An organoid culture medium, kit, and method of culturing an organoid

Abstract

The application discloses an organoid culture medium, a kit and a method for culturing organoids, and relates to the technical field of organoid culture.The organoid culture medium provided by the application can improve the proliferation activity of organoids, and can obtain relatively complete organoid morphology after culture, and reduces the cost of the culture medium.The tumor cell organoids obtained by using the organoid culture medium provided by the application have more complete tumor cell surface markers and less cell damage.

Description

Cross-references to related applications

[0001] This application claims priority to Chinese Patent Application No. 202610100143.2, filed on January 26, 2026, entitled "An organoid culture medium, a reagent kit and a method for culturing organoids", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of organoid culture technology, and more specifically, to an organoid culture medium, a reagent kit, and a method for culturing organoids. Background Technology

[0003] Organoid technology is a revolutionary three-dimensional in vitro modeling technology in the life sciences field in recent years. It refers to the use of stem cells, organ-specific progenitor cells, cancer cells, and other cells, which, under specific in vitro culture conditions, self-assemble and differentiate to form miniature simplified structures that mimic the structure, cell type, and function of real organs.

[0004] The key culture condition is the support of three-dimensional matrix gel. In three-dimensional matrix gel, various signaling molecules, including small molecule compounds, are precisely added to precisely regulate key signaling pathways, thereby reproducing the process of organogenesis in vitro.

[0005] Currently, organoid culture media suffer from problems such as high culture costs, poor organoid proliferation activity, inability to maintain organoid morphology integrity, limited number of passages, and slow proliferation.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide an organoid culture medium, a reagent kit, and a method for culturing organoids to solve the above-mentioned technical problems.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides an organoid culture medium comprising the following ingredients added to a basal culture medium: 0.8-1.5×GlutaMAX, 8-10 mM HEPES, 0.8-1× penicillin-streptomycin bispecific antibiotic solution, 0.8-1.25× B-27 serum-free culture supplement, 10-100 ng / ml Wnt3a protein, 1-1.25 mM N-acetyl-L-cysteine, 20-500 ng / ml RS-246204, 2-10 μM DMH1, 10-100 ng / mL EGF, 10-20 nM human gastrin-I, 0.5-2 μM TGF-β receptor inhibitor, 3-10 μM p38 MAPK inhibitor, 10-50 nM prostaglandin E2, 10-50 mM nicotinamide, and 80-100 μg / ml of other ingredients. 5-100 ng / mL of Primocin™ primary cell antibiotic, 1-1.25× N2 serum-free culture supplement, and 8-10 μM of ROCK kinase inhibitor.

[0009] Secondly, the present invention also provides a kit comprising the organoid culture medium described above.

[0010] Thirdly, the present invention also provides a method for culturing organoids, comprising the following steps: placing the cells to be cultured on a cell matrix or embedding them in an extracellular matrix, adding the above-mentioned organoid culture medium, culturing, and obtaining organoids of tumor cells.

[0011] Fourthly, the present invention also provides a method for evaluating or screening the efficacy of cancer drugs, comprising the following steps: (1) culturing organoids of tumor cells using the above-described method for culturing organoids; (2) adding the drug to be tested to the organoids and performing organoid function and / or activity tests.

[0012] The present invention has the following beneficial effects: The organoid culture medium provided by this invention can enhance the proliferative activity of organoids, and more complete organoid morphology can be obtained after culture. By replacing R-vertebral protein 1 with RS-246204 and neurogenesis inhibitor (Noggin) with DMH1, the cost of the culture medium is significantly reduced. Tumor cell organoids cultured using the organoid culture medium provided by this invention have more complete tumor cell surface markers and less cell damage. The organoid culture medium provided by this invention helps to reduce the difference between organoids and primary tissues and has higher stability. Attached Figure Description

[0013] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 Microscopic images of organoids cultured in different culture media under a microscope; Figure 2 The results of cell integrity testing of colorectal cancer cell organoids in different organoid culture media are shown in the figure. Figure 3 Bright field images of D7 and D9 after passage of the colorectal cancer organoid culture medium provided in Example 2; Figure 4 Bright field images of D7 and D9 after passage of the colorectal cancer organoid culture medium provided in Example 3; Figure 5 Bright field images of D7 and D9 after passage of the colorectal cancer organoid culture medium provided in Comparative Example 3; Figure 6 The image shows the cell viability results of colorectal cancer organoids cultured in the organoid culture medium of Example 1 and Comparative Example 4 in Experiment 3, after enzymatic digestion and mixing with Matrigel, and then adding medium containing different concentrations of 5-Fu uracil (0, 2 nM and 20 nM). Figure 7 The image shows the transcriptomic results of colorectal cancer organoids cultured in the organoid culture media of Example 1, Comparative Example 1, and Comparative Example 4, respectively. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0016] In this specification, "organoid" refers to a three-dimensional, organ-like cellular tissue formed by the spontaneous organization and aggregation of cells in a controlled space at high density.

[0017] In a first aspect, the present invention provides an organoid culture medium comprising the following ingredients added to a basal culture medium: 0.8-1.5×GlutaMAX, 8-10 mM HEPES, 0.8-1× penicillin-streptomycin bispecific antibiotic solution, 0.8-1.25× B-27 serum-free culture supplement, 10-100 ng / ml Wnt3a protein, 1-1.25 mM N-acetyl-L-cysteine, 20-500 ng / ml RS-246204, 2-10 μM DMH1, 10-100 ng / mL EGF, 10-20 nM human gastrin-I, 0.5-2 μM TGF-β receptor inhibitor, 3-10 μM p38 MAPK inhibitor, 10-50 nM prostaglandin E2, 10-50 mM nicotinamide, and 80-100 μg / ml of other ingredients. 5-100 ng / mL of Primocin™ primary cell antibiotic, 1-1.25× N2 serum-free culture supplement, and 8-10 μM of ROCK kinase inhibitor.

[0018] Wnt signaling pathway additives (Wnt3a protein) in organoid culture media are crucial for initiating and maintaining the proliferation of many organoids; Inhibitors of the BMP signaling pathway (DMH1) in organoid culture media are often used to maintain stemness and promote differentiation of specific lineages.

[0019] Inhibitors of the TGF-β / Activin signaling pathway can regulate cell differentiation.

[0020] Factors in the FGF signaling pathway can promote cell survival and proliferation.

[0021] GlutaMAX offers high stability and provides sustained nutrition to cells while reducing the accumulation of toxic byproducts, thus helping to improve cell viability and growth.

[0022] The penicillin-streptomycin bispecific antibiotic solution refers to a bispecific antibiotic solution containing 100 U / mL penicillin and 100 µg / mL streptomycin. When using it, it is added directly to the culture medium at a volume ratio of 0.8-1:100, which is 0.8× or 1×.

[0023] N-acetyl-L-cysteine ​​is added to the culture medium as a precursor to glutathione to reduce oxidative stress-induced cell damage, especially in primary cell culture or under stress conditions.

[0024] RS-246204 (CAS number 878451-87-7) can effectively initiate and maintain the initial formation and subsequent growth of small intestinal organoids in the absence of R-spondin-1 protein. It enhances cell proliferation or anti-apoptotic effects, thereby promoting intestinal epithelial regeneration.

[0025] The functions of EGF are to promote cell proliferation, differentiation, survival and migration.

[0026] The function of human gastrin-I is that it can specifically induce stem cells to differentiate into parietal cells with acid-secreting function and help form complex glandular structures.

[0027] p38 MAPK inhibitors inhibit the production of pro-inflammatory cytokines by binding to the ATP-binding site or other allosteric sites of the p38 protein, preventing its phosphorylation and activation of downstream targets.

[0028] Nicotinamide promotes organoid survival, proliferation, and maintains their stemness by influencing cellular energy metabolism, signaling pathways, and gene expression.

[0029] The synergistic effect of the above components enhances the proliferative activity of organoids, resulting in more complete organoid morphology after culture. Tumor cell organoids cultured using the organoid culture medium provided by this invention exhibit more complete tumor cell surface markers and less cell damage. Furthermore, by replacing R-vertebral protein 1 with RS-246204 and neurogenesis inhibitory protein (Noggin) with DMH1, the cost of the culture medium is significantly reduced.

[0030] In a preferred embodiment of the present invention, the TGF-β receptor inhibitor is A83-01, SB-431542, LY2157299, RepSox, LY2109761, TEW-7197, SD-208, SB525334, LY3200882, pirfenidone, or GC1008.

[0031] In a preferred embodiment of the present invention, the p38 MAPK inhibitor is selected from SB202190, SB 203580, BIRB 796, VX-745, SCIO-469, Ro4402257, LY2228820, PH-797804, Skepinone-L, MW-150, or PF-07265803.

[0032] In a preferred embodiment of the present invention, the ROCK kinase inhibitor is selected from Y-27632, fasudil, or H-1152.

[0033] In a preferred embodiment of the present invention, the basal culture medium is provided with the following ingredients in the following amounts: 1×GlutaMAX, 10 mM HEPES, 1× penicillin-streptomycin bispecific antibiotic solution, 1× B-27 serum-free culture supplement, 100 ng / ml Wnt3a protein, 1 mM N-acetyl-L-cysteine, 500 ng / ml RS-246204, 10 μM DMH1, 10 ng / mL EGF, 10 nM human gastrin-I, 2 μM TGF-β receptor inhibitor, 10 μM p38 MAPK inhibitor, 50 nM prostaglandin E2, 50 mM nicotinamide, 100 μg / mL Primocin™ primary cell antibiotic, 100 ng / mL FGF10, 1× N2 serum-free culture supplement, and 10 μM ROCK kinase inhibitor.

[0034] Secondly, the present invention also provides a kit comprising the above-described organoid culture medium. This kit is used for the culture of organoids.

[0035] Thirdly, the present invention also provides a method for culturing organoids, comprising the following steps: placing the cells to be cultured on a cell matrix or embedding them in an extracellular matrix, adding the above-mentioned organoid culture medium, culturing, and obtaining organoids of tumor cells.

[0036] In a preferred embodiment of the present invention, the culture is carried out under conditions of 5% ± 0.5% CO2.

[0037] Fourthly, the present invention also provides a method for evaluating or screening the efficacy of cancer drugs, comprising the following steps: (1) culturing organoids of tumor cells using the above-mentioned method for culturing organoids; (2) adding the drug to be tested to the organoids and performing organoid function and / or activity tests.

[0038] If the drug under test can improve organoid function and / or activity, then the drug under test is evaluated as having good efficacy against cancer.

[0039] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0040] Example 1 This embodiment provides a colorectal cancer organoid culture medium (i.e., formulation 3), which includes the following reagents added to the basal culture medium Advanced DMEM / F12: 1×GlutaMAX, 10mM HEPES, 1× penicillin-streptomycin dual antibiotic solution, 1× B-27 serum-free culture supplement, 100ng / ml Wnt3a protein, 1 mM N-acetyl-L-cysteine, 500ng / ml RS-246204, 10μM DMH1, 10 ng / mL EGF, 10nM human gastrin-I, 2μM TGF-β receptor inhibitor A83-01, 10 μM p38 MAPK inhibitor SB202190, 50 nM prostaglandin E2, 50 mM nicotinamide, 100 μg / mL Primocin™ primary cell antibiotic, 100ng / mL FGF10, 1× N2 serum-free culture supplement, and 10μM ROCK kinase inhibitor Y-27632.

[0041] Example 2 This embodiment provides a colorectal cancer organoid culture medium, which includes the following reagents added to the basal culture medium AdvancedDMEM / F12: 1×GlutaMAX, 10mM HEPES, 1× penicillin-streptomycin dual antibiotic solution, 1× B-27 serum-free culture supplement, 25 ng / ml Wnt3a protein, 1 mM N-acetyl-L-cysteine, 100 ng / ml R-spondin1, 20 ng / ml noggin, 50 ng / mL EGF, 10 nM human gastrin-I, 0.5 μM TGF-β receptor inhibitor A83-01, 3 μM p38 MAPK inhibitor SB202190, 10 nM prostaglandin E2, 10 mM nicotinamide, 100 μg / mL Primocin™ primary cell antibiotic, 20 ng / mL FGF10, 1× N2 serum-free culture supplement, and 10 μM ROCK kinase inhibitor Y-27632.

[0042] Example 3 This embodiment provides a colorectal cancer organoid culture medium, which includes the following reagents added to the basal culture medium AdvancedDMEM / F12: 1×GlutaMAX, 10mM HEPES, 1× penicillin-streptomycin dual antibiotic solution, 1× B-27 serum-free culture supplement, 25 ng / ml Wnt3a protein, 1 mM N-acetyl-L-cysteine, 500 ng / ml R-spondin1, 40 ng / ml noggin, 50 ng / mL EGF, 10 nM human gastrin-I, 0.5 μM TGF-β receptor inhibitor A83-01, 3 μM p38 MAPK inhibitor SB202190, 10 nM prostaglandin E2, 10 mM nicotinamide, 100 μg / mL Primocin™ primary cell antibiotic, 100 ng / mL FGF10, 1× N2 serum-free culture supplement, and 10 μM ROCK kinase inhibitor Y-27632.

[0043] Comparative Example 1 Compared to Example 1, the colorectal cancer organoid culture medium (Formula 1) provided in this example contains 500 ng / ml of R-Spondin1 and does not contain RS-246404. It contains 100 ng / ml of Noggin and does not contain DMH1.

[0044] Comparative Example 2 Compared to Example 1, the colorectal cancer organoid culture medium (Formulation 2) provided in this example contains 50 ng / ml of R-Spondin1 and does not contain RS-246404. It contains 100 ng / ml of Noggin and does not contain DMH1.

[0045] Comparative Example 3 Compared with Example 1, the colorectal cancer organoid culture medium provided in this example contains 100 ng / ml of Noggin and does not contain DMH1.

[0046] Comparative Example 4 The organoid culture medium used was Bozhen's, catalog number Colorectal Cancer Organoid Kit K2103-CR-100.

[0047]

[0048] Experimental Example 1 Add matrix gel to colorectal cancer cell precipitate (organoids from patient-derived colorectal cancer tissue that has been dissected and successfully library-built) and mix thoroughly. Add 50 μL of the cell precipitate mixture to each well of a 24-well plate, cure at 37°C for 30 min, and then add the culture medium of Examples 1-3 and Comparative Examples 1-3 respectively for culture. Observe and record the organoid status under a bright field microscope at D3, D7 and D9 respectively.

[0049] The results of Example 1 and Comparative Examples 1-2 are referenced. Figure 1 As shown, the culture medium formulations of Examples 1 and Comparative Examples 1-2 can maintain organoid morphology and proliferation similar to those of commercial culture medium (Bozhen).

[0050] The cost of the culture medium: Compared with Comparative Example 1, the cost per 100 mL of Example 1 is reduced by approximately 600 yuan, and compared with Comparative Example 2, the cost per 100 mL of Comparative Example 2 is reduced by approximately 500 yuan. Therefore, the organoid culture medium provided by the embodiments of the present invention has a significant technical advantage of lower cost.

[0051] Bright-field plots of colorectal cancer organoid culture medium after passage on days 7 and 9, as shown in Example 2. Figure 3 As shown, the results indicate that, compared to the organoid culture medium provided in Example 1, the organoids cultured in the organoid culture medium provided in Example 2 grow more slowly.

[0052] Bright-field plots of colorectal cancer organoid culture medium after passage on days 7 and 9, as shown in Example 3. Figure 4 As shown, the results indicate that, compared to the organoid culture medium provided in Example 1, the organoids cultured in the organoid culture medium provided in Example 3 grow more slowly.

[0053] Bright-field plots of colorectal cancer organoid culture medium after passage in Comparative Example 3 at D7 and D9 are referenced. Figure 5 As shown, the results indicate that, compared to the organoid culture medium provided in Example 1, the organoid growth rate cultured in the organoid culture medium provided in Comparative Example 3 was not significantly different, but the culture medium was expensive and could not meet the needs of large-scale organoid culture.

[0054] Experiment Example 2 This experiment tested the cell integrity of colorectal cancer cell organoids on different organoid culture media and detected tumor cell surface markers.

[0055] Cell integrity of colorectal cancer cell organoids cultured in organoid culture media of Comparative Examples 1-2 and Example 1 was detected. The immunofluorescence assay kits used to detect tumor markers (CDX2, CK20, Ki67) on the surface of organoids were purchased from Cell Signaling Technology. The product catalog number for CDX2 detection was 12306, the product catalog number for CK20 detection was 13063, and the product catalog number for Ki67 detection was 9449. Figure 2 The results showed that the organoid morphology and biomarker expression after culture in the organoid culture medium of Comparative Examples 1-2 and Example 1 were similar.

[0056] Experimental Example 3 Colorectal cancer organoids cultured in the organoid culture medium of Example 1 and Comparative Example 1 were enzymatically digested and mixed with matrix gel. 10 μL of the mixture was seeded in 96-well plates and cultured for 4 days. Then, medium containing different concentrations of 5-Fu uracil (0, 2 nM and 20 nM) was added. Cell viability was detected after 5 days.

[0057] Figure 6 The results showed that the colorectal cancer organoids cultured in the organoid culture medium provided in this embodiment of the invention exhibited concentration-dependent changes in cell viability after trypsin digestion, which was basically consistent with the results of the Bozhen culture medium. Therefore, the method for culturing organoids provided by this invention can be used for efficacy evaluation or screening of cancer drugs.

[0058] Experiment Example 4 This experiment tests the effect of the organoid culture medium provided in Example 1 and the organoid culture medium in Comparative Example 4 on the stability of tumor cell organoids.

[0059] The experimental method is as follows: Add matrix gel to the colorectal cancer cell pellet (organoids from patient-derived colorectal cancer tissue that have been dissociated and successfully library-constructed) and mix thoroughly. Add 50 μL of the cell pellet mixture to each well of a 24-well plate and cure at 37°C for 30 min. Then add the culture medium of Example 1, Comparative Example 1 and Comparative Example 4 respectively for culture. After passage 5 times, perform transcriptomics analysis to analyze the differences between colorectal cancer organoids and primary tissues.

[0060] Results reference Figure 7 As shown, the tumor cell organoids obtained from the organoid culture medium in Example 1 showed less transcriptomic difference and better stability compared to primary tissues. In Comparative Example 4, the tumor cell organoids obtained from the organoid culture medium showed greater difference (lighter color indicates a larger fold change) and poorer stability compared to primary tissues.

[0061] In other words, the organoid culture medium provided by this invention helps to reduce the differences between organoids and primary tissues and has higher stability.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An organoid culture medium, characterized in that, It includes adding the following ingredients to the basal culture medium: 1×GlutaMAX, 10 mM HEPES, 1× penicillin-streptomycin bispecific antibiotic solution, 1× B-27 serum-free culture supplement, 100 ng / ml Wnt3a protein, 1 mM N-acetyl-L-cysteine, 500 ng / ml RS-246204, 10 μM DMH1, 10 ng / mL EGF, 10 nM human gastrin-I, 2 μM A83-01, 10 μM SB202190, 50 nM prostaglandin E2, 50 mM nicotinamide, 100 μg / mL Primocin™ primary cell antibiotic, 100 ng / mL FGF10, 1× N2 serum-free culture supplement, and 10 μM ROCK kinase inhibitor; the basal culture medium is DMEM / F-12 medium.

2. A reagent kit, characterized in that, It includes the organoid culture medium as described in claim 1.

3. A method for culturing organoids, characterized in that, It includes the following steps: placing the cells to be cultured on a cell matrix or embedding them in an extracellular matrix, adding the organoid culture medium as described in claim 1, culturing, and obtaining organoids of tumor cells.

4. The method for culturing organoids according to claim 3, characterized in that, The culture was carried out under conditions of 5% ± 0.5% CO2.

5. A method for evaluating or screening the efficacy of cancer drugs, characterized in that, Includes the following steps: (1) Culture tumor cell organoids using the method for culturing organoids according to any one of claims 3-4; (2) Add the drug to be tested to the organoids and perform organoid function and / or activity tests.

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