Serum replacements, culture media and their applications in tumor organoid culture

Abstract

The present application relates to serum substitutes, culture media and their application in tumor organoid culture, and belongs to the field of biomedicine. Among them, the aforementioned serum substitutes include: cholesterol, L-ascorbic acid, arachidonic acid, myristic acid, (±)-α-lipoic acid, superoxide dismutase, sodium selenite, catalase, L-carnitine hydrochloride, putrescine dihydrochloride, L-reduced glutathione, D-(+)-galactose, transferrin, linolenic acid, retinol acetate, (±)-α-tocopherol acetate, corticosterone, DL-α-tocopherol, triiodothyronine sodium salt, insulin and bovine serum albumin. The aforementioned serum substitute can effectively simulate the nutritional environment for the growth of tumor organoids, and can replace serum for tumor organoid culture, and the organoids obtained by culture have good proliferation and differentiation results. The use of the aforementioned serum substitute for tumor organoid culture is lower in cost and suitable for industrial application.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically, relates to serum substitutes, culture media and their applications in tumor organoid culture. Background Art

[0002] Tumor organoids are three-dimensional cell structures cultured in vitro, derived from the patient's primary tumor tissue or metastatic tumor tissue. Tumor organoids can simulate the growth and behavior of primary tumor tissue, including cell heterogeneity, cell-cell interactions, cell-matrix interactions and other characteristics, providing an in vitro research platform that is closer to real tumors, and can be used to study the biological characteristics of tumors, drug sensitivity and personalized treatment. At present, bovine serum must be added to the culture of tumor organoids. Traditional animal serum has many problems in the development and production of biological agents, including batch-to-batch variability, unstable supply, high cost and potential risk of infectious diseases.

[0003] Serum substitutes refer to substitutes that are used to replace traditional animal serum and play similar or identical functions in the development and production of biological agents. Compared with traditional methods, the use of serum substitutes has many advantages: first, it reduces the use of animal serum, avoiding batch differences and potential risks; second, it improves the quality of cell culture and optimizes the cell culture environment by providing precise and controllable nutrients and growth factors; third, it promotes the development of biotechnology and provides new possibilities for the biopharmaceutical industry; fourth, it improves production efficiency and reduces costs, simplifies production processes, and reduces production costs; fifth, it complies with the concept of environmental protection and sustainable development, and helps reduce the consumption of natural resources. However, there is currently a lack of serum substitutes that can fully meet the needs of tumor organoid culture. Summary of the invention

[0004] The present application aims to solve at least one of the problems of the prior art. To this end, the present application proposes a serum substitute for tumor organoid culture.

[0005] Specifically, this application provides the following technical solutions:

[0006] In the first aspect of the present application, the present application proposes a serum substitute. According to the embodiments of the present application, the serum substitute includes: cholesterol, L-ascorbic acid, arachidonic acid, myristic acid, (±)-α-lipoic acid, superoxide dismutase, sodium selenite, catalase, L-carnitine hydrochloride, putrescine dihydrochloride, L-reduced glutathione, D-(+)-galactose, transferrin, linolenic acid, retinol acetate, (±)-α-tocopherol acetate, corticosterone, DL-α-tocopherol, triiodothyronine sodium salt, insulin and bovine serum albumin. In some examples of the present application, the inventor unexpectedly found that adding cholesterol, L-ascorbic acid, arachidonic acid, myristic acid and (±)-α-lipoic acid to the commonly used components of serum substitutes can effectively simulate the nutritional environment for the growth of tumor organoids, can replace serum for tumor organoid culture, and the obtained organoid proliferation and differentiation results are good. In the culture of some tumor organoids, the growth rate of organoids was faster when the above serum substitutes were used compared with fetal bovine serum. More importantly, the use of the above serum substitutes for tumor organoid culture is lower in cost and suitable for industrial application.

[0007] According to an embodiment of the present application, the serum substitute may further include at least one of the following additional technical features:

[0008] In some examples of the present application, the concentration ratio of cholesterol, L-ascorbic acid, arachidonic acid, myristic acid, (±)-α-lipoic acid, superoxide dismutase, sodium selenite, catalase, L-carnitine hydrochloride, putrescine dihydrochloride, L-reduced glutathione, D-(+)-galactose, transferrin, linolenic acid, retinol acetate, (±)-α-tocopheryl acetate, corticosterone, DL-α-tocopherol, triiodothyronine sodium salt, insulin and bovine serum albumin contained in the aforementioned serum substitute is (20-500): (25 -1000): (10-100): (10-500): (0.1-20): (10-500): (0.1-10): (25-1000): (40-800): (500-10000): (10-500): (200-10000): (300-8000): (10-500): (0.1-50): (10-500): (0.1-10): (10-500): (0.01-1): (50-5000): (10-500). The serum substitutes with the above mass ratio components can improve the culture efficiency and stability of tumor organoids, and can better maintain cell heterogeneity and biological functions. Compared with traditional animal serum, the serum substitutes with this ratio have the advantages of better stability, reproducibility and mass production, and can reduce the ethical and infectious risks of using animal-derived serum.

[0009] In some examples of the present application, the serum substitute includes: 20mg / L-500mg / L cholesterol, 25mg / L-1000 mg / L L-ascorbic acid, 10mg / L-100mg / L arachidonic acid, 10mg / L-500mg / L myristic acid, 0.1mg / L-20mg / L (±)-α-lipoic acid, 10mg / L-500mg / L superoxide dismutase, 0.1mg / L-10mg / L sodium selenite, 25mg / L-1000mg / L catalase, 40mg / L-800mg / L L-carnitine hydrochloride, 500mg / L-10000mg / L putrescine dihydrochloride, 10mg / L-500mg / L L-reduced glutathione, 200mg / L-10000mg / L D-(+)-galactose, 300mg / L-8000mg / L transferrin, 10mg / L-500mg / L linolenic acid, 0.1mg / L-50mg / L retinol acetate, 10mg / L-500mg / L (±)-α-tocopherol acetate, 0.1mg / L-10mg / L corticosterone, 10mg / L-500mg / L DL-α-tocopherol, 0.01mg / L-1mg / L triiodothyronine sodium salt, 50mg / L-5000mg / L insulin and 10g / L-500g / L bovine serum albumin. The serum replacement components at the above concentrations can not only support the growth and proliferation of tumor organoids, but also maintain their cellular heterogeneity and biological functions. Compared with traditional animal serum, serum replacements in this concentration range have the advantages of better stability, reproducibility and mass production, and can reduce the ethical and infectious risks of using animal-derived serum.

[0010] In some examples of the present application, the serum substitute includes: 50mg / L-200mg / L cholesterol, 50mg / L-500 mg / L L-ascorbic acid, 20mg / L-60mg / L arachidonic acid, 30mg / L-300mg / L myristic acid, 1mg / L-10mg / L (±)-α-lipoic acid, 20mg / L-150mg / L superoxide dismutase, 0.5mg / L-5mg / L sodium selenite, 50mg / L-500mg / L catalase, 100mg / L-500mg / L L-carnitine hydrochloride, 1000mg / L-5000mg / L putrescine dihydrochloride, 20mg / L-200mg / L L-reduced glutathione, 500mg / L-5000mg / L D-(+)-galactose, 500mg / L-3000mg / L transferrin, 30mg / L-300mg / L linolenic acid, 1mg / L-30mg / L retinol acetate, 20mg / L-200mg / L (±)-α-tocopherol acetate, 0.5mg / L-5mg / L corticosterone, 50mg / L-200mg / L DL-α-tocopherol, 0.05mg / L-0.5mg / L triiodothyronine sodium salt, 200mg / L-2000mg / L insulin and 50g / L-300g / L bovine serum albumin. The serum replacement components of the above concentrations can not only support the growth and proliferation of tumor organoids, but also maintain their cellular heterogeneity and biological functions. Compared with traditional animal serum, this ratio of serum replacement has the advantages of better stability, reproducibility and mass production, and can reduce the ethical and infectious risks of using animal-derived serum.

[0011] In some examples of the present application, the serum substitute includes: 60 mg / L cholesterol, 80 mg / L L-ascorbic acid, 30 mg / L arachidonic acid, 80 mg / L myristic acid, 8 mg / L (±)-α-lipoic acid, 20 mg / L superoxide dismutase, 4 mg / L sodium selenite, 300 mg / L catalase, 300 mg / L L-carnitine hydrochloride, 3000 mg / L putrescine dihydrochloride, 150 mg / L L-reduced glutathione, 2000 mg / L D-(+)-galactose, 2500 mg / L transferrin, 40 mg / L linolenic acid, 25 mg / L retinol acetate, 30 mg / L (±)-α-tocopherol acetate, 3 mg / L corticosterone, 60 mg / L DL-α-tocopherol, 0.06 mg / L Triiodothyronine sodium salt, 300mg / L insulin and 80g / L bovine serum albumin. After experimental verification, the serum substitutes with the above concentration components were used for tumor organoid culture, and the organoid proliferation and differentiation results were good.

[0012] In some examples of the present application, the serum substitute includes: 150 mg / L cholesterol, 50 mg / L L-ascorbic acid, 30 mg / L arachidonic acid, 200 mg / L myristic acid, 8 mg / L (±)-α-lipoic acid, 100 mg / L superoxide dismutase, 4 mg / L sodium selenite, 300 mg / L catalase, 200 mg / L L-carnitine hydrochloride, 1000 mg / L putrescine dihydrochloride, 150 mg / L L-reduced glutathione, 3000 mg / L D-(+)-galactose, 2500 mg / L transferrin, 40 mg / L linolenic acid, 30 mg / L retinol acetate, 40 mg / L (±)-α-tocopherol acetate, 4 mg / L corticosterone, 60 mg / L DL-α-tocopherol, 0.06 mg / L Triiodothyronine sodium salt, 1000mg / L insulin and 50g / L bovine serum albumin. After experimental verification, the serum substitutes with the above concentration components were used for tumor organoid culture, and the organoid proliferation and differentiation results were good.

[0013] In some examples of the present application, the serum substitute includes: 150 mg / L cholesterol, 80 mg / L L-ascorbic acid, 30 mg / L arachidonic acid, 200 mg / L myristic acid, 8 mg / L (±)-α-lipoic acid, 20 mg / L superoxide dismutase, 4 mg / L sodium selenite, 300 mg / L catalase, 300 mg / L L-carnitine hydrochloride, 1000 mg / L putrescine dihydrochloride, 150 mg / L L-reduced glutathione, 3000 mg / L D-(+)-galactose, 2500 mg / L transferrin, 40 mg / L linolenic acid, 25 mg / L retinol acetate, 100 mg / L (±)-α-tocopherol acetate, 4 mg / L corticosterone, 60 mg / L DL-α-tocopherol, 0.07 mg / L Triiodothyronine sodium salt, 300mg / L insulin and 80g / L bovine serum albumin. After experimental verification, the serum substitutes with the above concentration components were used for tumor organoid culture, and the organoid proliferation and differentiation results were good.

[0014] In a second aspect of the present application, the present application proposes a culture medium. According to an embodiment of the present application, the culture medium includes: any of the serum replacements in the above examples. In some examples of the present application, the above culture medium can be used for efficient and stable culture of tumor organoids.

[0015] According to an embodiment of the present application, the serum substitute may further include at least one of the following additional technical features:

[0016] In some examples of the present application, the aforementioned culture medium further includes: at least one of auxiliary factors and basal culture medium. The addition of auxiliary factors promotes cell proliferation and differentiation while maintaining the morphology and function of tumor organoids; the basal culture medium provides the basic nutrients and environment required for cell growth, such as carbon sources, nitrogen sources, inorganic salts, etc.

[0017] In some examples of the present application, the aforementioned auxiliary factors include: IGF1, gremlin 1, EGF, R-Spondinl, A8301, Y-27632, SB202190, N2, PGE2, HEPES, Glutamax, penicillin, streptomycin and DMEM / F12 culture medium.

[0018] In some examples of the present application, the aforementioned auxiliary factors include: 100ng / ml IGF1, 200ng / ml gremlin1, 50ng / ml EGF, 1μg / ml R-Spondin1, 500nM A8301, 10μM Y-27632, 10μM SB202190, 1×N2, 0.5μM PGE2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin and 500U / mL streptomycin. The auxiliary factors based on this concentration can better promote the proliferation and differentiation of colorectal cancer organoids.

[0019] In some examples of the present application, the aforementioned auxiliary factors include: BMP2, gremlin1, IGF2, LTBP2, EGF, HGF, Y27632, N2, HEPES, Glutamax, penicillin, streptomycin, and DMEM / F12 culture medium.

[0020] In some examples of the present application, the aforementioned auxiliary factors include: 50ng / ml BMP2, 50ng / ml gremlin1, 50ng / ml IGF2, 100ng / ml LTBP2, 100ng / ml EGF, 50ng / ml HGF, 5μM Y27632, 1X N2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium. The auxiliary factors based on this concentration can better promote the proliferation and differentiation of head and neck squamous cell carcinoma organoids.

[0021] In some examples of the present application, the aforementioned basal culture medium is selected from Advanced DMEM / F12.

[0022] In the third aspect of the present application, the present application proposes the use of the culture medium described in the second aspect in the preparation of a kit for culturing tumor organoids. In some examples of the present application, the kit can be used to proliferate and / or differentiate tumor cells, and further obtain tumor organoids. The obtained tumor organoids can simulate the actual situation of tumor growth in terms of morphology, transcriptional characteristics, and secretory functions, and scientific research based on this model has high reliability.

[0023] In the fourth aspect of the present application, the present application proposes a method for constructing a tumor organoid. According to an embodiment of the present application, the method includes: a step of culturing tumor cells using the culture medium described in the second aspect. The tumor organoid obtained based on the specific method of the embodiment of the present application can simulate the actual situation of tumor growth in morphology, transcriptional characteristics, and secretory function, and scientific research based on this model has high reliability.

[0024] According to an embodiment of the present application, the above-mentioned method for constructing a tumor organoid may further include at least one of the following additional technical features:

[0025] In some examples of the present application, the aforementioned tumor cells include at least one of colorectal cancer and human head and neck squamous cell carcinoma cells. Experimental verification shows that the serum replacement or culture medium based on the present application can achieve the construction of colorectal cancer and human head and neck squamous cell carcinoma organoids.

[0026] In the fifth aspect of the present application, the present application proposes a tumor organoid. According to the embodiments of the present application, the tumor organoid is constructed using the method described in the fourth aspect. In some examples of the present application, the tumor organoid obtained by the above method can simulate the actual situation of tumor growth in terms of morphology, transcriptional characteristics, and secretory function, and scientific research based on this model has high reliability.

[0027] In the sixth aspect of the present application, the present application proposes a method for screening drugs. According to an embodiment of the present application, the method comprises: contacting the drug to be screened with the tumor organoid described in the fifth aspect; comparing the disease state of the tumor organoid before and after contact, and determining whether the drug to be screened is a target drug. In some examples of the present application, based on the aforementioned method for screening drugs, drugs that effectively treat or alleviate tumor diseases can be obtained.

[0028] According to an embodiment of the present application, the above-mentioned method for screening drugs may also include at least one of the following additional technical features:

[0029] According to an embodiment of the present invention, the improvement of the disease state of the tumor organoid after contact is an indication that the drug to be screened is a target drug. In some examples of the present application, the obtained target drug can inhibit tumor proliferation, such as the gene expression of tumor cells returns to normal levels after contact.

[0030] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0032] Figure 1 Schematic diagram of colorectal cancer organoids under different culture conditions provided in the embodiments of the present application; wherein A is a morphological diagram of colorectal cancer organoids obtained by culture in the culture medium of the experimental group 1; B is a morphological diagram of colorectal cancer organoids obtained by culture in the culture medium of the control group;

[0033] Figure 2 Schematic diagram of optical microscope of head and neck squamous cell carcinoma organoids under different culture conditions provided in the examples of the present application; wherein A is a morphological diagram of head and neck squamous cell carcinoma organoids obtained by culture in the culture medium of the control group; B is a morphological diagram of head and neck squamous cell carcinoma organoids obtained by culture in the culture medium of the experimental group 1;

[0034] Figure 3 Schematic diagram of optical microscope of lung adenocarcinoma organoids under different culture conditions provided in the embodiments of the present application; wherein A is the morphological diagram of lung adenocarcinoma organoids obtained by culture with culture medium of the control group; and B is the morphological diagram of lung adenocarcinoma organoids obtained by culture with culture medium of experimental group 1. DETAILED DESCRIPTION

[0035] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0036] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of this application, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0037] The term "optionally" is used for descriptive purposes only and cannot be understood as indicating or implying relative importance. Thus, a feature defined as "optionally" may explicitly or implicitly include or exclude the feature.

[0038] In this article, drug screening is not particularly limited, and any drug that can be screened using the tumor organoids described in this application is included in the scope of drug screening described in this application, for example, high-throughput drug screening. "High-throughput drug screening" refers to a screening model based on the molecular level or the cellular level, using a microplate as a reaction carrier, combined with an automated operating system and a sensitive and rapid detection method, to efficiently complete the detection of tens of millions of samples to be tested to obtain the target drug.

[0039] Herein, the "disease status improves" means that after the tumor organoids that have been pre-treated for disease modeling come into contact with the target drug, the abnormal state of the tumor organoids gradually disappears, and the structure, function, and metabolism of the tumor organoids are repaired or completely restored to normal. It should be noted that the improvement of the disease status is not particularly limited, and any tumor abnormality detection indicator that is significantly improved or restored to normal levels is considered an improvement in the disease status.

[0040] 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.

[0041] Example 1 Colorectal cancer organoid culture

[0042] This example aims to verify whether the serum substitute based on the ratio of this application can replace fetal bovine serum for colorectal cancer organoid culture.

[0043] Serum replacement combination 1: 60mg / L cholesterol, 80 mg / L L-ascorbic acid, 30mg / L arachidonic acid, 80mg / L myristic acid, 8mg / L (±)-α-lipoic acid, 20mg / L superoxide dismutase, 4mg / L sodium selenite, 300mg / L catalase, 300mg / L L-carnitine hydrochloride, 3000mg / L putrescine dihydrochloride, 150mg / L L-reduced glutathione, 2000mg / L D-(+)-galactose, 2500mg / L transferrin, 40mg / L linolenic acid, 25mg / L retinol acetate, 30mg / L (±)-α-tocopherol acetate, 3mg / L corticosterone, 60mg / L DL-α-tocopherol, 0.06mg / L triiodothyronine sodium salt, 300mg / L Insulin and 80 g / L bovine serum albumin;

[0044] Serum replacement combination 2: 150mg / L cholesterol, 50 mg / L L-ascorbic acid, 30mg / L arachidonic acid, 200mg / L myristic acid, 8mg / L (±)-α-lipoic acid, 100mg / L superoxide dismutase, 4mg / L sodium selenite, 300mg / L catalase, 200mg / L L-carnitine hydrochloride, 1000mg / L putrescine dihydrochloride, 150mg / L L-reduced glutathione, 3000mg / L D-(+)-galactose, 2500mg / L transferrin, 40mg / L linolenic acid, 30mg / L retinol acetate, 40mg / L (±)-α-tocopherol acetate, 4mg / L corticosterone, 60mg / L DL-α-tocopherol, 0.06mg / L triiodothyronine sodium salt, 1000 mg / L insulin, and 50 g / L bovine serum albumin;

[0045] Serum replacement combination 3: 150mg / L cholesterol, 80 mg / L L-ascorbic acid, 30mg / L arachidonic acid, 200mg / L myristic acid, 8mg / L (±)-α-lipoic acid, 20mg / L superoxide dismutase, 4mg / L sodium selenite, 300mg / L catalase, 300mg / L L-carnitine hydrochloride, 1000mg / L putrescine dihydrochloride, 150mg / L L-reduced glutathione, 3000mg / L D-(+)-galactose, 2500mg / L transferrin, 40mg / L linolenic acid, 25mg / L retinol acetate, 100mg / L (±)-α-tocopherol acetate, 4mg / L corticosterone, 60mg / L DL-α-tocopherol, 0.07mg / L triiodothyronine sodium salt, 300mg / L Insulin and 80 g / L bovine serum albumin;

[0046] Experimental group 1: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0047] The mass ratio of the basic culture medium Advanced DMEM / F12 and sterile water is 99:1; the auxiliary factors include: 100ng / ml IGF1, 200ng / ml gremlin1, 50ng / ml EGF, 1ug / ml R-Spondin1, 500nM A8301, 10μM Y-27632, 10μM SB202190, 1X N2, 0.5μM PGE2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of the basic culture medium and sterile water, and 2% serum substitute combination 1 is added;

[0048] Experimental group 2: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0049] The mass ratio of the basic culture medium Advanced DMEM / F12 and sterile water is 99:1; the auxiliary factors include: 100ng / ml IGF1, 200ng / ml gremlin1, 50ng / ml EGF, 1ug / ml R-Spondinl, 500nM A8301, 10μM Y-27632, 10μM SB202190, 1X N2, 0.5μM PGE2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basic culture medium and sterile water, and 2% serum substitute combination 2 is added;

[0050] Experimental group 3: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0051] The mass ratio of the basic culture medium Advanced DMEM / F12 and sterile water is 99:1; the auxiliary factors include: 100ng / ml IGF1, 200ng / ml gremlin1, 50ng / ml EGF, 1ug / ml R-Spondinl, 500nM A8301, 10μM Y-27632, 10μM SB202190, 1X N2, 0.5μM PGE2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basic culture medium and sterile water, and 2% serum substitute combination 3 is added;

[0052] Control group: basal medium Advanced DMEM / F12, auxiliary factors and sterile water;

[0053] Among them, the mass ratio of the basic culture medium Advanced DMEM / F12 and sterile water is: 99:1; the auxiliary factors include: 100ng / ml IGF1, 200ng / ml gremlin1, 50ng / ml EGF, 1ug / ml R-Spondinl, 500nM A8301, 10μM Y-27632, 10μM SB202190, 1X N2, 0.5μM PGE2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basic culture medium and sterile water, and 10% fetal bovine serum is added.

[0054] The culture medium of the above experimental groups 1 to 3 and the control group was used to culture colorectal cancer organoids. The specific experimental steps are as follows:

[0055] 1) Fresh colorectal cancer surgical resection specimens were placed in the prepared Advanced DMEM / F12 medium containing 5% double antibody and sent to the laboratory for pretreatment within 12 hours;

[0056] 2) Sample washing: transfer the tissue into a 15 ml centrifuge tube, then wash with 5 ml of Advanced DMEM / F12 medium containing 5% double antibody for 30 seconds, remove the supernatant, and add 5 ml of Advanced DMEM / F12 medium containing 5% double antibody for washing. Repeat the above method for 3 times to remove impurities on the tissue surface;

[0057] 3) Sample cutting: In a biosafety cabinet, transfer the sample to a 6 cm culture dish and cut the tissue into pieces of 1-3 mm in size using sterilized surgical scissors on ice. 3 , the shearing process should not exceed 10 minutes to avoid cell damage;

[0058] 4) First digestion of tissue: transfer the minced tissue to a 15 ml centrifuge tube, add 10 ml of digestion solution and shake and digest at 37°C for 60 minutes. After the first digestion, add a final concentration of 5% FBS to terminate the digestion, then centrifuge at 300 rcf for 3 minutes, retain the precipitate and remove the supernatant;

[0059] 5) Cell filtration: Filter the digestion solution in step 4) with a 100 μm filter to remove large undigested tissue pieces. Centrifuge the filtered cell solution at 300 rcf for 3 min, and carefully remove the supernatant to obtain the cell pellet;

[0060] 6) Red blood cell lysis: Add 1 ml of red blood cell lysis buffer to the cell pellet in step 5), gently pipette to resuspend the cell pellet, and lyse at room temperature for 2-3 minutes;

[0061] 7) Cell collection: Centrifuge the liquid in step 6) at 300 rcf for 3 min, carefully remove the supernatant to obtain the cell pellet for later use;

[0062] 8) Take an appropriate amount of matrix gel to resuspend the cell pellet obtained in step 7), and then use a pipette to drop the gel mixed with cells into a 24-well plate, about 25 μl per well;

[0063] 9) Place the culture plate inoculated with the gel droplets in a CO2 incubator and let it stand for 15-20 minutes to allow it to fully solidify;

[0064] 10) Adding the two culture media into the culture dish and culturing at the same time, and then placing it in a constant temperature incubator at 37° C. and 5% CO2 concentration;

[0065] 11) The culture medium was replaced every 3 days. After 10 days of culture, colorectal cancer organoids were obtained.

[0066] Results Analysis

[0067] The morphological structure of colorectal cancer organoids was observed under an ordinary optical microscope. Figure 1 As shown, it can be seen that when the experimental group 1 culture medium was used for culture, the number of colorectal cancer organoids was the largest and the organoid culture diameter was larger ( Figure 1 A) The morphology of colorectal cancer organoids cultured in the experimental group 1 medium is similar to that of colorectal cancer organoids cultured in the fetal bovine serum medium, indicating that the culture effect of the experimental group 1 medium is better than that of fetal bovine serum and can replace the control group medium for colorectal cancer organoid culture. The results of experimental groups 2 and 3 are the same as those of experimental group 1 and will not be repeated here.

[0068] Example 2 Head and Neck Squamous Cell Carcinoma Organoid Culture

[0069] This example aims to verify whether the serum substitute based on the ratio of this application can replace fetal bovine serum for head and neck squamous cell carcinoma organoid culture.

[0070] Serum replacement combinations 1 to 3 are the same as in Example 1;

[0071] Experimental group 1: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0072] Among them, the mass ratio of the basic culture medium Advanced DMEM / F12 and sterile water is: 99:1; the auxiliary factors include: 50ng / ml BMP2, 50ng / ml gremlin1, 50ng / ml IGF2, 100ng / ml LTBP2, 100ng / ml EGF, 50ng / ml HGF, 5μM Y27632, 1X N2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basic culture medium and sterile water, and 2% serum substitute combination 1 is added.

[0073] Experimental group 2: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0074] Among them, the mass ratio of the basal culture medium Advanced DMEM / F12 and sterile water is: 99:1; the auxiliary factors include: 50ng / ml BMP2, 50ng / ml gremlin1, 50ng / ml IGF2, 100ng / ml LTBP2, 100ng / ml EGF, 50ng / ml HGF, 5μM Y27632, 1X N2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basal culture medium and sterile water, and 2% serum substitute combination 2 is added.

[0075] Experimental group 3: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0076] The mass ratio of the basal culture medium Advanced DMEM / F12 and sterile water is 99:1; the auxiliary factors include: 50ng / ml BMP2, 50ng / ml gremlin1, 50ng / ml IGF2, 100ng / ml LTBP2, 100ng / ml EGF, 50ng / ml HGF, 5μM Y27632, 1X N2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basal culture medium and sterile water, and 2% serum substitute combination 3 is added.

[0077] Control group: basal medium Advanced DMEM / F12, auxiliary factors and sterile water;

[0078] Among them, the mass ratio of the basal culture medium Advanced DMEM / F12 and sterile water is: 99:1; the auxiliary factors include: 50ng / ml BMP2, 50ng / ml gremlin1, 50ng / ml IGF2, 100ng / ml LTBP2, 100ng / ml EGF, 50ng / ml HGF, 5μM Y27632, 1X N2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basal culture medium and sterile water, and 10% serum is added.

[0079] The culture medium of experimental groups 1 to 3 and the control group were used to culture human head and neck squamous cell carcinoma organoids, respectively. The specific experimental steps were the same as those in Example 1 and will not be repeated here.

[0080] Results Analysis

[0081] Growth of human head and neck squamous cell carcinoma organoids Figure 2 As shown, it can be seen that when cultured with the experimental group 1 culture medium (serum replacement combination 1), the number of head and neck squamous cell carcinoma organoids is greater and the organoid culture diameter is larger ( Figure 2 A). The morphology of the head and neck squamous cell carcinoma organoids cultured in the experimental group 1 medium was similar to that of the head and neck squamous cell carcinoma organoids cultured in the control group medium, indicating that the culture effect of the experimental group 1 medium was better than that of fetal bovine serum and could replace the control group medium for the culture of head and neck squamous cell carcinoma organoids. The results of experimental groups 2 and 3 were the same as those of experimental group 1 and will not be repeated here.

[0082] Example 3 Lung Adenocarcinoma Organoid Culture

[0083] This example aims to verify whether the serum substitute based on the ratio of this application can replace fetal bovine serum for lung adenocarcinoma organoid culture.

[0084] Serum replacement combinations 1 to 3 are the same as in Example 1;

[0085] Experimental group 1: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0086] Among them, the mass ratio of the basal culture medium Advanced DMEM / F12 and sterile water is: 99:1; the auxiliary factors include: 250ng / mL R-Spondin 1, 500ng / mL Wnt3a, 200nM CHIR99021, 200ng / mL Noggin, 100ng / μL FGF-7, 500ng / mL FGF-10, 500ng / mL EGF, 500nM A83-01, 5μM Y-27632, 10μM SB202190, 1XN2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basal culture medium and sterile water, and 2% serum replacement combination 1 is added.

[0087] Experimental group 2: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0088] Among them, the mass ratio of the basal culture medium Advanced DMEM / F12 and sterile water is: 99:1; the auxiliary factors include: 250ng / mL R-Spondin 1, 500ng / mL Wnt3a, 200nM CHIR99021, 200ng / mL Noggin, 100ng / μL FGF-7, 500ng / mL FGF-10, 500ng / mL EGF, 500nM A83-01, 5μM Y-27632, 10μM SB202190, 1XN2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basal culture medium and sterile water, and 2% serum replacement combination 2 is added.

[0089] Experimental group 3: basal medium Advanced DMEM / F12, cofactors, and sterile water;

[0090] Among them, the mass ratio of the basal culture medium Advanced DMEM / F12 and sterile water is: 99:1; the auxiliary factors include: 250ng / mL R-Spondin 1, 500ng / mL Wnt3a, 200nM CHIR99021, 200ng / mL Noggin, 100ng / μL FGF-7, 500ng / mL FGF-10, 500ng / mL EGF, 500nM A83-01, 5μM Y-27632, 10μM SB202190, 1XN2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basal culture medium and sterile water, and 2% serum substitute combination 3 is added.

[0091] Control group: basal medium Advanced DMEM / F12, auxiliary factors and sterile water;

[0092] Among them, the mass ratio of the basal culture medium Advanced DMEM / F12 and sterile water is: 99:1; the auxiliary factors include: 250ng / mL R-Spondin 1, 500ng / mL Wnt3a, 200nM CHIR99021, 200ng / mL Noggin, 100ng / μL FGF-7, 500ng / mL FGF-10, 500ng / mL EGF, 500nM A83-01, 5μM Y-27632, 10μM SB202190, 1X N2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin, 500U / mL streptomycin, DMEM / F12 culture medium; the concentration of each component of the above auxiliary factors is based on its concentration in the mixture of basal culture medium and sterile water, and 10% serum is added.

[0093] The culture medium of experimental groups 1 to 3 and the control group was used to culture human lung adenocarcinoma organoids, respectively. The specific experimental steps were the same as those in Example 1 and will not be repeated here.

[0094] Results Analysis

[0095] The morphological structure of lung adenocarcinoma organoids was observed under an ordinary optical microscope. Figure 1 As shown, it can be seen that when the experimental group 1 culture medium was used, the number of lung adenocarcinoma organoids was the largest and the diameter was larger ( Figure 3A), and the morphology of lung adenocarcinoma organoids cultured in the experimental group 1 medium is similar to that of lung adenocarcinoma organoids cultured in the fetal bovine serum medium, indicating that the alternative medium is better than fetal bovine serum for the culture of lung adenocarcinoma organoids, so the replacement of fetal bovine serum medium is feasible. The results of experimental groups 2 and 3 are the same as those of experimental group 1, and will not be repeated here.

[0096] In summary, the serum substitute component for tumor organoid culture of the present application is widely applicable and can be used to culture various types of tumor tissues derived from humans; compared with traditional animal-derived serum, the use of serum substitutes can reduce issues involving ethical and infectious disease risks. This serum substitute has stronger stability, controllability and reproducibility, which helps to improve the efficiency and success rate of tumor organoid culture.

[0097] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0098] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A serum replacement, characterized in that It is composed of cholesterol, L-ascorbic acid, arachidonic acid, myristic acid, (±)-α-lipoic acid, superoxide dismutase, sodium selenite, catalase, L-carnitine hydrochloride, putrescine dihydrochloride, L-reduced glutathione, D-(+)-galactose, transferrin, linolenic acid, retinyl acetate, (±)-α-tocopheryl acetate, corticosterone, DL-α-tocopherol, triiodothyronine sodium salt, insulin and bovine serum albumin; The serum substitute comprises 60mg / L-150mg / L cholesterol, 50mg / L-80 mg / L L-ascorbic acid, 30mg / L arachidonic acid, 80mg / L-200mg / L myristic acid, 8mg / L (±)-α-lipoic acid, 20mg / L-100mg / L superoxide dismutase, 4mg / L sodium selenite, 300mg / L catalase, 200mg / L-300mg / L L-carnitine hydrochloride, 1000mg / L-3000mg / L putrescine dihydrochloride, 150mg / L L-reduced glutathione, 2000mg / L-3000mg / L D-(+)-galactose, 2500mg / L transferrin, 40mg / L It is composed of linolenic acid, 25mg / L-30mg / L retinol acetate, 30mg / L-100mg / L (±)-α-tocopherol acetate, 3mg / L-4mg / L corticosterone, 60mg / L DL-α-tocopherol, 0.06mg / L-0.07mg / L triiodothyronine sodium salt, 300mg / L-1000mg / L insulin and 50g / L-80g / L bovine serum albumin.

2. The serum substitute according to claim 1, characterized in that The serum substitute comprises 60 mg / L cholesterol, 80 mg / L L-ascorbic acid, 30 mg / L arachidonic acid, 80 mg / L myristic acid, 8 mg / L (±)-α-lipoic acid, 20 mg / L superoxide dismutase, 4 mg / L sodium selenite, 300 mg / L catalase, 300 mg / L L-carnitine hydrochloride, 3000 mg / L putrescine dihydrochloride, 150 mg / L L-reduced glutathione, 2000 mg / L D-(+)-galactose, 2500 mg / L transferrin, 40 mg / L linolenic acid, 25 mg / L retinol acetate, 30 mg / L (±)-α-tocopherol acetate, 3 mg / L corticosterone, 60 mg / L DL-α-tocopherol, 0.06 mg / L triiodothyronine sodium salt, 300 mg / L It is composed of insulin and 80g / L bovine serum albumin.

3. The serum substitute according to claim 1, characterized in that The serum substitute comprises 150 mg / L cholesterol, 50 mg / L L-ascorbic acid, 30 mg / L arachidonic acid, 200 mg / L myristic acid, 8 mg / L (±)-α-lipoic acid, 100 mg / L superoxide dismutase, 4 mg / L sodium selenite, 300 mg / L catalase, 200 mg / L L-carnitine hydrochloride, 1000 mg / L putrescine dihydrochloride, 150 mg / L L-reduced glutathione, 3000 mg / L D-(+)-galactose, 2500 mg / L transferrin, 40 mg / L linolenic acid, 30 mg / L retinol acetate, 40 mg / L (±)-α-tocopherol acetate, 4 mg / L corticosterone, 60 mg / L DL-α-tocopherol, 0.06 mg / L triiodothyronine sodium salt, 1000 mg / L It is composed of insulin and 50g / L bovine serum albumin.

4. The serum substitute according to claim 1, characterized in that The serum substitute comprises 150 mg / L cholesterol, 80 mg / L L-ascorbic acid, 30 mg / L arachidonic acid, 200 mg / L myristic acid, 8 mg / L (±)-α-lipoic acid, 20 mg / L superoxide dismutase, 4 mg / L sodium selenite, 300 mg / L catalase, 300 mg / L L-carnitine hydrochloride, 1000 mg / L putrescine dihydrochloride, 150 mg / L L-reduced glutathione, 3000 mg / L D-(+)-galactose, 2500 mg / L transferrin, 40 mg / L linolenic acid, 25 mg / L retinol acetate, 100 mg / L (±)-α-tocopherol acetate, 4 mg / L corticosterone, 60 mg / L DL-α-tocopherol, 0.07 mg / L triiodothyronine sodium salt, 300 mg / L It is composed of insulin and 80g / L bovine serum albumin.

5. A culture medium, characterized in that include: The serum replacement according to any one of claims 1 to 4.

6. The culture medium according to claim 5, characterized in that The basal medium of the culture medium is selected from Advanced DMEM / F12.

7. The culture medium according to claim 5 or 6, characterized in that Further including: Other cofactors.

8. The culture medium according to claim 7, characterized in that The other cofactors include: IGF1, gremlin1, EGF, R-Spondin1, A8301, Y-27632, SB202190, N2, PGE2, HEPES, Glutamax, penicillin and streptomycin.

9. The culture medium according to claim 8, characterized in that The auxiliary factors include: 100ng / ml IGF1, 200ng / ml gremlin 1, 50ng / ml EGF, 1μg / ml R-Spondin1, 500nM A8301, 10μM Y-27632, 10μM SB202190, 1× N2, 0.5μM PGE2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin and 500U / mL streptomycin.

10. The culture medium according to claim 7, characterized in that The other cofactors include: BMP2, gremlin1, IGF2, LTBP2, EGF, HGF, Y27632, N2, HEPES, Glutamax, penicillin and streptomycin.

11. The culture medium according to claim 10, characterized in that The other cofactors included: 50ng / ml BMP2, 50ng / ml gremlin1, 50ng / ml IGF2, 100ng / ml LTBP2, 100ng / ml EGF, 50ng / ml HGF, 5μM Y27632, 1X N2, 10mM HEPES, 2mM Glutamax, 500U / mL penicillin and 500U / mL streptomycin.

12. Use of the culture medium according to any one of claims 5 to 11 in preparing a kit for culturing tumor organoids.

13. A method for constructing tumor organoids, characterized in that: include: The step of culturing tumor cells using the culture medium according to any one of claims 5 to 11.

14. The method according to claim 13, characterized in that The tumor cells include at least one of colorectal cancer and human head and neck squamous cell carcinoma cells.

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