WO2021088847A1 - 一种妇科肿瘤原代细胞的培养方法及配套培养基 - Google Patents

一种妇科肿瘤原代细胞的培养方法及配套培养基 Download PDF

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WO2021088847A1
WO2021088847A1 PCT/CN2020/126391 CN2020126391W WO2021088847A1 WO 2021088847 A1 WO2021088847 A1 WO 2021088847A1 CN 2020126391 W CN2020126391 W CN 2020126391W WO 2021088847 A1 WO2021088847 A1 WO 2021088847A1
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final concentration
solution
recombinant protein
human recombinant
medium
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PCT/CN2020/126391
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French (fr)
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张函槊
尹申意
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北京基石生命科技有限公司
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Priority claimed from CN201911069291.9A external-priority patent/CN111621479A/zh
Priority claimed from CN201911069251.4A external-priority patent/CN111621478A/zh
Application filed by 北京基石生命科技有限公司 filed Critical 北京基石生命科技有限公司
Priority to EP20885048.7A priority Critical patent/EP4056684A4/en
Priority to JP2022525687A priority patent/JP2022554336A/ja
Priority to AU2020381037A priority patent/AU2020381037A1/en
Priority to US17/772,203 priority patent/US20220403342A1/en
Publication of WO2021088847A1 publication Critical patent/WO2021088847A1/zh

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Definitions

  • the invention relates to the field of biotechnology, in particular to a method for culturing primary gynecological tumor cells and a supporting culture medium.
  • gynecological tumors include breast cancer, ovarian cancer and endometrial cancer. According to statistics from the National Cancer Center in 2018, in 2014, breast cancer in my country accounted for 16.5% of the incidence of female malignant tumors, and the mortality rate reached 7.8%, ranking first and fifth in female tumors respectively. At present, the 5-year survival rate of breast cancer in my country is only 73.1% vs 90% (in the United States), which is still far behind the developed countries. In addition, the incidence of ovarian cancer in my country accounts for 2.5% of female malignant tumors, which has increased by 30% in the past ten years. These gynecological tumors pose a severe challenge to the lives and health of Chinese women.
  • Gynecological tumors are a type of complex disease. Its occurrence and development are a dynamic process, involving the interaction of many signal molecules, forming a complex molecular regulatory network, and being affected by external environmental factors. The etiology, occurrence and development of gynecological tumors have strong individual differences and cannot be generalized. Therefore, it is a trend in the field of gynecological tumor research and even the field of gynecological tumor diagnosis and treatment to use primary cell cultures of gynecological solid tumors as models for individualized and precise research.
  • the existing primary tumor cell culture technologies mainly include 2D culture, 3D culture, and reprogramming culture. These methods all face the problems of extremely long culture cycle, low culture success rate, and difficult removal of foreign cells to varying degrees.
  • the present invention provides a new primary cell culture technology and supporting reagents for gynecological solid tumor solid tumors.
  • the core of the technology is: (1) Treat gynecological solid tumor tissues with gentle cell dissociation reagents, To ensure the vitality of tumor cells in the tissue to the greatest extent; (2) Prepare a special serum-free medium, and use a suspension culture system to culture primary gynecological tumor cells in vitro to ensure the normal expansion of tumor cells while maximizing normal elimination Cell interference.
  • the present invention claims a culture medium.
  • the culture medium claimed in the present invention is medium A or medium B.
  • the medium A is composed of antibacterial and antifungal agents (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, cortex Alcohol (hydrocortisone), N-2 Supplement, Y-27632, Progesterone, ⁇ -Estradiol and basic medium for human cell culture (such as Advanced DMEM/F12 medium) .
  • antibacterial and antifungal agents penicillin-streptomycin-amphotericin B
  • HEPES HEPES
  • GlutaMax human recombinant protein EGF
  • human bFGF human recombinant protein HGF
  • human recombinant protein MSP cortex Alcohol (hydrocortisone), N-2 Supplement, Y-27632, Progesterone, ⁇ -Estradiol
  • basic medium for human cell culture such as Advanced DMEM/F
  • the final concentration of penicillin in the antibacterial and antifungal agent is 100-200 U/mL (such as 100 U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 ⁇ g/mL mL (such as 100 ⁇ g/mL); the final concentration of amphotericin B in the antibacterial and antifungal third antibody is 250-250ng/mL (such as 250ng/mL); the final concentration of HEPES is 8-12mM (such as 10mM); The final concentration of GlutaMax is 0.8-1.2% (such as 1%,% means volume percentage); the final concentration of the human recombinant protein EGF is 10-100ng/mL; the final concentration of the human recombinant protein bFGF The final concentration is 10-50ng/mL; the final concentration of the human recombinant protein HGF is 5-25ng/mL; the final concentration of the human recombinant protein MSP is 5-25ng/mL; the final concentration of the hydrocor
  • the medium B consists of antibacterial and antifungal agents (penicillin-streptomycin-amphotericin B), HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, N -N-acetyl-L-cysteine (N-acetyl-L-cysteine), N-2 Supplement, Y-27632, Progesterone, ⁇ -Estradiol and for human cell culture
  • the basic medium (such as Advanced DMEM/F12 medium) composition.
  • the final concentration of penicillin in the antibacterial and antifungal agent is 100-200 U/mL (such as 100 U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 ⁇ g/mL mL (such as 100 ⁇ g/mL); the final concentration of amphotericin B in the antibacterial and antifungal third antibody is 250-250ng/mL (such as 250ng/mL); the final concentration of HEPES is 8-12mM (such as 10mM); The final concentration of GlutaMax is 0.8-1.2% (such as 1%,% means volume percentage); the final concentration of the human recombinant protein EGF is 10-100ng/mL; the final concentration of the human recombinant protein bFGF The final concentration is 10-50ng/mL; the final concentration of the human recombinant protein HGF is 5-25ng/mL; the final concentration of the human recombinant protein MSP is 5-25ng/mL; the N-acetyl
  • composition of the third antibacterial and antifungal agent is as follows: each milliliter contains 10,000 units of penicillin (base), 10,000 ⁇ g of streptomycin (base) and 25 ⁇ g of amphotericin B.
  • the third antibacterial and antifungal agent is "Antibiotic-Antimycotic, 100X” (such as Gibco#15240062, or other products with the same composition).
  • the "Antibiotic-Antimycotic, 100X” contains 10,000 units of penicillin (alkali), 10,000 ⁇ g of streptomycin (alkali) and 25 ⁇ g of amphotericin B per milliliter, using penicillin G (sodium salt) and streptomyces sulfate in the form of 0.85% saline solution. And amphotericin B as Antifungal agent.
  • the GlutaMAX is an advanced cell culture additive that can directly replace L-glutamine in cell culture media.
  • the GlutaMAX is "GlutaMAX TM Supplement” (such as Gibco#35050061, or other products with the same composition).
  • the ingredient of the "GlutaMAX TM Supplement” is L-alanyl-L-glutamine, which is a substitute for L-glutamine, the concentration is 200 nM, and the solvent is 0.85% NaCl solution.
  • the N-2 Supplement is "N-2 Supplement (100X)” (such as Gibco#17502001, or other products with the same composition).
  • the "N-2 Supplement (100X)” contains a final concentration of 1 mM human transferrin (Holo), 500 mg/L of recombinant insulin full chain (Insulin Recombinant Full Chain), 0.63 mg/L L progesterone (Progesterone), 10mM putrescine (Putrescine), 0.52mg/L selenite (Selenite).
  • the Y-27632 is "Y-27632dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, Ki is 220nM and 300nM, respectively)" (such as MCE#129830-38-2, or its composition other products).
  • the brand product number of the third antibacterial and antifungal agent is Gibco#15240062; the brand product number of the HEPES is Gibco#15630080; The brand product number of GlutaMAX is Gibco#35050061; the brand product number of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand product number of the human recombinant protein bFGF is Peprotech AF-100-18B-50; The brand product number of the recombinant protein HGF is Peprotech AF-100-39-100; the brand product number of the human recombinant protein MSP is R&D#352-MS-050; the brand product number of the hydrocortisone is Selleck#S1696; the N-acetyl -The brand product number of L-cysteine is Sigma#A9165; the brand product number of the N-2 Supplement is Gibco#17502001
  • the medium for culturing primary gynecological tumor cells can exist in two forms:
  • the medium A contains the antibacterial and antifungal agent three antibodies (penicillin-streptomycin-amphotericin B), the HEPES, the GlutaMax, the human recombinant protein EGF, the human recombinant protein Protein bFGF, the human recombinant protein HGF, the human recombinant protein MSP, the cortisol, the N-2 Supplement, the Y-27632, the progesterone (Progesterone), the ⁇ -estradiol ( ⁇ -Estradiol) and a solution of the basic medium for human cell culture (such as Advanced DMEM/F12 medium).
  • the basic medium for human cell culture such as Advanced DMEM/F12 medium.
  • the medium B contains the third antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B), the HEPES, the GlutaMax, the human recombinant protein EGF, the human recombinant protein bFGF, The human recombinant protein HGF, the human recombinant protein MSP, the N-acetyl-L-cysteine (N-acetyl-L-cysteine), the N-2 Supplement, the Y-27632, the A solution of the progesterone, the ⁇ -Estradiol and the basic medium for human cell culture (such as Advanced DMEM/F12 medium).
  • penicillin-streptomycin-amphotericin B penicillin-streptomycin-amphotericin B
  • HEPES the HEPES
  • GlutaMax the human recombinant protein EGF
  • human bFGF the human recombinant protein bFGF
  • the medium After the medium is prepared, it needs to be filtered and sterilized with a 0.22 ⁇ M syringe filter (Millipore SLGP033RS), and can be stored at 4° C. for two weeks.
  • a 0.22 ⁇ M syringe filter (Millipore SLGP033RS)
  • each component in the culture medium exists alone, and is prepared according to the formula when used.
  • human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, and human recombinant protein MSP can be stored in the form of a stock solution (mother solution) (long-term storage at -80°C), specifically, it can be a 1000-fold stock solution (mother solution).
  • N-acetyl-L-cysteine, cortisol (Hydrocortisone) and Y-27632 can be stored in the form of a stock solution (mother solution) (long-term storage at -20°C), specifically 1000 times the stock solution (mother solution).
  • Progesterone and ⁇ -Estradiol can be stored in the form of a stock solution (mother solution) (long-term storage at -20°C), specifically, it can be a 100,000-fold stock solution (mother solution).
  • the 1000 ⁇ human recombinant protein EGF stock solution is composed of human recombinant proteins EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 ⁇ g/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is both PBS.
  • the 1000 ⁇ human recombinant protein bFGF stock solution is composed of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 ⁇ g/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is both PBS.
  • the 1000 ⁇ human recombinant protein HGF stock solution is composed of human recombinant proteins HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 ⁇ g/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is PBS.
  • the 1000 ⁇ human recombinant protein MSP stock solution is composed of human recombinant proteins MSP, BSA and PBS, wherein the final concentration of the human recombinant protein MSP is 20 ⁇ g/mL, the final concentration of the BSA is 0.01 g/mL, and the balance is both PBS.
  • the BSA can exist in the form of a 100-fold stock solution (mother solution) (prepared for current use), and is specifically composed of BSA and PBS, and the final concentration of BSA (Sigma#A1933) is 0.1g /mL, the remainder is PBS.
  • the 1000 ⁇ N-acetyl-L-cysteine stock solution is composed of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine is 0.5 M, the balance is ultrapure water.
  • 1000 ⁇ Y-27632 is composed of Y-27632 and ultrapure water.
  • the final concentration of Y-27632 is 10mM, and the balance is ultrapure water.
  • the 100000 ⁇ Progesterone stock solution is composed of Progesterone and absolute ethanol.
  • the final concentration of Progesterone is 1 mM, and the balance is absolute ethanol.
  • the 100000 ⁇ -Estradiol stock solution is composed of ⁇ -Estradiol and absolute ethanol.
  • the final concentration of ⁇ -Estradiol is 1 mM, and the balance is absolute ethanol.
  • 1000 ⁇ cortisol is composed of cortisol and ultrapure water, wherein the final concentration of the cortisol is 0.5M, and the balance is all ultrapure water.
  • the medium is a medium for culturing primary cells of gynecological tumors.
  • the “final concentration” is the final concentration in the medium.
  • the present invention claims a kit of reagents for culturing primary cells of gynecological tumors.
  • the kit of reagents for culturing primary cells of gynecological tumors contains the aforementioned medium and at least one of the following reagents: sample dissociation solution, sample preservation solution, cell separation buffer, cell digestion solution, Sample cleaning solution, digestion stop solution, cell cryopreservation solution and 1% CYTOP solution.
  • the sample dissociation fluid is composed of collagenase I, collagenase III, collagenase IV and PBS; wherein the final concentration of the collagenase I in the sample dissociation fluid is 150-250 U/mL (such as 200 U/mL). mL); the final concentration of the collagenase III in the sample dissociation solution is 250-350 U/mL (such as 290 U/mL); the final concentration of the collagenase IV in the sample dissociation solution is 150 -250U/mL (such as 200U/mL); the remainder is PBS.
  • the unit U of collagenase (the collagenase I, the collagenase III or the collagenase IV) is defined by the enzyme activity of the protease: at 37°C and pH 7.5, the collagenase is treated with 1 U of protease (The collagenase I, the collagenase III, or the collagenase IV) can release 1 ⁇ mol of L-leucine in 5 hours.
  • the brand product number of the collagenase I is Gibco#17100-017; the brand product number of the collagenase III is Solarbio#C8490; the brand product number of the collagenase IV is Gibco#17104- 019; The brand product number of the PBS is Gibco#21-040-CVR.
  • the sample preservation solution is composed of fetal bovine serum, double antibodies P/S (penicillin-streptomycin), HEPES and HBSS (Hank's balanced salt solution); wherein the final concentration of the fetal bovine serum is 1-5% (for example, 2%,% means volume percentage content); the final concentration of penicillin in the double antibody P/S is 100-200 U/mL (such as 100 U/mL); the streptomycin in the double antibody P/S The final concentration of HEPES is 100-200 ⁇ g/mL (such as 100 ⁇ g/mL); the final concentration of HEPES is 8-12 mM (such as 10 mM); the balance is HBSS.
  • the brand product number of the double antibody P/S is Gibco#15140122; the brand product number of the PBS is Gibco#21-040-CVR.
  • the cell separation buffer is composed of double antibody P/S (penicillin-streptomycin), heparin sodium and PBS; wherein the final concentration of penicillin in the double antibody P/S (penicillin-streptomycin) is 100 -200U/mL (such as 100U/mL); the final concentration of streptomycin in the double antibody P/S (penicillin-streptomycin) is 100-200 ⁇ g/mL (such as 100 ⁇ g/mL); the heparin sodium The final concentration is 10IU/mL; the rest is PBS.
  • the brand product number of the double antibody P/S penicillin-streptomycin
  • the brand product number of the heparin sodium is Solarbio#H8270
  • the brand product number of the PBS is Gibco#21-040-CVR.
  • each 10mL of the cell digestion solution contains 4-6mL (such as 5mL) Accutase, a final concentration of 5mM EDTA (that is, 10 ⁇ L 0.5M EDTA), 1.5-2.5mL (such as 2mL) TrypLE Express ,
  • the balance is PBS.
  • the Accutase is "StemPro TM Accutase TM Cell Dissociation Reagent” (such as Gibco#A11105-01, or other products with the same composition).
  • the Accutase is a single-component enzyme, dissolved in D-PBS, 0.5 mM EDTA solution.
  • the TrypLE Express is "TrypLE TM Express Enzyme (1X), no phenol red” (such as Gibco#12604013, or other products with the same composition).
  • the "TrypLE TM Express Enzyme (1X), no phenol red” contains 200 mg/L KCl, 200 mg/L KH 2 PO 4 , 8000 mg/L NaCl, 2160 mg/L Na 2 HPO 4 ⁇ 7H 2 O , 457.6mg/L EDTA; also contains recombinant protease.
  • the brand product number of the Accutase is Gibco#A11105-01; the brand product number of the 0.5M EDTA is Invitrogen#AM9261; the brand product number of the TrypLE Express is Gibco#12604013; the PBS The brand item number is Gibco#21-040-CVR.
  • the sample cleaning solution is composed of double antibodies P/S (penicillin-streptomycin) and PBS; wherein the final concentration of penicillin in the double antibodies P/S is 100-200 U/mL (such as 100 U/mL); The final concentration of streptomycin in the double antibody P/S is 100-200 ⁇ g/mL (such as 100 ⁇ g/mL); the balance is PBS.
  • P/S penicillin-streptomycin
  • PBS penicillin-streptomycin
  • the brand product number of the double antibody P/S is Gibco#15140122; the brand product number of the PBS is Gibco#21-040-CVR.
  • the digestion termination solution is composed of fetal bovine serum, double antibody P/S (penicillin-streptomycin) and DMEM medium; wherein the final concentration of the fetal bovine serum is 8-12% (such as 10%, expressed by %) Volume percentage content); the final concentration of penicillin in the double antibody P/S is 100-200 U/mL (such as 100 U/mL); the final concentration of streptomycin in the double antibody P/S is 100- 200 ⁇ g/mL (such as 100 ⁇ g/mL); the remainder is DMEM medium.
  • the brand product number of the double antibody P/S is Gibco#15140122; the brand product number of the PBS is Gibco#21-040-CVR.
  • the cell cryopreservation solution is composed of Advanced DMEM/F12 medium, DMSO and 1% methylcellulose solution; wherein, the Advanced DMEM/F12 medium, the DMSO and the 1% methylcellulose solution are The volume ratio is 20:2:(0.8-1.2), such as 20:2:1; the 1% methylcellulose solution is a methylcellulose aqueous solution with a concentration of 1g/100ml.
  • the brand product number of the Advanced DMEM/F12 medium is Gibco#12634010; the brand product number of the DMSO is Sigma#D2438; and the brand product number of the methylcellulose is Sigma#M7027.
  • composition of the 1% CYTOP solution is as follows: every 100 mL of the 1% CYTOP solution contains 1 mL of CYTOP, and the balance is fluorine oil.
  • the CYTOP is perfluoro (1-butenylvinylether) polymer.
  • the fluorine oil can be a fluorine oil with the brand product number 3M#FC40, or other products with the same composition.
  • the brand product number of the CYTOP is specifically Asashi glass#CTL-809M; the brand product number of the fluorine oil is specifically 3M#FC40.
  • the sample preservation solution can be used for the temporary preservation of the sample after in vitro, and can maintain the activity of the cells in the sample in a short time after the sample has been in vitro.
  • the sample preservation solution can be stored for 1 month at 4°C after preparation.
  • the sample cleaning solution can be used for cleaning and disinfecting samples.
  • the sample cleaning solution needs to be prepared and used.
  • the sample dissociation solution can be used for the dissociation of the sample, and can dissociate the primary gynecological tumor cells in the sample from the tissue.
  • the sample dissociation solution needs to be prepared for current use, and the collagenase I, collagenase III, and collagenase IV can be stored in the form of a stock solution (mother solution) at -20°C for a long time, specifically, it can be a 10-fold stock solution (mother solution).
  • the 10 ⁇ collagenase I stock solution is composed of the collagenase I and PBS; wherein the final concentration of the collagenase I is 2000 U/mL; the 10 ⁇ collagenase III stock solution is composed of the collagenase III and PBS; The final concentration of the collagenase III is 2000 U/mL; the 10 ⁇ collagenase IV stock solution is composed of the collagenase IV and PBS; wherein the final concentration of the collagenase IV is 2000 U/mL; the balance is PBS.
  • the enzymatic activities of the collagenase I, the collagenase III and the collagenase IV are defined in the foregoing.
  • the cell separation buffer is used to suspend cells in the pleural and ascites samples of gynecological tumors. After the cell separation buffer is prepared, it can be stored at 4°C for 1 month.
  • the cell digestion solution can be used for digestion and passage of cell masses, and can digest gynecological tumor masses into single cells.
  • the cell digestion solution needs to be prepared and used.
  • the digestion termination solution can be used to terminate the process of sample dissociation or cell digestion. After the digestion termination liquid is prepared, it can be stored at 4°C for one month.
  • the gynecological tumor primary cell culture medium can be used for the cultivation of gynecological tumor primary cells.
  • the gynecological tumor primary cell culture medium needs to be filtered and sterilized with a 0.22 ⁇ M syringe filter (Millipore SLGP033RS) after preparation, and can be stored at 4°C for two weeks.
  • the cell cryopreservation solution needs to be prepared and used immediately.
  • the 1% methyl cellulose solution can be stored for a long time at 4°C.
  • the "final concentration” is the final concentration of the corresponding component in the corresponding solution.
  • the present invention claims the application of the culture medium or set of reagents in culturing primary cells of gynecological tumors.
  • the present invention claims a method for culturing primary cells of gynecological tumors.
  • the method for culturing primary gynecological tumor cells as claimed in the present invention includes the following steps: using the medium described above to suspension culture primary gynecological tumor cells.
  • the gynecological tumor primary cells may be gynecological solid tumor primary cells or gynecological tumor pleural and ascites sample primary tumor cells.
  • the gynecological tumor primary cells are gynecological solid tumor primary cells
  • the gynecological solid tumor primary cells can be obtained after dissociating the gynecological solid tumor tissue with the sample dissociation solution described above.
  • the sample dissociation solution can be used to dissociate the gynecological solid tumor tissue according to a method including the following steps: 0.1-0.3 mL (such as 0.1 mL) of the sample dissociation solution per mg of tissue ,
  • the cut up the gynecological solid tumor tissue (for example, cut into small pieces of 0.8-1.2mm 3 ) is treated with the sample dissociation solution preheated at 37°C, and the sample is dissociated at 37°C.
  • Dissociation the dissociation time is 15 minutes to 3 hours. Observe the dissociation of the sample under the microscope every 15 minutes until a large number of single cells are observed.
  • the gynecological tumor primary cells are primary tumor cells of the gynecological tumor pleural and ascites fluid sample
  • the primary tumor cells of the gynecological tumor pleural and ascites fluid sample can be separated from the gynecological tumor pleural and ascites fluid sample using the cell separation buffer described above.
  • the separation buffer can be used to separate the gynecological tumor pleural and ascites sample according to the method including the following steps: use the cell separation buffer to suspend the cells in the gynecological tumor pleural and ascites sample, and then pass the density gradient Centrifugation (using Ficoll lymphocyte separation solution) to obtain the primary tumor cells of the gynecological tumor pleural and ascites sample.
  • the separation buffer before using the separation buffer to separate the gynecological tumor pleural and ascites fluid sample, it may further include a step of pre-separating the gynecological tumor pleural and ascites fluid sample: removing the gynecological tumor pleural and ascites fluid sample Impurities, clots and other components that affect cell density gradient separation.
  • the gynecological tumor primary cell culture medium can be used to suspend the gynecological tumor primary cell culture medium according to the method including the following steps: use the cell culture vessel M to suspend the gynecological tumor primary cell culture medium Cultivate the primary gynecological tumor cells at 37°C and 5% CO 2 and change the medium every 2-4 days (such as 3 days) until the cells form a mass of 80-120 ⁇ m (such as 100 ⁇ m) in diameter .
  • the initial seeding density of 10 5 / cm 2 container bottom area an example in six-well plates, at a density of 106 per well were plated cells.
  • the cell culture container M can be any one of the following: (1) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a COC resin material The cell culture container, the cell culture container made of cycloolefin polymer, or the cell culture container with low adsorption surface; (II) The cell culture container in (I) is modified by CYTOP.
  • the cell culture container is a cell culture dish, a cell culture well plate, or a micro well plate chip for cell culture (such as the micro well plate chip shown in FIG. 5 in Example 15) and the like.
  • the cell culture container in (I) can be modified by CYTOP according to the method including the following steps: the cell culture container in (I) is etched with pure oxygen, and the etching conditions are The power is 20W and the etching time is 3 minutes; then the surface of the cell culture container is covered with the 1% CYTOP solution described above, and the 1% CYTOP solution is dried to complete the CYTOP modification.
  • it may further include the following step of pre-dissociating the gynecological solid tumor tissue: washing the gynecological tumor solid with ethanol with a volume percentage of 70-75% (such as 75%) The tumor tissue sample surface for 10 to 30 seconds; wash the gynecological solid tumor tissue sample 10-20 times (such as 10 times) with the sample cleaning solution described above, and clean the gynecological solid tumor tissue with a sterile PBS solution Sample 5-10 times (such as 5 times); then remove impurities, connective tissue, adipose tissue, necrotic tissue and other components that affect primary cell culture in the gynecological solid tumor tissue sample.
  • the steps of pre-processing the gynecological solid tumor tissue for dissociation need to be operated on ice, and the entire operation steps need to be completed within 10 minutes.
  • the gynecological solid tumor tissue sample that undergoes the pre-dissociation treatment needs to be isolated within 2 hours, and it has been stored in the aforementioned sample preservation solution before the pre-dissociation treatment .
  • the method after dissociating the gynecological solid tumor tissue with the sample dissociation solution, it may further include the following steps: using 8-15 times (such as 10 times) the volume as described above Stop the dissociation reaction and collect the cell suspension; filter the cell suspension with a 100 ⁇ m or 40 ⁇ m sterile cell strainer to remove tissue debris and adherent cells; centrifuge at 800-1000g (such as 800g) at room temperature for 10-15 minutes (E.g. 10 minutes), discard the supernatant; then resuspend the cells with 3-5mL (e.g. 5mL) sterile PBS; then centrifuge at 800-1000g (e.g. 800g) at room temperature for 10-15 minutes (e.g. 10 minutes) and discard. Clear; then resuspend the cell pellet with the primary cell culture medium of the gynecological solid tumor solid tumor, observe the cell status under a microscope, and count the cells.
  • 8-15 times such as 10 times
  • the method may further include the following step: when the primary gynecological tumor cells form a mass with a diameter of 80-120 ⁇ m (such as 100 ⁇ m), the primary gynecological tumor cells are passaged.
  • the digestion temperature used during the passage is 37°C.
  • the digestion termination solution used when performing the passage is the digestion termination solution described above.
  • the step of passage collect the cell clumps to be passaged, wash the cell clumps with sterile PBS solution after centrifugation, centrifuge again, and then resuspend the cell clumps with the cell digestion solution.
  • the method may further include the steps of freezing and/or resuscitating the primary gynecological tumor cells after 2-3 passages and amplification.
  • the cell cryopreservation solution used when performing the cryopreservation is the cell cryopreservation solution described above.
  • the specific steps of the freezing storage are carried out: collecting the cell clumps to be frozen, washing the cell clumps with sterile PBS solution after centrifugation, then centrifuging, and then resuspending the cell clumps with the cell digestion solution , Perform digestion at 37°C until the cell clumps are digested into single cells, stop the digestion reaction with the digestion stop solution (the amount can be 5-10 times, such as 10 times the volume), and collect the cell suspension; After centrifugation, use the cell cryopreservation solution to resuspend the cell pellet at a density of 0.5-2 ⁇ 10 6 /mL (for example, 10 6 /mL), and transfer to liquid nitrogen for long-term storage after freezing in a gradient cooling box overnight. All centrifugation in the above freezing step can specifically be 800-1000g (such as 800g) centrifugation at room temperature for 10-20 minutes (such as 10 minutes).
  • the cryotube containing the cells to be resuscitated from liquid nitrogen quickly thaw the cells in 37-39°C (e.g. 37°C) sterile water; centrifuge (e.g., 800-1000g) , Such as 800g room temperature centrifugation for 5-10 minutes, such as 10 minutes), resuspend the cell pellet with the gynecological tumor primary cell culture medium, and then use a culture vessel with a low adsorption surface to culture the cells (the initial seeding density can be 10 5 Cells/cm 2 container bottom area), each tube of cells (10 6 cells) was recovered to a 3.5 cm petri dish), and the culture condition was 37°C, 5% CO 2 .
  • the initial seeding density can be 10 5 Cells/cm 2 container bottom area
  • each tube of cells (10 6 cells) was recovered to a 3.5 cm petri dish
  • the culture condition was 37°C, 5% CO 2 .
  • the gynecological tumor primary cells are isolated from surgical samples or needle biopsy samples or pleural and ascites samples (pleural fluid or ascites) of patients with gynecological tumors.
  • gynecological solid tumor tissue samples obtained from surgical samples should weigh more than 20 mg, more than 4 needle biopsy samples (belonging to solid tumor samples), and more than 100 mL of pleural and ascites samples.
  • PBSs can be 1 ⁇ PBS, pH 7.3-7.5.
  • the specific composition is as follows: the solvent is water, the solute and the concentration are: KH 2 PO 4 144 mg/L, NaCl 9000 mg/L, and Na 2 HPO 4 ⁇ 7H 2 O 795 mg/L.
  • Figure 1 shows the single cells obtained after treatment of breast cancer tissue.
  • the scale is 100 ⁇ m, 100 times magnification.
  • a and B are two parallel treatments respectively.
  • Figure 2 shows the cell mass obtained after primary culture of breast cancer tissue.
  • the scale is 100 ⁇ m, 100 times magnification.
  • A is cultured in medium A; B is cultured in medium B.
  • Figure 3 is a HE staining image of gynecological tumor cells obtained after primary culture of breast cancer tissue.
  • the scale is 100 ⁇ m, 200 times magnification.
  • A is cultured in medium A;
  • B is cultured in medium B.
  • Figure 4 is an immunohistochemical staining image of a paraffin section of tumor cell mass obtained after primary culture of breast cancer tissue.
  • the scale is 100 ⁇ m, 200 times magnification.
  • A is cultured in medium A;
  • B is cultured in medium B.
  • Figure 5 is a design diagram of the microplate chip of the present invention.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.
  • the quantitative experiments in the following examples are all set to repeat the experiment three times, and the results are averaged.
  • Example 1 Preparation of reagents for culturing primary cells of gynecological tumors
  • sample preservation solution After the sample preservation solution is prepared, use 15mL centrifuge tubes for aliquoting, 5mL per tube. After aliquoting, it can be stored at 4°C for 1 month.
  • the sample cleaning solution needs to be prepared for immediate use.
  • sample dissociation solution is prepared for immediate use.
  • the unit U of collagenase (the collagenase I, the collagenase III, or the collagenase IV) is defined by the enzyme activity of the protease: at 37°C and pH 7.5, use Treating collagenase (the collagenase I, the collagenase III or the collagenase IV) with 1 U protease for 5 hours can release 1 ⁇ mol of L-leucine.
  • the cell digestion solution is ready for use.
  • the digestion stop solution After the digestion stop solution is prepared, it can be stored at 4°C for one month.
  • Gynecological tumor primary cell culture medium 100mL
  • the specific formula of the medium A is shown in Table 9.
  • the specific formula of the medium B is shown in Table 10.
  • the preparation of the gynecological tumor primary cell culture medium is completed, it is filtered and sterilized with a 0.22 ⁇ M syringe filter (Millipore SLGP033RS), and can be stored at 4°C for two weeks.
  • a 0.22 ⁇ M syringe filter (Millipore SLGP033RS)
  • Tables 9 and 10 the preparation of the human recombinant protein stock solution is shown in Tables 12-15, the preparation of the hydrocortisone stock solution is shown in Table 16, and the preparation of the N-acetyl-L-cysteine stock solution is shown in Table 17.
  • the preparation of Y-27632 stock solution is shown in Table 18; the preparation of Progesterone stock solution is shown in Table 19; the preparation of ⁇ -Estradiol stock solution is shown in Table 20.
  • the preparation of 100 ⁇ BSA solution required when preparing these stock solutions is shown in Table 11.
  • the 100 ⁇ BSA solution is prepared for immediate use.
  • the 10000 ⁇ -Estradiol stock solution After the preparation of the 10000 ⁇ -Estradiol stock solution, it is divided into 0.5mL sterile centrifuge tubes. The stock solution can be stored at -20°C for a long time.
  • the cell cryopreservation solution is now prepared for immediate use.
  • the 1% methyl cellulose solution can be stored for a long time at 4°C after preparation.
  • the 1% CYTOP solution After the 1% CYTOP solution is prepared, it can be stored for a long time at room temperature.
  • the cell separation buffer After the cell separation buffer is prepared, it can be stored at 4°C for 1 month.
  • Example 2 Obtaining of postoperative specimens/biopsy puncture specimens/pleural and ascites samples of gynecological tumors
  • the attending doctor selects patients to be enrolled in accordance with the clinical indications specified in the medical guidelines, and selects appropriate samples for in vitro culture according to the intraoperative clinical indications.
  • the selection criteria for samples are: primary breast cancer, ovarian cancer, and uterus Endometrial cancer, cervical cancer or its metastatic lesions, surgical specimens weighing more than 20 mg, or pleural and ascites fluid samples exceeding 100 mL, or needle biopsy specimens exceeding 4 samples.
  • the attending doctor provides basic clinical information such as the patient's gender, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc.
  • the patient’s name, ID number and other information related to the patient’s privacy are concealed and replaced with a uniform experiment number.
  • the naming principle of the experiment number is the eight-digit date of the collection of the sample + the last four digits of the patient’s hospitalization number. For example, if the sample provided on January 1, 2018, the hospitalization number of the patient is T001512765, the sample experiment number is 201801012765.
  • the surgeon will collect fresh postoperative specimens/biopsy puncture specimens in a sterile environment in the operating room, and place them in the pre-prepared sample preservation solution (see Example 1). After the sample is isolated, it is temporarily stored on ice and transported to the laboratory for the next step within two hours. The pleural and ascites fluid samples were transported to the laboratory within 48 hours for the next step.
  • the surgical equipment used in the following operations must be sterilized in advance by high temperature and high pressure, and can be used after drying.
  • Example 4 Dissociation of solid tumor tissue samples from gynecological tumors
  • the surgical instruments used in the following embodiments need to be sterilized by high temperature and high pressure in advance, and can be used after being dried.
  • sample dissociation solution (see Example 1) per mg of tissue, treat the cut tissue sample with the sample dissociation solution preheated at 37°C, and perform the sample dissociation at 37°C. 15 minutes to 3 hours away. Observe the dissociation of the sample under the microscope every 15 minutes until a large number of single cells are observed.
  • Example 6 Density gradient centrifugation of pleural and ascites samples of gynecological tumors
  • Example 7 Primary cell culture of gynecological tumors
  • the medium used is the primary gynecological tumor cell culture medium A or medium B in Example 1.
  • the six-well plate is used as embodiment, a density of 106 per well were plated cells at 37 °C, 5% CO 2 condition cultured in cell culture incubator.
  • the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; the final concentration of human recombinant protein HGF is 20ng/mL; The final concentration of the recombinant protein MSP is 20ng/mL; the final concentration of hydrocortisone is 10 ⁇ g/mL; the final concentration of Y-27632 is 10 ⁇ M; the final concentration of Progesterone is 100nM; the final concentration of ⁇ -Estradiol is 10nM.
  • the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; the final concentration of human recombinant protein HGF is 20ng/mL; The final concentration of recombinant protein MSP is 20ng/mL; the final concentration of N-acetyl-L-cysteine is 1mM; the final concentration of Y-27632 is 10 ⁇ M; the final concentration of Progesterone is 100nM; the final concentration of ⁇ -Estradiol is 10nM.
  • tumor cells expanded in large numbers to form cell clusters with a diameter of 100 ⁇ m.
  • the total number of tumor cells could exceed 10 7 , and the number of other types of cells was significantly reduced or even disappeared.
  • This method has been tested on a large number of samples, and the success rate of in vitro culture of primary tumor cells of gynecological tumors can reach 70%.
  • the medium used is the gynecological tumor primary cell culture medium in Example 1. Taking a six-well plate as an example, press 10 per well. The density of 6 cells is plated and cultured in a cell incubator at 37°C and 5% CO 2.
  • the primary gynecological tumor cells in suspension culture can be cryopreserved after 2-3 passages and expansion:
  • Gynecological tumor primary cells preserved in liquid nitrogen can be resuscitated:
  • Example 11 HE staining identification of primary gynecological tumor cells
  • Figure 3 shows the HE staining effect of primary gynecological tumor cells cultured in vitro. It can be seen that these cells generally have high nuclear to cytoplasmic ratio, deep nuclear staining, intranuclear chromatin agglutination, multinucleation, and uneven cell size. feature.
  • Paraformaldehyde (Beijing Chemical Reagent Company, Analytical Pure), dissolve the paraformaldehyde powder in ultrapure water to prepare a 4% (4g/100mL) paraformaldehyde solution;
  • Test medium the two primary gynecological tumor cell culture media in Example 1, namely medium A or medium B.
  • the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; the final concentration of human recombinant protein HGF is 20ng/mL; The final concentration of the recombinant protein MSP is 20ng/mL; the final concentration of hydrocortisone is 10 ⁇ g/mL; the final concentration of Y-27632 is 10 ⁇ M; the final concentration of Progesterone is 100nM; the final concentration of ⁇ -Estradiol is 10nM.
  • the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of human recombinant protein bFGF is 20ng/mL; the final concentration of human recombinant protein HGF is 20ng/mL; The final concentration of recombinant protein MSP is 20ng/mL; the final concentration of N-acetyl-L-cysteine is 1mM; the final concentration of Y-27632 is 10 ⁇ M; the final concentration of Progesterone is 100nM; the final concentration of ⁇ -Estradiol is 10nM.
  • the dried sections can be observed or photographed under a microscope.
  • Figure 4 shows the effect of immunohistochemical staining of primary breast cancer cell masses cultured in vitro. It can be seen that the cells constituting the cell masses are positive for ER, which is consistent with the patient's pathological results, confirming that the culture obtained by this method is pure Higher tumor cells.
  • Example 13 In vitro culture of primary tumor cells from different types of gynecological tumor samples
  • this method can achieve a very high success rate for in vitro culture of primary tumor cell samples for various types of gynecological solid tumor samples.
  • Example 14 Using CYTOP modified cell culture consumables for primary tumor cell culture of gynecological tumors
  • the CYTOP modification method is as follows: firstly, the cell culture container is etched with pure oxygen, the etching condition is 20W power, and the etching time is 3 minutes. Then cover the surface of the petri dish or the culture plate with an appropriate amount (in a 96-well plate, 20 ⁇ L per well, an appropriate amount means completely covering the bottom of the petri dish) 1% CYTOP solution, and use it after the CYTOP solution is completely dried.
  • Polystyrene Polystyrene, abbreviated PS.
  • Embodiment 15 Microplate chip processing
  • microplate chips for culturing primary cells of gynecological tumors of the present invention.
  • the chip can be used for primary gynecological tumor cell culture and in vitro drug sensitivity testing experiments.
  • the design drawing of the microplate chip is shown in Figure 5.
  • the PMMA material (or PS, PC, COC, COP, LAS and other materials) is used to prepare the design drawing of the microplate chip structure as shown in Figure 5, and then the above-mentioned CYTOP modification method (see implementation Example 14) The surface was modified with CYTOP to obtain the microplate chip that can be used for primary cell culture of gynecological tumors.
  • the present invention provides a method and supporting reagents for extracting and culturing primary cells of gynecological tumors from fresh gynecological tumor surgical samples or needle biopsy samples or pleural and ascites samples.
  • the method has the following advantages:
  • tissue samples are small, only about 20mg of gynecological solid tumor surgical samples;
  • the culture stability is high, and the success rate of in vitro culture of qualified gynecological solid tumor surgical specimens with this method is as high as 70%;
  • the cell purity is high.
  • the proportion of tumor cells can reach 70%-95%, and the interference of miscellaneous cells is less.
  • the primary cell culture of gynecological tumors obtained by the method of the present invention can be used for in vitro experiments at a variety of cell levels, second-generation sequencing, construction of animal models, construction of cell lines, and the like. It is foreseeable that this culture method has broad application prospects in the research and clinical diagnosis and treatment of gynecological tumors.

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Abstract

提供了妇科肿瘤原代细胞培养方法及配套试剂，该技术核心是：用温和细胞解离试剂处理妇科肿瘤实体瘤组织，最大程度保证了组织中肿瘤细胞活力；配制特殊无血清培养基，利用悬浮培养体系对妇科肿瘤来源的实体瘤细胞进行体外培养，保证肿瘤细胞正常扩增的同时最大限度的排除正常细胞的干扰。利用该方法得到的妇科肿瘤原代细胞培养物可用于多种细胞水平体外实验、二代测序、构建动物模型、构建细胞系等等。可预见，这种培养方法在妇科肿瘤的研究和临床诊断治疗领域具有广泛的应用前景。

Description

一种妇科肿瘤原代细胞的培养方法及配套培养基 技术领域
本发明涉及生物技术领域，具体涉及一种妇科肿瘤原代细胞的培养方法及配套培养基。
背景技术
常见的妇科肿瘤有乳腺癌、卵巢癌和子宫内膜癌等。据国家癌症中心2018年数据统计，2014年，我国乳腺癌占女性恶性肿瘤发病率的16.5％，死亡率达7.8％，分别排女性肿瘤的第一位和第五位。当前，我国乳腺癌5年生存率仅73.1％vs 90％(美国)，与发达国家还有较大差距。此外，我国卵巢癌发病率占据女性恶性肿瘤的2.5％，过去十年增低加了30％。这些妇科肿瘤对我国女性的生命健康提出了严峻的挑战。
尽管世界各国的科研和医疗机构对妇科肿瘤的病因以及发生发展过程的研究都有很大力度的投入，但是人类对这种疾病仍然知之甚少。妇科肿瘤是一类复杂疾病，其发生、发展是一个动态的过程，涉及到诸多信号分子相互作用，形成了一个复杂的分子调控网络，同时还受到外界环境因素的影响。妇科肿瘤的病因和发生发展过程有很强的个体差异性，不能一概而论。因此将妇科肿瘤实体瘤原代细胞培养物作为模型进行个体化精准研究是妇科肿瘤研究领域乃至妇科肿瘤诊断治疗领域的趋势。
现有的原代肿瘤细胞培养技术主要有2D培养，3D培养，重编程培养等几类，这些方法都不同程度的面临培养周期极长，培养成功率低，杂细胞难以去除等问题。
发明公开
为了有效解决上述技术问题，本发明提供了一种新的妇科肿瘤实体瘤原代细胞培养技术及配套试剂，该技术的核心是：(1)用温和细胞解离试剂处理妇科肿瘤实体瘤组织，最大程度的保证了组织中肿瘤细胞的活力；(2)配制特殊的无血清培养基，利用悬浮培养体系对妇科肿瘤原代细胞进行体外培养，保证肿瘤细胞正常扩增的同时最大限度的排除正常细胞的干扰。
第一方面，本发明要求保护一种培养基。
本发明所要求保护的培养基为培养基A或培养基B。
所述培养基A由抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)、HEPES、GlutaMax、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白MSP、皮质醇(hydrocortisone)、N-2 Supplement、Y-27632、孕酮(Progesterone)、β-雌二醇(β-Estradiol)和用于人细胞培养的基础培养基(如Advanced DMEM/F12培养基)组成。其中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述抗菌抗真菌剂三抗中的两性霉素B的终浓 度为250-250ng/mL(如250ng/mL)；所述HEPES的终浓度为8-12mM(如10mM)；所述GlutaMax的终浓度为0.8-1.2％(如1％，％表示体积百分含量)；所述人重组蛋白EGF的终浓度为10-100ng/mL；所述人重组蛋白bFGF的终浓度为10-50ng/mL；所述人重组蛋白HGF的终浓度为5-25ng/mL；所述人重组蛋白MSP的终浓度为5-25ng/mL；所述皮质醇(hydrocortisone)的终浓度为1-10μg/mL；所述N-2 Supplement的终浓度为1％(体积百分含量)；所述Y-27632的终浓度为5-20μM；所述孕酮的终浓度为50-100nM；所述β-雌二醇的终浓度为10-50nM；余量均为用于人细胞培养的基础培养基(如Advanced DMEM/F12培养基)。
所述培养基B由抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)、HEPES、GlutaMax、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白MSP、N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine)、N-2 Supplement、Y-27632、孕酮(Progesterone)、β-雌二醇(β-Estradiol)和用于人细胞培养的基础培养基(如Advanced DMEM/F12培养基)组成。其中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为250-250ng/mL(如250ng/mL)；所述HEPES的终浓度为8-12mM(如10mM)；所述GlutaMax的终浓度为0.8-1.2％(如1％，％表示体积百分含量)；所述人重组蛋白EGF的终浓度为10-100ng/mL；所述人重组蛋白bFGF的终浓度为10-50ng/mL；所述人重组蛋白HGF的终浓度为5-25ng/mL；所述人重组蛋白MSP的终浓度为5-25ng/mL；所述N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine)的终浓度为0.5-2mM；所述N-2 Supplement的终浓度为1％(体积百分含量)；所述Y-27632的终浓度为5-20μM；所述孕酮的终浓度为50-100nM；所述β-雌二醇的终浓度为10-50nM；余量均为用于人细胞培养的基础培养基(如Advanced DMEM/F12培养基)组成。
进一步地，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)组成如下：每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B。所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)为“Antibiotic-Antimycotic,100X”(如Gibco#15240062，或与其组成相同的其他产品)。所述“Antibiotic-Antimycotic,100X”每毫升包含10000单位青霉素(碱)、10000μg链霉素(碱)和25μg两性霉素B，利用0.85％盐液形式的青霉素G(钠盐)、硫酸链霉素和两性霉素B作为
Figure PCTCN2020126391-appb-000001
抗真菌剂。所述GlutaMAX是一种高级细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。所述GlutaMAX为“GlutaMAX TM Supplement”(如Gibco#35050061，或与其组成相同的其他产品)。所述“GlutaMAX TM Supplement”的成分为L-alanyl-L-glutamine，是L-glutamine的替代物，浓度为200nM，溶剂为0.85％NaCl溶液。所述N-2 Supplement为“N-2 Supplement(100X)”(如Gibco#17502001，或与其组成相同的其他产品)。所述“N-2 Supplement(100X)”中 含有终浓度为1mM的人全铁转铁蛋白(Human Transferrin(Holo))、500mg/L的重组胰岛素全链(Insulin Recombinant Full Chain)、0.63mg/L的孕酮(Progesterone)、10mM的腐胺(Putrescine)、0.52mg/L的亚硒酸盐(Selenite)。所述Y-27632为“Y-27632dihydrochloride(一种ATP竞争性的ROCK-I和ROCK-II抑制剂，Ki分别为220nM和300nM)”(如MCE#129830-38-2，或与其组成相同的其他产品)。
在本发明的具体实施例中，所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)的品牌货号为Gibco#15240062；所述HEPES的品牌货号为Gibco#15630080；所述GlutaMAX的品牌货号为Gibco#35050061；所述人重组蛋白EGF的品牌货号为Peprotech AF-100-15-100；所述人重组蛋白bFGF的品牌货号为Peprotech AF-100-18B-50；所述人重组蛋白HGF的品牌货号为Peprotech AF-100-39-100；所述人重组蛋白MSP的品牌货号为R&D#352-MS-050；所述hydrocortisone的品牌货号为Selleck#S1696；所述N-acetyl-L-cysteine的品牌货号为Sigma#A9165；所述N-2 Supplement的品牌货号为Gibco#17502001；所述Y-27632的品牌货号为MCE#129830-38-2；所述孕酮(Progesterone)的品牌货号为Sigma#V900699；所述β-雌二醇(β-Estradiol)的品牌货号为Sigma#E2758；所述Advanced DMEM/F12培养基的品牌货号为Gibco#12634010。
进一步地，所述用于培养妇科肿瘤原代细胞的培养基的存在形式可为两种：
其一，所述培养基A为含有所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)、所述HEPES、所述GlutaMax、所述人重组蛋白EGF、所述人重组蛋白bFGF、所述人重组蛋白HGF、所述人重组蛋白MSP、所述皮质醇、所述N-2 Supplement、所述Y-27632、所述孕酮(Progesterone)、所述β-雌二醇(β-Estradiol)和所述用于人细胞培养的基础培养基(如Advanced DMEM/F12培养基)的溶液。
所述培养基B为含有所述抗菌抗真菌剂三抗(青霉素-链霉素-两性霉素B)、所述HEPES、所述GlutaMax、所述人重组蛋白EGF、所述人重组蛋白bFGF、所述人重组蛋白HGF、所述人重组蛋白MSP、所述N-乙酰-L-半胱氨酸(N-acetyl-L-cysteine)、所述N-2 Supplement、所述Y-27632、所述孕酮(Progesterone)、所述β-雌二醇(β-Estradiol)和所述用于人细胞培养的基础培养基(如Advanced DMEM/F12培养基)的溶液。
所述培养基配制好后需用0.22μM针头式滤器(Millipore SLGP033RS)过滤除菌，在4℃可以保存两周。
其二，所述培养基中的各组分单独存在，使用时按照配方进行配制。
更进一步地，其中的人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白MSP可以储液(母液)形式保存(-80℃长期保存)，具体可为1000倍储液(母液)。N-乙酰-L-半胱氨酸、皮质醇(Hydrocortisone)和Y-27632可以储液(母液)形式保存(-20℃长期保存)，具体可为1000倍储液(母液)。孕酮(Progesterone)和β-雌二醇(β-Estradiol)可以储液(母液)形式保 存(-20℃长期保存)，具体可为100000倍储液(母液)。
1000×人重组蛋白EGF储液由人重组蛋白EGF、BSA和PBS组成，其中所述人重组蛋白EGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
1000×人重组蛋白bFGF储液由人重组蛋白bFGF、BSA和PBS组成，其中所述人重组蛋白bFGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
1000×人重组蛋白HGF储液由人重组蛋白HGF、BSA和PBS组成，其中所述人重组蛋白HGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
1000×人重组蛋白MSP储液由人重组蛋白MSP、BSA和PBS组成，其中所述人重组蛋白MSP的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS。
上述四种1000倍储液中，所述BSA是可以100倍储液(母液)形式存在(现配现用)，具体由BSA和PBS组成，其中BSA(Sigma#A1933)的终浓度为0.1g/mL，余量均为PBS。
另外，1000×N-乙酰-L-半胱氨酸储液由N-乙酰-L-半胱氨酸和超纯水组成，其中所述N-乙酰-L-半胱氨酸的浓度为0.5M，余量均为超纯水。
1000×Y-27632由Y-27632和超纯水组成，其中Y-27632的终浓度为10mM，余量均为超纯水。
100000×Progesterone储液由Progesterone和无水乙醇组成，其中Progesterone的终浓度为1mM，余量均为无水乙醇。
100000×β-Estradiol储液由β-Estradiol和无水乙醇组成，其中β-Estradiol的终浓度为1mM，余量均为无水乙醇。
1000×皮质醇由皮质醇和超纯水组成，其中所述皮质醇的终浓度为0.5M，余量均为超纯水。
所述培养基为用于培养妇科肿瘤原代细胞的培养基。
上述培养基中，所述“终浓度”为在所述培养基中的终浓度。
第二方面，本发明要求保护一种用于培养妇科肿瘤原代细胞的成套试剂。
本发明所提供的用于培养妇科肿瘤原代细胞的成套试剂，含有前文所述培养基和如下试剂中的至少一种：样本解离液、样本保存液、细胞分离缓冲液、细胞消化液、样本清洗液、消化终止液、细胞冻存液和1％CYTOP溶液。
所述样本解离液由胶原酶I、胶原酶III、胶原酶IV和PBS组成；其中，所述胶原酶I在所述样本解离液中的终浓度为150-250U/mL(如200U/mL)；所述胶原酶III在所述样本解离液中的终浓度为250-350U/mL(如290U/mL)；所述胶原酶IV在所述样本解离液中的终浓度为150-250U/mL(如200U/mL)；余量均为PBS。
其中，用蛋白酶的酶活来定义胶原酶(所述胶原酶I、所述胶原酶III或所述胶原酶IV)的单位U：在37℃，pH 7.5的条件下，用1U蛋白酶处理胶原酶(所述胶原酶I、所述胶原酶III或所述胶原酶IV)5小时，可以释放L-亮氨 酸1μmol。
在本发明的具体实施例中，所述胶原酶I的品牌货号为Gibco#17100-017；所述胶原酶III的品牌货号为Solarbio#C8490；所述胶原酶IV的品牌货号为Gibco#17104-019；所述PBS的品牌货号为Gibco#21-040-CVR。
所述样本保存液由胎牛血清、双抗P/S(青霉素-链霉素)、HEPES和HBSS(Hank's平衡盐溶液)组成；其中，所述胎牛血清的终浓度为1-5％(如2％，％表示体积百分含量)；所述双抗P/S中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述双抗P/S中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述HEPES的终浓度为8-12mM(如10mM)；余量均为HBSS。
在本发明的具体实施例中，所述双抗P/S的品牌货号为Gibco#15140122；所述PBS的品牌货号为Gibco#21-040-CVR。
所述细胞分离缓冲液由双抗P/S(青霉素-链霉素)、肝素钠和PBS组成；其中，所述双抗P/S(青霉素-链霉素)中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述双抗P/S(青霉素-链霉素)中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；所述肝素钠的终浓度为10IU/mL；余量均为PBS。
在本发明的具体实施例中，所述双抗P/S(青霉素-链霉素)的品牌货号为Gibco#15140122；所述肝素钠的品牌货号为Solarbio#H8270；所述PBS的品牌货号为Gibco#21-040-CVR。
所述细胞消化液组成如下：每10mL所述细胞消化液中含有4-6mL(如5mL)Accutase，终浓度为5mM的EDTA(即10μL 0.5M EDTA)，1.5-2.5mL(如2mL)TrypLE Express，余量为PBS。
进一步地，所述Accutase为“StemPro TM Accutase TM Cell Dissociation Reagent”(如Gibco#A11105-01，或与其组成相同的其他产品)。所述Accutase是一种单一成分的酶，在D-PBS，0.5mM EDTA溶液中溶解。所述TrypLE Express为“TrypLE TM Express Enzyme(1X),no phenol red”(如Gibco#12604013，或与其组成相同的其他产品)。所述“TrypLE TM Express Enzyme(1X),no phenol red”中含有200mg/L的KCl、200mg/L的KH 2PO 4、8000mg/L的NaCl、2160mg/L的Na 2HPO 4·7H 2O、457.6mg/L的EDTA；还含有重组蛋白酶。
在本发明的具体实施例中，所述Accutase的品牌货号为Gibco#A11105-01；所述0.5M EDTA的品牌货号为Invitrogen#AM9261；所述TrypLE Express的品牌货号为Gibco#12604013；所述PBS的品牌货号为Gibco#21-040-CVR。
所述样本清洗液由双抗P/S(青霉素-链霉素)和PBS组成；其中，所述双抗P/S中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述双抗P/S中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；余量均为PBS。
在本发明的具体实施例中，所述双抗P/S的品牌货号为Gibco#15140122；所述PBS的品牌货号为Gibco#21-040-CVR。
所述消化终止液由胎牛血清、双抗P/S(青霉素-链霉素)和DMEM培养基组 成；其中，所述胎牛血清的终浓度为8-12％(如10％，％表示体积百分含量)；所述双抗P/S中的青霉素的终浓度为100-200U/mL(如100U/mL)；所述双抗P/S中的链霉素的终浓度为100-200μg/mL(如100μg/mL)；余量均为DMEM培养基。
在本发明的具体实施例中，所述双抗P/S的品牌货号为Gibco#15140122；所述PBS的品牌货号为Gibco#21-040-CVR。
所述细胞冻存液由Advanced DMEM/F12培养基、DMSO和1％甲基纤维素溶液组成；其中，所述Advanced DMEM/F12培养基、所述DMSO和所述1％甲基纤维素溶液的体积配比为20:2:(0.8-1.2)，如20:2:1；所述1％甲基纤维素溶液是浓度为1g/100ml的甲基纤维素水溶液。
在本发明的具体实施例中，所述Advanced DMEM/F12培养基的品牌货号为Gibco#12634010；所述DMSO的品牌货号为Sigma#D2438；所述甲基纤维素的品牌货号为Sigma#M7027。
所述1％CYTOP溶液的组成如下：每100mL所述1％CYTOP溶液中含有1mL CYTOP，余量为氟油。
其中，所述CYTOP为perfluoro(1-butenylvinylether)polymer。所述氟油可为品牌货号为3M#FC40的氟油，或与其组成相同的其他产品。
在本发明的具体实施方式中，所述CYTOP的品牌货号具体为Asashi glass#CTL-809M；所述氟油的品牌货号具体为3M#FC40。
所述样本保存液可用于样本离体后的暂时保存，可以在有样本离体后，短时间内维持样本中细胞的活性。所述样本保存液配制好后4℃可保存1个月。
所述样本清洗液可用于样本的清洗和消毒。所述样本清洗液需现配现用。
所述样本解离液可用于样本的解离，可以将样本中的妇科肿瘤原代细胞从组织中解离出来。所述样本解离液需现配现用，其中的胶原酶I、胶原酶III和胶原酶IV可以储液(母液)形式-20℃长期保存，具体可为10倍储液(母液)。10×胶原酶I储液由所述胶原酶I和PBS组成；其中所述胶原酶I的终浓度为2000U/mL；10×胶原酶III储液由所述胶原酶III和PBS组成；其中所述胶原酶III的终浓度为2000U/mL；10×胶原酶IV储液由所述胶原酶IV和PBS组成；其中所述胶原酶IV的终浓度为2000U/mL；余量均为PBS。所述胶原酶I、所述胶原酶III和所述胶原酶IV的酶活定义见前文。
所述细胞分离缓冲液，用于悬浮妇科肿瘤胸腹水样本中的细胞。所述细胞分离缓冲液配制完成后，可于4℃保存1个月。
所述细胞消化液可用于细胞团块的消化和传代，可以将妇科肿瘤肿瘤团块消化成单个细胞。所述细胞消化液需现配现用。
所述消化终止液可用于终止样本解离或细胞消化过程。所述消化终止液配制好后可在4℃保存一个月。
所述妇科肿瘤原代细胞培养基可用于妇科肿瘤原代细胞的培养。所述妇科 肿瘤原代细胞培养基配制好后需用0.22μM针头式滤器(Millipore SLGP033RS)过滤除菌，在4℃可以保存两周。
所述细胞冻存液需现配现用。其中，所述1％甲基纤维素溶液可在4℃长期保存。
在本发明中，所述“终浓度”均为相应组分在相应溶液中的终浓度。
第三方面，本发明要求保护所述培养基或成套试剂在培养妇科肿瘤原代细胞中的应用。
第四方面，本发明要求保护一种培养妇科肿瘤原代细胞的方法。
本发明所要求保护的培养妇科肿瘤原代细胞的方法，包括如下步骤：利用前文所述的培养基悬浮培养妇科肿瘤原代细胞。
其中，所述妇科肿瘤原代细胞可为妇科肿瘤实体瘤原代细胞或妇科肿瘤胸腹水样本原代肿瘤细胞。
当所述妇科肿瘤原代细胞为妇科肿瘤实体瘤原代细胞时，所述妇科肿瘤实体瘤原代细胞可用前文所述样本解离液对妇科肿瘤实体瘤组织进行解离处理后获得。
进一步地，可按照包括如下步骤的方法用所述样本解离液对所述妇科肿瘤实体瘤组织进行解离：按0.1-0.3mL(如0.1mL)所述样本解离液每mg组织的用量，将剪碎后的所述妇科肿瘤实体瘤组织(如剪成0.8-1.2mm 3的小块)用事先37℃预热的所述样本解离液进行处理，在37℃条件下进行样本解离，解离时间15分钟至3小时。每15分钟在显微镜下观察样本的解离情况，直到观察到大量的单个细胞。
当所述妇科肿瘤原代细胞为妇科肿瘤胸腹水样本原代肿瘤细胞时，所述妇科肿瘤胸腹水样本原代肿瘤细胞可用前文所述的细胞分离缓冲液从妇科肿瘤胸腹水样本中分离获得。
进一步地，可按照包括如下步骤的方法用所述分离缓冲液对所述妇科肿瘤胸腹水样本进行分离：用所述细胞分离缓冲液悬浮所述妇科肿瘤胸腹水样本中的细胞，然后通过密度梯度离心(利用Ficoll淋巴细胞分离液)获得所述妇科肿瘤胸腹水样本原代肿瘤细胞。
更进一步地，在用所述分离缓冲液对所述妇科肿瘤胸腹水样本进行分离之前还可包括对所述妇科肿瘤胸腹水样本进行分离前处理的步骤：去除所述妇科肿瘤胸腹水样本中的杂质、凝血块等影响细胞密度梯度分离的成分。
在所述方法中，可按照包括如下步骤的方法用所述妇科肿瘤原代细胞培养基悬浮培养所述妇科肿瘤原代细胞：使用细胞培养容器M，利用所述妇科肿瘤原代细胞培养基悬浮培养所述妇科肿瘤原代细胞，37℃，5％CO 2条件下进行培养，每2-4天(如3天)更换一次培养基，直至细胞形成直径80-120μm(如100μm)的团块。
其中，初始接种密度可为10 5个/cm 2容器底面积，以六孔板为例，按每孔10 6 个细胞的密度铺板。
其中，所述细胞培养容器M可为如下任一：(I)聚苯乙烯材质的细胞培养容器、聚碳酸酯材质的细胞培养容器、聚甲基丙烯酸甲酯材质的细胞培养容器、COC树脂材质的细胞培养容器、环烯烃聚合物材质的细胞培养容器或低吸附表面的细胞培养容器；(II)对(I)中的细胞培养容器进行CYTOP修饰后的细胞培养容器。
进一步地，所述细胞培养容器为细胞培养皿、细胞培养孔板或用于细胞培养的微孔板芯片(如实施例15中图5所示的微孔板芯片)等。
所述(II)中，可按照包括如下步骤的方法对所述(I)中的细胞培养容器进行CYTOP修饰：对所述(I)中的细胞培养容器进行纯氧刻蚀，刻蚀条件为功率20W，刻蚀时间为3分钟；然后用前文所述的1％CYTOP溶液覆盖所述细胞培养容器表面，晾干所述1％CYTOP溶液即完成CYTOP修饰。
进一步地，在所述方法中，还可包括如下对所述妇科肿瘤实体瘤组织进行解离前处理的步骤：用体积百分含量为70-75％(如75％)的乙醇清洗妇科肿瘤实体瘤组织样本表面10到30秒；用前文所述的样本清洗液清洗所述妇科肿瘤实体瘤组织样本10-20次(如10次)，用无菌的PBS溶液清洗所述妇科肿瘤实体瘤组织样本5-10次(如5次)；然后除去所述妇科肿瘤实体瘤组织样本中的杂质、结缔组织、脂肪组织、坏死组织等影响原代细胞培养的成分。
对所述妇科肿瘤实体瘤组织进行解离前处理的步骤需要在冰上操作，整个操作步骤需要在10分钟内完成。
进一步地，进行所述解离前处理的所述妇科肿瘤实体瘤组织样本的离体时间需为2小时以内，且在进行所述解离前处理之前一直保存于前文所述的样本保存液中。
进一步地，在所述方法中，用所述样本解离液对所述妇科肿瘤实体瘤组织进行解离处理后还可包括如下步骤：用8-15倍(如10倍)体积的前文所述的消化终止液终止解离反应，收集细胞悬液；用100μm或40μm无菌细胞滤网过滤所述细胞悬液，去除组织残片和粘连细胞；800-1000g(如800g)室温离心10-15分钟(如10分钟)，弃去上清；后用3-5mL(如5mL)无菌PBS重悬细胞；再800-1000g(如800g)室温离心10-15分钟(如10分钟)，弃去上清；然后用所述妇科肿瘤实体瘤原代细胞培养基重悬细胞沉淀，在显微镜下观察细胞状态，进行细胞计数。
进一步地，在所述方法中，还可包括如下步骤：待所述妇科肿瘤原代细胞形成直径80-120μm(如100μm)的团块时，对所述妇科肿瘤原代细胞进行传代。
更进一步地，进行所述传代时采用的消化温度为37℃。
更进一步地，进行所述传代时采用的消化终止液即为前文所述的消化终止液。
更加具体地，进行所述传代的步骤：收集待传代的细胞团块，离心后用无菌的PBS溶液清洗细胞团块，再离心，然后用所述细胞消化液重悬细胞团块，在37℃条件下进行消化，直到细胞团块都被消化为单个细胞，用所述消化终止液(其用量可为5-10倍，如10倍体积)终止消化反应，收集细胞悬液；离心后用所述妇科肿瘤原代细胞培养基重悬细胞沉淀，计数，然后使用具有低吸附表面的培养容器悬浮培养细胞(初始接种密度可为10 5个/cm 2容器底面积，以六孔板为例，按每孔10 6个细胞的密度铺板)，培养条件为37℃，5％CO 2。上述传代步骤中的所有离心均具体可为800-1000g(如800g)室温离心10-20分钟(如10分钟)。
进一步地，所述方法还可包括对经过2-3次传代扩增后的所述妇科肿瘤原代细胞进行冻存和/或复苏的步骤。
其中，进行所述冻存时采用的细胞冻存液为前文所述的细胞冻存液。
更进一步地，进行所述冻存的具体步骤：收集待冻存的细胞团块，离心后用无菌的PBS溶液清洗细胞团块，再离心，然后用所述细胞消化液重悬细胞团块，在37℃条件下进行消化，直到细胞团块都被消化为单个细胞，用所述消化终止液(其用量可为5-10倍，如10倍体积)终止消化反应，收集细胞悬液；离心后用所述细胞冻存液，按0.5-2×10 6/mL(如10 6/mL)的密度重悬细胞沉淀，梯度降温盒过夜冻存后转移至液氮中长期保存。上述冻存步骤中的所有离心均具体可为800-1000g(如800g)室温离心10-20分钟(如10分钟)。
更进一步地，进行所述复苏的具体步骤：将装有待复苏细胞的冻存管从液氮中取出，在37-39℃(如37℃)无菌水中迅速融化细胞；离心(如800-1000g，如800g室温离心5-10分钟，如10分钟)后用所述妇科肿瘤原代细胞培养基重悬细胞沉淀，然后使用具有低吸附表面的培养容器悬浮培养细胞(初始接种密度可为10 5个/cm 2容器底面积)，每管细胞(10 6个)复苏至3.5cm培养皿)，培养条件为37℃，5％CO 2。
在上述各方面中，所述妇科肿瘤原代细胞分离自妇科肿瘤患者的手术样本或穿刺活检样本或胸腹水样本(胸水或腹水)。其中，手术样本获得的妇科肿瘤实体瘤组织标本最好重量超过20mg，穿刺活检样本(属于实体瘤样本)超过4条，胸腹水样本超过100mL。
在本发明中，以上所有的所述PBS均可为1×PBS，pH7.3-7.5。其具体组成如下：溶剂为水，溶质及浓度为：KH 2PO 4 144mg/L，NaCl 9000mg/L，Na 2HPO 4·7H 2O795mg/L。
附图说明
图1为乳腺癌组织经过处理后得到的单细胞。标尺为100μm，100倍放大。A和B分别为两个平行处理。
图2为乳腺癌组织原代培养后得到的细胞团块。标尺为100μm，100倍放大。A为培养基A培养所得；B为培养基B培养所得。
图3为乳腺癌组织原代培养后得到的妇科肿瘤细胞HE染色图。标尺为100μm，200倍放大。A为培养基A培养所得；B为培养基B培养所得。
图4为乳腺癌组织原代培养后得到的肿瘤细胞团块石蜡切片免疫组化染色图。标尺为100μm，200倍放大。A为培养基A培养所得；B为培养基B培养所得。
图5为本发明微孔板芯片设计图。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1、配制用于培养妇科肿瘤原代细胞的试剂
1、样本保存液(100mL)
样本保存液(100mL)的具体配方如表1所示。
表1、样本保存液(100mL)
Figure PCTCN2020126391-appb-000002
样本保存液配制完成后，用15mL离心管进行分装，每管5mL。分装后可于4℃保存1个月。
2、样本清洗液(100mL)
样本清洗液(100mL)的具体配方如表2所示。
表2、样本清洗液(100mL)
Figure PCTCN2020126391-appb-000003
样本清洗液需现配现用。
3、样本解离液(10mL)
样本解离液(10mL)的具体配方如表3所示。
表3、样本解离液(10mL)
Figure PCTCN2020126391-appb-000004
Figure PCTCN2020126391-appb-000005
注：样本解离液现配现用。
表3中，胶原酶储液的配制如表4-表6所示。
表4、10×胶原酶I储液(100mL)
Figure PCTCN2020126391-appb-000006
10×胶原酶I储液配制后，用1.5mL无菌离心管分装，每管1mL。该储液可在-20℃长期保存。
表5、10×胶原酶III储液(100mL)
Figure PCTCN2020126391-appb-000007
10×胶原酶III储液配制后，用1.5mL无菌离心管分装，每管1mL。该储液可在-20℃长期保存。
表6、10×胶原酶IV储液(100mL)
Figure PCTCN2020126391-appb-000008
10×胶原酶IV储液配制后，用1.5mL无菌离心管分装，每管1mL。该储液可在-20℃长期保存。
表4-表6中，用蛋白酶的酶活来定义胶原酶(所述胶原酶I、所述胶原酶III或所述胶原酶IV)的单位U：在37℃，pH 7.5的条件下，用1U蛋白酶处理胶原酶(所述胶原酶I、所述胶原酶III或所述胶原酶IV)5小时，可以释放L-亮氨酸1μmol。
4、细胞消化液(10mL)
细胞消化液(10mL)的具体配方如表7所示。
表7、细胞消化液(10mL)
Figure PCTCN2020126391-appb-000009
细胞消化液现配现用。
5、消化终止液(100mL)
消化终止液(100mL)的具体配方如表8所示。
表8、消化终止液(100mL)
Figure PCTCN2020126391-appb-000010
消化终止液配制后，可在4℃保存一个月。
6、妇科肿瘤原代细胞培养基(100mL)
妇科肿瘤原代细胞培养基(100mL)有两种，分别记为培养基A和培养基B。所述培养基A的具体配方如表9所示。所述培养基B的具体配方如表10所示。
表9、妇科肿瘤原代细胞培养基A(100mL)
Figure PCTCN2020126391-appb-000011
表10、妇科肿瘤原代细胞培养基B(100mL)
Figure PCTCN2020126391-appb-000012
Figure PCTCN2020126391-appb-000013
妇科肿瘤原代细胞培养基配制完成后，用0.22μM针头式滤器(Millipore SLGP033RS)过滤除菌，在4℃可以保存两周。
表9和表10中，人重组蛋白储液的配制如表12-表15所示，hydrocortisone储液的配制如表16所示，N-acetyl-L-cysteine储液的配制如表17所示，Y-27632储液的配制如表18所示；Progesterone储液的配制如表19所示；β-Estradiol储液的配制如表20所示。配制这些储液时所需的100×BSA溶液配制如表11所示。
表11、100×BSA溶液(1mL)
Figure PCTCN2020126391-appb-000014
100×BSA溶液现配现用。
表12、1000×人重组蛋白EGF储液(5mL)
Figure PCTCN2020126391-appb-000015
1000×人重组蛋白EGF储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表13、1000×人重组蛋白bFGF储液(2.5mL)
Figure PCTCN2020126391-appb-000016
1000×人重组蛋白bFGF储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表14、1000×人重组蛋白HGF储液(5mL)
Figure PCTCN2020126391-appb-000017
1000×人重组蛋白HGF储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表15、1000×人重组蛋白MSP储液(2.5mL)
Figure PCTCN2020126391-appb-000018
1000×人重组蛋白MSP储液配制后，用1.5mL无菌离心管分装，该储液可在-80℃长期保存。
表16、1000×Hydrocortisone储液(10mL)
Figure PCTCN2020126391-appb-000019
1000×N-acetyl-L-cysteine储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表17、1000×N-acetyl-L-cysteine储液(5mL)
Figure PCTCN2020126391-appb-000020
1000×N-acetyl-L-cysteine储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表18、1000×Y-27632储液(3.125mL)
Figure PCTCN2020126391-appb-000021
1000×Y-27632储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表19、100000×Progesterone储液(15.9mL)
Figure PCTCN2020126391-appb-000022
100000×Progesterone储液配制后，用0.5mL无菌离心管分装，该储液可在-20℃长期保存。
表20、10000×β-Estradiol储液(18.36mL)
Figure PCTCN2020126391-appb-000023
10000×β-Estradiol储液配制后，用0.5mL无菌离心管分装，该储液可 在-20℃长期保存。
7、细胞冻存液
细胞冻存液的具体配方如表21所示。
表21、细胞冻存液
Figure PCTCN2020126391-appb-000024
细胞冻存液现配现用。
表21中，1％甲基纤维素溶液的配制如表22所示。
表22、1％甲基纤维素溶液(10mL)
Figure PCTCN2020126391-appb-000025
1％甲基纤维素溶液配制后可在4℃长期保存。
8、1％CYTOP溶液
表23、1％CYTOP溶液(100mL)
Figure PCTCN2020126391-appb-000026
1％CYTOP溶液配制完成后，可于常温长期保存。
9、细胞分离缓冲液(100mL)
细胞分离缓冲液(100mL)的具体配方如表24所示：
表24、细胞分离缓冲液(100mL)
Figure PCTCN2020126391-appb-000027
细胞分离缓冲液配制完成后，可于4℃保存1个月。
表24中，肝素钠溶液的配制如表25所示。
表25、1000×肝素钠(1mL)
Figure PCTCN2020126391-appb-000028
1000×肝素钠溶液现配现用。
实施例2、妇科肿瘤术后标本/活检穿刺标本/胸腹水样本的获取
1、与三甲医院合作，合作的开展通过了正规的医学伦理审查。
2、主治医生医生按照医学指南规定的临床指征选择入组患者，并根据术中临床指征选择合适的样本用于体外培养，样本的选取标准为：原发性乳腺癌、卵巢癌、子宫内膜癌、宫颈癌或其转移病灶，手术标本重量超过20mg的样本，或胸腹水样本超过100mL的样本，或穿刺活检标本超过4条的样本。
3、主治医生提供患者的性别、年龄、病史、家族史、吸烟史、病理分期分型、临床诊断等基本临床信息。隐去患者的姓名、身份证号等与病人隐私相关的信息，用统一的实验编号代替，实验编号的命名原则为采集样本的八位数字日期+患者住院号后四位。例如2018年1月1日提供的样本，患者住院号为T001512765，则样本实验编号为201801012765。
4、术中由外科医生，在手术室无菌环境中采集新鲜术后标本/活检穿刺标本，置于事先准备好的样本保存液(见实施例1)中。样本离体后在冰上暂存，两小时内运输到实验室进行下一步操作。胸腹水样本48小时内运输到实验室进行下一步操作。
实施例3、妇科肿瘤实体瘤组织样本解离前处理
下述操作需要在冰上操作，整个操作步骤需要在10分钟内完成。
下述操作中用到的手术器材，均需事先高温高压灭菌，烘干后才能使用。
1、样本称重。
2、用75％(体积百分含量)乙醇清洗样本表面10到30秒。
3、用样本清洗液清洗样本5次，用无菌的PBS溶液清洗样本5次。
4、用眼科剪、眼科镊、手术刀等器材，小心将样本中的脂肪组织、结缔组织、坏死组织剥离。
实施例4、妇科肿瘤实体瘤组织样本解离
下述实施例中用到的手术器材，均需事先高温高压灭菌，烘干后才能使用。
1、用眼科剪将组织剪碎成1mm 3左右的小块。
2、按0.1mL样本解离液(见实施例1)每mg组织的用量，用事先37℃预热的样本解离液处理剪碎的组织样本，在37℃条件下进行样本解离，解离时间15分钟至3小时。每15分钟在显微镜下观察样本的解离情况，直到观察到大量的单个细胞。
3、用10倍体积的消化终止液(见实施例1)终止解离反应，收集细胞悬液。
4、用100μm无菌细胞滤网过滤细胞悬液，去除组织残片和粘连细胞。
5、800g室温离心10分钟，弃去上清。
6、用5mL无菌PBS重悬细胞，800g室温离心10分钟，弃去上清。
7、用妇科肿瘤原代细胞培养基(见实施例1)重悬细胞沉淀，在显微镜下 观察细胞状态，进行细胞计数。
如图1所示，解离得到的单细胞悬液中，除了肿瘤细胞以外还混杂着大量各种类型的其他细胞，如红细胞，淋巴细胞，纤维细胞等等。本方法的优势之一就是在后续的培养过程中，只有癌细胞可以进行大量扩增，而其他细胞的比例逐渐减少甚至消失，最终获得纯度较高的妇科肿瘤原代肿瘤细胞。
实施例5、妇科肿瘤胸腹水样本预处理
下述操作需要在冰上操作，整个操作步骤需要在10分钟内完成。
1、妇科肿瘤胸腹水样本冰上静置30分钟左右，使样本中的凝血块和大的不溶固体沉降至样本管底部；
2、小心将上清液转移至50mL无菌离心管中，加入一倍体积预冷的PBS混匀；
3、2000g，4℃离心5分钟，弃去上清；
4、用细胞分离缓冲液(见实施例1)重悬细胞沉淀，2000g，4℃离心5分钟，弃去上清；
5、用细胞分离缓冲液(见实施例1)重悬细胞沉淀，调整细胞浓度至10 7/mL。
实施例6、妇科肿瘤胸腹水样本密度梯度离心
1、用50mL无菌离心管取与细胞悬液等体积的Ficoll细胞分离液(MP#50494)。
2、小心将细胞悬液加在细胞分离液上层，使两者之间形成一个清晰的界面。
3、2000g水平式常温离心20分钟。
4、吸取中间层白膜，至新管中。
5、用20mL无菌的PBS重悬细胞沉淀，1500g常温离心10分钟，弃去上清。
6、用妇科肿瘤原代细胞培养基(见实施例1)重悬细胞沉淀，在显微镜下观察细胞状态，进行细胞计数。
结果所示，分离得到的单细胞悬液中，除了肿瘤细胞以外还混杂着大量各种类型的其他细胞，如红细胞，淋巴细胞，纤维细胞等等。本方法的优势之一就是在后续的培养过程中，只有癌细胞可以进行大量扩增，而其他细胞的比例逐渐减少甚至消失，最终获得纯度较高的妇科肿瘤原代细胞。
实施例7、妇科肿瘤原代细胞培养
1、使用低吸附表面(low-attachment-surface)进行妇科肿瘤原代细胞悬浮培养，所用培养基即为实施例1中的妇科肿瘤原代细胞培养基A或培养基B，以六孔板为例，按每孔10 6个细胞的密度铺板，37℃，5％CO 2条件下在细胞培养箱中进行培养。
当所述培养基为培养基A时，其中，人重组蛋白EGF的终浓度为50ng/mL； 人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白MSP的终浓度为20ng/mL；hydrocortisone的终浓度为10μg/mL；Y-27632的终浓度为10μM；Progesterone的终浓度为100nM；β-Estradiol的终浓度为10nM。
当所述培养基为培养基B时，其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白MSP的终浓度为20ng/mL；N-acetyl-L-cysteine的终浓度为1mM；Y-27632的终浓度为10μM；Progesterone的终浓度为100nM；β-Estradiol的终浓度为10nM。
2、每天观察细胞状态，每3天更换一次培养基，直至细胞形成直径100μm左右的团块。
如图2所示，经过3-10天的培养，肿瘤细胞大量扩增形成直径100μm大小的细胞团块，肿瘤细胞总数量可以超过10 7，其他类型的细胞数量明显减少甚至消失。本方法经过大量样本测试，妇科肿瘤原代肿瘤细胞体外培养成功率可以达到70％。
实施例8、妇科肿瘤原代细胞传代
1、收集培养皿中的细胞团块，800g室温离心10分钟，弃去上清。
2、用无菌的PBS溶液清洗细胞团块，800g室温离心10分钟，弃去上清。
3、用细胞消化液(见实施例1)重悬细胞团块，在37℃条件下进行消化。每5分钟在显微镜下观察细胞团块消化的情况，直到细胞团块都被消化为单个细胞。
4、用10倍体积的消化终止液(见实施例1)终止解离反应，收集细胞悬液。
5、800g室温离心10分钟，弃去上清。
6、用妇科肿瘤原代细胞培养基(见实施例1)重悬细胞沉淀，细胞计数。
7、使用低吸附表面(low-attachment-surface)进行妇科肿瘤原代细胞培养，所用培养基即为实施例1中的妇科肿瘤原代细胞培养基，以六孔板为例，按每孔10 6个细胞的密度铺板，37℃，5％CO 2条件下在细胞培养箱中进行培养。
实施例9、妇科肿瘤原代细胞的冻存
悬浮培养的妇科肿瘤原代细胞经过2-3次传代扩增后，可以进行冻存：
1、收集培养皿中的细胞团块，800g室温离心10分钟，弃去上清。
2、用无菌的PBS溶液清洗细胞团块，800g室温离心10分钟，弃去上清。
3、用细胞消化液(见实施例1)重悬细胞团块，在37℃条件下进行消化。每15分钟在显微镜下观察细胞团块消化的情况，直到细胞团块都被消化为单个细胞。
4、用10倍体积的消化终止液(见实施例1)终止解离反应，收集细胞悬液， 细胞计数。
5、800g室温离心10分钟，弃去上清。
6、用细胞冻存液(见实施例1)，按10 6/mL的密度重悬细胞沉淀，2mL冻存管每管1mL细胞悬液，梯度降温盒过夜冻存后转移至液氮中长期保存。
实施例10、妇科肿瘤原代细胞的复苏
液氮中保存的妇科肿瘤原代细胞可以进行复苏：
1、提前五分钟准备37℃无菌水。
2、将冻存管从液氮中取出，在37℃无菌水中迅速融化细胞。
3、800g室温离心10分钟，弃去上清。
4、用妇科肿瘤原代细胞培养基(见实施例1)重悬细胞沉淀，使用低吸附表面进行妇科肿瘤原代细胞培养，每管细胞复苏至3.5cm培养皿中，37℃，5％CO 2条件下在细胞培养箱中进行培养。
实施例11、妇科肿瘤原代细胞的HE染色鉴定
下述实施例中用到的试剂耗材说明：
HE染色试剂盒(北京索莱宝生物科技有限公司，#G1120)；
阳离子防脱玻片(北京中杉金桥生物科技有限公司)；
二甲苯、甲醇、丙酮(北京化学试剂公司，分析纯)；
中性树脂胶(北京益利精细化学品有限公司)。
1、将悬浮细胞制成浓度为10 4/mL的细胞悬液，滴加10μL于阳离子防脱玻片上，自然晾干。
2、在风干的细胞上小心滴加50μL经4℃预冷过的甲醇/丙酮混合液(体积比1:1)，然后将玻片放入4℃冰箱固定10mins。
3、取出固定细胞的玻片，室温自然晾干。
4、用200μL PBS清洗玻片两次。
5、待玻片上水分微干时加入100μL苏木精染液染色1mins。
6、吸去苏木精染液，用200μL自来水清洗玻片3次。
7、滴加100μL分化液分化1mins。
8、吸去分化液，依次用自来水清洗玻片2次，蒸馏水清洗玻片1次。
9、吸去玻片表面水分，滴加200μL伊红染液染色40s。
10、吸去伊红染液，依次用75％、80％、90％、100％乙醇漂洗脱水20s、20s、40s、40s。
11、等乙醇晾干后，滴加50μL二甲苯进行细胞通透。
12、等二甲苯晾干完全后，滴加一滴中性树脂胶，用盖玻片封片，在显微镜下观察并拍照。
图3展示了体外培养得到的妇科肿瘤原代肿瘤细胞HE染色效果图，可以看 到这些细胞普遍具有核质比高、核深染、核内染色质凝集、多核、细胞大小不均一等肿瘤细胞特征。
实施例12、妇科肿瘤原代细胞的免疫组化染色鉴定
下述实施例中用到的试剂说明：
多聚甲醛(北京化学试剂公司，分析纯)，用超纯水溶解多聚甲醛粉末，制成4％(4g/100mL)多聚甲醛溶液；
双氧水(北京化学试剂公司，35％)；
封闭用正常山羊血清(Solarbio，SL038)；
免疫组化一抗抗体(福建迈新，kit-0012)；
免疫组化二抗抗体(Abcam，ab205719)；
EDTA修复液(Abcam，ab93684)；
DAB显色液(
Figure PCTCN2020126391-appb-000029
DAB Substrate Kit，8059S)
供试培养基：实施例1中的两种妇科肿瘤原代细胞培养基，即培养基A或培养基B。
当所述培养基为培养基A时，其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白MSP的终浓度为20ng/mL；hydrocortisone的终浓度为10μg/mL；Y-27632的终浓度为10μM；Progesterone的终浓度为100nM；β-Estradiol的终浓度为10nM。
当所述培养基为培养基B时，其中，人重组蛋白EGF的终浓度为50ng/mL；人重组蛋白bFGF的终浓度为20ng/mL；人重组蛋白HGF的终浓度为20ng/mL；人重组蛋白MSP的终浓度为20ng/mL；N-acetyl-L-cysteine的终浓度为1mM；Y-27632的终浓度为10μM；Progesterone的终浓度为100nM；β-Estradiol的终浓度为10nM。
收集实施例1中的妇科肿瘤原代细胞培养基培养得到的妇科肿瘤细胞团块进行石蜡切片，按以下步骤操作：
1.将切片依次浸入二甲苯Ⅰ10min，二甲苯Ⅱ(10min)。
2.再次浸入无水乙醇Ⅰ(5min)-无水乙醇Ⅱ(5min)-95％酒精(5min)-80％酒精(5min)-70％酒精(5min)，然后去离子水冲洗2次，每次2min。
3.将组织切片放入修复盒，然后加入适量稀释好的EDTA修复液(pH 9.0)，液面要浸没组织。
4.微波中档修复10min(液体沸腾时开始计时)，此过程中勿使组织干片。
5.将修复盒从微波炉中拿出，自然冷却降温，当修复液降至室温后取出玻片，PBS(pH 7.4)冲洗3遍，每次3min(冲洗过程中切勿对着组织冲洗，以免弄破组织)。
6.将配制好的3％的过氧化氢(去离子水稀释30％过氧化氢)滴加于切片 组织上以阻断内源性过氧化物酶，室温孵育15min，PBS冲洗3次，每次3min。
7.吸水纸吸干PBS，在玻片上滴加10％的山羊血清(与二抗种属来源一致或相似)，37℃封闭60min。
8.用吸水纸擦干玻片组织周围的液体，用油性笔在组织周围画圈，然后滴加稀释好的一抗，于湿盒中4℃孵育过夜。
9.PBS冲洗切片3次，每次3min，吸水纸擦干切片后滴加辣根过氧化物酶标记二抗，室温孵育60min。
10.PBS冲洗切片3次，每次3min，甩去PBS液体后吸水纸擦干切片，每张切片滴加新鲜配制的DAB显色液，显微镜下观察，阳性信号后自来水冲洗切片终止显色。
11.苏木素复染，1min，水洗后使用酸性乙醇分化液分化，再用自来水冲洗返蓝。
12.将切片置于水中冲洗后，将切片依次放入：70％酒精-80％酒精-90％酒精-95％酒精-无水乙醇Ⅰ-无水乙醇Ⅱ-二甲苯Ⅰ-二甲苯Ⅱ中脱水透明，每个试剂中放置2min，最后在通风橱中风干切片。
13.使用中性树胶封片，用盖玻片盖上。置于通风橱中晾干。
14.晾干的切片可以在显微镜下观察或拍照。
图4展示了体外培养的乳腺癌原代肿瘤细胞团块免疫组化染色的效果图，可以看到组成细胞团块的细胞ER阳性，与患者病理结果一致，证实了本方法培养得到的是纯度较高的肿瘤细胞。
实施例13、不同类型妇科肿瘤样本原代肿瘤细胞体外培养
本实施例中所有样本原代培养的操作方法流程均完全一致(参照前文所述)，仅样本病理类型有区别。进行测试的各样本情况见表26。
表26多种病理类型妇科肿瘤原代肿瘤细胞体外培养情况
Figure PCTCN2020126391-appb-000030
可以看到，本方法对各种类型的妇科肿瘤实体瘤样本进行原代肿瘤细胞体 外培养，均可以达到非常高的成功率。
实施例14、用CYTOP修饰的细胞培养耗材进行妇科肿瘤原代肿瘤细胞培养
本实施例中所有样本原代培养的操作方法流程均完全一致(参照前文所述)，CYTOP的修饰方法均完全一致，仅细胞培养耗材材质有所区别(表27)。
其中CYTOP修饰的方法为：首先对细胞培养容器进行纯氧刻蚀，刻蚀条件为功率20W，刻蚀时间为3分钟。然后用适量(以96孔板为例，每孔20μL，适量是指完全覆盖培养皿底部)1％CYTOP溶液覆盖培养皿或培养平板表面，待CYTOP溶液完全晾干后即可使用。
表27 CYTOP修饰耗材对妇科肿瘤原代细胞培养的影响
Figure PCTCN2020126391-appb-000031
注：聚苯乙烯(Polystyrene，缩写PS)。
由表27可见：可以看到CYTOP修饰后可以大幅度提高样本培养成功率。
实施例15、微孔板芯片加工
本实施例中使用注塑加工的方式，用PMMA材料(或者PS、PC、COC、COP、LAS等材料)加工得到用于培养本发明妇科肿瘤原代细胞所用的微孔板芯片。该芯片可以用于原代妇科肿瘤细胞培养以及体外药敏检测实验。微孔板芯片设计图纸见图5。
实际应用过程中，具体是采用PMMA材料(或者PS、PC、COC、COP、LAS等材料)制备得到设计图纸见图5的微孔板芯片的结构，然后再通过上述的CYTOP修饰方法(见实施例14)对其表面进行CYTOP修饰，就得到了这里所说的可用于妇科肿瘤原代细胞培养的微孔板芯片。
工业应用
本发明提供了一种从新鲜妇科肿瘤手术样本或穿刺活检样本或胸腹水样本中提取培养妇科肿瘤原代细胞的方法和配套试剂，该方法具有以下优点：
1、组织样本用量少，仅需20mg左右的妇科肿瘤实体瘤手术样本；
2、培养周期短，仅需3-10天即可获得10 7数量级的原代肿瘤细胞；
3、培养稳定性高，用本方法对合格的妇科肿瘤实体瘤手术标本进行体外培养的成功率高达70％；
4、细胞纯度高，利用本方法得到的妇科肿瘤原代细胞培养物中，肿瘤细胞的比例可以达到70％-95％，杂细胞干扰少。
利用本发明方法得到的妇科肿瘤原代细胞培养物可以用于多种细胞水平的体外实验、二代测序、构建动物模型、构建细胞系等等。可以预见，这种培养方法在妇科肿瘤的研究和临床诊断治疗领域具有广泛的应用前景。

Claims (29)

  1. 一种培养基，为培养基A或培养基B；
    所述培养基A由抗菌抗真菌剂三抗、HEPES、GlutaMax、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白MSP、皮质醇、N-2 Supplement、Y-27632、孕酮、β-雌二醇和用于人细胞培养的基础培养基组成；其中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为250-250ng/mL；所述HEPES的终浓度为8-12mM；所述GlutaMax的终浓度为0.8-1.2％(体积百分含量)；所述人重组蛋白EGF的终浓度为10-100ng/mL；所述人重组蛋白bFGF的终浓度为10-50ng/mL；所述人重组蛋白HGF的终浓度为5-25ng/mL；所述人重组蛋白MSP的终浓度为5-25ng/mL；所述皮质醇的终浓度为1-10μg/mL；所述N-2 Supplement的终浓度为1％(体积百分含量)；所述Y-27632的终浓度为5-20μM；所述孕酮的终浓度为50-100nM；所述β-雌二醇的终浓度为10-50nM；余量均为用于人细胞培养的基础培养基；
    所述培养基B由抗菌抗真菌剂三抗、HEPES、GlutaMax、人重组蛋白EGF、人重组蛋白bFGF、人重组蛋白HGF、人重组蛋白MSP、N-乙酰-L-半胱氨酸、N-2 Supplement、Y-27632、孕酮、β-雌二醇和用于人细胞培养的基础培养基组成；其中，所述抗菌抗真菌剂三抗中的青霉素的终浓度为100-200U/mL；所述抗菌抗真菌剂三抗中的链霉素的终浓度为100-200μg/mL；所述抗菌抗真菌剂三抗中的两性霉素B的终浓度为250-250ng/mL；所述HEPES的终浓度为8-12mM；所述GlutaMax的终浓度为0.8-1.2％(体积百分含量)；所述人重组蛋白EGF的终浓度为10-100ng/mL；所述人重组蛋白bFGF的终浓度为10-50ng/mL；所述人重组蛋白HGF的终浓度为5-25ng/mL；所述人重组蛋白MSP的终浓度为5-25ng/mL；所述N-乙酰-L-半胱氨酸的终浓度为0.5-2mM；所述N-2 Supplement的终浓度为1％(体积百分含量)；所述Y-27632的终浓度为5-20μM；所述孕酮的终浓度为50-100nM；所述β-雌二醇的终浓度为10-50nM；余量均为用于人细胞培养的基础培养基。
  2. 根据权利要求1所述的培养基，其特征在于：所述培养基A为含有所述抗菌抗真菌剂三抗、所述HEPES、所述GlutaMax、所述人重组蛋白EGF、所述人重组蛋白bFGF、所述人重组蛋白HGF、所述人重组蛋白MSP、所述皮质醇、所述N-2 Supplement、所述Y-27632、所述孕酮、所述β-雌二醇和所述用于人细胞培养的基础培养基的溶液；
    所述培养基B为含有所述抗菌抗真菌剂三抗、所述HEPES、所述GlutaMax、所述人重组蛋白EGF、所述人重组蛋白bFGF、所述人重组蛋白HGF、所述人重组蛋白MSP、所述N-乙酰-L-半胱氨酸、所述N-2 Supplement、所述Y-27632、所述孕酮、所述β-雌二醇和所述用于人细胞培养的基础培养基的溶液。
  3. 根据权利要求1所述的培养基，其特征在于：所述培养基中的各组分单独存在。
  4. 根据权利要求3所述的培养基，其特征在于：在所述培养基A中，所述人重组蛋白EGF、所述人重组蛋白bFGF、所述人重组蛋白HGF、所述人重组蛋白MSP、所述皮质醇、所述Y-27632、所述孕酮、所述β-雌二醇以母液形式存在；
    在所述培养基B中，所述人重组蛋白EGF、所述人重组蛋白bFGF、所述人重组蛋白HGF、所述人重组蛋白MSP、所述N-乙酰-L-半胱氨酸、所述Y-27632、所述孕酮、所述β-雌二醇以母液形式存在。
  5. 根据权利要求4所述的培养基，其特征在于：所述母液为1000-100000倍母液；
    1000×人重组蛋白EGF储液由人重组蛋白EGF、BSA和PBS组成，其中所述人重组蛋白EGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS；
    1000×人重组蛋白bFGF储液由人重组蛋白bFGF、BSA和PBS组成，其中所述人重组蛋白bFGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS；
    1000×人重组蛋白HGF储液由人重组蛋白HGF、BSA和PBS组成，其中所述人重组蛋白HGF的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS；
    1000×人重组蛋白MSP储液由人重组蛋白MSP、BSA和PBS组成，其中所述人重组蛋白MSP的终浓度为20μg/mL，所述BSA的终浓度为0.01g/mL，余量均为PBS；
    1000×N-乙酰-L-半胱氨酸储液由N-乙酰-L-半胱氨酸和水组成，其中所述N-乙酰-L-半胱氨酸的浓度为0.5M，余量均为水；
    1000×Y-27632由Y-27632和水组成，其中Y-27632的终浓度为10mM，余量均为水；
    100000×孕酮储液由孕酮和无水乙醇组成，其中孕酮的终浓度为1mM，余量均为无水乙醇；
    100000×β-雌二醇储液由β-雌二醇和无水乙醇组成，其中β-雌二醇的终浓度为1mM，余量均为无水乙醇；
    1000×皮质醇由皮质醇和水组成，其中所述皮质醇的终浓度为0.5M，余量均为水。
  6. 根据权利要求5所述的培养基，其特征在于：在所述1000×人重组蛋白EGF储液、所述1000×人重组蛋白bFGF储液、所述1000×人重组蛋白HGF储液、所述1000×人重组蛋白MSP储液中，所述BSA以母液形式存在。
  7. 根据权利要求6所述的培养基，其特征在于：所述BSA以100倍母液形式存在；100×BSA溶液由BSA和PBS组成；其中BSA的终浓度为0.1g/mL，余 量均为PBS。
  8. 根据权利要求1-7中任一所述的培养基，其特征在于：所述用于人细胞培养的基础培养基为Advanced DMEM/F12培养基。
  9. 根据权利要求1-8中任一所述的培养基，其特征在于：所述培养基为用于培养妇科肿瘤原代细胞的培养基。
  10. 用于培养妇科肿瘤原代细胞的成套试剂，含有权利要求1-9中任一所述的培养基和如下试剂中的至少一种：样本解离液、样本保存液、细胞分离缓冲液、细胞消化液、样本清洗液、消化终止液、细胞冻存液和1％CYTOP溶液；
    所述样本解离液由胶原酶I、胶原酶III、胶原酶IV和PBS组成；其中，所述胶原酶I在所述样本解离液中的终浓度为150-250U/mL；所述胶原酶III在所述样本解离液中的终浓度为250-350U/mL；所述胶原酶IV在所述样本解离液中的终浓度为150-250U/mL；余量均为PBS；
    所述样本保存液由胎牛血清、双抗P/S、HEPES和HBSS组成；其中，所述胎牛血清的终浓度为1-5％(体积百分含量)；所述双抗P/S中的青霉素的终浓度为100-200U/mL；所述双抗P/S中的链霉素的终浓度为100-200μg/mL；所述HEPES的终浓度为8-12mM；余量均为HBSS；
    所述细胞分离缓冲液由双抗P/S、肝素钠和PBS组成；其中，所述双抗P/S中的青霉素的终浓度为100-200U/mL；所述双抗P/S中的链霉素的终浓度为100-200μg/mL；所述肝素钠的终浓度为10IU/mL；余量均为PBS；
    所述细胞消化液组成如下：每10mL所述细胞消化液中含有4-6mL Accutase，终浓度为5mM的EDTA，1.5-2.5mL TrypLE Express，余量为PBS；
    所述样本清洗液由双抗P/S和PBS组成；其中，所述双抗P/S中的青霉素的终浓度为100-200U/mL；所述双抗P/S中的链霉素的终浓度为100-200μg/mL；余量均为PBS；
    所述消化终止液由胎牛血清、双抗P/S和DMEM培养基组成；其中，所述胎牛血清的终浓度为8-12％；所述双抗P/S中的青霉素的终浓度为100-200U/mL；所述双抗P/S中的链霉素的终浓度为100-200μg/mL；余量均为DMEM培养基；
    所述细胞冻存液由Advanced DMEM/F12培养基、DMSO和1％甲基纤维素溶液组成；其中，所述Advanced DMEM/F12培养基、所述DMSO和所述1％甲基纤维素溶液的体积配比为20:2:(0.8-1.2)；所述1％甲基纤维素溶液是浓度为1g/100ml的甲基纤维素水溶液；
    所述1％CYTOP溶液的组成如下：每100mL所述1％CYTOP溶液中含有1mL CYTOP，余量为氟油。
  11. 权利要求1-9中任一所述的培养基或权利要求10所述的成套试剂在培养妇科肿瘤原代细胞中的应用。
  12. 一种培养妇科肿瘤原代细胞的方法，包括如下步骤：利用权利要求1-9中任一所述的培养基悬浮培养妇科肿瘤原代细胞。
  13. 根据权利要求12所述的方法，其特征在于：所述妇科肿瘤原代细胞为妇科肿瘤实体瘤原代细胞或妇科肿瘤胸腹水样本原代肿瘤细胞。
  14. 根据权利要求13所述的方法，其特征在于：所述妇科肿瘤实体瘤原代细胞是用权利要求10中所述的样本解离液对妇科肿瘤实体瘤组织进行解离处理后获得的。
  15. 根据权利要求14所述的方法，其特征在于：在所述方法中，是按照包括如下步骤的方法用所述样本解离液对所述妇科肿瘤实体瘤组织进行解离的：按0.1-0.3mL所述样本解离液每mg组织的用量，将剪碎后的所述妇科肿瘤实体瘤组织用事先37℃预热的所述样本解离液进行处理，在37℃条件下进行样本解离，解离时间15分钟至3小时。
  16. 根据权利要求13所述的方法，其特征在于：所述妇科肿瘤胸腹水样本原代肿瘤细胞是用权利要求8中所述的细胞分离缓冲液从妇科肿瘤胸腹水样本中分离获得的。
  17. 根据权利要求16所述的方法，其特征在于：在所述方法中，是按照包括如下步骤的方法用所述分离缓冲液对所述妇科肿瘤胸腹水样本进行分离的：用所述细胞分离缓冲液悬浮所述妇科肿瘤胸腹水样本中的细胞，然后通过密度梯度离心获得所述妇科肿瘤胸腹水样本原代肿瘤细胞。
  18. 根据权利要求12-17中任一所述的方法，其特征在于：所述方法中，是按照包括如下步骤的方法用所述妇科肿瘤原代细胞培养基悬浮培养所述妇科肿瘤原代细胞的：使用细胞培养容器M，利用所述妇科肿瘤原代细胞培养基悬浮培养所述妇科肿瘤原代细胞，37℃，5％CO 2条件下进行培养，每2-4天更换一次培养基；
    所述细胞培养容器M为如下任一：(I)聚苯乙烯材质的细胞培养容器、聚碳酸酯材质的细胞培养容器、聚甲基丙烯酸甲酯材质的细胞培养容器、COC树脂材质的细胞培养容器、环烯烃聚合物材质的细胞培养容器或低吸附表面的细胞培养容器；(II)对(I)中的细胞培养容器进行CYTOP修饰后的细胞培养容器。
  19. 根据权利要求18所述的方法，其特征在于：所述细胞培养容器为细胞培养皿、细胞培养孔板或用于细胞培养的微孔板芯片。
  20. 根据权利要求18或19所述的方法，其特征在于：所述(II)中，是按照包括如下步骤的方法对所述(I)中的细胞培养容器进行CYTOP修饰的：对所述(I)中的细胞培养容器进行纯氧刻蚀，刻蚀条件为功率20W，刻蚀时间为3分钟；然后用权利要求10中所述的1％CYTOP溶液覆盖所述细胞培养容器表面，晾干所述1％CYTOP溶液即完成CYTOP修饰。
  21. 根据权利要求14-20中任一所述的方法，其特征在于：所述方法中，还包括如下对所述妇科肿瘤实体瘤组织进行解离前处理的步骤：用体积百分含量为70-75％的乙醇清洗妇科肿瘤实体瘤组织样本表面；用权利要求10中所述的样本清洗液和无菌的PBS溶液先后清洗所述妇科肿瘤实体瘤组织样本。
  22. 根据权利要求21所述的方法，其特征在于：进行所述解离前处理的所述妇科肿瘤实体瘤组织样本的离体时间为2小时以内，且在进行所述解离前处理之前一直保存于权利要求10中所述的样本保存液中。
  23. 根据权利要求14-22中任一所述的方法，其特征在于：所述方法中，用所述样本解离液对所述妇科肿瘤实体瘤组织进行解离处理后还包括如下步骤：用权利要求10中所述的消化终止液终止解离反应，收集细胞悬液；过滤所述细胞悬液，去除组织残片和粘连细胞；离心后用无菌PBS重悬细胞；再离心，然后用所述培养基重悬细胞沉淀。
  24. 根据权利要求12-23中任一所述的方法，其特征在于：在用所述培养基对所述妇科肿瘤原代细胞进行培养的过程中，还包括如下步骤：待所述妇科肿瘤原代细胞形成直径80-120μm的团块时，对所述妇科肿瘤原代细胞进行传代。
  25. 根据权利要求24所述的方法，其特征在于：进行所述传代时采用的细胞消化液为权利要求10中所述的细胞消化液。
  26. 根据权利要求24或25所述的方法，其特征在于：进行所述传代时采用的消化终止液为权利要求10中所述的消化终止液。
  27. 根据权利要求24-26中任一所述的方法，其特征在于：所述方法还包括对经过2-3次传代扩增后的所述妇科肿瘤原代细胞进行冻存和/或复苏的步骤。
  28. 根据权利要求27所述的方法，其特征在于：进行所述冻存时采用的细胞冻存液为权利要求10中的所述细胞冻存液。
  29. 根据权利要求1-9中任一所述的培养基或权利要求10所述的成套试剂或权利要求11所述应用或权利要求12-28中任一所述方法，其特征在于：所述妇科肿瘤为乳腺癌、卵巢癌、子宫内膜癌、宫颈癌或其转移病灶。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113957036A (zh) * 2021-09-06 2022-01-21 创芯国际生物科技(广州)有限公司 一种子宫内膜类器官培养基及培养方法

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104546868A (zh) * 2014-11-04 2015-04-29 上海交通大学医学院附属瑞金医院 基于中国人群的原代人源乳腺癌移植瘤模型的建立方法及模型
CN106414707A (zh) * 2014-01-23 2017-02-15 日产化学工业株式会社 培养基组合物的制造方法
US20170226472A1 (en) * 2014-08-04 2017-08-10 Universidad De Granada Culture medium and method for enriching and maintaining cancer stem cells (cscs) using said medium
CN107208026A (zh) * 2014-12-22 2017-09-26 美国圣戈班性能塑料公司 气体可渗透性材料
CN108884445A (zh) * 2016-03-09 2018-11-23 北京智康博药肿瘤医学研究有限公司 肿瘤细胞悬浮培养物和相关方法
CN111621479A (zh) * 2019-11-05 2020-09-04 北京基石生命科技有限公司 一种用于培养妇科肿瘤原代细胞的培养基
CN111621478A (zh) * 2019-11-05 2020-09-04 北京基石生命科技有限公司 一种妇科肿瘤原代细胞的培养方法
CN111808815A (zh) * 2019-04-11 2020-10-23 北京基石生命科技有限公司 一种胃癌实体瘤原代细胞的培养方法
CN111808816A (zh) * 2019-04-11 2020-10-23 北京基石生命科技有限公司 一种用于培养胃癌实体瘤原代细胞的培养基

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652376B (zh) * 2019-01-08 2021-10-15 创芯国际生物科技(广州)有限公司 一种用于卵巢癌组织3d培养的培养基

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106414707A (zh) * 2014-01-23 2017-02-15 日产化学工业株式会社 培养基组合物的制造方法
US20170226472A1 (en) * 2014-08-04 2017-08-10 Universidad De Granada Culture medium and method for enriching and maintaining cancer stem cells (cscs) using said medium
CN104546868A (zh) * 2014-11-04 2015-04-29 上海交通大学医学院附属瑞金医院 基于中国人群的原代人源乳腺癌移植瘤模型的建立方法及模型
CN107208026A (zh) * 2014-12-22 2017-09-26 美国圣戈班性能塑料公司 气体可渗透性材料
CN108884445A (zh) * 2016-03-09 2018-11-23 北京智康博药肿瘤医学研究有限公司 肿瘤细胞悬浮培养物和相关方法
CN111808815A (zh) * 2019-04-11 2020-10-23 北京基石生命科技有限公司 一种胃癌实体瘤原代细胞的培养方法
CN111808816A (zh) * 2019-04-11 2020-10-23 北京基石生命科技有限公司 一种用于培养胃癌实体瘤原代细胞的培养基
CN111621479A (zh) * 2019-11-05 2020-09-04 北京基石生命科技有限公司 一种用于培养妇科肿瘤原代细胞的培养基
CN111621478A (zh) * 2019-11-05 2020-09-04 北京基石生命科技有限公司 一种妇科肿瘤原代细胞的培养方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4056684A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113957036A (zh) * 2021-09-06 2022-01-21 创芯国际生物科技(广州)有限公司 一种子宫内膜类器官培养基及培养方法
CN113957036B (zh) * 2021-09-06 2022-07-05 创芯国际生物科技(广州)有限公司 一种子宫内膜类器官培养基及培养方法

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