STANDARD ORGANOID PRODUCTION METHOD

Abstract

A method of producing a standard-type organoid.

Claims

1. A method of producing an organoid, the method comprising: forming an organoid by culturing cells in a 3D cell culture plate, wherein in the forming of the organoid, the cell culture plate comprises 0 to 2 vol % of an extracellular matrix-based hydrogel, and wherein the 3D cell culture plate comprises: a well plate comprising a plurality of main wells and a plurality of sub wells formed at lower portions of the main wells to be injected with a cell culture solution and comprising recessed parts on a bottom surface thereof; and a connector for large-capacity and high-speed high content screening (HCS), which supports the well plate, and the connector for high content screening (HCS) comprises a base equipped with a fixing means so as to be attached to and detached from a lower end of the well plate and a cover positioned on an upper portion of the well plate to be coupled to the base, the main well has a step formed so as to be tapered at a predetermined site, and the step has an inclination angle (θ) ranging from 10 to 60° with respect to a wall of the main well.

2. The method of claim 1, wherein the extracellular matrix-based hydrogel is Matrigel.

3. The method of claim 1, wherein the cells are normal cells or cancer cells, and the cell culture period is 1 to 14 days.

4. The method of claim 1, wherein a size of the organoids is 300 to 500 μm in diameter.

5. The method of claim 1, wherein the sub well has an inclined surface formed so as to taper toward a recessed part.

6. The method of claim 1, wherein the sub well has an inclined surface formed so as to taper toward the recessed part, the sub wells have an upper end diameter ranging from 3.0 to 4.5 mm, the recessed parts have an upper end diameter ranging from 0.45 to 1.5 mm, an inclined surface (θ.sub.2) between the sub well and the recessed part ranges from 40 to 50°, and a length ratio of the diameter of the sub wells to the diameter of the recessed parts ranges from 1:0.1 to 0.5.

7. The method of claim 1, wherein the main well has an individual volume ranging from 100 to 300 μl, the recessed part has an individual volume ranging from 20 to 50 μl, and an individual volume ratio of the main well to the recessed part is 1:0.1 to 0.5 on average.

8. The method of claim 1, wherein the main well comprises a space part between the step and the sub well, the space part has a height (a.sub.h) ranging from 2.0 to 3.0 mm on average, the sub well has a height (b.sub.h) ranging from 1.0 to 2.0 mm on average, and a height ratio (a.sub.h:b.sub.h) of the space part to the sub well ranges from 1:0.3 to 1.

9. The method of claim 1, wherein the cells are seeded in the sub wells of the cell culture plate at 100 to 300 cells/well.

10. An organoid having a diameter of 300 to 500 um, produced according to claim 1.

Description

DESCRIPTION OF DRAWINGS

[0083] FIG. 1A is a front view of a cell culture plate according to an exemplary embodiment of the present invention, and FIG. 1B is a cross-sectional view of the cell culture plate according to an exemplary embodiment of the present invention.

[0084] FIG. 2 is a view illustrating, in detail, a main well formed in the cell culture plate according to an exemplary embodiment of the present invention.

[0085] FIG. 3 is a view illustrating a well plate, a base, and a cover of the cell culture plate according to an exemplary embodiment of the present invention ((A) a cover, (B) a base, and (C) a fixing means of a microplate and a base).

[0086] FIG. 4 is a view illustrating the high-speed mass imaging results of Example 1 and Comparative Example 1 ((A) Example 1, (B) Comparative Example 1).

[0087] FIG. 5 illustrates the results of culturing organoids in which Matrigel is included at 2 vol % according to an exemplary embodiment of the present invention and in which Matrigel is not used.

[0088] FIG. 6 illustrates the results of immunofluorescence staining of organoids including Matrigel at 2 vol % according to an exemplary embodiment of the present invention and in which Matrigel is not used.

[0089] FIG. 7A is a set of photographs illustrating the high-speed mass imaging results of Example 1, and FIG. 7B is a graph illustrating the area of the organoids cultured in Example 1.

[0090] FIG. 8A is a set of photographs illustrating the high-speed mass imaging results of Comparative Example 1, and FIG. 7B is a graph illustrating the area of the organoids cultured in Comparative Example 1.

[0091] FIG. 9 illustrates imaging results (left) and an organoid size distribution (right) when colorectal cancer cells according to an exemplary embodiment of the present invention are cultured for 14 days.

[0092] FIG. 10 illustrates organoid stained images (left) and organoid survival rates (right) obtained by culturing colorectal cancer cells according to an exemplary embodiment of the present invention for 14 days.

[0093] FIGS. 11 and 12 are a set of photographs and a graph illustrating the high-speed mass imaging results of Examples 1 to 3, respectively ((A) Example 1, (B) Example 2, and (C) Example 3).

MODES OF THE INVENTION

[0094] Since the present invention may be modified into various forms and include various exemplary embodiments, specific exemplary embodiments will be illustrated in the drawings and described in detail in the Detailed Description. However, the description is not intended to limit the present invention to the specific exemplary embodiments, and it is to be understood that all the changes, equivalents, and substitutions belonging to the spirit and technical scope of the present invention are included in the present invention. When it is determined that the detailed description of the related publicly known art in describing the present invention may obscure the gist of the present invention, the detailed description thereof will be omitted.

EXAMPLES

Experimental Methods

[0095] 1. Production of Organoid

[0096] After an existing colorectal cancer organoid was put into a 15 ml tube from a plate by pipetting and the tube was centrifuged at 2000 rpm for 3 minutes, the supernatant was removed, and then a PBS washing process was performed once, and the tube was again centrifuged at 2000 rpm for 3 minutes. Then, the cells were treated with Accutase for 7 minutes and completely separated into single cells. These single cells were seeded in sub wells of a cell culture plate at about 100 cells/well, and an organoid was produced by culturing the single cells for a total of 14 days. In this case, the culture solution is a DMEM/F12-based culture solution, and B27, N2, GlutaMAX, penicillin streptomycin, nicotinamide, N-acetyl, gastrin, A-83-01, EGF, noggin, R-spondin1, and WNT3A are included in the corresponding culture solution, and organoids were produced under culture conditions in which Matrigel was not contained or 2 vol % of Matrigel was contained.

[0097] 2: Measurement of Size and Number of Organoids

[0098] The ImageJ program was used for the size analysis of the organoids. Specifically, by selecting a region of interest in a phase image and applying a threshold in the ImageJ program, a part, which was not needed, was overwritten and a part, which was not properly drawn, was filled with black. An area to which the threshold filled with black was applied was calculated using an outer periphery.

[0099] 3: Immunofluorescence Staining Method

[0100] LGR5, which is an organoid stem cell, was stained and confirmed through immunofluorescence staining. First, a standard-type organoid according to the present invention was stored in a 4% paraformaldehyde solution at room temperature for 1 hour, and then stained with PBS. Then, after the standard-type organoid was refrigerated in 15% sucrose for one day and in 30% sucrose for one day, a cryo-block was manufactured using liquid nitrogen. Using the manufactured cryo-block, the block was cut to a thickness of 10 μm, and the cut cross-section was attached to a slide glass. The slide glass was treated with 0.1% TritonX for 10 minutes, and then washed twice with PBS. After the slide glass was stored in 3% BSA at room temperature for 1 hour, the slide glass was washed twice with PBS, and then an LGR5 primary antibody was maintained at room temperature for 2 hours. After washing with PBS, a secondary antibody was treated at room temperature for 2 hours, a mounting solution was added thereto, and measurement was performed under a fluorescence microscope.

[0101] In the case of FIG. 10, the cultured standard-type organoid is taken out, and Live/Dead fluorescence staining is performed. In the case of fluorescent staining, 1 mM calcein and 2 mM EtdH-1 are respectively stored at 2 μl per 1 ml and 1 μl per 1 ml in an incubator for 30 to 60 minutes, and then measurement is performed under a fluorescence microscope.

EXAMPLES

Example 1

[0102] Organoids were produced by the method described in Experimental Method 1. Organoids were produced under a condition in which 2 vol % of Matrigel was contained in the culture solution (Example 1-1) and under a condition in which Matrigel was not contained (Example 1-2).

Example 2

[0103] Cells were cultured in the same manner as in Example 1-1, except that the cells were seeded in sub wells at about 200 cells/well.

Example 3

[0104] Cells were cultured in the same manner as in Example 1-1, except that the cells were seeded in sub wells at about 300 cells/well.

Comparative Example 1

[0105] Organoids were cultured in Matrigel, which is a method widely used in the related art, and high-speed mass imaging was performed. However, in the comparative example, a 96-well plate, which is a typically used cell culture plate, was used, and an organoid was produced by seeding cells in Matrigel.

EXPERIMENTAL EXAMPLES

Experimental Example 1. Analysis of Organoid Images

[0106] The cells cultured in Example 1-1 and Comparative Example 1 were photographed, and the sizes of cell spheres were compared. Spheroids were subjected to imaging by an automated plate device, and in this case, the device was allowed to perform imaging by automatically focusing. Image size analysis was performed using a macro program of the ImageJ program.

[0107] Moreover, the results are illustrated in FIG. 4. FIG. 4 is a view illustrating the high-speed mass imaging results of Example 1-1 and Comparative Example 1 ((A) Example 1-1, (B) Comparative Example 1).

[0108] Referring to FIG. 4, it was confirmed that in the case of Example 1-1, the diameters of the cells cultured on the cell culture plate of the present invention were almost uniform. Specifically, when cells were seeded in each sub well at 100 cells/well on average, a uniform organoid that could be comparatively analyzed could be manufactured. In this case, the error range for the organoid size was around 20 μm. Through this, it can be seen that a standard organoid can be produced using the organoid culture method according to the present invention.

[0109] In contrast, it could be confirmed that in the case of Comparative Example 1 using a well plate in the related art, cell spheres were formed to have different sizes. The error range of the size of organoids generated due to the growth of multiple cells in the dome morphology of one Matrigel appeared with a deviation of 150 μm or more, and the cells were grown while overlapping, such that it was impossible to perform uniform high-speed mass imaging and experiments.

[0110] The base and well plate of the present invention include a convex part and a concave part to fix each other, respectively, and the convex part and the concave part may be connected to each other for the base to firmly fix the well plate, showing that an image in the well plate can be uniformly captured.

[0111] In contrast, it can be seen that the size and shape of the organoids cultured in Comparative Example 1 are not uniform. This seems to make an analysis of images difficult because the focal deviation of the imaging is increased in the absence of a plate base.

[0112] Further, referring to FIG. 5, it was confirmed that when the cell culture plate of the present invention was used even without containing Matrigel, organoids were formed well.

[0113] FIG. 6 confirms the expression level by staining LGR5, which is the most important marker for the formation of colorectal cancer organoids in order to confirm whether the cultured organoids were successfully formed. In addition, the presence of a colon-specific structure in the colorectal cancer organoids of a low-concentration Matrigel group or a group in which Matrigel was not used, formed through F-actin staining, was confirmed.

Experimental Example 2. Manufacture of Standard-Type Organoid

[0114] The cells cultured in Example 1-1 and Comparative Example 1 were subjected to high-speed mass imaging.

[0115] The organoids produced in Example 1-1 and Comparative Example 1 were subjected to imaging by an automated plate device, and in this case, the device was allowed to perform imaging by automatically focusing. Image size analysis was performed using a macro program of the ImageJ program.

[0116] Moreover, the results are illustrated in FIGS. 7 and 8.

[0117] FIG. 7A is a set of photographs illustrating the high-speed mass imaging results of Example 1-1, FIG. 7B is a graph illustrating the area of the organoids cultured for a certain period of time in Example 1, FIG. 8A is a set of photographs illustrating the high-speed mass imaging results of Comparative Example 1, and FIG. 7B is a graph illustrating the area of the organoids cultured for a certain period of time in Comparative Example 1.

[0118] Referring to FIG. 7, it can be confirmed that when the organoid prepared in Example 1-1 was subjected to automatic imaging, imaging can be performed without a large error because an imaging height is uniform, and due to this fact, an error range is very small when the actual area is measured.

[0119] In particular, when the organoid is cultured using the cell culture plate of the present invention, the organoid is cultured in a uniform size. That is, standardization is possible. As a result of standardization, the focus was automatically determined during image measurement, and a deviation for the measured height was minimized by a connector structure. Accordingly, when the screening image is measured, the deviation is around 20 μm, which is shown to be very small.

[0120] Referring to FIG. 8, it could be confirmed that in the case of Comparative Example 1, the organoids grew while overlapping each other, and it could be seen that the size and distribution position of the organoids were different, and thus could not be standardized. Therefore, during the measurement of the screening image, an error range was up to 150 μm was exhibited, which is large.

[0121] The results as described above are because when organoids are cultured by methods in the related art, since the organoids are grown randomly in Matrigel, it is difficult to uniformly culture a desired organoid, and a measured height is also variable, and thus, there is a limit in which it is difficult to apply the measured height to organoid screening imaging. Therefore, when the area of the organoid cultured for a certain period of time is analyzed, the deviation appears to be very large.

[0122] In the case of FIG. 9, in the standard-type organoids produced as a whole, the diameter of each organoid was measured using the ImageJ program. A total of 864 wells were subjected to high-speed mass imaging, it was confirmed that a uniform diameter was obtained by analyzing each of the images by ImageJ.

[0123] FIG. 9 illustrates the imaging results and the size of the organoid when the colorectal cancer cells of Example 1-1 were cultured for 14 days. It can be seen that it is possible to manufacture a standard organoid because the size of the organoids is uniform at 300 to 50 μm.

[0124] FIG. 10 is a set of image photographs illustrating the organoids according to Examples 1-1 and 1-2 over time (left) and a set of organoid viabilities according to Example 1-1 (right). In the case of FIG. 10, it was confirmed how much the cultured organoids were actually maintained by Live/Dead staining of the cultured organoids. First, the cultured standard-type organoid was washed with PBS, and then incubated with Accutase for about 10 minutes.

[0125] And then, the standard-type organoid was fragmented into single cells, and after Live/Dead reagents Calcein and EtdH-1 were stored in an incubator for about 30 to 60 minutes, how much the reagents were each present by placing the reagents in a C-Chip was confirmed under a fluorescence microscope, and the results thereof are illustrated in FIG. 10.

[0126] Through FIG. 10, it can be seen that organoid formation occurs very well in the absence of Matrigel, and that organoid viability is very high when colorectal cancer cells are cultured for 14 days.

[0127] FIG. 11 is a set of photographs illustrating the results of high-speed mass imaging of Examples 1-1, 2, and 3, and FIG. 9 illustrates the imaging results of colon cancer cells cultured for 14 days in Examples 1-1, 2 and 3 ((A) Example 1-1, (B) Example 2, and (C) Example 3).

[0128] Referring to FIG. 11A, when cells were seeded in each sub well at 100 cells/well on average, a uniform organoid that could be analyzed and compared could be manufactured (error range: around 20 μm).

[0129] In contrast, referring to FIGS. 11B and 11C, it can be confirmed that when cells are seeded in sub wells at more than 100 cells/well, organoids overflow from the sub wells, and thus non-uniform organoids are produced.

[0130] FIG. 12 is a graph illustrating the results of high-speed mass imaging of Examples 1-1, 2, and 3, and FIG. 12 illustrates the results of colon cells cultured for 7 days or 14 days in Examples 1-1, 2, and 3 ((A) Example 1-1, (B) Example 2, and (C) Example 3).

[0131] Referring to FIG. 12, it shows that the most desirable organoids can be manufactured when cells are cultured at 100 cells/well on average for 14 days. That is, it is considered that an optimum organoid can be differentiated and grown when cells are cultured at 100 cells/well or less on average are cultured for 14 days. In contrast, it could be confirmed that the organoid performance deteriorated when the number of cells seeded in the sub well was increased and the number of culture days was reduced.

[0132] For reference, the dotted line in FIG. 12 means the maximum space of the sub well of the cell culture plate of the present invention, and means a space in which cells can be cultured. This is considered to be capable of culturing cells at 100 cells/well or less on average.

[0133] Although a specific part of the present invention has been described in detail, it will be obvious to those skilled in the art that such a specific description is just a preferred embodiment and the scope of the present invention is not limited thereby. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

DESCRIPTION OF REFERENCE NUMERALS AND SYMBOLS

[0134] 100: Well plate [0135] 101: Step [0136] 110: Main well [0137] 120: Sub well [0138] 121: Recessed part [0139] 130: Space part [0140] 140: Concave part [0141] 200: Connector for large-capacity and high-speed HCS [0142] 210: Base [0143] 220: Cover [0144] 240: Convex part