WO2023060774A1 - Milieu de culture pour organoïdes de cancer du col utérin, procédé de culture et application associée - Google Patents

Milieu de culture pour organoïdes de cancer du col utérin, procédé de culture et application associée Download PDF

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WO2023060774A1
WO2023060774A1 PCT/CN2021/142274 CN2021142274W WO2023060774A1 WO 2023060774 A1 WO2023060774 A1 WO 2023060774A1 CN 2021142274 W CN2021142274 W CN 2021142274W WO 2023060774 A1 WO2023060774 A1 WO 2023060774A1
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cervical cancer
alkyl
culture
medium
organoids
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PCT/CN2021/142274
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Chinese (zh)
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刘青松
胡洁
黄涛
陈程
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合肥中科普瑞昇生物医药科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

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  • the invention belongs to the field of biotechnology, and in particular relates to a culture medium for cervical cancer organoids and a method for cultivating cervical cancer organoids using the medium.
  • Cervical cancer is a malignant tumor that originates in the cervix and is the most common malignant tumor in the female reproductive system. Cervical cancer ranks fourth among female malignancies worldwide. The highest incidence of cervical cancer is between 30 and 55 years old, and the incidence rate in my country is only behind breast cancer. Due to the popularity of cervical cancer screening and the promotion of HPV vaccine, cervical cancer has largely become a preventable disease, but its 5-year survival rate is still only about 60%. For early non-metastatic cervical cancer, surgery, radiotherapy and chemotherapy are the main treatment methods, but for metastatic or recurrent cervical cancer, traditional treatment methods have not achieved satisfactory results. With the use of targeted therapy and immunotherapy drugs such as anti-angiogenic drugs and immune checkpoint inhibitors, the survival time of these patients has been significantly prolonged, but the final cure effect has not yet been achieved. Cells serve as research models to discover new drug targets and guide personalized therapy.
  • Organoids which belong to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells and adult stem cells with differentiation ability. Endogenous tissue stem cells exist in different tissues and organs, and play an important role in maintaining the functional morphology of various organs. Under certain induction conditions in vitro, these stem cells can self-organize to form a miniature structure with a diameter of only a few millimeters.
  • Tumor organoids are obtained from primary tumors in patients, and some miniature 3D tumor cell models are cultivated in the laboratory. Tumor organoids highly simulate the characteristics of the source tumor tissue, retain the tumor heterogeneity among individuals, and can be used for functional testing, such as drug screening and individualized precision therapy.
  • cervical cancer organoid culture methods mostly use expensive protein factors such as R-spondin-1, WNT3A, and Noggin, resulting in high cost of organoid culture; and this technology is complicated to operate and technically difficult, leading to its large-scale commercialization Application is limited. Therefore, there is a need to develop a low-cost, simple and high success rate organoid culture method and medium.
  • the present invention provides a culture medium and a culture method for rapid expansion of cervical cancer organoids in vitro.
  • One aspect of the present invention is to provide a culture medium for cervical cancer organoids, said culture medium comprising an MST1/2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, hepatocyte growth factor, SB202190, Y27632 , A83-01, Epidermal Growth Factor, Fibroblast Growth Factor 10, Keratinocyte Growth Factor, GlutaMAX, and Niacinamide.
  • the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof,
  • R 1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted by 1-2 independent R (such as phenyl and naphthyl, etc.), aryl C1-C6 alkyl (such as benzyl, etc.) and heteroaryl (such as thienyl, etc.);
  • R 2 and R 3 are each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
  • R 4 and R 5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxyl, C1-C6 haloalkyl, C1-C6 Alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyran base, etc.);
  • R is selected from halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoro methyl).
  • halogen preferably fluorine and chlorine, more preferably fluorine
  • C1-C6 alkyl preferably methyl
  • C1-C6 alkoxy preferably methoxy
  • C1-C6 haloalkyl preferably trifluoro methyl
  • the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt thereof, or a solvate,
  • R is selected from C1-C6 alkyl, phenyl optionally substituted by 1-2 independently R6 , thienyl optionally substituted by 1-2 independently R6 , and optionally substituted by 1 -2 independently R6 substituted benzyl, R1 is more preferably optionally 1-2 independently R6 substituted phenyl;
  • R 5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 is more preferably hydrogen;
  • R 6 is each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, and R 6 is more preferably fluorine, methyl or trifluoromethyl.
  • the MST1/2 inhibitor is at least one selected from the following compounds or pharmaceutically acceptable salts or solvates thereof.
  • the MST1/2 kinase inhibitor of the present invention is Compound 1.
  • the content of each component in the culture medium of the present invention satisfies any one or more or all of the following:
  • the concentration of the MST1/2 kinase inhibitor is preferably 2.5-10 ⁇ M
  • the concentration of hepatocyte growth factor is preferably 5-40 ng/mL
  • the concentration of SB202190 is preferably 200-1000nM
  • the concentration of Y27632 is preferably 2.5-10 ⁇ M
  • the concentration of A83-01 is preferably 200-1000nM
  • the concentration of epidermal growth factor is preferably 1-40 ng/mL;
  • the concentration of fibroblast growth factor 10 is preferably 10-100 ng/mL
  • the concentration of keratinocyte growth factor is preferably 2-40 ng/mL;
  • the volume ratio of GlutaMAX to the medium is preferably 1:50 to 1:200;
  • the concentration of nicotinamide is preferably 1 to 10 mM.
  • the medium also contains an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one selected from streptomycin/penicillin, amphotericin B and Primocin one or more antibiotics.
  • the streptomycin concentration ranges from 25 to 400 ⁇ g/mL, and the penicillin concentration ranges from 25 to 400 U/mL; when the antibiotic is selected from amphotericin B, The concentration range is 0.25-4 ⁇ g/mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 ⁇ g/mL.
  • the invention also provides a method for culturing cervical cancer organoids.
  • cervical cancer organoids are cultured using the cervical cancer organoid culture medium of the present invention.
  • the method for culturing cervical cancer organoids of the present invention includes the following steps.
  • the process includes the following steps:
  • tissue samples Separate cervical cancer tissue samples, add basal culture medium and tissue digestion solution at a ratio of 1:1 (the amount of tissue digestion solution is about 10 mL of tissue digestion solution per 1 g of tumor tissue) and place in a constant temperature shaker for digestion , the digestion temperature is 4-37°C, the shaker speed is 200rpm-300rpm, and the digestion time is 3-6 hours;
  • the basal medium formula includes an initial medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin/penicillin, amphotericin B and Primocin.
  • the formula of tissue digestion solution includes 1640 medium, collagenase II (1-2 mg/mL), collagenase IV (1-2 mg/mL), DNase (50-100 U/mL), hyaluronidase (0.5-1 mg /mL), calcium chloride (1 ⁇ 5mM), bovine serum albumin BSA (5 ⁇ 10mg/mL).
  • the cervical cancer primary cells obtained in the above step 1 Resuspend the cervical cancer primary cells obtained in the above step 1 with the cervical cancer organoid culture medium of the present invention and count them, dilute the cell density to 5-10 ⁇ 105 cells/mL, take out the diluted cell suspension and add Mix in an equal volume of Matrigel matrigel, then inoculate the mixture on a multi-well plate, put the inoculated multi-well plate in the incubator for 30-60 minutes, wait until the Matrigel is completely solidified, and then add cervical cancer organoid medium for expansion culture.
  • a method for assessing or screening a drug for the treatment of cervical cancer characterized in that it comprises the following steps:
  • the culture cost is controllable, and the medium does not need to add expensive Wnt agonists, R-spondin family proteins and Noggin proteins;
  • the number of cervical cancer organoids obtained by the technique is large, which is suitable for high-throughput screening of candidate compounds and providing patients with high-throughput drug sensitivity functional tests in vitro.
  • 1A-1K are graphs showing the effects of different concentrations of factors added to the cervical cancer organoid medium of the present invention on the proliferation of cervical cancer organoids.
  • Figures 2A-2D are photographs of cervical cancer organoids cultured using the cervical cancer organoid medium of the present invention observed under a microscope, wherein Figure 2A shows photos of organoids obtained from sample OC1 cultured for 5 days; Figure 2B shows photos obtained from sample OC1 Photos of organoids obtained from OC1 after 14 days of culture; Figure 2C and 2D show photos of different fields of view of organoids obtained from sample OC2 after 7 days of culture.
  • Fig. 3 is the result of pathological and immunohistochemical identification of the original tissue sample OC4 and the cervical cancer organoid obtained by using the cervical cancer organoid culture medium culture sample OC4 of the present invention.
  • Figures 4A and 4B are the comparison results of using the cervical cancer organoid culture medium of the present invention and the culture medium of the prior art to culture cervical cancer organoids, wherein Figure 4A shows the photos after culturing for 10 days with the COM medium of the present invention; Fig. 4B shows photographs after 10 days of culture with literature medium ROM.
  • Fig. 5 is a graph showing the results of drug concentration sensitivity testing of cervical cancer organoids cultured using the cervical cancer organoid medium of the present invention.
  • an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signal transduction.
  • MST1/2 kinase inhibitors for example, bind to MST1/2 kinase and reduce its activity. Due to the similarity in the structures of MST1 and MST2, MST1/2 kinase inhibitors may also be, for example, compounds that bind to MST1 or MST2 and reduce their activity.
  • 2-Amino-2-(2,6-difluorophenyl)acetic acid methyl ester (A2): In a round bottom flask was added 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) Methanol (30 mL) was then added, followed by the dropwise addition of thionyl chloride (1.2 mL) under ice-cooling. The reaction system was reacted overnight at 85°C. After the reaction, the system was evaporated to dryness under reduced pressure to obtain a white solid, which was directly used in the next step.
  • MST1/2 inhibitor compounds of the present invention were synthesized according to a method similar to compound 1, and their structures and mass spectrometry data are shown in the table below.
  • the initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art.
  • the formulation of the basal medium is: DMEM/F12 medium (purchased from Corning Company)+100 ⁇ g/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercially available product concentration 50mg/ml ).
  • Intraoperative samples were obtained from patients by professional medical staff of professional medical institutions, and all patients signed informed consent. Intraoperative samples of 5-10 mm 3 were stored and transported in commercial tissue preservation solution (manufacturer: Miltenyi Biotec).
  • Tissue digestion solution formula 1640 medium (Corning, 10-040-CVR), collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3mM), bovine serum albumin BSA (10mg/mL).
  • Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma; calcium chloride and BSA were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.
  • the medium added with this additive has the effect of promoting the proliferation of at least two cases of cervical cancer organoids isolated from cervical cancer tissue;
  • “-” means that the medium with this additive added
  • the culture medium has an inhibitory effect on at least one case of cervical cancer organoids isolated from cervical cancer tissues;
  • “ ⁇ ” indicates that the medium added with this additive has an effect on at least one of the cervical cancer organoids Proliferation was not significantly affected in both cases.
  • FGF10 prepared on the basis of formula 7 is added to the 96-well plate inoculated with organoids, and the final concentrations of FGF10 are 10 ng/mL, 20 ng/mL, and 100 ng respectively. /mL; and use the medium of formula 7 to set up control wells (BC).
  • a ratio greater than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds has a better effect on promoting proliferation than the culture medium of the control well; a ratio less than 1 indicates that the prepared medium containing different concentrations of factors or small molecular compounds promotes proliferation The effect was weaker than that of the culture medium in the control well.
  • the volume concentration of B27 is preferably 1:25-1:100; the content of hepatocyte growth factor HGF is preferably 5-40 ng/mL; the content of SB202190 is preferably 200-1000 nM; the content of Y27632 is preferably The content of A83-01 is preferably 200-1000nM; the content of epidermal growth factor EGF is preferably 1-40ng/mL; the content of fibroblast growth factor 10 is preferably 10-100ng/mL; The content of growth factors is preferably 2-40ng/mL; the volume concentration of GlutaMAX is preferably 1:50-1:200; the content of MST1/2 kinase inhibitor compound 1 is preferably 2.5-10 ⁇ M; the content of nicotinamide is preferably 1- 10mM.
  • the cervical cancer primary cells (OC1, OC2 and OC4) obtained according to the method described in (2) of Example 1 were resuspended and counted with the cervical cancer organoid medium COM of the present invention, and the cell density was diluted to 5-10 ⁇ 10 cells/mL, take 400 ⁇ L of the diluted cell suspension and add it to an equal volume of Matrigel (Corning) to mix gently, and then inoculate the mixture in a 24-well plate at 40 ⁇ L/well. Put the inoculated culture plate into the incubator for 30 minutes, wait until the Matrigel is completely solidified, then add the cervical cancer organoid medium COM that has been returned to room temperature in advance, 500 ⁇ L per well, and expand the culture by replacing the medium every five days.
  • Figures 2A-2D are the cervical cancers obtained after culture of samples OC1 (day 5), OC1 (day 14), OC2 (day 7), and OC2 (day 7, another field of view) under a 4x objective lens Photos of organs. As shown, organoids grow in size during culture; different types of organoids can be formed from the same sample, which can mimic tumor heterogeneity in vitro.
  • Pathological and immunohistochemical identifications were performed on the cultured cervical cancer organoids, and the corresponding original tissue samples were also identified pathologically and immunohistochemically to compare the consistency of organoids and histopathological indicators.
  • Figure 3 is the results of pathological and immunohistochemical identification of the original tissue sample OC4 and the cervical cancer organoids obtained after its in vitro culture, which are pictures taken under a 20x objective lens.
  • the results show that the structural morphology of the organoid is a cancerous tissue morphology; according to the immunohistochemical indicators, it is judged that the cells obtained after culturing the organoids in this case are cervical cancer cells.
  • This result shows that the cervical cancer organoid cultured using the medium COM of the present invention is consistent with the diagnostic result of the cervical cancer tissue before culture.
  • Figures 4A and 4B are photographs of organoids cultured in COM medium and ROM medium for 10 days under a 4x objective lens.
  • COM medium can significantly promote the formation and expansion of cervical cancer organoids.
  • Example 5 Using the culture medium of the present invention to amplify the cervical cancer organoids for drug screening
  • Cervical cancer primary cells were isolated from the cervical cancer intraoperative sample (CCa5) according to the method of (2) of Example 1, and the organoids were cultured in COM medium, and drug screening was performed when the diameter of the cervical cancer organoids exceeded 50 ⁇ m.
  • cisplatin was prepared into 6 different concentrations of additives, the highest concentration was 9.5 ⁇ M, and then diluted with a 2-fold dilution ratio to obtain 4.75 ⁇ M, 2.38 ⁇ M, 1.19 ⁇ M, 0.59 ⁇ M, 0.3 ⁇ M ⁇ M of different concentrations of additives.
  • Carboplatin was prepared into 6 different concentrations of storage solution, the highest concentration was 137.92 ⁇ M, and then diluted by 2 times dilution ratio to obtain 68.96 ⁇ M, 34.48 ⁇ M, 17.24 ⁇ M, 8.62 ⁇ M, 4.31 ⁇ M ⁇ M of different concentrations of additives.
  • Paclitaxel was prepared into 6 different concentrations of additive solutions, the highest concentration was 11.51 ⁇ M, and then diluted with a 2-fold dilution ratio to obtain different concentrations of 5.76 ⁇ M, 2.88 ⁇ M, 1.44 ⁇ M, 0.72 ⁇ M, and 0.36 ⁇ M. concentration of additives.
  • bortezomib was prepared into 6 different concentrations of additives, the highest concentration was 2 ⁇ M, and then diluted by 2 times dilution ratio to obtain 1 ⁇ M, 0.5 ⁇ M, 0.25 ⁇ M, 0.125 ⁇ M, 0.0625 ⁇ M ⁇ M of different concentrations of additives.
  • step (1) Take out the prepared drug additive solution and place it at room temperature. Take out the organoids obtained by culturing according to step (1) from the incubator, remove the medium in the culture wells, and slowly pour the additives containing different concentrations of drugs into the 96-well transparent culture plate along the well wall at 100 ⁇ L per well. After the drug addition, the surface of the 96-well plate was sterilized and moved to the incubator to continue culturing, and the viability of the organoids was measured 5 days later.
  • the drug inhibition rate (%) 100%-(the chemiluminescence value drug treatment group of the culture well on the fifth day/the chemiluminescence value drug treatment group of the culture well on the zero day)/(the chemiluminescence value DMSO of the culture well on the fifth day/the The chemiluminescent value of the zero-day culture wells (DMSO )*100% was calculated to obtain the inhibition rates of different drugs, and the results are shown in FIG. 5 .
  • Fig. 5 is the inhibition rate curve of different concentrations of test drugs inhibiting the growth of cervical cancer organoids.
  • bortezomib has a strong inhibitory effect on organoid growth at 6 concentrations
  • paclitaxel has the same inhibitory efficiency at 6 concentrations
  • the inhibitory effects of different concentrations of cisplatin and carboplatin are significant. Certain differences were dose-dependent, suggesting that organoids from the same patient have different effectiveness and sensitivity to different drugs. According to the results, the effectiveness and effective dosage of the drug can be judged when the cervical cancer patients are clinically used.
  • the invention provides a culture medium and a culture method for cervical cancer organoids, and the cultured organoids can be applied to the efficacy evaluation and screening of drugs.
  • the present invention is suitable for industrial applications.

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Abstract

Milieu de culture pour organoïdes de cancer du col utérin, comprenant un inhibiteur de kinase MST1/2, au moins un additif de culture cellulaire choisi parmi le N2 et le B27, le facteur de croissance des hépatocytes, le SB202190, le Y27632, le A83-01, le facteur de croissance épidermique, le facteur de croissance des fibroblastes 10, le facteur de croissance des kératinocytes, le GlutaMAX et le niacinamide. La présente invention concerne également un procédé de culture d'organoïdes de cancer du col utérin. En utilisant le milieu de culture pour organoïdes de cancer du col utérin, il est possible d'obtenir une multiplication efficace et rapide des organoïdes de cancer du col utérin, les organoïdes multipliés conservent les caractéristiques pathologiques des patients, le taux de réussite de la culture et le taux de multiplication des organoïdes de cancer du col utérin sont améliorés, et une base de recherche peut être constituée pour le traitement personnalisé des patients.
PCT/CN2021/142274 2021-10-15 2021-12-29 Milieu de culture pour organoïdes de cancer du col utérin, procédé de culture et application associée WO2023060774A1 (fr)

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