CN112592896A

CN112592896A - Culture solution and culture method for lung adenocarcinoma organoid

Info

Publication number: CN112592896A
Application number: CN202011316097.9A
Authority: CN
Inventors: 黄卫人; 陈巍; 李志超
Original assignee: Shenzhen Second Peoples Hospital
Current assignee: Shenzhen Second Peoples Hospital
Priority date: 2020-11-22
Filing date: 2020-11-22
Publication date: 2021-04-02

Abstract

The invention provides a culture solution of organoid and its culture method, the said culture solution includes basal medium and specific additive factor, the said culture solution can improve the culture success rate and passable times of organoid, especially culture success rate and passable times of lung pancreatic cancer organoid, have solved the problem such as being low in culture success rate and passable times of organoid in the prior art. The culture method has high success rate, and the subculture frequency of the obtained organoid is high.

Description

Culture solution and culture method for lung adenocarcinoma organoid

Technical Field

The invention relates to the technical field of biological medicines, in particular to a culture solution and application thereof, and more particularly relates to a culture solution for lung adenocarcinoma organoids and a culture method thereof.

Background

Organoids are three-dimensional structures formed by the self-organization of various types of cells derived from stem cells, and can mimic the structure and function of the organ in which the cells reside. Compared with the traditional 2D cell culture and animal models, organoids have incomparable advantages, and the cellular composition and the function of organoids are very similar to those of in-vivo tissues. The great advantage of many organoids that can be cultured in vitro for a long period of time while maintaining their genomic stability makes it possible to more accurately reflect the effects of drugs. Organoids can reduce the complexity of the experiment compared to animal models, for real-time imaging techniques, which enable one to use such models to study mechanistic problems of human development and disease that are not easily or accurately studied in animal models.

Tumor organoids, also known as "cancer surrogate", "tumor-like", and the like, are primarily cultured in vitro in 3D using tumor tissue from a patient to mimic the biological characteristics of tumor tissue in vivo. While the pre-cancerous lesions (such as intraepithelial neoplasia) derived organoids are mainly used for simulating the occurrence and development of tumors and analyzing tumor-related omics changes. Tumor organoids highly recapitulate the characteristics of the source tumor tissue, preserve heterogeneity between individuals, and thus have application value in transformed medicine, and can be used for functional tests such as high-throughput drug screening, and even personalized treatment protocol formulation.

Lung adenocarcinoma is one of the lung cancers and belongs to the non-small cell carcinomas. Unlike squamous cell lung cancer, lung adenocarcinoma is more likely to occur in women and those who are not smoking. Originating from the bronchial mucosal epithelium, and a few originating from the mucous glands of the large bronchi. The onset age is small, and women are relatively common. Most adenocarcinomas originate in the smaller bronchi, peripheral lung cancer.

Currently, tumor organoids are obtained mainly by enzymatic digestion of tissues using digestive enzymes to obtain organoid-forming cell aggregates. However, the digestion of tissues by the enzymatic hydrolysis method destroys intercellular junctions, and the digestion time is long, so that the cell activity is reduced, the success rate of sample treatment is low, the number of obtained organoids is small, and the requirements of clinical application and scientific research are difficult to meet.

Because of the differences between different classes of cancer organoids, a culture medium or method that achieves a high success rate in the culture of one class of cancer organoids does not necessarily mean that a high success rate in the culture of another class of cancer organoids is achieved.

Patent CN110452877A discloses a culture medium for primary cells of lung cancer solid tumor and a culture method thereof, but the primary cells of lung cancer solid tumor cultured by the culture medium disclosed in the patent have the problems of low culture success rate, low passable times and the like.

Patent CN111411083B discloses a culture medium and a culture method for gastric cancer organoids, but the inventors found that the success rate of lung adenocarcinoma organoids cultured with the culture medium is low and the number of passable times is small.

Patent CN109837242A discloses a breast cancer organoid culture solution and a culture method thereof, but the inventor researches show that the success rate of lung adenocarcinoma organoids obtained by using the culture medium disclosed by the patent is low and the passable times are few.

Patent CN108396010A discloses an in vitro culture method of colorectal cancer organoid, but the inventor finds that the success rate of lung adenocarcinoma organoid obtained by culture with the culture medium is low and the passable times are few.

Patent CN111876386A discloses a culture medium for breast cancer organoids and a culture method thereof, but the inventors have found that the success rate of lung adenocarcinoma organoids obtained by culture with the culture medium is low and the passable times are small.

Therefore, there is still a need to develop a culture medium with high culture success rate for lung adenocarcinoma organoid and a culture method thereof.

Disclosure of Invention

Summary of The Invention

The first purpose of the invention is to provide a culture solution for organoid, which comprises a basic culture medium and a specific additive factor, and the culture solution can improve the culture success rate and passable times of organoid, especially the culture success rate and passable times of lung pancreatic cancer organoid, and solve the problems of low culture success rate and less passable times of organoid in the prior art.

Furthermore, by screening appropriate specific addition factors and proportion thereof, the invention obtains the organoid culture solution capable of improving the culture success rate of the lung pancreatic cancer organoid and the number of passable times, and solves the problems of low culture success rate of the lung pancreatic cancer organoid, less passable times and the like in the prior art.

It is a second object of the present invention to provide a use of the organoid culture broth for organoid culture or passaging.

The third purpose of the invention is to provide a culture method of organoid, the culture method has high success rate, and the subculture frequency of the organoid obtained by culture is high.

Detailed Description

In a first aspect, the present invention provides an organoid culture solution.

A culture solution of organoid, the culture solution comprising a basal medium and a specific additive factor; the specific addition factor comprises: a first antibiotic solution, a biological buffer, GlutaMAX, B27 serum-free additive, fibroblast growth factor, human noggin, R-Spondin1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- β type I receptor inhibitor, and optionally a ROCK inhibitor.

In some embodiments, the specific additive factor may consist of: a first antibiotic solution, a biological buffer, GlutaMAX, B27 serum-free additive, fibroblast growth factor, human noggin, R-Spondin1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- β type I receptor inhibitor, and optionally a ROCK inhibitor.

The basal medium may be Advanced DMEM/F-12.

The first antibiotic solution may comprise at least one of penicillin, streptomycin, or amphotericin B. In some embodiments, the first antibiotic solution comprises or is penicillin, streptomycin, and amphotericin B.

The biological buffer may include HEPES. In some embodiments, the biological buffer is HEPES.

The fibroblast growth factor may include or be at least one of fibroblast growth factor 7 or fibroblast growth factor 10. In some embodiments, the fibroblast growth factor is at least one of fibroblast growth factor 7 or fibroblast growth factor 10. In some embodiments, the fibroblast growth factors are fibroblast growth factor 7 and fibroblast growth factor 10.

The ROS inhibitor may include or be N-acetylcysteine.

The p38 MAPK inhibitor may include or be SB202190 or a salt or hydrate thereof. In some embodiments, the p38 MAPK inhibitor comprises or is SB202190 monohydrochloride hydrate.

The TGF- β type I receptor inhibitor may include or be A83-01.

The ROCK inhibitor may comprise or be Y-27632 or a salt thereof.

The final concentration of penicillin may be 5000-. In some embodiments, the final concentration of penicillin is 7000-12000 units/mL, based on the total volume of the first antibiotic solution. In some embodiments, the final concentration of penicillin is 10000 units/mL based on the total volume of the first antibiotic solution.

The final concentration of streptomycin may be 5000-15000 units/mL based on the total volume of the first antibiotic solution. In some embodiments, the final concentration of streptomycin is 7000-12000 units/mL, based on the total volume of the first antibiotic solution. In some embodiments, the final concentration of streptomycin is 10000 units/mL based on the total volume of the first antibiotic solution.

The final concentration of amphotericin B may be 10-35 μ g/mL based on the total volume of the first antibiotic solution. In some embodiments, the final concentration of amphotericin B is 15-30 μ g/mL based on the total volume of the first antibiotic solution. In some embodiments, the final concentration of amphotericin B is 25 μ g/mL based on the total volume of the first antibiotic solution.

The volume fraction of the first antibiotic solution may be 0.5% to 1.5% based on the total volume of the culture solution. In some embodiments, the volume fraction of the first antibiotic solution is 0.7% to 1.3% based on the total volume of the culture solution. In some embodiments, the volume fraction of the first antibiotic solution is 1.0% based on the total volume of the culture fluid.

The volume fraction of the biological buffer may be 0.5% to 1.5% based on the total volume of the culture fluid. In some embodiments, the volume fraction of the biological buffer is 0.7% to 1.3% based on the total volume of the culture fluid. In some embodiments, the volume fraction of the biological buffer is 1.0% based on the total volume of the culture fluid.

The final concentration of GlutaMAX may be 15-25nmol/L based on the total volume of the culture medium. In some embodiments, the final concentration of GlutaMAX is 17-22nmol/L based on the total volume of the culture. In some embodiments, the final concentration of GlutaMAX is 20nmol/L based on the total volume of the culture.

The volume fraction of the B27 serum-free additive can be 0.5-1.5% based on the total volume of the culture solution. In some embodiments, the B27 serum-free additive has a volume fraction of 0.7% to 1.3% based on the total volume of the culture fluid. In some embodiments, the B27 serum-free additive is present in a volume fraction of 1.0% based on the total volume of the culture fluid.

The final concentration of the fibroblast growth factor may be 20-80ng/mL based on the total volume of the culture solution. In some embodiments, the final fibroblast growth factor concentration is 30-60ng/mL based on the total volume of the culture broth. In some embodiments, the final fibroblast growth factor concentration is 40ng/mL based on the total volume of the culture broth.

The final concentration of fibroblast growth factor 7 may be 10-40ng/mL based on the total volume of the culture broth. In some embodiments, the final concentration of fibroblast growth factor 7 is 15-30ng/mL based on the total volume of the culture broth. In some embodiments, the final concentration of fibroblast growth factor 7 is 20ng/mL based on the total volume of the culture broth.

The final concentration of fibroblast growth factor 10 may be 10-40ng/mL based on the total volume of the culture medium. In some embodiments, the final concentration of fibroblast growth factor 10 is 15-30ng/mL based on the total volume of the culture medium. In some embodiments, the final concentration of fibroblast growth factor 10 is 20ng/mL based on the total volume of the culture medium.

The content of the human noggin may be 50-150ng/mL based on the total volume of the culture solution. In some embodiments, the human noggin content is 70-130ng/mL based on the total volume of the culture medium. In some embodiments, the human noggin content is 100ng/mL based on the total volume of the culture medium.

The final concentration of the ROS inhibitor may be 1.00-1.50mM based on the total volume of the culture. In some embodiments, the final concentration of the ROS inhibitor is 1.25mM, based on the total volume of the culture.

The final concentration of nicotinamide can be 8-12mM based on the total volume of the culture. In some embodiments, the final concentration of nicotinamide is 10mM based on the total volume of the culture broth.

The final concentration of the p38 MAPK inhibitor may be 5-15. mu.M based on the total volume of the culture. In some embodiments, the final concentration of the p38 MAPK inhibitor is 7-12 μ M based on the total volume of the culture. In some embodiments, the final concentration of the p38 MAPK inhibitor is 10 μ M based on the total volume of the culture.

The final concentration of R-spondin1 may be 250-750ng/mL based on the total volume of the culture solution. In some embodiments, the final concentration of R-spondin1 is 400-600ng/mL based on the total volume of the culture. In some embodiments, the final concentration of R-spondin1 is 500ng/mL based on the total volume of the culture.

The final concentration of the TGF-. beta.type I receptor inhibitor may be 250-750nM, based on the total volume of the culture. In some embodiments, the final concentration of the TGF- β type I receptor inhibitor is 400-600nM, based on the total volume of the culture. In some embodiments, the TGF- β type I receptor inhibitor is provided at a final concentration of 500nM, based on the total volume of the culture.

The final concentration of the ROCK inhibitor may be 5-15 μ M based on the total volume of the culture broth. In some embodiments, the ROCK inhibitor is present at a final concentration of 7 to 12 μ M based on the total volume of the culture medium. In some embodiments, the final concentration of the ROCK inhibitor is 10 μ Μ, based on the total volume of the culture broth.

The volume fraction of the basal medium can be 95.0-98.0% based on the total volume of the culture solution.

According to some embodiments of the invention, a culture solution of organoid, the culture solution consisting of a basal medium and specific addition factors; the specific additive factor consists of the following components: a first antibiotic solution, a biological buffer, GlutaMAX, B27 serum-free additive, fibroblast growth factor, human noggin, R-Spondin1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- β type I receptor inhibitor, and optionally a ROCK inhibitor.

According to some embodiments of the invention, a culture solution of organoid, the culture solution consisting of a basal medium and specific addition factors; the specific additive factor consists of: a first antibiotic solution, a biological buffer, GlutaMAX, B27 serum-free additive, fibroblast growth factor, human noggin, R-Spondin1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF-beta type I receptor inhibitor, and optionally a ROCK inhibitor; the volume fraction of the first antibiotic solution is 0.5-1.5% based on the total volume of the culture solution; the volume fraction of the biological buffer solution is 0.5% -1.5%; the final concentration of the GlutaMAX is 15-25 nmol/L; the volume fraction of the B27 serum-free additive is 0.5-1.5%; the final concentration of the fibroblast growth factor is 20-80 ng/mL; the content of the human noggin is 50-150 ng/mL; the final concentration of the ROS inhibitor is 1.00-1.50 mM; the final concentration of nicotinamide is 8-12 mM; the final concentration of the p38 MAPK inhibitor is 5-15 mu M; the final concentration of the R-spondin1 is 250-750 ng/mL; the final concentration of the TGF-beta type I receptor inhibitor is 250-750 nM; and the final concentration of the ROCK inhibitor is 5-15 μ M; the balance is the basic culture medium.

According to some embodiments of the invention, a culture solution of organoid, the culture solution consisting of a basal medium and specific addition factors; the specific additive factor consists of: a first antibiotic solution, a biological buffer, GlutaMAX, B27 serum-free additive, fibroblast growth factor, human noggin, R-Spondin1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF-beta type I receptor inhibitor, and optionally a ROCK inhibitor; the volume fraction of the first antibiotic solution is 0.7-1.3% based on the total volume of the culture solution; the volume fraction of the biological buffer solution is 0.7% -1.3%; the final concentration of the GlutaMAX is 17-22 nmol/L; the volume fraction of the B27 serum-free additive is 0.7-1.3%; the final concentration of the fibroblast growth factor is 30-60 ng/mL; the content of the human noggin is 70-130 ng/mL; the final concentration of the ROS inhibitor is 1.25 mM; the final concentration of nicotinamide is 10 mM; the final concentration of the p38 MAPK inhibitor is 7-12 mu M; the final concentration of the R-spondin1 is 400-600 ng/mL; the final concentration of the TGF-beta type I receptor inhibitor is 400-600 nM; the final concentration of the ROCK inhibitor is 7-12 mu M; the balance is the basic culture medium.

According to some embodiments of the invention, a culture solution of organoid, the culture solution consisting of a basal medium and specific addition factors; the specific additive factor consists of: a first antibiotic solution, a biological buffer, GlutaMAX, B27 serum-free additive, fibroblast growth factor, human noggin, R-Spondin1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF-beta type I receptor inhibitor, and optionally a ROCK inhibitor; the volume fraction of the first antibiotic solution is 1.0 percent based on the total volume of the culture solution; the volume fraction of the biological buffer solution is 1.0%; the final concentration of the GlutaMAX is 20 nmol/L; the volume fraction of the B27 serum-free additive is 1.0%; the final concentration of the fibroblast growth factor is 40 ng/mL; the content of the human noggin is 100 ng/mL; the final concentration of the ROS inhibitor is 1.25 mM; the final concentration of nicotinamide is 10 mM; the final concentration of the p38 MAPK inhibitor is 10 μ M; the final concentration of the R-spondin1 is 500 ng/mL; the final concentration of the TGF-beta type I receptor inhibitor is 500 nM; the final concentration of the ROCK inhibitor is 10 μ M; the balance is the basic culture medium.

In a second aspect, the present invention provides a use of the culture solution according to any one of the first aspect.

Use of a culture solution according to any one of the first aspect in organoid culture or passaging. In some embodiments, the organoid comprises a tumor organoid. In some embodiments, the organoid is a lung pancreatic cancer organoid.

In a third aspect, the present invention provides a method for culturing organoids.

A method for culturing organoids, comprising the steps of:

A) taking cancer tissue blocks, cutting into pieces, adding a first digestive juice for digestion, centrifuging to obtain a first precipitate, washing the first precipitate, and centrifuging to obtain a second precipitate;

B) digesting the second precipitate with a second digestive juice, terminating digestion, centrifuging to obtain a third precipitate, washing the third precipitate, and centrifuging to obtain a fourth precipitate;

C) resuspending the fourth pellet with media and filtering to remove large pieces of tissue; counting the cells, and centrifuging to obtain a fifth precipitate; resuspending the fifth pellet with the culture medium of any one of claims 1-4 and matrigel, inoculating;

D) after the matrigel inoculated in the step C) is solidified, adding the culture solution of any one of claims 1 to 4 for culture to obtain the organoid.

The culture solution in the step D) is replaced every 2 to 3 days; and culturing the medium containing the ROCK inhibitor for 1 week, and culturing the medium containing no ROCK inhibitor for 1-2 weeks.

The first digestive fluid comprises a second antibiotic solution, GlutaMAX, HEPES, Advanced DMEM/F-12, collagenase type II, DNAse I, and a ROCK inhibitor.

The ROCK inhibitor comprises or is Y-27632 or a salt thereof.

The second antibiotic solution comprises at least one of penicillin, streptomycin, or amphotericin B. In some embodiments, the second antibiotic solution comprises penicillin, streptomycin, and amphotericin B.

The final concentration of collagenase type II is 2.5-10mg/mL based on the total volume of the first digest. In some embodiments, the final concentration of collagenase type II is 4-7mg/mL, based on the total volume of the first digest solution. In some embodiments, the final concentration of collagenase type II is 5mg/mL based on the total volume of the first digest solution.

The final concentration of DNase I is 50-150 μ g/mL based on the total volume of the first digest. In some embodiments, the final concentration of DNase I is 70-120 μ g/mL based on the total volume of the first digest. In some embodiments, the final concentration of dnase I is 100 μ g/mL based on the total volume of the first digest.

The final concentration of the ROCK inhibitor in the first digest is 5-15 μ M based on the total volume of the first digest. In some embodiments, the final concentration of the ROCK inhibitor in the first digest is 7-12 μ Μ, based on the total volume of the first digest. In some embodiments, the final concentration of the ROCK inhibitor in the first digest is 10 μ Μ, based on the total volume of the first digest.

The final concentration of penicillin is 50-150 units/mL based on the total volume of the first digestive juice. In some embodiments, the final concentration of penicillin is 70-120 units/mL based on the total volume of the first digest. In some embodiments, the final concentration of penicillin is 100 units/mL based on the total volume of the first digest solution.

The final concentration of streptomycin is 50-150 units/mL based on the total volume of the first digestive juice. In some embodiments, the final concentration of streptomycin is 70-120 units/mL based on the total volume of the first digest solution. In some embodiments, the final concentration of streptomycin is 100 units/mL based on the total volume of the first digest.

The final concentration of amphotericin B is 10-35 μ g/m based on the total volume of the first digestive juice. In some embodiments, the final concentration of amphotericin B is 15-30 μ g/m based on the total volume of the first digest. In some embodiments, the final concentration of amphotericin B is 25 μ g/mL based on the total volume of the first digest solution.

The final concentration of the GlutaMAX in the first digestive juice is 15-25nmol/L based on the total volume of the first digestive juice.

The final concentration of HEPES in the first digest solution is 5-15mM based on the total volume of the first digest solution.

The second digest comprises TrypLE Express and a ROCK inhibitor.

The final concentration of the ROCK inhibitor in the second digest is 5-15 μ M based on the total volume of the second digest. In some embodiments, the final concentration of the ROCK inhibitor in the second digest is 7-12 μ Μ, based on the total volume of the second digest. In some embodiments, the final concentration of the ROCK inhibitor in the second digest is 10 μ Μ, based on the total volume of the second digest.

The organoids include or are lung cancer organoids. In some embodiments, the organoid is a lung adenocarcinoma organoid.

The cancer tissue is lung cancer tissue or lung adenocarcinoma tissue. In some embodiments, the cancerous tissue is lung adenocarcinoma tissue.

Advantageous effects

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, the specific addition factor is added into the culture solution, so that the culture success rate and passable times of the organoid can be improved, and the problems of low culture success rate, low passable times and the like of the organoid in the prior art are solved.

(2) Furthermore, by screening proper specific addition factors and contents thereof, the components of the specific addition factors in the content range disclosed by the invention can play a synergistic role, the culture success rate of the lung pancreatic cancer organoid (the success rate is more than or equal to 85 percent, and the more preferable proportion can ensure that the success rate is more than or equal to 90 percent and even can reach 95 percent) and the organoid culture solution with passable times (the minimum can reach 9 continuous passages and even can reach not less than 10 continuous passages) can be improved, and the problems of low culture success rate, less passable times and the like of the lung pancreatic cancer organoid in the prior art are solved.

(3) The culture solution used in the 1 st week of organoid culture contains ROCK inhibitor, which can reduce cell anoikis caused by tissue dissociation, and the culture solution without ROCK inhibitor is used in the 2 nd to 3 rd week, which can avoid the change of ROCK inhibitor to cytoskeleton and improve the similarity between organoid and tumor tissue.

Drawings

FIG. 1 is an image of the organoid culture of example 4 during operation; wherein, A is the minced lung pancreatic cancer tissue; panel B is an image of the second precipitate after addition of the digestive juice before undigestion; panel C is an image of the second precipitate after digestion with added digestive juice; and D is a diagram of the shape of the inoculated gel drops.

FIG. 2 is a morphological diagram under a light microscope of lung adenocarcinoma organoids cultured in step D) for 1 to 7 days in example 4 using the culture solution described in example 2, and the scale is 100 μm.

FIG. 3 is a morphogram of lung adenocarcinoma organoids before and after digestion in example 6; wherein, A is a morphological diagram of lung adenocarcinoma organoid before digestion; and B is a diagram of the morphology of the digested lung adenocarcinoma organoids, wherein the scale is 100 mu m.

FIG. 4 is a photograph of hematoxylin-eosin staining of the cancer tissue described in example 4 and organoids cultured with the culture solution described in example 2, on a scale of 50 μm.

FIG. 5 is an immunofluorescent stained image of the cancer tissue described in example 4, with a scale of 50 μm.

FIG. 6 is an immunofluorescent-stained image of organoids obtained by culturing in example 4 in the culture solution described in example 2, with a scale bar of 50 μm.

Description of the terms

In the present invention, TTF-1 is a thyroid transcription factor; napsin a is aspartic protease a; thyroid transcription factor and aspartic protease A are the most commonly used molecular markers in the diagnosis of lung adenocarcinoma, and are expressed in most lung adenocarcinomas.

In the invention, CK5 is cytokeratin 5; cytokeratin 5 and p40, common molecular markers for squamous lung carcinoma, are hardly expressed in lung adenocarcinoma. DAPI is 4', 6-diamidino-2-phenylindole, which is commonly used to stain cell nuclei.

In the present invention, rpm means revolutions per minute; μ M means micromoles per liter; μ g means μ g; μ L means μ L; ng/mL represents nanograms per milliliter; mL means mL; g at the time of centrifugation is a centrifugal force unit, and 200g represents a centrifugal force of 200 times the gravitational acceleration.

In the present invention, "optional" means that the component described therein may or may not be present.

In the description of the present invention, it is to be understood that the terms "first", "second" and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, some non-limiting examples are further disclosed below, and the present invention is further described in detail.

The reagents used in the present invention are either commercially available or can be prepared by the methods described herein.

Reagents and sources thereof used in the present invention:

example 1: organoid culture solution

Organoid culture media were prepared as in table 1:

table 1: formula of organoid culture solution

Example 2: organoid culture solution

Organoid culture broth was prepared as in table 2:

table 2: formula of organoid culture solution

Example 3: organoid culture solution

Organoid culture was prepared as in table 3:

table 3: formula of organoid culture solution

Comparative example 1: organoid culture medium (refer to CN111411083B medium formula)

A culture medium for gastric cancer organoid comprises a basic culture medium 1640, specific additive factors and sterile water; wherein the mass ratio of the basic culture medium 1640 to the sterile water is 99: 1; the specific addition factors comprise: vitamin a-free B27, 2 ×; n-acetyl cysteine, 0.5. mu.M; EGF, 50 ng/mL; noggin, 100 ng/mL; r-spondin1, 800 ng/mL; wnt3a, 100 ng/mL; CHIR99021, 8 μ M; 1.5 μ M of thiazovivin; gastrin I, 25 ng/mL; valproic acid, 0.5 mM; penicillin streptomycin mixed solution, 1.2 x; amphotericin B, 0.8. mu.g/mL; primocin, 1 mg/mL; the final concentration of each component of the specific additive factor is based on the final concentration of each component in the mixed solution of the basic culture medium and the sterile water.

Comparative example 2: organoid culture medium (refer to CN109837242A medium formula)

An organoid culture comprising 1% penicillin, 1% streptomycin, 50ng/mL EGF, 50ng/mL fibroblast growth factor, 50ng/mL human noggin, 20mM HEPES, 500nM A83-01, 1.0 μ g/mL R Spondin 3, 5 μ M Y-27632, 5 μ M SB202190, 1% B27, 3mM Glutamax, 8% FBS Advanced DMEM/F12 medium.

Comparative example 3: organoid culture medium (refer to CN108396010A medium formula)

An organoid culture comprising 50ng/mL EGF, 100ng/mL Wnt-3a, 1. mu.g/mL R-Spondin1, 500nM A83-01, 10. mu.M Y-27632, 50ng/mL human noggin, 12. mu.M SB202190, 1 XN 2, 1 XB 27 serum-free additive, 10mM HEPES, 2mM Glutamax, 500 units/mL penicillin, 500 units/mL streptomycin, 12.5. mu.g/mL amphotericin, 10% FBS DMEM/F12 culture.

Comparative example 4: organoid culture medium (refer to CN111876386A medium formula)

Organoid culture was prepared as in table 4:

table 4: formula of organoid culture solution

Reagent	Final concentration
		R-Spondin1 conditioned medium	250ng/mL
Neuregulin
	1	5nM
EGF			5ng/mL
	Human noggin	100ng/mL
A83-01			500nM
	Y-27632	5μM
SB202190			500nM
	B27supplement(50×)	1×
N-acetylcysteine			1.25mM
	Nicotinamide	5mM
GlutaMAX(100×)			1×
	HEPES(100×)	1×
Penicillin/streptomycin			1×
	Primocin	50mg/mL
Advanced DMEM/F-12			1×

Example 4: organoid culture

Reagent:

first digestive juice: the formulation is shown in table 5.

Table 5: first digestive juice formula

Reagent	Final concentration
		antibiotic-antimycotic(100×)	1×
GlutaMAX(100×)	1×
		HEPES(100×)	1×
Advanced DMEM/F-12	1×
		Collagenase type II	5mg/mL
DNase I	100μg/mL
		Y-27632 hydrochloride	10μM

And (3) second digestive juice: contains TrypLE Express and 10 μ M of Y-27632 hydrochloride.

The operation is as follows: the organoid culture medium of examples 1-3 was used to culture organoids according to the following procedure:

A) taking a lung pancreatic cancer tissue block, cutting the lung pancreatic cancer tissue block into fragments with the diameter less than 2mm, adding a first digestive juice, digesting for 1h in a shaker (120rpm) at 37 ℃, centrifuging for 5 minutes at 200g, discarding the supernatant to obtain a first precipitate, washing the first precipitate with 10mL of room-temperature Advanced DMEM/F-12, centrifuging for 5 minutes at 200g, discarding the supernatant to obtain a second precipitate;

B) digesting the second precipitate with the second digestion solution at 37 ℃ for 10 minutes in a shaker (120rpm), adding 10mL of cold Advanced DMEM/F-12 containing 20% FBS to terminate the digestion, centrifuging at 4 ℃ and 200g for 5 minutes, discarding the supernatant to obtain a third precipitate, adding 2mL of cold Advanced DMEM/F-12 to wash the third precipitate, centrifuging at 4 ℃ and 200g for 5 minutes, and discarding the supernatant to obtain a fourth precipitate;

C) resuspend the fourth pellet with 10mL of cold Advanced DMEM/F-12 and filter to remove large pieces of tissue with a 70 μm cell strainer; counting cells, collecting 100,000 cells, centrifuging at 4 deg.C and 200g for 5 min, and discarding supernatant to obtain fifth precipitate; resuspending the fifth pellet with 30 μ L organoid culture medium and 300 μ L matrigel, and inoculating;

D) c), after the inoculated matrigel is solidified, adding a culture solution of organoids for culture, and replacing the culture solution every 2-3 days; and culturing the organoids in the culture medium containing the ROCK inhibitor for 1 week, and culturing the organoids in the culture medium containing no ROCK inhibitor for 1-2 weeks.

Taking the lung pancreatic cancer tissues and the obtained organoids to carry out hematoxylin-eosin staining and immunofluorescence staining, wherein the results are shown in figures 4-6, the obtained organoids are similar to the pathomorphic characteristics of the lung pancreatic cancer tissues according to the results, and the obtained organoids can accurately reduce the pathomorphic characteristics of the tumor tissues from which the organoids are derived.

Comparative example 5: organoid culture

Reagent: the same as in example 4.

The operation is as follows: the organoid culture medium of comparative examples 1-4 was taken and organoid cultured according to the following procedure:

D) c), after the inoculated matrigel is solidified, adding a culture solution of organoids for culture, and replacing the culture solution every 2-3 days; culturing for 2-3 weeks to obtain organoid.

Example 5: comparison of culture success rates of culture solutions of different organoids

The culture was performed 20 times according to the culture method of example 4, and the organoid culture success rates of the culture solutions of the organoids of examples 1 to 3 were examined, respectively; the culture was performed 20 times according to the culture method of comparative example 5, and the success rates of culture of the organoid culture solutions of the organoids of comparative examples 1 to 4 were examined, respectively. The success rate of culture of the culture medium for each organ is shown in Table 6.

Table 6: success rate of culture solution for different organs

Culture solution	Success rate of cultivation
		Example 1	90％
Example 2	95％
		Example 3	85％
Comparative example 1	65％
		Comparative example 2	80％
Comparative example 3	75％
		Comparative example 4	75％

Example 6: organoid passage

First, passage of organoids obtained in example 4

Reagent:

and (3) second digestive juice: the same as in example 1.

Organoid culture: any of examples 1 to 3 (the culture solution of organoids used for passaging corresponds to the culture solution used when organoids were cultured).

The operation is as follows:

centrifuging matrigel containing obtained organoid after culturing for 2-3 weeks at 4 deg.C and 200g for 5 min, discarding supernatant to obtain sixth precipitate, adding second digestive juice, and digesting at 37 deg.C for 5 min with shaking table (120 rpm); adding cold Advanced DMEM/F-12 containing 20% FBS to stop digestion; centrifuging for 5 minutes at 4 ℃ under 200g, discarding the supernatant to obtain a seventh precipitate, resuspending the fourth precipitate with 10mL of cold Advanced DMEM/F-12, pipetting with a 10mL pipette, pipetting organoids into smaller cell clusters (see B picture in figure 3), centrifuging for 5 minutes at 4 ℃ under 200g, discarding the supernatant to obtain an eighth precipitate, adding 30 μ L of precooled organoid culture medium to resuspend the cell precipitates, adding 600-; and culturing the organoids by using the culture solution containing the ROCK inhibitor for 1 week, and culturing the organoids by using the culture solution not containing the ROCK inhibitor for 1-2 weeks to obtain the subcultured organoids.

Second, passage of organoids obtained in comparative example 5

Reagent:

and (3) second digestive juice: the same as in example 1.

Organoid culture: any of comparative examples 1 to 4 (the culture solution of organoids used for passaging corresponds to the culture solution used when organoids were cultured).

Centrifuging matrigel containing obtained organoid after culturing for 2-3 weeks at 4 deg.C and 200g for 5 min, discarding supernatant to obtain sixth precipitate, adding second digestive juice, and digesting at 37 deg.C for 5 min with shaking table (120 rpm); adding cold Advanced DMEM/F-12 containing 20% FBS to stop digestion; centrifuging for 5 minutes at 4 ℃ under the condition of 200g, discarding the supernatant to obtain a seventh precipitate, resuspending the fourth precipitate by using 10mL of cold Advanced DMEM/F-12, blowing by using a 10mL pipette, blowing the organoid into a smaller cell mass, centrifuging for 5 minutes at 4 ℃ under the condition of 200g, discarding the supernatant to obtain an eighth precipitate, adding 30 mu L of precooled organoid culture medium to suspend the cell precipitate, adding 600 mu L of matrigel, inoculating, adding the organoid culture solution to culture after the inoculated matrigel is solidified, and replacing the culture solution once every 2-3 days; culturing for 2-3 weeks to obtain subcultured organoids.

Third, statistics of minimum passable times

The organoids obtained in example 4 and comparative example 5 were serially passaged as above, and passaged (no more than 10 times) each time 30% of the organoids expanded to form a cell mass with a diameter exceeding 200 μm, and the minimum passable number was recorded. The statistical results are shown in Table 7.

Table 7: minimum passable times statistical table

While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention within the context, spirit and scope of the invention. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention.

Claims

1. The culture solution of the organoid is characterized by comprising a basic culture medium and a specific additive factor; the specific additive factor comprises or is: a first antibiotic solution, a biological buffer, GlutaMAX, B27 serum-free additive, fibroblast growth factor, human noggin, R-Spondin1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- β type I receptor inhibitor, and optionally a ROCK inhibitor.

2. The culture solution of claim 1, wherein the basal medium is Advanced DMEM/F-12; and/or the first antibiotic solution comprises penicillin, streptomycin, and amphotericin B; and/or the biological buffer comprises or is HEPES; and/or the fibroblast growth factor comprises or is at least one of fibroblast growth factor 7 or fibroblast growth factor 10; and/or the ROS inhibitor comprises or is N-acetylcysteine; and/or the p38 MAPK inhibitor comprises or is SB202190 or a salt or hydrate thereof; or the p38 MAPK inhibitor comprises or is SB202190 monohydrochloride hydrate; and/or said TGF- β type I receptor inhibitor comprises or is a 83-01; and/or said ROCK inhibitor comprises or is Y-27632 or a salt thereof.

3. The broth of any one of claims 1-2, the first antibiotic solution comprising penicillin, streptomycin, and amphotericin B; the final concentration of the penicillin is 5000-15000 units/mL, 7000-12000 units/mL or 10000 units/mL, and the final concentration of the streptomycin is 5000-15000 units/mL, 7000-12000 units/mL or 10000 units/mL based on the total volume of the first antibiotic solution; the final concentration of amphotericin B is 10-35. mu.g/mL, 15-30. mu.g/mL or 25. mu.g/mL.

4. The broth according to any one of claims 1-3, wherein the first antibiotic solution has a volume fraction of 0.5% to 1.5%, 0.7% to 1.3%, or 1.0%, based on the total volume of the broth; and/or the volume fraction of the biological buffer is 0.5% -1.5%, 0.7% -1.3% or 1.0%; and/or the final concentration of the GlutaMAX is 15-25nmol/L, 17-22nmol/L or 20 nmol/L; and/or the volume fraction of the B27 serum-free additive is 0.5-1.5%, 0.7-1.3% or 1.0%; and/or the final concentration of the fibroblast growth factor is 20-80ng/mL, 30-60ng/mL or 40 ng/mL; and/or the content of the human noggin is 50-150ng/mL, 70-130ng/mL or 100 ng/mL; and/or the final concentration of the ROS inhibitor is 1.00-1.50mM or 1.25 mM; and/or the final concentration of nicotinamide is 8-12mM or 10 mM; and/or the final concentration of the p38 MAPK inhibitor is 5-15 μ M, 7-12 μ M or 10 μ M; and/or the final concentration of the R-spondin1 is 250-750ng/mL, 400-600ng/mL or 500 ng/mL; and/or the final concentration of the TGF-beta type I receptor inhibitor is 250-750nM, 400-600nM or 500 nM; and/or the final concentration of the ROCK inhibitor is 5-15. mu.M, 7-12. mu.M or 10. mu.M; and/or the volume fraction of the basal medium is 95.0-98.0%; or the balance is the basic culture medium.

5. Use of a culture solution according to any one of claims 1 to 4 for organoid culture or passaging.

6. A method for culturing organoids, comprising the steps of:

7. The method according to claim 6, wherein the culture solution in step D) is replaced every 2-3 days; and culturing the medium containing the ROCK inhibitor for 1 week, and culturing the medium containing no ROCK inhibitor for 1-2 weeks.

8. The method of any one of claims 6-7, wherein the first digestive fluid comprises a first antibiotic solution, GlutaMAX, HEPES, Advanced DMEM/F-12, collagenase type ii, dnase I, and a ROCK inhibitor; and/or the second digest comprises a TrypLE Express and a ROCK inhibitor; and/or said ROCK inhibitor comprises or is Y-27632 or a salt thereof.

9. The method of claim 8, wherein the final concentration of collagenase type ii is 2.5-10mg/mL, 4-7mg/mL, or 5mg/mL, based on the total volume of the first digest solution; and/or the final concentration of DNase I is 50-150. mu.g/mL, 70-120. mu.g/mL or 100. mu.g/mL; and/or the final concentration of the ROCK inhibitor in the first digest is 5-15 μ Μ, 7-12 μ Μ or 10 μ Μ; and/or the final concentration of the ROCK inhibitor in the second digest is 5-15 μ M, 7-12 μ M, or 10 μ M, based on the total volume of the second digest.

10. The method of any one of claims 6-9 or the use of claim 5, wherein the organoid comprises or is a lung cancer organoid or a lung adenocarcinoma organoid; and/or the cancer tissue is lung cancer tissue or lung adenocarcinoma tissue.