Technique for generating cancer organoid using gelatin
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- THE ASAN FOUND
- Filing Date
- 2026-01-21
- Publication Date
- 2026-07-30
Smart Images
Figure KR2026001211_30072026_PF_FP_ABST
Abstract
Description
Cancer organoid generation technology using gelatin
[0001] The present invention relates to a technology for generating cancer organoids using gelatin.
[0002] The present invention claims priority based on Korean Patent Application No. 10-2025-0011836 filed on January 24, 2025, and all contents disclosed in the specification and drawings of said applications are incorporated by reference into the present application.
[0003] Currently, drug evaluation for new drug development is conducted using cell lines or animal models. While the cell line method is the easiest to use as it utilizes established cell lines, it has the disadvantage of not being able to observe specific pathogenic mechanisms or drug responses due to the genetic characteristics of individual patients. Mice, rats, rabbits, dogs, pigs, and monkeys are commonly used as animal models, but they exhibit structural or interspecies differences compared to human tissues. Such characteristics present a disadvantage, for example, in that the interaction between the virus and the host cannot be accurately analyzed when developing treatments for viral infections.
[0004] To address these drawbacks, organoids are garnering attention. Organoids are organ-like structures produced by three-dimensionally culturing cells derived from tissues or organs. Cell-derived organoids can be cultured in vitro for longer periods, allowing for an increased number of passages. Furthermore, compared to conventional methods, organoids can yield a larger number of cells from single cells or cells in the initiation stage, making them more suitable for research. For instance, they can be utilized in various fields, such as pharmacological screening, embryological research, and functional recovery through transplantation. Moreover, organoids, which are cancer tissue analogs formed from harvested cancerous tissue from patients, can serve as a key foundation for the recent development of personalized anticancer drugs and diagnostic markers.
[0005] Meanwhile, cancer is one of the incurable diseases that humanity must address, and massive capital is currently being invested globally in the development of a cure. In Korea, it is the leading cause of death from disease, with over 100,000 people diagnosed and more than 60,000 dying annually. Although various anticancer therapies have advanced dramatically in cancer diagnosis and treatment over the past decade, the mortality rate from cancer remains high. Furthermore, side effects associated with the application of various anticancer drugs and therapies persist. Research aimed at reducing these side effects is currently being actively conducted. Accordingly, a new evaluation model is needed to minimize side effects and maximize efficacy during the development of cancer treatments or diagnostic methods.
[0006] Organoids are emerging as experimental models capable of long-term culture and cryopreservation. Unlike conventional cell lines, they do not undergo an immortalization process, thereby preserving the cells' inherent characteristics. Furthermore, by reproducing hierarchical and histological structures previously observed only in vivo, they allow for the study of physiological phenomena at a higher level than cells. Consequently, they are being recognized as a promising new model for cancer research. Organoids enable drug evaluation with higher accuracy compared to immortalized cell lines with altered intrinsic characteristics or animal models with structures distinct from the human body. In particular, because they utilize patient-derived tissues, they offer the advantage of verifying both the safety and efficacy of drugs prior to clinical trials in humans. By allowing for the systematic study of the pathological characteristics and drug responsiveness of cancer cells—which were difficult to observe with existing cell lines or animal models—organoids can serve as a crucial foundation for enhancing the accuracy and effectiveness of cancer treatment and diagnosis, as well as for establishing personalized treatment strategies.
[0007] However, to date, the technology for producing organoids derived from colorectal cancer or sarcoma cells remains in the early stages, and culture medium compositions for effectively manufacturing them have not yet been commercialized.
[0008] The object of the present invention is to provide a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, comprising gelatin as an active ingredient as a substitute for basement membrane extract (BME).
[0009] Another object of the present invention is to provide a method for producing a colorectal cancer or sarcoma cell-derived organoid, comprising the step of culturing colorectal cancer or sarcoma cells in the culture medium composition of the present invention.
[0010] Another objective of the present invention is to provide a method for preparing a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, comprising the step of adding gelatin as a substitute for basement membrane extract (BME).
[0011]
[0012] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.
[0013] To achieve the above objectives, the present invention provides a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, comprising gelatin as an active ingredient as a substitute for basement membrane extract (BME).
[0014] In one embodiment of the present invention, the colorectal cancer or sarcoma cells may be one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue, but are not limited thereto.
[0015] In another embodiment of the present invention, the gelatin may be contained in an amount of 5 to 15 weight percent relative to the total weight of the medium, but is not limited thereto.
[0016] In another embodiment of the present invention, the gelatin may influence the promotion of organoid formation, the promotion of cancer marker-related expression, the improvement of structural stability, or the improvement of metabolic activity, but is not limited thereto.
[0017] The present invention provides a method for producing a colorectal cancer or sarcoma cell-derived organoid, comprising the step of culturing colorectal cancer or sarcoma cells in the culture medium composition of the present invention.
[0018] In one embodiment of the present invention, the colorectal cancer or sarcoma cells may be one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue, but are not limited thereto.
[0019] The present invention provides a method for preparing a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, comprising the step of adding gelatin as a substitute for basement membrane extract (BME).
[0020] In one embodiment of the present invention, the colorectal cancer or sarcoma cells may be one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue, but are not limited thereto.
[0021] In another embodiment of the present invention, the gelatin may be contained in an amount of 5 to 15 weight percent relative to the total weight of the medium, but is not limited thereto.
[0022] In another embodiment of the present invention, the gelatin may influence the promotion of organoid formation, the promotion of cancer marker-related expression, the improvement of structural stability, or the improvement of metabolic activity, but is not limited thereto.
[0023] In addition, the present invention provides a medium composition comprising gelatin as an active ingredient as a substitute for basement membrane extract (BME) for forming organoids derived from colorectal cancer or sarcoma cells.
[0024] In addition, the present invention provides a medium composition comprising gelatin as an active ingredient as a substitute for basement membrane extract (BME) for preparing a medium for forming organoids derived from colorectal cancer or sarcoma cells.
[0025] In this invention, it was confirmed that when gelatin is used as a substitute for basement membrane extract (BME), organoid formation is promoted, the expression of cancer markers is enhanced, and structural stability is improved compared to when conventional BME is used. Furthermore, metabolic activity is also improved through the observation that the oxygen consumption of the organoids increases. Additionally, the organoid formation medium of this invention is more economical than BME in terms of cost and the time required for organoid formation. Since the limitations of reproducing the biological characteristics of tumors in conventional 2D cell culture methods are overcome by the medium composition of this invention utilizing gelatin, thereby providing a physiologically similar model, this patent can be utilized as a useful platform for future preclinical studies, diagnosis, and therapeutic evaluation.
[0026] Figure 1 shows the process of culturing organoids from a tumor specimen.
[0027] Figure 2a shows the overall experimental procedure for confirming whether organoids are formed in colorectal cancer cell lines and sarcoma-derived cells.
[0028] Figure 2b shows 2D images of colorectal cancer cell lines (HT29 and CT26) and sarcoma patient-derived cells (Patient 1 and Patient 2).
[0029] Figure 2c shows the results of organoid formation using a colorectal cancer cell line (CT26) and sarcoma patient-derived cells (Patient 1 and Patient 2) when BME was used.
[0030] Figure 2d shows the results of organoid formation using colorectal cancer cell lines (HT29 and CT26) and sarcoma patient-derived cells (Patient 1 and Patient 2) when gelatin was used.
[0031] Figure 2e shows the quantitative results for markers related to colorectal cancer (HT29 and CT26) and sarcoma (Patient 1) expressed in the formed organoids after forming organoids using BME and gelatin.
[0032] Figure 3a shows the difference in the degree of aggregation of sarcoma patient-derived cells when different concentrations of gelatin were used. The solid line in the lower right corner represents a length of 100 μm.
[0033] Figure 3b shows the results of confirming organoid formation and organoid structure formation when BME and gelatin were used.
[0034] Figure 3c shows the results of confirming the oxygen consumption of the formed organoids when BME and gelatin were used.
[0035] The inventors used gelatin, an extracellular matrix, to develop a method for generating cancer organoids that can replace conventional basement membrane extract (BME). As a result, not only were structurally more stable organoids than conventional BME established in sarcoma or colorectal cancer, but it was also confirmed that the expression of cancer marker-related markers increased. Furthermore, the present invention can overcome the limitation that BME cannot be applied when forming organoids from conventional sarcoma-derived cells and colorectal cancer-derived cells.
[0036] Including all claims below, gelatin is an extracellular matrix component used in organoid culture and is a cost-effective choice among other culture materials, thereby reducing the economic burden. In addition, cancer organoids can be efficiently produced by minimizing the cancer organoid formation period (BME: 21 days, gelatin: 10 days).
[0037] The present invention provides a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, comprising gelatin as an active ingredient as a substitute for basement membrane extract (BME).
[0038] In all claims below, the “basement membrane” is a continuous sheet-like structure composed of a specialized extracellular matrix that forms a boundary between endothelial cells, epithelial cells, muscle cells, or neurons and their adjacent stroma, and may play a role in tissue structure formation by influencing cell adhesion, migration, proliferation, and differentiation, but is not limited thereto.
[0039] In all claims below, the term “Basement Membrane Extract (BME)” in this specification may be a composition that mimics the in vivo microenvironment and promotes the formation and growth of organoids, but is not limited thereto. It may be a broad concept including all basement membrane extracts that can be readily manufactured, purchased, or obtained by a person skilled in the art.
[0040] Additionally, in all claims below, the “basement membrane extract” may be a soluble form of basement membrane purified from Engelbreth-Holm-Swarm (EHS) tumors, a natural extracellular matrix hydrogel that forms a reconstituted basement membrane by polymerizing at 37°C, and may include, but is not limited to, laminin, entactin, and heparan sulfate proteoglycans as major components.
[0041] In all claims below, the “basement membrane extract” may be Cultrex Reduced Growth Factor Basement Membrane Extract, PathClear (BME, R&D SYSTEMS biotechne, Cat# 3533-010-02), but is not limited thereto.
[0042] In all claims below, the term “gelatin” in this specification refers to a type of derived protein obtained by breaking down and purifying natural proteins constituting animal hides, tendons, cartilage, etc., and may be a biomatrix material, but is not limited thereto. It may be a broad concept including all gelatins that can be easily manufactured, purchased, or obtained by a person skilled in the art.
[0043] Including all claims below, the gelatin comprises 1 to 19 weight% based on the total weight of the medium, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 6 to 19, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 8 to 19, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 19, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 19, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 10 to 12, 10 to 11, less than 20, less than 19, less than 18, Less than 17, less than 16, less than 15, less than 14, less than 13,It may be contained in an amount of less than 12, less than 11, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 weight%, and specifically, gelatin may be contained in an amount of 5 to 15 weight% relative to the total weight of the medium, but is not limited thereto.
[0044] Including all claims below, gelatin may influence, but is not limited to, the promotion of organoid formation, the promotion of cancer marker-related expression, the improvement of structural stability, or the improvement of metabolic activity.
[0045] Including all claims below, the promotion of organoid formation in this specification may mean that organoids derived from colorectal cancer or sarcoma cells are not formed when a medium containing BME is used, whereas organoids derived from colorectal cancer or sarcoma cells are formed when a medium containing gelatin of the present invention is used.
[0046] Including all claims below, the promotion of cancer marker expression may mean that when a medium containing the gelatin of the present invention is used, the expression of biomarkers such as AKT (Protein kinase B, PKB), P-AKT (Phosphorylated AKT), and mTOR (mammalian target of rapamycin), which play a central role in regulating the epithelial-mesenchymal transition (EMT) process of colorectal cancer tumor cells is increased compared to when a medium containing BME is used.
[0047] In addition, as per the entire claim below, the promotion of expression of cancer indicator-related markers in this specification may mean that when using a medium containing gelatin of the present invention, the expression of one or more selected from the group consisting of CDK4 (cyclin-dependent kinase 4), MDM2 (mouse double minute 2), and Vimentin, which are biomarkers whose expression increases in liposarcoma among sarcomas, is increased more than when using a medium containing BME. Since the expression of colorectal cancer and sarcoma indicator-related markers is promoted, it implies that organoids formed in the medium containing gelatin of the present invention preserve the molecular characteristics of sarcoma or colorectal cancer well.
[0048] Including all claims below, the improvement in structural stability may mean that when a medium containing the gelatin of the present invention is used, the colorectal cancer or sarcoma cell-derived organoid formed therefrom has higher cohesion and its structure becomes clearer than when a medium containing BME is used.
[0049] Including all claims below, in this specification, improvement in metabolic activity may mean an increase in oxygen consumption as a result of measuring the oxygen consumption rate (OCR) of the cell. This implies that there is also high metabolic activity.
[0050] In this specification, including all claims below, “colorectal cancer” means a tumor that occurs anywhere on the mucosa of the colon or rectum; specifically, tumors that occur in the rectum, sigmoid colon, descending colon, transverse colon, ascending colon, cecum, and anal canal, etc., depending on the location of occurrence, are collectively referred to as colorectal cancer and may be used interchangeably with colon cancer, rectal cancer, rectal cancer, and rectal-colon cancer.
[0051] In this specification, including all claims below, “sarcoma” refers to a tumor that arises in non-epithelial connective tissues such as bone, cartilage, muscle, fat, nerve, and blood vessel, and is broadly classified into osteosarcoma, which arises in bone, and soft tissue sarcoma, which arises in soft tissues such as skin, fat, nerve, blood vessel, and muscle. Furthermore, in one embodiment of the present invention, the sarcoma may be one or more selected from the group consisting of Kaposi's sarcoma, fibrosarcoma, liposarcoma, chondrosarcoma, leiomyosarcoma, rhabdomyosarcoma, soft tissue sarcoma, angiosarcoma, and lymphangiosarcoma, but is not limited thereto.
[0052] In the entirety of the following claims, colorectal cancer or sarcoma cells may be one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue, but are not limited thereto.
[0053] In the entire specification including the following claims, “primary cells” may mean cells obtained by directly isolating and extracting from living biological tissue, but is not limited thereto. In addition, in one embodiment of the present invention, the primary cells may be cells derived from a sarcoma patient or cells derived from a colorectal cancer patient, but are not limited thereto.
[0054] In all claims below, the “cell line derived from cancerous tissue” may be a cell line that has been carcinogenicated to be able to grow continuously, but is not limited thereto. In addition, in one embodiment of the present invention, the cell line derived from cancerous tissue may be one or more selected from the group consisting of HT29, CT26, HCT116, SW480, Caco-2, LoVo, MC38, Colon26, and CMT-93, and may be one or more selected from the group consisting of Saos-2, U-2 OS, MG-63, HT1080, SW872, RD, SK-LMS-1, and K7M2, but is not limited thereto.
[0055] In this specification, including all claims below, "organoid" refers to a mass of cells having a three-dimensional structure. It is defined as a reduced and simplified version of a tissue or organ produced through an artificial culture process without being collected, acquired, or harvested from an individual or the like. Organoids have the advantages of being capable of long-term culture and cryopreservation, as well as being easy to manipulate and observe. At the same time, since immortalization is not required, the original characteristics of the cells are maintained, and by reproducing the hierarchical and histological structures of cells that could only be observed in vivo, they serve as experimental models capable of studying physiological phenomena at a higher level than cells. In one embodiment of the present invention, the organoid may be a cancer organoid, but is not limited thereto.
[0056] In all claims below, the term "cancer organoid" refers to an organoid having a structure, cellular composition, and function identical or similar to cancer in vivo, and the term "cancer organoid" may be used interchangeably with "tumor organoid" or "tumoroid." The cancer may be colorectal cancer or sarcoma, but is not limited thereto.
[0057] Including all claims below, the organoids in this specification may be colorectal cancer or sarcoma organoids; or organoids that are analogues of colorectal cancer tissue or sarcoma tissue, but are not limited thereto.
[0058] In all claims below, the term “formation” in this specification means forming a cancer organoid from cancer-derived cells and may mean exhibiting organ or tissue characteristics to perform the inherent functions of cancer, and in the present invention, “formation” may be used interchangeably with “manufacturing,” “culture,” or “proliferation,” but is not limited thereto.
[0059] Including all claims below, the “medium composition for forming an organoid” may be a medium composition for manufacturing an organoid, a medium composition for culturing an organoid, and a medium composition for expanding an organoid.
[0060] Including all claims below, "expansion" in this specification may mean increasing the number of formed organoids to obtain a sufficient amount for use in an intended purpose.
[0061] In all claims below, the term “medium” means a mixture of nutrient substances that enables the growth, survival, expansion, or differentiation of cells or organoids in vitro, and includes all appropriate conventional media used in the art. Depending on the type of cell or organoid, the type of medium and culture conditions may be selected at the technical level of the art. The medium may be a basal media for cell culture containing a carbon source, a nitrogen source, and trace elements, but is not limited thereto.
[0062] Including all claims below, the medium composition for forming organoids derived from colorectal cancer or sarcoma cells may further comprise, but is not limited to, one or more selected from the group consisting of antibiotics, fetal bovine serum, B27 supplement, Epidermal Growth Factor (EGF), human fibroblast growth factor, R-Spondin protein, Noggin protein, n-Acetyl Cysteine, Nicotinamide, A83-01, SB202190, primocin, DMEM / F12, and Roswell Park Memorial Institute (RPMI) 1640 medium.
[0063] Including all claims below, the antibiotic may be one or more selected from the group consisting of penicillin and streptomycin, the B27 supplement may be a B27 supplement excluding vitamin A, the EGF may be human EGF, the fibroblast growth factor may be human fibroblast growth factor, the R-spondin protein may be recombinant human R-spondin protein, the Noggin protein may be recombinant human Noggin protein, and the DMEM / F12 may be Advanced DMEM / F12, but is not limited thereto.
[0064] The present invention provides a method for producing a colorectal cancer or sarcoma cell-derived organoid, comprising the step of culturing colorectal cancer or sarcoma cells in the culture medium composition of the present invention.
[0065] In all claims below, the colorectal cancer or sarcoma cells may be cancer cells isolated from an individual, but are not limited thereto.
[0066] In all claims below, the term "individual" in this specification includes all individuals that have never developed cancer, individuals that are likely to develop cancer, individuals that have developed cancer, or individuals that have been cured after developing cancer, and may include, without limitation, human or non-human primates, mice, rats, mammals such as dogs, cats, horses, and cattle. Furthermore, in the present invention, the term "individual" may be used interchangeably with "subject" or "patient." Additionally, cancer cells isolated from the individual may include stem cells.
[0067] In the entirety of the following claims, “cancer cells isolated from an individual” may be isolated through a process of cutting cancer tissue and an enzymatic degradation process. Specifically, the cutting may include both physical cutting and mechanical cutting and may be performed by general tissue cutting methods known in the art. Additionally, the enzymatic degradation may be performed under general enzymatic degradation conditions known in the art and may be performed, for example, using one or more enzymes selected from the group consisting of dispase II, DNA degrading enzyme (DNase) I, and collagenase II.
[0068] In all claims below, the culture conditions of “culture,” such as the culture day, culture temperature, and culture substrate, may be performed under conditions commonly used in the art for the culture of organoids, and a person skilled in the art may use a method suitable for the purpose of the present invention.
[0069] Specifically, the step of culturing in this specification, including the entire claim below, is 4 to 20 days, 4 to 18 days, 4 to 16 days, 4 to 14 days, 4 to 12 days, 4 to 10 days, 6 to 20 days, 6 to 18 days, 6 to 16 days, 6 to 14 days, 6 to 12 days, 6 to 10 days, 8 to 20 days, 8 to 18 days, 8 to 16 days, 8 to 14 days, 8 to 12 days, 8 to 10 days, 10 to 20 days, 10 to 18 days, 10 to 16 days, 10 to 14 days, 10 to 12 days, 1 day, 2 days, 3 days, 4 days, 5 days, It may be performed on the 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th, but is not limited thereto.
[0070] In the entirety of the following claims, colorectal cancer or sarcoma cells may be one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue, but are not limited thereto.
[0071] The present invention provides a method for preparing a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, comprising the step of adding gelatin as a substitute for basement membrane extract (BME).
[0072] In the entirety of the following claims, colorectal cancer or sarcoma cells may be one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue, but are not limited thereto.
[0073] Including all claims below, the gelatin comprises 1 to 19 weight% based on the total weight of the medium, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 5 to 19, 5 to 18, 5 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 6 to 19, 6 to 18, 6 to 17, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, 6 to 10, 7 to 19, 7 to 18, 7 to 17, 7 to 16, 7 to 15, 7 to 14, 7 to 13, 7 to 12, 7 to 11, 7 to 10, 8 to 19, 8 to 18, 8 to 17, 8 to 16, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 9 to 19, 9 to 18, 9 to 17, 9 to 16, 9 to 15, 9 to 14, 9 to 13, 9 to 12, 9 to 11, 9 to 10, 10 to 19, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 10 to 12, 10 to 11, less than 20, less than 19, less than 18, Less than 17, less than 16, less than 15, less than 14, less than 13,It may be contained in an amount of less than 12, less than 11, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 weight%, and specifically, gelatin may be contained in an amount of 5 to 15 weight% relative to the total weight of the medium, but is not limited thereto.
[0074] Including all claims below, gelatin may influence, but is not limited to, the promotion of organoid formation, the promotion of cancer marker-related expression, the improvement of structural stability, or the improvement of metabolic activity.
[0075] In addition, the present invention provides a medium composition comprising gelatin as an active ingredient as a substitute for basement membrane extract (BME) for forming organoids derived from colorectal cancer or sarcoma cells.
[0076] In addition, the present invention provides a medium composition comprising gelatin as an active ingredient as a substitute for basement membrane extract (BME) for preparing a medium for forming organoids derived from colorectal cancer or sarcoma cells.
[0077]
[0078] The present invention provides a method for evaluating the efficacy of an anticancer drug, comprising the steps of: preparing an organoid by culturing colorectal cancer or sarcoma cells isolated from an individual in the culture medium composition of the present invention; and treating the organoid with an anticancer drug.
[0079] Including all claims below, the organoids described herein retain stem cell capabilities and possess histological composition and function very similar to human colorectal cancer or sarcoma, making them useful for screening or evaluating the efficacy of therapeutic agents or anticancer drugs against tumors. In particular, when the organoids are manufactured using patient-derived cells or tissues, they can be used to pre-identify patient-specific anticancer drug resistance, refractoryness, or resistance. Since most cancer patients have a short remaining lifespan, it is necessary to find and receive the most effective anticancer drug for them as soon as possible. To this end, personalized medicine and precision medicine have recently been developed, and organoids derived from the cancer tissue of a specific patient according to the present invention can be utilized very effectively. Furthermore, by using the organoids to observe gene expression regulation following drug treatment, it can contribute to research on molecular mechanisms for the purpose of cancer treatment.
[0080] Including all claims below, the organoids derived from patient cancer tissue are very similar to actual cancer tissue and have the advantage of maintaining the patient's unique genotype and phenotype well in vitro, so they can be usefully utilized for evaluating the efficacy of treatments for tumors or anticancer drugs to overcome resistance.
[0081] In this specification, including all claims below, "anticancer drug resistance" is also referred to as "anticancer drug intolerance" or "anticancer drug refractory," and means that when treating a cancer patient with an anticancer drug, there is no effect from the beginning of treatment, or there is an effect initially but the effect decreases or is lost during the course of continuous treatment, or the response to treatment does not persist for a long period of time.
[0082] The present invention provides an anticancer drug screening method comprising: a step of producing an organoid by culturing colorectal cancer or sarcoma cells isolated from an individual in the culture medium composition of the present invention; and a step of treating the organoid with an anticancer drug candidate substance.
[0083] Including all claims below, the organoid (derived from patient cancer tissue) is very similar to actual cancer tissue and has the advantage of maintaining the patient's unique genotype and phenotype well in vitro. Therefore, the organoid can be usefully utilized in an anticancer drug screening platform by treating the organoid with an anticancer drug candidate and then measuring the organoid's proliferative capacity, metastatic capacity, and anticancer drug resistance.
[0084] In this specification, including all claims below, "candidate substance" means a substance expected to be able to treat cancer. Specifically, any substance expected to inhibit or improve the growth, migration, or metastasis of cancer cells, or to increase the death of cancer cells, may be used without limitation and includes all expected therapeutic substances such as compounds, genes, or proteins.
[0085]
[0086] The above-described details of the present invention may have the same meaning as applicable to the composition and manufacturing method of the present invention, but are not limited thereto.
[0087] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.
[0088]
[0089] [Example]
[0090]
[0091] Example 1. Culture process of primary cells from tumor specimens and organoid culture process
[0092]
[0093] In this embodiment, cancer tissue derived from a sarcoma patient obtained during surgery was transferred to a clean bench. The cancer tissue was washed with sterile physiological saline to remove residues and prepare a tumor specimen. The cancer tissue transferred to a Petri dish was finely crushed to a size of 1–2 mm using sterile scissors or a scalpel, and the resulting minced tumor was transferred to a C-tube, after which enzymes H, R, A (Cat # 130-095-929, Miltenyi Biotec) were added. The enzyme-added C-tube was transferred to a tumor dissociation device (gentleMACS, miltenyiibiotec) to separate the cancer tissue into cancer cells. After separation, the separated cancer cells were filtered through a 100 μm cell strainer to remove large tissue clumps and isolate primary cells. The separated cancer cells were cultured for about one week. The culture medium for sarcoma primary cells contained 1% Penicillin-Streptomycin (Cat # 15140122, gibco), 10% fetal bovine serum (Cat # A5670701, ThermoFisher), 1X B27 supplement minus vitamin A (Cat # 12587010, gibco), 50 ng / mL human EGF (Cat # GMP-C029, novoprotein), and 100 ng human fibroblast growth factor-basic (Cat # 100-18B-50UG, gibco). The cultured cancer cells were 5 x 10⁶ 5 I prepared as many as there are cells.
[0094] In addition, the culture medium for colorectal cancer cell lines (HT29 and CT26) used Roswell Park Memorial Institute (RPMI) 1640 medium (Cat # LM011-01, Welgene) supplemented with 10% Fetal bovine serum (Cat # A5670701, ThermoFisher) and 1% Penicillin-Streptomycin (Cat # SV30010, Cytiva).
[0095] A 10% gelatin solution (Cat # 9000-70-8, SAMCHUN) dissolved in the culture medium of the prepared cancer cells, namely sarcoma primary cells (Fig. 1) and colorectal cancer cell lines (HT29 and CT26), was added to a Petri dish and incubated at room temperature for 30 minutes. Afterward, organoid medium was added and the cells were cultured at 37°C for 10 days. The colorectal cancer organoid medium used was the same medium as the culture medium for the colorectal cancer cell lines presented above. Breeding organoid medium contains 1% Penicillin-Streptomycin (Cat # 15140122, gibco), 10% fetal bovine serum (Cat # A5670701, ThermoFisher), 500 ng / mL recombinant human R-Spondin protein (Cat # 4645-RS, bio-techne R&D systems), 100 ng / mL human Noggin Recombinant Protein (Cat # 120-10C-20UG, gibco), 1 human EGF (Cat # GMP-C029, A solution containing 100 ng human fibroblast growth factor-basic (Cat # 100-18B-50UG, gibco), 500 nM A83-01 (Cat # HY-10432, MeChemExpress), 10 μM SB202190 (Cat # S1077, Selleckchem), and 100 mg / mL primocin (Cat # ant-pm-05, InvivoGen) in advanced DMEM / F12 (Cat#12634028, gibco) was used.
[0096]
[0097] Example 2.1. Confirmation of organoid formation in colorectal cancer cell lines and sarcoma-derived cells
[0098]
[0099] In this embodiment, the formation of organoids in colorectal cancer cell lines and sarcoma-derived cells was confirmed (Fig. 2a). Specifically, primary cells of cancer cells (Patient 1 and Patient 2) isolated from colorectal cancer cell lines (HT29 and CT26) and cancer tissue derived from sarcoma patients in the same manner as in Example 1 were used (Fig. 2b).
[0100] When organoids were formed using Cultrex Reduced Growth Factor Basement Membrane Extract, PathClear (BME, R&D SYSTEMS biotechne, Cat# 3533-010-02) and gelatin, it was confirmed that organoids could not be formed in CT26 cell lines and sarcoma patient-derived cells when using the conventional BME (Fig. 2c). On the other hand, it was confirmed that organoids were formed in both colorectal cancer cell lines (HT29 and CT26) and sarcoma patient-derived cells when using gelatin (Fig. 2d).
[0101] From the above results, it was confirmed that, contrary to the inability to form organoids when using conventional BME, organoids can be formed from sarcoma or colorectal cancer-derived cells when using gelatin.
[0102]
[0103] Example 2.2. Confirmation of cancer marker-related expression in organoids formed from colorectal cancer cell lines and sarcoma-derived cells
[0104]
[0105] In this embodiment, quantitative expression results of markers related to cancer indicators were confirmed for organoids formed from colorectal cancer cell lines and sarcoma-derived cells. Specifically, primary cells (Patient 1) of cancer cells isolated from colorectal cancer cell lines (HT29 and CT26) and cancer tissue derived from a sarcoma patient in the same manner as in Example 1 were used.
[0106] Organoids were formed using Cultrex Reduced Growth Factor Basement Membrane Extract (BME) and gelatin, and the cancer markers expressed in the formed organoids were quantitatively compared using Western Blot. Specifically, AKT, P-AKT, and mTOR biomarkers, which play a central role in regulating the epithelial-mesenchymal transition (EMT) process in colorectal cancer tumor cells, were identified, and it was confirmed that the expression of these markers (AKT, P-AKT, and mTOR) increased when gelatin was used compared to when BME was used. Additionally, utilizing the CDK4, MDM2, and Vimentin biomarkers, which show increased expression in liposarcoma among sarcomas, it was confirmed that the expression of these biomarkers (CDK4, MDM2, and Vimentin) was further increased when gelatin was used compared to when BME was used. It was confirmed that cancer marker-related markers showed higher expression levels when gelatin was used compared to when conventional BME was used (Fig. 2e).
[0107] This suggests that the specific molecular characteristics of colorectal cancer as well as sarcoma are better preserved when gelatin is used compared to when conventional BME is used.
[0108]
[0109] Example 3.1. Confirmation of the degree of aggregation according to gelatin concentration in organoids formed from sarcoma-derived cells
[0110]
[0111] In this embodiment, changes in the degree of aggregation of organoids according to changes in gelatin concentration were confirmed for organoids formed from sarcoma-derived cells. Specifically, organoids were formed using primary cancer cells isolated from cancer tissue derived from a sarcoma patient in the same manner as in Example 1.
[0112]
[0113] As a result of culturing organoids with gelatin concentrations of 10% by weight and 20% by weight relative to the total weight of the medium, it was confirmed that the cells exhibited higher aggregation at a gelatin concentration of 10% by weight compared to a gelatin concentration of 20% by weight (Fig. 3a).
[0114] This suggests that gelatin concentration can have a significant effect on organoid formation.
[0115]
[0116] Example 3.2. Confirmation of the clear structure of organoids formed from sarcoma-derived cells
[0117]
[0118] In this embodiment, the formation of an organoid structure was confirmed when BME and gelatin were used for an organoid formed from sarcoma-derived cells. Specifically, an organoid was formed using primary cancer cells isolated from cancer tissue derived from a sarcoma patient in the same manner as in Example 1.
[0119]
[0120] As a result of forming organoids using BME and gelatin, when BME was used, no organoid was formed, making it difficult to confirm the structure of the organoid, whereas when gelatin was used, not only was an organoid formed, but a clear organoid structure could also be confirmed (Fig. 3b).
[0121] This suggests that gelatin is a decisive factor in organoid formation and structural stability.
[0122]
[0123] Example 3.3. Confirmation of increased oxygen consumption in organoids formed from sarcoma-derived cells
[0124]
[0125] In this embodiment, the oxygen consumption of the organoid was determined when BME and gelatin were used for an organoid formed from sarcoma-derived cells. Specifically, an organoid was formed using primary cancer cells (Patient 1) isolated from cancer tissue derived from a sarcoma patient in the same manner as in Example 1.
[0126]
[0127] The Oxygen Demand Rate (OCR) of the cells was directly measured using the Seahorse XF Cell Mito Stress Test (Cat # 103015-100, Agilent). As a result of forming organoids using BME and gelatin, it was confirmed that oxygen consumption increased when gelatin was used compared to when BME was used (Fig. 3c).
[0128] From the above results, it was confirmed that gelatin can influence the increase in organoid metabolic activity, suggesting that using gelatin can lead to the formation of metabolically active organoids.
[0129]
[0130] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.
[0131] In this invention, it was confirmed that when gelatin is used as a substitute for basement membrane extract (BME), organoid formation is promoted, the expression of cancer markers is enhanced, and structural stability is improved compared to when conventional BME is used. Furthermore, metabolic activity is also improved through an increase in the oxygen consumption of the organoids. Additionally, the organoid formation medium of this invention is more economical than BME in terms of cost and the time required for organoid formation. Since the limitations of reproducing the biological characteristics of tumors in conventional 2D cell culture methods are overcome by the medium composition of this invention utilizing gelatin, thereby providing a physiologically similar model, this patent has industrial applicability as it can be utilized as a useful platform for future preclinical studies, diagnosis, and treatment evaluation.
Claims
1. A culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, comprising gelatin as an active ingredient as a substitute for basement membrane extract (BME).
2. In Paragraph 1, A culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, characterized in that the colorectal cancer or sarcoma cells are one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue.
3. In Paragraph 1, A culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, characterized in that the gelatin is contained in an amount of 5 to 15 weight percent relative to the total weight of the culture medium.
4. In Paragraph 1, A culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, characterized in that the above gelatin influences the promotion of organoid formation, the promotion of cancer marker-related expression, the improvement of structural stability, or the improvement of metabolic activity.
5. A method for producing a colorectal cancer or sarcoma cell-derived organoid, comprising the step of culturing colorectal cancer or sarcoma cells in the culture medium composition of claim 1.
6. In Paragraph 5, A method for producing a colorectal cancer or sarcoma cell-derived organoid, characterized in that the colorectal cancer or sarcoma cells are one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue.
7. A method for preparing a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, comprising the step of adding gelatin as a substitute for basement membrane extract (BME).
8. In Paragraph 7, A method for preparing a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, characterized in that the colorectal cancer or sarcoma cells are one or more selected from the group consisting of primary cells and cell lines derived from cancerous tissue.
9. In Paragraph 7, A method for preparing a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, characterized in that the gelatin is contained in an amount of 5 to 15 weight percent relative to the total weight of the culture medium.
10. In Paragraph 7, A method for preparing a culture medium composition for forming organoids derived from colorectal cancer or sarcoma cells, characterized in that the above gelatin influences the promotion of organoid formation, the promotion of cancer marker-related expression, the improvement of structural stability, or the improvement of metabolic activity.
11. Use of a culture medium composition containing gelatin as an active ingredient as a substitute for basement membrane extract (BME) for forming organoids derived from colorectal cancer or sarcoma cells.
12. Use for preparing a medium for forming colorectal cancer or sarcoma cell-derived organoids, comprising a medium composition containing gelatin as an active ingredient as a substitute for basement membrane extract (BME).