KR102333275B1 - Method of providing information for patient-specific drug selection - Google Patents

Abstract

The present invention provides an information providing method for selecting a drug tailored to a subject with a disease.

Description

Method of providing information for patient-specific drug selection

The present invention relates to a method for providing information for patient-specific drug selection. More particularly, it relates to a method of providing information for individual customized drug selection to a subject having a disease using the organoid cultured according to the present invention.

Organoids, also called 'organ analogues' or 'organ-like organs', are organ-specific cell aggregates manufactured by re-aggregating and recombination of cells isolated from stem cells or organ origin cells using a three-dimensional culture method. Organoids contain specific cells of the organ as a model, reproduce the specific function of the organ, and can be spatially organized in a form similar to that of an actual organ. It has been reported that patient-derived tumor organoids represent the characteristics of cancer cells and cancer tissues of the patient as they are, and can also reproduce the genetic variation characteristics of the patient's cancer tissues.

Organoids can be used in cell therapy, biotissue engineering, drug development, toxicology, and even precision medicine. In order to increase the utilization of organoids, a large amount of comparable quantitative organoids and their analysis methods are needed. However, there is still no method for quantitatively culturing organoids. The reason is that after hardening Matrigel, which is the most important element among the elements for growing organoids, into a dome shape, the organoids are grown in it, so the organoids grow differently.

Also, since organoids grown in Matrigel can grow on top of each other in a three-dimensional support, the limitations are clear.

Also, recently, high-throughput screening techniques are being developed in combination with much more stable and physiological patient-derived organoids for use in early drug discovery programs and toxicity 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 section, the second culture section, and the third culture section are formed for each layer, and the degree of cell growth can be checked for each layer. However, the cell culture chip of Patent Document 1 has a problem in that organoids cannot be obtained in high yield.

In addition, there are cases where the pipetting operation of changing the culture medium is performed during cell culture. In the case of corning spheroid microplates capable of 3D cell culture, the spheroids or organoids in cell culture are affected, so they are sucked up during the pipetting operation, There is a case where a change in position or the like occurs, and there is a problem that is not good for the cell culture environment.

Organoids have the advantage of low-cost, high-dose testing of human diseases that are difficult to make in animal models. However, in almost all cases, the conventional three-dimensional (Matrigel organoid, MOs) culture is generally performed by embedding cells in bulk volume Matrigel?? arranged and physiologically irregular. Although these culture methods have enabled numerous insightful studies in the laboratory, they have important limitations for high-dose screening: scalability, consistency, uniformity, organoid production and reproducibility of multicellular interactions. In particular, since the Matrigel organoid has a structure embedded in the solidified Matrigel, a difference in space and time occurs in the focal plane, and the mass transfer from the outside to the inside of the Matrigel dome is not performed accurately. cause

With respect to tissue-level complexity, tumors are heterogeneous cell populations that include cancer cells and endothelial cells, invasive immune cells, stromal cells, and complex extracellular matrix (ECM) networks and different cell types. This tumor heterogeneity lies between cancers in different patients (inter-tumor heterogeneity) and single tumors (intra-tumoral heterogeneity). The latter include cell surface marker phenotypic diversity, (epigenetic) genetic abnormalities, growth rates, cell death and other features of cancer-induced disease progression and treatment failure. Recent observations show that a single tumor contains different subclones with respect to karyotype, cancer stem and sensitivity to chemotherapy. Moreover, it has been reported that the nature of this heterogeneity can be reversibly acquired through the interaction of a given tumor cell's subclones and/or the tumor microenvironment with the tumor microenvironment, indicating that both the cellular interactions and the microenvironment of the tumor cells are important for cancer therapy. suggest that it may be a factor.

However, the specific relationship between the clonal diversity of MOs and their functions related to drug response is poorly understood due to the random display and physiological irregularities of cultured MOs. To overcome these limitations, Hydro Plate, a scalable, homogeneous organoid culture and screening platform, was developed.

Accordingly, the present inventors completed the present invention by continuing research on organoids having a uniform size while not using or minimizing the use of an extracellular matrix-based hydrogel (eg, Matrigel).

1. Republic of Korea Patent No. 10-1756901

An object of the present invention is to provide information for selecting a drug tailored to a subject with a disease.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, 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 includes

Provided is an informational method for personalized drug selection for a subject with a disease:

isolating diseased tissue from a diseased subject;

classifying the disease tissue by genetic mutation;

isolating cells from the classified disease tissue;

Culturing the isolated cells as disease organoids in a three-dimensional cell culture plate:

treating the cultured disease organoid with a drug; and

Including; measuring the activity of the disease organoid

As a method of providing information for a subject-specific drug selection with a disease,

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

The three-dimensional cell culture plate,

a well plate including a plurality of main wells and a plurality of sub wells formed under each of the main wells to inject a cell culture solution and having a recess on the bottom surface; and a connector for high-capacity high-speed HCS (High contents screening) supporting the well plate;

The large-capacity and high-speed HCS (High contents screening) connector includes a bottom of the well plate and a base provided with a fixing means to be detachable from each other, and a cover positioned on the top of the well plate and coupled to the base, and the main well A step is formed so as to be tapered at a predetermined portion, and the step has an inclination angle (θ) in the range of 10 to 60° with respect to the wall of the main well.

The subject may include any animal, preferably a mammal, more preferably a human.

The “disease tissue” and the diseased tissue refer to those separated from the tissue or organ.

In the step of separating the diseased tissue by genetic mutation, the diseased tissue isolated from the subject with the disease is tested by a genetic testing method widely known in the art to find out the mutation in the disease-causing gene, and then isolating it by mutation is a step

For example, when mutations occur in KRAS, TP53, PIK3C, etc. known as cancer-causing mutant genes, it can be classified as a mutation. As another example, there is a high/low variation called microsatellite instability (MSI). In the case of such a mutation, the mismatch repair system of DNA does not work properly, and the erroneously transcribed DNA is not removed and continues to divide and replicate, resulting in disease (eg, cancer). As such, various mutations exist in cancer, and there are no drugs that target all of these mutations, so continuous development is required, and tissues can be classified according to each disease, for example, a genetic mutation in colorectal cancer.

Single cells can be isolated from the classified disease tissue and cultured as disease organoids in the three-dimensional cell culture plate according to the present invention. As used herein, the term "disease organoid" refers to an organoid isolated from a tissue of a subject having a disease.

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

The cell may be a cancer cell.

The cell may be a single cell.

The cells may be obtained by isolation from a tissue or an organoid already made from a subject having cancer. Since a method of cellularizing a tissue and separating it into a single cell is a known technique, a detailed description thereof will be omitted.

The cell culture period is preferably 1 to 14 days.

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

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

The "three-dimensional cell culture plate" of the present invention is a plate optimized for organoid culture, which will be described in detail below. Hereinafter, the "three-dimensional cell culture plate" is also referred to as a "hydro plate", and the organoids cultured in this hydro plate are described as "hydro organoids" or "HO" or "HOs". Also, as a control, Matrigel-based plates are described as "Matrigel plates" (basic 24-well plates used in the past), and organoids cultured in these Matrigel plates are referred to as "Matrigel Organiods" or "MO " or "MOs".

As will be described in detail below, by using the three-dimensional cell culture plate of the present invention, patient-derived disease organoids can be mass-produced.

The sub-well of the three-dimensional cell culture plate has an inclined surface that is tapered toward the concave portion, the diameter of the upper end 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 concave portion 121 is 0.45 to 1.5 _{mm, and the slope (θ 2} ) of the sub-well and the concave portion may be in the range of 40 to 50°, and the length ratio of the diameter of the sub-well and the diameter of the concave portion 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 on average 1: 0.1 to 0.5 days can

The main well includes a space between the step and the sub-well, and the height of the space (a _h ) 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 (a h} :b _h ) of the space portion and the sub-well may be in the range of 1:0.3 to 1.

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

In an embodiment of the present invention, the drug may be one drug or two or more drugs.

The activity of the organoid may mean the viability of the organoid.

After measuring the activity of the disease organoid,

When the activity of the disease organoid decreases after treatment with the drug compared to before the drug treatment, it is determined that the drug is effective, and the step of selecting a drug tailored to the subject having the disease may be further added.

If more than one drug is

When the activity of the disease organoids after treatment with the two or more drugs is lower than when each drug is treated alone, it is judged that there is an effect when the two or more drugs are treated in combination; a subject having a disease A further step of selecting as a customized concomitant drug may be added.

In one embodiment of the present invention, after the step of measuring the activity of the disease organoid, the step of determining the dose of the drug by deriving the IC50 value of the drug may be further added.

IC50 refers to the maximum concentration at the moment when the cell activity (enzyme/protein activity) is halved when the drug is administered. This can help determine the effective drug dose for treatment.

In one embodiment of the present invention, the disease may be cancer, and specifically, the cancer is colon cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, brain tumor, ovarian cancer, skin cancer, prostate cancer, breast cancer, cervical cancer, thyroid cancer, fiber It may be, but is not limited to, a sarcoma, uterine sarcoma, or blood cancer.

In addition, the drug may be an anticancer agent, and specifically, it may be one or more selected from the group consisting of a cytotoxic anticancer agent, an immune anticancer agent, a targeted anticancer agent, an anticancer agent and a new anticancer agent, but is not necessarily limited thereto.

Hereinafter, the present invention will be described in detail.

Since the present invention can have various changes and can have various embodiments, specific embodiments are 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 it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

The terms used in the present application are only used to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise.

In the present invention, terms such as "comprises" or "have" are intended to designate that the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification exist, but one or more other features It should be understood that this does not preclude the possibility of the presence or addition of numbers, steps, operations, components, parts, or combinations thereof.

Hydrogels are commonly used to serve as an extracellular matrix when culturing organoids. For example, after Metrigel is hardened into a dome shape on the bottom of a cell culture plate, organoids are grown in it. Organoids grow in different sizes and shapes, and as a result, their functions grow differently, making it difficult to standardize. .

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

In one embodiment, the present invention uses a three-dimensional cell culture plate comprising:

a well plate including a plurality of main wells and a plurality of sub wells formed under each of the main wells to inject a cell culture solution and having a recess on the bottom surface; and

It includes a connector for high-capacity high-speed HCS (High contents screening) that supports the well plate,

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

A step is formed in the main well to be tapered at a predetermined portion, and the step has an inclination angle (θ) in the range of 10 to 60° with respect to the wall of the main well.

In the case of a conventional 96-well plate, since experiments and analyzes have to be performed several times or more in order to evaluate drug efficacy in high yield, there is a problem in that it takes a lot of time and money. In addition, there are often cases where a pipetting operation of replacing the culture medium is performed during cell culture. In the case of a conventional corning spheroid microplate, the spheroids or organoids in cell culture are affected, so pipetting Spheroids or organoids are sucked up during operation, or there is a case where a change in position occurs, and there is a problem that is not good for the cell culture environment.

Accordingly, the present invention is to solve the above-mentioned problems, and it is possible to manufacture spheroids/organoids with high yield by including a plurality of sub-wells in a plurality of main wells formed in a well plate, and high-capacity and high-speed for supporting the plate. By including a connector for HCS (High Contents Screening), we provide a cell culture plate that can uniformly capture images in the well plate by reducing tolerances when shooting large-capacity, high-speed images. Furthermore, by the tanteok of the main well, the cultured cells are provided with a cell culture plate capable of minimizing the influence of the pipetting operation when replacing the media.

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 conventional or dictionary meanings, and the inventor should properly understand the concept of the term in order to best describe his invention. Based on the principle that it can be defined, it should be interpreted as meaning and concept consistent with the technical idea of the present invention.

Accordingly, the embodiments described in this specification and the configurations shown in the drawings are only the most preferred embodiment of the present invention and do not represent all the technical spirit of the present invention, so at the time of the present application, various 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 a chamber of the present invention It is a view showing in detail the main well formed in the cell culture plate according to the example, and FIG. 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) microplate and fixing means of the base).

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 under each of a plurality of main wells 110 and the main well 110, so that the cell culture solution is a well plate 100 that is injected and includes a plurality of sub-wells 120 including a recess 121 on the bottom surface; And it is configured to include a high-capacity high-capacity HCS (High contents screening) connector 200 for supporting the well plate (100).

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

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

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

Conventionally, there is a case of performing a pipetting operation to change the culture medium during cell culture in a microplate. In this case, the spheroids or organoids in cell culture are affected, and the spheroids or organoids are sucked up during the pipetting operation. Or, there is a case where a change in position occurs, and there is a problem in the cell culture environment that is not good, but the step 101 is to prevent such a problem.

The step 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° with respect to 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 the inclination angle of the step 101 is less than 10°, the inclination angle in the main well 110 is too small and there is not enough space to apply the pipette. A spheroid or an organoid may be sucked up by sliding into the sub-well 120 , or a change in position 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 step 101 is too large, so it may be difficult to sufficiently suck the culture solution, and When seeding cells, a problem in that the cells do not enter all the sub-wells 120 and are seeded in the step 101 may occur. Therefore, it is preferable to have an inclination angle in the above-mentioned range.

Meanwhile, 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 to be described later. Specifically, the space 130 is a space into which the culture solution is injected, and is a space in which the cells in the sub-well 120 can share the same culture solution.

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

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

In this case, the height ratio (a _h :b _h ) of the space portion and the sub-well 120 may be in the range of 1:0.3 to 1, and in more detail, the height ratio (a _h : b _h ) 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 compared to the height of the space, when the media of the sub-well 120 is exchanged, cells being cultured inside may pop out with even a little force, and the sub-well ( If the height of 120) exceeds 1:1 compared to the height of the space part, the culture medium required for the cells is not sufficiently converted, which may cause cell death. Accordingly, the space 130 and the sub-well 120 preferably have the above-described height range and height ratio.

Next, the sub-well 120 is formed under each of the main well 110 , and includes a concave portion 121 on the bottom surface. In a specific embodiment, the sub-well 120 may include a plurality of sub-wells 120 under the main well 110 .

Each of the sub-wells 120 included in the lower portion of the main well 110 has the same size and shape, so that organoids with uniform conditions can be generated.

The sub-well 120 may have an inclined surface that is tapered toward the concave portion 121 . Specifically, the horizontal width of the upper end of the sub-well 120 may decrease as it descends in the vertical direction. For example, the upper end of the sub-well 120 may have an inverted pyramid shape. In the illustrated embodiment, the upper end of the sub-well 120 has a pyramid shape, but may be configured in a shape such that the horizontal width decreases as it descends in the vertical direction, such as a funnel shape.

In particular, by including a plurality of sub-wells 120 having the same size and shape, the cell culture plate can produce a large amount of spheroids or organoids under uniform conditions.

As a specific embodiment, one main well 110 may include 4 to 25 sub-wells 120 of the same size, and 96 to 1,728 sub-wells 120 may be included in the entire microplate 100. can Accordingly, the same and precise size control is possible. Therefore, it is possible to mass-produce standard organoids with uniform size and shape.

In addition, the sub-well 120 includes a concave portion 121, and the concave portion 121 has a space formed at the lower end of the concave portion so that 3D spheroids or organoids can be cultured. Specifically, the concave part 121 may have a 'U' shape, a 'V' shape, or a 'Ц' shape. For example, the recess 121 may have a 'U' shape. .

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

In addition, the ratio of the diameter of the sub-well 120 to the diameter of the concave portion 121 may be in a range of 1:0.1 to 0.5, and preferably, the diameter of the sub-well 120 and the concave portion 121 are in the range of 1:0.1 to 0.5. 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, Cells may come out, and if the top diameter of the concave portion 121 exceeds 0.5 compared to the top diameter 1 of the sub-well 120, it is impossible to replace the sufficient culture medium required for the cells to stably culture Difficult problems may 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), 43 to the inclination angle of 47 ° range (θ _2), or the average angle of inclination of 45 ° may have a (θ _2).

The above-described sub-well 120 may be capable of culturing cells of 100 cells/well or less, and has the advantage of stably controlling the organoid size when cells of 100 cells/well or less are seeded.

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, and the individual volume of the concave portion 121 is in the range of 20 to 50 μl, and the main well 110 and the concave volume are in the range of 20 to 50 μl. The individual volume ratio of the portion 121 is on average 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 concave portion may be in the range of 25 to 35 μl, and the main well 110 and the recess ( 121) may have an average volume ratio of 1:0.11.

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

In addition, the recess 121 is a space in which cells are actually cultured, and when the volume is less than 20 μl, there is not enough cell culture space, which may cause a problem that cells escape, and when it exceeds 50 μl, cells, etc. Difficulty in culturing stably may occur. Accordingly, it is preferable that the main well 110 and the concave portion 121 have a volume within the above-described range.

Due to the above-mentioned configuration of the cell culture plate of the present invention, even if it does not contain hydrogel (Matrigel) or contains less than 2% by volume, organoids can be well formed.

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

More specifically, the large-capacity and high-speed HCS (High contents screening) connector includes a base 210 and a well plate 100 provided with fixing means 140 and 240 so as to be detachable from the lower end of the well plate 100 . It is located on the upper portion, and includes a cover 220 coupled to the base (210). And, the upper end of the base 210 and the lower end of the well plate 100 are characterized in that they include fixing means (140, 240) that can be fixed to each other to be detachable.

At this time, the base includes a convex portion 240 for supporting the well plate 100 , and the well plate 100 has a concave portion opposite to the convex portion 240 of the base 210 . A unit 140 may be included. The well plate 100 is fixed by the fixing means so that an image can be uniformly taken during screening.

The base is 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 thereto.

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

Preclinical trials using animals usually take several months. However, in the present invention, this time can be dramatically shortened by using the hydroorganoids cultured in the aforementioned hydroplate. Specifically, it is possible to find the optimal drug for a patient by using a patient-derived disease organoid with known genetic mutation, cultured in a hydro plate, and it takes only 2-3 weeks.

For example, in the case of colorectal cancer, drugs used for each type of colon are different. For the general type, a single treatment such as 5-FU or Oxaliplatin may be applied, or a synthetic drug such as FOLFOX (Irinocan + cetubximeb) may be used. For the MSI type mutation described above, Xelox (Capecitabine + Oxaliplatin), and for the KRAS mutation, RO5126766; IDF-11774; and azd3965.

In one embodiment according to the present invention, after analyzing the RNA expression of each colorectal cancer tissue at the genomic level through sequencing, the mutations found based on this are classified and organoidized colorectal cancer based on the mutation. Candidate drug groups to be treated on organoids could be accurately determined in a short period of time. In addition, it was possible to determine which drug was the most effective by actually treating these candidate drugs for colorectal cancer organoids.

The organoid manufacturing method of the present invention is economical because it can minimize the use of Matrigel. In addition, the method for producing organoids according to the present invention provides the effect of mass-producing diseased organoids.

Through the present invention, when large-capacity drug screening is performed, organoids are formed in a uniform size that is comparable to each other, unlike in the past, so that the effect and quantitative analysis of the drug are possible. Through this, it is possible to select a drug suitable for the specificity of each modified gene, and more effective drug treatment is possible.

1 (a) is a front view of the cell culture plate according to an embodiment of the present invention, Figure 1 (b) is a cross-sectional view of the 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) fixing means for microplate and base).
4 is a view showing the high-speed mass imaging results of Example 1 and Comparative Example 1 ((a) Example 1, (b) Comparative Example 1).
Figure 5 (a) is the result of organoid culture containing 2% by volume of Matrigel and not using Matrigel according to an embodiment of the present invention, (b) is the result of immunofluorescence staining.
Figure 6 (a) is a photograph showing the high-speed mass imaging results of Example 1, Figure 6 (b) is a graph showing the area of the organoid cultured in Example 1.
FIG. 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 in Comparative Example 1. Referring to FIG.
8 shows imaging results (left) and organoid size distribution (right) when colon cancer cells according to an embodiment of the present invention are cultured for 14 days.
9 shows an organoid staining image (left) and organoid survival rate (right) in which colon cancer cells were cultured for 14 days according to an embodiment of the present invention.
10 is a photograph and graph showing the high-speed mass imaging results of Examples 1 to 3 (100 cells/well-Example 1, 200 cells/well-Example 2, 300 cells/well-Example 3).
11 is a schematic diagram of platform development using a hydroorganoid culture plate . (a) U-shaped micro-well strip platform design for organoid culture (hydro plate). upper left; Top and side views of a single U-shaped microwell strip, top right; 96-well plate for U-shaped microwell strips. Scale bar, 9 mm. floor; Time course schematic of organoid formation in microwells of the strip. A single U-well of the strip consists of 9 microwells and the strip can be inserted into a 96 well plate. Scale bar, 9 mm. (b) Flowchart of organoid generation. (cd) Representative brightfield images showing platform-derived organoids (Matrigel Org; Matrigel-embedded organoid, Hydro Org; Hydrogel-based organoid). Scale bar, (c) 200 μm, (d) 500 μm. (e) Representative immunofluorescence images showing the expression of KRT20, F-actin, mucin 2, chromogranin A, lysozyme, E-cadherin and β-catenin in hydroorganoids. Lower panel is a large magnified image of the inset of the upper panel, nuclear counter staining (blue); 4,6-diamidino-2-phenylindole (DAPI), scale bar, top 100 μm, bottom 50 μm.
12 is data showing consistent and standardized organoid production in hydroplates of the present invention. (a) Schematic diagram of organoid generation. Primary organoids have been established from patient-derived colorectal cancer tissues in traditional Matrigel domes. Organoids were dissociated into single cells and generated on Matrigel gut or hydrogel platforms in culture media containing Matrigel, 100% or 2%, respectively. (b, c) Time-lapse images of organoids generated in (b) Matrigel and (c) Hydro culture platform. Scale bar, 100 μm. Individual morphologies of platform-derived organoids (top panel) and corresponding figures (bottom panel). Matrigel Org; Matrigel-embedded organoid, Hydro Org; Hydrogel-based organoid. (d) Quantitative measurements of diameter distribution and (e) mean area of organoids generated (mean ± sdm, n = 9). (f) Representative fluorescence microscopy images and (g) viability of the generated organoids. Organoids were stained with live/dead assay dyes 7 and 14 days after plating on each platform. The relative proportions of live and dead cells per organoid were analyzed. (mean ± SEM, n = 6). Scale bar, 200 μm.
13 shows the characterization of intracellular composition in hydro-organoids. (a) Whole mount confocal images for KRT20, chromogranin A, LGR5, β-catenin, CD44, mucin 2, lysozyme and F-actin. Scale bar, 50 μm for MO, 100 μm for HO. (bc) Quantification of cancer stem cell subsets in each platform-derived organoid. (b) Histogram plots showing expression of representative cancer stem cell markers in the indicated organoids. (c) Representative density plot gating for CD44 + EpCAM + CD166 + CD133 + LGR5 + cells in the indicated organoids. red box; The relative total percentage of each subset per organoid. (d) Relative expression levels of ki67 (proliferative marker), LGR5, β-catenin (stem cell marker), Sox9, Chromogranin A and Villin (differentiation marker) in each organoid. The fold expression of each indicated gene in hydroorganoids (HO, n = 3) compared to Matrigel organoids (MO, n = 3) was determined by RQ-PCR (3 experiments, * p < 0.05, ns , p > 0.05). .
14 shows a method for evaluating patient-derived hydroorganoids as a preclinical drug screening model.　(a) Scheme of the experimental workflow. (b) Dose-response curves with _{IC 50} values for first-line CRC therapy. The established hydroorganoids were treated with a vehicle (DMSO), doxifluridine, oxaliplatin, or a combination of doxyfluridine and oxaliplatin for 6 days, once every 2 days, a total of 3 times, and drug reaction was measured by CCK8 analysis (mean ± sem). (c) Relative expression levels of ki67 (proliferative marker) and caspase 3 (apoptotic marker) in drug-treated hydroorganoids. Fold changes in expression in the indicated drug-treated groups relative to the levels in DMSO-treated groups as determined by RQ-PCR are shown PCR (mean ± sdmns P >0.05, * P <0.05, ** P <0.01, *** P <=0.001). (df) Summary of preclinical evidence for the XELOX combination (Capecitabine + Oxaliplatin). (d) Representative brightfield images of hydroorganoids treated with oxaliplatin or XELOX combination. Scale bar, 500 μm (e) The combined effect of capecitabine and oxaliplatin (XELOX). Response rate improvement rates were analyzed for the indicated concentrations in each condition (mean ± sdm). (f) To replace the existing animal test model, the expression level of TP (thymidine phosphorylase) measured in an actual animal experiment was also measured in the hydroorganoid to confirm how similar it was. This means that when oxaliplatin is treated, more TP is expressed in organoid colorectal cancer, which can maximize the effect of the combined drug by replacing more doxyfluridine with 5-FU. Therefore, thymidine phosphorylase (TP) expression in oxaliplatin-treated hydroorganoids was measured by enzyme-linked immunosorbent assay, and a monoclonal antibody specific for human TP was used (mean ± sdm. ns. P > 0.05, * P). <0.05, ** P <0.01, *** P <= 0.001).
15 is the differentiated drug sensitivity of patient-derived hydroorganoids compared to patient-derived Matrigel organoids.　(　a) Schematic diagram of the experiment. Matrigel or hydroorganoids established from one parent organoid were treated with vehicle (DMSO), doxyfluridine, oxaliplatin or a combination of doxyfluridine and oxaliplatin every other day for 6 days. (bc) Representative live/dead images of (b) Matrigel or (c) hydroorganoids under the indicated conditions. Organoids were stained with live/dead assay dye on day 6 after drug treatment. Scale bar, 500 μm for MO, 250 μm for HO. (df) Comparative analysis of drug sensitivity and reproducibility for combination therapy (Doxifluridine + Oxaliplatin) in organoids from each platform. Three independent experimental batches were analyzed using CCK8 assay for drug response. Relative dose-response curves (mean ± sdm.) of (d) Matrigel or (e) hydroorganoids after 6 days of treatment with combination therapy. (f) Graph showing _{IC 50 values per experiment.} Relative fold differences per experiment indicate reproducibility of drug response. (IC50 values are mean ± SD of each condition analyzed in triplicate).
16 is a comparison result of transcripts of patient-derived hydro and Matrigel organoids. (a) Heat map for hierarchical clustering using Z-scores for normalized values (log2-based). 892 genes satisfying fc≥&p<0.05. HO; hydroorganoids, MO; Matrigel Organoids. (b) Multidimensional Scaling (MDS) plot. (c) Pearson's coefficient. (d) Total number of upstream or downstream genes in hydroorganoids compared to Matrigel organoids (fc≥ & p <0.05) (ef) Top 15 enriched GO terms of genes differentially expressed in Matrigel or hydroorganoids . (G-I) Gene set enrichment analysis of differentially expressed genes, ECM receptor interactions, adhesion junctions, and basal transcription factor gene sets in Matrigel or hydroorganoids. The y-axis represents the enrichment score (ES) and the x-axis is the gene represented by the gene set (vertical black line). The green line connects the point of the ES and gene.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are 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 describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Example]

experimental method

1: Organoid Preparation

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

All samples used for organoid establishment (Table 1) and biological analysis were obtained from Yonsei University Severance Hospital (IRB # 0000-0003-0180-8565). This study was approved by the ethics committees of both institutions. Healthy and neoplasmic colonic tissues were obtained from endoscopic biopsy or surgically excised specimens. For human tissue, information on whether or not genetic mutations such as K-RAS, N-RAS, MSI (microsatellite instability), and MSS (microsatellite stable) were obtained along with tissue samples.

[Table 1]

2: Hydro plate preparation and HO incubation

The hydro plate of the present invention made of a silicone-based material (LUMISIL886, Barkers) was manufactured by Lee & Lee Hightech (Bucheon, Korea). After sterilization and defoaming, hydroplates were pre-coated with 10% BSA in PBS (A9418, Sigma Aldrich) for 12 h to improve low adhesion at the bottom of the subwells. Finally, enzymatically dissociated single cells were inoculated with a complete culture medium containing 2% Matrigel at a density of 100 cells per sub-well to enable suspension culture.

3: Determination of organoid size and number

The size of the organoids was analyzed using the Image J program. Specifically, by selecting a region of interest in the phase image and applying a threshold with the Image J program, the unnecessary areas are overwritten with white and the areas not drawn properly are filled with black. The area to which the titer filled in black was applied was obtained using the outline.

4: Drug screening

Organoids were harvested from Matrigel and dissociated into single cells using TrypLE (supplemented with ROCKi) and mechanical dispersion. The cell suspension was resuspended in 2% Matrigel/98% complete medium. 100 cells per well of a 96-well hydro plate were seeded and grown for 7 days. A series of concentration dilutions of Doxifluridine (S2045, Selleckchem), Oxaliplatin (S1224, Selleckchem) and XELOX (a combination of Doxifluridine and Oxaliplatin) or vehicle (DMSO) control were applied to the organoid cultures every 2 days. did. To analyze the drug response of organoids, CCK-8 assay (CK04, Dojindo) and live/dead staining (L3224, Invitrogen) were used to analyze cell viability after 6 days of treatment according to the manufacturer's instructions. Data analysis and IC50 value determination was performed with GraphPad Prism (version 8.0.1).

5: Viability analysis (live/dead staining and CCK-8 analysis)

For live/dead staining, organoids were incubated with 50 mM calcein-AM and 25 mg/mL EthD-1 (ethidium homodimer-1; Molecular Probes, USA) at 37 °C for 40 min and then under confocal microscopy ( Olympus, Japan). Calcein-AM (green, for live cells) or EthD-1 (red, for dead cells) signals were quantified with ImageJ software (NIH, Bethesda) (NIH, Bethesda, MD, USA). In addition, the survival rate of organoids was quantitatively analyzed using the WST tetrazolium salt method (CCK-8, CK04, Dojindo). The response by the CCK-8 substrate solution at the indicated time points after drug treatment was measured at 450 nm using a microplate reader (Thermo Fisher, USA) and presented as a dose-response curve.

6: Immunofluorescence staining and flow cytometry

It was confirmed by staining LGR5, an organoid stem cell, through immunofluorescence staining. First, standard organoids according to the present invention were stored in 4% paraformaldehyde solution at room temperature for 1 hour and then stained with PBS. Then, after refrigeration for one day in 15% sucrose and one day in 30% sucrose, a cryo-block was made using liquid nitrogen. Using the frozen block made, the section was processed to a thickness of 10 μm, and the cut section was attached to a slide glass. After treatment with tritonX 0.1% for 10 minutes, it was washed twice with PBS. After storage at room temperature for 1 hour with 3% BSA, LGR5 immunostaining was performed after washing with PBS twice. Thereafter, the mounting solution was added and measured with a fluorescence microscope.

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

For IF staining, organoids were fixed in 4% paraformaldehyde (PFA) for 30 min at room temperature, permeabilized, and blocking solution (6% BSA + 0.2% Triton X-100 + 0.01% Sodium Azide in PBS). , blocked by 　. Then, it was stained with a primary antibody and a fluorophore-conjugated secondary antibody (Table 2) at room temperature for 48 hours. Nuclei were counterstained with DAPI (4,6-diamidino-2-phenylindole dihydrochloride, Invitrogen), and images were obtained with a confocal microscope (Olympu　s, Japan). For whole mount staining, organoids were rinsed with PBST and immersed in a clearing solution (25% UREA in distilled water + 65% sucrose in distilled water) to adjust RI values and stained with antibodies as previously described. For flow cytometry, enzymatically dissociated single cells were resuspended in HBSS containing 2% FBS and ROCKi (HF2-i buffer) and labeled with a fluorophore-conjugated antibody (Table 2) on ice for 30 min. Appropriately matched isotype controls were also used to identify non-specific labeling. After resuspension in HF buffer with 7-AAD, cells were analyzed using a FACSVerse flow cytometer (BD Biosciences). Data were evaluated using FlowJo software (Tree Star, Ashland, OR, USA).

[Table 2]

7: ELISA

Thymidine phosphorylase protein was measured using a human TP ELISA kit (EKU07660, Biomatik) according to the manufacturer's instructions.

8: RNA preparation, quantitative RT-PCR and RNA-seq analysis

CT 값은,

Ct는 비히클 처리 오가노이드에 정규화 값으로 주어진, 하우스 키핑 유전자 GAPDH로 정규화되었고, 상대적 발현 값은 최종적으로 2(

C(T)) 방법을 사용하여 결정하였다.　전체 전사체 시퀀싱에 의한 유전자 발현 분석을 위해 TruSeq Stranded Total RNA Sample　Preparation 가이드에 따라 오가노이드의 RNA 라이브러리를　준비했다.　정렬된 판독은 HTSeq-count를 사용하여 정량화되었다 (Anders et al., 2015).　차별적으로 발현된 유전자, 폴드 비율, p-값 및 오류 발견 비율은 각 피험자에 대한 edgeR 알고리즘에 의해 확인되었다.　강화된 KEGG 경로는 GSEA-P에 의해 확인되었다.Total mRNA was isolated from organoids using RNeasy Plus Mini Kits (Qiagen, Hilden, Germany) and reverse transcribed using RNA in a cDNA kit (4387406, Applied Biosystems) according to the manufacturer's protocol. Then, quantitative PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) using the primers listed in Table 3.

The CT value is

Ct was normalized to the housekeeping gene GAPDH, given as the normalized value to vehicle-treated organoids, and the relative expression value was finally 2(

C(T)) method. For gene expression analysis by whole transcriptome sequencing, an RNA library of organoids was prepared according to the TruSeq Stranded Total RNA Sample Preparation guide. Aligned reads were quantified using HTSeq-count (Anders et al., 2015). Differentially expressed genes, fold rates, p-values and error detection rates were identified by the edgeR algorithm for each subject. The enhanced KEGG pathway was confirmed by GSEA-P.

[Table 3]

9: Statistical analysis

All quantitative data are presented as the mean ± SEM over at least 3 independent replicates and the sample size “n” for each experiment is indicated in the legend of each figure. In statistical analysis, all p-values were calculated by one-way ANOVA using Prism 7 Student's t-test (NS: insignificant difference, 　　*p <0.05, 　**　p <0.01 and 　***　p <0.001 ).

Example 1.

Organoids were prepared by the method mentioned in Experimental Method 1 above. Organoids were prepared under conditions in which 2% by volume of Matrigel was contained in the culture medium (Example 1-1) and under conditions in which Matrigel was not added (Example 1-2).

Example 2.

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

Example 3.

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

<Comparative example>

Comparative Example 1.

Organoids were cultured inside Matrigel, a method widely used in the past, 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 cells were seeded in Matrigel to prepare organoids.

<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 cell spheres was compared. The spheroid is imaged in an automated plate device, and at this time, the device automatically focuses and proceeds. Image size analysis was performed using a macro program called imageJ.

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

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 was almost uniform. Specifically, when cells were seeded at an average of 100 cells/well in each sub-well, a uniform organoid that could be analyzed for comparison could be prepared. At this time, the error range for the organoid size was about 20 μm. Through this, it can be seen that standard organoid production is possible using the organoid culture method according to the present invention.

On the other hand, in the case of Comparative Example 1 using a conventional well plate, it was confirmed that the cell spheres were formed differently in size. This is because multiple cells grow in the dome shape of one matregel, so the error range of the generated organoid size is 150 μm or more, and it overlaps, making it impossible to perform uniform high-speed mass imaging and experiments.

The base and the well plate of the present invention each include a convex part and a concave part to fix each other, and the convex part and the concave part are coupled to each other so that the base can firmly fix the well plate, It shows that images 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. If there is no plate base, it seems that image analysis becomes difficult because the focal deviation of imaging increases.

In addition, referring to FIG. 5( a ), it was confirmed that organoid formation was well achieved when the cell culture plate of the present invention was used even without Matrigel.

Figure 5 (b) shows the expression level was confirmed by staining LGR5, which is the most important marker for the formation of colon cancer organoids, in order to confirm that the cultured organoids were successfully formed. In addition, it was confirmed that a colon-specific structure was present in the colon cancer organoids of the group not using the low concentration Matrigel or Matrigel formed by F-actin staining.

Experimental Example 2. Organoid production

The cells cultured in Example 1-1 and Comparative Example 1 were imaged at high speed.

The organoids prepared in Example 1-1 and Comparative Example 1 were imaged in an automated plate device, and at this time, the device automatically focused and proceeded. Image size analysis was performed using a macro program called imageJ.

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

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

Referring to FIG. 6 , when the organoid prepared in Example 1-1 is automatically imaged, the imaging height is uniform and imaging is possible without a large error. can

In particular, when culturing an organoid 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 is automatically set when measuring the image, and the deviation of 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 the case of Comparative Example 1, it can be seen 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 inside and outside.

This is because when organoids are cultured in the conventional method, it is difficult to uniformly culture the desired organoids because the organoids grow randomly in Matrigel, and the measurement height is also variable, making it difficult to apply to organoid screening imaging. has exist. Therefore, when the area of the organoids cultured for a certain period is analyzed, it is judged 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 standard organoids made entirely. A total of 864 wells were subjected to high-speed mass imaging, and it was confirmed that uniform diameters were obtained by analyzing each of them with ImageJ.

8 shows the imaging results and organoid size when the colorectal cancer cells of Example 1-1 were cultured for 14 days. It can be seen that standard organoid production is possible because the organoid size is uniform at 300-50 um.

9 is a photograph showing the organoids according to Examples 1-1 and 1-2 over time (left) and the organoid survival rate according to Example 1-1 (right). In the case of FIG. 9, Live/Dead staining of the cultured organoids was performed to confirm how much the cultured organoids were actually maintained. First, the cultured standard organoids are washed with PBS, and then incubated with accutase for about 10 minutes. After fragmenting into single cells, Calcein and EtdH-1, which are Live/Dead reagents, were stored in an incubator for about 30-60 minutes, and the amount of Live/Dead each was checked with a fluorescence microscope.

It can be seen from Figure 9 that even without Matrigel, organoid formation occurs very well, and when cultured for 14 days, it can be seen that 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. 10 (a) is when the colon cancer cells were cultured for 14 days in Examples 1-1, 2, and 3 shows the imaging results of ((a) Example 1-1, (b) Example 2, (c) Example 3).

Referring to FIG. 10( a ) , when cells were applied to each sub-well at an average of 100 cells/well, a uniform organoid that could be compared and analyzed could be prepared (error range: about 20 μm).

Figure 10 (b) is a graph showing the high-speed mass imaging results of Examples 1-1, 2 and 3, Figure 10 (b) is Example 1-1, 2 and 3 colon cancer cells 7 days or 14 days Shows the results of liver culture ((a) Example 1-1, (b) Example 2, (c) Example 3).

Referring to FIG. 10(b), it shows that the most desirable organoids can be produced when cells are cultured on average of 100 cells/well for an average of 14 days. That is, when cells with an average of 100 cells/well or less are cultured for 14 days, it is likely that optimal organoids can be differentiated and grown. On the other hand, when increasing the number of cells seeded in the subwell and reducing the number of culture days, it was confirmed that the organoid performance deteriorated.

For reference, the dotted line in FIG. 10 (b) means the maximum space of the sub-well of the cell culture plate of the present invention, which means a space for culturing cells. It is considered possible

Experimental Example 3. Patient-specific drug screening

3.1　Homogeneous hydroorganoids summarizing the histologic complexity of colorectal cancer

According to the contents described in Experimental Methods 1 and 2, patient-derived colorectal cancer organoids (HOs) were prepared using hydroplates. The hydro plate according to the present invention has 9 sub-wells (500 μm diameter) in each well (6.3 mm diameter) and is suitable for the existing 96-well plate format (11a). Eight wells form one strip and a total of 12 strips form a complete hydroplate. 864 HOs can be generated simultaneously, and the number of strips inserted into the plate can be adjusted to meet the production of any number of organoids. In one subwell, single cells were seeded and aggregated using complete culture medium containing 2% Matrigel and cultured in suspension (11b) . The 9 sub-wells in the well also had the same culture conditions. In particular, the hydroplate consistently produces uniform HOs (size and shape) in self-organized form, reproducing multicellular interactions in subwells (Fig. 11c-e).

To compare the morphological and histological properties of hydroorganoids (HOs) with Matrigel organoids (MOs), surgically resected colorectal cancer (CRC) tissue was isolated into individual cells or cell clusters, embedded in Matrigel, and primary established as parent organoids (Fig. 12a). 100 cells in each subwell aggregated into one clump on day 3 of culture and continued to grow for 14 days of total culture (Fig. 12b). Interestingly, unlike MO, which showed wide morphological and diameter variations between and within wells, HO was relatively uniform and homogeneous (Fig. 12f-g) with no significant differences in viability (Fig. 12c-e).

Because low Matrigel concentrations, convective and diffusive transport, and multicellular interactions of hydroplates may influence the cellular and functional properties of organoids, the developmental stages and characteristics of CRC MOs and HOs were histologically analyzed (Fig. 13a). , b, d). The histological characteristics of undifferentiated parent organoids expressing mucin are highly conserved in both organoids. However, cancer stem cell subsets such as CD44 + EpCAM +, CD44 + EpCAM + CD166 + CD133 + and CD44 + EpCAM + CD166 + CD133 + LGR5 + cells were higher in HO than in MO. (Fig. 13c). This biomarker expression shows that the hydroplate of the present invention can enhance the expansion or maintenance of cancer stem cells in organoids.

3.2　 Hydroorganoids as a consistent and reproducible platform for preclinical cancer drug screening

To demonstrate the potential of HOs for drug screening, CRC HOs were exposed to various concentrations of three drugs, Doxifluridine (converted from capecitabine in liver), Oxaliplatin, and their combination for 6 days. (3 treatments with an interval of 2 days). Live/dead imaging and CCK-8 analysis were applied to analyze dose-response curves and half-maximum inhibitory concentration (IC50) values (Fig. 14). HOs showed obvious toxicity in a window defined between 0.03 μM and 0.07 μM for each drug treatment (Fig. 14b, c). The synergistic effect of combination therapy (XELOX, Capecitabine + Oxaliplatin) compared to oxaliplatin alone, as reported in patient-derived xenografts (PDX) (Jim Cassidy et. al., Journal of Clinical Oncology, Volume 22, Issue 11) , appeared through a mechanism that induces thymidine kinase (TP) expression (Fig. 14d-f). Specifically, the effectiveness and accuracy of HOs for drug screening were confirmed through comparative screening with MOs (Fig. 15). In particular, clear visualization of contraction and apoptosis of HOs was detected after drug treatment (Fig. 15b,c). Interestingly, HOs responded more sensitively to the same drug than MOs, and the IC values of experiments with HOs were continuously measured, demonstrating high reproducibility during replicates of three experiments (Fig. 15d–f).

Distinct gene expression pattern of 3.3　hydroorganoids

The molecular features of MO and HO were characterized and compared by RNA-seq analysis. 892 genes (DEG, fc≥2, p <0.05) were differentially expressed, and the two organoids were classified into two distinct subtrees according to hierarchical clustering of up- and down-regulated genes (625, 267, respectively). , Figure 16a-d). Unbiased transcriptome analysis with principal component analysis showed that the two organoids are distinct but closely linked (Fig. 16b-c). Unbiased transcriptome analysis with principal component analysis showed that the two organoids are distinct but closely linked (Fig. 16b-c). Expression of genes involved in ECM receptor interactions and adhesive splicing was highly downregulated in MOs (Fig. 16e–i). That is, these results show that HOs are very sensitive to drugs.

Organoid, stem cell-derived, organ-mimicking multicellular systems have recently been highlighted as important preclinical models for biomedical applications such as drug discovery and regenerative medicine. Inevitably, large-scale culture of organoids has become a prerequisite for industrial and clinical applications. However, current organoid culture systems that rely on embedding single cells or clusters in ECM hydrogels (e.g. Matrigel®) provide true in vivo functions (intratumoral heterogeneity) as well as precise and standardized control of organoid generation. There are limits to what can be achieved. As a result, culture techniques have been developed to meet the problem-solving needs, but fundamental biological differences between culture techniques have not been elucidated. In the present invention, a 96-well plate-compatible hydroplate was developed for culturing uniform HOs. Unlike conventional, Matrigel culture, the generation of HOs in hydroplates is synchronized into aggregates by heterogeneous populations of individual tumor cells and cultured in the absence of Matrigel or low Matrigel content. This method controlled the initial conditions of HOs by limiting the size, geometry and microenvironment of cell aggregates, and led to uniform and homogeneous organoid formation. HOs are functionally similar to MOs as a preclinical model, but differ in terms of the transcriptome and subcellular composition of cancer cells and differentially contribute to organoid properties and therapeutic responsiveness.

MOs and HOs The underlying mechanisms that can be considered as the reasons for these cellular and functional differences are as follows. The first major parameter is the ECM dynamics of HOs.

The HO culture medium can be supplemented with 2% Matrigel as a minimal environment to support organoid formation and complex morphogenesis. This minimal environment may limit the formation of structures that are highly dependent on ECM dynamics (mechanical transformation by stiffness), but allow for mass transfer kinetics of drugs. This is consistent with the result of increased drug sensitivity of HOs compared to MOs to the same drug. Geometry and cell density may also contribute to functional differences in HOs. The geometric control of hydroplates enables precise spatiotemporal modulation of biophysical and biochemical signals for cells grown within HOs, resulting in molecularly and functionally uniform and reproducible organoids and morphologies. This possibility is confirmed by the constant and reproducible IC values of HOs in three replicates. Moreover, in stem cell-based aggregates such as HOs, superficial and centrally located cells can be attributed to cell-cell and cell-ECM interactions, mechanical tension, cytoskeletal rearrangement, and diffusion gradients (i.e., oxygen gradients) of endogenous and exogenous molecules. They are exposed to different physical parameters, which in turn influence the differentiation and maintenance of cells related to functions such as drug responsiveness.

Indeed, in the present invention, we observed an increase in gene expression related to ECM receptor interaction and adhesion junctions in HOs and a decrease in genes related to differentiation and development through comparative transcriptome analysis. We also observed quantitative differences in cancer stem cell subsets in the two organoids (HOs and MOs) by flow cytometry. These results indirectly indicate a functional correlation between geometry and biological effects in HOs.

In summary, the present invention shows another type of scalable organoid culture method for application in biological research and high-dose assays. These results show that organoid culture types and methods can influence distinct properties, chemical resistance, and assay accuracy.

Using the organoid culture method of the present invention, accurate drug screening is possible by producing patient-specific disease organoids. Therefore, the present invention provides accurate drug information in the preclinical stage without using an animal model.

The specific part of the present invention has been described in detail above, and for those of ordinary skill in the art, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be obvious. Accordingly, 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 140: recess
200: Connector for high-capacity high-speed HCS
210: base 220: cover
240: iron part

<110> Korea University Research and Business Foundation Next & Bio Inc. <120> Method of providing information for patient-specific drug selection <130> DHP20-188P <150> KR 20/0022912 <151> 2020-02-25 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> GapDH forward primer <400> 1 aacagcgaca cccatcctc 19 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GapDH reverse primer <400> 2 cataccagga aatgagcttg acaa 24 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> LGR5 forward primer <400> 3 ttccccaggc cccttcaac 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LGR5 reverse primer <400> 4 cactgctgcg atgaccccaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Chromogranin A forward primer <400> 5 acactttcca agcccagccc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Chromogranin A reverse primer <400> 6 agagcctccg accgaccttt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Villin forward primer <400> 7 gctgaggtca caagccccaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Villin reverse primer <400> 8 gcagagaagg cagctggagt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta catenin forward primer <400> 9 tgtggtcacc tgtgcagctg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Beta catenin reverse primer <400> 10 tgcttcttgg tgtcggctgg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KI67 forward primer <400> 11 agctgactct gccactaagc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> KI67 reverse primer <400> 12 gtccagctgt agtgcccaat 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> SOX9 forward primer <400> 13 atgaagatgc cgacgagca 19 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SOX9 reverse primer <400> 14 tcctcgctct ccttcttcag at 22

Claims (16)

isolating diseased tissue from a diseased subject;
classifying the diseased tissue by genetic mutation;
isolating cells from the classified disease tissue;
Culturing the isolated cells as disease organoids in a three-dimensional cell culture plate:
treating the cultured disease organoid with a drug; and
Including; measuring the activity of the disease organoid
As a method of providing information for a subject-specific drug selection with a disease,
In the disease organoid culture step, the three-dimensional cell culture plate contains 0 to 2% by volume of an extracellular matrix-based hydrogel,
The three-dimensional cell culture plate,
a well plate including a plurality of main wells and a plurality of sub wells formed under each of the main wells to inject a cell culture solution and having a recess on the bottom surface; and a connector for high-capacity high-speed HCS (High contents screening) supporting the well plate;
The large-capacity and high-speed HCS (High contents screening) connector includes a bottom of the well plate and a base provided with a fixing means to be detachable from each other, and a cover positioned on the top of the well plate and coupled to the base, and the main well A step is formed so as to be tapered in a predetermined area, and the step is a cell culture plate having an inclination angle (θ) in the range of 10 to 60° with respect to the wall of the main well,
A method of providing information for personalized drug selection for a subject with a disease.
According to claim 1,
The extracellular matrix-based hydrogel is Matrigel, a method for providing information for a subject-specific drug selection with a disease.
According to claim 1,
the cell is a cancer cell;
The incubation period is 1 to 14 days, a method for providing information for a subject-specific drug selection with a disease.
According to claim 1,
The method for providing information for a subject-specific drug selection with a disease, characterized in that the sub-well has an inclined surface that is tapered toward the concave portion.
According to claim 1,
The sub-well has an inclined surface that is tapered toward the concave portion;
The top diameter of the sub-well 120 is in the range of 3.0 to 4.5 mm,
The diameter of the upper end of the concave portion 121 is in the range of 0.45 to 1.5 mm,
_{The slope (θ 2} ) of the sub-well and the recess is in the range of 40 to 50°,
The information providing method for selecting a drug tailored to a subject having a disease, characterized in that the ratio of the length of the diameter of the sub-well to the diameter of the concave portion is in the range of 1:0.1 to 0.5.
According to claim 1,
The individual volumes of the main wells range from 100 to 300 μl,
The individual volumes of said recesses range from 20 to 50 μl,
An information providing method for selecting a drug tailored to a subject having a disease, characterized in that the individual volume ratio of the main well and the concave portion is on average 1: 0.1 to 0.5.
According to claim 1,
The main well includes a space between the step and the sub well,
The height of the space (a _h ) is in the range of 2.0 to 3.0 mm on average,
The height of the sub-well (b _h ) ranges from 1.0 to 2.0 mm on average,
_{The height ratio (a h} :b _h ) of the space portion and the sub-well is in the range of 1:0.3 to 1. The method for providing information for selecting a drug tailored to a subject having a disease.
According to claim 1,
Wherein the drug is one drug or two or more drugs, a method for providing information for a subject-specific drug selection with a disease.
According to claim 1,
The activity of the organoid is the viability (viability) of the organoid, the method for providing information for a subject-specific drug selection with a disease.
According to claim 1,
After measuring the activity of the disease organoid,
When the activity of the disease organoid after treatment with the drug falls compared to before the drug treatment, it is determined that the effect of the drug is effective, and further adding a step of selecting a drug tailored to the subject with the disease,
A method of providing information for personalized drug selection for a subject with a disease.
According to claim 1,
If more than one drug is
When the activity of the disease organoids after treatment with the two or more drugs is lower than when each drug is treated alone, it is judged that there is an effect when the two or more drugs are treated in combination; a subject having a disease To further add the step of selecting as a customized combination drug,
A method of providing information for personalized drug selection for a subject with a disease.
According to claim 1,
After measuring the activity of the disease organoid,
Which further adds the step of determining the dose of the drug by deriving the IC50 value of the drug,
A method of providing information for personalized drug selection for a subject with a disease.
According to claim 1,
The disease is cancer, a method for providing information for a subject-specific drug selection with a disease.
14. The method of claim 13,
The cancer is colorectal cancer, lung cancer, stomach cancer, pancreatic cancer, kidney cancer, brain tumor, ovarian cancer, skin cancer, prostate cancer, breast cancer, cervical cancer, thyroid cancer, fibrosarcoma, uterine sarcoma, or blood cancer. How to provide information for
According to claim 1,
The drug is an anticancer agent, a method for providing information for a subject-specific drug selection with a disease.
16. The method of claim 15,
The anti-cancer agent is one or more anti-cancer agents selected from the group consisting of cytotoxic anti-cancer agents, immune anti-cancer agents, targeted anti-cancer agents, anti-cancer agents, and new anti-cancer agents, information providing method for customized drug selection for a subject having a disease.

Images