CN112522201A - Culture medium and culture method for bladder cancer organoid - Google Patents
Culture medium and culture method for bladder cancer organoid Download PDFInfo
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- CN112522201A CN112522201A CN202011495937.2A CN202011495937A CN112522201A CN 112522201 A CN112522201 A CN 112522201A CN 202011495937 A CN202011495937 A CN 202011495937A CN 112522201 A CN112522201 A CN 112522201A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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Abstract
The invention provides a culture medium of bladder cancer organoid and a culture method thereof, wherein the culture method adopts two different organoid culture mediums for culture, and discloses the composition and proportion of the two organoid culture mediums, the method and the culture medium can improve the culture success rate, passable times, growth speed and growth form of the bladder cancer organoid, and solve the problems of low culture success rate, less passable times, less organoid growth, poorer cell form, slow growth and the like of the bladder cancer organoid in the prior art.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a culture medium and application thereof, and more particularly relates to a bladder cancer organoid culture medium and a culture method thereof.
Background
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.
Bladder cancer is a common malignant tumor, and the incidence and mortality of the cancer are in the first place of urinary system tumors and in the 9 th place of male malignant tumors. Clinically, about 70% of bladder cancers are non-muscle invasive bladder cancers and 30% are muscle invasive bladder cancers. The 5-year recurrence rate of non-muscle invasive bladder cancer is over 50%. The main treatment mode of non-muscle invasive bladder cancer is transurethral cystectomy (TURBT), but the recurrence rate of tumors after TURBT operation is high, and the simple TURBT can not solve the problems of continuous development and high recurrence rate after carcinoma in situ operation, and the estimated recurrence rate of high-risk bladder cancer patients is 50% within 1 year and 90% within 5 years.
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.
Although the successful culture of tumor organoid sources including breast cancer, colon cancer, lung cancer, esophageal cancer and the like is reported at present, wherein the culture success rate of breast cancer and colon cancer organoid is as high as more than 80%, the required culture medium and culture method are different because the 'micro-environments' required for organoid culture in vitro are different, and the culture medium with high success rate for culturing organoid of breast cancer, colon cancer, lung cancer, esophageal cancer and the like is not necessarily suitable for the culture of bladder cancer organoid.
Patent application CN110452877A discloses a culture medium for primary cells of lung cancer solid tumors and a culture method thereof, but the primary cells of lung cancer solid tumors cultured by the culture medium disclosed by the patent have the problems of low culture success rate, small passable times and the like.
Patent application CN111411083B discloses a stomach cancer organoid culture medium and a culture method, but the inventor researches show that the success rate of obtaining the bladder cancer organoid by using the culture medium disclosed by the patent is low and the passable times are few.
Patent application CN109837242A discloses a breast cancer organoid culture medium and a culture method thereof, but the inventor researches show that the success rate of obtaining bladder cancer organoids by using the culture medium disclosed by the patent is low and the passable times are few.
Patent application CN108396010A discloses an in vitro culture method of colorectal cancer organoid, but the inventor finds that the success rate of bladder cancer organoid obtained by culture with the culture medium of the patent is low and the passable times are few.
Patent application CN111876386A discloses a breast cancer organoid culture medium and a culture method thereof, but the inventor researches show that the success rate of obtaining bladder cancer organoids by using the culture medium disclosed by the patent is low and the passable times are few.
Patent application CN111197030A discloses a culture medium and a culture method for bladder cancer organoids, but the inventors have found that the success rate of obtaining bladder cancer organoids by using the culture medium is low and the passable times are small.
Therefore, there is still a need to develop a culture medium with high success rate for culturing bladder cancer organoids and a culture method thereof.
Disclosure of Invention
Summary of The Invention
The invention aims to provide a bladder cancer organoid culture method, which can improve the culture success rate and passable times of bladder cancer organoids and solve the problems of less growth, poorer cell morphology, slow growth and the like of bladder cancer organoids in the prior art.
The invention also aims to provide a culture medium for the bladder cancer organoids, and the culture medium culture method can improve the culture success rate and passable times of the bladder cancer organoids, and solves the problems of low culture success rate, low passable times and the like of the bladder cancer organoids in the prior art.
Detailed Description
The invention provides a method for culturing bladder cancer organoids.
A method for culturing a bladder cancer organoid, comprising the steps of:
A) taking bladder cancer tissue blocks, cutting into pieces, adding a first digestive juice for digestion, centrifuging to obtain a first precipitate, digesting the first precipitate with a second digestive juice, centrifuging to obtain a second precipitate, re-suspending the second precipitate with a neutralization culture solution, filtering to remove large tissue blocks, and centrifuging filtrate to obtain a third precipitate;
B) resuspending the third precipitate with organoid culture medium A and matrigel under ice bath, inoculating, and incubating;
C) and C) after the matrigel inoculated in the step B) is solidified, adding the organoid culture medium A, culturing for 3 days, and then culturing for 7-12 days by using the organoid culture medium B to obtain the bladder cancer organoid.
The filtration was performed using a 70 μm cell screen.
The organoid medium A or organoid medium B in step C) is replaced every 2-3 days.
The organoid medium a may comprise the following components and final concentrations: based on the total volume of the organoid culture medium A, the volume percent is 0.5-1.5% of antimicrobial-antimicrobial 100 x, the volume percent is 0.5-1.5% of GlutaMAX100 x, the volume percent is 0.5-1.5% of HEPES buffer solution 100 x, and the epidermal growth factor is 0-5 ng/ml; 5-15ng/mL fibroblast growth factor, 5-20ng/mL basic fibroblast growth factor, 0-750nM A83-01, 0-15 μ M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 5-15 μ M Y-27632, 5-15mM nicotinamide and 1-3% by volume B27 supplement 50X diluted in basal medium Advanced DMEM/F12.
In some embodiments, the organoid medium a can include the following components and final concentrations: 1% by volume of antimicrobial-antimicrobial 100 x, 1% by volume of GlutaMAX100 x, and 1% by volume of HEPES buffer 100 x, based on the total volume of organoid medium a; 7-12ng/mL fibroblast growth factor 10, 7-12ng/mL basic fibroblast growth factor, 250-750nM A83-01, 5-7 μ M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 5-15 μ M Y-27632, 8-12mM nicotinamide and 2% by volume B27 supplement 50X, diluted with basal medium Advanced DMEM/F12, said organoid medium A being free of epidermal growth factor.
In some embodiments, the organoid medium a may consist of the following components and final concentrations: based on the total volume of the organoid culture medium A, the volume percent is 0.5-1.5% of antimicrobial-antimicrobial 100 x, the volume percent is 0.5-1.5% of GlutaMAX100 x, the volume percent is 0.5-1.5% of HEPES buffer solution 100 x, and the volume percent is 0-5ng/ml of epidermal growth factor; 5-15ng/mL fibroblast growth factor, 5-20ng/mL basic fibroblast growth factor, 0-750nM A83-01, 0-15 μ M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 5-15 μ M Y-27632, 5-15mM nicotinamide and 1-3% by volume percent B27 supplement 50X, diluted with basal medium Advanced DMEM/F12.
In some embodiments, the organoid medium a may consist of the following components and final concentrations: 1% by volume of antimicrobial-antimicrobial 100 x, 1% by volume of GlutaMAX100 x, and 1% by volume of HEPES buffer 100 x, based on the total volume of organoid medium a; 7-12ng/mL fibroblast growth factor 10, 7-12ng/mL basic fibroblast growth factor, 250-750nM A83-01, 5-7 μ M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 5-15 μ M Y-27632, 8-12mM nicotinamide and 2% by volume B27 supplement 50X, diluted with basal medium Advanced DMEM/F12, said organoid medium A being free of epidermal growth factor.
The organoid medium B may comprise the following components and final concentrations: based on the total volume of the organoid culture medium B, the volume percent of the organoid culture medium B is 0.5-1.5 percent of antimicrobial-antimicrobial 100 x, the volume percent of the organoid culture medium B is 0.5-1.5 percent of GlutaMAX100 x, the volume percent of the organoid culture medium B is 0.5-1.5 percent of HEPES buffer solution 100 x, and the volume percent of the organoid culture medium B is 30-75ng/ml of epidermal growth factor; 5-15ng/mL fibroblast growth factor 10, 5-20ng/mL basic fibroblast growth factor, 0-750nM A83-01, 0-15 μ M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 8-12mM nicotinamide and 1-3% by volume percent B27 supplement 50X, diluted with basal medium Advanced DMEM/F12, said organoid medium B containing no ROCK inhibitor.
In some embodiments, the organoid medium B may include the following components and final concentrations: 1% by volume of antimicrobial-antimicrobial 100 x, 1% by volume of GlutaMAX100 x, 1% by volume of HEPES buffer 100 x, 45-55ng/ml of epidermal growth factor, based on the total volume of organoid medium B; 5-7ng/mL fibroblast growth factor 10, 12-17ng/mL basic fibroblast growth factor, 250-750nM A83-01, 5-15. mu.M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 8-12mM nicotinamide and 2% by volume B27 supplement 50X, diluted in basal medium Advanced DMEM/F12, said organoid B medium being free of ROCK inhibitors.
The organoid medium B may consist of the following components and final concentrations: based on the total volume of the organoid culture medium B, the volume percent of the organoid culture medium B is 0.5-1.5 percent of antimicrobial-antimicrobial 100 x, the volume percent of the organoid culture medium B is 0.5-1.5 percent of GlutaMAX100 x, the volume percent of the organoid culture medium B is 0.5-1.5 percent of HEPES buffer solution 100 x, and the volume percent of the organoid culture medium B is 30-75ng/ml of epidermal growth factor; 5-15ng/mL fibroblast growth factor 10, 5-20ng/mL basic fibroblast growth factor, 0-750nM A83-01, 0-15 μ M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 8-12mM nicotinamide and 1-3% by volume percent B27 supplement 50X, diluted with basal medium Advanced DMEM/F12, said organoid medium B containing no ROCK inhibitor.
In some embodiments, the organoid medium B may consist of the following components and final concentrations: 1% by volume of antimicrobial-antimicrobial 100 x, 1% by volume of GlutaMAX100 x, 1% by volume of HEPES buffer 100 x, 45-55ng/ml of epidermal growth factor, based on the total volume of organoid medium B; 5-7ng/mL fibroblast growth factor 10, 12-17ng/mL basic fibroblast growth factor, 250-750nM A83-01, 5-15. mu.M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 8-12mM nicotinamide and 2% by volume B27 supplement 50X, diluted with basal medium Advanced DMEM/F12, said organoid medium B containing no ROCK inhibitor.
The neutralization medium may comprise: based on the total volume of the neutralization medium, the mixture is diluted with 0.5-1.5% by volume of antimicrobial-antimicrobial 100X, 0.5-1.5% by volume of GlutaMAX 100X, 0.5-1.5% by volume of HEPES buffer 100X, 7-13. mu.M of Y-27632 and 10-25% by volume of fetal bovine serum by Advanced DMEM/F12 medium. In some embodiments, the neutralization medium comprises: based on the total volume of the neutralization medium, 1% by volume of antimicrobial-antimicrobial 100X, 1% by volume of GlutaMAX 100X, 1% by volume of HEPES buffer 100X, 10. mu.M of Y-27632 and 20% by volume of fetal bovine serum were diluted with Advanced DMEM/F12 medium.
The Antibiotic-Antibiotic 100 x may comprise at least one of penicillin, streptomycin, or amphotericin B. In some embodiments, the Antibiotic-Antibiotic 100 x comprises penicillin, streptomycin, and amphotericin B.
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 antibiotic solution. In some embodiments, the final concentration of penicillin is 10000 units/mL based on the total volume of the antibiotic solution.
The final concentration of streptomycin may be 5000-15000 units/mL in total volume of Antibiotic-Antibiotic 100 ×. In some embodiments, the final concentration of streptomycin is 7000-12000 units/mL, based on a total volume of Antibiotic-Antibiotic 100 ×. In some embodiments, the final concentration of streptomycin is 10000 units/mL, based on a total volume of Antibiotic-Antibiotic 100 ×.
The final concentration of amphotericin B may be 10-35. mu.g/mL in total volume of Antibiotic-Antibiotic 100 ×. In some embodiments, the final concentration of amphotericin B is 15-30 μ g/mL in total volume of Antibiotic-Antibiotic 100 ×. In some embodiments, the final concentration of amphotericin B is 25 μ g/mL in total volume of Antibiotic-Antibiotic 100 ×.
The first digest may be Advanced DMEM/F12 medium containing collagenase type II and Y-27632.
The incubating may include: incubating at 37 deg.C for 1-2min, turning, and incubating for 8-10 min.
The final concentration of collagenase type II in the first digest solution may be 5 mg/ml.
The final concentration of Y-27632 in the first digest solution may be 10. mu.M.
The second digestive fluid may be a TrypLE Express pancreatin substitute solution comprising Y-27632.
In the second digest, the final concentration of Y-27632 may be 10. mu.M.
The culture conditions for the culture in step C) may be 37 ℃ and contain 5% by volume of CO2In an incubator.
Advantageous effects
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts two organoid culture mediums with different components and component contents to culture the bladder cancer organoid, can improve the culture success rate and passable times of the bladder cancer organoid, and solves the problems of less organoid growth, poorer cell morphology, slow growth and the like in the prior art.
(2) Furthermore, the invention screens the components and the contents of organoid culture medium; the components in the quasi-culture medium A and the quasi-culture medium B can play a synergistic role in the content range of the invention, can improve the culture success rate of bladder cancer organoids and organoid culture medium with passable times, and solves the problems of low culture success rate of bladder cancer organoids, less passable times and the like in the prior art.
(3) The culture medium is added with Antibiotic-Antibiotic, GlutaMAX and HEPES buffer solution, so that the risk of microbial contamination in the culture of bladder cancer organoids can be effectively reduced, and the pH stability of the environment can be maintained.
(4) The organoid culture medium and the method of the invention are adopted to culture the bladder cancer organoid, the obtained bladder cancer organoid is similar to the pathological morphological characteristics of bladder cancer tissues, and the obtained organoid can accurately restore the pathological morphological characteristics of tumor tissues from which the organoid is derived.
(5) The organoid culture medium and the method for culturing bladder cancer organoids have high culture success rate and more passage times.
Drawings
FIG. 1 shows the results of light microscopic observation of organoids of bladder cancer obtained by the organoid culture medium A and organoid culture medium B of example 2 and the culture method of example 4; as the culture time is prolonged, the volume of the bladder cancer organoids is gradually increased; the scale in the figure is 50 μm.
FIG. 2 is a diagram of the morphological structure of bladder cancer organoids obtained using organoid medium A and organoid medium B described in example 2 and the culturing method described in example 4; wherein Tissue-HE represents a hematoxylin-eosin staining pattern of bladder cancer Tissue; Organoid-BF shows a brightfield microscopic picture of bladder cancer organoids; Organoid-HE represents the hematoxylin-eosin staining pattern of bladder cancer organoids; the scale in the figure is 50 μm.
FIG. 3 is a graph showing the results of immunofluorescent staining identification of bladder cancer tissues of patients. The scale in the figure is 100. mu.m.
FIG. 4 is a graph showing the results of immunofluorescence staining identification of bladder cancer organoids obtained using organoid medium A and organoid medium B described in example 2 and the culture method described in example 4.
FIG. 5 is a graph of growth of passaged bladder cancer organoids using organoid culture medium A and organoid culture medium B described in example 2 and the passaging method described in example 6.
FIG. 6 is a graph showing the growth of a bladder cancer organoid obtained in comparative example 6; the scale in the figure is 50 μm.
FIG. 7 is a graph showing the growth of a bladder cancer organoid obtained in comparative example 7; the scale in the figure is 50 μm.
FIG. 8 is a graph showing the growth of a bladder cancer organoid obtained in comparative example 8; the scale in the figure is 50 μm.
Description of the terms
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 invention, DAPI is 4', 6-diamidino-2-phenylindole and is used for marking cell nucleus; CK5 is cytokeratin 5, used to label basal cells; CK7 is cytokeratin 7, used to label luminal cells; CK20 is cytokeratin 20, used to label luminal cells; p53 is P53 gene coding protein for marking important cancer suppressor protein; UPII is urosoluble protein and is used for marking urothelial cells; ECAD is E-cadherin, used to label epithelial cells; GATA3 is GATA binding protein 3, used to label luminal cells; ki67 is a mitosis-associated antigen used to label cell proliferation.
In the present invention, the ice bath condition means an ice-water mixture condition of about 0 ℃. The balance is the remaining content.
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 medium
Organoid media were prepared as in table 1:
table 1: formula of organoid culture medium
Example 2: organoid culture medium
Organoid media were prepared as in table 2:
table 2: formula of organoid culture medium
Example 3: organoid culture medium
Organoid media were prepared as in table 3:
table 3: formula of organoid culture medium
Example 4: culture of bladder cancer organoids
The composition of the neutralization medium was: based on the total volume of the neutralized culture solution, 1% by volume of antimicrobial-antimicrobial 100X, 1% by volume of GlutaMAX 100X, 1% by volume of HEPES buffer 100X, 10. mu.M of Y-27632 and 20% by volume of fetal bovine serum were diluted with Advanced DMEM/F12 medium.
The composition of the first digestive juice is: advanced DMEM/F12 medium containing collagenase type II at a final concentration of 5mg/ml and 10. mu.M of Y-27632.
The composition of the second digestive juice is: TrypLE Express pancreatin substitute solution containing 10. mu.M of Y-27632.
The operation is as follows: organoid culture medium A and organoid culture medium B described in examples 1-3 were taken separately and organoid cultured according to the following procedure:
A) taking bladder cancer tissue blocks, cutting the bladder cancer tissue blocks into minced meat, adding a first digestive juice, digesting for 45min at 37 ℃, centrifuging to obtain a first precipitate, digesting the first precipitate for 3 min by using a second digestive juice, centrifuging to obtain a second precipitate, re-suspending the second precipitate by using a neutralization culture solution, filtering by using a 70 mu m cell screen, and centrifuging filtrate to obtain a third precipitate;
B) resuspending the third precipitate with organoid medium A and matrigel in an ice bath, inoculating into a pre-warmed 6-well plate at 37 deg.C, and incubating again at 37 deg.C for 1-2 min; turning over immediately after the matrigel microdroplet is slightly formed, and incubating for 8-10min again;
C) and B), after the inoculated matrigel is solidified, adding an organoid culture medium A, culturing for 3 days, and then culturing for 7-12 days by using the organoid culture medium B to obtain the bladder cancer organoid, wherein the organoid culture medium B is replaced every 2-3 days.
The bladder cancer tissues and the obtained organoids are taken to be observed by an optical microscope, hematoxylin-eosin staining and immunofluorescence staining, the results are shown in figures 1-4, the results show that the obtained organoids are similar to the pathological morphological characteristics of the bladder cancer tissues, and the obtained organoids can accurately restore the pathological morphological characteristics of the tumor tissues from which the organoids are derived; the bladder cancer organoids cultured by the method of example 4 have a high growth rate, good cell morphology, and a high growth rate.
Comparative example 1: organoid culture medium (refer to CN111411083B medium formula)
A gastric cancer organoid culture medium 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 organ-like medium comprising 1% penicillin, 1% streptomycin, 50ng/mL EGF, 50ng/mL fibroblast growth factor, 50ng/mL Noggin recombinant protein, 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 medium comprising 50ng/mL EGF, 100ng/mL Wnt-3a, 1. mu.g/mL R-Spondin1, 500nM A83-01, 10. mu.M Y-27632, 50ng/mL Noggin recombinant protein, 12. mu.M SB202190, 1 XN 2, 1 XB 27 supplement, 10mM HEPES, 2mM Glutamax, 500 units/mL penicillin, 500 units/mL streptomycin, 12.5. mu.g/mL amphotericin, 10% FBS DMEM/F12 medium.
Comparative example 4: organoid culture medium (refer to CN111876386A medium formula)
Organoid media were prepared as in table 4:
table 4: formula of organoid culture medium
| Reagent | Final concentration |
| R-Spondin1 conditioned medium | 250ng/mL |
| Neuregulin 1 | 5nM |
| EGF | 5ng/mL |
| Noggin recombinant 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× |
Comparative example 5: organoid culture
Reagent: the same as in example 4.
The operation is as follows: organoid culture media described in comparative examples 1-4 were taken, and organoids were cultured according to the following procedure:
A) taking bladder cancer tissue blocks, cutting the bladder cancer tissue blocks 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 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 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 5min, and discarding supernatant to obtain fifth precipitate; resuspending the fifth pellet with 30 μ L organoid medium and 300 μ L matrigel, and inoculating;
D) c), after the inoculated matrigel is solidified, adding an organoid culture medium for culture, and replacing the culture medium every 2-3 days; culturing for 2-3 weeks to obtain organoid.
Example 5: culture success rate of different organ culture media
Bladder cancer tissues of different patients were collected and cultured 10 times according to the culture method of example 4, and the culture media of the bladder cancer organoids of examples 1 to 3 were examined. The success rates of the various organ culture media and the culture methods are shown in Table 4.
Table 4: culture success rate of different organ culture media
| Culture medium | Culture method | Success rate of cultivation |
| Example 1 | Example 4 | 85％ |
| Example 2 | Example 4 | 95％ |
| Example 3 | Example 4 | 90％ |
| Comparative example 1 | Example 5 | 65％ |
| Comparative example 2 | Example 5 | 80％ |
| Comparative example 3 | Example 5 | 75％ |
| Comparative example 4 | Example 5 | 75％ |
Example 6: organoid passage
First, passage of organoids
Reagent:
and (3) second digestive juice: the same as in example 1.
Organoid culture medium: any of examples 1-3 (the organoid medium used for passaging was the same as that used for organoid culture).
The operation is as follows:
centrifuging matrigel containing the obtained bladder cancer organoid in example 4 at 4 deg.C and 200g for 5min, discarding supernatant to obtain fourth precipitate, adding second digestive juice into the fourth precipitate, and digesting at 37 deg.C for 3-5 min with shaking table (120 rpm); the neutralization medium described in example 4 was added to stop digestion; centrifuging for 5 minutes at 4 ℃ under 200g, discarding the supernatant to obtain a fifth precipitate, resuspending the fifth precipitate by 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 200g, discarding the supernatant to obtain a sixth precipitate, adding 30 muL of precooled organoid culture medium A to resuspend the cell precipitate, adding 600 muL of matrigel, inoculating, solidifying the inoculated matrigel, adding organoid culture medium A to culture for 3 days, and culturing for 7-12 days by organoid culture medium B to obtain the subcultured bladder cancer organoid, wherein the organoid culture medium B is replaced once every 2-3 days.
Second, minimum passable number statistics
The organoids obtained in example 4 and comparative examples 1-5 were serially passaged as described above, and each time 30% of the organoids expanded to form a cell mass with a diameter exceeding 200 μm, the minimum passable number was recorded. The statistical results are shown in Table 5.
Table 5: minimum passable times statistical table
| Culture medium | Minimum passable number of times |
| Example 1 | 15 times of |
| Example 2 | 20 times (twice) |
| Example 3 | 13 times (twice) |
| Comparative example 1 | 4 times (twice) |
| Comparative example 2 | 6 times of |
| Comparative example 3 | 5 times (twice) |
| Comparative example 4 | 6 times of |
Comparative example 6: organoid culture medium without epidermal growth factor and culture result thereof
Organoid media were prepared as in table 6:
table 6: formula of organoid culture medium
Culture of bladder cancer organoids: the organoid medium A and organoid medium B of comparative example 6 were used to culture bladder cancer organoids according to the procedure described in example 4, and the resulting bladder cancer organoids were observed by light microscopy.
And (3) culturing results: referring to FIG. 6, when the culture medium of comparative example 6 was used to culture bladder cancer organoids according to the procedure of example 4, it was found that the organoids grew less, the cell morphology was poor, and the growth was slow.
Comparative example 7: organoid culture medium without B27 supplement and culture results thereof
Organoid media were prepared as in table 7:
table 7: formula of organoid culture medium
Culture of bladder cancer organoids: the organoid medium A and organoid medium B of comparative example 7 were used to culture bladder cancer organoids according to the procedure described in example 4, and the resulting bladder cancer organoids were observed by light microscopy.
And (3) culturing results: referring to FIG. 7, when the culture medium of comparative example 7 was used and cultured according to the procedure of example 4, organoids were found to grow less, the morphology of cells was poor, and the growth was slow.
Comparative example 8: r-spondin 1-free organoid culture medium and culture results thereof
Organoid media were prepared as per table 8:
table 8: formula of organoid culture medium
Culture of bladder cancer organoids: the organoid medium A and organoid medium B of comparative example 8 were used to culture bladder cancer organoids according to the procedure described in example 4, and the resulting bladder cancer organoids were observed by light microscopy.
And (3) culturing results: referring to FIG. 8, when the culture medium of comparative example 8 was used to culture bladder cancer organoids according to the procedure of example 4, it was found that the organoids grew less, the cell morphology was poor, and the growth was slow.
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 (10)
1. A method for culturing a bladder cancer organoid, comprising the steps of:
A) taking bladder cancer tissue blocks, cutting into pieces, adding a first digestive juice for digestion, centrifuging to obtain a first precipitate, digesting the first precipitate with a second digestive juice, centrifuging to obtain a second precipitate, re-suspending the second precipitate with a neutralization culture solution, filtering to remove large tissue blocks, and centrifuging filtrate to obtain a third precipitate;
B) resuspending the third precipitate with organoid culture medium A and matrigel under ice bath, inoculating, and incubating;
C) and C) after the matrigel inoculated in the step B) is solidified, adding the organoid culture medium A, culturing for 3 days, and then culturing for 7-12 days by using the organoid culture medium B to obtain the bladder cancer organoid.
2. The method of claim 1, wherein organoid medium B in step C) is replaced every 2-3 days.
3. The method of any one of claims 1-2, wherein organoid medium a comprises the following components and final concentrations: based on the total volume of the organoid culture medium A, the volume percent is 0.5-1.5% of antimicrobial-antimicrobial 100 x, the volume percent is 0.5-1.5% of GlutaMAX100 x, the volume percent is 0.5-1.5% of HEPES buffer solution 100 x, and the volume percent is 0-5ng/ml of epidermal growth factor; 5-15ng/mL fibroblast growth factor, 5-20ng/mL basic fibroblast growth factor, 0-750nM A83-01, 0-15 μ M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 5-15 μ M Y-27632, 5-15mM nicotinamide and 1-3% by volume percent B27 supplement 50X, diluted with basal medium Advanced DMEM/F12.
4. The method of claim 3, wherein organoid medium A comprises the following components and final concentrations: 1% by volume of antimicrobial-antimicrobial 100 x, 1% by volume of GlutaMAX100 x, and 1% by volume of HEPES buffer 100 x, based on the total volume of organoid medium a; 7-12ng/mL fibroblast growth factor 10, 7-12ng/mL basic fibroblast growth factor, 250-750nM A83-01, 5-7. mu.M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 5-15. mu.M Y-27632, 8-12mM nicotinamide and 2% by volume B27 supplement, diluted with basal medium Advanced DMEM/F12, said organoid medium A being free of epidermal growth factor.
5. The method of claim 1, wherein organoid medium B comprises the following components and final concentrations: based on the total volume of the organoid culture medium B, the volume percent of the organoid culture medium B is 0.5-1.5 percent of antimicrobial-antimicrobial 100 x, the volume percent of the organoid culture medium B is 0.5-1.5 percent of GlutaMAX100 x, the volume percent of the organoid culture medium B is 0.5-1.5 percent of HEPES buffer solution 100 x, and the volume percent of the organoid culture medium B is 30-75ng/ml of epidermal growth factor; 5-15ng/mL fibroblast growth factor 10, 5-20ng/mL basic fibroblast growth factor, 0-750nM A83-01, 0-15 μ M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 8-12mM nicotinamide and 1-3% by volume percent B27 supplement 50X, diluted with basal medium advanced DMEM/F12, said organoid medium B being free of ROCK inhibitors.
6. The method of claim 5, wherein organoid medium B comprises the following components and final concentrations: 1% by volume of antimicrobial-antimicrobial 100 x, 1% by volume of GlutaMAX100 x, 1% by volume of HEPES buffer 100 x, 45-55ng/ml of epidermal growth factor, based on the total volume of organoid medium B; 5-7ng/mL fibroblast growth factor 10, 12-17ng/mL basic fibroblast growth factor, 250-750nM A83-01, 5-15. mu.M SB202190, 50-150ng/mL Noggin recombinant protein, 250-750ng/mL Rspondin-1 recombinant protein, 0.8-1.2mM N-acetylcysteine, 8-12mM nicotinamide and 2% by volume B27 supplement 50X, diluted in basal medium Advanced DMEM/F12.
7. The method of claim 1, wherein the neutralization medium comprises: based on the total volume of the neutralization medium, 0.5-1.5% by volume of antimicrobial-antimicrobial 100X, 0.5-1.5% by volume of GlutaMAX 100X, 0.5-1.5% by volume of HEPES buffer 100X, 7-13. mu.M of Y-27632 and 10-25% by volume of fetal bovine serum are diluted with Advanced DMEM/F12 medium, and the organoid medium B does not contain ROCK inhibitor.
8. The method of claim 1, wherein said first digest is Advanced DMEM/F12 medium comprising collagenase type ii and Y-27632.
9. The method of claim 1, said second digestive fluid is a TrypLE Express pancreatin substitute solution comprising Y-27632.
10. The method according to claim 1, wherein the culturing in step C) is carried out at 37 ℃ and contains 5% by volume of CO2In an incubator.
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