was examined. RS-246204 was added to the culture solution of the small intestinal crypts, respectively, at a final concentration of 6.25 M, 12.5 M, 25 M, 50 M, 100 M, and 200 M, and then was incubated in the culture medium for 4 days. After 4 days, it was observed that the small intestinal organoids grown in the culture solution containing 25 M and 50 M of RS-246024 showed similar morphology and growth to the small intestinal organoids grown of the ENR culture solution (see FIG. 2A).

3.2. WST Analysis

[0044] For more accurate confirmation, RS-246204 was treated at the same concentration, and after 4 days, 10 l of WST was added to each well. WST is a tetrazolium salt which reacts with dehydrogenase to produce formazan, thereby causing the culture solution to become orange color. The dehydrogenase is an enzyme which exists only in a living cell, and thus it is possible to check the survival rate of cell when treated with WST. 3 hours after adding WST, only the culture solution was taken, and the absorbance was measured at 450 nm. When the survival rate in the EN culture solution, which is a negative control group, is set to 100%, the culture solution containing 25 M and 50 M of RS-246204 showed an organoid survival rate similar to that in the ENR culture solution, which is a positive control group (see FIG. 2B).

[0045] As a result, it was observed that when the small intestinal organoids were cultured using the isolated crypts, the small intestinal organoids grown in the culture solution in which R-spondin was replaced with RS-246204 grew in a manner similar to the small intestinal organoids grown in the conventional culture solution containing R-spondin.

3.3. Budding Characteristics and Appearance Analysis

[0046] The morphological characteristic of the small intestinal organoids is that they grow while budding. In order to check that the budding rate of the small intestinal organoids grown in the RS-246204-added culture solution (hereinafter, RS-246204 culture solution) is similar to that grown in ENR culture solution, the total number of organoids, the number of budding organoids, and the number of non-budding organoids were counted in the wells culturing for 4 days. The rate of budding organoids and non-budding organoids grown in the culture solution containing 50 M of RS-246204 is about 1:1, which is similar to the rate of organoids grown in the ENR culture solution (see FIG. 2C). It was observed that the rate of non-budding organoids increased at a concentration lower than 50 M, and most organoids were killed at a concentration higher than 50 M.

[0047] In order to check whether the growth efficiency of the small intestinal organoids grown in the RS-246204 culture solution has a difference, the small intestinal organoids cultured in each culture solution were photographed by an optical microscope every day. The circumference of the individual organoid according to the condition of culture medium and date was measured, and converted into percentage. As a result, it was observed that not only the growth efficiency but also the increase rate of the circumference according to date were also similarly observed (see FIG. 2D).

3.4. Subculture

[0048] It was confirmed that it is possible to subculture small intestinal organoids grown in RS-246204 culture solution in the same manner as small intestinal organoids grown in ENR culture solution, and it was observed that the growth and maintenance were also possible even after the subculture (see FIG. 2E). The crypts cultured in a culture solution which does not comprise R-spondin or RS-246204 could not be cultured to the small intestinal organoids.

Example 4. Analysis of Gene Expression of Small Intestinal Organoids Cultured by RS-246204

4.1. RT-PCR Analysis

[0049] In order to confirm whether lineage markers specific to the small intestinal organoids are expressed when cultured with RS-246204, RNA analysis was performed. The small intestinal organoids cultured in ENR and RS-246204 culture solutions, respectively, for 4 days were collected. RNA was extracted and cDNA was synthesized, and then RT-PCR was performed. As a marker for intestinal stem cells, goblet cells, paneth cells, enteroendocrine cells and enterocyte, RT-PCR was performed using RNA primers for Lgr5, muc-1 and muc-2, defesing-5, chromogranin A (ChgA), and villin, respectively. As a result, it was confirmed that all these cells were present in the small intestinal organoid cultured by RS-246204, and that Olfactomedin-4 (Olfm4) and CD44, which are genes located downstream of Lgr5 signaling, were also expressed (see FIG. 3A).

[0050] For quantitative analysis, qRT-PCR analysis was performed. AccuPower 2X Greenstar qPCR MasterMix (Bioneer) and Thermal Cycler Dice Real Time System III (Takara, Japan) were used, and the reaction was performed for 10 seconds at 95 C. (denaturation), 15 seconds at 57 C. (annealing), and 20 seconds at 72 C. (extension). The RNA primers excluded Muc1 among those used in the RT-PCR test, changed the enterocyte marker from villin to Intestinal Alkaline Phosphatase (TAP), and used the same sequence for the rest. The qRT-PCR results showed that the relative amount of markers expressed showed little difference between the ENR and RS-246204 culture solutions (see FIG. 3B).

4.2. Immunofluorescence Analysis

[0051] In addition, immunofluorescence staining was performed on the small intestinal organoids cultured in the ENR and RS-246204 culture solutions to confirm the expression of Muc-2, lysozyme, and Ki67, which is a marker of proliferating cells (see FIG. 3C).

Example 5. Function Maintenance of Small Intestinal Organoids Cultured by STK611777

[0052] In order to check whether functions of small intestinal epithelial cells are maintained in the small intestinal organoids cultured using the RS-246204 culture solution, CFTR agonist Forskolin analysis was performed. The forskolin is a compound which stimulates ion channels to open, thereby promoting the release of moisture. In case of small intestinal organoids, moisture gathers into the lumen by the forskolin stimulation and changes into a large spherical shape, thereby confirming the maintenance of the function of epithelial cells. After culturing for 4 days in RS-246204 culture solution and ENR culture solution, respectively, it was replaced with a culture solution to which forskolin was added at a concentration of 5 M. Photographs were taken by an optical microscope at an interval of 10 minutes for 1 hour immediately after the replacement. Based on the optical micrographs, the free curve tool of Dixi eXcope (Korea) program was used to measure the circumference of each organoid per hour and analyze the change in circumference.

[0053] As a result, it was observed that the small intestinal organoids grown in RS-246204 culture solution performed well as epithelial cells, and showed a similar change in circumference to those grown in ENR culture medium (see FIG. 4).

[0054] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. Such equivalents are intended to be encompassed by the following claims.