CN111621478A

CN111621478A - Culture method of gynecological tumor primary cells

Info

Publication number: CN111621478A
Application number: CN201911069251.4A
Authority: CN
Inventors: 尹申意; 张函槊
Original assignee: Beijing Genex Health Technology Co ltd
Current assignee: Beijing Genex Health Technology Co ltd
Priority date: 2019-11-05
Filing date: 2019-11-05
Publication date: 2020-09-04

Abstract

The invention discloses a culture method of gynecological tumor primary cells. The invention provides a culture method of primary gynecological tumor cells and a matched reagent, and the technical core is as follows: the gynecological tumor solid tumor tissue is treated by a mild cell dissociation reagent, so that the activity of tumor cells in the tissue is ensured to the maximum extent; preparing a special serum-free culture medium, and culturing solid tumor cells derived from gynecological tumors in vitro by using a suspension culture system, so that the interference of normal cells is eliminated to the maximum extent while the normal amplification of the tumor cells is ensured. The gynecological tumor primary cell culture obtained by the method can be used for various cell level in vitro experiments, next generation sequencing, animal model construction, cell line construction and the like. The culture method has wide application prospect in the fields of research of gynecological tumors and clinical diagnosis and treatment.

Description

Culture method of gynecological tumor primary cells

Technical Field

The invention relates to the technical field of biology, in particular to a culture method of gynecological tumor primary cells.

Background

Common gynecological tumors include breast cancer, ovarian cancer, endometrial cancer and the like. According to data statistics of 2018 by the national cancer center, in 2014, the breast cancer accounts for 16.5% of the incidence rate of female malignant tumors in China, the mortality rate reaches 7.8%, and the first and fifth female tumors are ranked respectively. Currently, the 5-year survival rate of breast cancer in China is only 73.1% vs 90% (in the United states), and a large gap exists between the breast cancer and developed countries. In addition, the incidence rate of ovarian cancer in China accounts for 2.5 percent of that of female malignant tumors, and the incidence rate is increased by 30 percent in the last decade. These gynecological tumors pose a serious challenge to the health of women in our country.

Although research on the etiology and development of gynecological tumors by scientific and medical institutions in various countries of the world is heavily invested, human beings are still poorly aware of the disease. Gynecological tumor is a kind of complex disease, the occurrence and development of which are dynamic processes, involving the interaction of many signal molecules, forming a complex molecular regulation network, and being affected by external environmental factors. The etiology, occurrence and development process of gynecological tumor have strong individual difference, which cannot be concluded in a whole. Therefore, the individual precise research of the gynecological tumor solid tumor primary cell culture as a model is a trend in the gynecological tumor research field and even the gynecological tumor diagnosis and treatment field.

The existing primary tumor cell culture technology mainly comprises 2D culture, 3D culture, reprogramming culture and the like, and the methods all face the problems of extremely long culture period, low culture success rate, difficult removal of mixed cells and the like in different degrees.

Disclosure of Invention

In order to effectively solve the technical problems, the invention provides a novel gynecological tumor solid tumor primary cell culture technology and a matched reagent, and the core of the technology is as follows: (1) the gynecological tumor solid tumor tissue is treated by a mild cell dissociation reagent, so that the activity of tumor cells in the tissue is ensured to the maximum extent; (2) preparing a special serum-free culture medium, and culturing the gynecological tumor primary cells in vitro by using a suspension culture system, thereby ensuring normal amplification of tumor cells and eliminating the interference of normal cells to the maximum extent.

In a first aspect, the invention claims a method for culturing primary gynecological tumor cells.

The method for culturing the gynecological tumor primary cells claimed by the invention can comprise the following steps:

suspending and culturing the gynecological tumor primary cells by using a gynecological tumor primary cell culture medium;

the gynecological tumor primary cell culture medium consists of antibacterial antifungal agents three-antibody (penicillin-streptomycin-amphotericin B), HEPES (HEPES), GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, cortisol (hydrocortisone), N-2Supplement, Y-27632, Progesterone (Progesterone), beta-Estradiol (beta-Estradiol) and Advanced DMEM/F12 culture medium. Wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-200 mu g/mL (such as 100 mu g/mL); the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250ng/mL (such as 250 ng/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the final concentration of GlutaMax is 0.8-1.2% (e.g., 1%,% represents volume percent); the final concentration of the human recombinant protein EGF is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein MSP is 5-25 ng/mL; the final concentration of the cortisol (hydrocortisone) is 1-10 mug/mL; the final concentration of the N-2Supplement is 1 percent (volume percentage); the final concentration of the Y-27632 is 5-20 mu M; the final concentration of the progesterone is 50-100 nM; the final concentration of the beta-estradiol is 10-50 nM; the balance is Advanced DMEM/F12 medium.

Further, the composition of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is as follows: each ml contains 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B. The antimicrobial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) is "antibacterial-antibacterial, 100X" (e.g., Gibco #15240062, or other products of the same composition). The "Antibiotic-Antibiotic, 100X" contained 10000 units of penicillin (base), 10000. mu.g of streptomycin (base) and 25. mu.g of amphotericin B per ml, using penicillin G (sodium salt), streptomycin sulfate and amphotericin B in the form of 0.85% saline as the active ingredients

An antifungal agent. The GlutaMAX is GlutaMAX^TMSupplement "(e.g., Gibco #35050061, or other products of the same composition). The "GlutaMAX^TMThe Supplement "was composed of L-allyl-L-glutamine as a substitute for L-glutamine at a concentration of 200nM in a 0.85% NaCl solution. The N-2Supplement is "N-2 Supplement (100X)" (e.g., Gibco #17502001,or other products of the same composition as it). The "N-2 Supplement (100X)" contained Human total Transferrin (Human Transferrin (Holo)) at a final concentration of 1mM, 500mg/L recombinant Insulin whole Chain (Insulin recombinant full Chain), 0.63mg/L Progesterone (Progesterone), 10mM Putrescine (Putrescine), and 0.52mg/L Selenite (Selenite). The GlutaMAX is a high-grade cell culture additive and can directly replace L-glutamine in a cell culture medium. The GlutaMAX is GlutaMAX^TMSupplement "(e.g., Gibco #35050061, or other products of the same composition). Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor with Ki of 220nM and 300nM, respectively)" (e.g. MCE #129830-38-2, or other products of the same composition).

In a specific embodiment of the invention, the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is under the brand code Gibco # 15240062; the brand of HEPES is Gibco # 15630080; the brand name of GlutaMAX is Gibco # 35050061; the brand of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand of the human recombinant protein bFGF is Peprotech AF-100-18B-50; the brand of the human recombinant protein HGF is Peprotech AF-100-39-100; the brand goods number of the human recombinant protein MSP is R & D # 352-MS-050; the brand name of the hydrocortisone is Selleck # S1696; the brand goods number of the N-2Supplement is Gibco # 17502001; the brand goods number of the Y-27632 is MCE # 129830-38-2; the brand of the Progesterone (Progesterone) has a brand code of Sigma # V900699; the brand code of the beta-Estradiol (beta-Estradiol) is Sigma # E2758; the brand of the Advanced DMEM/F12 medium is Gibco # 12634010.

Wherein, the gynecological tumor primary cells can be gynecological tumor solid tumor primary cells or gynecological tumor breast and abdominal water sample primary tumor cells.

When the gynecological tumor primary cells are gynecological tumor solid tumor primary cells, the gynecological tumor solid tumor primary cells can be obtained by dissociating gynecological tumor solid tumor tissues by using a sample dissociation solution.

The sample dissociation liquid consists of collagenase I, collagenase III, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250U/mL (such as 200U/mL); the final concentration of collagenase III is 250-350U/mL (such as 290U/mL); the final concentration of collagenase IV is 150-250U/mL (such as 200U/mL); the balance being PBS.

Wherein the unit U of collagenase (said collagenase I, said collagenase III or said collagenase IV) is defined by the enzymatic activity of a protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I, said collagenase III or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

In a specific embodiment of the present invention, the brand name of collagenase I is Gibco # 17100-017; the brand code of collagenase III is Solarbio # C8490; the brand goods number of the collagenase IV is Gibco # 17104-; the PBS was branded under Gibco # 21-040-CVR.

Further, the sample dissociation liquid can be used for dissociating the gynecological tumor solid tumor tissue according to the method comprising the following steps: shearing the gynecological tumor solid tumor tissue (e.g. into 0.8-1.2 mm) according to the dosage of 0.1-0.3mL (e.g. 0.1mL) of the sample dissociation solution per mg of tissue³Small pieces of (a) were treated with the sample dissociation solution preheated at 37 ℃ in advance, and sample dissociation was performed at 37 ℃ for 15 minutes to 3 hours. The dissociation of the samples was observed under the microscope every 15 minutes until a large number of single cells were observed.

When the gynecological tumor primary tumor cells are gynecological tumor pleural effusion and peritoneal ascites sample primary tumor cells, the gynecological tumor pleural effusion and peritoneal ascites sample primary tumor cells can be obtained by separating the gynecological tumor pleural effusion and peritoneal ascites sample with a cell separation buffer solution.

The cell separation buffer solution consists of double-antibody P/S (penicillin-streptomycin), heparin sodium and PBS; wherein the final concentration of penicillin in the double-resistant P/S (penicillin-streptomycin) is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-resistant P/S (penicillin-streptomycin) is 100-200 mug/mL (such as 100 mug/mL); the final concentration of the heparin sodium is 10 IU/mL; the balance being PBS.

In a specific embodiment of the invention, the bis-anti P/S (penicillin-streptomycin) is under the brand code Gibco # 15140122; the brand code of the heparin sodium is Solarbio # H8270; the PBS was branded under Gibco # 21-040-CVR.

Further, the isolation buffer can be used for isolating the gynecological tumor pleural effusion and peritoneal fluid sample according to the method comprising the following steps: suspending the cells in the gynecological tumor pleural effusion sample by using the cell separation buffer solution, and then obtaining the primary tumor cells of the gynecological tumor pleural effusion sample by density gradient centrifugation (using Ficoll lymphocyte separation solution).

Furthermore, before the isolation of the gynecological tumor hydrothorax and ascites sample by using the isolation buffer, the method can further comprise the step of performing pre-isolation treatment on the gynecological tumor hydrothorax and ascites sample: and removing impurities, clotted blood and other components which influence the cell density gradient separation in the gynecological tumor pleural effusion and peritoneal fluid sample.

In the method, the gynecological tumor primary cells may be cultured in suspension with the gynecological tumor primary cell culture medium according to a method comprising the following steps: using a cell culture container M to culture the gynecological tumor primary cells in a suspension manner by using the gynecological tumor primary cell culture medium at 37 ℃ and 5% CO₂Culturing is carried out under conditions in which the medium is changed every 2-4 days (e.g., 3 days) until the cells form clumps of 80-120 μm (e.g., 100 μm) in diameter.

Wherein the initial seeding density may be 10⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density of individual cells was plated.

Wherein, the cell culture container M can be any one of the following: (I) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a cell culture container made of COC resin, a cell culture container made of cyclic olefin polymer, or a cell culture container with a low adsorption surface; (II) subjecting the cell culture vessel of (I) to CYTOP modification.

Further, the cell culture vessel is a cell culture dish, a cell culture well plate, or a microplate chip for cell culture (such as the microplate chip shown in fig. 5 in example 15) or the like.

In the (II), the cell culture vessel in the (I) may be subjected to CYTOP modification according to a method comprising the steps of: carrying out pure oxygen etching on the cell culture container in the step (I), wherein the etching condition is that the power is 20W, and the etching time is 3 minutes; the cell culture vessel surface was then covered with a 1% CYTOP solution and the CYTOP modification was completed by air drying the 1% CYTOP solution.

Wherein the composition of the 1% CYTOP solution is as follows: each 100mL of the 1% CYTOP solution contained 1mL of LCYTOP, the balance being fluoro oil.

Wherein the CYTOP is perfluoro (1-butenylvinylether) polymer. The fluoro oil may be a fluoro oil of brand 3M # FC40, or other product of the same composition.

In a particular embodiment of the invention, the CYTOP brand code is specifically Asashi glass # CTL-809M; the brand goods number of the fluorine oil is specifically 3M # FC 40.

Further, the method can also comprise the following step of carrying out dissociation pretreatment on the gynecological tumor solid tumor tissue: cleaning the surface of the gynecological tumor solid tumor tissue sample for 10 to 30 seconds by using ethanol with the volume percentage of 70 to 75 percent (such as 75 percent); washing the gynecological tumor solid tumor tissue sample 10-20 times (such as 10 times) with a sample washing solution, and washing the gynecological tumor solid tumor tissue sample 5-10 times (such as 5 times) with a sterile PBS solution; then removing impurities, connective tissues, adipose tissues, necrotic tissues and other components which influence the culture of the primary cells in the gynecological tumor solid tumor tissue sample.

Wherein the sample washing solution consists of double-antibody P/S (penicillin-streptomycin) and PBS; wherein the final concentration of penicillin in the double-resistant P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL (such as 100 mug/mL); the balance being PBS.

In a specific embodiment of the invention, the brand of the double antibody P/S is Gibco # 15140122; the PBS was branded under Gibco # 21-040-CVR.

The step of performing dissociation pretreatment on the gynecological tumor solid tumor tissue needs to be operated on ice, and the whole operation step needs to be completed within 10 minutes.

Furthermore, the time for the gynecological tumor solid tumor tissue sample to be subjected to the pre-dissociation treatment needs to be within 2 hours in vitro, and the gynecological tumor solid tumor tissue sample is preserved in a sample preservation solution before the pre-dissociation treatment.

Wherein the sample preservation solution consists of fetal calf serum, double-antibody P/S (penicillin-streptomycin), HEPES and HBSS (Hank' S balanced salt solution); wherein the final concentration of fetal calf serum is 1-5% (such as 2%,% represents volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL (such as 100 mug/mL); the final concentration of HEPES is 8-12mM (e.g., 10 mM); the balance being HBSS.

Further, in the method, after the dissociation treatment of the gynecological tumor solid tumor tissue by the sample dissociation liquid, the method may further include the following steps: terminating the dissociation reaction with 8-15 (e.g., 10) times the volume of the digestion stop solution, and collecting the cell suspension; filtering the cell suspension with a 100 μm or 40 μm sterile cell strainer to remove tissue debris and adherent cells; 800-1000g (e.g., 800g) of the suspension is centrifuged at room temperature for 10-15 minutes (e.g., 10 minutes), and the supernatant is discarded; then resuspend the cells in 3-5mL (e.g., 5mL) sterile PBS; centrifuging at room temperature for 10-15 min (such as 10min) again at 800-; then, the gynecological tumor solid tumor primary cell culture medium is used for resuspending the cell sediment, and the cell state is observed under a microscope for cell counting.

Wherein the digestion stop solution consists of fetal calf serum, double-antibody P/S (penicillin-streptomycin) and a DMEM medium; wherein the final concentration of fetal calf serum is 8-12% (such as 10%,% represents volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL (such as 100 mug/mL); the balance is DMEM medium.

Further, in the method, the following steps may be further included: when the gynecological tumor primary cells form a lump with the diameter of 80-120 mu m (such as 100 mu m), the gynecological tumor primary cells are subjected to passage.

Wherein, the cell digestive juice adopted when the passage is carried out consists of the following components: each 10mL of the cell digest contained 4-6mL (e.g., 5mL) of Accutase, a final concentration of 5mM EDTA (i.e., 10. mu.L of 0.5M EDTA), 1.5-2.5mL (e.g., 2mL) of TrypLE Express, and the balance PBS.

Further, the Accutase is StemPro^TMAccutase^TMCell DissociationReagent "(e.g., Gibco # A11105-01, or other product of the same composition). The Accutase is a single-component enzyme, and is dissolved in D-PBS, 0.5mM EDTA solution. The TrypLE Express is' TrypLE^TMExpressEnzyme (1X), no phenol red "(e.g., Gibco #12604013, or other products of the same composition). The TrypLE^TMExpress Enzyme (1X), no phenol red "contains 200mg/L KCl and 200mg/L KH₂PO₄8000mg/L NaCl, 2160mg/L Na₂HPO₄·7H₂O, 457.6mg/L EDTA; also contains recombinant protease.

In a specific embodiment of the invention, the brand name of the Accutase is Gibco # A11105-01; the brand name of the 0.5M EDTA is Invitrogen # AM 9261; the brand goods number of the TrypLE Express is Gibco # 12604013; the PBS was branded under Gibco # 21-040-CVR.

Further, the digestion temperature used for the passage was 37 ℃.

Further, the digestion stop solution used in the passage is the digestion stop solution described above.

More specifically, the step of performing said passaging is carried out: collecting the culture medium to be passedCentrifuging, washing the cell pellet with sterile PBS solution, centrifuging, suspending the cell pellet with the cell digestive fluid, digesting at 37 deg.C until the cell pellet is digested into single cell, stopping digestion reaction with the digestion stop fluid (which can be 5-10 times (such as 10 times) volume), and collecting cell suspension; resuspending the cell pellet with the gynecological tumor primary cell culture medium after centrifugation, counting, and then suspension culturing the cells using a culture vessel with a low adsorption surface (initial seeding density can be 10)⁵Per cm²Bottom area of the container, e.g. six-well plate, 10 per well⁶Density plating of individual cells), culture conditions were 37 ℃ and 5% CO₂. All the centrifugation in the above-mentioned passaging step may be specifically 800-1000g (e.g., 800g) at room temperature for 10-20 minutes (e.g., 10 minutes).

Further, the method can also comprise the step of performing cryopreservation and/or resuscitation on the gynecological tumor primary cells after the gynecological tumor primary cells are subjected to passage expansion for 2-3 times.

Wherein the cell freezing solution adopted during freezing is composed of Advanced DMEM/F12 culture medium, DMSO and 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20:2 (0.8-1.2), such as 20:2: 1; the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

In a specific embodiment of the invention, the Advanced DMEM/F12 medium is under the brand code Gibco # 12634010; the brand code of the DMSO is Sigma # D2438; the brand of methylcellulose is Sigma # M7027.

Further, the freezing step is carried out by collecting the cell pellet to be frozen, centrifuging, washing the cell pellet with sterile PBS solution, centrifuging, suspending the cell pellet with the cell digest, digesting at 37 deg.C until the cell pellet is digested into single cells, terminating the digestion reaction with the digestion terminating solution (which may be used in an amount of 5-10 times, for example, 10 times, volume), collecting the cell suspension, centrifuging, and freezing the cell pellet with the cell lysate in an amount of 0.5-2 × 10 times⁶/mL (e.g., 10)⁶mL), and transferring the cell sediment to liquid nitrogen for long-term storage after the cell sediment is frozen and stored overnight by a gradient cooling box. All the centrifugation in the above freezing step may be specifically 800-1000g (e.g., 800g) at room temperature for 10-20 minutes (e.g., 10 minutes).

Further, the specific steps of performing the resuscitation are: taking out the freezing tube containing the cells to be rescued from the liquid nitrogen, and rapidly thawing the cells in sterile water at 37-39 deg.C (such as 37 deg.C); suspending the cell pellet with the gynecological tumor primary cell culture medium after centrifugation (e.g. 800-⁵Per cm²Bottom area of container), cells per tube (10)⁶Respectively) reviving to 3.5cm culture dish), culturing at 37 deg.C and 5% CO₂。

In a second aspect, the invention claims a kit for culturing primary cells of a gynecological tumor.

The kit for culturing gynecological tumor primary cells provided by the invention specifically comprises the gynecological tumor primary cell culture medium and at least one of the following reagents: the sample dissociation solution, the sample preservation solution, the cell digestion solution, the sample washing solution, the cell separation buffer solution, the digestion stop solution, the cell cryopreservation solution and the 1% CYTOP solution described above.

The sample preservation solution can be used for temporarily preserving a sample after the sample is separated, and can maintain the activity of cells in the sample in a short time after the sample is separated. The sample preservation solution can be preserved for 1 month at 4 ℃ after being prepared.

The sample washing solution can be used for washing and disinfecting a sample. The sample cleaning solution needs to be ready for use.

The sample dissociation liquid can be used for dissociation of a sample, and gynecological tumor primary cells in the sample can be dissociated from tissues. The sample dissociation solution needs to be prepared at present, wherein collagenase I, collagenase III and collagenase IV can be stored for a long time at the temperature of-20 ℃ in a stock solution (mother solution), and specifically, the stock solution (mother solution) can be 10 times. The 10 × collagenase I stock consists of the collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL; a 10 × collagenase III stock consists of the collagenase III and PBS; wherein the final concentration of collagenase III is 2000U/mL; the 10 × collagenase IV stock consists of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance being PBS. The enzyme activities of collagenase I, collagenase III and collagenase IV are defined above.

The cell separation buffer is used for suspending the cells in the gynecological tumor pleural effusion and peritoneal fluid sample. After the preparation of the cell separation buffer solution is finished, the cell separation buffer solution can be stored at 4 ℃ for 1 month.

The cell digestive juice can be used for digesting and passaging cell masses and can digest gynecological tumor masses into single cells. The cell digestive juice is required to be prepared immediately.

The digestion stop solution can be used for stopping the dissociation of the sample or the digestion process of the cells. The prepared digestion stop solution can be stored for one month at 4 ℃.

The gynecological tumor primary cell culture medium can be used for culturing gynecological tumor primary cells. The gynecological tumor primary cell culture medium needs to be sterilized by filtration through a 0.22 mu M needle filter (Millipore SLGP033RS) after being prepared, and can be stored for two weeks at 4 ℃. The human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF and the human recombinant protein MSP can be stored for a long time at the temperature of-80 ℃ in a stock solution (mother solution) form, and particularly can be stored in a stock solution (mother solution) of 1000 times. Cortisol (Hydrocortisone) and Y-27632 can be stored in stock solution (mother solution) at-20 deg.C for a long time, specifically 1000 times of stock solution (mother solution). Progesterone (Progesterone) and beta-Estradiol (beta-Estradiol) can be stored for a long time at-20 ℃ in a stock solution (mother liquor), and specifically can be 100000 times of stock solution (mother liquor).

The 1000 Xhuman recombinant protein EGF stock solution consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The stock solution of 1000 Xhuman recombinant protein bFGF consists of human recombinant protein bFGF, BSA and PBS, wherein the final concentration of the human recombinant protein bFGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The 1000 Xhuman recombinant protein HGF stock solution consists of human recombinant proteins HGF, BSA and PBS, wherein the final concentration of the human recombinant proteins HGF is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. The stock solution of 1000 Xhuman recombinant protein MSP consists of human recombinant protein MSP, BSA and PBS, wherein the final concentration of the human recombinant protein MSP is 20 μ g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS. In the four 1000-fold stock solutions, the BSA can be present (as ready-to-use) as a 100-fold stock solution (stock solution), and specifically consists of BSA and PBS, wherein the final concentration of BSA (Sigma # A1933) is 0.1g/mL, and the balance is PBS. In addition, a 1000 × Hydrocortisone stock solution was composed of Hydrocortisone at a concentration of 0.5M and ultrapure water as the rest. 1000 XY-27632 consists of Y-27632 and ultrapure water, wherein the final concentration of Y-27632 is 10mM, and the balance is ultrapure water. The 100000 XProgesterone stock solution consisted of Progesterone and absolute ethanol, with a final concentration of Progesterone of 1mM and the balance being absolute ethanol. The 100000 × beta-Estradiol stock solution consists of beta-Estradiol and absolute ethyl alcohol, wherein the final concentration of the beta-Estradiol is 1mM, and the balance is the absolute ethyl alcohol.

The cell freezing medium needs to be prepared at present. Wherein the 1% methylcellulose solution can be stored for a long period of time at 4 ℃.

In a third aspect, the invention claims the use of the kit of parts as described hereinbefore for culturing primary cells of a gynaecological tumour.

The culture medium for the gynecological tumor primary cells and the application of the culture medium in culturing the gynecological tumor primary cells also belong to the protection scope of the invention.

In the above aspects, the gynecological tumor may be a primary gynecological tumor or a metastatic focus thereof. The gynecological tumor can be breast cancer, ovarian cancer, endometrial cancer, cervical cancer or metastasis thereof.

In the above aspects, the gynecological tumor primary cells are isolated from a surgical sample or a needle biopsy sample or a pleural and peritoneal fluid sample (pleural fluid or ascites) of a patient with a gynecological tumor. Wherein, the best weight of gynecological tumor solid tumor tissue specimens obtained from the operation samples exceeds 20mg, the number of the puncture biopsy samples (belonging to the solid tumor samples) exceeds 4, and the number of the hydrothorax and ascites samples exceeds 100 mL.

In the present invention, all of the above PBS's may be 1 × PBS, pH7.3-7.5, and the specific composition is that the solvent is water, the solute is KH₂PO₄144mg/L，NaCl 9000mg/L，Na₂HPO₄·7H₂O 795mg/L。

The invention provides a method for extracting and culturing gynecological tumor primary cells from a fresh gynecological tumor operation sample or a puncture biopsy sample or a hydrothorax and ascites sample and a matched reagent, and the method has the following advantages:

1. the dosage of the tissue sample is less, and only about 20mg of gynecological tumor solid tumor operation sample is needed;

2. the culture period is short, and only 3-10 days are needed to obtain 10⁷An order of magnitude of primary tumor cells;

3. the culture stability is high, and the success rate of in vitro culture of qualified gynecological tumor solid tumor operation specimens by using the method is up to 70 percent;

4. the purity of the cells is high, the proportion of the tumor cells in the gynecological tumor primary cell culture obtained by the method can reach 70-95%, and the interference of the mixed cells is less.

The gynecological tumor primary cell culture obtained by the method can be used for in vitro experiments, next generation sequencing, animal model construction, cell line construction and the like of various cell levels. It is expected that the culture method has wide application prospect in the fields of research and clinical diagnosis and treatment of gynecological tumors.

Drawings

FIG. 1 shows single cells obtained after treatment of breast cancer tissue. The scale is 100 μm, 100 times magnification.

FIG. 2 shows the cell mass obtained after primary culture of breast cancer tissue. The scale is 100 μm, 100 times magnification.

FIG. 3 is a HE staining diagram of gynecological tumor cells obtained after primary culture of breast cancer tissues. The scale is 100 μm, 200 times magnification.

FIG. 4 is a photograph of immunohistochemical staining of paraffin sections of tumor cell mass obtained after primary culture of breast cancer tissues. The scale is 100 μm, 200 times magnification.

FIG. 5 is a diagram of a microplate chip design of the invention.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of reagents for culturing Primary cells of gynecological tumors

1. Sample preservation solution (100mL)

The specific formulation of the specimen preservation solution (100mL) is shown in table 1.

TABLE 1 sample preservation solution (100mL)

After the preparation of the sample preservation solution is completed, the sample preservation solution is subpackaged by 15mL centrifuge tubes, and each tube is 5 mL. Can be stored at 4 deg.C for 1 month after subpackaging.

2. Sample cleaning solution (100mL)

The specific formulation of the sample rinse (100mL) is shown in table 2.

TABLE 2 sample rinse (100mL)

The sample cleaning solution needs to be prepared for use.

3. Sample dissociation liquid (10mL)

The specific formulation of the sample dissociation solution (10mL) is shown in table 3.

TABLE 3 sample dissociation solution (10mL)

Note: the sample dissociation liquid is prepared for use.

In table 3, the formulation of collagenase stock solutions is shown in tables 4-6.

TABLE 410 collagenase I stock solution (100mL)

After preparing the 10 Xcollagenase I stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 510 collagenase III stock solution (100mL)

After preparing the 10 Xcollagenase III stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

TABLE 610 collagenase IV stock solution (100mL)

After preparing the 10 Xcollagenase IV stock solution, the solution was dispensed into 1.5mL sterile centrifuge tubes, 1mL each. The stock solution can be stored at-20 deg.C for a long period.

In tables 4-6, the unit U of collagenase (said collagenase I, said collagenase III or said collagenase IV) is defined by the enzymatic activity of the protease: 1 μmol of L-leucine can be released by treating collagenase (said collagenase I, said collagenase III or said collagenase IV) with 1U of protease at 37 ℃ and pH 7.5 for 5 hours.

4. Cell digestive juice (10mL)

The specific formulation of the cell digest (10mL) is shown in Table 7.

TABLE 7 cell digest (10mL)

The cell digestive juice is prepared for use.

5. Digestive stop solution (100mL)

The specific formulation of the digestion-stopping solution (100mL) is shown in Table 8.

TABLE 8 digestive stop solution (100mL)

The digestion stop solution can be stored for one month at 4 ℃ after being prepared.

6. Gynecological tumor primary cell culture medium (100mL)

The specific formulation of gynecological tumor primary cell culture medium (100mL) is shown in table 9.

TABLE 9 Primary cell culture media for gynecological tumors (100mL)

Gynecological tumor Primary cell culture Medium preparation was completed, followed by filtration sterilization using 0.22. mu.M needle filter (Millipore SLGP033RS), and storage at 4 ℃ for two weeks was possible.

In Table 9, the preparation of human recombinant protein stocks is shown in tables 11-14, the preparation of hydrocortisone stocks is shown in Table 15, and the preparation of Y-27632 stocks is shown in Table 16; the formulation of the progrestasterone stock solutions is shown in table 17; the formulation of the β -Estradiol stock solution is shown in Table 18. The 100 × BSA solutions required to formulate these stock solutions are shown in table 10.

TABLE 10100 XBSA solution (1mL)

The 100 × BSA solution is ready for use.

TABLE 111000 × stock solution of human recombinant protein EGF (5mL)

After 1000 Xhuman recombinant protein EGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 121000 × stock solution of human recombinant protein bFGF (2.5mL)

After 1000 Xhuman recombinant protein bFGF stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with the volume of 1.5mL, and the stock solution can be preserved at the temperature of minus 80 ℃ for a long time.

TABLE 131000 Xhuman recombinant protein HGF stock solution (5mL)

1000 Xthe human recombinant protein HGF stock solution is prepared and subpackaged by a sterile centrifuge tube of 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 141000 × stock solution of human recombinant protein MSP (2.5mL)

1000 Xthe human recombinant protein MSP stock solution is prepared and then subpackaged by a sterile centrifuge tube with 1.5mL, and the stock solution can be preserved for a long time at the temperature of minus 80 ℃.

TABLE 151000 Hydrocortisone stock solution (10mL)

After preparing a stock solution of 1000 XN-acetyl-L-cysteine, subpackaging the stock solution by using a sterile centrifuge tube with the volume of 0.5mL, and storing the stock solution at the temperature of 20 ℃ below zero for a long time.

TABLE 161000 XY-27632 stock solution (3.125mL)

After preparing the stock solution of 1000 XY-27632, the stock solution is subpackaged by a sterile centrifuge tube of 0.5mL and can be stored for a long time at the temperature of minus 20 ℃.

TABLE 17100000 XProgesterone stock solutions (15.9mL)

After 100000 XProgesterone stock solution is prepared, the stock solution is subpackaged by a sterile centrifuge tube with 0.5mL, and the stock solution can be preserved at the temperature of minus 20 ℃ for a long time.

TABLE 1810000 XSeta-Estradiol stock solution (18.36mL)

10000 times beta-Estradiol stock solution is prepared and then is subpackaged by a sterile centrifuge tube of 0.5mL, and the stock solution can be preserved for a long time at the temperature of 20 ℃ below zero.

7. Cell cryopreservation liquid

The specific formulation of the cell culture medium is shown in Table 19.

TABLE 19 cell cryopreservation solution

The cell frozen stock solution is prepared for use at present.

In table 19, the preparation of the 1% methylcellulose solution is shown in table 20.

TABLE 201% methylcellulose solution (10mL)

The 1% methyl cellulose solution can be stored for a long time at 4 ℃ after being prepared.

8. 1% CYTOP solution

TABLE 211% CYTOP solution (100mL)

After the 1% CYTOP solution is prepared, the product can be stored for a long time at normal temperature.

9. Cell separation buffer (100mL)

The specific formulation of cell isolation buffer (100mL) is shown in Table 22:

TABLE 22 cell isolation buffer (100mL)

After the preparation of the cell separation buffer, the cells can be stored at 4 ℃ for 1 month.

In table 22, the preparation of the heparin sodium solution is shown in table 23.

TABLE 231000 Xheparin sodium (1mL)

1000 Xheparin sodium solution is prepared for use.

Example 2 acquisition of gynecological tumor postoperative specimen/biopsy puncture specimen/pleural and peritoneal fluid sample

1. In cooperation with the Hospital, the cooperative development passed a formal medical ethical examination.

2. The attending physician selects patients to be grouped according to clinical indications specified by medical guidelines and selects appropriate samples for in vitro culture according to the clinical indications in surgery, the selection criteria of the samples are as follows: primary breast cancer, ovarian cancer, endometrial cancer, cervical cancer or metastasis thereof, a sample with the weight of a surgical specimen exceeding 20mg, or a sample with a pleural effusion and ascites sample exceeding 100mL, or a sample with a puncture biopsy specimen exceeding 4.

3. The primary physician provides basic clinical information such as sex, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc. of the patient. The name, the identification card number and other information of the patient related to the privacy of the patient are hidden and replaced by a uniform experiment number, and the naming principle of the experiment number is eight-digit numerical date of the collected sample plus four digits after the patient is hospitalized. For example, if the sample is provided on 1/2018, the hospitalization number of the patient is T001512765, and the sample experiment number is 201801012765.

4. During surgery, the surgeon collects fresh post-operative/biopsy specimens in a sterile operating room environment and places them in a previously prepared specimen preservation solution (see example 1). The samples were kept temporarily on ice after being isolated and transported to the laboratory within two hours for further processing. The pleural effusion samples were transported to the laboratory for further processing within 48 hours.

Example 3 pretreatment of tissue sample for solid tumor of gynecological tumor

The following operations required working on ice and the entire procedure required completion within 10 minutes.

The surgical instruments used in the following operations all need to be sterilized in advance at high temperature and high pressure and can be used after being dried.

1. The samples were weighed.

2. The sample surface was rinsed with 75% (volume percent) ethanol for 10 to 30 seconds.

3. The samples were washed 5 times with sample wash and 5 times with sterile PBS solution.

4. The fat tissue, connective tissue and necrotic tissue in the sample are carefully stripped off with the aid of an ophthalmic scissors, an ophthalmic forceps, a scalpel and the like.

Example 4 tissue sample dissociation of solid tumors of gynaecological tumors

The surgical instruments used in the following examples were sterilized at high temperature and high pressure in advance and dried before use.

1. Cutting the tissue into pieces of 1mm by using an ophthalmic scissors³The left and right small blocks.

2. The minced tissue samples were treated with a sample dissociation solution preheated at 37 ℃ in advance at a dose of 0.1mL of the sample dissociation solution (see example 1) per mg of tissue, and dissociation was carried out at 37 ℃ for 15 minutes to 3 hours. The dissociation of the samples was observed under the microscope every 15 minutes until a large number of single cells were observed.

3. The dissociation reaction was stopped with 10 volumes of a digestion stop solution (see example 1) and the cell suspension was collected.

4. The cell suspension was filtered through a 100 μm sterile cell strainer to remove tissue debris and adherent cells.

5. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

6. The cells were resuspended in 5mL sterile PBS, centrifuged at 800g for 10 minutes at room temperature, and the supernatant discarded.

7. Resuspend the cell pellet with gynecological tumor primary cell culture medium (see example 1), observe the cell state under microscope, and count the cells.

As shown in FIG. 1, the dissociated single cell suspension contains a large amount of various types of cells, such as erythrocytes, lymphocytes, and fibroblasts, in addition to tumor cells. One of the advantages of the method is that in the subsequent culture process, only cancer cells can be greatly amplified, and the proportion of other cells is gradually reduced or even disappears, so that gynecological tumor primary tumor cells with higher purity are finally obtained.

Example 5 pretreatment of gynecological tumor thoracic and abdominal Water samples

1. The gynecological tumor chest and abdomen water sample is kept still for about 30 minutes on ice, so that the blood clots and large insoluble solids in the sample are settled to the bottom of the sample tube;

2. carefully transferring the supernatant into a 50mL sterile centrifuge tube, adding one volume of precooled PBS and mixing uniformly;

3. 2000g, centrifuging for 5 minutes at 4 ℃, and removing supernatant;

4. resuspending the cell pellet in cell isolation buffer (see example 1), centrifuging at 2000g and 4 ℃ for 5 minutes, and discarding the supernatant;

5. resuspending the cell pellet with cell isolation buffer (see example 1) and adjusting the cell concentration to 10⁷/mL。

Example 6 Density gradient centrifugation of thoracic and abdominal Water samples for gynecological tumors

1. An equal volume of Ficoll cell separation (MP #50494) was taken from the cell suspension using a 50mL sterile centrifuge tube.

2. The cell suspension is carefully applied to the upper layer of the cell separation medium, so that a clear interface is formed between the two.

3. 2000g of the suspension were centrifuged horizontally at room temperature for 20 minutes.

4. Sucking the middle layer white film into a new tube.

5. The cell pellet was resuspended in 20mL sterile PBS, 1500g was centrifuged at RT for 10min, and the supernatant was discarded.

6. Resuspend the cell pellet with gynecological tumor primary cell culture medium (see example 1), observe the cell state under microscope, and count the cells.

As a result, the isolated single cell suspension contains a large amount of various types of other cells, such as erythrocytes, lymphocytes, fibroblasts, and the like, in addition to tumor cells. One of the advantages of the method is that in the subsequent culture process, only cancer cells can be greatly amplified, and the proportion of other cells is gradually reduced or even disappears, so that gynecological tumor primary cells with higher purity are finally obtained.

Example 7 culture of Primary cells for gynecological tumors

1. Gynecological tumor primary cell suspension culture is carried out by using a low-adsorption surface (low-adsorption surface), namely the gynecological tumor primary cell culture medium in example 1 (wherein 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 human recombinant protein MSP is 20ng/mL, the final concentration of hydrocortisone is 10 mug/mL, the final concentration of Y-27632 is 10 muM, the final concentration of Progesterone is 100nM, the final concentration of β -Estradiol is 10nM), and by taking a six-well plate as an example, according to the concentration of 10nM in each well⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

2. The cell status was observed every day, and the medium was changed every 3 days until the cells formed clumps of about 100 μm in diameter.

As shown in FIG. 2After 3-10 days of culture, the tumor cells are greatly expanded to form cell masses with the diameter of 100 mu m, and the total number of the tumor cells can exceed 10⁷The number of other types of cells is significantly reduced or even eliminated. According to the method, through a large number of sample tests, the success rate of in vitro culture of the primary tumor cells of the gynecological tumor can reach 70%.

Example 8 passage of Primary cells of gynecological tumors

1. The cell pellet was collected from the dish, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

2. The cell pellet was washed with sterile PBS solution, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

3. The cell pellet was resuspended in cell digest (see example 1) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 5 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of a digestion stop solution (see example 1) and the cell suspension was collected.

6. Resuspend the cell pellet with gynecological tumor primary cell culture medium (see example 1) and count the cells.

7. Culturing the gynecological tumor primary cells by using a low-adsorption surface (low-adsorption-surface), wherein the culture medium is the gynecological tumor primary cell culture medium in example 1, and the culture medium is a six-well plate 10 times per well as an example⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

Example 9 cryopreservation of Primary gynecological tumor cells

After the suspension culture of the gynecological tumor primary cells is subjected to passage amplification for 2-3 times, the gynecological tumor primary cells can be frozen:

3. The cell pellet was resuspended in cell digest (see example 1) and digested at 37 ℃. The digestion of the cell pellet was observed under a microscope every 15 minutes until the cell pellet was digested into single cells.

4. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 1), and the cell suspension was collected and counted.

6. Cell cryopreservation (see example 1) at 10⁶Resuspending the cell sediment at a density of/mL, freezing 1mL of cell suspension in each tube of a 2mL freezing tube, freezing overnight by using a gradient cooling box, and transferring the cell sediment into liquid nitrogen for long-term storage.

Example 10 recovery of Primary cells from gynecological tumors

The gynecological tumor primary cells preserved in liquid nitrogen can be recovered:

1. sterile water at 37 ℃ was prepared five minutes in advance.

2. The vial was removed from the liquid nitrogen and the cells were rapidly thawed in sterile water at 37 ℃.

3. 800g were centrifuged at room temperature for 10 minutes and the supernatant discarded.

4. Resuspending the cell pellet with gynecological tumor primary cell culture medium (see example 1), culturing gynecological tumor primary cells using low adsorption surface, resuscitating each tube of cells in a 3.5cm dish at 37 deg.C and 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

Example 11 HE staining identification of Primary gynecological tumor cells

The reagent consumables used in the following examples are illustrated:

HE staining kit (beijing solibao biotechnology limited, # G1120);

cation anticreep slide (Beijing China fir Jinqiao Biotech limited);

xylene, methanol, acetone (Beijing chemical reagent company, analytical pure);

neutral resin adhesive (fine chemicals, GmbH, Beijing).

1. Suspension cells were made to a concentration of 10⁴cells/mLAnd (5) dripping 10 mu L of suspension on a cation anti-falling glass slide, and naturally drying.

2. 50 μ L of a methanol/acetone mixture (volume ratio 1:1) pre-cooled at 4 ℃ was carefully added dropwise to the air-dried cells, and then the slide was fixed in a refrigerator at 4 ℃ for 10 mins.

3. And taking out the cell-fixed slide, and naturally drying at room temperature.

4. Slides were washed twice with 200 μ L PBS.

5. When the water on the slide is slightly dry, 100 mu L of hematoxylin staining solution is added for staining for 1 mins.

6. The hematoxylin stain was aspirated and the slides were washed 3 times with 200 μ L of tap water.

7. 100 mu L of differentiation solution is added dropwise for differentiation for 1 mins.

8. The differentiation medium was aspirated off, and the slides were washed sequentially 2 times with tap water and 1 time with distilled water.

9. The water on the surface of the slide is sucked off, and 200 mu L of eosin dye solution is dripped to stain the slide for 40 s.

10. Absorbing eosin dye solution, rinsing and dehydrating with 75%, 80%, 90% and 100% ethanol for 20s, 40s and 40 s.

11. After the ethanol was dried, 50. mu.L of xylene was added dropwise for cell permeation.

12. After xylene is completely dried, a drop of neutral resin adhesive is added dropwise, and the piece is mounted by a cover glass, observed under a microscope and photographed.

FIG. 3 shows the HE staining effect of primary tumor cells of gynecological tumors obtained by in vitro culture, and it can be seen that these cells generally have the characteristics of tumor cells such as high nuclear-mass ratio, deep nuclear staining, chromatin condensation in nuclei, multinuclear, and uneven cell size.

Example 12 immunohistochemical staining identification of gynecological tumor Primary cells

The reagents used in the following examples are illustrative:

paraformaldehyde (Beijing chemical reagent company, analytical pure) was dissolved in ultrapure water to prepare a 4% (4g/100mL) paraformaldehyde solution;

hydrogen peroxide (beijing chemicals, 35%);

blocking with normal goat serum (Solarbio, SL 038);

immunohistochemical primary anti-antibody (Fujianmei, kit-0012);

immunohistochemical secondary antibodies (Abcam, ab 205719);

EDTA repair solution (Abcam, ab 93684);

DAB color-developing liquid (

DAB Substrate Kit，8059S)

The gynecological tumor primary cell culture medium in example 1 (wherein 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 human recombinant protein MSP is 20ng/mL, the final concentration of hydrocortisone is 10 μ g/mL, the final concentration of Y-27632 is 10 μ M, the final concentration of Progesterone is 100nM, and the final concentration of beta-Estradiol is 10nM) is collected, and the obtained gynecological tumor cell mass is cultured to be sliced with paraffin, and the operation is carried out according to the following steps:

1. the slices were sequentially immersed in xylene I for 10min and xylene II (10 min).

2. Soaking in anhydrous ethanol I (5min) -anhydrous ethanol II (5min) -95% ethanol (5min) -80% ethanol (5min) -70% ethanol (5min), and washing with deionized water for 2 times, each for 2 min.

3. The tissue slices were placed in a repair box, and then a suitable amount of diluted EDTA repair solution (pH 9.0) was added, the surface of the solution being submerged in the tissue.

4. Microwave medium-grade repair for 10min (time is started when liquid boils), during which time no tissue dry-slices are allowed.

5. The repairing box is taken out of the microwave oven, naturally cooled and cooled, when the repairing liquid is cooled to room temperature, the slide is taken out, and the PBS (pH 7.4) is washed for 3 times and 3min each time (the tissue is not washed against the tissue during the washing process so as to avoid breaking the tissue).

6. Prepared 3% hydrogen peroxide (30% hydrogen peroxide diluted with deionized water) was added dropwise to the sliced tissue to block endogenous peroxidase, incubated at room temperature for 15min, and washed 3 times with PBS, 3min each.

7. The PBS was blotted on absorbent paper, 10% goat serum (from the same or similar source as the secondary antibody species) was added dropwise to the slide, and the slide was blocked at 37 ℃ for 60 min.

8. The liquid surrounding the slide tissue was wiped dry with absorbent paper, a circle was drawn around the tissue with an oil pen, then diluted primary antibody was added dropwise and incubated overnight in a wet box at 4 ℃.

And 9, washing the slices with PBS for 3 times, each time for 3min, wiping the slices with absorbent paper, dripping horseradish peroxidase-labeled secondary antibody, and incubating at room temperature for 60 min.

And (10) washing the slices with PBS for 3 times, 3min each time, throwing away PBS liquid, wiping the slices with absorbent paper, dripping a freshly prepared DAB color developing solution into each slice, observing under a microscope, and washing the slices with tap water after positive signals to stop color development.

11. And (3) performing hematoxylin counterstaining for 1min, washing with water, then differentiating with an acidic ethanol differentiation solution, and washing with tap water to turn blue.

12. Placing the slices into water for washing, and then sequentially placing the slices into: dehydrating 70% ethanol-80% ethanol-90% ethanol-95% ethanol-absolute ethanol I-absolute ethanol II-xylene I-xylene II, standing each reagent for 2min, and air drying in a fume hood.

13. The slides were mounted using neutral gum and covered with a coverslip. Placing in a fume hood for air drying.

14. The dried sections can be viewed under a microscope or photographed.

FIG. 4 shows the effect of immunohistochemical staining of breast cancer primary tumor cell masses cultured in vitro, and it can be seen that ER cells constituting the cell masses are positive and consistent with the pathological results of patients, confirming that the tumor cells cultured by the method have higher purity.

Example 13 in vitro culture of Primary tumor cells in different types of gynecological tumor samples

The procedures of all primary culture procedures of the samples in this example are completely identical (see the above description), and only the pathological types of the samples are different. The samples tested are shown in Table 24.

TABLE 24 in vitro culture of Primary tumor cells of gynecological tumors of various pathological types

As can be seen, the method can achieve very high success rate for the in vitro culture of the primary tumor cells of various gynecological tumor solid tumor samples.

EXAMPLE 14 culture of gynecological tumor Primary tumor cells with CYTOP-modified cell culture consumables

In this example, the procedures of all primary cultures were identical (see above), the CYTOP modification method was identical, and only the materials of the cell culture consumables were different (table 25).

The CYTOP modification method comprises the following steps: firstly, pure oxygen etching is carried out on the cell culture container, the etching condition is 20W, and the etching time is 3 minutes. Then, the surface of the culture dish or the culture plate is covered with an appropriate amount of 1% CYTOP solution (taking a 96-well plate as an example, 20 mu L of each well, and the appropriate amount refers to the condition of completely covering the bottom of the culture dish), and the CYTOP solution can be used after being completely dried.

TABLE 25 Effect of CYTOP-modified consumables on gynecological tumor Primary cell culture

Note: polystyrene (Polystyrene, abbreviated PS).

As can be seen from table 25: it can be seen that the success rate of sample culture can be greatly improved after CYTOP modification.

Example 15 microplate chip processing

In this embodiment, a way of injection molding is used, and a PMMA material (or PS, PC, COC, COP, LAS, etc.) is used to process a microplate chip for culturing the primary gynecological tumor cells of the present invention. The chip can be used for primary gynecological tumor cell culture and in-vitro drug sensitivity detection experiments. The microplate chip design is shown in FIG. 5.

In the practical application process, the PMMA material (or PS, PC, COC, COP, LAS and other materials) is used to prepare the structure of the microplate chip shown in the design drawing of FIG. 5, and then the surface of the microplate chip is subjected to CYTOP modification by the CYTOP modification method (see example 14), so that the microplate chip for culturing the primary gynecological tumor cells is obtained.

Claims

1. A method for culturing primary gynecological tumor cells comprises the following steps: suspending and culturing the gynecological tumor primary cells by using a gynecological tumor primary cell culture medium;

the gynecological tumor primary cell culture medium consists of an antibacterial antifungal agent three-antibody, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, cortisol, N-2Supplement, Y-27632, progesterone, beta-estradiol and an Advanced DMEM/F12 culture medium; wherein the final concentration of penicillin in the three-antibody of the antibacterial antifungal agent is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent is 100-; the final concentration of amphotericin B in the three-antibody of the antibacterial antifungal agent is 250 ng/mL; the final concentration of the HEPES is 8-12 mM; the final concentration of the GlutaMax is 0.8-1.2% (volume percentage); the final concentration of the human recombinant protein EGF is 10-100 ng/mL; the final concentration of the human recombinant protein bFGF is 10-50 ng/mL; the final concentration of the human recombinant protein HGF is 5-25 ng/mL; the final concentration of the human recombinant protein MSP is 5-25 ng/mL; the final concentration of the cortisol is 1-10 mug/mL; the final concentration of the N-2Supplement is 1 percent (volume percentage); the final concentration of the Y-27632 is 5-20 mu M; the final concentration of the progesterone is 50-100 nM; the final concentration of the beta-estradiol is 10-50 nM; the balance was Advanced DMEM/F12 medium.

2. The method of claim 1, wherein: the gynecological tumor primary cells are gynecological tumor solid tumor primary cells or gynecological tumor breast and abdominal water sample primary tumor cells;

further, the gynecological tumor solid tumor primary cells are obtained by dissociating gynecological tumor solid tumor tissues by using a sample dissociation solution;

the sample dissociation liquid consists of collagenase I, collagenase III, collagenase IV and PBS; wherein the final concentration of the collagenase I in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase III in the sample dissociation liquid is 250-350U/mL; the final concentration of the collagenase IV in the sample dissociation liquid is 150-250U/mL; the balance being PBS;

further, the gynecological tumor solid tumor tissue is dissociated by the sample dissociation liquid according to the method comprising the following steps: treating the sheared solid tumor tissue of the gynecological tumor by using the sample dissociation liquid preheated at 37 ℃ in advance according to the dosage of 0.1-0.3mL of the sample dissociation liquid per mg of the tissue, and dissociating the sample at 37 ℃ for 15 minutes to 3 hours;

and/or

Further, the primary tumor cells of the gynecological tumor hydrothorax and ascites sample are obtained by separating the gynecological tumor hydrothorax and ascites sample with a cell separation buffer solution;

the cell separation buffer solution consists of double-antibody P/S, heparin sodium and PBS; wherein the final concentration of penicillin in the double-resistant P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL; the final concentration of the heparin sodium is 10 IU/mL; the balance being PBS;

further, the gynecological tumor pleural and peritoneal fluid sample is separated by the separation buffer according to the method comprising the following steps: suspending the cells in the gynecological tumor pleural effusion sample by using the cell separation buffer solution, and then obtaining the primary tumor cells of the gynecological tumor pleural effusion sample by density gradient centrifugation.

3. The method according to claim 1 or 2, characterized in that: in the method, the gynecological tumor primary cells are cultured in a suspension manner by using the gynecological tumor primary cell culture medium according to the following steps: use ofA cell culture container M for suspension culture of the gynecological tumor primary cells by using the gynecological tumor primary cell culture medium at 37 ℃ and 5% CO₂Culturing under the condition, and replacing the culture medium every 2-4 days;

the cell culture vessel M is any one of: (I) a cell culture container made of polystyrene, a cell culture container made of polycarbonate, a cell culture container made of polymethyl methacrylate, a cell culture container made of COC resin, a cell culture container made of cyclic olefin polymer, or a cell culture container with a low adsorption surface; (II) subjecting the cell culture vessel of (I) to CYTOP modification;

further, the cell culture container is a cell culture dish, a cell culture pore plate or a micropore plate chip for cell culture;

further, in the (II), the cell culture vessel in the (I) is subjected to CYTOP modification according to a method comprising the following steps: carrying out pure oxygen etching on the cell culture container in the step (I), wherein the etching condition is that the power is 20W, and the etching time is 3 minutes; then covering the surface of the cell culture container with 1% CYTOP solution, and airing the 1% CYTOP solution to finish the CYTOP modification;

still further, the composition of the 1% CYTOP solution is as follows: each 100mL of the 1% CYTOP solution contained 1mL of LCYTOP, the balance being fluoro oil.

4. A method according to any one of claims 1-3, characterized in that: the method also comprises the following step of carrying out dissociation pretreatment on the gynecological tumor solid tumor tissue: cleaning the surface of a gynecological tumor solid tumor tissue sample by using ethanol with the volume percentage of 70-75%; sequentially cleaning the gynecological tumor solid tumor tissue samples by using a sample cleaning solution and a sterile PBS solution;

specifically, the sample cleaning solution consists of double-antibody P/S and PBS; wherein the final concentration of penicillin in the double-resistant P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL; the balance being PBS.

5. The method of claim 4, wherein: the in vitro time of the gynecological tumor solid tumor tissue sample subjected to the dissociation pretreatment is within 2 hours, and the gynecological tumor solid tumor tissue sample is preserved in a sample preservation solution before the dissociation pretreatment;

specifically, the sample preservation solution consists of fetal bovine serum, double-antibody P/S, HEPES and HBSS; wherein the final concentration of the fetal calf serum is 1-5% (volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL; the final concentration of the HEPES is 8-12 mM; the balance being HBSS.

6. The method according to any one of claims 1-5, wherein: in the method, the dissociation treatment of the gynecological tumor solid tumor tissue by using the sample dissociation solution further comprises the following steps: terminating the dissociation reaction by using a digestion termination solution, and collecting cell suspension; filtering the cell suspension to remove tissue debris and adherent cells; resuspending the cells with sterile PBS after centrifugation; re-centrifuging, and then re-suspending the cell sediment by using the gynecological tumor solid tumor primary cell culture medium;

specifically, the digestion stop solution consists of fetal calf serum, double-antibody P/S and a DMEM medium; wherein the final concentration of the fetal calf serum is 8-12% (volume percentage content); the final concentration of penicillin in the double-resistant P/S is 100-200U/mL; the final concentration of streptomycin in the double-antibody P/S is 100-200 mug/mL; the balance is DMEM medium.

7. The method according to any one of claims 1-6, wherein: in the process of culturing the gynecological tumor primary cells by using the gynecological tumor primary fine culture medium, the method also comprises the following steps: when the gynecological tumor primary cells form lumps with the diameter of 80-120 mu m, carrying out passage on the gynecological tumor primary cells;

specifically, the cell digest used for the passage was composed as follows: every 10mL of the cell digestive juice contains 4-6mL of Accutase, EDTA with the final concentration of 5mM, 1.5-2.5mL of TrypLE Express and the balance of PBS; and/or

The use of a digestion stopping solution for said passaging, which is the digestion stopping solution according to claim 6;

and/or

The method also comprises the step of performing cryopreservation and/or resuscitation on the gynecological tumor primary cells after 2-3 times of passage amplification;

specifically, the cell cryopreservation solution adopted in the cryopreservation process consists of an Advanced DMEM/F12 culture medium, DMSO and a 1% methylcellulose solution; wherein the volume ratio of the Advanced DMEM/F12 culture medium to the DMSO to the 1% methylcellulose solution is 20:2 (0.8-1.2); the 1% methylcellulose solution is an aqueous solution of methylcellulose having a concentration of 1g/100 ml.

8. A kit for culturing primary gynecological tumor cells, comprising the culture medium of gynecological tumor cells according to any one of claims 1 to 7 and at least one of the following reagents: the sample dissociation solution, the sample preservation solution, the cell separation buffer solution, the cell digestion solution, the sample washing solution, the digestion stop solution, the cell cryopreservation solution and the 1% CYTOP solution according to any one of claims 1 to 7.

9. Use of a kit according to claim 8 for culturing primary gynecological tumor cells.

10. The method or kit or use according to any one of claims 1 to 9, wherein: the gynecological tumor is breast cancer, ovarian cancer, endometrial cancer, cervical cancer or metastasis focus thereof.