CN112592896B

CN112592896B - Culture solution for lung adenocarcinoma organoid and culture method thereof

Info

Publication number: CN112592896B
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: 2024-06-25
Anticipated expiration: 2040-11-22
Also published as: CN112592896A

Abstract

The invention provides a culture solution of an organoid and a culture method thereof, wherein the culture solution comprises a basic culture medium and specific additive factors, and can improve the culture success rate and passaging times of the organoid, in particular to the culture success rate and passaging times of the organoid of lung adenocarcinoma, and solve the problems of low culture success rate and few passaging times of the organoid in the prior art. The culture method has high success rate and the subculture times of the organoids obtained by culture are high.

Description

Culture solution for lung adenocarcinoma organoid and culture method thereof

Technical Field

The invention relates to the technical field of biological medicine, 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 multiple types of cells originating from stem cells, capable of mimicking the structure and function of the organ in which the cells are located. Compared with the traditional 2D cell culture and animal model, the organoids have incomparable advantages, and the cell composition and the function of the organoids are quite similar to those of in-vivo tissues. Many classes of organ functions are capable of maintaining their genome stability over prolonged culture in vitro, and this great advantage enables them to more accurately reflect the effects of drugs. Organoids can reduce the complexity of experiments compared to animal models for real-time imaging techniques, which enable one to use such models to study the mechanistic problems of human development and disease that are not easily or precisely studied in animal models.

Tumor organoids, also known as "cancer substitutes", "tumor-like", and the like, are primarily cultured in vitro 3D using patient tumor tissue to mimic the biological characteristics of tumor tissue in vivo. The organoids derived from pre-cancerous lesions (e.g., intraepithelial neoplasias) are used primarily to simulate the development and progression of tumors and to analyze the histological changes associated with the tumors. The tumor organoids highly summarize the characteristics of the source tumor tissue and preserve the heterogeneity among individuals, so that the organoids have application value in transformation medicine, and can be used for functional tests such as high-throughput drug screening and even establishment of personalized treatment schemes.

Lung adenocarcinoma is one type of lung cancer, which belongs to non-small cell carcinoma. Unlike squamous cell lung carcinoma, lung adenocarcinoma is more likely to occur in women and non-smoky persons. A few mucous glands originating from the bronchial mucosal epithelium and a few originating from the large bronchi. The age of onset is relatively small and women are relatively frequent. Most adenocarcinomas originate in the smaller bronchi and are peripheral lung cancers.

Currently, the approach to obtaining tumor organoids is mainly enzymatic digestion, using digestive enzymes to treat tissue to obtain a cell mass capable of forming organoids. However, as the enzymolysis method digests tissues to destroy intercellular connection, and the digestion time is long, the cell activity is reduced, so that the success rate of sample treatment is low, the obtained organoids are few in quantity, and the requirements of clinical application and scientific research are difficult to meet.

Due to the differences between different types of cancerous organoids, a medium or method that can achieve a high success rate on the culture of one type of cancerous organoid does not necessarily mean that a high success rate can be achieved on the culture of another type of cancerous organoid.

Patent CN110452877A discloses a culture medium and a culture method of lung cancer solid tumor primary cells, but the lung cancer solid tumor primary cells cultured by the culture medium have the problems of low success rate of culture, less passaging times and the like.

Patent CN111411083B discloses a culture medium and a culture method of gastric cancer organoids, but the inventor researches and discovers that the success rate of culturing lung adenocarcinoma organoids obtained by using the culture medium is low and the passaging times are less.

Patent CN109837242A discloses a breast cancer organoid culture solution and a culture method thereof, but the inventor researches and discovers that the success rate of obtaining lung adenocarcinoma organoids by using the culture medium of the patent is low and the passaging times are less.

Patent CN108396010a discloses an in vitro culture method of colorectal cancer organoids, but researches of the inventor find that the success rate of obtaining lung adenocarcinoma organoids by culture with the culture medium of the patent is low and the passable times are small.

Patent CN111876386A discloses a culture medium of breast cancer organoids and a culture method thereof, but the inventor researches and discovers that the success rate of obtaining lung adenocarcinoma organoids by using the culture medium is low and the passaging times are less.

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

Disclosure of Invention

Summary of The Invention

The first object of the present invention is to provide a culture solution for organoids, which comprises a basal medium and specific additive factors, and which can improve the success rate of organoid culture and passaging times, especially for lung adenocarcinoma organoids, and solve the problems of low success rate of organoid culture and low passaging times in the prior art.

Furthermore, by screening proper specific additive factors and proportion thereof, the invention obtains the culture solution of the organoid capable of improving the success rate of the culture of the organoid of the lung adenocarcinoma and the passaging times, and solves the problems of low success rate of the culture of the organoid of the lung adenocarcinoma, less passaging times and the like in the prior art.

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

A third object of the present invention is to provide a method for culturing an organoid which has a high success rate and a high passaging number of the organoid obtained by culturing.

Detailed Description

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

An organoid culture broth comprising a basal medium and a specific additive factor; the specific additive factors comprise: a first antibiotic solution, a biological buffer, a glamax, B27 serum-free additive, a fibroblast growth factor, human noggin, R-Spondin 1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- βi receptor inhibitor, and optionally a ROCK inhibitor.

In some embodiments, the specific additive factor may consist of: a first antibiotic solution, a biological buffer, a glamax, B27 serum-free additive, a fibroblast growth factor, human noggin, R-Spondin 1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- βi 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 comprise 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 factor is fibroblast growth factor 7 and fibroblast growth factor 10.

The ROS inhibitor may include or be N-acetylcysteine.

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

The TGF-beta type I receptor inhibitor may comprise or be A83-01.

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

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

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

The first antibiotic solution may have a volume fraction of 0.5% -1.5% based on the total volume of the culture solution. In some embodiments, the first antibiotic solution has a volume fraction of 0.7% to 1.3% based on the total volume of the culture broth. In some embodiments, the first antibiotic solution has a volume fraction of 1.0% based on the total volume of the culture broth.

The volume fraction of the biological buffer may be 0.5% -1.5% based on the total volume of the culture solution. In some embodiments, the biological buffer is present in a volume fraction of 0.7% to 1.3% based on the total volume of the culture broth. In some embodiments, the biological buffer is present in a volume fraction of 1.0% based on the total volume of the culture broth.

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

The volume fraction of the B27 serum-free additive may be 0.5% -1.5% based on the total volume of the culture broth. In some embodiments, the volume fraction of the B27 serum-free additive is 0.7% -1.3% based on the total volume of the culture broth. In some embodiments, the volume fraction of the B27 serum-free additive is 1.0% based on the total volume of the culture broth.

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

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

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

The human noggin may be present in an amount of 50-150ng/mL based on the total volume of the culture broth. In some embodiments, the human noggin is present in an amount of 70-130ng/mL based on the total volume of the culture broth. In some embodiments, the human noggin is present in an amount of 100ng/mL based on the total volume of the culture broth.

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

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

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

The final concentration of R-spondin 1 may be 250-750ng/mL based on the total volume of the culture broth. In some embodiments, the final concentration of R-spondin 1 is 400-600ng/mL based on the total volume of the culture broth. In some embodiments, the final concentration of R-spondin 1 is 500ng/mL based on the total volume of the culture broth.

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

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

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

According to some embodiments of the invention, an organoid culture broth is comprised of a basal medium and a specific additive factor; the specific additive factors consist of the following components: a first antibiotic solution, a biological buffer, a glamax, B27 serum-free additive, a fibroblast growth factor, human noggin, R-Spondin 1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- βi receptor inhibitor, and optionally a ROCK inhibitor.

According to some embodiments of the invention, an organoid culture broth is comprised of a basal medium and a specific additive factor; the specific additive factors consist of: a first antibiotic solution, a biological buffer, a glamax, B27 serum-free additive, a fibroblast growth factor, human noggin, R-Spondin 1, a ROS inhibitor, nicotinamide, a p38MAPK inhibitor, a TGF- βi 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 15-25nmol/L; the volume fraction of the serum-free additive B27 is 0.5-1.5%; the final concentration of the fibroblast growth factor is 20-80ng/mL; the content of the human cephalin is 50-150ng/mL; the final concentration of the ROS inhibitor is 1.00-1.50mM; the final concentration of nicotinamide is 8-12mM; the final concentration of the p38MAPK inhibitor is 5-15 mu M; the final concentration of the R-spondin 1 is 250-750ng/mL; the final concentration of the TGF-beta type I receptor inhibitor is 250-750nM; and the final concentration of the ROCK inhibitor is 5-15 μm; the balance being the basal medium.

According to some embodiments of the invention, an organoid culture broth is comprised of a basal medium and a specific additive factor; the specific additive factors consist of: a first antibiotic solution, a biological buffer, a glamax, B27 serum-free additive, a fibroblast growth factor, human noggin, R-Spondin 1, a ROS inhibitor, nicotinamide, a p38MAPK inhibitor, a TGF- βi 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 is 0.7% -1.3%; the final concentration of the Glutamax is 17-22nmol/L; the volume fraction of the serum-free additive B27 is 0.7-1.3%; the final concentration of the fibroblast growth factor is 30-60ng/mL; the content of the human noggin is 70-130ng/mL; the final concentration of the ROS inhibitor is 1.25mM; the final concentration of nicotinamide is 10mM; the final concentration of the p38MAPK inhibitor is 7-12 mu M; the final concentration of the R-spondin 1 is 400-600ng/mL; the final concentration of the TGF-beta type I receptor inhibitor is 400-600nM; the final concentration of the ROCK inhibitor is 7-12 mu M; the balance being the basal medium.

According to some embodiments of the invention, an organoid culture broth is comprised of a basal medium and a specific additive factor; the specific additive factors consist of: a first antibiotic solution, a biological buffer, a glamax, B27 serum-free additive, a fibroblast growth factor, human noggin, R-Spondin 1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- βi receptor inhibitor, and optionally a ROCK inhibitor; the volume fraction of the first antibiotic solution is 1.0% based on the total volume of the culture solution; the volume fraction of the biological buffer is 1.0%; the final concentration of the Glutamax is 20nmol/L; the volume fraction of the serum-free additive B27 is 1.0%; the final concentration of the fibroblast growth factor is 40ng/mL; the content of the human noggin is 100ng/mL; the final concentration of the ROS inhibitor is 1.25mM; the final concentration of nicotinamide is 10mM; the final concentration of the p38 MAPK inhibitor was 10 μm; the final concentration of the R-spondin 1 is 500ng/mL; the final concentration of the TGF-beta type I receptor inhibitor is 500nM; the final concentration of the ROCK inhibitor is 10 mu M; the balance being the basal medium.

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

Use of a culture broth according to any of the first aspects for organoid culture or passaging. In some embodiments, the organoid comprises a tumor organoid. In some embodiments, the organoid is a lung adenocarcinoma organoid.

In a third aspect, the present invention provides a method of culturing an organoid.

A method of culturing an organoid comprising the steps of:

A) Cutting cancer tissue blocks, 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, stopping digestion, centrifuging to obtain a third precipitate, washing the third precipitate, and centrifuging to obtain a fourth precipitate;

C) Re-suspending the fourth precipitate with a medium, and filtering to remove large tissues; counting cells, and centrifuging to obtain a fifth precipitate; resuspending the fifth pellet with the culture broth and matrigel of any of the above claims, inoculating;

D) And C), after the matrix gel inoculated in the step C) is solidified, adding the culture solution of any one of the above steps for culturing to obtain the organoids.

The culture solution in the step D) is replaced every 2-3 days; and culturing for 1 week with the culture solution containing the ROCK inhibitor, and then culturing for 1-2 weeks with the culture solution without the ROCK inhibitor.

The first digestive juice comprises a second antibiotic solution, glutaMAX, HEPES, advancedDMEM/F-12, collagenase type II, deoxyribonuclease I and a ROCK inhibitor.

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

The second antibiotic solution contains 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 digestive juice. In some embodiments, the final concentration of collagenase type II is from 4 to 7mg/mL based on the total volume of the first digestive juice. In some embodiments, the final concentration of collagenase type ii is 5mg/mL based on the total volume of the first digestive juice.

The final concentration of DNase I is 50-150 μg/mL based on the total volume of the first digest. In some embodiments, the final concentration of deoxyribonuclease I is from 70-120 μg/mL based on the total volume of the first digestive juice. In some embodiments, the final concentration of deoxyribonuclease I is 100 μg/mL based on the total volume of the first digestive juice.

The final concentration of the ROCK inhibitor in the first digestive juice is 5-15 μm based on the total volume of the first digestive juice. In some embodiments, the ROCK inhibitor is present in the first digestive juice at a final concentration of 7 to 12 μm based on the total volume of the first digestive juice. In some embodiments, the ROCK inhibitor is present in the first digestive juice at a final concentration of 10 μm based on the total volume of the first digestive juice.

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 digestive juice. In some embodiments, the final concentration of penicillin is 100 units/mL based on the total volume of the first digestive juice.

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

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

And 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 digestive juice is 5-15mM based on the total volume of the first digestive juice.

The second digestive juice comprises a TrypLE Express and ROCK inhibitor.

The final concentration of the ROCK inhibitor in the second digestive juice is 5-15 μm based on the total volume of the second digestive juice. In some embodiments, the ROCK inhibitor is present in the second digestive juice at a final concentration of 7 to 12 μm based on the total volume of the second digestive juice. In some embodiments, the ROCK inhibitor is present in the second digestive juice at a final concentration of 10 μm based on the total volume of the second digestive juice.

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 cancer tissue is lung adenocarcinoma tissue.

Advantageous effects

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

(1) The specific additive factors are added into the culture solution, so that the success rate of the organoid culture and the passaging times can be improved, and the problems of low success rate of the organoid culture, small passaging times and the like in the prior art are solved.

(2) Furthermore, the components of the specific additive factors within the content range disclosed by the invention can play a synergistic role by screening the proper specific additive factors and the content thereof, so that the culture success rate (the success rate is more than or equal to 85%, more preferable proportion can lead the success rate to be more than or equal to 90%, even more preferable proportion can lead the success rate to be more than or equal to 95%) and the culture solution of the organs with passaging times (the minimum number can reach 9 continuous passages, even can reach not less than 10 continuous passages) can be improved, and the problems of low culture success rate, low passaging times and the like of the organs with lung adenocarcinoma in the prior art are solved.

(3) The culture solution used in week 1 of the organoid culture contains the ROCK inhibitor, so that the anoikis of cells caused by tissue dissociation can be reduced, and the culture solution without the ROCK inhibitor is used in weeks 2-3, so that the change of the ROCK inhibitor to the cytoskeleton can be avoided, and the similarity of the organoid and tumor tissue can be improved.

Drawings

FIG. 1 is an image of a culture of the organoids of example 4 during operation; wherein, a graph a is a sheared lung adenocarcinoma tissue; panel B is an image of the second precipitate before it is undigested after addition of digestive juice; panel C is an image of the second precipitate after digestion with addition of digestive juice; panel D is a gel drop morphology after inoculation.

FIG. 2 is a schematic diagram of a lung adenocarcinoma organoid cultured in step D) for 1-7 days using the culture broth described in example 2, with a scale of 100 μm, in example 4.

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

FIG. 4 is a hematoxylin-eosin stained image of the cancerous tissue described in example 4 and of an organoid obtained by culture using the culture broth described in example 2, on a scale of 50. Mu.m.

FIG. 5 is an immunofluorescence staining image of the cancerous tissue described in example 4, with a scale bar of 50 μm.

FIG. 6 is an immunofluorescent-stained image of an organoid obtained in example 4 using the culture broth described in example 2, the scale of which is 50. Mu.m.

Description of the terms

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

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

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

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

In the description of the present invention, it should 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 a relative importance or number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. In the description of the present invention, the meaning of "a plurality" is two or more, unless explicitly defined otherwise.

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means 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 present invention. In this specification, schematic representations of the above terms are not necessarily directed 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, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

Detailed Description

In order to better understand the technical solution of the present invention, the following further discloses some non-limiting examples, which are further described in detail.

The reagents used in the present invention are all 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 solutions were prepared as in table 1:

table 1: formula of organoid culture solution

Example 2: organoid culture solution

Organoid culture solutions were prepared as in table 2:

Table 2: formula of organoid culture solution

Example 3: organoid culture solution

Organoid culture solutions were prepared as in table 3:

table 3: formula of organoid culture solution

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Comparative example 1: organoid culture (see formulation in CN 111411083B)

A gastric cancer organoid culture medium comprising basal medium 1640, specific additive factors and sterile water; wherein the mass ratio of the basal medium 1640 and the sterile water is 99:1; the specific additive factors include: B27,2×;N-acetylcysteine,0.5μM;EGF,50ng/mL;Noggin,100ng/mL;R-spondin 1,800ng/mL;Wnt3a,100ng/mL;CHIR99021,8μM;thiazovivin,1.5μM;Gastrin I,25ng/mL; valproic acid without vitamin a, 0.5mM; penicillin streptomycin mixture, 1.2×; amphotericin B,0.8 μg/mL; primocin,1mg/mL; the final concentration of each component of the specific additive factors 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 (see formulation in CN 109837242A)

An organoid culture broth comprising 1% penicillin, 1% streptomycin, 50ng/mL EGF, 50ng/mL fibroblast growth factor, 50ng/mL human noggin, 20mM HEPES, 500nM a83-01, 1.0 μg/mL R Spondin 3, 5 μΜ Y-27632, 5 μΜ SB202190, 1% B27, 3mM Glutamax, ADVANCED DMEM/F12 medium of 8% fbs.

Comparative example 3: organoid culture (see formulation in CN 108396010A)

An organoid culture broth comprising 50ng/mL EGF, 100ng/mL Wnt-3a, 1 μg/mL R-Spondin1, 500nM A83-01, 10 μM Y-27632, 50ng/mL human noggin, 12 μMSB202190, 1 XN 2, 1 XB 27 serum free additive, 10mM HEPES, 2mM Glutamax, 500 units/mL penicillin, 500 units/mL streptomycin, 12.5 μg/mL amphotericin, 10% FBS DMEM/F12 broth.

Comparative example 4: organoid culture (see formulation in CN 111876386A)

Organoid culture solutions were prepared as in table 4:

table 4: formula of organoid culture solution

Reagent(s)	Final concentration
		R-Spondin 1 conditioned Medium	250ng/mL
Neuregulin 1	5nM
		EGF	5ng/mL
Human head protein	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(s)	Final concentration
		antibiotic-antimycotic(100×)	1×
GlutaMAX(100×)	1×
		HEPES(100×)	1×
Advanced DMEM/F-12	1×
		Collagenase type II	5mg/mL
Deoxyribonuclease I	100μg/mL
		Y-27632 hydrochloride	10μM

Second digestive juice: y-27632 hydrochloride containing TrypLE Express and 10. Mu.M.

The operation is as follows: the organoids of examples 1-3 were cultured by the following procedure:

A) Cutting lung adenocarcinoma tissue blocks into fragments with the diameter less than 2mm, adding a first digestion solution, digesting for 1h by using a shaking table (120 rpm) at 37 ℃, centrifuging for 5 minutes by 200g, discarding the supernatant to obtain a first precipitate, washing the first precipitate with 10mL of ADVANCED DMEM/F-12 at room temperature, centrifuging for 5 minutes by 200g, and discarding the supernatant to obtain a second precipitate;

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

C) The fourth pellet was resuspended with 10mL of cold ADVANCED DMEM/F-12 and the bulk tissue removed by filtration through a 70 μm cell strainer; counting cells, collecting 100,000 cells, centrifuging at 4deg.C under 200g for 5 min, and discarding supernatant to obtain fifth precipitate; resuspension of the fifth pellet with 30 μl of organoid culture and 300 μl of matrigel, inoculation;

d) After the matrix gel inoculated in the step C) is solidified, adding the organoid culture solution for culturing, and replacing the culture solution every 2-3 days; and culturing for 1 week with the culture solution containing the ROCK inhibitor, and culturing for 1-2 weeks with the culture solution without the ROCK inhibitor to obtain organoids.

The lung adenocarcinoma tissue and the obtained organoid are taken for hematoxylin-eosin staining and immunofluorescence staining, the results are shown in figures 4-6, and the result shows that the obtained organoid has similar pathological morphological characteristics with the lung adenocarcinoma tissue, and the obtained organoid can accurately reduce the pathological morphological characteristics of the tumor tissue from which the organoid is derived.

Comparative example 5: organoid culture

Reagent: same as in example 4.

The operation is as follows: the organoids of comparative examples 1-4 were cultured in the following steps:

d) After the matrix gel inoculated in the step C) is solidified, adding the organoid culture solution for culturing, and replacing the culture solution every 2-3 days; culturing for 2-3 weeks to obtain organoids.

Example 5: comparison of success rates of culture solutions for different classes of organs

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

Table 6: success rate of culture solution of different organs

Culture solution	Success rate of culture
		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: passage of organoids

1. Passage of organoids obtained in example 4

Reagent:

second digestive juice: as in example 1.

Organoid culture broth: any of examples 1-3 (the organoid culture medium used for passaging corresponds to the culture medium used in culturing organoids).

The operation is as follows:

Taking matrix gel containing the obtained organoid after 2-3 weeks of culture, centrifuging at 4deg.C and 200g for 5 min, removing supernatant to obtain sixth precipitate, adding second digestive juice, and digesting at 37deg.C with shaking table (120 rpm) for 5 min; cold ADVANCED DMEM/F-12 containing 20% FBS was added to terminate digestion; centrifuging at 4deg.C for 5 min under 200g condition, discarding supernatant to obtain seventh precipitate, re-suspending fourth precipitate with 10mL cold ADVANCED DMEM/F-12, blowing with 10mL pipette, blowing organoid into smaller cell mass (see figure 3B), centrifuging at 4deg.C for 5 min under 200g condition, discarding supernatant to obtain eighth precipitate, adding 30 μl of precooled organoid culture medium to re-suspend cell precipitate, adding 600-900 μl of matrigel, inoculating, solidifying the matrix gel after inoculation, adding organoid culture solution for culturing, and replacing culture solution every 2-3 days; and culturing for 1 week with the culture solution containing the ROCK inhibitor, and culturing for 1-2 weeks with the culture solution without the ROCK inhibitor to obtain the subcultured organoids.

2. Passage of the organoids obtained in comparative example 5

Reagent:

second digestive juice: as in example 1.

Organoid culture broth: any of comparative examples 1 to 4 (the organoid culture broth used for passaging corresponds to the culture broth used for culturing organoids).

Taking matrix gel containing the obtained organoid after 2-3 weeks of culture, centrifuging at 4deg.C and 200g for 5 min, removing supernatant to obtain sixth precipitate, adding second digestive juice, and digesting at 37deg.C with shaking table (120 rpm) for 5 min; cold ADVANCED DMEM/F-12 containing 20% FBS was added to terminate digestion; centrifuging at 4deg.C under 200g for 5 min, discarding supernatant to obtain seventh precipitate, re-suspending fourth precipitate with 10mL cold ADVANCED DMEM/F-12, blowing with 10mL pipette, blowing organoid into small cell mass, centrifuging at 4deg.C under 200g for 5 min, discarding supernatant to obtain eighth precipitate, adding 30 μl of precooled organoid culture medium to re-suspend cell precipitate, adding 600-900 μl matrigel, inoculating, solidifying after matrigel, adding organoid culture solution, culturing, and replacing culture solution every 2-3 days; culturing for 2-3 weeks to obtain the subcultured organoids.

3. Minimum passable count

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 organoids were amplified to form cell masses with diameters exceeding 200 μm, the minimum passable number was recorded. The statistical results are shown in Table 7.

Table 7: statistics of minimum passable times

While the methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and combinations of the methods and applications described herein can be made and applied within the spirit and scope of the invention. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention.

Claims

1. A method for culturing lung adenocarcinoma organoids, comprising the steps of:

A) Taking lung adenocarcinoma tissue blocks, shearing, adding a first digestive juice for digestion, centrifuging to obtain a first precipitate, washing the first precipitate, and centrifuging to obtain a second precipitate;

C) Re-suspending the fourth precipitate with a medium, and filtering to remove large tissues; counting cells, and centrifuging to obtain a fifth precipitate; resuspending the fifth precipitate with the culture medium and matrigel, inoculating;

D) After the matrigel inoculated in the step C) is solidified, adding a culture solution of the organoid for culturing, wherein the culture solution is replaced every 2-3 days, and is firstly cultured for 1 week by using the culture solution containing the ROCK inhibitor, and then is cultured for 1-2 weeks by using the culture solution without the ROCK inhibitor, so that the organoid for lung adenocarcinoma is obtained;

The culture solution consists of a basic culture medium and specific additive factors; the specific additive factors consist of the following components: a first antibiotic solution, a biological buffer, a glamax, B27 serum-free additive, a fibroblast growth factor, human noggin, R-Spondin1, a ROS inhibitor, nicotinamide, a p38 MAPK inhibitor, a TGF- βi receptor inhibitor, and optionally a ROCK inhibitor; the basal culture medium is ADVANCED DMEM/F-12; the first antibiotic solution comprises penicillin, streptomycin, and amphotericin B; the biological buffer is HEPES; the fibroblast growth factors are fibroblast growth factor 7 and fibroblast growth factor 10; the ROS inhibitor is N-acetylcysteine; the p38 MAPK inhibitor is SB202190; the TGF-beta I type receptor inhibitor is A83-01; the ROCK inhibitor is Y-27632 hydrochloride;

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 15-25nmol/L; the volume fraction of the serum-free additive B27 is 0.5-1.5%; the final concentration of the fibroblast growth factor is 20-80ng/mL; the content of the human cephalin is 50-150ng/mL; the final concentration of the ROS inhibitor is 1.00-1.50mM; the final concentration of nicotinamide is 8-12mM; the final concentration of the p38 MAPK inhibitor is 5-15 mu M; the final concentration of the R-spondin 1 is 250-750ng/mL; the final concentration of the TGF-beta type I receptor inhibitor is 250-750nM; the final concentration of the ROCK inhibitor is 5-15 mu M; the balance being the basal medium.

2. The method of claim 1, wherein the first antibiotic solution comprises penicillin, streptomycin, and amphotericin B; the final concentration of penicillin is 5000-15000 units/mL and the final concentration of streptomycin is 5000-15000 mug/mL based on the total volume of the first antibiotic solution; the final concentration of amphotericin B is 10-35 μg/mL.

3. The method of claim 2, wherein the final concentration of penicillin is 7000-12000 units/mL and the final concentration of streptomycin is 7000-12000 μg/mL; the final concentration of amphotericin B is 15-30 μg/mL.

4. The method of claim 3, wherein the final concentration of penicillin is 10000 units/mL and the final concentration of streptomycin is 10000 μg/mL; the final concentration of amphotericin B was 25 μg/mL.

5. The method of claim 1, wherein the first antibiotic solution has a volume fraction of 0.7% to 1.3% based on the total volume of the culture broth; and/or the biological buffer has a volume fraction of 0.7% -1.3%; and/or the final concentration of the Glutamax is 17-22nmol/L; and/or the volume fraction of the B27 serum-free additive is 0.7% -1.3%; and/or the final concentration of the fibroblast growth factor is 30-60ng/mL; and/or the content of the human noggin is 70-130ng/mL; and/or the final concentration of the ROS inhibitor is 1.25mM; and/or the final concentration of nicotinamide is 10mM; and/or the final concentration of the p38 MAPK inhibitor is 7-12. Mu.M; and/or the final concentration of R-spondin 1 is 400-600ng/mL; and/or the final concentration of the TGF-beta type I receptor inhibitor is 400-600nM; and/or the final concentration of the ROCK inhibitor is 7-12 μm; and/or the volume fraction of the basal medium is 95.0% -98.0%; or the balance of the basal medium.

6. The method of claim 5, wherein the first antibiotic solution has a volume fraction of 1.0% based on the total volume of the culture broth; and/or the volume fraction of the biological buffer is 1.0%; and/or the final concentration of the Glutamax is 20nmol/L; and/or the volume fraction of the B27 serum-free additive is 1.0%; and/or the final concentration of the fibroblast growth factor is 40ng/mL; and/or the content of the human noggin is 100ng/mL; and/or the final concentration of the ROS inhibitor is 1.25mM; and/or the final concentration of nicotinamide is 10mM; and/or the final concentration of the p38 MAPK inhibitor is 10 μm; and/or the final concentration of R-spondin 1 is 500ng/mL; and/or the final concentration of the TGF-beta type I receptor inhibitor is 500nM; and/or the final concentration of the ROCK inhibitor is 10 μm; and/or the volume fraction of the basal medium is 95.0% -98.0%; or the balance of the basal medium.

7. The method of claim 1, wherein the first digestive juice comprises a first antibiotic solution, glutaMAX, HEPES, ADVANCED DMEM/F-12, collagenase type ii, dnase I, and a ROCK inhibitor; the ROCK inhibitor in the first digestive juice is Y-27632 hydrochloride.

8. The method of claim 7, wherein the final concentration of collagenase type ii is 2.5-10mg/mL based on the total volume of the first digestive juice; and/or the final concentration of DNase I is 50-150 μg/mL; and/or the final concentration of the ROCK inhibitor in the first digestive juice is 5-15 μm.

9. The method of claim 8, wherein the final concentration of collagenase type ii is 4-7mg/mL based on the total volume of the first digestive juice; and/or the final concentration of the DNase I is 70-120 mug/mL; and/or the final concentration of the ROCK inhibitor in the first digestive juice is 7-12 μm.

10. The method of claim 9, wherein the final concentration of collagenase type ii is 5mg/mL based on the total volume of the first digestive juice; and/or the final concentration of deoxyribonuclease I is 100 μg/mL; and/or the ROCK inhibitor is present in the first digestive juice at a final concentration of 10 μm.

11. The method of claim 1, wherein the second digestive juice comprises a TrypLE Express and ROCK inhibitor; the ROCK inhibitor in the second digestive juice is Y-27632 hydrochloride.

12. The method of claim 11, wherein the ROCK inhibitor is present in the second digestive juice at a final concentration of 5-15 μm based on the total volume of the second digestive juice.

13. The method of claim 12, wherein the ROCK inhibitor is present in the second digestive juice at a final concentration of 7-12 μm based on the total volume of the second digestive juice.

14. The method of claim 13, wherein the ROCK inhibitor is present in the second digestive juice at a final concentration of 10 μm based on the total volume of the second digestive juice.

15. Use of a culture method according to any one of claims 1-14 for the culture or passage of lung adenocarcinoma organoids.