WO2023274338A1

WO2023274338A1 - Culture medium, method, and reagent kit for rapidly culturing tumor organoids

Info

Publication number: WO2023274338A1
Application number: PCT/CN2022/102599
Authority: WO
Inventors: 富国祥; 李远闯; 林汉卿; 程春燕; 李俊强
Original assignee: 丹望医疗科技(上海)有限公司
Priority date: 2021-07-01
Filing date: 2022-06-30
Publication date: 2023-01-05
Also published as: CN113481162A; CN113481162B

Abstract

Provided are a culture medium, method, and reagent kit for rapidly culturing tumor organoids. The culture medium comprises a culture medium component and an additive component, wherein the culture medium component contains a sterile albumen extraction solution. The culture medium can shorten the time for culturing the tumor organoids. The reagent kit comprises the culture medium for culturing the tumor organoids, a tissue preservation solution, a tissue digestion solution, and a cell mass collection solution. Using the reagent kit to culture the tumor organoids can increase the success rate and survival rate of culturing the tumor organoids, and an organoid model can be rapidly established within 2-5 days, and can be applied to subsequent experimental tests.

Description

Media, methods and kits for rapidly growing tumor organoids

priority information

This application claims priority and rights to the patent application No. 202110744566.5 filed with the State Intellectual Property Office of China on July 01, 2021, and is hereby incorporated by reference in its entirety.

technical field

The present disclosure relates to the field of organoid culture, in particular, the present disclosure relates to a medium, a method and a kit for rapidly culturing tumor organoids.

Background technique

At present, the morbidity and mortality of cancer are increasing year by year. In 2020, there will be 4.57 million new cancer cases and 3 million cancer deaths in China. The huge economic and social burden caused by tumors is very heavy.

Surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy and endocrine therapy are the main means of tumor treatment. About 70% of the patients need radiotherapy and chemotherapy, but due to individual differences in patients, tumor drug resistance or radiation resistance is an important factor restricting the successful treatment of tumors. At present, the overall effective rate of clinical tumor drugs is low (about 30%), and unnecessary and ineffective treatments for tumors cause huge waste. Therefore, the personalized treatment of tumor is imminent.

Organoids are micro-organs with a three-dimensional structure grown in an in vitro environment. They have complex structures similar to real organs and can partially simulate the physiological functions of source tissues or organs. At present, multiple organs including colorectum, prostate, taste buds, esophagus, fallopian tubes, liver, pancreas, stomach, salivary glands and breasts have successfully obtained normal tissue or tumor organoids in vitro.

The traditional method of constructing tumor organoids needs to extract tumor cells from tumor tissue. The initial culture often starts with a single cell or a cell cluster smaller than 30 μm, and after 9-14 days of culture, an organoid model larger than 100 μm meets the experimental requirements. At the same time, the growth factor composition of the medium and its diffusion rate in Matrigel are also the key factors affecting the growth of organoids.

Although a variety of tumor tissues can be successfully cultured into organoids in vitro using different methods and under different culture conditions, it currently takes 1-3 months to construct a tumor organoid model in vitro and use this model for drug or radiation testing. The clinical demand window period is only 2-3 weeks. This requires accelerating the construction of organoids in vitro to meet clinical needs.

Therefore, it is of great scientific research and commercial value to develop a medium and a kit that can be cultured efficiently and quickly, and is universally applicable to the culture of various tumor organoids.

Contents of the invention

The present disclosure aims to solve one of the technical problems in the related art at least to a certain extent. Therefore, an object of the present disclosure is to provide a medium, a method and a kit for rapidly culturing tumor organoids. The medium for culturing tumor organoids provided by the present disclosure can greatly shorten the culturing time of tumor organoids. The kit for rapidly culturing tumor organoids provided by the present disclosure includes medium for culturing tumor organoids, tissue preservation solution, tissue digestion solution, and cell mass collection solution. Using the kit for culturing tumor organoids can improve the growth rate of tumor organoids. The success rate and survival rate of organ culture, and the organoid model can be quickly established within 2-5 days, which can be used for subsequent experimental tests.

To this end, the first aspect of the present disclosure provides a medium for culturing tumor organoids. According to an embodiment of the present disclosure, the medium includes a medium component and an additive component, wherein the medium component contains sterile egg white extract.

The existing traditional method to construct tumor organoids needs to extract tumor cells from tumor tissue. The initial culture often starts with a single cell or a cell cluster smaller than 30 μm, and after 9-14 days of culture, an organoid model larger than 100 μm meets the experimental requirements. At the same time, the growth factor composition of the medium and its diffusion rate in Matrigel are also the key factors affecting the growth of organoids. Although a variety of tumor tissues can be successfully cultured into organoids in vitro using different methods and under different culture conditions, it currently takes 1-3 months to construct a tumor organoid model in vitro and use this model for drug or radiation testing. The clinical demand window period is only 2-3 weeks. This requires accelerating the construction of organoids in vitro to meet clinical needs. The inventors found that adding sterile egg white extract to the medium for culturing tumor organoids can help promote the growth of organoids and increase the growth rate of organoids. Using the medium for culturing tumor organoids provided in the present disclosure, it is possible to culture tumor tissues from various sources such as the digestive system, the respiratory system, the urinary system, and the reproductive system.

According to an embodiment of the present disclosure, in the culture medium, the concentration of the sterile egg white extract is 10-30% (v/v).

According to an embodiment of the present disclosure, the concentration of the sterile egg white extract is 20-30% (v/v). As a result, the organoid growth rate can be further increased.

According to an embodiment of the present disclosure, the sterile egg white extract is obtained by:

(1) separating the egg whites of the eggs, and filtering the egg whites through a filter screen to obtain the collected liquid, wherein the filter screen pore size is 40-100 μm;

(2) The collected solution is subjected to negative pressure suction filtration, so as to obtain the sterile egg white extract.

According to an embodiment of the present disclosure, in step (1), the egg white is subjected to at least one filter screen filtration, wherein the filter screen pore size decreases successively during the at least one filter screen filter.

For example, a filter with a pore size of 0.22 μm can be used for the negative pressure suction filtration.

According to an embodiment of the present disclosure, the additive component further includes bFGF, FGF4, and FGF10.

The addition of bFGF, FGF4, and FGF10 promoted the differentiation of cell clusters to obtain organoids with the desired tissue morphology.

According to an embodiment of the present disclosure, the concentration of bFGF is 10-500 ng/ml, preferably 10-100 ng/ml.

According to an embodiment of the present disclosure, the concentration of FGF4 is 10-500 ng/ml, preferably 10-100 ng/ml.

According to an embodiment of the present disclosure, the concentration of FGF10 is 10-500 ng/ml, preferably 10-100 ng/ml.

According to an embodiment of the present disclosure, the additive component further includes HEPES, GlutaMAX, B27, N2, N-acetylcysteine, EGF, gastrin I, A83-01, Forskolin, Y-27632dihydrochloride, and optionally Wnt -3a, HGF, R-spondin-1, Noggin, Nicotinamide, Prostaglandin E2, SB202190, CHIR99021.

According to an embodiment of the present disclosure, the concentration of HEPES is 1-10 mM.

According to an embodiment of the present disclosure, the concentration of GlutaMAX is 1-5 mM.

According to an embodiment of the present disclosure, the concentration of B27 is 1%-2% (v/v).

According to an embodiment of the present disclosure, the concentration of N2 is 1%-2% (v/v).

According to an embodiment of the present disclosure, the concentration of N-acetylcysteine is 0.1-1 mM.

According to an embodiment of the present disclosure, the concentration of R-spondin-1 is 100-500 ng/ml.

According to an embodiment of the present disclosure, the concentration of Noggin is 10-500 ng/ml, preferably 10-100 ng/ml.

According to an embodiment of the present disclosure, the concentration of Wnt-3a is 10-500 ng/ml, preferably 10-100 ng/ml.

According to an embodiment of the present disclosure, the concentration of the EGF is 10-200 ng/ml.

According to an embodiment of the present disclosure, the concentration of nicotinamide is 1-10 mM.

According to an embodiment of the present disclosure, the gastrin I concentration is 1-20 nM, preferably 5-10 nM.

According to an embodiment of the present disclosure, the concentration of A83-01 is 100-1000 nM, preferably 200-500 nM.

According to an embodiment of the present disclosure, the concentration of the prostaglandin E2 is 1-100 nM, preferably 1-20 nM.

According to an embodiment of the present disclosure, the concentration of SB202190 is 1-20 μM, preferably 5-10 μM.

According to an embodiment of the present disclosure, the concentration of CHIR99021 is 1-10 μM.

According to an embodiment of the present disclosure, the concentration of the Forskolin is 1-50 μM, preferably 1-20 μM.

According to an embodiment of the present disclosure, the concentration of said HGF is 10-500 ng/ml, preferably 10-100 ng/ml.

According to an embodiment of the present disclosure, the concentration of Y-27632dihydrochloride is 1-20 μM, preferably 5-10 μM.

According to an embodiment of the present disclosure, the medium component further comprises medium Advanced DMEM/F12.

By using the medium for culturing tumor organoids of the present disclosure, each component in the medium can accelerate the growth rate of the organoids, which can save the construction speed of the organoid models. Shorten patient in vitro drug or radiation testing time.

According to an embodiment of the present disclosure, the additive component further includes a mixed solution of penicillin, streptomycin and amphotericin B.

According to an embodiment of the present disclosure, the concentration of penicillin in the mixture of penicillin, streptomycin and amphotericin B is 100-200 U/mL; the concentration of streptomycin is 100-200 μg/ml; the amphotericin The concentration of vitamin B was 250–500 ng/mL.

According to an embodiment of the present disclosure, the additive component accounts for 3%-5% of the total volume of the medium.

According to an embodiment of the present disclosure, the tumor originates from the digestive system, respiratory system, urinary system and reproductive system.

The tumors are common tumors such as colon cancer, liver cancer, lung cancer, kidney cancer, and breast cancer.

The second aspect of the present disclosure provides the use of the medium described in the first aspect in culturing tumor organoids. According to an embodiment of the present disclosure, the tumor organoid is selected from tumor organoids related to the digestive system, respiratory system, urinary system or reproductive system.

A third aspect of the present disclosure provides a method for culturing tumor organoids. According to an embodiment of the present disclosure, the method comprises:

1) Digesting the tumor sample to obtain tumor cell clusters;

2) cultivating the tumor cell mass using the medium described in the first aspect, so as to obtain tumor organoids.

According to an embodiment of the present disclosure, the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.

According to an embodiment of the present disclosure, the digestion treatment includes digesting the tumor sample with a tissue digestion fluid, so as to obtain a cell suspension containing tumor cell clusters,

Wherein, the tissue digestion fluid includes hyaluronidase and Disspace type II.

According to an embodiment of the present disclosure, the tissue digestion fluid further includes collagenase IV.

According to an embodiment of the present disclosure, the concentration of the hyaluronidase is 10-100 μg/mL.

According to an embodiment of the present disclosure, the concentration of the Disspace type II is 100-2000 μg/mL, preferably 50-200 μg/mL.

According to an embodiment of the present disclosure, the concentration of the collagenase IV is 100-1000 U/mL.

According to an embodiment of the present disclosure, the tissue digestion solution further includes the culture medium Advanced DMEM/F12, Y-27632dihydrochloride and optionally a mixture of penicillin, streptomycin and amphotericin B.

According to an embodiment of the present disclosure, the concentration of Y-27632dihydrochloride is 1-20 μM.

According to an embodiment of the present disclosure, the method further includes storing the tumor sample in a tissue preservation solution before digesting the tumor sample, wherein the tissue preservation solution includes B27 and Y-27632dihydrochloride.

According to an embodiment of the present disclosure, the tissue preservation solution further includes HEPES, GlutaMAX, medium Advanced DMEM/F12, and optionally a mixed solution of penicillin, streptomycin and amphotericin B.

According to an embodiment of the present disclosure, the method further includes in step 1), after obtaining the cell suspension containing the tumor cell mass, filtering the cell suspension using a collection filter with a pore size of 40-100 μm, And using the cell mass collection liquid to backwash the collection filter, so as to obtain the tumor cell mass.

The disclosure uses a cell strainer to filter and use a cell mass collection solution to obtain larger cell mass, maintain cell activity, and accelerate the construction of tumor organoids.

According to an embodiment of the present disclosure, the cell mass collection fluid includes BSA.

According to an embodiment of the present disclosure, the cell mass collection solution further includes Y-27632dihydrochloride, phosphate buffer, and optionally a mixed solution of penicillin, streptomycin and amphotericin B.

According to an embodiment of the present disclosure, the concentration of the BSA is 0.1%-1% (v/v).

The fourth aspect of the present disclosure provides a tissue digestion solution. According to an embodiment of the present disclosure, the tissue digestion fluid includes hyaluronidase and Disspace type II.

The tissue digestion liquid of the present disclosure contains a variety of digestive enzymes, which can reduce the time for tissue digestion, improve cell activity, and increase the yield of cell clusters.

According to an embodiment of the present disclosure, the penicillin concentration in the penicillin, streptomycin and amphotericin B mixed solution is 100-200 U/mL; the streptomycin concentration is 100-200 μg/ml; the amphotericin The concentration of mycin B was 250–500 ng/mL.

The fifth aspect of the present disclosure provides the use of the tissue digestion solution described in the fourth aspect in digesting tumor samples. According to an embodiment of the present disclosure, the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.

The sixth aspect of the present disclosure provides a tissue preservation solution. According to an embodiment of the present disclosure, the tissue preservation solution includes B27 and Y-27632 dihydrochloride.

The tissue preservation solution provided by the present disclosure contains basic nutrients of tumor tissue, which can maintain the activity of the tissue during in vitro preservation; at the same time, the preservation solution contains Y-27632 dihydrochloride, which can inhibit apoptosis and increase the survival rate of tumor tissue.

The seventh aspect of the present disclosure provides the use of the tissue preservation solution described in the sixth aspect in preserving tumor samples. According to an embodiment of the present disclosure, the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.

The eighth aspect of the present disclosure provides a cell mass collection solution. According to an embodiment of the present disclosure, the cell mass collection fluid includes BSA.

The ninth aspect of the present disclosure provides the use of the cell mass collection liquid described in the eighth aspect in collecting tumor cell mass obtained by digesting a tumor sample with the tissue digestion liquid described in the fourth aspect. According to an embodiment of the present disclosure, the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.

The tenth aspect of the present disclosure provides a kit for culturing tumor organoids. According to an embodiment of the present disclosure, the kit includes the medium described in the first aspect, and the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.

According to an embodiment of the present disclosure, the kit further includes at least one of a digestion solution, a preservation solution, and a collection solution.

According to an embodiment of the present disclosure, the digestive fluid is the tissue digestive fluid described in the fourth aspect.

According to an embodiment of the present disclosure, the preservation solution is the tissue preservation solution described in the sixth aspect.

According to an embodiment of the present disclosure, the collection liquid is the cell mass collection liquid described in the eighth aspect.

The eleventh aspect of the present disclosure provides a kit for culturing tumor organoids. According to an embodiment of the present disclosure, the kit includes the tissue digestion fluid described in the fourth aspect, and the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.

According to an embodiment of the present disclosure, the kit further includes at least one of a culture medium, a preservation solution, and a collection solution.

According to an embodiment of the present disclosure, the medium is the medium described in the first aspect.

The twelfth aspect of the present disclosure provides a kit for culturing tumor organoids. According to an embodiment of the present disclosure, the kit includes the tissue preservation solution according to the sixth aspect, and the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.

According to an embodiment of the present disclosure, the kit further includes at least one of culture medium, digestion solution, and collection solution.

The thirteenth aspect of the present disclosure provides a kit for culturing tumor organoids. According to an embodiment of the present disclosure, the kit includes the cell mass collection liquid according to the eighth aspect, and the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.

According to an embodiment of the present disclosure, the kit further includes at least one of culture medium, digestion solution, and preservation solution.

The kit provided in the present disclosure greatly shortens the time required for culturing tumor organoids, improves the success rate and survival rate of tumor organoid culturing, and can rapidly establish tumor organoid models. The kit for rapidly culturing tumor organoids provided by the present disclosure includes medium for culturing tumor organoids, tissue preservation solution, tissue digestion solution, and cell mass collection solution. Using the kit for culturing tumor organoids can improve the growth rate of tumor organoids. The success rate and survival rate of organ culture, and the organoid model can be quickly established within 2-5 days, which can be used for subsequent experimental tests.

Additional aspects and advantages of the disclosure will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosure.

Description of drawings

The above and/or additional aspects and advantages of the present disclosure will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, in which:

Figure 1 shows the morphological structure diagram of intestinal cancer organoids in Example 1 of the present disclosure;

Figure 2 shows the morphological structure diagram of intestinal cancer organoids in Example 2 of the present disclosure;

Fig. 3 shows the histological structure diagram of liver cancer organoids in Example 3 of the present disclosure;

Fig. 4 shows the histological structure diagram of lung cancer organoids in Example 4 of the present disclosure;

Fig. 5 shows the histological structure diagram of kidney cancer organoids in Example 5 of the present disclosure;

Fig. 6 shows the histological structure diagram of breast cancer organoids in Example 6 of the present disclosure;

Figure 7 shows the immunohistochemical diagram of intestinal cancer organoid cdx2 in Example 9 of the present disclosure;

Figure 8 shows the immunohistochemical profile of intestinal cancer organoid ck20 in Example 9 of the present disclosure.

Detailed description of the invention

Embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary only for explaining the present disclosure and should not be construed as limiting the present disclosure.

In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

According to some specific embodiments of the present disclosure, the method for rapid establishment and rapid culture of tumor organoids provided by the present disclosure includes the following steps:

1) Store fresh tumor surgical resection specimens or biopsy tissues in pre-cooled tissue preservation solution at 4°C, and send them to the laboratory for pretreatment within 24 hours;

2) Cut the tumor tissue into ^5-10mm3 size with a scalpel, put it into the tissue digestion solution and digest it with shaking at 37°C for 20-60 minutes;

3) Pass the cell suspension after digestion in step 2) through a 100 μm cell strainer and a 40 μm cell strainer in sequence, and use the cell mass collection solution to backwash the 40 μm cell strainer to obtain a cell mass with a diameter of 40-100 μm, and remove it by centrifugation Supernatant, cell pellets for later use;

4) Mix the rapid proliferation medium and Matrigel at a ratio of 1:1-1:2, and then resuspend the cell mass pellet obtained in step 3) to obtain a gel mixed with cells, and inoculate the gel;

5) After the inoculation gel in step 4) is fully solidified, add a rapid proliferation medium, and culture at 37° C. and 5% CO ₂ concentration;

6) The rapid proliferation medium was replaced every 2-3 days, and cultured for 2-5 days to obtain tumor organoids.

According to a specific embodiment of the present disclosure, the rapid proliferation medium includes sterile egg white extract 10-30% (v/v), preferably 20-30% (v/v); medium Advanced DMEM/F12; bFGF, 10-500ng/ml, preferably 10-100ng/ml; FGF4, 10-500ng/ml, preferably 10-100ng/ml; FGF10, 10-500ng/ml, preferably 10-100ng/ml; HEPES, 1-10mM; GlutaMAX , 1-5mM; B27, 1%-2% (v/v); N2, 1%-2% (v/v); N-acetylcysteine, 0.1-1mM; EGF, 10-200ng/ml ; gastrin I, 1-20nM, preferably 5-10nM; A83-01, 100-1000nM, preferably 200-500nM; Forskolin, 1-50μM, preferably 1-20μM; Y-27632dihydrochloride, 1-20μM, preferably 5-10μM; A mixture of penicillin, streptomycin and amphotericin B at a penicillin concentration of 100-200 U/mL, a streptomycin concentration of 100-200 μg/ml, an amphotericin B concentration of 250–500 ng/mL, and optionally Wnt-3a, 10-500ng/ml, preferably 10-100ng/ml; optional HGF, 10-500ng/ml, preferably 10-100ng/ml; optional R-spondin-1, 100-500ng/ml; optional Noggin , 10-500ng/ml, preferably 10-100ng/ml; optional nicotinamide, 1-10mM; optional prostaglandin E2, 1-100nM, preferably 1-20nM; optional SB202190, 1-20nM, preferably 5-10 nM; optionally CHIR99021, 1-10 μM.

According to a specific embodiment of the present disclosure, the tissue preservation solution includes B27, 1%-2% (v/v); Y-27632dihydrochloride, 1-20μM; HEPES, 1-10mM; GlutaMAX, 1-5mM; Advanced DMEM/ F12: Penicillin, streptomycin and amphotericin B mixture, penicillin concentration 100-200U/mL, streptomycin concentration 100-200μg/ml, amphotericin B concentration 250-500ng/mL.

According to a specific embodiment of the present disclosure, the tissue digestion fluid includes hyaluronidase, 10-100 μg/mL; Disspace type II, 100-2000 μg/mL, preferably 50-200 μg/mL; collagenase IV, 100-1000 U/mL mL; culture medium Advanced DMEM/F12; Y-27632dihydrochloride, 1-20μM; penicillin, streptomycin and amphotericin B mixture, penicillin concentration 100-200U/mL, streptomycin concentration 100-200μg/ml, amphotericin The concentration of vitamin B was 250–500 ng/mL.

According to a specific embodiment of the present disclosure, the cell mass collection solution includes BSA, 0.1%-1% (v/v); Y-27632dihydrochloride, 1-20 μM; phosphate buffer saline; penicillin, streptomycin and amphotericin B mixed solution, penicillin concentration 100-200U/mL, streptomycin concentration 100-200μg/ml, amphotericin B concentration 250-500ng/mL.

"Optional" and "optionally" in the present disclosure mean that subsequent components may or may not be added.

The factors "bFGF" and "FGF basic" can be substituted equivalently in this disclosure.

The specific composition and dosage of rapid proliferation medium, tissue preservation solution, tissue digestion solution, and cell mass collection solution can be appropriately adjusted according to the type of tumor organoids to be cultured.

According to a specific embodiment of the present disclosure, the sterile egg white extract is obtained in the following manner:

(2) performing negative pressure suction filtration on the collected liquid, so as to obtain the sterile egg white extract,

Wherein, in step (1), egg white is subjected to at least one strainer filtration, and the strainer aperture decreases successively in at least one strainer filtration; When carrying out described negative pressure suction filtration, the aperture of the filter used is 0.2- 0.3 μm.

Take 1-3 days old fresh eggs, separate egg whites and pass through 100 μm, 70 μm, 40 μm cell strainers respectively; the collected liquid is sucked through a 0.22 μm filter by negative pressure to obtain a sterile egg white extract.

The eggs used in the preparation of the sterile egg white extract in the present disclosure are preferably fresh eggs, that is, eggs that are 1-3 days old.

According to an embodiment of the present disclosure, the filter screen used for separating egg whites is preferably a cell filter with a pore size of 40-100 μm, and the egg whites are preferably filtered multiple times in order of decreasing pore size of the filter screens, so as to remove microorganisms in the egg whites.

Embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary only for explaining the present disclosure and should not be construed as limiting the present disclosure. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Reagent source:

The HEPES used in the embodiments of the present disclosure was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.

The GlutaMAX used in the embodiments of the present disclosure was purchased from Thermofisher Company of the United States.

The B27 used in the embodiments of the present disclosure was purchased from Thermofisher Company of the United States.

The Y-27632dihydrochloride used in the examples of the present disclosure was purchased from SellecK, USA.

Collagenase IV (collagenase IV) used in the examples of the present disclosure was purchased from Thermofisher Company of the United States.

The Hyaluronidase (hyaluronidase) used in the examples of the present disclosure was purchased from Thermofisher Company of the United States.

The Disspace type II used in the embodiments of the present disclosure was purchased from American thermofisher company.

The N2 used in the embodiments of the present disclosure was purchased from Thermofisher Company of the United States.

N-acetylcysteine (N-acetylcysteine) used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

R-spondin-1 used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

Noggin used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

The Wnt-3a used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

The EGF used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

Nicotinamide (nicotinamide) used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

The gastrin I used in the embodiments of the present disclosure was purchased from MedChemExpress, USA.

The A83-01 used in the embodiments of the present disclosure was purchased from MedChemExpress, USA.

Prostaglandin E2 (prostaglandin E2) used in the examples of the present disclosure was purchased from MedChemExpress, USA.

The SB202190 used in the embodiments of the present disclosure was purchased from MedChemExpress, USA.

The CHIR99021 used in the embodiments of the present disclosure was purchased from MedChemExpress, USA.

The bFGF (FGF basic) used in the embodiments of the present disclosure was purchased from Sigma-Aldrich Company.

Forskolin used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

The HGF used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

FGF4 used in the examples of the present disclosure was purchased from Sigma-Aldrich.

The FGF10 used in the examples of the present disclosure was purchased from Sigma-Aldrich Company.

The cdx2 antibody used in the examples of the present disclosure was purchased from Novus.

The ck20 used in the embodiments of the present disclosure was purchased from Abcam.

Example 1 Colon cancer organoid culture

1. Reagent preparation

1) Rapid proliferation medium for tumor organoids: composed of medium components Advanced DMEM/F12, 20% sterile egg white extract and specific additive factors; the composition of specific additive factors: HEPES, 10mM; GlutaMAX, 2mM; 1X B27, 1:100(v/v); 1X N2, 1:100(v/v); N-acetylcysteine, 1mM; R-spondin-1, 500ng/ml; Noggin, 100ng/ml; EGF, 50ng/ml ml; Nicotinamide, 10mM; gastrin I, 10nM; A83-01, 500nM; ProstaglandinE2, 10nM; SB202190, 5μM; FGF basic, 50ng/ml; FGF4, 50ng/ml; Amphotericin B mixture (100 U/mL penicillin, 100 μg/ml streptomycin, 250 ng/mL amphotericin B), 1X; Y-27632dihydrochloride, 10 μM. Factors were added at 4% of the total volume of the medium.

2) Sterile egg white extract

The preparation method of the aseptic egg white extract is as follows: take 1-3 days old fresh eggs, separate the egg whites and pass through 100 μm, 70 μm, and 40 μm cell strainers respectively; the collected solution is suctioned through a 0.22 μm filter to obtain a sterile egg white extract.

3) Tissue preservation solution

The composition of tissue preservation solution: Advanced DMEM/F12, 10mM HEPES; 2mM GlutaMAX; 1X B27 (1:100 (v/v)); 10μM Y-27632dihydrochloride; 1X penicillin, streptomycin and amphotericin B mixed solution (100U /mL penicillin, 100 μg/ml streptomycin, 250 ng/mL amphotericin B).

4) Tissue digestive juice

Tumor tissue digestion solution: including basal medium Advanced DMEM/F12, specific additive factors and digestive enzymes; among them, specific additive factors include: 10μM Y-27632dihydrochloride; penicillin, streptomycin and amphotericin B mixed solution (100U/ mL penicillin, 100 μg/mL streptomycin, 250 ng/mL amphotericin B), 1X; digestive enzymes include: 500 U/mL collagenase IV; 20 μg/mL hyaluronidase; 100 μg/mL Disspace type II.

5) Cell mass collection solution

Cell mass collection solution: phosphate buffer saline, 1% BSA and specific addition factors. Among them, specific addition factors: 10 μM Y-27632dihydrochloride; penicillin, streptomycin and amphotericin B mixture (100 U/mL penicillin, 100 μg/ml streptomycin, 250 ng/mL amphotericin B).

2. Culture method of intestinal cancer organoids

This embodiment provides a method for culturing intestinal cancer organoids, comprising:

1) Store fresh colorectal cancer resection specimens in pre-cooled tissue preservation solution at 4°C, and send them to the laboratory for pretreatment within 24 hours;

2) Sample cleaning: transfer the tissue into a 50ml centrifuge tube, then wash with 20ml of phosphate buffered saline solution containing 1% penicillin, streptomycin and amphotericin B mixture for 5 minutes to remove impurities on the tissue surface;

3) Mince the sample: In the biological safety cabinet, transfer the sample to a 10cm petri dish, and use a scalpel on ice to cut the tissue to a size of ^5-10mm3 ;

4) Transfer the chopped tissue to a 15ml centrifuge tube, add 10ml of tissue digestion solution and shake and digest at 37°C for 30 minutes;

5) After the digestion, add 5ml of phosphate buffer to stop the digestion, and then filter through a 100μm cell strainer into a 50ml centrifuge tube;

6) The filtered cell solution was centrifuged at 600 rpm at 4°C for 5 minutes, and the supernatant was removed to obtain a cell pellet;

7) Add 5 ml of phosphate buffer to the cell pellet to resuspend the cell pellet, and filter the cell pellet suspension through a 40 μm cell strainer;

8) Put the 40μm cell strainer upside down on the mouth of a 50ml centrifuge tube, wash the cell strainer from above with 20ml of cell mass collection solution, and collect the cell mass;

9) Centrifuge the 50ml centrifuge tube of the cell mass suspension collected in step 8) at 600rpm at 4°C for 5 minutes, remove the supernatant to obtain the cell mass pellet for later use;

10) Take an appropriate amount of rapid proliferation medium of tumor organoids and mix it with Matrigel 1:2, then resuspend the cell pellet obtained in step 9) with the mixture, and then use a pipette to drop the gel mixed with cells to 96 In a well plate, 10 μl per drop;

11) Put the petri dish inoculated with the gel drops into the _CO2 incubator and let it stand for 10 minutes to make the gel solidify;

12) Add the rapid proliferation medium of the tumor organoids into the culture dish, and then place it in a constant temperature incubator to culture at 37°C and 5% _CO2 concentration;

13) The culture medium was changed every 2-3 days, and intestinal cancer organoids were obtained after culturing for 2-5 days. The tissue morphology and structure were observed under a common optical microscope as spherical cell clusters, as shown in FIG. 1 .

Example 2 Culture of intestinal cancer organoids

1. Reagent preparation

The composition of sterile egg white extract, tissue preservation solution, tissue digestion solution, and cell mass collection solution is the same as in Example 1.

2. Culture method of intestinal cancer organoids

1) Store fresh colonoscopic biopsy specimens of colon cancer in pre-cooled tissue preservation solution at 4°C, and send them to the laboratory for pretreatment within 24 hours;

13) The culture medium was changed every 2-3 days, and intestinal cancer organoids were obtained after culturing for 2-5 days. The tissue morphology and structure were observed under an ordinary optical microscope as spherical cell clusters, as shown in FIG. 2 .

Example 3 Culture of Liver Cancer Organoids

1. Reagent preparation

1) Rapid proliferation medium for tumor organoids: composed of medium components Advanced DMEM/F12, 30% sterile egg white extract and specific additive factors; the composition of specific additive factors: HEPES, 10mM; GlutaMAX, 2mM; 1X B27, 1:100(v/v); 1X N2, 1:100(v/v); N-acetylcysteine, 1mM; R-spondin-1, 500ng/ml; EGF, 50ng/ml; gastrin I, 10nM ; A83-01, 500nM; Forskolin, 10μM; FGF basic, 50ng/ml; FGF4, 50ng/ml; FGF10, 100ng/ml; penicillin, streptomycin and amphotericin B mixed solution (100U/mL ml streptomycin, 250 ng/mL amphotericin B), 1X; Y-27632dihydrochloride, 10 μΜ. Factors were added at 4% of the total volume of the medium.

2. Culture method of liver cancer organoids

This embodiment provides a method for culturing liver cancer organoids, comprising:

1) Store fresh liver cancer resection specimens in pre-cooled tissue preservation solution at 4°C, and send them to the laboratory for pretreatment within 24 hours;

13) The culture medium was changed every 2-3 days, and the liver cancer organoids were obtained by culturing for 2-5 days. The tissue morphology was observed under an ordinary optical microscope as a spherical cell cluster structure, as shown in FIG. 3 .

Example 4 Culture of lung cancer organoids

1. Reagent preparation

1) Rapid proliferation medium for tumor organoids: composed of medium components Advanced DMEM/F12, 20% sterile egg white extract and specific additive factors; the composition of specific additive factors: HEPES, 10mM; GlutaMAX, 2mM; 1X B27, 1:100(v/v); 1X N2, 1:100(v/v); N-acetylcysteine, 1mM; EGF, 50ng/ml; gastrin I, 10nM; A83-01, 500nM; CHIR990215μM; SB202190 , 5μM; FGF basic, 100ng/ml; FGF4, 50ng/ml; FGF10, 50ng/ml; penicillin, streptomycin and amphotericin B mixture (100U/mL penicillin, 100μg/ml streptomycin, 250ng/mL Amphotericin B), 1X; Y-27632dihydrochloride, 10 μΜ. Factors were added at 4% of the total volume of the medium.

2. Culture method of lung cancer organoids

This embodiment provides a method for culturing lung cancer organoids, comprising:

1) Store fresh lung cancer surgical resection specimens in pre-cooled tissue preservation solution at 4°C, and send them to the laboratory for pretreatment within 24 hours;

10) Take an appropriate amount of rapid proliferation medium of tumor organoids and mix it with Matrigel 1:2, then resuspend the cell pellet obtained in step 9) with the mixture, and then use a pipette to drop the gel mixed with cells to 96 In a well plate, 10 μl per drop.

13) The medium was replaced every 2-3 days, and the lung cancer organoids were obtained after culturing for 2-5 days. The tissue morphology was observed under a common light microscope, showing a spherical cell cluster structure, as shown in FIG. 4 .

Example 5 Culture of Renal Cancer Organoids

1. Reagent preparation

1) Rapid proliferation medium for tumor organoids: composed of medium components Advanced DMEM/F12, 30% sterile egg white extract and specific additive factors; the composition of specific additive factors: HEPES, 10mM; GlutaMAX, 2mM; B27, 1X; N2, 1X; N-acetylcysteine, 1mM; EGF, 50ng/ml; gastrin I, 10nM; A83-01, 500nM; FGF basic, 50ng/ml; FGF4, 50ng/ml; FGF10, 50ng/ml; Mixture of penicillin, streptomycin and amphotericin B (100 U/mL penicillin, 100 μg/ml streptomycin, 250 ng/mL amphotericin B), 1X; Y-27632dihydrochloride, 10 μM. Factors were added at 4% of the total volume of the medium.

2. Culture method of kidney cancer organoids

This embodiment provides a method for culturing kidney cancer organoids, comprising:

1) Store fresh kidney cancer surgical resection specimens in pre-cooled tissue preservation solution at 4°C, and send them to the laboratory for pretreatment within 24 hours;

9) Centrifuge the 50ml centrifuge tube of the cell mass suspension collected in step 8) at 600 rpm at 4°C for 5 minutes, remove the supernatant to obtain a cell mass pellet for later use.

13) The culture medium was changed every 2-3 days, and kidney cancer organoids were obtained by culturing for 2-5 days. The morphology and structure of the tissue were observed under an ordinary optical microscope as spherical cell clusters, as shown in FIG. 5 .

Example 6 Culture of Breast Cancer Organoids

1. Reagent preparation

1) Rapid proliferation medium for tumor organoids: composed of medium components Advanced DMEM/F12, 30% sterile egg white extract and specific additive factors; the composition of specific additive factors: HEPES, 10mM; GlutaMAX, 2mM; 1X B27, 1:100(v/v); 1X N2, 1:100(v/v); N-acetylcysteine, 1mM; R-spondin-1, 500ng/ml; Noggin, 10ng/ml; EGF, 50ng/ml ml; Nicotinamide, 10mM; gastrin I, 10nM; A83-01, 500nM; ProstaglandinE2, 10nM; +, 10μM; FGF basic, 10ng/ml; FGF4, 50ng/ml; Amphotericin B mixture (100 U/mL penicillin, 100 μg/ml streptomycin, 250 ng/mL amphotericin B), 1X; Y-27632dihydrochloride, 10 μM. Factors were added at 4% of the total volume of the medium.

2. Culture method of breast cancer organoids

This embodiment provides a method for culturing breast cancer organoids, comprising:

1) Store fresh breast cancer surgical resection specimens in pre-cooled tissue preservation solution at 4°C, and send them to the laboratory for pretreatment within 24 hours;

13) The medium was changed every 2-3 days, and the breast cancer organoids were obtained by culturing for 2-5 days. The tissue morphology and structure were observed under an ordinary optical microscope as spherical cell clusters, as shown in FIG. 6 .

Example 7 Detection of cell activity after intestinal cancer tissue digestion

The method for detecting cell viability after intestinal cancer tissue digestion is as follows:

1) Store the fresh surgical resection specimen of intestinal cancer in the tissue preservation solution of Example 1 pre-cooled at 4°C, and send it to the laboratory for pretreatment within 24 hours;

4) Transfer the chopped tissue to a 15ml centrifuge tube, add 10ml of the tissue digestion solution of Example 1, and shake and digest at 37°C for 30 minutes;

6) The filtered cell solution was centrifuged at 600 rpm at 4°C for 5 minutes, and the supernatant was removed to obtain a cell pellet.

7) The cell mass was resuspended in 1 ml of phosphate buffer, 20 μL of the cell mass suspension was added to 20 μL equal volume containing 5 μM calcein and 5 μM propidium iodide, and the mixture was detected by a fluorescence cytometer.

Example 8 Detection of cell mass size after intestinal cancer tissue digestion:

The method for detecting the size of cell clusters after intestinal cancer tissue digestion is as follows:

8) Put the 40 μm cell strainer upside down on the mouth of a 50 ml centrifuge tube, use 20 ml of the cell mass collection solution of Example 1 to wash the cell strainer from above, and collect the cell mass;

9) Centrifuge the 50ml centrifuge tube of the cell mass suspension collected in step 8) at 600 rpm at 4°C for 5 minutes, remove the supernatant to obtain the cell mass pellet;

10) Resuspend the cell mass obtained in step 9) with 1 ml of phosphate buffer to obtain a cell suspension.

11) Take three drops of 10 μL of cell suspension on a glass slide and take pictures under an optical microscope, use Image J software for image analysis, and record the size and number of individual cells and cell clusters.

Example 9 Identification of bowel cancer organoids

The intestinal cancer organoids obtained in Example 2 were prepared for paraffin-embedded sectioning. The embedded organoids were sliced and observed by immunohistochemical staining.

1) Fixation: Put it into the pre-prepared fixative solution (4% formaldehyde fixation) and fix it for 0.5 hours. Absorb 4% formaldehyde fixative solution in the culture well, add 500ul 70% alcohol to soak for 10 minutes;

2) Dehydration: Absorb the 70% alcohol solution, use ophthalmic forceps to pick out the matrigel containing intestinal cancer organoids, and put them into the embedding and fixing box. Put the embedding and fixing box into the automatic dehydrator in sequence: 70% alcohol twice for 30 minutes, 95% alcohol twice for 30 minutes, 100% alcohol twice for 30 minutes, xylene twice for 30 minutes, 60°C paraffin solution for 2 hours Second-rate;

3) Embedding: Encapsulate the organoids with embedding molds, then cut into 3-5 μm slices with a microtome and stick them on the detachment-proof glass slides;

4) Slicing: put the slide on the slicing machine, 42°C, 8 hours, 56°C, 1 hour;

5) Dewaxing: slides were dewaxed by xylene twice for 5 minutes, 100% alcohol twice, 95% alcohol twice, and 70% alcohol twice;

6) Antigen retrieval: After the slides are dewaxed, rinse them in clean water for 2 minutes, add 3% H ₂ O ₂ to soak them for 10 minutes, wash them twice in clean water for 2 minutes, add citric acid buffer, and steam them in a steamer for 20 minutes. minutes, cooled to room temperature;

7) Blocking: Pour off the citric acid buffer, place the slide in PBS for 5 minutes and wash twice, dry the PBS around the tissue, and add donkey serum;

8) Add primary antibody: absorb donkey serum, add primary antibody (cdx2, 1:100; ck20, 1:100) and incubate overnight in 4°C refrigerator;

9) Add secondary antibody: Take the slides out of the refrigerator, put them into PBS and wash 3 times, 5 minutes each time, dry the PBS around the tissue, add secondary antibody and incubate at room temperature for 30 minutes;

10) Color development: Put the glass slide into PBS and wash 3 times, wipe off the PBS around the tissue for 5 minutes each time, and then add the color developer. Wash in PBS for 5 minutes after color development;

11) Counterstaining: soak slides in hematoxylin for 5 minutes;

12) Dehydration: Rinse the counterstained slides in water, put the slides in 70% alcohol twice for 2 minutes, 95% alcohol twice for 2 minutes, 100% alcohol twice for 2 minutes, and xylene for 2 minutes. 2 times per minute;

13) Seal the slide: drop neutral gum next to the tissue, cover it with a cover glass, and place it on a 37°C oven for 2 hours to dry;

14) Photographing: The slides were observed and photographed under a microscope, as shown in Figure 7 and Figure 8, the tissue markers cdx2 and ck20 were positive, indicating that intestinal cancer organoids were successfully obtained using the culture method of the present disclosure.

Comparative Example 1 Exploration on the Composition of Tissue Preservation Solution

B27 was removed from the tissue preservation solution provided in this comparative example, and other compositions were the same as in Example 1.

According to the method of Example 7, the viability of intestinal cancer cells after digestion was detected, and the cell viability in the intestinal cancer tissues preserved in the composition preservation solution of Example 1 and Comparative Example 1 was compared. The results in Table 1 show that the viable cell rate of the tissue preservation solution in Example 1 is significantly better than that of Comparative Example 1, which shows that adding B27 to the tissue preservation solution helps to improve cell viability.

Table 1

对比项Comparison item	细胞总数total number of cells	活细胞数量number of living cells	活细胞比例live cell ratio
实施例1Example 1	5.2×10 ⁵个 5.2×10 ⁵ pieces	4.5×10 ⁵个 4.5×10 ⁵ pieces	86.54％86.54%
对比例1Comparative example 1	5.1×10 ⁵个 5.1×10 ⁵ pieces	3.7×10 ⁵个 3.7×10 ⁵ pieces	72.55％72.55%

Comparative Example 2 Exploration on the Composition of Tissue Preservation Solution

In the tissue preservation solution provided in this comparative example, Y-27632dihydrochloride was removed, and other compositions were the same as in Example 1.

According to the method of Example 7, the viability of intestinal cancer cells after digestion was detected, and the cell viability in the intestinal cancer tissues preserved in the composition preservation solution of Example 1 and Comparative Example 2 was compared. The results in Table 2 show that the viable cell rate of Example 1 is significantly better than that of Comparative Example 2, which shows that adding Y-27632dihydrochloride to the tissue preservation solution helps to improve cell viability.

Table 2

对比项Comparison item	细胞总数total number of cells	活细胞数量number of living cells	活细胞比例live cell ratio
实施例1Example 1	5.2×10 ⁵个 5.2×10 ⁵ pieces	4.5×10 ⁵个 4.5×10 ⁵ pieces	86.54％86.54%
对比例2Comparative example 2	4.9×10 ⁵个 4.9×10 ⁵ pieces	2.8×10 ⁵个 2.8×10 ⁵ pieces	57.14％57.14%

Comparative Example 3 Exploration on Composition of Digestive Juice of Tissues

Hyaluronidase is removed from the tissue digestion solution provided in this comparative example, and other compositions are the same as in Example 1.

According to the method in Example 8, the size of the cell cluster after digestion of the intestinal cancer tissue was detected. Compare the size of cell clusters preserved in the tissue preservation solution of Example 1 and Comparative Example 3. The results in Table 3 show that the number of cell clusters in Example 1 is significantly better than that in Comparative Example 3, which shows that adding hyaluronidase to the tissue digestion solution helps to increase the yield of cell clusters.

table 3

对比项Comparison item	细胞团数量number of cell clusters	平均大小average size
实施例1Example 1	84个84	65μm65μm
对比例3Comparative example 3	27个27	52μm52μm

Comparative Example 4 Exploration on Composition of Digestive Juice of Tissues

Disspace type II was removed from the tissue digestion solution provided in this comparative example, and the other compositions were the same as in Example 1.

According to the method in Example 8, the size of the cell cluster after digestion of the intestinal cancer tissue was detected. Compare the size of the cell clusters preserved in the tissue preservation solution of Example 1 and Comparative Example 4. The results in Table 4 show that the number of cell clusters in Example 1 is significantly better than that in Comparative Example 4, which shows that adding Disspace type II to the tissue preservation solution helps to increase the yield of cell clusters.

Table 4

对比项Comparison item	细胞团数量number of cell clusters	平均大小average size
实施例1Example 1	84个84	65μm65μm
对比例4Comparative example 4	19个19	48μm48μm

Comparative Example 5 Exploration of the composition of the cell mass collection fluid

BSA was removed from the cell mass collection solution provided in this comparative example, and the other compositions were the same as in Example 1.

According to the method in Example 8, the size of the cell cluster after digestion of the intestinal cancer tissue was detected. The cell mass sizes of Example 1 and Comparative Example 5 were compared. The results in Table 5 show that the number of cell clusters in Example 1 is significantly better than that in Comparative Example 5, which shows that adding BSA to the cell cluster collection solution helps to increase the yield of cell clusters.

table 5

对比项Comparison item	细胞团数量number of cell clusters	平均大小average size
实施例1Example 1	75个75	72μm72μm
对比例5Comparative example 5	59个59	55μm55μm

Comparative Example 6 Exploration on the Composition of the Medium for Rapid Proliferation of Tumor Organoids

The tumor organoid rapid proliferation medium provided in this comparative example removed the sterile egg white extract, bFGF, FGF4, and FGF10, and the other compositions were the same as the tumor organoid rapid proliferation medium in Example 1.

Comparative Example 7 Exploration on the Composition of the Medium for Rapid Proliferation of Tumor Organoids

bFGF, FGF4, and FGF10 were removed from the tumor organoid rapid proliferation medium provided in this comparative example, and the other compositions were the same as the tumor organoid rapid proliferation medium in Example 1.

Comparative Example 8 Exploration on the Composition of Rapid Proliferation Medium of Tumor Organoids

The sterile egg white extract was removed from the tumor organoid rapid proliferation medium provided in this comparative example, and the other compositions were the same as the tumor organoid rapid proliferation medium in Example 1.

Intestinal cancer organoids were cultured according to the method of Example 1 using the tumor organoid rapid proliferation medium of Example 1 and Comparative Examples 6-8. After 3 days of culture, 3 duplicate wells were photographed under an optical microscope, and the number and size of organoids were measured with Image J software.

The results show that comparative example 6 is a traditional colon cancer medium formula, on this basis, a sterile egg white extract is added to obtain the medium in comparative example 7, and the organoid size of comparative example 7 is significantly better than that of comparative example 6, which shows that in the medium Adding sterilized egg white extract can help to increase the growth rate of organoids.

The morphology of organoids in Comparative Example 7 is dedifferentiated and grows rapidly, which is different from that of Comparative Example 6 and tumor tissue in vivo. On this basis, the medium in Example 1 is obtained by adding differentiation factors bFGF (FGF basic), FGF4, and FGF10. The cultured organoids were similar to Comparative Example 6 and in vivo tissue morphology.

In Comparative Example 8, the addition of bFGF, FGF4, and FGF10 to the traditional intestinal cancer medium showed that the growth of intestinal cancer organoids was inhibited.

Table 6

对比项Comparison item	类器官平均数量Average number of organoids	平均大小average size
实施例1Example 1	66个66	102μm102μm
对比例6Comparative example 6	57个57	84μm84μm
对比例7Comparative example 7	68个68	105μm105μm
对比例8Comparative example 8	45个45	77μm77μm

In summary, the tumor organoid rapid culture kit disclosed in the present disclosure is widely applicable, and can culture tumor tissues from various sources such as the digestive system, the respiratory system, the urinary system, and the reproductive system. Each component in the kit can increase the yield of tumor cell clusters and accelerate the growth rate of organoids. Can save organoid model construction speed. Shorten patient in vitro drug or radiation testing time.

In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the described specific features, structures, materials or characteristics may be combined in any suitable manner in any one or more embodiments or examples. In addition, those skilled in the art can combine and combine different embodiments or examples and features of different embodiments or examples described in this specification without conflicting with each other.

Although the embodiments of the present disclosure have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations on the present disclosure, and those skilled in the art can understand the above-mentioned embodiments within the scope of the present disclosure. The embodiments are subject to changes, modifications, substitutions and variations.

Claims

A medium for culturing tumor organoids, comprising medium components and additive components, wherein the medium components contain sterile egg white extract.
The medium according to claim 1, wherein the concentration of the sterile egg white extract is 10-30% (v/v);

Optionally, the concentration of the sterile egg white extract is 20-30% (v/v).
The culture medium according to claim 1 or 2, wherein the sterile egg white extract is obtained in the following manner:

(1) separating the egg whites of the eggs, and filtering the egg whites through a filter screen to obtain the collected liquid, wherein the filter screen pore size is 40-100 μm;

(2) performing negative pressure suction filtration on the collected liquid, so as to obtain the sterile egg white extract;

Optionally, in step (1), the egg white is subjected to at least one sieve filtration, wherein in the at least one sieve filtration, the pore size of the filter decreases successively.
The medium according to any one of claims 1-3, wherein the additive components further comprise bFGF, FGF4 and FGF10;

Optionally, the concentration of bFGF is 10-500ng/ml, preferably 10-100ng/ml;

Optionally, the concentration of FGF4 is 10-500ng/ml, preferably 10-100ng/ml;

Optionally, the concentration of FGF10 is 10-500 ng/ml, preferably 10-100 ng/ml.
The medium according to any one of claims 1-4, wherein the additive component further comprises HEPES, GlutaMAX, B27, N2, N-acetylcysteine, EGF, gastrin I, A83-01, Forskolin, Y-27632 dihydrochloride, and optionally Wnt-3a, HGF, R-spondin-1, Noggin, Nicotinamide, Prostaglandin E2, SB202190, CHIR99021;

Optionally, the concentration of HEPES is 1-10mM;

Optionally, the concentration of GlutaMAX is 1-5mM;

Optionally, the concentration of B27 is 1%-2% (v/v);

Optionally, the concentration of N2 is 1%-2% (v/v);

Optionally, the concentration of N-acetylcysteine is 0.1-1 mM;

Optionally, the concentration of R-spondin-1 is 100-500ng/ml;

Optionally, the concentration of Noggin is 10-500ng/ml, preferably 10-100ng/ml;

Optionally, the concentration of Wnt-3a is 10-500ng/ml, preferably 10-100ng/ml;

Optionally, the concentration of the EGF is 10-200ng/ml;

Optionally, the concentration of the nicotinamide is 1-10mM;

Optionally, the gastrin I concentration is 1-20nM, preferably 5-10nM;

Optionally, the concentration of the A83-01 is 100-1000nM, preferably 200-500nM;

Optionally, the concentration of the prostaglandin E2 is 1-100nM, preferably 1-20nM;

Optionally, the concentration of SB202190 is 1-20 μM, preferably 5-10 μM;

Optionally, the concentration of CHIR99021 is 1-10 μM;

Optionally, the concentration of the Forskolin is 1-50 μM, preferably 1-20 μM;

Optionally, the concentration of HGF is 10-500ng/ml, preferably 10-100ng/ml;

Optionally, the concentration of Y-27632 dihydrochloride is 1-20 μM, preferably 5-10 μM.
The medium according to any one of claims 1-5, wherein said medium component further comprises medium Advanced DMEM/F12;

Optionally, the additive component further includes a mixed solution of penicillin, streptomycin and amphotericin B;

Optionally, the penicillin concentration in the penicillin, streptomycin and amphotericin B mixed solution is 100-200U/mL; the streptomycin concentration is 100-200 μg/ml; the amphotericin B The concentration is 250–500 ng/mL.
The medium according to any one of claims 1-6, wherein the additive component accounts for 3%-5% of the total volume of the medium;

Optionally, the tumor originates from the digestive system, respiratory system, urinary system and reproductive system.
The use of the medium according to any one of claims 1-7 in culturing tumor organoids, wherein the tumor organoids are selected from tumor organoids related to the digestive system, respiratory system, urinary system or reproductive system.
A method of cultivating tumor organoids, the method comprising:

1) Digesting the tumor sample to obtain tumor cell clusters;

2) cultivating the tumor cell mass using the medium according to any one of claims 1-7, so as to obtain tumor organoids;

Optionally, the tumor is selected from tumors associated with the digestive system, respiratory system, urinary system or reproductive system.
The method according to claim 9, wherein the digestion process comprises digesting the tumor sample with a tissue digestion fluid, so as to obtain a cell suspension containing tumor cell clusters,

Wherein, the tissue digestion fluid includes hyaluronidase and Disspace type II;

Optionally, the tissue digestion solution further includes collagenase IV;

Optionally, the concentration of the hyaluronidase is 10-100 μg/mL;

Optionally, the concentration of the Disspace type II is 100-2000 μg/mL, preferably 50-200 μg/mL;

Optionally, the concentration of the collagenase IV is 100-1000 U/mL.
The method according to claim 10, wherein the tissue digestion solution further comprises medium Advanced DMEM/F12, Y-27632 dihydrochloride and optional penicillin, streptomycin and amphotericin B mixed solution;

Optionally, the concentration of Y-27632 dihydrochloride is 1-20 μM;

Optionally, the penicillin concentration in the penicillin, streptomycin and amphotericin B mixed solution is 100-200U/mL; the streptomycin concentration is 100-200 μg/ml; the amphotericin B The concentration is 250–500 ng/mL.
The method according to any one of claims 9-11, wherein the method further comprises storing the tumor sample in a tissue preservation solution before digesting the tumor sample, wherein the tissue The preservation solution includes B27 and Y-27632dihydrochloride;

Optionally, the tissue preservation solution further includes HEPES, GlutaMAX, medium Advanced DMEM/F12 and optional penicillin, streptomycin and amphotericin B mixed solution;

Optionally, the concentration of B27 is 1%-2% (v/v);

Optionally, the concentration of Y-27632 dihydrochloride is 1-20 μM;

Optionally, the concentration of HEPES is 1-10mM;

Optionally, the concentration of GlutaMAX is 1-5mM;

Optionally, the penicillin concentration in the penicillin, streptomycin and amphotericin B mixed solution is 100-200U/mL; the streptomycin concentration is 100-200 μg/ml; the amphotericin B The concentration is 250–500 ng/mL.
The method according to any one of claims 9-12, wherein the method further comprises in step 1), after obtaining the cell suspension containing the tumor cell mass, using the cell suspension with a pore size of filter through a 40-100 μm collection filter, and backwash the collection filter with the cell mass collection liquid, so as to obtain the tumor cell mass;

Optionally, the cell mass collection solution includes BSA;

Optionally, the cell mass collection solution further includes Y-27632 dihydrochloride, phosphate buffer and optional penicillin, streptomycin and amphotericin B mixed solution;

Optionally, the concentration of the BSA is 0.1%-1% (v/v);

Optionally, the concentration of Y-27632 dihydrochloride is 1-20 μM;

Optionally, the penicillin concentration in the penicillin, streptomycin and amphotericin B mixed solution is 100-200U/mL; the streptomycin concentration is 100-200 μg/ml; the amphotericin B The concentration is 250–500 ng/mL.
A tissue digestion solution including hyaluronidase and Disspace type II.
The tissue digestion solution according to claim 14, wherein the tissue digestion solution further comprises collagenase IV;

Optionally, the concentration of the hyaluronidase is 10-100 μg/mL;

Optionally, the concentration of the Disspace type II is 100-2000 μg/mL, preferably 50-200 μg/mL;

Optionally, the concentration of the collagenase IV is 100-1000 U/mL.
The tissue digestion solution according to claim 14 or 15, wherein the tissue digestion solution further comprises the culture medium Advanced DMEM/F12, Y-27632 dihydrochloride, and optional penicillin, streptomycin and amphotericin B mixed solution;

Optionally, the concentration of Y-27632 dihydrochloride is 1-20 μM;

Optionally, the penicillin concentration in the penicillin, streptomycin and amphotericin B mixed solution is 100-200U/mL; the streptomycin concentration is 100-200 μg/ml; the amphotericin B The concentration is 250–500 ng/mL.
The use of the tissue digestion solution according to any one of claims 14-16 in digesting tumor samples, wherein the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.
A tissue preservation solution comprising B27 and Y-27632 dihydrochloride.
The tissue preservation solution according to claim 18, wherein the tissue preservation solution further comprises HEPES, GlutaMAX, culture medium Advanced DMEM/F12 and optional penicillin, streptomycin and amphotericin B mixed solution;

Optionally, the concentration of B27 is 1%-2% (v/v);

Optionally, the concentration of Y-27632 dihydrochloride is 1-20 μM;

Optionally, the concentration of HEPES is 1-10mM;

Optionally, the concentration of GlutaMAX is 1-5mM;

Optionally, the penicillin concentration in the penicillin, streptomycin and amphotericin B mixed solution is 100-200U/mL; the streptomycin concentration is 100-200 μg/ml; the amphotericin B The concentration is 250–500 ng/mL.
The use of the tissue preservation solution according to claim 18 or 19 in preserving tumor samples, wherein the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system.
A cell mass collection liquid, wherein the cell mass collection liquid includes BSA;

Optionally, the cell mass collection solution further includes Y-27632 dihydrochloride, phosphate buffer and optional penicillin, streptomycin and amphotericin B mixed solution;

Optionally, the concentration of the BSA is 0.1%-1% (v/v);

Optionally, the concentration of Y-27632 dihydrochloride is 1-20 μM;

Optionally, the penicillin concentration in the penicillin, streptomycin and amphotericin B mixed solution is 100-200U/mL; the streptomycin concentration is 100-200 μg/ml; the amphotericin B The concentration is 250–500 ng/mL.
The use of the cell mass collection liquid according to claim 21 in collecting tumor cell mass obtained by digesting tumor samples with the tissue digestion liquid according to any one of claims 14-16, wherein the tumor is selected from the group consisting of , respiratory system, urinary system or reproductive system related tumors.
A kit for culturing tumor organoids, wherein the kit includes the medium according to any one of claims 1-7, and the tumor is selected from the group of tumors related to the digestive system, respiratory system, urinary system or reproductive system tumors;

Optionally, the kit further includes at least one of digestion solution, preservation solution and collection solution.
The kit according to claim 23, wherein the digestive fluid is the tissue digestive fluid according to any one of claims 14-16;

Optionally, the preservation solution is the tissue preservation solution according to claim 18 or 19;

Optionally, the collection liquid is the cell mass collection liquid according to claim 21.
A kit for culturing tumor organoids, wherein the kit includes the tissue digestion solution according to any one of claims 14-16, and the tumor is selected from the group consisting of the digestive system, the respiratory system, the urinary system or the reproductive system associated tumors;

Optionally, the kit further includes at least one of culture medium, preservation solution and collection solution.
The kit according to claim 25, wherein the medium is the medium according to any one of claims 1-7,

Optionally, the preservation solution is the tissue preservation solution according to claim 18 or 19;

Optionally, the collection liquid is the cell mass collection liquid according to claim 21.
A kit for culturing tumor organoids, wherein the kit includes the tissue preservation solution according to claim 18 or 19, and the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system;

Optionally, the kit further includes at least one of culture medium, digestion solution and collection solution.
The kit according to claim 27, wherein the medium is the medium according to any one of claims 1-7,

Optionally, the digestive fluid is the tissue digestive fluid according to any one of claims 14-16;

Optionally, the collection liquid is the cell mass collection liquid according to claim 21.
A kit for culturing tumor organoids, wherein the kit includes the cell mass collection fluid according to claim 21, and the tumor is selected from tumors related to the digestive system, respiratory system, urinary system or reproductive system;

Optionally, the kit further includes at least one of culture medium, digestion solution and preservation solution.
The kit according to claim 29, wherein the medium is the medium according to any one of claims 1-7,

Optionally, the digestive fluid is the tissue digestive fluid according to any one of claims 14-16;

Optionally, the preservation solution is the tissue preservation solution according to claim 18 or 19.