Organ-like basic culture medium and method for maintaining and promoting growth of organ-like

Abstract

The invention provides an organoid basic culture medium and a method for maintaining and promoting organoid growth, and the organoid basic culture medium comprises a basic culture medium and an additive, the additives comprise taurine sodium 3-hydroxybutyrate, taurine, sodium hypoxanthate, creatinine, L-ornithine, urea, L-sodium lactate, putrescine dihydrochloride, anhydrous citric acid, ethanolamine, N-acetylglycine, alpha ketoglutaric acid, formic acid, malic acid, malonic acid, succinic acid, uric acid, ammonium metavanadate, manganese dichloride, sodium selenite, creatine, glycerol and uridine. Anhydrous sodium acetate and para aminobenzoic acid. When the organ-like basic culture medium is used for treating the organ-like, the activity of the organ-like can be effectively maintained, and basic nutrition and metabolism requirements of the organ-like can be provided, so that the organ-like has the advantages of high activity, fast proliferation and long-term stable passage, and is suitable for various types of organs and wide in application.

Description

Technical Field

[0001] The present application relates to the biological field, and in particular, to an organoid basal culture medium and a method for maintaining and promoting the growth of organoids. Background Art

[0002] Organoids refer to tissue analogs with a certain spatial structure formed by three-dimensional (3D) culture of adult stem cells or pluripotent stem cells in vitro. Although organoids are not real human organs, they can simulate real organs in structure and function, can simulate the structure and function of in vivo tissues to the greatest extent, and can be cultured stably for a long time.

[0003] Before or during organoid culture, organoids need to be treated with organoid basal culture medium to maintain cell activity, provide basic nutritional and metabolic needs of organoids, and remove cell debris, metabolic waste or organoid treatment reagent residues, etc., to prevent these impurities from affecting the growth environment of organoids, inhibiting cell activity, and even causing culture failure. However, some existing organoid basal culture media will affect cell activity during the processing process and cannot provide sufficient basic nutritional and metabolic needs of organoids.

[0004] Therefore, the current basal culture medium for organoids remains to be studied. Summary of the invention

[0005] The present application aims to solve the technical problems existing in the prior art at least to a certain extent. To this end, the present application proposes an organoid basal medium and a method for maintaining and promoting the growth of organoids. Treating organoids with the organoid basal medium of the present application can effectively maintain the activity of organoids, provide the basic nutritional and metabolic needs of organoids, and make organoids have the advantages of high activity, rapid proliferation, and long-term stable passage. It is suitable for various types of organoids and has a wide range of applications.

[0006] In one aspect of the present application, the present application proposes an organoid basal culture medium. According to an embodiment of the present application, the organoid basal culture medium comprises: a basal culture medium and an additive, wherein the additive comprises: Taurine sodium 3-hydroxybutyrate, taurine, sodium hypoxanthine, creatinine, L-ornithine, urea, sodium L-lactate, putrescine dihydrochloride, anhydrous citric acid and ethanolamine.

[0007] According to the embodiment of the present application, the organoid basal culture medium is rich in various components such as various water-soluble acids and non-proteinogenic amino acids. Using it to treat organoids can not only remove cell debris, metabolic waste or organoid treatment reagent residues (such as digestive fluid), but also maintain the activity of organoids and provide the basic nutritional and metabolic needs of organoids, so that the organoids have the advantages of high activity, rapid proliferation and long-term stable passage. It is suitable for various types of organoids and has a wide range of applications.

[0008] According to an embodiment of the present application, the above-mentioned organoid basal culture medium may also have the following additional technical features: According to an embodiment of the present application, based on the total volume of the basal medium, the additive includes: 4.41~8.2μg / ml taurine sodium 3-hydroxybutyrate, 7.88~14.64μg / ml taurine, 1.67~3.1μg / ml sodium hypoxanthine, 5.94~11.03μg / ml creatinine, 8.26~15.34μg / ml L-ornithine, 210.21~390.39μg / ml urea, 125.5~233.08μg / ml sodium L-lactate, 0.057~0.11μg / ml putrescine dihydrochloride, 17.48~32.47μg / ml anhydrous citric acid, 1.33~2.47μg / ml ethanolamine.

[0009] Therefore, the use of additives with the above concentration composition can further maintain the activity of organoids and provide the basic nutritional and metabolic needs of organoids, thereby facilitating long-term stable passaging.

[0010] According to an embodiment of the present application, based on the total volume of the basal medium, the additive includes: 6.31μg / ml taurine sodium 3-hydroxybutyrate, 11.26μg / ml taurine, 2.39μg / ml sodium hypoxanthine, 8.48μg / ml creatinine, 11.8μg / ml L-ornithine, 300.3μg / ml urea, 179.3μg / ml sodium L-lactate, 0.081μg / ml putrescine dihydrochloride, 24.98μg / ml anhydrous citric acid, 1.9μg / ml ethanolamine.

[0011] Therefore, the use of additives with the above concentration composition can further maintain the activity of organoids, provide the basic nutritional and metabolic needs of organoids, thereby further increasing the cell number and viability, and facilitating long-term stable passaging.

[0012] According to an embodiment of the present application, the additive further includes: N-acetylglycine, alpha-ketoglutaric acid, formic acid, malic acid, malonic acid, succinic acid, uric acid, ammonium metavanadate, manganese dichloride, sodium selenite, creatine, glycerol, uridine, anhydrous sodium acetate and p-aminobenzoic acid.

[0013] By further adding the above-mentioned multiple components and using them to maintain organoids, the activity of organoids can be maintained, and the basic nutritional and metabolic needs of organoids can be provided, so that the organoids have the advantages of high activity, rapid proliferation and long-term stable propagation.

[0014] According to an embodiment of the present application, based on the total volume of the basal medium, the additive further includes: 7.38~13.7μg / ml N-acetylglycine, 0.51~0.95μg / ml α-ketoglutaric acid, 1.61~2.99μg / ml formic acid, 0.47~0.87μg / ml malic acid, 0.73~1.35μg / ml malonic acid, 1.65~3.07μg / ml succinic acid, 41.19~76.49μg / ml uric acid, 0.0001~0.0039μg / ml ammonium metavanadate, 0.000035~0.000065μg / ml manganese dichloride, 0.0035~0.0065μg / ml sodium selenite, 3.67~6.82μg / ml creatine, 7.73~14.37μg / ml glycerol, 0.51~0.96μg / ml uridine, 2.29~4.27μg / ml anhydrous sodium acetate and 0.7~1.3μg / ml p-aminobenzoic acid.

[0015] According to an embodiment of the present application, based on the total volume of the basal medium, the additive further includes: 10.54μg / ml N-acetylglycine, 0.73μg / ml α-ketoglutarate, 2.3μg / ml formic acid, 0.67μg / ml malic acid, 1.04μg / ml malonic acid, 2.36μg / ml succinic acid, 58.84μg / ml uric acid, 0.0003μg / ml ammonium metavanadate, 0.00005μg / ml manganese dichloride, 0.005μg / ml sodium selenite, 5.25μg / ml creatine, 11.05μg / ml glycerol, 0.733μg / ml uridine, 3.281μg / ml anhydrous sodium acetate and 1μg / ml p-aminobenzoic acid.

[0016] Therefore, the use of additives with the above concentration composition can further maintain the activity of organoids, provide basic nutritional and metabolic needs of organoids, increase cell number and viability, and facilitate long-term stable passaging.

[0017] According to an embodiment of the present application, the additive further includes: Glutamic acid, isoleucine, lysine hydrochloride, phenylalanine, proline, ammonium chloride, calcium chloride aldehyde, calcium nitrate tetrahydrate, potassium dihydrogen phosphate, sodium bicarbonate, disodium phosphate anhydrous, acetyl carnitine, betaine, L-amino-N-butyric acid, L-carnitine hydrochloride, L-citrulline, L-hydroxyproline, acetone, fructose and galactose.

[0018] By further adding the above-mentioned non-proteinogenic amino acids, inorganic salts and other components, and using them to treat organoids, not only can cell debris, metabolic waste or organoid processing reagent residues (such as digestive fluid) be removed, but also the basic nutritional and metabolic needs of organoids can be provided, and the activity of organoids can be improved, so that organoids have the advantages of high activity, rapid proliferation and long-term stable passage.

[0019] According to an embodiment of the present application, based on the total volume of the basal medium, the additive further includes: 8.24~15.3μg / ml glutamic acid, 6.42~12μg / ml isoleucine, 25.5~47.5μg / ml lysine hydrochloride, 9.25~17.2μg / ml phenylalanine, 16~30μg / ml proline, 1.49~2.79μg / ml ammonium chloride, 182.56~339.05μg / ml calcium chloride aldehyde, 6.61~12.28μg / ml calcium nitrate tetrahydrate, 1.4~2.6μg / ml potassium dihydrogen phosphate, 1411.37~2621.11μg / ml sodium bicarbonate, 94.5~175.5μg / ml anhydrous disodium phosphate, 0.83~1.56μg / ml acetylcarnitine, 5.74~10.66μg / ml betaine, 1.44~2.68μg / ml L-amino-N-butyric acid, 5.54~10.3μg / ml L-carnitine hydrochloride, 4.9~9.11μg / ml L-citrulline, 1.84~3.41μg / ml L-hydroxyproline, 2.44~4.53μg / ml acetone, 5.04~9.37μg / ml fructose and 7.57~14.05μg / ml galactose.

[0020] Therefore, the use of additives with the above concentration composition can further maintain the activity of organoids and provide the basic nutritional and metabolic needs of organoids, thereby facilitating long-term stable passaging.

[0021] According to an embodiment of the present application, based on the total volume of the basal medium, the additive further includes: 11.77μg / ml glutamic acid, 9.18μg / ml isoleucine, 36.53μg / ml lysine hydrochloride, 13.215μg / ml phenylalanine, 23.026μg / ml proline, 2.14μg / ml ammonium chloride, 260.803μg / ml calcium chloride aldehyde, 9.446μg / ml calcium nitrate tetrahydrate, 2μg / ml potassium dihydrogen phosphate, 2016.24μg / ml sodium bicarbonate, 135.005μg / ml anhydrous disodium phosphate, 1.199μg / ml acetylcarnitine, 8.201μg / ml betaine, 2.062μg / ml L-amino-N-butyric acid, 7.92μg / ml L-carnitine hydrochloride, 7.008μg / ml L-citrulline, 2.623μg / ml L-hydroxyproline, 3.485 μg / ml acetone, 7.206 μg / ml fructose and 10.81 μg / ml galactose.

[0022] Therefore, the use of additives with the above concentration composition can further maintain the activity of organoids and provide the basic nutritional and metabolic needs of organoids, thereby facilitating long-term stable passaging.

[0023] According to an embodiment of the present application, based on the total volume of the basal medium, the additive includes: 6.31μg / ml taurine 3-hydroxybutyrate sodium, 11.26μg / ml taurine, 2.39μg / ml hypoxanthine sodium, 8.48μg / ml creatinine, 11.8μg / ml L-ornithine, 300.3μg / ml urea, 179.3μg / ml sodium L-lactate, 0.081μg / ml putrescine dihydrochloride, 24.98μg / ml anhydrous citric acid, 1.9μg / ml ethanolamine, 10.54μg / ml N-acetylglycine, 0.73μg / ml α-ketoglutaric acid, 2.3μg / ml formic acid, 0.67μg / ml malic acid, 1.04μg / ml malonic acid, 2.36μg / ml succinic acid, 58.84μg / ml uric acid, 0.0003μg / ml ammonium metavanadate, 0.00005μg / ml manganese dichloride, 0.005μg / ml sodium selenite, 5.25μg / ml creatine, 11.05μg / ml glycerol, 0.733μg / ml uridine, 3.281μg / ml anhydrous sodium acetate, 1μg / ml p-aminobenzoic acid, 11.77μg / ml glutamic acid acid, 9.18μg / ml isoleucine, 36.53μg / ml lysine hydrochloride, 13.215μg / ml phenylalanine, 23.026μg / ml proline, 2.14μg / ml ammonium chloride, 260.803μg / ml calcium chloride aldehyde, 9.446μg / ml calcium nitrate tetrahydrate, 2μg / ml potassium dihydrogen phosphate, 2016.24μg / ml sodium bicarbonate, 135.005μg / ml anhydrous disodium phosphate, 1.199μg / ml acetylcarnitine, 8.201μg / ml betaine, 2.062μg / ml L-amino-N-butyric acid, 7.92 μg / ml L-carnitine hydrochloride, 7.008 μg / ml L-citrulline, 2.623 μg / ml L-hydroxyproline, 3.485 μg / ml acetone, 7.206 μg / ml fructose and 10.81 μg / ml galactose.

[0024] Therefore, the supplement having the above composition can further maintain the activity of organoids, thereby facilitating long-term stable passaging.

[0025] According to an embodiment of the present application, the basal culture medium includes DMEM / F12 culture medium.

[0026] According to the embodiments of the present application, the organoid culture medium is a serum-free culture medium. The serum composition is complex and varies greatly between batches. It may contain unknown contaminants or inhibitors, which may affect cell growth and the stability of experimental results. In addition, animal-derived components in serum may cause immune reactions or ethical issues. Serum-free culture medium can avoid these problems and provide a purer and easier to standardize growth environment, thereby obtaining more reliable and consistent experimental data.

[0027] According to an embodiment of the present application, the organoid culture medium does not contain protein. Because the protein component may introduce batch-to-batch differences, immunogenicity problems and potential microbial contamination, and the decomposition products of the protein may be toxic to the organoids or affect their normal functions. Therefore, the use of a protein-free organoid culture medium can reduce the complexity and unpredictability of the culture process, provide a purer and more controllable growth environment, help improve the stability and reliability of organoid culture, and reduce potential interference with organoid function. Exemplarily, the additive can be supplemented with a biogenic G-27 supplement (Bozhen Bio, 50X, Catalog No. S223152-10) and appropriate growth factors to further enhance organoid activity.

[0028] In another aspect of the present application, the present application proposes a method for maintaining or promoting organoids. According to an embodiment of the present application, the method comprises: treating the organoids with the aforementioned organoid basal medium. Thus, the method of the present application can not only remove cell debris, metabolic waste or organoid treatment reagent residues (such as digestive fluid), but also maintain the activity of the organoids, provide the basic nutrition and metabolic needs of the organoids, so that the organoids have the advantages of high activity, rapid proliferation and long-term stable passage, and are suitable for various types of organoids and are widely used.

[0029] In some embodiments, the processing includes: culturing the organoids with an organoid complete medium made from the organoid basal medium after plating, and / or, washing the organoids with the organoid basal medium, and / or, diluting the digested organoid suspension with the organoid basal medium to terminate the digestion reaction.

[0030] It should be noted that the characteristics and advantages described above for the organoid basal culture medium are also applicable to the method for processing and maintaining organoids and will not be repeated here.

[0031] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. DETAILED DESCRIPTION

[0032] The following is a detailed description of the embodiments of the technical solution of the present application. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0033] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0034] "Scope" disclosed in the present application is limited in the form of lower limit and upper limit, and a given range is limited by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a special range. The scope limited in this way can be including end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a scope. For example, if the scope of 60-120 and 80-110 is listed for a specific parameter, it is understood that the scope of 60-110 and 80-120 is also expected. In addition, if the minimum range values ​​1 and 2 listed, and if the maximum range values ​​3,4 and 5 are listed, the following scope can all be expected: 1-3, 1-4, 1-5, 2-3, 2-4 and 2-5. In the present application, unless otherwise specified, the numerical range "ab" represents the abbreviation of any real number combination between a and b, wherein a and b are real numbers, and the scope limited in this way can be including end values ​​a and b. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0035] If not otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0036] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the literature in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained commercially.

[0037] Example 1 In this example, the organoid basal medium and complete medium were prepared according to the following method: 1. Formulation Basic culture medium: DMEM / F12 medium.

[0038] Supplements: 6.31 μg / ml taurine sodium 3-hydroxybutyrate, 11.26 μg / ml taurine, 2.39 μg / ml sodium hypoxanthine, 8.48 μg / ml creatinine, 11.8 μg / ml L-ornithine, 300.3 μg / ml urea, 179.3 μg / ml sodium L-lactate, 0.081 μg / ml putrescine dihydrochloride, 24.98 μg / ml anhydrous citric acid, 1.9 μg / ml ethanolamine, based on the total volume of the basal medium.

[0039] 2. Mix the components of the additive, add the resulting additive to the culture medium, mix well, and obtain the organoid basal culture medium.

[0040] 3. Taking colorectal cancer as an example, add G-27 supplement (Bozhen Bio, 50X, product number S223152-10, the volume ratio of G-27 supplement to basal culture medium is 1:50), 50ng / ml EGF, 100ng / ml Noggin, 500ng / ml R-Spondin1, 0.5μM A-83-01, 5μM Y27632, 3μM SB202190 (all of the above are final concentrations) to the basal culture medium of the organoid obtained in the previous step, mix well, and obtain the complete culture medium for colorectal cancer organoids.

[0041] Example 2 In this example, the organoid basal medium was prepared as follows: 1. Formulation Basic culture medium: DMEM / F12 medium.

[0042] Additives: 6.31μg / ml taurine 3-hydroxybutyrate sodium, 11.26μg / ml taurine, 2.39μg / ml hypoxanthine sodium, 8.48μg / ml creatinine, 11.8μg / ml L-ornithine, 300.3μg / ml urea, 179.3μg / ml sodium L-lactate, 0.081μg / ml putrescine dihydrochloride, 24.98μg / ml anhydrous citric acid, 1.9μg / ml ethanolamine, 10.54μg / ml N-acetylglycine, 0.73μg / ml α-ketoglutaric acid, 2.3μg / ml formic acid, 0.67μg / ml malic acid, 1.04μg / ml malonic acid, 2.36μg / ml succinic acid, 58.84μg / ml uric acid, 0.0003μg / ml ammonium metavanadate, 0.00005μg / ml manganese dichloride, 0.005μg / ml sodium selenite, 5.25μg / ml creatine, 11.05μg / ml glycerol, 0.733μg / ml uridine, 3.281μg / ml anhydrous sodium acetate, 1μg / ml p-aminobenzoic acid, 11.77μg / ml glutamic acid acid, 9.18μg / ml isoleucine, 36.53μg / ml lysine hydrochloride, 13.215μg / ml phenylalanine, 23.026μg / ml proline, 2.14μg / ml ammonium chloride, 260.803μg / ml calcium chloride aldehyde, 9.446μg / ml calcium nitrate tetrahydrate, 2μg / ml potassium dihydrogen phosphate, 2016.24μg / ml sodium bicarbonate, 135.005μg / ml anhydrous disodium phosphate, 1.199μg / ml acetylcarnitine, 8.201μg / ml betaine, 2.062μg / ml L-amino-N-butyric acid, 7.92 μg / ml L-carnitine hydrochloride, 7.008 μg / ml L-citrulline, 2.623 μg / ml L-hydroxyproline, 3.485 μg / ml acetone, 7.206 μg / ml fructose and 10.81 μg / ml galactose, based on the total volume of the basal medium.

[0043] Mix the components of the additives, add the resulting additives to the culture medium, mix well, and obtain an organoid basal culture medium. Taking colorectal cancer as an example, add G-27 supplement (Bozhen Bio, 50X, product number S223152-10, the volume ratio of G-27 supplement to basal culture medium is 1:50), 50ng / ml EGF, 100ng / ml Noggin, 500ng / ml R-Spondin1, 0.5μM A-83-01, 5μM Y27632, 3μM SB202190 (all of the above are final concentrations) to the organoid basal culture medium obtained in the previous step, mix well, and obtain a colorectal cancer organoid complete culture medium. Example 3 The difference from Example 1 is that the added amounts of 6.31 μg / ml sodium taurine 3-hydroxybutyrate, 11.26 μg / ml taurine, 2.39 μg / ml sodium hypoxanthine, 8.48 μg / ml creatinine, 11.8 μg / ml L-ornithine, 300.3 μg / ml urea, 179.3 μg / ml sodium L-lactate, 0.081 μg / ml putrescine dihydrochloride, 24.98 μg / ml anhydrous citric acid, and 1.9 μg / ml ethanolamine are changed to 3.5 μg / ml sodium taurine 3-hydroxybutyrate, 6.3 μg / ml taurine, 1.33 μg / ml sodium hypoxanthine, 4.75 μg / ml creatinine, 6.6 μg / ml L-ornithine, 168.17 μg / ml urea, 100.4 μg / ml Sodium L-lactate, 0.046 μg / ml putrescine dihydrochloride, 13.98 μg / ml anhydrous citric acid, and 1.06 μg / ml ethanolamine, based on the total volume of the basal medium.

[0044] Comparative Example 1 The difference from Example 1 is that the organoid basal culture medium only contains the basal culture medium DMEM / F12 culture medium.

[0045] Comparative Example 2 The difference from Example 1 is that 6.31 μg / ml taurine sodium 3-hydroxybutyrate, 11.26 μg / ml taurine, 2.39 μg / ml hypoxanthine sodium, 8.48 μg / ml creatinine, and 11.8 μg / ml L-ornithine are not added. The addition amount of 300.3 μg / ml urea, 179.3 μg / ml L-sodium lactate, and 24.98 μg / ml anhydrous citric acid is changed to 320.54 μg / ml urea, 189.3 μg / ml L-sodium lactate, and 34.98 μg / ml anhydrous citric acid.

[0046] Comparative Example 3 The difference from Example 1 is that 6.31 μg / ml taurine 3-hydroxybutyrate sodium, 11.26 μg / ml taurine, 2.39 μg / ml hypoxanthine sodium, 8.48 μg / ml creatinine, and 11.8 μg / ml L-ornithine are replaced by 2.02 μg / ml nicotinamide, 8.98 μg / ml choline chloride, 17.29 μg / ml glycine, 4.45 μg / ml L-alanine, and 7.5 μg / ml L-asparagine.

[0047] Test Case Taking colorectal cancer as an example, the organoid basal medium and colorectal cancer complete medium prepared in Examples 1 to 3 and Comparative Examples 1 to 3 were used to treat human colon cancer organoids (CRC), and the specific steps were as follows: When the colorectal cancer organoids are cultured to a diameter of 100-200 μm, remove the culture medium and add an equal volume of organoid basal medium. Bozhen Biology E238002-100) gently scrape (or blow) the matrix gel ( Bozhen Biology M315066) and colorectal cancer organoid mixture, transfer to a 1.5 ml centrifuge tube, pipette 5-10 times to separate colorectal cancer organoids and matrix gel, and centrifuge at 200-300g for 3-5 minutes. After centrifugation, it is clearly visible that the culture medium, matrix gel, and organoid precipitation are divided into three layers from top to bottom, and the upper culture medium and matrix gel are removed.

[0048] Add 1 ml of organoid basal medium to resuspend the organoid pellet and centrifuge at 200-300 g for 3-5 minutes.

[0049] Discard the supernatant and add 5 times the amount of organoid digestion solution as the organoid pellet ( Bozhen Biology E238001-100), mix well and place in a 37°C incubator for digestion. During this period, use a pipette tip rinsed with organoid rinse solution to blow the tissue digestion suspension every 2 minutes to help fully digest until the colorectal cancer organoids are digested into small cell clumps.

[0050] After digestion is completed, add 5 times the volume of the digestion suspension to dilute the digested organoid suspension to terminate the digestion reaction.

[0051] After mixing, centrifuge at 200-300 g for 3-5 minutes. After centrifugation, wash twice with organoid basal medium to remove residual digestion fluid.

[0052] The washed cell pellet was resuspended in 1 ml of organoid basal medium, and 18 μl of the suspension (plus 2 μl of trypan blue staining reagent) was taken for live cell detection and counting.

[0053] After counting, the remaining cell suspension was centrifuged at 200-300 g for 3-5 minutes, the supernatant was aspirated and the precipitate was retained. The corresponding amount of organoid matrix gel was calculated based on the ratio of 8000-10000 cells per well to 20-30 μl matrix gel (taking a 24-well plate as an example), and the mixture was mixed on ice and placed on ice after mixing.

[0054] Use a pipette to absorb the mixture of matrix gel and colorectal cancer cells and transfer it to the cell culture well plate. The mixed suspension must be applied to the center of the culture well and should not touch the side wall of the culture well after spreading. Place the culture plate in a 37°C, 5% CO2 cell culture incubator to solidify for 20 minutes. After the matrix gel solidifies and no longer flows, slowly add colorectal cancer organoid complete culture medium prepared from organoid basal culture medium along the well wall (for example, add 500 μl complete culture medium for 24 wells), place the 24-well plate in a 37°C, 5% CO2 cell culture incubator, and replace the complete culture medium every 2 days.

[0055] The cell culture plate was placed in an incubator and cultured at 37°C. The growth state of the organoids was observed under an inverted microscope every 2 days, and photographs were taken under an inverted microscope to record the morphology and distribution of the organoids in multiple fields of view. After 6 days of culture, the diameter of the organoids was observed. The results are shown in Table 1. It can be seen that after 6 days of culture with the complete medium for organoids prepared in Examples 1 to 3, the diameter of the organoids was significantly larger than that of Comparative Examples 1 to 3, indicating that the basal medium prepared in accordance with Examples 1 to 3 can better maintain the activity of the organoids, provide basic nutrition and metabolic requirements, and promote the growth of organoids. Among them, the basal medium for organoids prepared in Example 2 has better effect.

[0056] Table 1 Organoid diameter (unit: μm)

[0057] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. An organoid basal culture medium, characterized in that include: The basic culture medium and additives include sodium taurine 3-hydroxybutyrate, taurine, sodium hypoxanthine, creatinine, L-ornithine, urea, sodium L-lactate, putrescine dihydrochloride, anhydrous citric acid and ethanolamine.

2. The organoid basal medium according to claim 1, characterized in that Based on the total volume of the basal medium, the additives include: 4.41-8.2 μg / ml taurine 3-hydroxybutyrate sodium, 7.88-14.64 μg / ml taurine, 1.67-3.1 μg / ml hypoxanthine sodium, 5.94-11.03 μg / ml creatinine, 8.26-15.34 μg / ml L-ornithine, 210.21-390.39 μg / ml urea, 125.5-233.08 μg / ml L-sodium lactate, 0.057-0.11 μg / ml putrescine dihydrochloride, 17.48-32.47 μg / ml anhydrous citric acid and 1.33-2.47 μg / ml ethanolamine.

3. The organoid basal medium according to claim 1 or 2, characterized in that Based on the total volume of the basal medium, the supplement includes: 6.31 μg / ml taurine 3-hydroxybutyrate sodium, 11.26 μg / ml taurine, 2.39 μg / ml hypoxanthine sodium, 8.48 μg / ml creatinine, 11.8 μg / ml L-ornithine, 300.3 μg / ml urea, 179.3 μg / ml L-sodium lactate, 0.081 μg / ml putrescine dihydrochloride, 24.98 μg / ml anhydrous citric acid and 1.9 μg / ml ethanolamine.

4. The organoid basal medium according to claim 1, characterized in that The additives further include: N-acetylglycine, α-ketoglutaric acid, formic acid, malic acid, malonic acid, succinic acid, uric acid, ammonium metavanadate, manganese dichloride, sodium selenite, creatine, glycerol, uridine, anhydrous sodium acetate and p-aminobenzoic acid.

5. The organoid basal medium according to claim 4, characterized in that Based on the total volume of the basal medium, the additive further includes: 7.38-13.7 μg / ml N-acetylglycine, 0.51-0.95 μg / ml α-ketoglutaric acid, 1.61~2.99μg / ml formic acid, 0.47~0.87μg / ml malic acid, 0.73~1.35μg / ml malonic acid, 1.65~3.07μg / ml succinic acid, 41.19~76.49μg / ml uric acid, 0.0001~0.0039μg / ml ammonium metavanadate, 0.000035~0.000065μg / ml manganese dichloride, 0.0035~0.0065μg / ml sodium selenite, 3.67~6.82μg / ml creatine, 7.73~14.37μg / ml glycerol, 0.51~0.96μg / ml uridine, 2.29~4.27μg / ml anhydrous sodium acetate and 0.7~1.3μg / ml p-aminobenzoic acid.

6. The organoid basal medium according to claim 4 or 5, characterized in that Based on the total volume of the basal medium, the supplement further includes: 10.54 μg / ml N-acetylglycine, 0.73 μg / ml α-ketoglutarate, 2.3 μg / ml formic acid, 0.67 μg / ml malic acid, 1.04 μg / ml malonic acid, 2.36 μg / ml succinic acid, 58.84 μg / ml uric acid, 0.0003 μg / ml ammonium metavanadate, 0.00005 μg / ml manganese dichloride, 0.005 μg / ml sodium selenite, 5.25 μg / ml creatine, 11.05 μg / ml glycerol, 0.733 μg / ml uridine, 3.281 μg / ml anhydrous sodium acetate and 1 μg / ml p-aminobenzoic acid.

7. The organoid basal medium according to claim 4, characterized in that The additives further include: glutamic acid, isoleucine, lysine hydrochloride, phenylalanine, proline, ammonium chloride, calcium chloride aldehyde, calcium nitrate tetrahydrate, potassium dihydrogen phosphate, sodium bicarbonate, anhydrous disodium phosphate, acetyl carnitine, betaine, L-amino-N-butyric acid, L-carnitine hydrochloride, L-citrulline, L-hydroxyproline, acetone, fructose and galactose.

8. The organoid basal medium according to claim 7, characterized in that Based on the total volume of the basal medium, the additive further includes: 8.24-15.3 μg / ml glutamic acid, 6.42-12 μg / ml isoleucine, 25.5-47.5 μg / ml lysine hydrochloride, 9.25-17.2 μg / ml phenylalanine, 16-30 μg / ml proline, 1.49-2.79 μg / ml ammonium chloride, 182.56-339.05 μg / ml calcium chloride aldehyde, 6.61~12.28μg / ml calcium nitrate tetrahydrate, 1.4~2.6μg / ml potassium dihydrogen phosphate, 1411.37~2621.11μg / ml sodium bicarbonate, 94.5~175.5μg / ml anhydrous disodium phosphate, 0.83~1.56μg / ml acetyl carnitine, 5.74~10.66μg / ml betaine, 1.44~2.68μg / ml L-amino-N-butyric acid, 5.54~10.3μg / ml L-carnitine hydrochloride, 4.9~9.11μg / ml L-citrulline, 1.84~3.41μg / ml L-hydroxyproline, 2.44~4.53μg / ml acetone, 5.04~9.37μg / ml fructose and 7.57~14.05μg / ml galactose.

9. The organoid basal medium according to claim 7 or 8, characterized in that Based on the total volume of the basal medium, the additive further includes: 11.77 μg / ml glutamic acid, 9.18 μg / ml isoleucine, 36.53 μg / ml lysine hydrochloride, 13.215 μg / ml phenylalanine, 23.026 μg / ml proline, 2.14 μg / ml ammonium chloride, 260.803 μg / ml calcium chloride aldehyde, 9.446 μg / ml calcium nitrate tetrahydrate, 2 μg / ml potassium dihydrogen phosphate, 2016.24 μg / ml sodium bicarbonate, 135.005 μg / ml anhydrous disodium phosphate, 1.199 μg / ml acetylcarnitine, 8.201 μg / ml betaine, 2.062 μg / ml L-amino-N-butyric acid, 7.92 μg / ml L-carnitine hydrochloride, 7.008 μg / ml L-citrulline, 2.623 μg / ml L-hydroxyproline, 3.485 μg / ml acetone, 7.206 μg / ml fructose and 10.81 μg / ml galactose.

10. The organoid basal medium according to claim 1, characterized in that Based on the total volume of the basal medium, the supplement includes: 6.31 μg / ml taurine 3-hydroxybutyrate sodium, 11.26 μg / ml taurine, 2.39 μg / ml hypoxanthine sodium, 8.48 μg / ml creatinine, 11.8 μg / ml L-ornithine, 300.3 μg / ml urea, 179.3 μg / ml L-sodium lactate, 0.081 μg / ml putrescine dihydrochloride, 24.98 μg / ml anhydrous citric acid, 1.9 μg / ml ethanolamine, 10.54 μg / ml N-acetylglycine, 0.73 μg / ml α-ketoglutaric acid, 2.3μg / ml formic acid, 0.67μg / ml malic acid, 1.04μg / ml malonic acid, 2.36μg / ml succinic acid, 58.84μg / ml uric acid, 0.0003μg / ml ammonium metavanadate, 0.00005μg / ml manganese dichloride, 0.005μg / ml sodium selenite, 5.25μg / ml creatine, 11.05μg / ml glycerol, 0.733μg / ml uridine, 3.281μg / ml anhydrous sodium acetate, 1μg / ml p-aminobenzoic acid, 11.77μg / ml glutamic acid acid, 9.18μg / ml isoleucine, 36.53μg / ml lysine hydrochloride, 13.215μg / ml phenylalanine, 23.026μg / ml proline, 2.14μg / ml ammonium chloride, 260.803μg / ml calcium chloride aldehyde, 9.446μg / ml calcium nitrate tetrahydrate, 2μg / ml potassium dihydrogen phosphate, 2016.24μg / ml sodium bicarbonate, 135.005μg / ml anhydrous disodium phosphate, 1.199μg / ml acetylcarnitine, 8.201μg / ml betaine, 2.062μg / ml L-amino-N-butyric acid, 7.92 μg / ml L-carnitine hydrochloride, 7.008 μg / ml L-citrulline, 2.623 μg / ml L-hydroxyproline, 3.485 μg / ml acetone, 7.206 μg / ml fructose and 10.81 μg / ml galactose.

11. The organoid basal medium according to claim 1, characterized in that The basic culture medium includes DMEM / F12 culture medium; the organoid culture medium is a serum-free culture medium; and the organoid culture medium does not contain protein.

12. A method for maintaining or promoting organoid growth, characterized in that: include: The organoid is treated with the organoid basal medium according to any one of claims 1 to 11.

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