WO2023060695A1 - 肝癌悬浮类器官的培养基和培养方法 - Google Patents

肝癌悬浮类器官的培养基和培养方法 Download PDF

Info

Publication number
WO2023060695A1
WO2023060695A1 PCT/CN2021/131963 CN2021131963W WO2023060695A1 WO 2023060695 A1 WO2023060695 A1 WO 2023060695A1 CN 2021131963 W CN2021131963 W CN 2021131963W WO 2023060695 A1 WO2023060695 A1 WO 2023060695A1
Authority
WO
WIPO (PCT)
Prior art keywords
liver cancer
alkyl
medium
concentration
culture
Prior art date
Application number
PCT/CN2021/131963
Other languages
English (en)
French (fr)
Inventor
刘青松
汪文亮
黄涛
陈程
Original Assignee
合肥中科普瑞昇生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 合肥中科普瑞昇生物医药科技有限公司 filed Critical 合肥中科普瑞昇生物医药科技有限公司
Publication of WO2023060695A1 publication Critical patent/WO2023060695A1/zh

Links

Images

Classifications

    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a culture medium for liver cancer suspension organoids, a method for cultivating liver cancer suspension organoids using the culture medium, and its application in drug efficacy evaluation and screening.
  • Liver cancer is the third most common malignant tumor in terms of mortality after gastric cancer and esophageal cancer. In 2020, more than 900,000 people worldwide will be newly diagnosed with liver cancer, and more than 830,000 people will die from liver cancer. The number of deaths is close to the number of new cases.
  • Organoids 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 high-throughput drug screening and individualized precision therapy.
  • R-spondin-1, Noggin and some expensive protein factors are mostly used in liver cancer organoid culture methods, resulting in high cost of organoid culture; and this technology needs to be pre-coated with Matrigel, making the culture and passage operations complicated And the technology is difficult; due to the coating of Matrigel, the cost of large-scale cultivation is high, the standardization is difficult, and large-scale commercial 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 liver cancer suspension organoids in vitro.
  • One aspect of the present invention is to provide a culture medium for liver cancer suspension organoids, said medium comprising MST1/2 kinase inhibitor, Y27632, at least one cell culture additive selected from N2 and B27, SB202190, insulin, fibroblast Cell Growth Factor 10, Cholera Toxin, ITS Cell Culture Supplement, Hepatocyte Growth Factor, GlutaMAX, and Non-Essential Amino Acids.
  • 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, or solvate thereof,
  • 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 2.5-40 ⁇ M, more preferably 2.5-20 ⁇ M;
  • the volume ratio of B27 or N2 cell culture supplement to the medium is 1:25 to 1:200, more preferably 1:25 to 1:100;
  • the concentration of hepatocyte growth factor is preferably 1.25-20 ng/mL, more preferably 1.25-10 ng/mL;
  • the volume ratio of the ITS cell culture supplement to the medium is preferably 1:12.5 to 1:200, more preferably 1:50 to 1:200;
  • the concentration of SB202190 is preferably 5-40nM, more preferably 5-20nM;
  • the concentration of fibroblast growth factor 10 is preferably 2.5-20 ng/mL, more preferably 2.5-10 ng/mL;
  • the concentration of cholera toxin is preferably 1.25-20 ng/mL, more preferably 2.5-10 ng/mL;
  • the concentration of insulin is preferably 2.5-40 ⁇ g/mL, more preferably 2.5-20 ⁇ g/mL;
  • the concentration of Y27632 is preferably 2.5-40 ⁇ M, more preferably 5-20 ⁇ M;
  • the volume ratio of GlutaMAX to the medium is preferably 1:12.5 to 1:200;
  • Non-essential amino acids are one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the concentration of non-essential amino acids is preferably 12.5- 200 ⁇ M, more preferably 100 to 200 ⁇ M.
  • 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 the liver cancer suspension organoid.
  • primary liver cancer cells are cultured in suspension using the liver cancer suspension organoid medium of the present invention.
  • Sppension culture refers to a tissue culture system in which single cells and small cell clusters are cultured in liquid medium or low-adsorption culture dishes that are constantly stirred or shaken. It is a culture method of non-anchorage-dependent cells.
  • the method for culturing liver cancer suspension organoids of the present invention includes the following steps.
  • the process includes the following steps:
  • 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 (1-2mg/mL), collagenase IV (1-2mg/mL), DNase (50-100U/mL), hyaluronidase (0.5-1mg/mL) mL), calcium chloride (1 ⁇ 5mM), bovine serum albumin BSA (5 ⁇ 10mg/mL).
  • liver cancer primary cells obtained in the above step 1 were resuspended and counted with the liver cancer suspension organoid medium of the present invention, the cells were seeded on a low-adsorption culture plate at a density of 1-5 ⁇ 10 5 /well, and a corresponding volume of liver cancer Suspension organoid medium for scale-up.
  • the present invention also provides a method for evaluating or screening a drug for treating liver cancer, which comprises the following steps:
  • liver cancer organoids using the method for culturing liver cancer suspension organoids of the present invention
  • liver cancer organoids can be rapidly cultured, and the amplified liver cancer organoids can also be continuously passaged;
  • the culture cost is controllable, and the medium does not need to add expensive Wnt agonists, R-spondin family proteins, Noggin proteins, BMP inhibitors and other factors;
  • This culture method does not need to use Matrigel, and the cultivation and passage operation is simple and convenient, and is suitable for large-scale cultivation;
  • 1A-1K are graphs showing the effects of different concentrations of factors added to the liver cancer organoid culture medium of the present invention on the proliferation of liver cancer organoids.
  • Figures 2A-2D are photographs of liver cancer suspension organoids cultured using the liver cancer suspension organoid medium of the present invention under a microscope, wherein Figure 2A shows photos of organoids cultured from sample GL-003 after 7 days; Figure 2B shows Photos of organoids cultured from sample GL-006 after 7 days; Figure 2C shows photos of organoids cultured from sample GL-008 after 7 days; Figure 2D shows photos of organoids cultured from sample GL-013 after 7 days .
  • Figure 3A is a view of the pathological and immunohistochemical identification of the liver cancer suspension organoids cultured with the liver cancer suspension organoid medium of the present invention for the sample GL-006;
  • Figure 3B is the pathological and immunohistochemical identification of the original tissue of the sample GL-006 Authenticated view.
  • Figures 4A-4C are graphs comparing the results of culturing liver cancer organoids using the liver cancer suspension organoid medium of the present invention and the existing medium, wherein Figure 4A shows the liver cancer obtained after 7 days of culture with the HC-XF medium of the present invention Photos of suspension organoids; Figure 4B shows photos of liver cancer suspension organoids cultured in Laura medium for 7 days; Figure 4C shows a comparison of the relative size of liver cancer suspension organoids cultured in HC-XF medium and Laura medium picture.
  • Figures 5A and 5B show the results of different drug sensitivity tests on liver cancer organoids cultured using the liver cancer suspension organoid medium of the present invention, wherein Figure 5A shows photos of organoid growth after 5 days of treatment with different concentrations of test drugs; Figure 5B shows a histogram of the inhibition rates of different concentrations of the test drugs inhibiting the growth of liver cancer organoids.
  • 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 from professional medical institutions, and all patients signed informed consent. Intraoperative samples of 0.25 cm 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 was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx.
  • the primary liver cancer cells obtained in the above steps were resuspended and counted in pre-cooled DMEM/F12, and the cells were seeded in 24-well low-adsorption culture plates at a density of 1-5 ⁇ 10 5 /well, and then added to the
  • the culture medium shown in Table 1 was expanded by changing the medium every three days. After 7 days, the cultured organoids were photographed, and the diameters of the organoids were measured and counted, and the promotion effects of various factors on the proliferation of liver cancer suspension organoids were compared.
  • As an experimental control a basal medium without any additives was used, and the experimental results are shown in Table 1.
  • “+” means that compared with the basal medium, the medium added with this additive has the effect of promoting the proliferation of at least two cases of liver cancer suspension organoids isolated from liver cancer tissue; “-” means that the culture medium added with this additive
  • the base has an inhibitory effect on at least one case of liver cancer suspension organoids isolated from liver cancer tissues; Proliferation was not significantly affected.
  • B27 hepatocyte growth factor (HGF), ITS cell culture supplement, Y27632, SB202190, fibroblast growth factor 10 (FGF10), insulin, cholera toxin (CTX), GlutaMAX, compound 1, non-essential Amino acids and other factors were further cultivated for experiments.
  • HGF hepatocyte growth factor
  • FGF10 fibroblast growth factor 10
  • CTX cholera toxin
  • GlutaMAX compound 1, non-essential Amino acids and other factors
  • HGF human growth factor
  • ITS cell culture supplement 200 ⁇ L was added to the 96-well plate inoculated with organoids on the basis of Formula 3, and the final concentration of ITS cell culture supplement was 1:200, 1:100, 1:50, 1:25, 1:12.5; and set up control wells (BC) using the medium of recipe 3.
  • Y27632 prepared on the basis of formula 4 was added to the 96-well plate inoculated with organoids, and the final concentrations of Y27632 were 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M, 20 ⁇ M, 40 ⁇ M; and set up control wells (BC) using the medium of recipe 4.
  • 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 to 1:200, more preferably 1:25 to 1:100; the content of hepatocyte growth factor is preferably 1.25 to 20 ng/mL, more preferably 1.25-10 ng/mL; the volume concentration of ITS cell culture additive is preferably 1:12.5-1:200, more preferably 1:50-1:200; the content of Y27632 is preferably 2.5-40 ⁇ M, more preferably 5-20 ⁇ M;
  • the content of SB202190 is preferably 5-40 nM, more preferably 5-20 nM; the content of fibroblast growth factor 10 is preferably 2.5-20 ng/mL, more preferably 2.5-10 ng/mL; the content of insulin is preferably 2.5-40 ⁇ g/mL mL, more preferably 2.5-20 ⁇ g/mL; the content of cholera toxin is preferably 1.25-20 ng/mL, more preferably 2.5-10 ng/mL; the volume concentration of GlutaMAX is
  • liver cancer primary cells (GL-003, GL-006, GL-008, GL-013) obtained according to the method described in (2) of Example 1 were resuspended with the liver cancer suspension organoid medium HC-XF of the present invention and counted, the cells were seeded in a 24-well low-adsorption culture plate at a density of 5 ⁇ 10 5 /well, 1 mL of liver cancer suspension organoid medium was added, and the medium was replaced every three days for expansion culture.
  • FIGS. 2A-2D are photographs of liver cancer suspension organoids obtained after 7 days of culture of samples GL-003, GL-006, GL-008, and GL-013 under a 10x objective lens. Under the microscope, the suspended liver cancer organoids were spherical in shape with a smooth surface.
  • Pathological and immunohistochemical identifications were performed on the cultured liver cancer suspension organoids, and the corresponding tissue samples were subjected to pathological and immunohistochemical identifications to compare the consistency of the results between organoids and original tissues.
  • Figure 3A is a view of the results of pathological and immunohistochemical identification of liver cancer suspension organoids obtained from sample GL-006 cultured in vitro, which are pictures taken under a 20x objective lens.
  • HE results showed that the structural morphology of organoids was cancerous; the expression of CK19, Heppar-1, and Ki67 suggested that the sample was liver cancer.
  • Figure 3B is a view of the pathological and immunohistochemical results of the corresponding tissue of GL-006 before culture. The results show that the diagnosis results of liver cancer organoids cultured using the medium HC-XF of the present invention are consistent with those of liver cancer tissues before culture.
  • the medium used in the preparation literature (Laura et al., Nat Med.2017,23(12):1424-1435), its formula is Advanced DMEM/F12 medium (purchased from Corning Company)+1:100 Penicillin/Streptomycin (purchased from Corning)+1:100GlutaMAX (purchased from Corning)+10mM HEPES (purchased from Thermo Fisher)+1:50B27 (purchased from Gibco)+1:100N2 (purchased from Gibco)+1.25mmol/L N -Acetyl cysteine (purchased from MCE company)+10mmol/L nicotinamide (purchased from MCE company)+10nM gastrin (purchased from MCE company)+50ng/ml epidermal growth factor (purchased from R&D company)+ 100ng/ml fibroblast growth factor 10 (purchased from Sino biological company)+25ng/ml hepatocyte growth factor (purchased from R&D company)+10 ⁇ mol/L Forskolin
  • the primary liver cancer cells were obtained from the intraoperative tissue sample GL-018 according to the method of (2) of Example 1, and cultured in suspension organoids using HC-XF medium and Laura medium according to the method of Example 3, respectively.
  • Figures 4A and 4B are photographs of organoids cultured in HC-XF medium and Laura medium respectively under a 4x objective lens
  • Figure 4C is a histogram comparing the relative sizes of organoids cultured in the two media.
  • HC-XF medium can significantly promote the expansion and culture of liver cancer organoids.
  • Example 5 The liver cancer suspension organoids amplified using the medium of the present invention are used for drug screening
  • Liver cancer primary cells were isolated from the intraoperative liver cancer sample (GL-006) according to the method of Example 1 (2), and cultured in suspension organoids using HC-XF medium, and carried out when the diameter of the liver cancer suspension organoids exceeded 50 ⁇ m drug screening.
  • HC-XF medium HC-XF medium
  • organoids cultured according to step (1) from the incubator, transfer the organoids to a 15ml centrifuge tube, centrifuge at 1500rpm for 5 minutes. Resuspend the organoids in HC-XF medium, count, adjust the concentration of organoids to 50/50 ⁇ L, add 50 ⁇ L of the adjusted organoid suspension into the 96-well, then add 50 ⁇ L of the diluted drug, and put the 96-well plate surface After disinfection, move to the incubator to continue culturing, and measure organoid viability 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 FIGS. 5A and 5B .
  • Figure 5A is a photo of organoid growth after 5 days of drug treatment taken under a microscope (Invitrogen EVOS M500) with a 4x objective lens
  • Figure 5B is a histogram of the inhibition rate of different concentrations of test drugs inhibiting the growth of liver cancer organoids.
  • FIG. 5A It can be confirmed from FIG. 5A that the growth of organoids cultured using the liver cancer suspension organoid medium of the present invention is significantly inhibited after high-concentration drug treatment.
  • Fig. 5B is a histogram of the inhibition rate of three test drugs at different concentrations to inhibit the growth of liver cancer organoids. It can be seen from Figure 5B that the data error value of the drug treatment group is very small, indicating that using this system for drug screening, the data between the repeated wells of the same drug are basically consistent.
  • the three anti-tumor drugs high concentrations have strong inhibitory effects on organoid growth, and low concentrations have relatively different inhibitory effects on organoid growth, which shows that organoids from the same patient are more effective against different drugs. different from sensitivity. According to the results, the effectiveness and effective dosage of the drug can be judged when the liver cancer patient is used clinically.
  • the invention provides a culture medium and a culture method for liver cancer suspension organoids, and the cultured organoids can be applied to the curative effect evaluation and screening of drugs.
  • the present invention is suitable for industrial applications.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种用于肝癌悬浮类器官培养的培养基，包括MST1/2激酶抑制剂、Y27632、选自N2和B27的至少一种细胞培养添加剂、SB202190、胰岛素、成纤维细胞生长因子10、霍乱毒素、ITS细胞培养添加剂、肝细胞生长因子、GlutaMAX、和非必需氨基酸。还涉及肝癌悬浮类器官的培养方法及其用途。通过使用所述肝癌悬浮类器官培养基，能够实现肝癌细胞的有效快速扩增，这样扩增得到的肝癌细胞保持了患者的病理特性，提高了肝癌细胞的培养成功率和扩增速率，可以为患者的个性化治疗提供研究基础。

Description

肝癌悬浮类器官的培养基和培养方法 技术领域
本发明属于生物技术领域，具体涉及一种用于肝癌悬浮类器官的培养基、使用该培养基培养肝癌悬浮类器官的方法、及其在药物的疗效评估和筛选中的应用。
背景技术
肝癌是死亡率仅次于胃癌、食道癌的第三大常见恶性肿瘤。2020年，全世界被新确诊为肝癌的人超过90万，因肝癌死亡的人超过83万，死亡人数接近新发病人数。
近几年来，肝癌术后辅助化疗作为一种新型辅助治疗方式已逐渐得到临床医生的重视与认可，包括术后TACE治疗，口服药物治疗等，但由于缺乏规范化疗方案，常规凭经验化疗，忽略个体差异，带有一定的盲目性，因此效果一直不佳，单药及联合用药的有效率低于20％(Jindal A,Thadi A,Shailubhai K.Hepatocellular Carcinoma:Etiology and Current and Future Drugs[J].J Clin Exp Hepatol,2019,9(2):221-232)。新兴的靶向药物虽然在一定程度上降低了毒副作用，但是数量太少，治疗费用昂贵，且有效率随个体差异，难以满足大多数患者的治疗需求。由于缺乏有效的肝癌药敏试验体系，无法做到精准化疗，因此将肝癌体外药敏结果与临床体内反应相对应就成为治疗关键。
传统临床药物敏感性检测大多采用二维细胞培养。然而，二维培养的细胞仅在有限程度上模拟组织生理条件，缺乏体内真实的组织结构，易导致低分化水平和细胞生理功能的丢失，进而导致获得的实验结果很难预测临床实际结果。类器官主要来源于人体具有分化能力的胚胎干细胞、诱导多潜能干细胞和成体干细胞。不同组织器官都存在内源组织干细胞，在维持各器官的功能形态方面发挥着重要作用。这些干细胞在体外一定的诱导条件下，可以自组织形成一个直径仅为几毫米的迷你结构。肿瘤类器官是用取自患者体内原发性肿瘤，在实验室中培养出一些微型的3D肿瘤细胞模型。肿瘤类器官高度模拟了来源肿瘤组织的特征，保留了个体之间的肿瘤异质性，可用于功能性的测 试，如进行高通量药物筛选和个体化精准治疗。
当前，肝癌类器官培养方法多采用R-spondin-1、Noggin以及一些昂贵的蛋白因子，导致类器官培养成本较高；且这项技术需要用基质胶预先进行包被处理，培养和传代操作复杂和技术难度大；由于基质胶的包被，导致其大规模培养成本较高，标准化难度大，大规模商业化应用受到限制。因此，需要开发一种低成本、简单且成功率高的类器官培养方法和培养基。
发明内容
为了解决上述技术问题，本发明提供了一种用于在体外快速扩增肝癌悬浮类器官的培养基及培养方法。
本发明的一个方面在于提供一种肝癌悬浮类器官的培养基，所述培养基包含MST1/2激酶抑制剂、Y27632、选自N2和B27的至少一种细胞培养添加剂、SB202190、胰岛素、成纤维细胞生长因子10、霍乱毒素、ITS细胞培养添加剂、肝细胞生长因子、GlutaMAX、和非必需氨基酸。其中，所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物，
Figure PCTCN2021131963-appb-000001
其中，
R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的芳基(例如苯基和萘基等)、芳基C1-C6烷基(例如苯甲基等)和杂芳基(例如噻吩基等)；
R 2和R 3各自独立地选自C1-C6烷基，优选C1-C3烷基，更优选甲基；
R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8 环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基(所述杂环基选自例如哌啶基、四氢吡喃基等)；
R 6选自卤素(优选氟和氯，更优选氟)、C1-C6烷基(优选甲基)、C1-C6烷氧基(优选甲氧基)、和C1-C6卤代烷基(优选三氟甲基)。
优选的实施方式中，MST1/2激酶抑制剂包括式(Ia)的化合物或其药学可接受的盐、或溶剂化物，
Figure PCTCN2021131963-appb-000002
其中，
R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基，R 1更优选为任选地被1-2个独立地R 6取代的苯基；
R 5选自氢、C1-C6烷基、和C3-C6环烷基，R 5更优选为氢；
R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基，R 6更优选为氟、甲基或三氟甲基。
优选地，所述MST1/2抑制剂是选自以下化合物或其药学可接受的盐、或溶剂化物中的至少一种。
Figure PCTCN2021131963-appb-000003
Figure PCTCN2021131963-appb-000004
Figure PCTCN2021131963-appb-000005
Figure PCTCN2021131963-appb-000006
Figure PCTCN2021131963-appb-000007
最优选地，本发明的MST1/2激酶抑制剂为化合物1。
在本发明的实施方式中，本发明的培养基中各成分的含量满足以下任意一项或多项或全部满足：
(1)MST1/2激酶抑制剂的浓度优选为2.5～40μM，更优选为2.5～20μM；
(2)B27或N2细胞培养添加剂相对于培养基的体积比为1:25～1:200，更优选为1:25～1:100；
(3)肝细胞生长因子的浓度优选为1.25～20ng/mL，更优选为1.25～10ng/mL；
(4)ITS细胞培养添加剂相对于培养基的体积比优选为1:12.5～1:200，更优选为1:50～1:200；
(5)SB202190的浓度优选为5～40nM，更优选为5～20nM；
(6)成纤维细胞生长因子10的浓度优选为2.5～20ng/mL，更优选为2.5～10ng/mL；
(7)霍乱毒素的浓度优选为1.25～20ng/mL，更优选为2.5～10ng/mL；
(8)胰岛素的浓度优选为2.5～40μg/mL，更优选为2.5～20μg/mL；
(9)Y27632的浓度优选为2.5～40μM，更优选为5～20μM；
(10)GlutaMAX相对于培养基的体积比优选为1:12.5～1:200；
(11)非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，非必需氨基酸的浓度优选为12.5～200μM，更优选为100～200μM。
在本发明的实施方式中，所述培养基还含有选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。
在优选的实施方式中，当抗生素选自链霉素/青霉素时，链霉素浓度范围为25～400μg/mL，青霉素浓度范围为25～400U/mL，当抗生素选自两性霉素B时，浓度范围为0.25～4μg/mL，当抗生素选自Primocin时，浓度范围为25～400μg/mL。
本发明还提供一种肝癌悬浮类器官的培养方法。在本发明的肝癌悬浮类器官的培养方法中，使用本发明的肝癌悬浮类器官培养基对肝癌原代细胞进行悬浮培养。“悬浮培养”是指在受到不断搅动或摇动的液体培养基里或低吸附培养皿里，培养单细胞及小细胞团的组织培 养系统，是非贴壁依赖性细胞的一种培养方式。
本发明的肝癌悬浮类器官培养方法包括以下步骤。
1.从肝癌实体瘤组织分离样本，获得肝癌原代细胞。该处理过程包括以下步骤：
(1)分离肝癌组织样本，加入1:3比例的基础培养基和组织消化液(组织消化液的加入量是每1g肿瘤组织使用约10mL组织消化液)中置于恒温摇床中进行消化，消化温度为4～37℃，摇床转速为200rpm～350rpm，消化时间为3～6小时；
(2)消化完成后，离心后弃去上清液，离心转速为1200～1600rpm，离心时间为2～6分钟。
其中，基础培养基配方包括选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。组织消化液配方包括1640培养基、胶原酶Ⅱ(1～2mg/mL)、胶原酶Ⅳ(1～2mg/mL)、DNA酶(50～100U/mL)、透明质酸酶(0.5～1mg/mL)、氯化钙(1～5mM)、牛血清白蛋白BSA(5～10mg/mL)。
2.配制本发明的肝癌悬浮类器官培养基，并对上述步骤获得的肝癌原代细胞进行培养。
将上述步骤1中获得的肝癌原代细胞用本发明的肝癌悬浮类器官培养基重悬并计数，将细胞按照1～5×10 5/孔密度接种于低吸附培养板，添加相应体积的肝癌悬浮类器官培养基进行扩大培养。
本发明还提供一种用于评估或筛选治疗肝癌疾病的药物的方法，其包括以下步骤：
(1)使用本发明的肝癌悬浮类器官的培养方法培养肝癌类器官；
(2)选定需要检测的药物并按照所需浓度梯度进行稀释；
(3)对(1)中培养得到的类器官添加稀释后的所述药物；
(4)进行类器官大小或类器官活力测试。
本发明的有益效果包括：
(1)提高肝癌类器官培养的成功率，成功率达到90％以上；
(2)保证体外原代培养的肝癌类器官能够保持病人的病理特性；
(3)扩增效率高，能快速培养出肝癌类器官，扩增出的肝癌类器 官还可以连续传代；
(4)培养成本可控，培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白、Noggin蛋白、BMP抑制剂等因子；
(5)本培养方法不需要使用基质胶，培养和传代操作简便，适合大规模培养；
(6)所述技术培养获得的肝癌类器官数量大，适合高通量筛选候选化合物和为病人提供高通量药物体外敏感性功能测试。
附图说明
图1A-1K为显示本发明的肝癌悬浮类器官培养基所添加因子的不同浓度对肝癌类器官增殖的影响的图。
图2A-2D为利用显微镜观察使用本发明的肝癌悬浮类器官培养基培养得到的肝癌悬浮类器官的照片，其中图2A显示7天后由样本GL-003培养获得的类器官的照片；图2B显示7天后由样本GL-006培养获得的类器官的照片；图2C显示7天后由样本GL-008培养获得的类器官的照片；图2D显示7天后由样本GL-013培养获得的类器官的照片。
图3A为对样本GL-006使用本发明的肝癌悬浮类器官培养基培养得到的肝癌悬浮类器官进行病理和免疫组化鉴定的视图；图3B为对样本GL-006原始组织进行病理和免疫组化鉴定的视图。
图4A-4C为使用本发明的肝癌悬浮类器官培养基与现有培养基对肝癌类器官进行培养的比较结果图，其中图4A显示用本发明的HC-XF培养基培养7天后得到的肝癌悬浮类器官的照片；图4B显示用Laura培养基培养7天后得到的肝癌悬浮类器官的照片；图4C显示HC-XF培养基和Laura培养基培养得到的肝癌悬浮类器官的相对大小的对比柱状图。
图5A和5B显示对使用本发明的肝癌悬浮类器官培养基培养得到肝癌类器官进行不同药物敏感性测试的结果，其中图5A显示不同浓度的测试药物加药处理5天后类器官生长的照片；图5B显示不同浓度的测试药物抑制肝癌类器官生长的抑制率柱状图。
具体实施方式
为更好地理解本发明，下面结合实施例及附图对本发明作进一步描述。以下实施例仅是对本发明进行说明而非对其加以限定。
[MST1/2激酶抑制剂的制备实施例]
本说明书中，MST1/2激酶抑制剂是指直接或间接地对MST1/2信号传导进行负调节的任意的抑制剂。一般来说，MST1/2激酶抑制剂例如与MST1/2激酶结合并降低其活性。由于MST1和MST2的结构具有相似性，MST1/2激酶抑制剂也可以是例如与MST1或MST2结合并降低其活性的化合物。
1.MST1/2激酶抑制剂化合物1的制备
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯 磺酰胺1
Figure PCTCN2021131963-appb-000008
2-氨基-2-(2,6-二氟苯基)乙酸甲酯(A2)：在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸(2.0克)后加入甲醇(30毫升)，随后冰浴下滴加二氯亚砜(1.2毫升)。反应体系在85℃反应过夜。反应结束后，体系在减压下蒸干溶剂，所得白色固体，直接用于下一步。
2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(A3)：在圆底烧瓶中加入2-氨基-2-(2,6-二氟苯基)乙酸甲酯(2克)后加入丙酮(30毫升)和碳酸钾(2.2克)，然后用冰盐浴使体系冷却到-10℃，接着缓慢加入2,4-二氯-5-硝基嘧啶(3.1克)的丙酮溶液。反应体系在室温搅拌过夜。反应结束后，过滤，滤液在减压下除去溶剂，残留物经加压硅胶柱层析提纯后得化合物A3。LC/MS：M+H 359.0。
2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(A4)：在圆底烧瓶中加入2-((2-氯-5-硝基嘧啶-4-基)氨基)-2-(2,6-二氟苯基)乙酸甲酯(2.5 克)后加入醋酸(50毫升)和铁粉(3.9克)。反应体系在60℃搅拌两小时。反应结束后，体系在减压下蒸干溶剂，所得物用饱和碳酸氢钠中和至碱性。乙酸乙酯萃取，有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤，减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A4。LC/MS：M+H 297.0。
2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(A5)：在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-7,8-二氢蝶啶-6(5H)-酮(2克)和N,N-二甲基乙酰胺(10毫升)，冷却至-35℃，加入碘甲烷(0.9毫升)，随后加入氢化钠(615毫克)，反应体系继续搅拌两小时。反应结束后，加水淬灭，乙酸乙酯萃取，有机相分别用水、饱和食盐水洗涤后用无水硫酸钠干燥。有机相经过滤，减压蒸干后得粗品。粗品经乙醚洗涤后得化合物A5。LC/MS：M+H 325.0。
4-((7-(2,6-二氟苯基)-5,8-二甲基-6-氧代-5,6,7,8-四氢蝶啶-2-基)氨基)苯磺酰胺(1)：在圆底烧瓶中加入2-氯-7-(2,6-二氟苯基)-5,8-二甲基-7,8-二氢蝶啶-6(5H)-酮(100毫克)、磺胺(53毫克)、对甲苯磺酸(53毫克)和仲丁醇(5毫升)。反应体系在120℃搅拌过夜。反应结束后，过滤，甲醇和乙醚洗涤得化合物1。LC/MS：M+H 461.1。
2.本发明的其他MST1/2抑制剂化合物的制备
本发明的其他MST1/2抑制剂化合物按照与化合物1类似的方法合成，其结构及质谱数据如下表所示。
Figure PCTCN2021131963-appb-000009
Figure PCTCN2021131963-appb-000010
Figure PCTCN2021131963-appb-000011
Figure PCTCN2021131963-appb-000012
Figure PCTCN2021131963-appb-000013
实施例1肝癌悬浮类器官培养基中各添加因子对肝癌悬浮类器官增殖的影响
(1)肝癌悬浮类器官培养基的配制
首先配制含有初始培养基的基础培养基。初始培养基可选自本领域常用的DMEM/F12、DMEM、F12或RPMI-1640。在本实施例中，基础培养基的配方为：DMEM/F12培养基(购自Corning公司)+100μg/mL Primocin(购自InvivoGen公司，0.2％(v/v)，市售产品浓度50mg/ml)。
在基础培养基内分别加入不同种类的添加剂(参见表1)配制成含有不同添加成分的肝癌悬浮类器官培养基。
(2)肝癌原代细胞的分离和处理
1样品选择
肝癌实体瘤组织样品(术中)由专业医疗机构的专业医务人员从患者获取，患者均签署了知情同意书。术中样本0.25cm 3，采用商品化组织保存液(生产厂家：Miltenyi Biotec)存储运输。
2材料准备
15mL无菌离心管、移液枪、10mL移液管、无菌枪头等表面消毒后放入超净工作台中紫外照射30分钟。提前30分钟从4℃冰箱取出基础培养基，提前30分钟从-20℃冰箱取出组织消化液。
组织消化液配方：1640培养基(Corning，10-040-CVR)、胶原酶Ⅱ(2mg/mL)、胶原酶Ⅳ(2mg/mL)、DNA酶(50U/mL)、透明质酸酶(0.75mg/mL)、氯化钙(3.3mM)、牛血清白蛋白BSA(10mg/mL)。
以上提及的胶原酶Ⅱ、胶原酶Ⅳ、DNA酶、透明质酸酶均购自Sigma公司；氯化钙购自生工生物工程(上海)股份有限公司；BSA购自Biofroxx公司。
3样品分离
3.1超净台中取组织样品于培养皿中，去除带血液的组织，用基础培养基冲洗2次，将组织转移至另一培养皿中用无菌手术刀进行机械分离，将组织块分割为1*1*1mm 3大小；
3.2将切割后的术中组织吸至15mL离心管中，加入5mL基础培养基，混匀，于1500rpm离心4分钟；
3.3弃上清，加入1:3比例的基础培养基和组织消化液(注：组织消化液的加入量是1g肿瘤组织使用约10mL组织消化液)，标记样 品名称及编号，用封口膜密封，在37℃下于300rpm摇床(知楚仪器ZQLY-180N)中进行消化，期间每30分钟观察消化是否完成，判断依据为无肉眼可见的颗粒物；
3.4消化完成后，经100μm滤网过滤掉未消化的组织团块，滤网上的组织团块用基础培养基冲洗入离心管中以减少细胞损失，于25℃下1500rpm离心4分钟；
3.5弃上清，观察是否有血细胞，若有血细胞，加8mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解20分钟，期间颠倒混匀一次，25℃下1500rpm离心4分钟；
3.6弃上清，加入2mL基础培养基重悬细胞，备用。
4细胞计数及处理
4.1镜下观察：移取少量重悬细胞平铺于培养皿中，显微镜(CNOPTEC，BDS400)下观察癌细胞密度和形态；
4.2活细胞计数：取重悬的细胞悬液12μL，12μL台盼蓝染液(生产厂家：生工生物工程(上海)股份有限公司)充分混合后，取20μL加入细胞计数板(生产厂家：Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)下计算出活的大细胞(细胞粒径>10μm)百分率＝活细胞数/总细胞数*100％。
(3)肝癌悬浮类器官的培养
将上述步骤中获得的肝癌原代细胞用预冷的DMEM/F12重悬并计数，将细胞按照1～5×10 5/孔密度接种于24孔低吸附培养板，然后分别加入事先恢复到室温的表1所示的培养基，按照每三天更换一次培养基进行扩大培养。7天后对所培养的类器官进行拍照，并测量统计类器官的直径大小，比较各因子对肝癌悬浮类器官增殖的促进作用。其中，作为实验对照，使用未添加任何添加剂的基础培养基，将实验结果示于表1。
表1培养基中的添加成分及促类器官增殖效果
序号 培养基添加剂种类 供应商 终浓度 促增殖程度分级
1 N2 Gibco 1:50 +
2 表皮细胞生长因子EGF R&D 5ng/mL ○
3 R-spondin1 R&D 20ng/mL ○
4 前列腺素E2 Tocris 0.5μM ○
5 胰岛素 Peprotech 10μg/mL +
6 B27 Gibco 1:50 +
7 A8301 MCE 100nM -
8 SB202190 MCE 10nM +
9 碱性成纤维细胞生长因子bFGF R&D 10ng/mL -
10 氢化可的松 Sigma 10ng/mL ○
11 Noggin R&D 30ng/mL ○
12 胎牛血清FBS Excell 5％ +
13 胰岛素样生长因子-1IGF-1 R&D 45ng/mL ○
14 角化细胞生长因子KGF R&D 5ng/mL ○
15 GlutaMAX Gibco 1:100 +
16 非必需氨基酸 Corning 100μM +
17 地塞米松 MCE 0.1μM ○
18 神经调节蛋白1NRG1 sino biological 5ng/mL ○
19 Y27632 MCE 10μM +
20 ITS细胞培养添加剂 Gibco 1:100 +
21 化合物1 制备例 5μM +
22 CHIR99021 MCE 2.5μM -
23 肝细胞生长因子HGF R&D 5ng/mL +
24 霍乱毒素CTX MCE 5ng/mL +
25 成纤维细胞生长因子10 R&D 5ng/mL +
其中，“+”表示与基础培养基相比，加入该添加剂的培养基对从肝癌组织分离出的肝癌悬浮类器官中的至少两例有促进增殖的作用；“－”表示添加该添加剂的培养基对从肝癌组织分离出的肝癌悬浮类器官中的至少一例显示有抑制增殖的作用；“○”表示添加该添加剂的培养基对从肝癌组织分离出的肝癌悬浮类器官中的至少两例的增殖没有明显的影响。
根据以上结果，拟选择B27、肝细胞生长因子(HGF)、ITS细胞培养添加剂、Y27632、SB202190、成纤维细胞生长因子10(FGF10)、胰岛素、霍乱毒素(CTX)、GlutaMAX、化合物1、非必需氨基酸等因子进行进一步培养实验。
实施例2培养基添加因子的不同浓度对肝癌悬浮类器官的增殖作用
按照实施例1之(2)的方法从术中组织样本(编号为GL-003、GL-004)获得肝癌原代细胞，并使用下表2中的培养基配方进行类器 官培养。
表2培养基配方(浓度为终浓度)
Figure PCTCN2021131963-appb-000014
在使用配方1的培养基时，在接种有悬浮类器官的96孔板中在配方1的基础上分别添加配制好的B27每孔200μL，B27的终浓度分别为1:200、1:100、1:50、1:25、1:12.5；并使用配方1的培养基设置对照孔(BC)。该系列的培养基中其他添加因子的终浓度与HC-XF培养基相同。以下配方1-11的实验也以同样的方式进行，不再赘述。
在使用配方2的培养基时，在接种有悬浮类器官的96孔板中在配方2的基础上分别添加配制好的HGF每孔200μL，HGF的终浓度分别为1.25ng/mL、2.5ng/mL、5ng/mL、10ng/mL、20ng/mL；并使用配方2的培养基设置对照孔(BC)。
在使用配方3的培养基时，在接种有类器官的96孔板中在配方3的基础上分别添加配制好的ITS细胞培养添加剂每孔200μL，ITS细胞培养添加剂终浓度分别为1:200、1:100、1:50、1:25、1:12.5；并使用配方3的培养基设置对照孔(BC)。
在使用配方4的培养基时，在接种有类器官的96孔板中在配方4的基础上分别添加配制好的Y27632每孔200μL，Y27632的终浓度分 别为2.5μM、5μM、10μM、20μM、40μM；并使用配方4的培养基设置对照孔(BC)。
在使用配方5的培养基时，在接种有类器官的96孔板中在配方5的基础上分别添加配制好的SB202190每孔200μL，SB202190的终浓度分别为2.5nM、5nM、10nM、20nM、40nM；并使用配方5的培养基设置对照孔(BC)。
在使用配方6的培养基时，在接种有类器官的96孔板中在配方6的基础上分别添加配制好的FGF10每孔200μL，FGF10终浓度分别为2.5ng/mL、5ng/mL、10ng/mL、20ng/mL、40ng/mL；并使用配方6的培养基设置对照孔(BC)。
在使用配方7的培养基时，在接种有类器官的96孔板中在配方7的基础上分别添加配制好的胰岛素每孔200μL，胰岛素的终浓度分别为2.5μg/mL、5μg/mL、10μg/mL、20μg/mL、40μg/mL；并使用配方7的培养基设置对照孔(BC)。
在使用配方8的培养基时，在接种有类器官的96孔板中在配方8的基础上分别添加配制好的CTX每孔200μL，CTX的终浓度分别为1.25ng/mL、2.5ng/mL、5ng/mL、10ng/mL、20ng/mL；并使用配方8的培养基设置对照孔(BC)。
在使用配方9的培养基时，在接种有类器官的96孔板中在配方9的基础上分别添加配制好的GlutaMAX每孔200μL，GlutaMAX的终浓度分别为1:200、1:100、1:50、1:25、1:12.5；并使用配方9的培养基设置对照孔(BC)。
在使用配方10的培养基时，在接种有类器官的96孔板中在配方10的基础上分别添加配制好的化合物1每孔200μL，化合物1的终浓度分别为2.5μM、5μM、10μM、20μM、40μM；并使用配方10的培养基设置对照孔(BC)。
在使用配方11的培养基时，在接种有类器官的96孔板中在配方11的基础上分别添加配制好的非必需氨基酸每孔200μL，非必需氨基酸的终浓度分别为12.5μM、25μM、50μM、100μM、200μM；并使用配方11的培养基设置对照孔(BC)。
14天后对所培养的类器官进行拍照，并测量统计类器官的直径大 小，比较各因子浓度对肝癌类器官增殖的促进作用。将2例样本收集的数据汇总示于图1A～1K。图1A～1K中，比值为使用各培养基培养14天得到的类器官直径与对应的BC对照孔培养14天得到的类器官直径的比。比值大于1说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果优于对照孔培养基；比值小于1，则说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果较对照孔培养基促增殖效果弱。
根据图1A～1K的结果，B27的体积浓度优选为1:25～1:200，更优选为1:25～1:100；肝细胞生长因子的含量优选为1.25～20ng/mL，更优选为1.25～10ng/mL；ITS细胞培养添加剂的体积浓度优选为1:12.5～1:200，更优选为1:50～1:200；Y27632的含量优选为2.5～40μM，更优选为5～20μM；SB202190的含量优选为5～40nM，更优选为5～20nM；成纤维细胞生长因子10的含量优选为2.5～20ng/mL，更优选为2.5～10ng/mL；胰岛素的含量优选为2.5～40μg/mL，更优选为2.5～20μg/mL；霍乱毒素的含量优选为1.25～20ng/mL，更优选为2.5～10ng/mL；GlutaMAX的体积浓度优选为1:12.5～1:200；MST1/2激酶抑制剂化合物1的含量优选为2.5～40μM，更优选为2.5～20μM；非必需氨基酸的含量优选为12.5～200μM，更优选为100～200μM。
实施例3肝癌悬浮类器官培养及鉴定
将按照实施例1之(2)所述方法获得的肝癌原代细胞(GL-003、GL-006、GL-008、GL-013)用本发明的肝癌悬浮类器官培养基HC-XF重悬并计数，将细胞按照5×10 5/孔密度接种于24孔低吸附培养板，添加1mL肝癌悬浮类器官培养基，按照每三天更换一次培养基进行扩大培养。
在第7天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的肝癌类悬浮器官。图2A-2D的是10倍物镜下拍摄样本GL-003、GL-006、GL-008、GL-013培养7天后得到的肝癌悬浮类器官的照片。肝癌悬浮类器官在镜下呈规则球状，表面较光滑。
对培养得到的肝癌悬浮类器官进行病理和免疫组化鉴定，同时将对应的组织样本进行病理和免疫组化鉴定，比较类器官和原始组织的 结果的一致性。
图3A为由样本GL-006体外培养后得到的肝癌悬浮类器官进行病理和免疫组化鉴定的结果视图，分别为20倍物镜下拍照的图片。如图3A所示，HE结果显示类器官的结构形态为癌组织形态；CK19、Heppar-1、Ki67表达，提示该样本为肝癌。图3B为培养前的GL-006对应组织的病理和免疫组化结果视图。结果表明使用本发明的培养基HC-XF培养的肝癌类器官与培养前的肝癌组织的诊断结果一致。
实施例4与现有类器官培养基培养效果的比较
(1)对照培养基的配制
配制文献(Laura等，Nat Med.2017,23(12):1424-1435)中使用的培养基，其配方为Advanced DMEM/F12培养基(购自Corning公司)+1:100Penicillin/Streptomycin(购自Corning公司)+1:100GlutaMAX(购自Corning公司)+10mM HEPES(购自赛默飞公司)+1:50B27(购自Gibco公司)+1:100N2(购自Gibco公司)+1.25mmol/L N-乙酰半胱氨酸(购自MCE公司)+10mmol/L烟酰胺(购自MCE公司)+10nM胃泌素(购自MCE公司)+50ng/ml表皮细胞生长因子(购自R&D公司)+100ng/ml成纤维细胞生长因子10(购自sino biological公司)+25ng/ml肝细胞生长因子(购自R&D公司)+10μmol/L Forskolin(购自MCE公司)+5μmol/L A8301(购自MCE公司)+10μmol/L Y27632(购自MCE公司)+3nmol/L地塞米松(购自MCE公司)。以下简称为Laura培养基。
(2)肝癌悬浮类器官培养
按照实施例1之(2)的方法从术中组织样本GL-018获得肝癌原代细胞，并分别用HC-XF培养基和Laura培养基按照实施例3的方法进行悬浮类器官培养。
在培养第7天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的肝癌悬浮类器官。图4A和4B是4倍物镜下拍摄分别由HC-XF培养基和Laura培养基培养得到的类器官的照片，图4C是两种培养基培养得到的类器官相对大小的对比柱状图。
根据图4A-4C的结果可知，与Laura培养基相比，HC-XF培养基 能显著促进肝癌类器官的扩增和培养。
实施例5使用本发明的培养基扩增得到的肝癌悬浮类器官用于药物筛选
(1)肝癌悬浮类器官培养
从肝癌术中样本(GL-006)按照实施例1之(2)的方法分离得到肝癌原代细胞，并使用HC-XF培养基进行悬浮类器官培养，待肝癌类悬浮器官直径超过50μm时进行药物筛选。
(2)筛选药物配制
按照下表配制2个浓度梯度的3种药物(表柔比星、柔红霉素、多柔比星；均购自MCE公司)，保存待用。
表3表柔比星、多柔比星、柔红霉素药物添加液的配制
表柔比星 配制浓度(mM) 2 0.2
多柔比星 配制浓度(mM) 2 0.2
柔红霉素 配制浓度(mM) 2 0.2
(3)加药
取出配制好的药物，置于室温，将药物用HC-XF培养基稀释1000倍后备用。从孵箱取出按照步骤(1)培养获得的类器官，将类器官转移至15ml离心管，1500rpm，离心5分钟。用HC-XF培养基重悬类器官，计数，调整类器官浓度为50个/50μL，在96孔中加入调整好的类器官悬液50μL，再加入50μL稀释后的药物，将96孔板表面消毒后移至培养箱中继续培养，5天后测定类器官活力。
(4)类器官活力测试
4℃冰箱取出CellTiter-Glo发光试剂(购自Promega公司)，取10毫升试剂于加样槽中，培养箱中取出待检测96孔板，每孔加入20μL CellTiter-Glo发光试剂，静置10分钟后混匀，使用多功能酶标仪(Perkin Elmer公司Envision)检测。
(5)数据处理
按照公式药物抑制率(％)＝100％-(第五天培养孔化学发光数值 药物处理组/第零天培养孔化学发光数值 药物处理组)/(第五天培养孔化学发 光数值 DMSO/第零天培养孔化学发光数值 DMSO)*100％，计算得到不同药物的抑制率，将结果示于图5A和5B。图5A为4倍物镜下显微镜(Invitrogen公司EVOS M500)拍摄的加药处理5天后类器官生长的照片，图5B为不同浓度的测试药物抑制肝癌类器官生长的抑制率柱状图。
由图5A可以确认，使用本发明的肝癌悬浮类器官培养基培养得到的类器官，在高浓度的药物处理之后类器官生长明显受到抑制。图5B是不同浓度的三种测试药物抑制肝癌类器官生长的抑制率柱状图。由图5B可以看出，药物处理组的数据误差值很小，表明利用此套体系进行药物筛选，相同药物重复孔之间数据基本保持一致。三个抗肿瘤药物中，高浓度下都具有较强的抑制类器官生长的效果，低浓度下对类器官生长的抑制效果具有较大差异，这表明同一病人的类器官对不同药物的有效性和敏感性不同。根据结果可以判断肝癌病人在临床使用该种药物时的有效性及有效用量。
工业应用性
本发明提供一种用于肝癌悬浮类器官培养的培养基及培养方法，可将培养得到的类器官应用于药物的疗效评估和筛选。因而，本发明适于工业应用。
尽管本文对本发明作了详细说明，但本发明不限于此，本技术领域的技术人员可以根据本发明的原理进行修改，因此，凡按照本发明的原理进行的各种修改都应当理解为落入本发明的保护范围。

Claims (11)

  1. 一种用于肝癌悬浮类器官的培养基，其特征在于包括MST1/2激酶抑制剂、Y27632、选自N2和B27的至少一种细胞培养添加剂、SB202190、胰岛素、成纤维细胞生长因子10、霍乱毒素、ITS细胞培养添加剂、肝细胞生长因子、GlutaMAX、和非必需氨基酸，
    其中，所述MST1/2激酶抑制剂包括式(I)的化合物或其药学可接受的盐、或溶剂化物，
    Figure PCTCN2021131963-appb-100001
    其中，
    R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的芳基、芳基C1-C6烷基和杂芳基；
    R 2和R 3各自独立地选自C1-C6烷基；
    R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、和C3-C6杂环基C1-C6烷基；
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
  2. 如权利要求1所述的培养基，其中
    R 1选自C1-C6烷基、C3-C6环烷基、C4-C8环烷基烷基、C2-C6螺环烷基、以及任选地被1-2个独立地R 6取代的苯基、萘基、苯甲基和噻吩基；
    R 2和R 3各自独立地选自C1-C3烷基；
    R 4和R 5各自独立地选自氢、C1-C6烷基、C3-C6环烷基、C4-C8 环烷基烷基、C1-C6烷基羟基、C1-C6卤代烷基、C1-C6烷基氨基C1-C6烷基、C1-C6烷氧基C1-C6烷基、哌啶基C1-C6烷基、和四氢吡喃基C1-C6烷基；
    R 6选自卤素、C1-C6烷基、C1-C6烷氧基、和C1-C6卤代烷基。
  3. 如权利要求1所述的培养基，其中所述MST1/2激酶抑制剂包括式(Ia)的化合物或其药学可接受的盐、或溶剂化物，
    Figure PCTCN2021131963-appb-100002
    其中，
    R 1选自C1-C6烷基、任选地被1-2个独立地R 6取代的苯基、任选地被1-2个独立地R 6取代的噻吩基、和任选地被1-2个独立地R 6取代的苯甲基；
    R 5选自氢、C1-C6烷基、和C3-C6环烷基；
    R 6各自独立地选自卤素、C1-C6烷基、和C1-C6卤代烷基。
  4. 如权利要求3所述的培养基，其中
    R 1为任选地被1-2个独立地R 6取代的苯基；
    R 5为氢；
    R 6优选为氟、甲基或三氟甲基。
  5. 如权利要求1所述的培养基，其中所述MST1/2激酶抑制剂选自以下化合物或其药学可接受的盐中的至少一种：
    Figure PCTCN2021131963-appb-100003
    Figure PCTCN2021131963-appb-100004
    Figure PCTCN2021131963-appb-100005
    Figure PCTCN2021131963-appb-100006
    Figure PCTCN2021131963-appb-100007
  6. 如权利要求1～5中任一项所述的培养基，其特征在于所述培养基中各成分的含量满足以下任意一项或多项或全部满足：
    所述MST1/2激酶抑制剂的浓度为2.5～40μM；
    所述B27或N2细胞培养添加剂相对于培养基的体积比为1:25～1:200；
    所述肝细胞生长因子的浓度为1.25～20ng/mL；
    所述ITS细胞培养添加剂相对于培养基的体积比为1:12.5～1:200；
    所述Y27632的浓度为2.5～40μM；
    所述SB202190的浓度为5～40nM；
    所述成纤维细胞生长因子10的浓度为2.5～20ng/mL；
    所述胰岛素的浓度为2.5～40μg/mL；
    所述霍乱毒素的浓度为1.25～20ng/mL；
    所述GlutaMAX相对于培养基的体积比为1:12.5～1:200；
    所述非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，其浓度为12.5～200μM。
  7. 如权利要求1～6中任一项所述的培养基，其特征在于所述培养基中各成分的含量满足以下任意一项或多项或全部满足：
    所述MST1/2激酶抑制剂的浓度为2.5～20μM；
    所述B27或N2细胞培养添加剂相对于培养基的体积比为1:25～1:100；
    所述肝细胞生长因子的浓度为1.25～10ng/mL；
    所述ITS细胞培养添加剂相对于培养基的体积比为1:50～1:200；
    所述Y27632的浓度为5～20μM；
    所述SB202190的浓度为5～20nM；
    所述成纤维细胞生长因子10的浓度为2.5～10ng/mL；
    所述胰岛素的浓度为2.5～20μg/mL；
    所述霍乱毒素的浓度为2.5～10ng/mL；
    所述GlutaMAX相对于培养基的体积比为1:12.5～1:200；
    所述非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，其浓度为100～200μM。
  8. 如权利要求1～7中任一项所述的培养基，其特征在于还包括：
    选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基；和
    选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种的抗生素。
  9. 如权利要求1～8中任一项所述的培养基，其特征在于所述培养基不含Wnt激动剂、R-spondin家族蛋白、Noggin蛋白、或BMP抑制剂。
  10. 一种肝癌悬浮类器官的培养方法，其特征在于包括以下步骤：
    (1)从肝癌实体瘤组织分离样本，获得肝癌原代细胞；
    (2)配制根据权利要求1～9中任一项所述的肝癌悬浮类器官的培养基，并对步骤(1)获得的肝癌原代细胞进行悬浮培养。
  11. 一种用于评估或筛选治疗肝癌的药物的方法，其特征在于，包括以下步骤：
    (1)使用根据权利要求10所述的肝癌悬浮类器官的培养方法培养肝癌悬浮类器官；
    (2)选定需要检测的药物并按照所需浓度梯度进行稀释；
    (3)对(1)中培养得到的悬浮类器官添加稀释后的所述药物；
    (4)进行类器官大小或类器官活力检测。
PCT/CN2021/131963 2021-10-14 2021-11-22 肝癌悬浮类器官的培养基和培养方法 WO2023060695A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111198660.1 2021-10-14
CN202111198660 2021-10-14

Publications (1)

Publication Number Publication Date
WO2023060695A1 true WO2023060695A1 (zh) 2023-04-20

Family

ID=85961093

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/131963 WO2023060695A1 (zh) 2021-10-14 2021-11-22 肝癌悬浮类器官的培养基和培养方法

Country Status (2)

Country Link
CN (1) CN115975937A (zh)
WO (1) WO2023060695A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111039944A (zh) * 2018-10-12 2020-04-21 中国科学院合肥物质科学研究院 Mst1激酶抑制剂及其用途
CN111411084A (zh) * 2020-04-28 2020-07-14 江苏信安佳医疗科技有限公司 一种用于构建肝肿瘤无支架类器官的培养基及培养方法
CN112210537A (zh) * 2020-09-28 2021-01-12 浙江科途医学科技有限公司 肝癌类器官及其培养方法、培养用培养基和用途
CN113528444A (zh) * 2020-04-15 2021-10-22 合肥中科普瑞昇生物医药科技有限公司 一种用于食管鳞癌上皮细胞的培养基、培养方法及其应用
CN113528425A (zh) * 2020-04-15 2021-10-22 合肥中科普瑞昇生物医药科技有限公司 一种用于乳腺上皮干细胞的培养基和培养方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111039944A (zh) * 2018-10-12 2020-04-21 中国科学院合肥物质科学研究院 Mst1激酶抑制剂及其用途
CN113528444A (zh) * 2020-04-15 2021-10-22 合肥中科普瑞昇生物医药科技有限公司 一种用于食管鳞癌上皮细胞的培养基、培养方法及其应用
CN113528425A (zh) * 2020-04-15 2021-10-22 合肥中科普瑞昇生物医药科技有限公司 一种用于乳腺上皮干细胞的培养基和培养方法
CN111411084A (zh) * 2020-04-28 2020-07-14 江苏信安佳医疗科技有限公司 一种用于构建肝肿瘤无支架类器官的培养基及培养方法
CN112210537A (zh) * 2020-09-28 2021-01-12 浙江科途医学科技有限公司 肝癌类器官及其培养方法、培养用培养基和用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG HAINA, WANG RUI, HUANG DAN, LI SIHAN, GAO BEIBEI, KANG ZHIJIE, TANG BO, XIE JIAJUN, YAN FANZHI, LIANG RUI, LI HUA, YAN JINSON: "Homoharringtonine Exerts Anti-tumor Effects in Hepatocellular Carcinoma Through Activation of the Hippo Pathway", FRONTIERS IN PHARMACOLOGY, vol. 12, XP093056583, DOI: 10.3389/fphar.2021.592071 *

Also Published As

Publication number Publication date
CN115975937A (zh) 2023-04-18

Similar Documents

Publication Publication Date Title
CN113528444B (zh) 一种用于食管鳞癌上皮细胞的培养基、培养方法及其应用
CN113528425B (zh) 一种用于乳腺上皮肿瘤细胞的培养基和培养方法
WO2021184408A1 (zh) 胃癌原代细胞的培养基及培养方法
WO2023060774A1 (zh) 宫颈癌类器官的培养基、及其培养方法和应用
WO2023035299A1 (zh) 一种用于肝癌类器官培养的培养基、及其培养方法和应用
WO2023004888A1 (zh) 一种用于口腔癌类器官培养的培养基、及其培养方法和应用
WO2023060764A1 (zh) 胃癌原代细胞的培养基和培养方法
WO2023060676A1 (zh) 卵巢癌类器官的培养基、培养方法及其应用
WO2023060695A1 (zh) 肝癌悬浮类器官的培养基和培养方法
WO2022241845A1 (zh) 肝癌原代细胞的培养基及培养方法
EP4368706A1 (en) Culture medium and culture method for lung cancer epithelial cells, and application thereof
WO2022227110A1 (zh) 口腔癌原代细胞的培养基及培养方法
WO2023060709A1 (zh) 食管癌类器官的培养基、培养方法及其应用
WO2023060684A1 (zh) 肺癌类器官的培养基、培养方法及其应用
WO2023060710A1 (zh) 胃癌类器官的培养基及培养方法
WO2023060711A1 (zh) 肺癌胸水来源类器官的培养基、培养方法及其应用
WO2023060642A1 (zh) 一种肠癌原代细胞的培养基及体外培养方法和用途
WO2023060643A1 (zh) 肠癌类器官的培养基及培养方法
WO2023060696A1 (zh) 卵巢癌原代细胞的培养基、培养方法及其应用
WO2023060820A1 (zh) 胃癌原代细胞的培养基和培养方法
WO2023060677A1 (zh) 卵巢癌原代细胞的培养基、培养方法及其应用
WO2022160368A1 (zh) 肠癌原代细胞的培养基及培养方法
WO2023060681A1 (zh) 一种用于宫颈癌原代细胞的培养基和培养方法
WO2023039999A1 (zh) 一种用于肺癌上皮细胞的培养基、培养方法及其应用
EP4159845A1 (en) Culture medium for laryngeal cancer epithelial cells, culture method, and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21960422

Country of ref document: EP

Kind code of ref document: A1