CN112760287A

CN112760287A - Culture medium for culturing primary cells of solid tumors of urinary tumors

Info

Publication number: CN112760287A
Application number: CN201911065995.9A
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: 2019-11-04
Filing date: 2019-11-04
Publication date: 2021-05-07

Abstract

The invention discloses a culture medium for culturing primary cells of solid tumors of urinary tumors. The culture medium for culturing the primary solid tumor cells of the urinary tumor is prepared into a special serum-free culture medium, and the culture medium is used for carrying out in-vitro suspension culture on the solid tumor cells from the urinary tumor, so that the interference of normal cells can be eliminated to the maximum extent while the normal amplification of the tumor cells is ensured. The primary cell culture of the solid tumor of the urinary tumor obtained by the method can be used for in vitro experiments of various cell levels, next generation sequencing, animal model construction, cell line construction and the like. The culture method has wide application prospect in the research and clinical diagnosis and treatment fields of the urinary tumor.

Description

Culture medium for culturing primary cells of solid tumors of urinary tumors

Technical Field

The invention relates to the technical field of biology, in particular to a culture medium for culturing primary cells of solid tumors of urinary tumors.

Background

Urological tumours are tumours that occur anywhere in the urinary system. Including tumors of the kidney, renal pelvis, ureter, bladder, and urethra. Bladder cancer is also most common due to the longest urine retention time in the bladder. Urinary system tumors often develop after age 40, with about one time more in men than women. According to data statistics of 2018 in the national cancer center, in 2014, the incidence rates of bladder cancer and kidney cancer in China respectively account for 4.5% and 2.7% of the incidence rate of male malignant tumors. In recent years, the incidence of urinary diseases in China has increased year by year, the average annual growth is about 6 percent,

although research and medical institutions in all countries of the world have a great deal of investment in the etiology and development of urinary tumors, human beings are still poorly aware of the disease. Urinary tumors are a complex disease, and the occurrence and development of the urinary tumors are dynamic processes, and involve the interaction of a plurality of signal molecules, so that a complex molecular regulation network is formed and is influenced by external environmental factors. The etiology, occurrence and development process of urinary tumor are highly variable among individuals and cannot be determined in a general way. In addition, the number of selectable treatment modes and medicines for urinary tumors is large, and how to select an optimal treatment scheme is always a great problem in clinical diagnosis and treatment. Therefore, the trend of individual precise research by using the primary cell culture of solid tumor of urinary tumor as a model is the research field of urinary tumor and the diagnosis and treatment field of urinary tumor.

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 urinary tumor solid tumor primary cell culture technology and a matched reagent.

In a first aspect, the invention claims a medium for culturing primary cells of solid tumors of urinary tumors.

The culture medium for culturing the primary cells of the solid tumor of the urinary tumor consists of three antibacterial 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-acetyl-L-cysteine (N-acetyl-L-cysteine), N-2Supplement, Y-27632, Dihydrotestosterone (Dihydrotestosterone), Prostaglandin E2(Prostaglandin E2) 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 N-acetyl-L-cysteine (N-acetyl-L-cysteine) is 0.5-2 mM; 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 dihydrotestosterone is 1-10 nM; the final concentration of prostaglandin E2 is 1-10 μ M; 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). The "N-2 Supplement (100X)" contains Human total Transferrin (Human Transferrin (Holo)) at a final concentration of 1mM, and 500mg/L recombinant Insulin whole chain (Insulin R)ecombinant Full Chain), 0.63mg/L Progesterone (Progesterone), 10mM Putrescine (Putrescine), 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). The Dihydrotestosterone is an endogenous androgenic steroid and hormone, is an agonist of androgen receptor, has 2-3 times higher affinity than testosterone, and has 3 times higher effect of inducing androgen receptor signaling than testosterone. The Prostaglandin E2 plays an important role in childbirth (softening the cervix, causing uterine contractions), and also stimulates osteoblasts to release factors that stimulate osteoclast resorption.

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 and cargo number of the N-acetyl-L-cysteine is Sigma # A9165; 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 Dihydrotesterone brand name is Selleck # S4757; the Prostaglandin E2 has a brand name of Selleck # S3003; the brand of the Advanced DMEM/F12 medium is Gibco # 12634010.

Further, the culture medium for culturing the primary cells of the solid tumor of urinary tumor can exist in two forms:

the culture medium for culturing the primary cells of the solid tumor of urinary tract is a solution containing the antimicrobial antifungal agent tris (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-2Supplement, the Y-27632, the Dihydrotestosterone (Dihydrotestosterone), the Prostaglandin E2(Prostaglandin E2) and the Advanced DMEM/F12 medium.

The media was prepared and sterilized by filtration through a 0.22 μ M needle filter (Millipore SLGP033RS) and stored at 4 ℃ for two weeks.

Secondly, each component in the culture medium for culturing the primary cells of the solid urinary tumor exists independently and is prepared according to a formula when in use.

Furthermore, the human recombinant protein EGF, the human recombinant protein bFGF, the human recombinant protein HGF and the human recombinant protein MSP can be stored in a stock solution (mother solution) form (long-term storage at the temperature of minus 80 ℃) and can be specifically 1000 times of the stock solution (mother solution). N-acetyl-L-cysteine, Y-27632 and Prostaglandin E2 can be stored in stock (mother liquor) form (long-term storage at-20 ℃), specifically 1000 times of stock (mother liquor). Dihydrotestosterone can be stored in a stock solution (mother liquor) form (long-term storage at-20 ℃), 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, the 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is the ultrapure water.

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.

1000 × Prostagladin E2 stock consisted of Prostagladin E2 and DMSO, with Prostagladin E2 at a final concentration of 10mM, the remainder being DMSO.

The 100000 × Dihydrotesterone stock solution consists of Dihydrotesterone and DMSO, wherein the final concentration of Dihydrotesterone is 100 μ M, and the balance is DMSO.

In a second aspect, the invention claims the use of said medium for culturing primary cells of solid tumors of urinary tumors.

In the application, a culture container with a low-adsorption surface (low-adsorption surface) is used, the primary urinary tumor solid tumor cells are cultured in suspension by using the 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.

Further, in the application, the primary cells of the solid tumor of the urinary tumor are passaged when the primary cells of the solid tumor of the urinary tumor form lumps with the diameter of 80-120 μm (such as 100 μm).

Wherein, the digestion stop solution adopted during the passage (can be stored for one month at 4 ℃ after being prepared) consists of fetal calf serum, double-antibody P/S (penicillin-streptomycin) and DMEM culture 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.

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.

More specifically, the step of performing said passaging is carried out: collecting cell mass to be passaged, centrifuging, washing the cell mass with sterile PBS solution, centrifuging, suspending the cell mass with the cell digestive juice, digesting at 37 deg.C until the cell mass is digested into single cell, stopping digestion reaction with the digestion stopping solution (the dosage can be 5-10 times, such as 10 times of volume), and collecting cell suspension; resuspending the cell pellet with the urological tumor solid 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, in the application, the method can also comprise the step of performing cryopreservation and/or resuscitation on the primary urinary tumor solid tumor cells after the primary urinary tumor solid tumor 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 specific steps of the cryopreservation are as follows: collecting cell mass to be frozen, centrifuging, washing the cell mass with sterile PBS solution, centrifuging, suspending the cell mass with the cell digestive juice, digesting at 37 deg.C until the cell mass is digested into single cell, terminating the digestion reaction with the digestion terminating solution (the dosage can be 5-10 times, such as 10 times of volume), and collecting cell suspension; centrifuging, and freezing the cells at 0.5-2 × 10⁶/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 primary cell culture medium of the solid tumor cell 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 each of the above aspects, the solid urological tumour tumor may be a primary solid urological tumour tumor. The pathological type is bladder cancer or prostate cancer.

In each of the above aspects, the primary urinary tumor solid tumor cells are isolated from a surgical sample or needle biopsy sample of a patient with urinary tumor. Wherein, the urinary tumor solid tumor tissue specimen obtained from the operation sample preferably has the weight of more than 20mg, and the number of the puncture biopsy samples exceeds 4.

In a particular embodiment of the invention, the urological tumour is in particular bladder cancer or prostate cancer.

In the present invention, all of the above PBS's may be 1 XPBS, pH 7.3-7.5. It is provided withThe body composition is as follows: the solvent is water, and the solute and the concentration are as follows: KH (Perkin Elmer)₂PO₄ 144mg/L，NaCl 9000mg/L，Na₂HPO₄·7H₂O 795mg/L。

The invention provides a method for extracting and culturing primary cells of solid urinary tumor from a fresh solid urinary tumor operation 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 urinary 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 solid tumor operation specimens or biopsy samples of urinary tumors by using the method is up to 70 percent;

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

The primary cell culture of the solid tumor of the urinary tumor 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 research and clinical diagnosis and treatment fields of solid tumor of urinary tumor.

Drawings

FIG. 1 shows single cells obtained after treatment of bladder cancer tissue. The scale is 200 μm, 100 times magnification.

FIG. 2 shows the cell masses obtained after primary culture of bladder cancer tissue. The scale is 200 μm, 100 times magnification.

FIG. 3 is a HE staining pattern of urinary tumor cells obtained after primary culture of bladder cancer tissue. The scale is 100 μm, 40 times magnification.

FIG. 4 is an immunofluorescence image of paraffin sections of tumor cell masses obtained after primary culture of bladder cancer tissue. The scale is 100 μm, 100 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 solid tumors of urological 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 II stock solution (100mL)

After preparing the 10 Xcollagenase II 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 II 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 II 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. Primary cell culture medium of solid tumor of urinary tumor (100mL)

The specific formulation of the primary cell culture medium (100mL) for solid tumors of urinary tract tumors is shown in table 9.

TABLE 9 Primary cell culture Medium for solid tumors of urinary tract tumors (100mL)

After preparation of the primary cell culture medium for solid tumors from urinary tumors, the cells were sterilized by filtration using a 0.22 μ M syringe filter (Millipore SLGP033RS) and stored at 4 ℃ for two weeks.

In Table 9, the preparation of human recombinant protein stocks is shown in tables 11 to 14, the preparation of N-acetyl-L-cysteine stocks is shown in Table 15, and the preparation of Y-27632 stocks is shown in Table 16; the formulation of the Dihydrotestosterone stock solution is shown in table 17; prostagladin E2 stock was formulated as shown in Table 18. The 100 × BSA solutions required to formulate these stocks are shown in table 9.

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 XN-acetyl-L-cysteine stock solution (5mL)

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 × Dihydrotesterone stock solution (34.4mL)

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

TABLE 181000 XSSTAglandin E2 stock solution (1.42mL)

1000 XSrostaglandin E2 stock solution was dispensed into 0.5mL sterile centrifuge tubes and the stock solution was stored for a long period at-20 ℃.

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.

Example 2 obtaining of post-operative/biopsy puncture specimens of solid tumors of urological tumors

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 bladder or prostate cancer, samples with surgical specimen weights exceeding 20mg, or samples with needle biopsy samples 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 the operation, fresh specimens are collected by the surgeon in a sterile environment in the operating room and placed 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.

Example 3 Pre-dissociation treatment of solid tumor tissue samples from urological tumors

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 urinary tumor solid tumor tissue sample dissociation

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 urological tumor solid tumor primary cell culture medium (see example 1), observe the cell status under microscope, and perform cell counting.

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 the primary tumor cells of the solid urinary tumor with higher purity are finally obtained.

Example 5 culturing of Primary cells from solid tumors of urological tumors

1. Urinary solid tumor cell suspension culture was performed using a low-adsorption surface (low-adsorption surface), which is the urinary solid tumor cell culture medium of 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 N-acetyl-L-cysteine is 1mM, the final concentration of Y-27632 is 10 μ M, the final concentration of dihydrate is 10nM, the final concentration of prostagladin E2 is 10 μ M), and a six-well plate is used as an example, and the concentration per well is 10 μ M⁶Individual 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 figure 2, after 3-10 days of culture, the tumor cells are greatly expanded to form cell masses with the diameter of 100 μm, and the total number of the tumor cells can exceed 10⁷The number of other types of cells is significantly reduced or even eliminated. Through a large number of sample tests, the success rate of in vitro culture of primary tumor cells of solid urinary tumor tumors can reach 70%.

Example 6 passage of Primary cells from solid tumors of urological 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 urological tumor solid tumor primary cell culture medium (see example 1) and count the cells.

7. Culturing primary cells of solid tumor of urinary tract by using low-adsorption surface (low-adsorption surface), wherein the culture medium is the culture medium of the primary cells of solid tumor of urinary tract in example 1, and the culture medium is prepared by taking a six-well plate as an example, and each well is 10 times⁶Individual cells were plated at 37 ℃ in density with 5% CO₂The culture was carried out in a cell culture incubator under the conditions.

Example 7 cryopreservation of Primary cells of solid tumors of urological tumors

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

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 10⁶Density resuspension fine/mLAnd (4) cell precipitation, wherein 1mL of cell suspension is stored in each tube of a 2mL freezing tube, and the cell suspension is transferred to liquid nitrogen for long-term storage after the cell suspension is frozen overnight by a gradient cooling box.

Example 8 Resuscitation of Primary cells of solid tumors of urological tumors

The primary cells of the solid tumor of the urinary tumor 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 the culture medium of solid tumor cells (see example 1), culturing the solid tumor cells using a low adsorption surface, resuscitating the cells in a 3.5cm dish at 37 deg.C and 5% CO/tube₂The culture was carried out in a cell culture incubator under the conditions.

Example 9 HE staining identification of Primary cells of solid tumors of urological tumors

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 10⁴And dripping 10 mu L of cell suspension on the 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 urinary tumors obtained by in vitro culture, which shows that these cells generally have the characteristics of high nuclear-mass ratio, deep nuclear staining, chromatin condensation in nuclei, multinuclear, uneven cell size and other tumor cells.

Example 10 immunofluorescence staining identification of urological 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;

methanol, dimethyl sulfoxide (Beijing chemical reagent company, analytical pure);

hydrogen peroxide (beijing chemicals, 35%);

mixing methanol, dimethyl sulfoxide and 35% hydrogen peroxide according to a volume ratio of 4:4:1 to prepare a Dan's rinsing solution;

bovine serum albumin (Sigma, # A1933) was dissolved in PBS to prepare a 3% (3g/100mL) BSA solution;

immunofluorescence primary anti-antibody (Abcam, # ab 17139);

immunofluorescent secondary antibody (CST, # 4408);

hoechst dye liquor (Beijing Sorleibao Biotech limited, # C0021);

immunofluorescent staining of colorectal cancer cell masses was performed as follows, primary antibody against CK8⁺Antibody to CK18, characterizing cells of epithelial origin.

1. The cell pellet was collected from the dish, washed once with PBS, resuspended in 4% paraformaldehyde and fixed overnight at 4 ℃.

2. The supernatant was discarded by centrifugation at 800g, and the cell pellet was resuspended in a precooled methanol solution and placed on ice for 1 hour.

3. Centrifuging at 800g, discarding supernatant, resuspending cell pellet with Dan's rinsing solution, and standing at room temperature for 2 hr.

4. The supernatant was discarded by centrifugation at 800g, and the cells were washed sequentially with 75%, 50%, 25% (volume percent) methanol diluted with PBS for 10 minutes each.

5. The supernatant was discarded by centrifugation at 800g, and the cell pellet was suspended in 3% BSA solution and blocked at room temperature for 2 hours.

6. According to the following steps: 500, primary antibody was diluted with 3% BSA solution and the cell pellet was resuspended with antibody dilution (3% BSA solution) and primary antibody was incubated overnight at 4 ℃.

7. The supernatant was discarded by centrifugation at 800g, and the cell pellet was washed 5 times with PBS solution for 20 minutes each.

8. According to the following steps: 2000, the secondary antibody was diluted with 3% BSA solution and the cell pellet was resuspended in antibody dilution (3% BSA solution) and the secondary antibody was incubated at room temperature for 2 hours.

9. The supernatant was discarded by centrifugation at 800g, and the cell pellet was washed 5 times with PBS solution for 20 minutes each.

10. Adding 100 Xhoechst dye solution according to the volume ratio of 1/100, and dyeing for 20 minutes at room temperature.

11. The cell pellet was washed 2 times with PBS solution for 10 minutes each. The staining of the cell mass was observed using a confocal laser microscope.

FIG. 4 shows the effect of immunofluorescence staining of primary tumor cell masses of bladder cancer cultured in vitro, and it can be seen that cells C constituting the cell massesK8⁺CK18 is positive and is tumor cells of epithelial origin, which proves that the tumor cells cultured by the method have higher purity.

Example 11 in vitro culture of Primary cells of solid tumors of different types of urological tumors

The flow of the operation method of primary culture of all samples in this example is completely consistent (refer to the above description), and only the type of the sample is different. The samples tested are shown in Table 22.

TABLE 22 in vitro culture of Primary tumor cells of various pathological urinary tumors

It can be seen that the method can achieve very high success rate for the in vitro culture of primary tumor cells of various types of solid tumor samples of urinary tumors.

Example 12 urological tumor solid tumor Primary tumor cell culture 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 23).

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 23 Effect of CYTOP-modified consumables on bladder and prostate cancer Primary tumor cell culture

Note: polystyrene (Polystyrene, abbreviated PS).

As can be seen from table 23: it can be seen that the success rate of sample culture can be greatly improved after CYTOP modification.

Example 13 microplate chip processing

In this example, a method of injection molding is used, and PMMA material (or PS, PC, COC, COP, LAS, etc.) is used to process to obtain a microplate chip for culturing the primary cells of solid tumor of urinary tumor. The chip can be used for primary urinary 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, etc.) is specifically used to prepare the structure of the microplate chip shown in fig. 5, and then the surface of the microplate chip is subjected to CYTOP modification by the CYTOP modification method (see example 11), so as to obtain the microplate chip for culturing primary cells of solid tumors of urinary tumors.

Claims

1. A culture medium for culturing primary cells of solid tumors of urinary tumors, comprising: the culture medium consists of three antibacterial antifungal agents, HEPES, GlutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, N-acetyl-L-cysteine, N-2Supplement, Y-27632, dihydrotestosterone, prostaglandin E2 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; 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 N-acetyl-L-cysteine is 0.5-2 mM; 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 dihydrotestosterone is 1-10 nM; the final concentration of prostaglandin E2 is 1-10 μ M; the balance is Advanced DMEM/F12 medium.

2. The culture medium according to claim 1, wherein: the culture medium is a solution containing the antibacterial antifungal agent triantion, 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, the N-2Supplement, the Y-27632, the dihydrotestosterone, the prostaglandin E2 and the Advanced DMEM/F12 culture medium.

3. The culture medium according to claim 1, wherein: the components of the medium are present separately.

4. The culture medium of claim 3, wherein: 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, the Y-27632, the dihydrotestosterone and the prostaglandin E2 exist in a mother liquor.

5. The culture medium according to claim 4, wherein: the mother liquor is 1000-fold 100000-fold mother liquor;

the 1000 multiplied human 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 multiplied human 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 protein HGF 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 MSP stock solution consists of human recombinant protein MSP, BSA and PBS, wherein the final concentration of the human recombinant protein MSP is 20 mu g/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS;

the 1000 XN-acetyl-L-cysteine stock solution consists of N-acetyl-L-cysteine and water, wherein the concentration of the N-acetyl-L-cysteine is 0.5M, and the balance is water;

the 1000 XY-27632 stock solution consists of Y-27632 and water, wherein the final concentration of Y-27632 is 10mM, and the balance is water;

1000 × prostaglandin E2 stock consists of prostaglandin E2 and DMSO, with prostaglandin E2 at a final concentration of 10mM, the remainder being DMSO;

the 100000 × dihydrotestosterone stock consists of dihydrotestosterone and DMSO, with the final concentration of dihydrotestosterone being 100 μ M and the balance being DMSO.

6. The culture medium of claim 5, wherein: in the stock solutions of 1000 × human recombinant protein EGF, 1000 × human recombinant protein bFGF, 1000 × human recombinant protein HGF and 1000 × human recombinant protein MSP, the BSA is present in a stock solution.

7. The culture medium of claim 6, wherein: the BSA exists in the form of 100 times of mother liquor; 100 × BSA solution consisted of BSA and PBS; wherein the final concentration of BSA was 0.1g/mL, and the balance was PBS.

8. Use of a culture medium according to any one of claims 1-7 for culturing primary cells of solid tumors of urinary tumors.

9. The culture medium according to any one of claims 1 to 7 or the use according to claim 8, wherein: the urinary tumor is bladder cancer or prostate cancer.