CN112680417A - Ovarian cancer organoid culture medium and culture method - Google Patents
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Abstract
The invention relates to a biological medicine technology, in particular to a culture medium and a culture method for ovarian cancer organoids. The culture medium comprises: basal medium DMEM/F12, B27, glutamine, HEPES, N-acetylcysteine, FGF2, A83-01, nicotinamide, Forskolin (Forskolin), beta-estradiol, Hydrocortisone (Hydrocortisone), R-spondin-1, Noggin (Noggin), FGF10, EGF, Heregulin beta 1, and at least one selected from PFBS, PFOA and PFOS. The culture medium and the culture method improve the success of establishing the ovarian cancer culture medium, increase the amplification efficiency of ovarian cancer organoids, stabilize the proliferation efficiency after passage, accelerate the drug screening process and reduce the cost of culture materials.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to an ovarian cancer organoid culture medium and a culture method.
Background
Organoids (Organoids) are a three-dimensional culture system in vitro in a mixed cell state, which has a self-renewing stem cell population, has individual differences and tissue specificities, has consistent spatial characteristics with the source tissue and reproduces part of the functions of the source tissue, thereby providing a highly physiologically relevant simulation system. Based on the characteristics, the method constructs tumor organoids (PDO) from tumor tissues, can be applied to the explanation of tumor occurrence mechanisms, the screening of high-flux medicines, the research and development of treatment targets and individualized treatment, and has extremely high scientific research value and clinical value.
Ovarian cancer is a common disease threatening the health of modern women, two-line chemotherapy schemes and operations are two important treatment means for ovarian malignant tumor at present, and the current two-line chemotherapy schemes and operations need auxiliary chemotherapy except for few early cancers which can be cured by simple operations. The choice of chemotherapeutic regimen has always been a clinical challenge and focus. Tumor organoids derived from tumor patients have proven to be valuable diagnostic tools in precision medical applications. For example, using organoid in vitro screening of drugs from patient samples can provide guidance for the care of cancer patients and predict the outcome of treatment with the drugs. Successful PDO establishment is mainly marked by two factors: firstly, the in vivo characteristics of the original tumor are stably maintained when the in vitro amplification is carried out in a large quantity; secondly, the appearance of the source tissue is consistent.
At present, the most advanced establishment and culture method of ovarian cancer organoids is a method published by researchers such as Hans Clevers and the like in 2019 and 4 months on Nature Medicine, and Neugulin-1 cytokine necessary for the growth of ovarian cancer is added to a conventional organoid culture medium (containing cytokines WNT, Noggin, EGF and Rspo1), and hormones required for the growth of ovarian cancer cells such as hydrocortisone, beta-estradiol and the like are supplemented, so that a plurality of ovarian cancer organoids are successfully established. Even so, ovarian cancer organoid culture still requires large amounts of ovarian cancer tissue, and several media at the same time for reagents in the primary setting. Therefore, the method has the problems of high culture cost and unsatisfactory culture success rate (mainly manifested by difficult passage and low initial establishment success rate).
Disclosure of Invention
In order to solve the problems, the invention relates to a culture medium formula for preparing ovarian cancer organoids, which can promote the rapid proliferation of tumor tissues, improve the culture success rate of the ovarian cancer organoids and reduce the culture cost.
In the process of forming tumor organoids, tumor cells with drying potential can self-assemble to form 3D structural tissues with certain spatial structures and certain physiological functions. Theoretically, an ideal ovarian cancer organoid can accurately mimic the epithelial structure of ovarian cancer and can be subcultured for a long period of time.
The reported establishment method of ovarian cancer organoids mainly has the following problems:
1. the establishment process is complicated, and tumor cells are damaged by multiple operations, so that the establishment success rate is low and the cost is high;
2. culture failure due to contamination by surgical-or patient-derived microorganisms;
3. the heterogeneity of the tumor causes low proliferation efficiency after organoid passage, and is difficult to passage for many times.
The invention patent of Sunzhijia et al in China "a method for preparing ovarian cancer organoid" (patent No. CN111040996A), human endometrial stem cells without proliferation function adopted by Sunzhijia et al are all used as feeder cells for culturing and establishing ovarian cancer organoid, because the feeder cells have more uncontrollable factors (sources and individual differences) and are more complicated, the method has high cost, complicated operation and high establishing difficulty, and can not replace the method for establishing ovarian cancer organoid by using matrigel.
Aiming at the problems, the technology optimizes the culture medium and the culture method to successfully establish the ovarian cancer organoid from the ascites and the tumor tissues of the ovarian cancer patient.
Supplement after claims are determined
The culture medium improves the success rate of establishing the ovarian cancer culture medium (the success rate is increased), increases the amplification efficiency of ovarian cancer organoids (the passage time is shortened), and has stable proliferation efficiency after passage, thereby accelerating the drug screening process and reducing the cost of culture materials when the culture medium is used for in vitro drug screening.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the establishment of ovarian cancer tumor organoids.
FIG. 2 is the establishment of ovarian cancer organoids from ascites or tissue from ovarian cancer patients.
FIG. 3 shows the result of culturing the ovarian cancer organoids in example 1.
Detailed Description
In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.
The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
ITS, insulin iron selenium transport protein, is a mixture of insulin, transferrin and selenium, and has been applied to the construction of tissue engineered cartilage, the expansion and redifferentiation of chondrocytes.
FGF, fibroblast growth factor, a polypeptide secreted by the pituitary and hypothalamus. Can promote the migration of endothelial cells and the proliferation of smooth muscle cells, promote the formation of new blood vessels, and repair damaged endothelial cells.
The dual-resistance added in the cleaning solution reduces the possibility of microbial infection during the primary culture of the organoid.
In a preferred embodiment of the present invention, the formulation of the reagent used in the present invention is shown in the following table:
the invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.
Example 1: one example of an ovarian cancer organoid was established from ascites in stage III epithelial ovarian cancer patients.
Step 1: separation of the cells is achieved. 50ml of ascites specimen is collected from the operation, after non-cell components are removed by filtering through a sterile aperture screen, the specimen is centrifuged at the rotating speed of 100-200g for 5-10 minutes at low temperature, and the supernatant is discarded.
Step 2: to achieve tumor cell aggregation: the cells obtained by centrifugation are lysed by 2-5ml of erythrocyte lysate to remove erythrocytes, and RPMI 1640 culture medium is added for neutralization and centrifugation to remove the lysate. Adding a cleaning solution①(sterile PBS solution with 100U/ml penicillin, 0.1mg/ml streptomycin) 8-10ml resuspended centrifuged cells and transferred to a 15ml centrifuge tube with 50-200g for 5 minutes, the supernatant discarded and the procedure repeated 2-5 times. A small amount of the solution was taken before the final centrifugation step and the number of cells was counted under a microscope.
And step 3: primary culture and replanting of organoids: dissolving the matrix glue on ice, mixing the tumor cells obtained by centrifugation into the matrix glue with proper volume for resuspension, dripping 25 mu L of matrix glue mixed liquor in the center of a 48-hole cell culture dish, and immediately placing the culture plate into an incubator for 0.5h after the sample addition is finished so as to polymerize the matrix glue into hemispheres. After multimerization 300. mu.L of Advanced DMEM/F12 medium mixed with various small molecule substances and cytokines was added to each well. The main components are cytokines and small molecular compounds such as 100U/mL penicillin, 0.1mg/mL streptomycin, 500nmol/L A83-01, 10mmol/L nicotinamide, 10nmol/L forskolin, 100nmol/L PFBS, 500ng/mL hydrocortisone, 100ng/mL cholera toxin, 100nmol/L beta-estradiol, 1 mu mol/L PEG2, 1000mg/mL R-spondin-1, 200ng/mL FGF2, 50ng/mL EGF and the like. Weighing the culture platePlacing into incubator, and replacing culture medium every 2 days③. And observing the morphological change of the cells under a microscope in the culture process, and embedding for subculture after budding reaches a certain length after about 7 days of culture.
The results are shown in FIG. 3, successful establishment of ovarian cancer organoids. Hans Clevers et al performed organoid culture on ovarian cancer tissues from 49 patients, and finally established 32 cases of ovarian cancer organoids successfully, with a success rate of 65% and a passage time of 1-4 weeks. The culture medium in the embodiment carries out organoid culture on the operation samples of 23 ovarian cancer patients, so that 18 ovarian cancer organoids are successfully established, the success rate is 78%, and the passage time is 7-15 days. Description of the culture Medium: 1) the success rate of establishing the ovarian cancer culture medium is improved (the success rate is increased); 2) the expansion efficiency of the ovarian cancer organoid is increased (the passage time is shortened); 3) because the amplification efficiency is increased and the proliferation efficiency after passage is stable, the drug screening process is accelerated and the cost of culture materials is reduced when the in vitro drug screening is performed.
Example 2: one example of an ovarian cancer organoid was established from in situ tumors of stage III epithelial ovarian cancer patients.
Step 1: pretreatment of tumor tissues: the epithelial ovarian cancer patient in-situ tissue sample is cut into pieces, washed by a cleaning solution (sterile PBS solution added with 100U/ml of penicillin and 0.1mg/ml of streptomycin) for multiple times, and cut into minced meat with the size of about 1-cubic millimeter in a sterile environment.
Step 2: preparing ovarian cancer single cells, adding 5-8ml of Advanced DMEM/F12 culture medium containing 5mg/ml collagenase type II and 5 mu M Y-27632, and placing in a constant temperature incubator at 37 ℃ for enzymolysis for 0.5-2 hours; sieving with 100 μm cell sieve after enzymolysis, adding Advanced DMEM/F12 culture medium to stop digestion, centrifuging for 5-10min under the condition of 100-200g centrifugation, discarding supernatant (repeating once), and centrifuging to obtain precipitate as ovarian cancer cells; a small amount of the solution was taken before the final centrifugation step and the number of cells was counted under a microscope.
And step 3: primary culture and replanting of organoids: dissolving the stroma glue on ice, mixing the tumor cells obtained by centrifugation into the stroma glue with proper volume for resuspension, and dripping the stroma glue into the center of a 48-hole cell culture dish25 mu L of matrigel mixed solution, and immediately placing the culture plate into an incubator for 0.5h after the sample is added, so that the matrigel is polymerized into a hemisphere shape. After multimerization 300. mu.L of Advanced DMEM/F12 medium mixed with various small molecule substances and cytokines (the feasible combinations are shown in the table below) was added to each well. Placing the culture plate into the incubator again, and replacing the culture medium every 2 days③. And observing the morphological change of the cells under a microscope in the culture process, and embedding for subculture after budding reaches a certain length after about 7 days of culture.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (11)
1. An ovarian cancer organoid culture medium comprising: basal medium DMEM/F12, B27, glutamine, HEPES, N-acetylcysteine, FGF2, A83-01, nicotinamide, Forskolin (Forskolin), beta-estradiol, Hydrocortisone (Hydrocortisone), R-spondin-1, Noggin (Noggin), FGF10, EGF, Heregulin beta 1, and at least one selected from PFBS, PFOA and PFOS.
2. The culture medium according to claim 1, comprising basal medium DMEM/F12 and the following concentrations of the components: 0.5X-2 XB 27, 1-3mM glutamine, 0.5-5% (w/v) HEPES, 0.5-5mmol/L N-acetylcysteine, 20-200ng/ml FGF2, 100-1000nmol/L A83-01, 5-20mmol/L nicotinamide, 5-20nmol/L Forskolin (Forskolin), 50-200nmol/L beta-estradiol, 100-1000ng/ml Hydrocortisone (Hydrocortisone), 200-1000mg/ml R-spondin-1, 50-200ng/ml Noggin (Noggin), 10-500ng/ml FGF10, 10-50ng/ml EGF, 30-100ng/ml Heregulin beta 1, and 10-1000nmol/L PFBS, PFOA or PFOS based on the total volume of the medium.
3. The culture medium of claim 1 or 2, further comprising one or more (e.g., 2, 3, 4, 5, or 6) of the following components: cholera toxin, Y-27632, PEG2, LIF, SB202190, and antibiotics;
preferably, the antibiotic is selected from: penicillin, streptomycin, Normocin, Primocin, or a combination thereof;
preferably, the culture medium further comprises cholera toxin, PEG2, and an antibiotic;
preferably, the culture medium further comprises cholera toxin, SB202190, LIF, and antibiotics;
preferably, the culture medium further comprises PEG2, LIF, and antibiotics;
preferably, the culture medium further comprises Y-27632, PEG2, LIF, and antibiotics;
preferably, the medium further comprises cholera toxin, Y-27632, SB202190, and an antibiotic;
preferably, the culture medium further comprises Y-27632, PEG2, SB202190, and an antibiotic;
preferably, the medium further comprises cholera toxin, Y-27632, PEG2, LIF, SB202190, and an antibiotic.
Preferably, the concentration of the cholera toxin is 50-200 ng/mL; the concentration of Y-27632 is 5-20 mu M; the concentration of PEG2 is 0.5-2 μmol/L; LIF concentration is 5-50 ng/ml; the concentration of SB202190 is 5-20 μmol/L; the concentration of the penicillin is 50-200U/ml; the concentration of streptomycin is 0.05-0.2 mg/ml; the concentration of Normocin is 50-200 mg/ml; and/or the concentration of Primocin is 50-200 mg/ml; based on the total volume of the culture medium.
4. The culture medium according to claim 1, comprising a basal medium DMEM/F12 and an additional factor selected from the group consisting of:
(a)100U/mL penicillin, 0.1mg/mL streptomycin, 1 XB 27, 2mM glutamine, 1% HEPES, 1.25mmol/L N-acetylcysteine, 500nmol/L A83-01, 10mmol/L nicotinamide, 10nmol/L forskolin, 100nmol/L PFBS, 500ng/mL hydrocortisone, 100ng/mL cholera toxin, 100nmol/L beta-estradiol, 1. mu. mol/L PEG2, 1000mg/mL R-spondin-1, 100ng/mL noggin, 100ng/mL FGF10, 200ng/mL FGF2, 50ng/mL EGF and 30ng/mL Heregulin beta 1;
(b)100U/mL penicillin, 0.1mg/mL streptomycin, 100mg/mL Normocin, 1 XB 27, 2mM glutamine, 1% HEPES, 1.25mmol/L N-acetylcysteine, 500nmol/L A83-01, 10nmol/L forskolin, 10mmol/L nicotinamide, 100nmol/L PFBS, 20. mu. mol/L SB202190, 100nmol/L beta-estradiol, 500ng/mL hydrocortisone, 100ng/mL cholera toxin, 1000mg/mL R-spondin-1, 100ng/mL noggin, 100ng/mL FGF10, 200ng/mL FGF2, 20ng/mL LIF, 50ng/mL EGF and 100ng/mL Heregulin beta 1;
(c)100mg/ml Primocin, 100mg/ml Normocin, 1 XB 27, 2mM glutamine, 1% HEPES, 1.25mmol/L N-acetylcysteine, 500nmol/L A83-01, 1.5 μmol/L PEG2, 10nmol/L forskolin, 10mmol/L nicotinamide, 100nmol/L PFBS, 100nmol/L β -estradiol, 500ng/ml hydrocortisone, 1000mg/ml R-spondin-1, 100ng/ml noggin, 100ng/ml FGF10, 50ng/ml FGF2, 50ng/ml EGF, 20ng/ml LIF, and 100ng/ml Heregulin β 1; or
(d)100mg/ml Primocin, 100mg/ml Normocin, 1 XB 27, 2mM glutamine, 1% HEPES, 1.25mmol/L N-acetylcysteine, 500nmol/L A83-01, 10mmol/L nicotinamide, 10. mu. mol/L SB 202190; 10nmol/L PFBS, 20. mu. M Y-27632, 100nmol/L beta-estradiol, 500ng/mL hydrocortisone, 100ng/mL cholera toxin, 1000mg/mL R-spondin-1, 100ng/mL noggin, 200ng/mL FGF10, 100ng/mL FGF2, 50ng/mL EGF and 100ng/mL Heregulin beta 1.
5. Use of a culture medium according to any one of claims 1 to 4 in the preparation of an ovarian cancer organoid and/or in the preparation of a reagent for an ovarian cancer organoid.
6. A method of culturing an ovarian cancer organoid, comprising: culturing ovarian cancer cells by mixing the ovarian cancer cells with the culture medium according to any one of claims 1 to 4 and the matrigel, thereby obtaining an ovarian cancer organoid.
7. The method of claim 6, wherein the method comprises the steps of:
(i) obtaining ovarian cancer cells and preparing ovarian cancer cell suspension; preferably, the ovarian cancer cells are derived from ascites or ovarian cancer tissue of an ovarian cancer patient;
(ii) resuspending the ovarian cancer cell suspension and matrigel, inoculating to a culture container (such as cell culture plate, cell culture dish), and solidifying; and
(iii) adding the culture medium of any one of claims 1 to 5 to a culture vessel, and culturing until the ovarian cancer organoids are obtained.
8. The method according to claim 6, wherein the ovarian cancer cells are derived from ovarian cancer tissue of an ovarian cancer patient, and step (i) comprises subjecting the ovarian cancer tissue to enzymatic digestion to obtain ovarian cancer cells in a DMEM/F12 medium containing 1-10 μ M Y-27632 and 1-10mg/ml collagenase.
9. The method of claim 7, wherein step (i) further comprises washing the ovarian cancer cells or ovarian cancer patient in situ tissue with a wash solution, wherein the wash solution is a PBS solution supplemented with penicillin (preferably 50-200U/ml) and streptomycin (preferably 0.05-0.2 mg/ml).
10. An ovarian cancer organoid prepared by the method of any one of claims 6 to 9.
11. Use of the ovarian cancer organoid of claim 10 for in vitro high throughput screening of ovarian cancer targeted drugs and evaluation of susceptibility to anti-ovarian cancer drugs.
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