KR20200081295A - A method for prepairing a standard organoid - Google Patents

Abstract

The present invention provides a standard organoid manufacturing method including a step of culturing cells in a three-dimensional cell culture plate to form organoids.

Description

A method for prepairing a standard organoid}

The present invention relates to a method for preparing a standard organoid. More specifically, it relates to a method of manufacturing a uniform size organoid.

Organoids, also called'long-term analogs' or'similar organs', are organ-specific cell aggregates produced by agglutinating and recombining cells isolated from stem cells or organ-derived cells by 3D culture. Organoids contain specific cells of a modeled organ, reproduce specific functions of the organ, and can be spatially organized in a form similar to a real organ. It has been reported that patient-derived tumor organoids represent the characteristics of the patient's cancer cells and cancer tissues and can reproduce the genetic variation characteristics of the patient's cancer tissues.

Organoids can be used in cell therapy, bio-tissue engineering, new drug development, toxicology, and even precision medicine. In order to increase the utilization of organoids, a large amount of comparable quantitative organoids and methods of analysis are required. However, there is no method for quantitatively cultivating organoids. The reason for this is that the most important element of the organoids is matrigel, which is hardened on the floor, and then the organoids grow in it.

In addition, the limitations are clear because organoids grown in the matrigel can grow on top of each other in a three-dimensional support.

In addition, high-throughput screening technology has recently been developed in combination with a much more stable and physiological patient-derived organoid for use in early drug discovery programs and toxic screens.

Korean Patent Registration No. 10-1756901 (Patent Document 1) discloses a cell culture chip capable of culturing three-dimensional tissue cells. In the cell culture chip of Patent Document 1, the first culture unit, the second culture unit, and the third culture unit are formed for each layer, and the growth progress of cells for each layer can be confirmed. However, the cell culture chip of Patent Document 1 has a problem that it is not possible to obtain organoids with high yield.

In addition, there is a case of pipetting to replace the culture medium during cell culture. In the case of a corning spheroid microplate capable of three-dimensional cell culture, spheroids or organoids in cell culture are affected and sucked up during pipetting, There are cases where a position change or the like occurs, and there is a problem in the cell culture environment.

Accordingly, the present inventors have completed the present invention by continuing to study the standard type organoid having a uniform size while not using or minimizing the use of an extracellular matrix-based hydrogel (eg, matrigel).

1. Korea Registered Patent No. 10-1756901

An object of the present invention is to provide a method for preparing a standard organoid.

Another object of the present invention is to provide a standard organoid having a uniform size and similar functionality of each organoid prepared by the above method.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention

A method of manufacturing an organoid comprising culturing cells in a 3D cell culture plate to form an organoid,

In the organoid formation step, the cell culture plate contains 0 to 2% by volume of the extracellular matrix-based hydrogel,

The three-dimensional cell culture plate,

A well plate including a plurality of main wells and cell culture fluids formed at the lower portions of the main wells and including a plurality of sub wells including recesses on the bottom surface; And a connector for high-capacity high-speed high contents screening (HCS) supporting the well plate.

The high-capacity high-speed HCS (High contents screening) connector is located on the top of the well plate and the base provided with fixing means to be detachable from the bottom of the well plate, and includes a cover coupled to the base, the main well A stepped plate is formed to taper at a predetermined site, and the stepped plate is a cell culture plate having an inclination angle θ in a range of 10 to 60° based on the wall of the main well.

The cells may be normal cells or cancer cells.

The cells may be single cells.

The cells can be obtained from normal tissues, cancer tissues or from organoids that have already been made. The method of separating tissues into single cells by cellization is a well-known technique, and thus detailed description thereof will be omitted.

The cell culture period is preferably 1 to 14 days

The extracellular matrix-based hydrogel may be Matrigel (product name).

The size of the organoid may be 300-500 um in diameter.

In the present invention, the term "standard type organoid" refers to an organoid having a size of 300-500 um in diameter.

The size of the organoids produced in the present invention is in the range of 300-500 um, which is particularly optimized for cancer diseases.

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

The sub-well of the three-dimensional cell culture plate is formed with an inclined surface to taper toward the recess, the upper diameter of the sub-well 120 is in the range of 3.0 to 4.5 mm, and the diameter of the upper end of the recess 121 is 0.45 to 1.5 mm range, the sub-well and the inclined surface (θ ₂ ) of the recess is in the range of 40 to 50°, and the length ratio for the diameter of the sub-well and the diameter of the recess may be in the range of 1:0.1 to 0.5.

The individual volume of the main well of the three-dimensional cell culture plate 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 and the recess is an average of 1: 0.1 to 0.5 days Can.

The main well includes a space between the step and the sub well, and the height (a _h ) of the space is in the range of 2.0 to 3.0 mm on average, and the height (b _h ) of the sub well is in the range of 1.0 to 2.0 mm on average. , The height ratio of the space portion and the sub well (a _h :b _h ) may range from 1:0.3 to 1.

The cells may be seeded at 100-300 cells/well in the cell culture plate.

Hereinafter, the present invention will be described in detail.

The present invention can be applied to various changes and can have various embodiments, and specific embodiments will be illustrated in the drawings and described in detail in the detailed description.

However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In the description of the present invention, when it is determined that a detailed description of known technologies related to the present invention may obscure the subject matter of the present invention, the detailed description will be omitted.

The terms used in this application are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise.

In the present invention, terms such as “comprises” or “have” are intended to indicate that there are features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, and one or more other features. It should be understood that the existence or addition possibilities of fields or numbers, steps, operations, components, parts or combinations thereof are not excluded in advance.

In general, when culturing organoids, hydrogels are used to provide the role of extracellular matrix. For example, after matrigel is hardened in a dome shape on the bottom of the cell culture plate, the organoids are grown therein, and the size and shape of the organoids grows in pieces, and as a result, the function grows in different ways, making it difficult to standardize. .

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

The present invention uses, in one embodiment, a three-dimensional cell culture plate comprising:

A well plate including a plurality of main wells and cell culture fluids formed at the lower portions of the main wells and including a plurality of sub wells including recesses on the bottom surface; And

Includes a connector for a high-capacity high-speed HCS (High contents screening) for supporting the well plate,

The high-capacity connector for high-speed HCS (High contents screening) is located on the top of the well plate and the base provided with fixing means to be detachable from the bottom of the well plate, and includes a cover coupled to the base,

The main well is formed with a stepped to taper a predetermined portion, and the stepped cell culture plate having an inclination angle (θ) in the range of 10 to 60° based on the wall of the main well.

In the case of the conventional 96-well plate, experiments and analyzes have to be conducted several times or more to evaluate the drug efficacy of a high yield, so there is a problem that it takes a lot of time and money. In addition, in the case of cell culture, pipetting is often performed to replace the culture medium. In the case of the conventional corning spheroid microplate, the spheroid or organoid in cell culture is affected, and thus, the spheroid or organoid during pipetting is affected. There is a case where it is sucked up or a position change occurs, and thus there is a problem in the cell culture environment.

Therefore, the present invention is to solve the above-mentioned problems, it is possible to manufacture a high yield of spheroid/organoid by including a plurality of sub-wells in a plurality of main wells formed in the well plate, high-capacity high-speed to support the plate By including a connector for HCS (High contents screening), it provides a cell culture plate capable of uniformly photographing an image in a well plate by reducing a tolerance when photographing a large-capacity high-speed image. Furthermore, by the tanch of the main well, the cultured cells provide a cell culture plate capable of minimizing the effect of pipetting during media replacement.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. Prior to this, the terms or words used in the present specification and claims should not be construed as being limited to ordinary or lexical meanings, and the inventor appropriately explains the concept of terms to explain his or her invention in the best way. Based on the principle that it can be defined, it should be interpreted as meanings and concepts consistent with the technical spirit of the present invention.

Therefore, the embodiments shown in the embodiments and the drawings described in this specification are only the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention, and thus can replace them at the time of application. It should be understood that there may be equivalents and variations.

Figure 1 (a) is a front view of a cell culture plate according to an embodiment of the present invention, Figure 1 (b) is a cross-sectional view of a cell culture plate according to an embodiment of the present invention, Figure 2 is one chamber of the present invention The main well formed in the cell culture plate according to an example is a view showing in detail, Figure 3 is a view showing the well plate, the base and the cover of the cell culture plate according to an embodiment of the present invention ((a) cover, (b ) Base, (c) Micro plate and base fixing means).

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.

1 to 3, the cell culture plate 10 according to an embodiment of the present invention is formed in a plurality of main wells (main well, 110), and each of the main well 110, the cell culture broth A well plate 100 that is injected and includes a plurality of sub wells 120 including a concave portion 121 on a bottom surface; And a connector 200 for high-capacity high-speed high contents screening (HSC) supporting the well plate 100.

First, referring to FIGS. 1 and 2, the well plate 100 according to an embodiment of the present invention will be described in detail.

The well plate 100 is made of a plastic injection molded plate shape through a mold. As described above, the main well 110 has a repetitive pattern as a well structure so that the production cost can be reduced and the size can be easily enlarged by using micro-machining for manufacturing a mold for plastic injection. Therefore, it is easy to mass-produce cells, and can be used in a variety of sizes according to user needs.

A plurality of main wells 110 are formed in the well plate 100, and each main well 110 includes a stepped 101. The stepped 101 is formed on a predetermined portion of the main well 110, and more specifically, the stepped 101 may be formed at a position of 1/3 to 1/2 of the total length of the main well 110. In addition, the stepped 101 may be formed at a position from 1/3 to 1/2 from the bottom of the main well 110.

Conventionally, when culturing a cell in a microplate, there is a case where a pipette is replaced to replace the culture medium. In this case, the spheroid or organoid being cultured is affected, and the spheroid or organoid is sucked up during pipetting. Or, there is a case where a position change or the like occurs, and there is a problem that is not good for the cell culture environment, but the stepped 101 is to prevent such a problem.

The stepped 101 may be a space to which a pipette is applied, and specifically, may have an inclination angle θ in the range of 10 to 60° based on the wall of the main well 110. Alternatively, 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, when the inclination angle of the stepped 101 is less than 10 °, the inclination angle in the main well 110 is too small, there is not enough space to apply the pipette, when inhaling the culture solution in the main well 110, pipette The spheroid or organoid may be sucked up by sliding inside the sub-well 120, or a position change may occur. In addition, when the inclination angle θ exceeds 60°, a space for applying a pipette is provided, but the inclination angle of the stepped 101 may be too large to make it difficult to sufficiently inhale the culture solution, and to the sub well 120 When seeding cells, a problem that the cells do not enter all the sub-wells 120 and seed on the stepped 101 may occur. Therefore, it is desirable to have an inclination angle in the above-described range.

On the other hand, the main well 110 according to an embodiment of the present invention may include a space 130 between the stepped 101 and the sub well 120, which will be described later. Specifically, the space part 130 is a space in which the culture solution is injected, and is a space in which cells inside the sub-well 120 can share the same culture solution.

More specifically, the height (a _h ) of the space portion 130 may range from an average of 2.0 to 3.0 mm, or may range from 2.2 to 2.8 mm, or may range from an average of 2.3 to 2.7 mm. In addition, the height (b _h ) of the sub-well 120 may range from an average of 1.0 to 2.0 mm, or may range from an average of 1.2 to 1.8 mm.

For example, the height a _h of the space portion 130 is 2.5 mm on average, and the height b _h of the sub-well may be 1.5 mm on average.

At this time, the height ratio of the space portion and the sub-well 120 (a _h :b _h ) may be in the range of 1:0.3 to 1, and more specifically, the height ratio of the space portion and the sub-well 120 (a _h : b _h ) may be 1:0.4 to 0.9 or 1: 0.5 to 0.8. If, when the height of the sub-well 120 is less than 1:0.3 compared to the height of the space, when exchanging the media of the sub-well 120, cells being cultured therein may pop out with a little force. When the height of 120) exceeds 1:1 with respect to the height of the space, the culture medium required for the cells is not sufficiently converted, which may cause cell death. Therefore, it is preferable that the space part 130 and the sub-well 120 have the above-described height range and height ratio.

Next, the sub wells 120 are formed at the bottom of each of the main wells 110 and include recesses 121 on the bottom surface. As a specific aspect, the sub-well 120 may include a plurality of lower portions of the main well 110.

The sub-wells 120 included in the lower portion of the main well 110 have the same size and shape, and accordingly, can generate spheroids and organoids under uniform conditions.

The sub-well 120 may be formed with an inclined surface to taper toward the concave portion 121. Specifically, as the upper end of the sub-well 120 descends based on the vertical direction, the horizontal width may decrease. For example, the upper portion of the sub-well 120 may be formed in an inverted pyramid shape. In the illustrated embodiment, the upper end of the sub-well 120 may have a pyramid shape or a funnel shape, such that a horizontal width decreases as it descends in the vertical direction.

In particular, the sub-well 120 includes a plurality of the same size and shape, so that the cell culture plate can generate a large amount of spheroid or organoid under uniform conditions.

As a specific aspect, one main well 110 may include 4 to 25 sub-wells 120 of the same size, and the entire micro-plate 100 may include 96 to 1,728 sub-wells 120. Can. Accordingly, it is possible to precisely and precisely control the size.

In addition, the sub-well 120 includes a concave portion 121, and a space is formed at the bottom of the concave portion so that the 3D spheroid or organoid can be cultured. Specifically, the concave portion 121 may have a'U' shape, a'V' shape, or a'Ц' shape, for example, the concave portion 121 may have a'U' shape. .

The upper diameter of the sub-well 120 may range from 3.0 to 4.5 mm, or may be from 3.5 to 4.3 mm, or average 4 mm. In addition, the diameter of the top of the concave portion 121 may be 0.45 to 1.5 mm, or may be 0.5 to 1.0 mm or an average of 0.5 mm.

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

When the upper diameter of the concave portion 121 is less than 0.1 compared to the upper diameter 1 of the sub-well 120, the cell culture space of the concave portion 121 is not sufficiently provided. When the problem that the cells come out may occur, and the upper diameter of the concave portion 121 exceeds 0.5 compared to the upper diameter 1 of the sub-well 120, it is impossible to replace sufficient culture solution required for the cells to stably culture Difficult problems can arise.

On the other hand, the slopes of relative to the wall of the main-well sub-well 120 and the recess 121 may have an inclination angle (θ ₂₎ of 40 to 50 °, 42 to 48 ° range of inclination angle of (θ _2), It may have an inclination angle θ _{2 in the} range of 43 to 47°, or an inclination angle θ ₂ of 45° on average.

The sub-well 120 described above is capable of cell culture of 100 to 1000 cells/well or less, and has the advantage of stably controlling the spheroid size.

Further, 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, the main well 110 and the concave The individual volume ratio of the part 121 is characterized by being an average of 1: 0.07 to 0.5. Preferably, the individual volume of the main well 110 according to the embodiment is in the range of 250 to 300 μl, and the individual volume of the recess may be in the range of 25 to 35 μl, and the main well 110 and the recess ( The individual volume ratio of 121) may be 1: 0.11 on average.

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

In addition, the concave portion 121 is a space in which substantial cells are cultured. If the volume is less than 20 μl, there may be insufficient cell culture space to cause cells to escape, and when it exceeds 50 μl, cells may be removed. Problems that are difficult to stably culture may occur. Therefore, it is preferable that the main well 110 and the concave portion 121 have a volume in the above-described range.

Due to the above-described configuration of the cell culture plate of the present invention, the cells are maintained in a spheroid form even if the hydrogel is not included, that is, the hydrogel is not coated on the cell culture plate, and reprogramming to induced pluripotent stem cells is prevented. It occurs with high efficiency and maintains its shape and function continuously after reprogramming.

The cell culture plate 10 according to an embodiment of the present invention includes a connector 200 for a high-capacity high-speed high contents screening (HCS) supporting the well plate 100. Here, the connector 200 for high-capacity high-speed high contents screening (HCS) means a connector 200 mounted on a high contents screening (HCS) system. Specifically, the connector 200 is a base in the present invention. It may mean the 210 and the cover 220.

More specifically, the high-capacity high-speed HCS (High contents screening) connector, the base 210 and the well plate 100 provided with fixing means 140 and 240 so as to be detachable from the bottom of the well plate 100 Located on the upper portion of the, includes a cover 220 coupled to the base 210. In addition, the upper end of the base 210 and the lower end of the well plate 100 are characterized by including fixing means 140 and 240 that can be fixed to be detachable from each other.

At this time, the base includes an iron portion 240 for supporting the well plate 100, and the well plate 100 is a yaw facing the iron portion 240 of the base 210 It may include a portion 140. The well plate 100 is fixed by the fixing means so that an image can be uniformly photographed during screening.

The base may be polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyamide, polyester, polyvinyl chloride, polyurethane, polycarbonate, polyvinylidene chloride, polytetrafluoroethylene, polyetheretherketone or polyetherimide. It may be made of a material, but is not necessarily limited to this.

The well plate may be made of polydimethylsilicon, high fat-modified silicone, methylchlorophenyl silicone, alkyl-modified silicone, methylphenylsilicone, silicone polyester, or amino-modified silicone material, but is not limited thereto.

The method of manufacturing an organoid of the present invention is economical because it can minimize the use of matrigel. In addition, the method for manufacturing an organoid according to the present invention provides an effect of mass-producing a standard organoid.

Through the present invention, when proceeding with large-scale drug screening, since the organoids are formed in a uniform size that can be compared with each other, the effect and quantitative analysis of the drug becomes possible. Through this, drugs suitable for each modified gene specificity can be selected, and more effective drug treatment is possible.

Figure 1 (a) is a front view of a cell culture plate according to an embodiment of the present invention, Figure 1 (b) is a cross-sectional view of a cell culture plate according to an embodiment of the present invention.
2 is a view showing in detail the main well formed in the cell culture plate according to an embodiment of the present invention.
3 is a view 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) microplate and fixing means of the base).
4 is a view showing the results of high-speed mass imaging of Example 1 and Comparative Example 1 ((a) Example 1, (b) Comparative Example 1).
5(a) is a result of culturing organoids containing matteligel in a volume of 2% by volume and using matrigel according to one embodiment of the present invention, and (b) is an immunofluorescence staining result.
Figure 6 (a) is a photograph showing the results of high-speed mass imaging of Example 1, Figure 6 (b) is a graph showing the area of the organoids cultured in Example 1.
Figure 7 (a) is a photograph showing the results of high-speed mass imaging of Comparative Example 1, Figure 7 (b) is a graph showing the area of the organoids cultured in Comparative Example 1.
8 shows imaging results (left) and organoid size distribution (right) when colon cancer cells are cultured for 14 days according to an embodiment of the present invention.
9 shows an organoid staining image (left) and organoid survival rate (right) cultured for 14 days of colorectal cancer cells according to an embodiment of the present invention.
10 and 11 are photographs and graphs showing the results of high-speed mass imaging of Examples 1 to 3 ((a) Example 1, (b) Example 2, (c) Example 3).

The present invention can be applied to various conversions and can have various embodiments. Hereinafter, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In the description of the present invention, when it is determined that a detailed description of known technologies related to the present invention may obscure the subject matter of the present invention, the detailed description will be omitted.

[Example]

<Experiment method>

1: Organoid production

Existing colon cancer organoids were pipetted from a plate into a 15 ml tube with 1 ml of culture medium, centrifuged for 3 minutes at 2000 rpm, and then the supernatant was removed, followed by PBS washing once and centrifuged at 2000 rpm for 3 minutes. Then, it was treated with acuutase for 7 minutes to completely separate it into single cells. The single cells were seeded in sub-wells of the cell culture plate at about 100 cells/well, and cultured for a total of 14 days to prepare organoids. At this time, the culture medium is a DMEM/F12-based culture medium, B27, N2, GlutaMAX, penicillin streptomycin, Nictoiamide, N-acetyl, gastrin, A-83-01, EGF, noggin, R-spondin1, WNT3A are contained in the culture medium. An organoid was prepared under the condition that it did not contain matrigel or contained 2% by volume.

2: Measurement of organoid size and number

The size analysis of organoids was performed using the Image J program. Specifically, by selecting the region of interest from the phase image and applying the threshold with the Image J program, the unnecessary parts are overwritten with white and the parts that are not properly drawn are filled with black. The area applied with the threshold filled with black was obtained by using the outline.

3: immunofluorescence staining method

It was confirmed by staining LGR5, an organoid stem cell, through immunofluorescence staining. First, the 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. Thereafter, after a day at 15% sucrose and a day at 30% sucrose, a cryo-block was made using liquid nitrogen. Using the frozen block made, the section was processed to a thickness of 10 um, and the cut section was attached to the slide glass. After treating tritonX 0.1% for 10 minutes, it was washed twice with PBS. After storing at room temperature for 1 hour with 3% BSA, after washing the PBS twice, the primary antibody of LGR5 is maintained at room temperature for 2 hours. After PBS washing, the secondary antibody was treated at room temperature for 2 hours, and the mounting solution was added and measured under a fluorescence microscope.

In the case of Fig. 9, the cultured standard organoid is taken out, and Live/Dead fluorescence staining is performed. For fluorescent staining, Calcein 1mM is 2ul per 1ml and EtdH-1 2mM is 1ul per 1ml, stored in an incubator for 30-60 minutes, and then measured with a fluorescence microscope.

<Example>

Example 1.

Organoids were prepared by the method mentioned in Experimental Method 1. Organoids were prepared under the conditions of containing 2% by volume of Matrigel in the culture solution (Example 1-1) and without Matrigel (Example 1-2).

Example 2.

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

Example 3.

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

Comparative Example 1.

Organoids were cultivated inside the metrichel, a widely used method, and high-speed mass imaging was performed. However, in the comparative example, a 96-well plate, which is a commonly used cell culture plate, was used, and organoids were prepared by seeding cells in Matrigel.

<Experimental Example>

Experimental Example 1. Organoid image analysis

The cells cultured in Example 1-1 and Comparative Example 1 were photographed, and the size of the cell cells was compared. The spheroid is imaged on an automated plate device, and the device automatically focuses and proceeds. Image size analysis was performed using a macro program of the imageJ program.

And, the results are shown in FIG. 4. 4 is a view showing the results of high-speed mass imaging of Example 1-1 and Comparative Example 1 ((a) Examples 1-1, (b) Comparative Example 1)

Referring to Figure 4, in the case of Example 1-1, it was confirmed that the diameter of the cells cultured in the cell culture plate of the present invention is almost uniform. Specifically, when cells were seeded in an average of 100 cells/well in each sub-well, a uniform organoid capable of comparative analysis could be prepared. At this time, the error range for the organoid size was about 20 μm. Through this, it can be seen that using the organoid culture method according to the present invention, it is possible to prepare a standard organoid.

On the other hand, in Comparative Example 1 using a conventional well plate, it was confirmed that the size of the cell cells is formed differently. This is because the multiple cells grow in a dome shape of a single matelle, the error range of the resulting organoid size is more than 150 μm, and even overlaps, making uniform high-speed mass imaging and experiments impossible.

The base and the well plate of the present invention include an iron part and a yaw part, respectively, in order to fix each other, and the iron part and the recess are combined with each other so that the base can securely fix the well plate. It shows that the image can be taken uniformly.

On the other hand, it can be seen that the size and shape of the organoids cultured in Comparative Example 1 are not uniform. It seems that the analysis of the image becomes difficult because the focal deviation of the imaging becomes large when there is no plate base.

5(a), it was confirmed that organoid formation was well achieved when the cell culture plate of the present invention was used even if it did not contain matrigel.

FIG. 5(b) shows the expression level by staining LGR5, the most important marker for the formation of colon cancer organoids, to confirm that the cultured organoids were successfully formed. In addition, colon-specific structures were confirmed in the colon cancer organoids of the group not using the low-concentration matrigel or metrigel formed by F-actin staining.

Experimental Example 2. Preparation of standard organoids

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

The organoids prepared in Example 1-1 and Comparative Example 1 were imaged in an automated plate device, and the device was automatically focused to proceed. Image size analysis was performed using a macro program of the imageJ program.

And, the results are shown in FIGS. 6 and 7.

Figure 6 (a) is a photograph showing the results of high-speed mass imaging of Example 1-1, Figure 6 (b) is a graph showing the area of the organoids cultured for a period of time in Example 1, Figure 7 (a) Is a photograph showing the results of high-speed mass imaging of Comparative Example 1, and FIG. 7(b) is a graph showing the area of the organoids cultured for a period of time in Comparative Example 1.

Referring to FIG. 6, in the case of automatically imaging the organoid prepared in Example 1-1, it is possible to perform imaging without a large error due to uniform imaging height, thereby confirming that an error range is very small when measuring an actual area. Can.

In particular, when the organoids are cultured using the cell culture plate of the present invention, the organoids are cultured to a uniform size. That is, standardization is possible. As a result of the standardization, the focus is automatically taken when measuring the image, and the variation in the measurement height is minimized by the connector structure. Accordingly, when measuring the screening image, the deviation is very small, around 20 μm.

Referring to FIG. 7, in Comparative Example 1, it can be confirmed that the organoids grow to overlap each other, and it can be seen that standardization is impossible because the organoids have different sizes and distribution positions. Therefore, when measuring the screening image, the error range is up to 150 μm, which is large in and out.

This is because when the organoid is cultured in a conventional method, since the organoid grows randomly in the metrigel, it is difficult to uniformly cultivate the desired organoid, and the measurement height is also variable, making it difficult to apply to the organoid screening imaging. has exist. Therefore, when analyzing the area of the organoids cultured for a certain period, it is determined that the deviation is very large.

In the case of FIG. 8, the diameter of each organoid was measured using the Image J program in the entire standard organoid. A total of 864 wells were subjected to high-speed mass imaging, and each of them was analyzed with ImageJ to confirm that a uniform diameter appeared.

8 shows imaging results and organoid size when colon cancer cells of Example 1-1 were cultured for 14 days. It can be seen that standard organoids can be manufactured because the organoids have a uniform size of 300-50 um.

9 is an image photograph showing the organoids according to Examples 1-1 and 1-2 over time (left) and the organoid survival rates according to Example 1-1 (right). In the case of Figure 9, the cultured organoids were subjected to Live/Dead staining, and it was confirmed how much the cultured organoids were maintained. First, the cultured standard organoids are washed with PBS, and then incubated for about 10 minutes with accutase. After fragmentation into single cells, Live/Dead reagents, Calcein and EtdH-1, were stored in an incubator for about 30-60 minutes, and then put in a C-Chip to confirm how much each was present with a fluorescence microscope.

It can be seen from FIG. 9 that organoid formation occurs very well without matteligel, and when cultured for 14 days, the organoid survival rate is very high.

10 is a photograph showing the results of high-speed mass imaging of Examples 1-1, 2, and 3, and FIG. 8 is an imaging result when colon cancer cells were cultured for 14 days in Examples 1-1, 2, and 3 ((A) Example 1-1, (b) Example 2, (c) Example 3).

Referring to FIG. 10(a), when cells were subjected to an average of 100 cells/well in each subwell, a uniform organoid capable of comparative analysis was produced (error range: about 20 μm).

On the other hand, referring to Figures 10b) and 10(c), when the cells are seeded in a subwell of 100 cells/well or more, it can be confirmed that the organoids overflow from the subwells, resulting in non-uniform organoids. have.

11 is a graph showing the results of high-speed mass imaging of Examples 1-1, 2, and 3, and FIG. 11 is a result of culturing colorectal cancer cells in Examples 1-1, 2, and 3 for 7 or 14 days. ((A) Example 1-1, (b) Example 2, (c) Example 3).

Referring to FIG. 11, when cells are cultured for an average of 100 cells/well for an average of 14 days, the most preferable organoids can be produced. That is, when cells with an average of 100 cells/well or less are cultured for 14 days, it seems that the optimal organoids can differentiate and grow. On the other hand, when the number of cells seeded in the subwell was increased and the number of culture days was reduced, it was confirmed that the organoid performance was poor.

For reference, the dotted line in FIG. 11 means the maximum space of the subwell of the cell culture plate of the present invention, and the space in which the cells can be cultured. This is considered to be able to culture cells with an average of 100 cells/well or less.

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. It will be obvious. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Since the specific parts of the present invention have been described in detail above, for those skilled in the art, this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. It will be obvious. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

100: well plate
101: step 110: main well
120: sub well 121: recess
130: space part 140: main part
200: High-capacity high-speed HCS connector
210: base 220: cover
240: iron

Claims (10)

A method of manufacturing an organoid comprising culturing cells in a 3D cell culture plate to form an organoid,
In the organoid formation step, the cell culture plate contains 0 to 2% by volume of the extracellular matrix-based hydrogel,
The three-dimensional cell culture plate,
A well plate including a plurality of main wells and cell culture fluids formed at the lower portions of the main wells and including a plurality of sub wells including recesses on the bottom surface; And a connector for high-capacity high-speed high contents screening (HCS) supporting the well plate.
The high-capacity high-speed HCS (High contents screening) connector is located on the top of the well plate and the base provided with fixing means to be detachable from the bottom of the well plate, and includes a cover coupled to the base, the main well The stepped is formed to taper a predetermined area, and the stepped is a cell culture plate having an inclination angle θ in the range of 10 to 60° based on the wall of the main well,
Method of manufacturing organoids.
According to claim 1,
The extracellular matrix-based hydrogel is matrigel, an organoid manufacturing method.
According to claim 1,
The cells are normal cells or cancer cells,
The culture period is 1 to 14 days, organoid manufacturing method.
According to claim 1,
The size of the organoid is 300-500 um in diameter, an organoid manufacturing method.
According to claim 1,
The sub-well is an organoid manufacturing method characterized in that the inclined surface is formed to taper toward the recess.
According to claim 1,
The sub-well is formed with an inclined surface to taper toward the concave portion,
The upper diameter of the sub-well 120 ranges from 3.0 to 4.5 mm,
The diameter of the top of the concave portion 121 ranges from 0.45 to 1.5 mm,
The inclined surface (θ ₂ ) of the sub-well and the recess is in the range of 40 to 50°
The organoid manufacturing method, characterized in that the length ratio for the diameter of the sub-well and the diameter of the recess is in the range of 1:0.1 to 0.5.
According to claim 1,
The individual volume of the main well ranges from 100 to 300 μl,
The individual volume of the recess is in the range of 20 to 50 μl,
The method of manufacturing an organoid, characterized in that the average volume ratio of the main well and the concave portion is 1: 0.1 to 0.5 on average.
According to claim 1,
The main well includes a space between the stepped and sub wells,
The height (a _h ) of the space portion ranges from an average of 2.0 to 3.0 mm,
The height (b _h ) of the sub wells ranges from 1.0 to 2.0 mm on average,
The method of manufacturing an organoid, wherein the height ratio of the space portion and the sub well (a _h :b _h ) is in the range of 1:0.3 to 1.
According to claim 1,
The cells are seeded at 100 to 300 cells/well on the cell culture plate, organoid manufacturing method.
An organoid having a size of 300-500 um in diameter, prepared according to any one of claims 1 to 9.

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