Method for producing standard organoids

Abstract

The present invention provides a method for producing standard organoids.

Description

Technical Field

[0001] The present invention relates to a method for producing standard organoids. More specifically, it relates to a method for producing organoids of uniform size.

Background Art

[0002] Organoids, also called "organ-like" or "mini-organs", are organ-specific cell masses produced by re-aggregating and recombining cells isolated from stem cells or organ-derived cells by a three-dimensional culture method. Organoids contain organ-specific cells as models, can reproduce specific functions of organs, and can be spatially organized in a shape similar to that of actual organs. It has been reported that patient-derived tumor organoids can show the characteristics of a patient's cancer cells and cancer tissues as they are, and can reproduce the genetic mutation characteristics of the patient's cancer tissues.

[0003] Organoids can be used in the fields of cell therapy, tissue engineering, new drug development, toxicology, and precision medicine. In order to increase the utilization rate of organoids, a method for quantifying and analyzing a large number of comparable organoids is required. However, there has been no method for quantitatively culturing organoids so far. The reason is that after Matrigel, which is the most important factor in growing organoids, is solidified in a dome shape on the bottom, and organoids are grown therein, the organoids grow irregularly.

[0004] In addition, organoids grown in Matrigel in this way may sometimes grow overlapping each other in a three-dimensional support, so it is clear that there are limitations.

[0005] Also, recently, high-throughput screening technology combined with more stable physiologically patient-derived organoids has been developed for use in early drug discovery programs and toxicity screening.

[0006] Republic of Korea Patent No. 10-1756901 (Patent Document 1) discloses a cell culture chip capable of culturing three-dimensional tissue cells. The cell culture chip of Patent Document 1 has a first culture part, a second culture part, and a third culture part formed in layers, respectively, and the degree of progress of cell growth can be confirmed for each layer. However, the cell culture chip of Patent Document 1 has a problem that organoids cannot be obtained at a high yield.

[0007] In addition, although pipetting operations for exchanging the culture solution may be performed during cell culture, in the case of a Corning spheroid microplate capable of three-dimensional cell culture, it may affect the spheroids or organoids during cell culture, and the spheroids or organoids may be sucked up or their positions may change during pipetting operations, so there is a problem that is not favorable for the cell culture environment.

[0008] Therefore, the present inventors have completed the present invention through repeated research on standard-type organoids having a uniform size without using or minimizing the use of a hydrogel (for example, Matrigel) based on the extracellular matrix.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] An object of the present invention is to provide a method for producing standard-type organoids.

[0011] Another object of the present invention is to provide standard organoids having a uniform size and similar functionality of each organoid produced by the above method.

[0012] However, the technical problems to be solved by the present invention are not limited to the above-described problems, and other problems not mentioned can be clearly understood by those of ordinary skill in the art from the following description.

Means for Solving the Problems

[0013] In order to solve the above problems, the present invention A method for producing an organoid including a step of culturing cells in a three-dimensional cell culture plate to form an organoid, In the step of forming the organoid, the cell culture plate contains 0 to 2% by volume of a hydrogel based on an extracellular matrix, The three-dimensional cell culture plate A well plate including a plurality of main wells and a plurality of sub wells formed at the bottom of each main well, into which a cell culture solution is injected and the bottom surface includes a concave portion, and a high content screening (HCS) connector for supporting the well plate, The high content screening (HCS) connector includes a base having fixing means detachably fixed to the lower end of the well plate and a cover disposed on the upper part of the well plate and coupled to the base. The main well has a step formed so that a predetermined portion is tapered, and the step has an inclination angle (θ) in the range of 10 to 60° with respect to the wall of the main well.

[0014] The cells may be normal cells or cancer cells.

[0015] The cells may be single cells.

[0016] The cells can be obtained by separating them from normal tissues, cancer tissues, or organoids that have already been prepared. Since the method of cellulating tissues and separating them into single cells is a known technique, specific description thereof is omitted.

[0017] The extracellular matrix-based hydrogel may be Matrigel (trade name).

[0018] The size of the organoids may be 300 to 500 μm in diameter.

[0019] In the present invention, the term "standard organoid" refers to a uniform organoid having a size of 300 to 500 μm in diameter.

[0020] The size of the organoids produced in the present invention is in the range of 300 to 500 μm, which is a size particularly optimized for cancer diseases.

[0021] As will be described in detail below, standard organoids can be mass-produced using the three-dimensional cell culture plate of the present invention.

[0022] The sub-wells of the three-dimensional cell culture plate are formed with inclined surfaces so as to be tapered toward the recesses. The upper end diameter of the sub-well 120 is in the range of 3.0 to 4.5 mm, the upper end diameter of the recess 121 is in the range of 0.45 to 1.5 mm, the inclined surface (θ2) of the sub-well and the recess is in the range of 40 to 50°, and the ratio of the length of the diameter of the sub-well to the diameter of the recess may be in the range of 1:0.1 to 0.5.

[0023] The individual volume of the main wells of the three-dimensional cell culture plate is in the range of 100 to 300 μl, the individual volume of the recesses is in the range of 20 to 50 μl, and the individual volume ratio of the main wells to the recesses may be on average 1:0.1 to 0.5.

[0024] The main well includes a space between the step and the sub-well, the height (ah) of the space is in the range of 2.0 to 3.0 mm on average, the height (bh) of the sub-well is in the range of 1.0 to 2.0 mm on average, and the ratio of the height of the space to the sub-well (ah:bh) may be in the range of 1:0.3 to 1.

[0025] The somatic cells can be seeded in the sub-wells of the cell culture plate at 100 to 1000 cells / well.

[0026] Hereinafter, the present invention will be described in detail.

[0027] Since the present invention can be modified in various ways and can have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the detailed description.

[0028] However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, or alternatives included in the spirit and technical scope of the present invention. In the description of the present invention, if it is determined that a detailed description of related known technologies may obscure the gist of the present invention, the detailed description will be omitted.

[0029] The terms used in this application are merely used to explain specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise.

[0030] In the present invention, terms such as "including" or "having" are used to specify the existence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and should not be construed as precluding the possibility of the existence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0031] Generally, when culturing organoids, hydrogels are used to play the role of the extracellular matrix. For example, after solidifying Matrigel in a dome shape on the bottom of a cell culture plate, organoids are grown therein. However, the organoids grow irregularly in size and shape, and thus there are problems that their functions are expressed in a fragmented manner and it is difficult to standardize them.

[0032] The present invention produces organoids using a three-dimensional cell culture plate that does not contain or contains a minimal amount of a hydrogel based on the extracellular matrix. A specific description of the three-dimensional cell culture plate of the present invention is as follows.

[0033] In one embodiment, the present invention uses a three-dimensional cell culture plate including the following.

[0034] The three-dimensional cell culture plate includes a well plate including a plurality of main wells and a plurality of sub wells formed at the lower part of each of the main wells, into which a cell culture solution is injected and which include recesses on the bottom surface, and a high content screening (HCS) connector for supporting the well plate. The high content screening (HCS) connector includes a base provided with fixing means detachably fixed to the lower end of the well plate and a cover disposed on the upper part of the well plate and coupled to the base. The main well is formed with a step so as to be tapered at a predetermined site, and the step has an inclination angle (θ) in the range of 10 to 60° with respect to the wall of the main well.

[0035] In the case of a conventional 96-well plate, in order to evaluate the efficacy of a high-yield drug, it was necessary to conduct experiments and analyses more than once, which was time-consuming and costly. Furthermore, pipetting operations for exchanging the culture medium are often performed during cell culture. However, in the case of a conventional Corning spheroid microplate, it may affect the spheroids or organoids during cell culture, such that the spheroids or organoids may be sucked up or their positions may change during pipetting operations, which is an unfavorable problem for the cell culture environment.

[0036] Therefore, the present invention has been made to solve the above-described problems, and includes a plurality of sub-wells in a plurality of main wells formed in a well plate, capable of producing high-yield spheroids / organs, and includes a connector for high content screening (HCS) that supports the plate, and provides a cell culture plate that can narrow the tolerance during high-capacity high-speed image capture and uniformly capture images within the well plate. Furthermore, due to the step of the main well, a cell culture plate is provided in which the cells to be cultured are minimally affected by the pipetting operation during medium replacement.

[0037] Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Prior to the description, terms or words used in this specification and the claims should not be construed as being limited to the ordinary or dictionary meanings, and the inventor should interpret them as meanings and concepts consistent with the technical idea of the present invention based on the principle that the concept of the terms can be appropriately defined in order to best explain his own invention.

[0038] Therefore, it should be understood that the examples described in this specification and the configurations shown in the drawings are only the most preferred example of the present invention and do not substitute all of the technical ideas of the present invention, and there may be various equivalents and modifications that can replace them at the time of this application.

[0039] FIG. 1(a) is a front view of a cell culture plate according to an embodiment of the present invention, FIG. 1(b) is a cross-sectional view of a cell culture plate according to an embodiment of the present invention, FIG. 2 is a diagram showing in detail a main well formed in a cell culture plate according to an embodiment of the present invention, and FIG. 3 is a diagram showing a well plate, a base, and a cover of a cell culture plate according to an embodiment of the present invention ((a) cover, (b) base, (c) fixing means for a microplate and a base).

[0040] Hereinafter, a cell culture plate according to an embodiment of the present invention will be described in detail with reference to FIGS. 1 to 3.

[0041] As shown in FIGS. 1 to 3, a cell culture plate 10 according to an embodiment of the present invention includes a well plate 100 including a plurality of main wells 110 and a plurality of sub-wells 120 formed at the lower part of each of the main wells 110, into which a cell culture solution is injected and which include recesses 121 on the bottom surface, and a high content screening (HCS) connector 200 for supporting the well plate 100.

[0042] First, with reference to FIGS. 1 and 2, the well plate 100 according to an embodiment of the present invention will be described in detail.

[0043] The well plate 100 is formed in a plate shape by plastic injection molding through a mold. Such a plastic injection mold can be manufactured by giving a repeating pattern to the main well 110 as a well structure, thereby reducing the production cost and facilitating the enlargement of the size by microfabrication. As a result, mass production of cells is easy, and it can be deformed into various sizes according to the needs of users and used.

[0044] A plurality of the main wells 110 are formed in the well plate 100, and each main well 110 includes a step 101. The step 101 is formed at a predetermined part of the main well 110. More specifically, the step 101 may be formed at a position of 1 / 3 to 1 / 2 of the total length of the main well 110, or may be formed at a position of 1 / 3 to 1 / 2 from the lower end of the main well 110.

[0045] Conventionally, when culturing cells in a microplate, pipetting operations for exchanging the culture medium may be performed. In this case, however, it may affect the spheroids or organoids during cell culture, and the spheroids or organoids may be sucked up or their positions may change during the pipetting operation. Therefore, there was a problem that was not favorable for the cell culture environment. The step 101 is provided to prevent such problems.

[0046] The step 101 may be a space to which a pipette is applied. Specifically, it may have an inclination angle (θ) in the range of 10° to 60° with respect to the wall of the main well 110. Or, it may have an inclination angle in the range of 20° to 50°, and preferably may have an inclination angle in the range of 30° to 45°. If the inclination angle of the step 101 is less than 10°, the space for applying the pipette is too small due to the small inclination angle in the main well 110, and when sucking the culture medium in the main well 110, the pipette may slip inside the sub-well 120, and the spheroids or organoids may be sucked up or their positions may change. Also, when the inclination angle (θ) exceeds 60°, although the space for applying the pipette is ensured, the inclination angle of the step 101 may be too large and it may be difficult to sufficiently suck the culture medium. When seeding cells into the sub-well 120, there may be a problem that the cells do not enter all the sub-wells 120 and are seeded on the step 101. Therefore, it is preferable to have an inclination angle within the above-described range.

[0047] In addition, the main well 110 according to an embodiment of the present invention can include a space portion 130 between the step 101 and the sub-well 120 described later. Specifically, the space portion 130 is a space into which a culture solution is injected, and is a space in which cells inside the sub-well 120 can share the same culture solution.

[0048] More specifically, the height (ah) of the space portion 130 may be in the range of 2.0 to 3.0 mm on average, may be in the range of 2.2 to 2.8 mm, or may be in the range of 2.3 to 2.7 mm on average. Further, the height (bh) of the sub-well 120 may be in the range of 1.0 to 2.0 mm on average, or may be in the range of 1.2 to 1.8 mm on average.

[0049] For example, the height (ah) of the space portion 130 may be 2.5 mm on average, and the height (bh) of the sub-well may be 1.5 mm on average.

[0050] At this time, the ratio of the height of the space portion to the sub-well 120 (ah:bh) may be in the range of 1:0.3 to 1. More specifically, the ratio of the height of the space portion to the sub-well 120 (ah:bh) may be 1:0.4 to 0.9 or 1:0.5 to 0.8. If the height of the sub-well 120 is less than 1:0.3 with respect to the height of the space portion, when exchanging the medium in the sub-well 120, the cells being cultured inside may escape even with a small force. If the height of the sub-well 120 exceeds 1:1 with respect to the height of the space portion, the culture solution necessary for the cells may not be sufficiently converted, and apoptosis may be induced. Therefore, it is preferable that the space portion 130 and the sub-well 120 have the above-described height range and height ratio.

[0051] Next, the sub-well 120 is formed at each lower portion of the main well 110 and includes a recess 121 on the bottom surface. As a specific aspect, the sub-well 120 can include a plurality of them at the lower portion of the main well 110.

[0052] The sub-wells 120 included in the lower part of the main well 110 are the same in size and shape, whereby spheroids and organoids under uniform conditions can be generated.

[0053] The sub-well 120 can form an inclined surface so as to be tapered toward the recess 121. Specifically, the upper end portion of the sub-well 120 may be such that the horizontal width becomes smaller as it descends with respect to the vertical direction. For example, the upper end portion of the sub-well 120 can be in the shape of an inverted pyramid. In the illustrated embodiment, the upper end portion of the sub-well 120 can be in a shape such that the horizontal width becomes smaller as it descends vertically, such as in the shape of a pyramid or a funnel. In particular, by including a plurality of sub-wells 120 that are the same in size and shape, the cell culture plate can generate a large amount of spheroids or organoids under uniform conditions.

[0054] As a specific mode, one main well 110 can include 4 to 25 sub-wells 120 of the same size, and the entire microplate 100 can include 96 to 1,728 sub-wells 120. Thereby, it is possible to be identical and precisely control the size.

[0055] In addition, the sub-well 120 includes a recess 121, and a space is formed at the lower end of the recess so that the recess 121 can culture 3D spheroids or organoids. Specifically, the recess 121 may be in a "U shape", "V shape", or "Ц shape". For example, the recess 121 may be in a "U shape".

[0056] The upper end diameter of the sub-well 120 may be in the range of 3.0 to 4.5 mm, may be 3.5 to 4.3 mm, or may be an average of 4 mm. Further, the upper end diameter of the recess 121 may be 0.45 to 1.5 mm, may be 0.5 to 1.0 mm, or may be an average of 0.5 mm.

[0057] In addition, the ratio of the diameter of the sub-well 120 to the length of the diameter of the recess 121 may be in the range of 1:0.1 to 0.5, and preferably the ratio of the diameter of the sub-well 120 to the length of the diameter of the recess 121 may be 1:0.12.

[0058] When the upper end diameter of the recess 121 is less than 0.1 with respect to the upper end diameter 1 of the sub-well 120, it is not possible to sufficiently secure the cell culture space of the recess 121, and when exchanging the culture solution, there may be a problem that cells may escape even with a small force. When the upper end diameter of the recess 121 exceeds 0.5 with respect to the upper end diameter 1 of the sub-well 120, there may be a problem that a sufficient amount of culture solution required for the cells cannot be exchanged and it is difficult to culture stably.

[0059] On the other hand, the inclined surfaces of the sub-well 120 and the recess 121 may have an inclination angle (θ2) of 40 to 50°, an inclination angle (θ2) in the range of 42 to 48°, an inclination angle (θ2) in the range of 43 to 47°, or an average inclination angle (θ2) of 45° with respect to the wall of the main well.

[0060] The above-described sub-well 120 has the advantage that cell culture of 100 to 1000 cells / well or less is possible and the size of the spheroid can be stably controlled.

[0061] Furthermore, the individual volume of the main well 110 according to an embodiment of the present invention is in the range of 100 to 300 μl, the individual volume of the recess 121 is in the range of 20 to 50 μl, and the individual volume ratio of the main well 110 to the recess 121 is characterized by being on average 1:0.07 to 0.5. Preferably, the individual volume of the main well 110 according to the above embodiment is in the range of 250 to 300 μl, the individual volume of the recess may be in the range of 25 to 35 μl, and the individual volume ratio of the main well 110 to the recess 121 may be on average 1:0.11.

[0062] Specifically, when the individual volume of the main well 110 is less than 100 μl, there may be a problem that a sufficient amount of culture solution cannot be accommodated during cell culture, and when it exceeds 300 μl, the culture efficiency may decrease.

[0063] In addition, the recess 121 is a space in which the cells are actually cultured, and if the volume is less than 20 μl, the cell culture space is insufficient, which may cause a problem of cells escaping, and if the volume exceeds 50 μl, there is a risk of a problem of it being difficult to stably culture the cells, etc. Therefore, it is preferable that the main well 110 and the recess 121 have volumes in the above-mentioned ranges.

[0064] Due to the configuration of the cell culture plate of the present invention described above, cells are maintained in a spheroid state even without containing a hydrogel, i.e., without coating the cell culture plate with a hydrogel, reprogramming into induced pluripotent stem cells occurs with high efficiency, and the morphology and function of the cells are sustained after reprogramming.

[0065] The cell culture plate 10 according to an embodiment of the present invention includes a connector 200 for high content screening (HCS) that supports a well plate 100. Here, the connector 200 for high content screening (HCS) refers to a connector 200 that is attached to an HCS (High Contents Screening) system, and specifically, the connector 200 may refer to a base 210 and a cover 220 in the present invention.

[0066] More specifically, the connector for high content screening (HCS) includes a base 210 having fastening means 140, 240 detachably attached to the lower end of a well plate 100, and a cover 220 disposed on the upper part of the well plate 100 and coupled to the base 210. The upper end of the base 210 and the lower end of the well plate 100 include fastening means 140, 240 detachably attached to each other.

[0067] At this time, the base may include a convex portion 240 for supporting the well plate 100, and the well plate 100 may include a concave portion 140 facing the convex portion 240 of the base 210. By fixing the well plate 100 with the fixing means, an image can be uniformly taken during screening.

[0068] The base can be made of polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, polyester, polyvinyl chloride, polyurethane, polycarbonate, polyvinylidene chloride, polytetrafluoroethylene, polyether ether ketone, or polyether imide material, but is not necessarily limited thereto.

[0069] The well plate can be made of polydimethylsilicone, high-fat modified silicone, methylchlorophenyl silicone, alkyl-modified silicone, methylphenyl silicone, silicone polyester, or amino-modified silicone material, but is not necessarily limited thereto.

Effects of the Invention

[0070] The method for producing an organoid of the present invention is economical because the use of Matrigel can be minimized. In addition, the method for producing an organoid according to the present invention provides an effect of mass-producing standard organoids.

[0071] According to the present invention, when performing high-throughput drug screening, organoids are formed in a uniform size that can be compared with each other, different from the prior art, so that the effect of the drug and quantitative analysis are possible. Thereby, a drug suitable for each modified gene specifically can be selected, and more effective drug treatment is possible.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0084] The present invention can be modified into various forms and can have various embodiments. Hereinafter, specific examples are illustrated in the figures and will be described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that all modifications, equivalents, and alternatives included in the spirit and technical scope of the present invention are included. In the description of the present invention, when it is determined that a specific description of related known technologies obscures the gist of the present invention, the detailed description thereof will be omitted.

Examples

[0085] Experimental Method

[0086] 1: Preparation of Organoids

[0087] Existing colorectal cancer organoids were pipetted into a 15-ml tube with 1 ml of medium on a plate, centrifuged at 2000 rpm for 3 minutes, and then the supernatant was removed. After that, the PBS washing process was carried out once, and it was centrifuged at 2000 rpm for 3 minutes in the same way. Then, it was treated with acuutase for 7 minutes to completely separate into single cells. These single cells were seeded at about 100 cells / well in the sub-wells of a cell culture plate and cultured for a total of 14 days to prepare organoids. At this time, the culture medium was a culture medium based on DMEM / F12, and this culture medium contained B27, N2, GlutaMAX, penicillin streptomycin, Nictoiamide, N-acetyl, gastrin, A-83-01, EGF, noggin, R-spondin1, and WNT3A. The organoids were prepared under conditions without or containing 2% by volume of Matrigel.

[0088] 2: Measurement of the size and number of organoids

[0089] Analysis of the size of the organoids was performed using the Image J program. Specifically, the region of interest was selected from the phase image, a threshold was applied with the Image J program, unnecessary parts were overwritten in white, and parts that were not properly drawn were filled in black. The area to which the threshold filled in black was applied was determined using the outer perimeter.

[0090] 3: Immunofluorescence staining method

[0091] The stem cells of the organoids, LGR5, were stained and confirmed by immunofluorescence staining. First, the standard organoids according to the present invention were stored in a 4% paraformaldehyde solution at room temperature for 1 hour, and then stained with PBS. Thereafter, they were stored refrigerated in 15% sucrose for 1 day and in 30% sucrose for 1 day, and then cryo-blocks were prepared using liquid nitrogen. Using the prepared cryo-blocks, they were cut to a thickness of 10 μm, and the cut surfaces were attached to glass slides. After treatment with 0.1% tritonX for 10 minutes, they were washed twice with PBS. After storage in 3% BSA at room temperature for 1 hour, after two PBS washes, the LGR5 primary antibody was held at room temperature for 2 hours. After PBS washing, the secondary antibody was treated at room temperature for 2 hours, a mounting solution was added, and measurement was performed with a fluorescence microscope.

[0092] In the case of Fig. 10, the cultured standard organoids are taken out and Live / Dead fluorescence staining is performed. In the case of fluorescence staining, Calcein 1 mM is 2 μl per 1 ml, and EtdH-1 2 mM is 1 μl per 1 ml. After storing in an incubator for 30 to 60 minutes, measurement is performed with a fluorescence microscope.

[0093] (Example)

[0094] Example 1.

[0095] Organoids were prepared by the method described in the above Experimental Method 1. Organoids were prepared under the condition that Matrigel was contained in the culture solution at 2% by volume (Example 1-1) and under the condition that Matrigel was not contained (Example 1-2).

[0096] Example 2.

[0097] 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.

[0098] Example 3.

[0099] Cells were cultured in the same manner as in Example 1-1, except that the cells were seeded into sub-wells at about 300 cells / well.

[0100] Comparative Example 1.

[0101] Conventionally, organoids were cultured in Matrigel, a widely used method, and high-speed large-scale imaging was performed. However, in the comparative example, a 96-well plate, which is a commonly used cell culture plate, was used, and cells were seeded into Matrigel to prepare organoids.

[0102] (Experimental Example)

[0103] Experimental Example 1. Analysis of organoid images

[0104] The cells cultured in Example 1-1 and Comparative Example 1 were photographed, and the sizes of the cell spheres were compared. The spheroids were imaged with an automated plate device, and at this time, the device was set to automatically focus. The analysis of the image size was performed using the macro program of the imageJ program.

[0105] Also, the results are shown in Fig. 4. It is a figure showing the high-speed large-scale imaging results of Example 1-1 and Comparative Example 1 ((a) Example 1-1, (b) Comparative Example 1).

[0106] Referring to Fig. 4, in the case of Example 1-1, it was confirmed that the diameters of the cells cultured in the cell culture plate of the present invention were almost uniform. Specifically, when the cells were seeded into each sub-well at an average of 100 cells / well, uniform organoids that could be comparatively analyzed could be prepared. At this time, the error range with respect to the size of the organoids was within about 20 μm. Thus, it can be seen that the preparation of standard organoids is possible using the organoid culture method according to the present invention.

[0107] In the case of Comparative Example 1 using a conventional well plate on the reverse side, it was confirmed that the sizes of the cell spheres were formed differently. This is because multiple cells grow within one dome-shaped Matrigel, resulting in a variation in the error range of the size of the generated organoids of 150 μm or more. There was also a possibility that the cells grew overlapping each other, making uniform high-speed large-scale imaging and experiments impossible.

[0108] In order to fix the base of the present invention and the well plate to each other, each includes a concave portion and a convex portion, and the concave portion and the convex portion are coupled to each other so that the base can firmly fix the well plate, indicating that an image within the well plate can be uniformly photographed.

[0109] On the other hand, it can be seen that the sizes and shapes of the organoids cultured in Comparative Example 1 are not uniform. This is presumably because when there is no plate base, the deviation of the imaging focus becomes large, making it difficult to analyze the image.

[0110] Also, as shown in FIG. 5, it was confirmed that when the cell culture plate of the present invention was used without containing Matrigel, organoids were well formed.

[0111] FIG. 6 shows that in order to confirm that the cultured organoids were normally formed, LGR5, which is the most important marker for the formation of colorectal cancer organoids, was stained and the expression level was confirmed. Also, it was confirmed that there are colon-specific structures in the colorectal cancer organoids of the low-concentration Matrigel group or the Matrigel-free group formed by F-actin staining.

[0112] Experimental Example 2. Preparation of standard organoids

[0113] The cells cultured in Example 1-1 and Comparative Example 1 were subjected to high-speed large-scale imaging. The organoids prepared in Example 1-1 and Comparative Example 1 were imaged using an automated plate instrument, and at this time, the instrument was automatically focused. The analysis of the image size was performed using the macro program of the imageJ program.

[0114] And the results are shown in FIGS. 7 and 8.

[0115] FIG. 7(a) is a photograph showing the high-speed mass imaging result of Example 1-1, FIG. 7(b) is a graph showing the area of the organoid cultured in Example 1 for a certain period, FIG. 8(a) is a photograph showing the high-speed mass imaging result of Comparative Example 1, and FIG. 8(b) is a graph showing the area of the organoid cultured in Comparative Example 1 for a certain period.

[0116] Referring to FIG. 7, when the organoid prepared in Example 1-1 was automatically imaged, it was confirmed that imaging was possible with a uniform height and very little error in imaging, and thus the error range was very small when the actual area was measured.

[0117] In particular, when culturing organoids using the cell culture plate of the present invention, the organoids are cultured in a uniform size. That is, standardization is possible. As a result of standardization, the focus was automatically adjusted during image measurement, and the deviation of the measurement height was minimized by the connector structure. Thereby, the deviation during the measurement of the screening image is very small, within about 20 μm.

[0118] Referring to FIG. 8, in the case of Comparative Example 1, it was confirmed that the organoids grew overlapping each other, and the size and distribution position of the organoids were different from each other, indicating that standardization was impossible. Therefore, it was shown that the error range was as large as about 150 μm at maximum when measuring the screening image.

[0119] Such results are because when culturing organoids by conventional methods, the organoids grow randomly within Matrigel, making it difficult to uniformly culture the desired organoids, and the height for measurement is also variable, thus there are limitations in applying them to organoid screening imaging. Therefore, when analyzing the area of organoids cultured for a certain period, a very large variation is shown.

[0120] In the case of Figure 9, for the standard-type organoids prepared overall, the diameter of each organoid was measured using the Image J program. A total of 864 wells were subjected to high-speed large-scale imaging and analyzed with ImageJ respectively, and it was confirmed that a uniform diameter could be obtained.

[0121] Figure 9 shows the imaging results and the size of organoids when the colorectal cancer cells of Example 1-1 were cultured for 14 days. Since the size of the organoids is uniform from 300 to 50 μm, it can be seen that it is possible to prepare standard organoids.

[0122] Figure 10 shows the image photos (left) of the organoids over time according to Examples 1-1 and 1-2 and the organoid survival rate according to Example 1-1 (right). In the case of Figure 10, the cultured organoids were subjected to Live / Dead staining to confirm the extent to which the actually cultured organoids were maintained. First, the cultured standard-type organoids were washed with PBS and then incubated with Accutase for about 10 minutes. Then, they were fragmented into single cells, incubated with Calcein and EtdH-1, which are Live / Dead reagents, in an incubator for about 30 to 60 minutes, and then the results of confirming the presence of each in a C-Chip with a fluorescence microscope are shown.

[0123] Through Figure 10, it can be seen that organoids can be formed very well even without Matrigel, and when cultured for 14 days, it can be seen that the organoid survival rate is very high.

[0124] Figure 11 is a photograph showing the high-speed mass imaging results of Examples 1-1, 2, and 3, and Figure 9 shows the imaging results when culturing colorectal cancer cells for 14 days in Examples 1-1, 2, and 3 ((a) Example 1-1, (b) Example 2, (c) Example 3).

[0125] Referring to Figure 11(a), when cells were seeded at an average of 100 cells / well in each sub-well, uniform organoids that could be comparatively analyzed were able to be produced (error range: within about 20 μm).

[0126] On the contrary, referring to Figure 11(b) and Figure 11(c), when cells were seeded at 100 cells / well or more in the sub-well, it was confirmed that the organoids overflowed from the sub-well and non-uniform organoids were generated.

[0127] Figure 12 is a graph showing the high-speed mass imaging results of Examples 1-1, 2, and 3, and Figure 12 shows the results when culturing colorectal cancer cells for 7 days or 14 days in Examples 1-1, 2, and 3 ((a) Example 1-1, (b) Example 2, (c) Example 3).

[0128] Referring to Figure 12, it shows that when cells were cultured at an average of 100 cells / well for an average of 14 days, the most preferable organoids could be produced. That is, when culturing cells of 100 cells / well or less for 14 days, it is considered that optimal organoids can differentiate and grow. On the other hand, when increasing the number of cells seeded in the sub-well and reducing the number of culture days, it was confirmed that the performance of the organoids decreased.

[0129] For reference, the dashed line in Figure 12 means the maximum space of the sub-well of the cell culture plate of the present invention and means the space where cells can be cultured. It is judged that cells of 100 cells / well or less can be cultured.

[0130] The specific parts of the content of the present invention have been described in detail above. However, for those with ordinary knowledge in this technical field, such specific technologies are merely preferred embodiments, and it is obvious that the scope of the present invention is not limited thereby. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Explanation of Reference Numerals

[0131] 100: Well plate 101: Step 110: Main well 120: Sub-well 121: Concave portion 130: Space portion 140: Drop-in portion 200: Connector for high-content screening 210: Base 220: Cover 240: Convex portion

Claims

1. A method for producing an organoid, comprising: a step of culturing cells in a three-dimensional cell culture plate to form an organoid, in the organoid formation step, the cell culture plate contains 0 to 2% by volume of a hydrogel based on an extracellular matrix, the three-dimensional cell culture plate includes a well plate including a plurality of main wells and a plurality of sub-wells formed at the bottom of each of the main wells, into which a cell culture solution is injected and which include recesses on the bottom surface, and a high-content screening (HCS) connector for supporting the well plate, the high-content screening (HCS) connector includes a base detachably fixed to the lower end of the well plate by fixing means and a cover disposed on the upper part of the well plate and coupled to the base, the main well has a step and includes a space between the step and the sub-well, the sub-well has an inclined surface formed to be tapered toward the recess in which cells are cultured, the step has an inclination angle (θ1) in the range of 10 to 60° with respect to the wall of the main well, the inclined surface (θ2) between the sub-well and the recess is in the range of 40 to 50°. A method for producing an organoid.

2. The hydrogel based on the extracellular matrix is Matrigel. The method for producing an organoid according to Claim 1.

3. The cells are normal cells or cancer cells, and the culture period is 1 to 14 days. The method for producing an organoid according to Claim 1.

4. The upper end diameter of the sub-well is in the range of 3.0 to 4.5 mm, the upper end diameter of the recess is in the range of 0.45 to 1.5 mm, and the ratio of the length to the diameter of the sub-well and the diameter of the recess is in the range of 1:0.1 to 0.

5. The method for producing an organoid according to Claim 1.

5. The individual volume of the main well is in the range of 100 to 300 μl, the individual volume of the recess is in the range of 20 to 50 μl, and the individual volume ratio of the main well to the recess is on average 1:0.1 to 0.

5. The method for producing an organoid according to Claim 1.

6. The height (ah) of the space part is in the range of 2.0 to 3.0 mm on average, The height (bh) of the sub-well is in the range of 1.0 to 2.0 mm on average, The method for producing an organoid according to claim 1, characterized in that the ratio of the height of the space part to the height of the sub-well (ah:bh) is in the range of 1:0.3 to 1.

7. The method for producing an organoid according to claim 1, wherein the cells are seeded in the concave portion of the cell culture plate at 100 to 300 cells / concave portion.

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