CN118028239A

CN118028239A - Culture medium and culture method for culturing micro-tumor model of urinary system tumor

Info

Publication number: CN118028239A
Application number: CN202410444015.0A
Authority: CN
Inventors: 张函槊; 尹申意
Original assignee: Beijing Cornerstone Jingzhun Diagnostic Technology Co ltd
Current assignee: Beijing Cornerstone Jingzhun Diagnostic Technology Co ltd
Priority date: 2024-04-15
Filing date: 2024-04-15
Publication date: 2024-05-14
Anticipated expiration: 2044-04-15
Also published as: CN118028239B

Abstract

The invention discloses a culture medium and a culture method for a micro-tumor model for culturing urinary system tumors. First, the present invention provides a medium for culturing a micro-tumor model of a urinary system tumor. Further, the invention also provides a method for culturing a micro-tumor model of the urinary system tumor by using the culture medium, which has the technical core that: firstly, treating solid tumor tissues of urinary system tumors by using a mild cell dissociation reagent, and ensuring the activity of various types of cells in the tissues to the greatest extent; and then, utilizing a suspension culture system containing the culture medium to self-assemble various types of cells separated from tumor tissues to form a micro-tumor model. The urinary system tumor micro-tumor model obtained by the method can accurately reflect various characteristics of the original focus of a patient, and is a scientific research experimental model and a preclinical experimental model in the field of tumor accurate diagnosis and treatment.

Description

Culture medium and culture method for culturing micro-tumor model of urinary system tumor

Technical Field

The invention relates to the technical field of biology, in particular to a culture medium and a culture method for a micro-tumor model for culturing urinary system tumors.

Background

Urinary tumors are tumors occurring in any part of the urinary system, including kidney, renal pelvis, ureter, bladder, urethra tumors. Bladder cancer is most common because urine stays within the bladder for the longest period of time. Urinary system tumors often occur after age 40, about one time more in men than in women.

Although scientific research and medical institutions in various countries of the world have great investment in research on the causes of urinary system tumors and the occurrence and development processes, people still have little knowledge of the disease. The urinary system tumor is a kind of complex disease, the occurrence and development of which are dynamic processes, and involve the interaction of a plurality of signal molecules, so that a complex molecular regulation network is formed, and the urinary system tumor is also influenced by external environmental factors.

With the progress of tumor diagnosis technology, the development of surgical technology and the advent of new methods for anti-tumor treatment in recent years, the overall therapeutic effect of urinary system tumors has been improved to a great extent, but the biological processes of occurrence, development, recurrence and metastasis of such diseases in human beings are still poorly understood, and the mechanisms of heterogeneity, drug resistance and tumor immunity are still poorly understood. Moreover, the etiology and occurrence and development processes of the urinary system tumor have strong individual variability, and cannot be roughly said. Therefore, the individuation and accurate research of the primary cell culture of the urinary system tumor solid tumor serving as a model is a trend in the research field of urinary system tumor and even in the diagnosis and treatment field of urinary system tumor. Therefore, it is important to develop a tumor model capable of accurately reflecting the characteristics of the original focus of a patient, and the existing primary tumor cell culture technology mainly comprises 2D culture, 3D culture, reprogramming culture and other types, and the methods have the problems of extremely long culture period, low culture success rate, difficult removal of mixed cells, incapability of reproducing tumor microenvironment and the like.

Disclosure of Invention

In order to effectively solve the technical problems, the invention provides a novel urinary system tumor micro-tumor model culture technology and a matched reagent, and the core of the technology is as follows: (1) The solid tumor tissue of the urinary system tumor is treated by using a mild cell dissociation reagent, so that the activity of various types of cells in the tissue is ensured to the greatest extent; (2) Preparing a special serum-free culture medium, and utilizing a suspension culture system to self-assemble various types of cells separated from urinary system tumor tissues to form a cell mass structure with various cell components, which is called as a micro tumor model of urinary system tumor.

In a first aspect, the invention claims a medium for culturing a micro-tumor model of a urinary system tumor.

The culture medium for culturing the micro-tumor model of the urinary system tumor, which is claimed by the invention, consists of an antibacterial and antifungal agent, such as three antibodies, HEPES, glutaMax, human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein IL-2, human recombinant protein IL-15, N-acetyl-L-cysteine (N-acetyl-L-cysteine), an N-2 additive (N-2 Supplement), Y-27632, dihydrotestosterone (Dihydrotestosterone), prostaglandin E2 (Prostaglandin E2), ITS-X and a basal culture medium; the antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B.

The final concentration of HEPES in the medium may be 8-12mM (e.g.10 mM); the final concentration of GlutaMax may be 0.8-1.2% (e.g., 1%) by volume; the final concentration of the human recombinant protein EGF may be 10-100ng/mL (e.g., 50 ng/mL); the final concentration of the human recombinant protein bFGF can be 10-50ng/mL (such as 20 ng/mL); the final concentration of the human recombinant protein HGF may be 5-25ng/mL (e.g., 20 ng/mL); the final concentration of the human recombinant protein MSP may be 5-25ng/mL (e.g., 20 ng/mL); the final concentration of the human recombinant protein IL-2 can be 10-100ng/mL (such as 20 ng/mL); the final concentration of the human recombinant protein IL-15 can be 10-100ng/mL (e.g., 20 ng/mL); the final concentration of the N-acetyl-L-cysteine may be 0.5-2mM (e.g., 1 mM); the final concentration of the N-2 additive may be 1% by volume; the final concentration of Y-27632 may be 5-20. Mu.M (e.g., 10. Mu.M); the final concentration of dihydrotestosterone may be 1-10nM (e.g., 5 nM); the final concentration of prostaglandin E2 may be 1-10. Mu.M (e.g., 10. Mu.M); the final concentration of ITS-X may be 1% by volume.

In a specific embodiment of the invention, the basal medium is ADVANCED DMEM/F12 medium.

Further, the final concentration of penicillin in the antibacterial and antifungal agent triple antibody in the culture medium may be 100-200U/mL (e.g., 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent three-antibody can be 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antimicrobial antifungal agent triple antibody may be 200-250ng/mL (e.g., 250 ng/mL).

Further, the antibacterial and antifungal agent triple antibody (penicillin-streptomycin-amphotericin B) consists of the following components: comprises 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml. The antibacterial and antifungal agent three antibodies are 'Antibiotic-Antimycotic, 100X' (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic,100X" contained 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, penicillin G (sodium salt) in the form of 0.85% salt solution, streptomycin sulfate and amphotericin B were used as antifungal agents. The GlutaMAX is "GlutaMAX ^TM supply" (e.g., gibco #35050061, or other product of the same composition). The component of the 'GlutaMAX ^TM supply' is L-alanyl-L-glutamine, which is an alternative to L-glutamine, the concentration is 200nM, and the solvent is 0.85% NaCl solution. The N-2 Supplement is "N-2 Supplement (100X)" (such as Gibco #17502001, or other products of the same composition). The "N-2 Supplement (100X)" contains 1mM final concentration of human whole iron transferrin (Human Transferrin (Holo)), 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 an advanced cell culture additive and can directly replace L-glutamine in a cell culture medium. The Y-27632 is "Y-27632 dihydrochloride (an ATP-competitive ROCK-I and ROCK-II inhibitor, ki 220nM and 300nM, respectively)" (e.g. MCE#129830-38-2, or other products of the same composition). The solvent of ITS-X is EBSS solution (Earle's balanced salt solution), and the solute and concentration are as follows: insulin 1g/L; transferrin 0.55g/L; 0.00067g/L of sodium selenite; ethanolamine 0.2g/L. Dihydrotestosterone is an endogenous androgenic steroid and hormone, is an agonist of the androgen receptor, has 2-3 times higher affinity than testosterone, and induces androgen receptor signaling 3 times higher potency than testosterone. The Prostaglandin E plays an important role in labor (softening the cervix, causing uterine contractions) and also stimulates osteoblasts to release factors that stimulate osteoclast uptake.

In a specific embodiment of the present invention, the antibacterial and antifungal agent, the third antibody (penicillin-streptomycin-amphotericin B), is identified as Gibco #15240062; the brand number of the HEPES is Gibco#15630080; the brand goods number of the Glutamax is Gibco#35050061; the brand goods number of the human recombinant protein EGF is Peprotech AF-100-15-100; the brand goods number of the human recombinant protein bFGF is Peprotech AF-100-18B-50; the brand goods number of the human recombinant protein HGF is Peprotech AF-100-39; the brand goods number of the human recombinant protein MSP is R & D#352-MS-050; the brand goods number of the human recombinant protein IL-2 is Peprotech 200-02; the brand goods number of the human recombinant protein IL-15 is Peprotech 200-15; the brand number of the N-acetyl-L-cysteine is Sigma#A9165; the brand number of the N-2 Supplement is Gibco #17502001; the brand goods number of the Y-27632 is MCE#129830-38-2; the brand number of Dihydrotestosterone is Abmole #m6033; the brand number of Prostaglandin E is Selleck #s3003; the brand number of the ITS-X is Gibco#51500056; the brand number of ADVANCED DMEM/F12 medium is Gibco #12634010.

Further, the culture medium may exist in two forms:

The culture medium is a solution formed by mixing the antibacterial and antifungal agent tri-antibody (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 human recombinant protein IL-2, the human recombinant protein IL-15, the N-acetyl-L-cysteine (N-acetyl-L-cysteine), the N-2 additive (N-2 Supplement), the Y-27632, the dihydrotestosterone (Dihydrotestosterone), the prostaglandin E2 (Prostaglandin E2), the ITS-X and the basic culture medium (such as ADVANCED DMEM/F12 culture medium).

The medium was prepared and sterilized by filtration using a 0.22. Mu.M needle filter (Millipore SLGP RS) and stored at 4℃for two weeks.

And secondly, each component in the culture medium exists independently, and the culture medium is prepared according to a formula when in use.

Furthermore, the human recombinant protein EGF, human recombinant protein bFGF, human recombinant protein HGF, human recombinant protein MSP, human recombinant protein IL-2 and human recombinant protein IL-15 can exist in the form of stock solution (mother solution) which can be stored for a long time at 80 ℃, and can be 1000 times of stock solution (mother solution). N-acetyl-L-cysteine, Y-27632 and Prostaglandin E2 may be present as stock solutions (mother liquor) which may be stored for a prolonged period of time at (-20 ℃), in particular 1000 times the stock solution (mother liquor). Dihydrotestosterone can exist in the form of stock solution (mother liquor), which can be stored at (-20deg.C for a long period of time), specifically 100000 times stock solution (mother liquor).

1000 Xstock solution of human recombinant protein EGF consists of human recombinant protein EGF, BSA and PBS, wherein the final concentration of the human recombinant protein EGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000 Xstock solution of 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 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000 Xstock solution of human recombinant protein HGF consists of human recombinant protein HGF, BSA and PBS, wherein the final concentration of the human recombinant protein HGF is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000 Xstock solution of human recombinant protein MSP consists of human recombinant protein MSP, BSA and PBS, wherein the final concentration of human recombinant protein MSP is 20 mug/mL, the final concentration of BSA is 0.01g/mL, and the balance is PBS.

1000X stock solution of human recombinant protein IL-2 consists of human recombinant protein IL-2, BSA and PBS, wherein the final concentration of the human recombinant protein IL-2 is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

1000X stock solution of human recombinant protein IL-15 comprises human recombinant protein IL-15, BSA and PBS, wherein the final concentration of the human recombinant protein IL-15 is 20 mug/mL, the final concentration of the BSA is 0.01g/mL, and the balance is PBS.

Of the seven 1000-fold stock solutions, BSA was present in 100-fold stock (stock solution) form (as-prepared), and consisted of BSA and PBS, with final concentration of BSA (Sigma#A1933) of 0.1g/mL and the balance PBS.

In addition, the 1000 XN-acetyl-L-cysteine stock solution consisted of N-acetyl-L-cysteine and ultrapure water, wherein the concentration of N-acetyl-L-cysteine was 0.5M, and the balance was 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× Prostaglandin E2 consists of Prostaglandin E and DMSO; wherein the final concentration of Prostaglandin E is 10mM, and the rest is DMSO.

100000 X Dihydrotestosterone consists of Dihydrotestosterone and DMSO; wherein the final concentration of Dihydrotestosterone is 100 mu M, and the rest is DMSO.

In a specific embodiment of the invention, the urinary system tumor is a kidney cancer, bladder cancer or ureter cancer.

In a second aspect, the invention claims a kit for culturing a micro-tumor model of a urinary system tumor.

The kit of parts for culturing a micro-tumor model of a urinary system tumor claimed in the present invention consists of all or part of the culture medium described in the first aspect above as follows: sample dissociation liquid, sample preservation liquid, sample washing liquid, cell digestion liquid, digestion termination liquid and cell freezing liquid.

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

The sample cleaning solution can be used for cleaning and disinfecting the sample. The sample cleaning liquid needs to be prepared at present.

The sample dissociation liquid can be used for dissociation of a sample. The sample dissociation solution needs to be prepared at present, wherein collagenase I, collagenase II and collagenase IV can be stored for a long time at the temperature of 20 ℃ below zero in a liquid storage (mother solution) form, and can be particularly 10 times of the liquid storage (mother solution). A 10 x collagenase I stock solution consisting of said collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL; the balance was PBS.10 x collagenase II stock solution consisting of the collagenase II and PBS; wherein the final concentration of collagenase II is 2000U/mL; the balance was PBS. A 10 x collagenase IV stock solution consisting of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance was PBS. The enzyme activities of collagenase I, collagenase II and collagenase IV are defined below.

The cell digestive juice can be used for digestion and passage of cell masses, and can digest colorectal cancer tumor masses into single cells. The cell digestive juice needs to be prepared at present.

The digestion stop solution may be used to terminate sample dissociation or cell digestion processes. The digestion stop solution can be stored at 4 ℃ for one month after being prepared.

The sample dissociation solution consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of collagenase I is 150-250U/mL (such as 200U/mL); the final concentration of collagenase II is 150-250U/mL (such as 200U/mL); the final concentration of collagenase IV is 150-250U/mL (such as 200U/mL); the balance was PBS.

Wherein the unit U of collagenase (said collagenase I or said collagenase II or said collagenase IV) is defined by the enzymatic activity of a protease: l-leucine can be released by treating collagenase (either said collagenase I or said collagenase II or said collagenase IV) with 1U protease at 37℃and pH7.5 for 5 hours.

In a specific embodiment of the invention, the brand number of the collagenase I is Gibco#17100-017; the brand goods number of the collagenase II is Gibco#17101-015; the brand goods number of the collagenase IV is Gibco#17104-019; the PBS was designated by the brand name Gibco #21-040-CVR.

The sample preservation solution consists of fetal bovine serum, an antibacterial and antifungal agent, namely, a triple antibody (penicillin-streptomycin-amphotericin B), HEPES and HBSS; wherein the final concentration of said fetal bovine serum is 1-5% (volume percent, e.g., 2%); the final concentration of penicillin in the antibacterial antifungal agent three antibody is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL (such as 250 ng/mL); the final concentration of HEPES is 8-12mM (e.g.10 mM); the balance is HBSS.

Further, the antibacterial and antifungal agent triple antibody (penicillin-streptomycin-amphotericin B) consists of the following components: each milliliter contains 10000 units of penicillin (alkali), 10000 mug of streptomycin (alkali) and 25 mug of amphotericin B. The antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is "Antibiotic-Antimycotic,100×" (such as Gibco #15240062, or other products of the same composition). The "Antibiotic-Antimycotic,100×" contains 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, and penicillin G (sodium salt) in the form of 0.85% salt solution, streptomycin sulfate and amphotericin B are used as Fungizone antifungals.

In a specific embodiment of the invention, the fetal bovine serum is given a brand number of Gibco #16000-044; the brand number of the antibacterial and antifungal agent three antibody (penicillin-streptomycin-amphotericin B) is Gibco #15240062; the brand number of the HEPES is Gibco#15630080; the brand number of the HBSS is Gibco#14170161.

The sample cleaning solution consists of an antibacterial and antifungal agent, namely, a third antibody (penicillin-streptomycin-amphotericin B) and PBS; wherein the final concentration of penicillin in the antibacterial and antifungal agent three antibodies is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL (such as 250 ng/mL); the balance was PBS.

Further, the antibacterial and antifungal agent triple antibody (penicillin-streptomycin-amphotericin B) consists of the following components: each milliliter contains 10000 units of penicillin (alkali), 10000 mug of streptomycin (alkali) and 25 mug of amphotericin B. The antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is "Antibiotic-Antimycotic,100X" (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic,100×" contains 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, and penicillin G (sodium salt) in the form of 0.85% salt solution, streptomycin sulfate and amphotericin B are used as Fungizone antifungals.

In a specific embodiment of the present invention, the antibacterial and antifungal agent, the third antibody (penicillin-streptomycin-amphotericin B), is identified as Gibco #15240062; the PBS was designated by the brand name Gibco #21-040-CVR.

The cell digestive juice comprises the following components: each 10mL of the cell digestive juice contains 4-6mL (such as 5 mL) of Ackutase, EDTA with the final concentration of 5mM, 1.5-2.5mL (such as 2 mL) of TrypLE Express, and the balance of PBS.

Further, the Ackutase is "StemPro ™ Ackutase ™ Cell Dissociation Reagent" (e.g., gibco #A11105-01, or other products having the same composition). The Ackutase is a single component enzyme dissolved in D-PBS,0.5mM EDTA solution. The TrypLE Express is "TrypLE ™ Express Enzyme (1×), no phenol red" (such as Gibco #12604013, or other products of the same composition). The "TrypLE ™ Express Enzyme (1X), no phenol red" contains 200mg/L KCl, 200mg/L KH ₂PO₄, 8000mg/L NaCl, 2160mg/L Na ₂HPO₄·7H₂ O and 457.6mg/L EDTA; also contains recombinant protease.

In the specific embodiment of the invention, the brand number of the Ackutase is Gibco#A11105-01; the brand name of the 0.5M EDTA is Invitrogen#AM9261; the brand goods number of the TrypLE Express is Gibco#12604013; the PBS was designated by the brand name Gibco #21-040-CVR.

The digestion stopping solution consists of fetal bovine serum, an antibacterial and antifungal agent, namely, a triple antibody (penicillin-streptomycin-amphotericin B) and a DMEM culture medium; wherein the final concentration of said fetal bovine serum is 8-12% (volume percent, e.g., 10%); the final concentration of penicillin in the antibacterial antifungal agent three antibody is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL (such as 100 mug/mL); the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL (such as 250 ng/mL); the balance being DMEM medium.

Further, the antibacterial and antifungal agent triple antibody (penicillin-streptomycin-amphotericin B) consists of the following components: each milliliter contains 10000 units of penicillin (alkali), 10000 mug of streptomycin (alkali) and 25 mug of amphotericin B. The antibacterial and antifungal agent (penicillin-streptomycin-amphotericin B) is "Antibiotic-Antimycotic,100X" (such as Gibco #15240062, or other products with the same composition). The "Antibiotic-Antimycotic,100X" contains 10000 units of penicillin (base), 10000 μg of streptomycin (base) and 25 μg of amphotericin B per ml, and penicillin G (sodium salt) in the form of 0.85% salt solution, streptomycin sulfate and amphotericin B are used as Fungizone antifungals.

In a specific embodiment of the invention, the fetal bovine serum is given a brand number of Gibco #16000-044; the brand number of the antibacterial and antifungal agent three antibody (penicillin-streptomycin-amphotericin B) is Gibco #15240062; the DMEM medium is given a brand number of Gibco #11965-092.

The cell freezing solution consists of ADVANCED DMEM/F12 culture medium, DMSO and 1% methyl cellulose solution; wherein the volume ratio of the ADVANCED DMEM/F12 culture medium, the DMSO and the 1% methylcellulose solution is 20:2 (0.8-1.2) (e.g. 20:2:1); the 1% methylcellulose solution is an aqueous methylcellulose solution having a concentration of 1g/100 ml.

In a specific embodiment of the invention, the ADVANCED DMEM/F12 medium is Gibco #12634010; the DMSO brand number is Sigma #d2438; the methylcellulose has a brand number of Sigma #M7027.

In a third aspect, the invention claims the use of a medium as described in the first aspect hereinbefore or a kit of reagents as described in the second aspect hereinbefore for culturing a microtumour model of a tumour of the urinary system.

In a fourth aspect, the invention claims a method of culturing a microtumor model of a urinary system tumor.

The method for culturing the micro-tumor model of the urinary system tumor, which is claimed by the invention, can comprise the following steps:

(a1) Dissociating solid tumor tissue of the urinary system tumor with the sample dissociation solution described in the second aspect;

(a2) And (3) performing suspension culture on the dissociated cells in the step (a 1) by using the culture medium in the first aspect to form cell clusters, thus obtaining the micro-tumor model of the urinary system tumor.

Further, in the step (a 1), the sample dissociation solution may be used to dissociate the solid tumor tissue of the urinary system tumor according to a method including the steps of: and (3) according to the dosage of not more than 0.5mg of tissue in 1mL of the sample dissociation solution, performing sample dissociation on the sheared solid tumor tissue of the urinary system tumor by using the sample dissociation solution at 37 ℃ for 30 minutes to 2 hours (such as1 hour).

Further, in step (a 2), the dissociated cells of (a 1) may be suspension-cultured with the medium according to a method comprising the steps of: the dissociated cells of (a 1) were cultured in suspension using the medium in a cell culture vessel having a low adsorption surface (low-adsorption-surface), and the culture was performed under conditions of 37℃and 5% CO ₂.

Wherein the initial seeding density may be 10 ⁵ cells/cm ² container bottom area, for example, a six well plate, plated at a density of 10 ⁶ cells per well.

Further, the time of the cultivation in the step (a 2) is 3 to 5 days.

Further, before step (a 1), the method may further comprise the step of performing dissociation pretreatment on the solid tumor tissue of the urinary system tumor as follows: washing the surface of a solid tumor tissue sample of the urinary system tumor with 70-75% (e.g. 75%) ethanol by volume for 10-30 seconds; washing a solid tumor tissue sample of the urinary system tumor 5-10 times (e.g., 5 times) with the sample washing solution described in the second aspect, and washing a solid tumor tissue sample of the urinary system tumor 5-10 times (e.g., 5 times) with a sterile PBS solution; and then removing impurities, connective tissues, adipose tissues, necrotic tissues and other components affecting primary cell culture in the solid tumor tissue sample of the urinary system tumor.

The step of subjecting the solid tumor tissue of the urinary system tumor to dissociation pretreatment needs to be performed on ice, and the whole operation step needs to be completed within 10 minutes.

Further, the dissociation pretreatment is performed on a solid tumor tissue sample of the urinary system tumor for an in vitro time of 12 hours or less, and the sample is stored in the sample storage solution in the second aspect before the dissociation pretreatment is performed.

Further, in the step (a 1), the dissociation treatment of the solid tumor tissue of the urinary system tumor by using the sample dissociation solution may further include the following steps: terminating the dissociation reaction with 8-15 (e.g. 10) volumes of the digestion stop solution as described in the second aspect above, and collecting the cell suspension; filtering the cell suspension with a100 μm or 40 μm sterile cell strainer to remove tissue debris and adherent cells; centrifuging at room temperature for 10-15 min (such as 10 min) at 800-1000g (such as 800 g), and discarding supernatant; cells were then resuspended with 3-5mL (e.g., 5 mL) of sterile PBS; centrifuging at room temperature for 10-15 min (such as 10 min) at 800-1000g (such as 800 g), and discarding supernatant; the cell pellet is then resuspended with the medium described in the first aspect above.

In a fifth aspect, the invention claims a method of obtaining solid tumor primary cells of a urinary system tumor.

The method for obtaining the primary cells of the solid tumor of the urinary system tumor, which is claimed in the present invention, is to isolate the primary cells of the solid tumor of the urinary system tumor from the micro-tumor model of the urinary system tumor obtained by the method described in the fourth aspect.

The method specifically comprises the following steps:

(b1) Digesting the micro-tumor model of the urinary system tumor by using a cell digestive juice (such as the cell digestive juice) to obtain single cells;

Further, the method comprises the step of terminating the digestion. Digestion was terminated as described above with the digestion stop solution.

(B2) And (3) separating CD326 positive cells from the single cells obtained in the step (b 1) to obtain the primary cells of the solid tumor of the urinary system tumor.

Further, CD326 positive cells can be sorted by CD326 magnetic beads.

In the above aspects, the pathological stage of the urinary system tumor is stage II or stage III, and the urinary system tumor or the urinary system tumor metastasis of various pathological types. Samples with surgical specimens weighing more than 20 mg.

In the present invention, all of the above-mentioned PBS's may be 1 XPBS, pH7.3-7.5. The concrete composition is as follows: the solvent is water, and the solute and the concentration are as follows: KH ₂PO₄144mg/L,NaCl 9000mg/L,Na₂HPO₄·7H₂ O795 mg/L.

The invention provides a method for extracting and culturing urinary system tumor micro-tumor models from fresh urinary system tumor solid tumor tissues and a matched reagent, and the method has the following advantages:

1. the tissue sample consumption is small, and only about 20mg of urinary system tumor operation sample is needed;

2. The culture period is short, and the urinary system tumor micro-tumor model of the order of magnitude of 10 ⁴-10⁶ can be obtained only by 3-5 days;

3. The culture stability is high, and the success rate of in vitro culture of qualified urinary system tumor operation specimens by the method is up to 80 percent;

4. the cell types are rich, the urinary system tumor micro-tumor model can store various cell types such as tumor cells, interstitial cells, immune cells and the like in the original focus, and the tumor micro-environment can be well reproduced;

5. The urinary system tumor microtumor model can accurately reproduce the pathological subtype of the original focus;

6. the urinary system tumor micro tumor model can accurately reproduce the genetic background of the original focus.

The urinary system tumor micro-tumor model obtained by the method can accurately reflect various characteristics of the original focus of a patient, and is a scientific research experimental model and a preclinical experimental model in the field of tumor accurate diagnosis and treatment. It is expected that the culture method has wide application prospect in the fields of research and clinical diagnosis and treatment of urinary system tumors.

Drawings

Fig. 1 is a bright field picture 48 hours prior to the urinary system tumor (bladder cancer) microtumor model formation process. The scale is 100 μm,100 times magnification.

Fig. 2 is a comparison graph of the effects of HE staining on a urinary tumor microtumor model and its corresponding primary lesion tissue sample. The scale is 50 μm,400 times magnification.

FIG. 3 is a graph showing the effect of multiple immunofluorescent staining of a urinary tumor microtumor model and its corresponding primary lesion tissue sample.

FIG. 4 shows the clear field pattern of the primary tumor cells of the urinary system tumor obtained by separation and purification, and the immunofluorescence staining result.

FIG. 5 shows the comparison of copy number variation analysis (CNV) of the urinary tumor micro tumor model obtained by culturing in two culture media according to scheme A, B and the corresponding original focus tissue sample.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1 preparation of reagents for use in culturing urinary tumor microtumor models

1. Sample preservation solution (100 mL)

The specific formulation of the sample storage solution (100 mL) is shown in Table 1.

After the sample preservation solution is prepared, split charging is carried out by using a 15mL centrifuge tube, and each tube is 5mL. Can be stored at 4deg.C for 1 month after packaging.

2. Sample cleaning solution (100 mL)

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

The sample cleaning liquid needs to be prepared at present.

3. Sample dissociation liquid (10 mL)

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

Note that: the sample dissociation solution is prepared at present.

In Table 3, the collagenase stock solutions were prepared as shown in tables 4 to 6.

After preparation of the 10 Xcollagenase I stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.

After preparation of the 10 Xcollagenase II stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.

After preparation of the 10 Xcollagenase IV stock solution, 1.5mL sterile centrifuge tubes were used for split charging, 1mL per tube. The stock solution can be stored at-20deg.C for a long period.

In tables 4, 5 and 6, the unit U of collagenase (either said collagenase I or said collagenase II or said collagenase IV) is defined by the enzymatic activity of the protease: l-leucine can be released by treating collagenase (either said collagenase I or said collagenase II or said collagenase IV) with 1U protease at 37℃and pH7.5 for 5 hours.

4. Cell digestive juice (10 mL)

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

The cell digestive juice is prepared at present.

5. Digestion stop liquid (100 mL)

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

After the digestion stop solution is prepared, the mixture can be stored at 4 ℃ for one month.

6. Urinary system tumor micro tumor model culture medium (100 mL)

The specific formulation of the urinary tumor microtumor model medium (100 mL) is shown in Table 9.

After the preparation of the urinary tumor microtumor model medium, the medium was sterilized by filtration through a 0.22 μm needle filter (Millipore SLGP033 RS) and stored at 4 ℃ for two weeks.

In Table 9, the formulations of the human recombinant protein stock solutions are shown in tables 11 to 16 (the formulation of the BSA stock solution is shown in Table 10), the formulation of the N-acetyl-L-cysteine stock solution is shown in Table 17, the formulation of the Y-27632 stock solution is shown in Table 18, the formulation of the Dihydrotestosterone stock solution is shown in Table 19, and the formulation of the Prostaglandin E stock solution is shown in Table 20.

The 100 XBSA solution was ready for use.

After 1000 Xstock solution of human recombinant protein EGF was prepared, it was sub-packaged with 1.5mL sterile centrifuge tubes, and the stock solution could be stored at-80℃for a long period of time.

After the 1000 Xhuman recombinant protein bFGF stock solution is prepared, the stock solution is split-packed by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.

After 1000 Xhuman recombinant protein HGF stock solution was prepared, split-packed with 1.5mL sterile centrifuge tubes, the stock solution could be stored at-80℃for a long period of time.

After 1000 Xhuman recombinant protein MSP stock solution is prepared, split charging is carried out by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.

After 1000 Xhuman recombinant protein IL-2 stock solution is prepared, the stock solution is split-packed by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.

After 1000 Xhuman recombinant protein IL-15 stock solution is prepared, the stock solution is split-packed by a 1.5mL sterile centrifuge tube, and the stock solution can be stored at-80 ℃ for a long time.

After 1000 XN-acetyl-L-cysteine stock solution was prepared, it was sub-packaged with 0.5mL sterile centrifuge tubes and the stock solution could be stored at-20℃for a long period of time.

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

After 100000X Dihydrotestosterone stock solution is prepared, the stock solution can be stored for a long period of time at-20 ℃ after being split-packed by a 0.5mL sterile centrifuge tube.

After the 1000X Prostaglandin E2 stock solution is prepared, the stock solution is split-packed by a 0.5mL sterile centrifuge tube, and the stock solution can be stored for a long time at-20 ℃.

7. Cell freezing solution

The specific formulation of the cell cryopreservation solution is shown in table 21.

The cell freezing solution is prepared for use at present.

In Table 21, the formulation of the 1% methylcellulose solution is shown in Table 22.

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

Example 2 acquisition of urinary System tumor post-operative specimens

1. In cooperation with a three-dimensional hospital, samples are obtained in the form of a clinical study initiated by a researcher, and the cooperation is developed through regular medical ethical examination.

2. The attending physician selects the group of patients according to clinical indications prescribed by the medical guidelines, and selects the appropriate sample for in vitro culture according to the intraoperative clinical indications, the criteria for selection of the sample are: primary renal cancer, bladder cancer or ureteral cancer, stage II or stage III pathology, and urinary system tumor samples weighing more than 20 mg.

3. All cases in the group are uniformly coded by adopting a mode of sample collection date and four positions after patient hospitalization number, for example, samples provided by 1 month and 1 day in 2020, and the patient hospitalization number is T001537474, and then the sample experiment number is 202001017474. And concealing information related to privacy of the patient, such as the name, the identification card number and the like of the patient. The hospital provides the sample with basic clinical information such as sex, age, medical history, family history, smoking history, pathological staging, clinical diagnosis, etc.

4. After tumor tissue is isolated in operation, a sample collection technician collects a fresh sample in an operating room sterile environment, and the collected sample needs to select a part with abundant fresh blood vessels, so that the part with poor cell activity such as necrotic tissue, adipose tissue, fibrotic tissue and the like is avoided. The collected sample was placed in a sample preservation solution (see example 1) pre-cooled at 4 ℃. The sample holding tube containing the sample is temporarily stored on ice and transported to a laboratory for the next operation within 12 hours, and the temperature is controlled to be 2-8 ℃ during the transportation.

Example 3 pretreatment for dissociation of tumor tissue samples of urinary System

The following operations are performed on ice, and the whole operation steps are completed within 10 minutes.

The surgical instruments used in the following operations all need to be sterilized by high-temperature steam (120 ℃ for 20 minutes) in advance and can be used after being dried.

1. After weighing the sample, the surface of the sample was rinsed with medical alcohol (75% by volume) for 10 to 30 seconds.

2. The sample was washed 5 times with the sample wash and 5 times with sterile PBS solution.

3. The adipose tissue, connective tissue and necrotic tissue in the sample are carefully peeled off by using an ophthalmic scissors, an ophthalmic forceps, a surgical knife and other devices.

Example 4 dissociation of urinary tumor tissue samples

The surgical instruments used in the following examples were sterilized by high-temperature steam (120 ℃ C., 20 minutes) and dried before use.

1. The tissue was cut into small pieces of about 0.5mm ³ with an ophthalmic scissors.

2. Tissue is treated with a sample dissociation solution (see example 1), 1mL of sample dissociation solution is used for tissue with a sample size of no more than 0.5mg, and 0.1mL of sample dissociation solution is required to increase each 0.1mg increase in tissue weight for tissue with a sample size of more than 0.5 mg. Sample dissociation solution treatment condition is 37 ℃, and dissociation time is 1 hour. Dissociation of the sample was observed under a microscope every 15 minutes during dissociation until most cells were observed to be shed from the tissue.

3. The dissociation reaction was stopped with 10 volumes of digestion stop solution (see example 1), and after the cell suspension was filtered through a 100 μm sterile cell screen to remove tissue debris and adherent cells, the supernatant was discarded by centrifugation at 800g for 10 minutes at room temperature.

4. Cells were resuspended in 5mL of sterile PBS, centrifuged at 800g for 10 min at room temperature, and the supernatant discarded.

5. Cell pellet was resuspended in urinary tumor microtumor model medium (see example 1), counted, trypan blue stained to determine cell viability, and isolated cell viability was greater than 70% for cell inoculation culture.

Example 5 urinary tumor microtumor model culture

1. The urinary tumor micro tumor model suspension culture was performed using a low adsorption surface (low-adsorption-surface), wherein the culture medium was the urinary tumor micro tumor model culture medium in Table 9 of example 1 (wherein the final concentration of human recombinant protein EGF was 50ng/mL, the final concentration of human recombinant protein bFGF was 20ng/mL, the final concentration of human recombinant protein HGF was 20ng/mL, the final concentration of human recombinant protein MSP was 20ng/mL, the final concentration of human recombinant protein IL-2 was 20ng/mL, the final concentration of human recombinant protein IL-15 was 20ng/mL, the final concentration of N-acetyl-L-cysteine was 1mM, the final concentration of Y-27632 was 10. Mu.M, the final concentration of Dihydrotestosterone nM, the final concentration of Prostaglandin E. Mu.M was 10. Mu.M), and the culture medium was plated in a six-well plate at a density of 10 ⁶ cells per well of 2-3mL. The inoculated cells were cultured at 37℃under 5% CO ₂ in a cell incubator.

2. The state of the cells was observed daily until the cells formed a mass with a diameter of about 100 μm, after which the medium was changed every 2-3 days to maintain the growth state of the urinary tumor microtumors.

As shown in FIG. 1, during the first 48 hours of culture, many different types of cells from cancer tissue spontaneously aggregate, self-assembling to form a 100 μm-sized cell mass structure, which we call a micro-tumor model. The total number of micro tumor cell clusters can reach 10 ⁵-10⁶. Through a large number of sample tests, the method can achieve 80% of success rate of the micro-tumor model culture of different urinary system tumor operation samples.

Example 6 HE staining comparative identification of urinary tumor microtumor model and primary lesions

Description of reagent consumables used in the following examples:

HE staining kit (bezier biotechnology limited, #ba 4027);

cation anticreep slide (fir gold bridge biotechnology limited in beijing);

xylene, methanol, acetone (beijing chemical company, analytical grade);

neutral resin gum (Beijing Yili Fine chemical Co., ltd.).

1. Collecting tissue blocks of urinary system tumor focus, and soybean size (5 mm square tissue block); the urinary system tumor micro tumor pellet obtained by the procedure of example 5 was collected and centrifuged at 2000rpm for 10min at room temperature to prepare a cell pellet.

2. The original focal Tissue mass and the micro tumor cell pellet mass were separately dehydrated and fixed (dehydrator standard procedure, sakura Tissue0Tek VIP 5Jr1 dehydrator).

3. Paraffin embedding (standard procedures of paraffin embedding machine, leica EG1150H paraffin embedding machine) is carried out on the dehydrated and fixed original focus tissue block and the micro tumor cell sediment block respectively.

4. The embedded original focus tissue block and the micro tumor cell precipitated block are respectively sliced with a slice thickness of 5 μm (Leica RM2245 semi-automatic rotary-wheel type slicer).

5. Spreading the slices (Leica HI1210 spreading machine), attaching onto cation anti-drop slide, and baking (Leica HI1220 baking machine) to obtain paraffin slice.

6. HE staining was performed on the original lesions and the microtomous sections with HE staining kit, respectively: after dewaxing gradient ethanol rehydration, hematoxylin dye solution is used for dyeing for 3 minutes, and the slide is washed 3 times by tap water. 100. Mu.L of the differentiation solution was differentiated for 1 minute, and the slide was washed 2 times with tap water and 1 time with distilled water. After dyeing with eosin dye liquor for 1 minute, gradient ethanol is dehydrated. After the ethanol is dried, 50 mu L of xylene is added dropwise to each slide for permeation. And (3) dropwise adding a drop of neutral resin adhesive after the xylene is completely dried, and sealing the sheet by using a cover glass.

7. And observing the dyeing effect under a mirror, and taking a picture.

Fig. 2 shows a comparison of the results of HE staining of a urinary tumor microtumor model and its corresponding primary lesion tissue sample obtained by culture. It can be seen that all the three micro tumor models of urinary system tumor can maintain the pathological structural characteristics of the original focus.

Example 7 multiple immunofluorescent staining identification of urinary tumor microtumor model and primary lesions

The reagents used in the following examples were as follows:

Paraformaldehyde (analytical grade, beijing chemical reagent Co.) was dissolved in ultrapure water to prepare a 4% (4 g/100 mL) paraformaldehyde solution;

Methanol, dimethyl sulfoxide (beijing chemical reagent company, analytical grade);

Hydrogen peroxide (Beijing chemical reagent Co., 35%);

Mixing methanol, dimethyl sulfoxide and 35% hydrogen peroxide according to the proportion of 4:4:1 (volume ratio) to prepare a Dandelion rinse solution;

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

Immunofluorescent primary antibodies (PanCK: abcam#ab215838, vim: abclonal #A19607, CD3: bioleged# 300434);

immunofluorescent secondary antibodies (CST#4408 s, CST#8889 s);

Hoechst dye liquor (Beijing Soy Biotechnology Co. # C0021);

using paraffin sections obtained in example 6 as materials, multiplex immunofluorescent staining was performed as follows, three antibodies were labeled respectively pan-CK for the characterization of epithelial-derived tumor cells, vimentin for the labeling of mesenchymal cells, CD3 for the labeling of T cells:

1. After dewaxing and rehydration of paraffin sections, the sections were washed once with PBS and treated with pre-chilled methanol solution for 1 hour.

2. The Dane rinse was treated at room temperature for 2 hours, followed by 75%, 50%, 25% (volume percent) of methanol diluted with PBS for 10 minutes each.

3. The 3% BSA solution was blocked for 2 hours at room temperature.

4. Primary antibody mixed dilutions (3% BSA solution) were used overnight at 4 ℃.

5. The sections were washed 5 times for 20 minutes in PBS.

6. Secondary antibody mixed dilutions (diluted with 3% bsa solution at 1:2000) were used for 2 hours at room temperature.

7. The sections were washed 5 times for 20 minutes in PBS.

8. Hoechst dye liquor and dyeing for 20 minutes at room temperature.

9. The quenched caplet was capped, and the staining was observed using a laser confocal microscope and photographed.

Fig. 3 shows the effect diagram of multiple immunofluorescence staining of the urinary system tumor microtumor model and the corresponding original focus tissue sample, and can be seen that in the urinary system tumor microtumor model, not only the panCK positive (green fluorescence) tumor cells in the original focus are reserved, but also the Vimentin positive interstitial cells (white) and immune cells (T cell red) are reserved, and the cell diversity and microenvironment of the original focus are maximally reproduced. The high similarity of the micro tumor model obtained by the culture of the method and the original focus in terms of cell composition is proved. The results of multiplex immunofluorescence staining of 15 urinary tumor microtumor samples (5 cases of kidney cancer, bladder cancer and ureter cancer) and corresponding primary focus tissue blocks are shown in Table 23.

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Example 8 isolation of primary tumor cells of urinary tumor from a urinary tumor microtumor model

The cell bead sorting procedure mentioned in the following examples uses the Methaemarrhena CD326 bead cationic selection kit (Methaemarrhena- # 130-061-101).

1. Urinary system tumor microtumor models suspension-cultured in example 5 were collected, centrifuged at 800g at room temperature for 10 minutes, and the supernatant was discarded.

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

3. Cell pellet was re-selected with cell digests (see example 1) and digested at 37 ℃ for 5-30 minutes, and cell pellet digestions were observed under a microscope every 5 minutes during digestion until most of the cell pellet was digested as single cells.

4. Digestion was stopped with 10 volumes of digestion stop solution (see example 1), and the cell suspension was centrifuged at 800g for 10 minutes at room temperature and the supernatant discarded.

5. The single cell suspension obtained by digestion is separated by using a meitian and gentle CD326 magnetic bead positive selection kit to obtain CD326 positive tumor cells: the cell pellet was resuspended in sorting buffer (formula see Table 24), and 20. Mu.L of CD326 magnetic beads were added to the system at cell amounts less than 10 ⁷ and incubated on ice for 30 minutes. The cell pellet was washed with 2mL of sorting buffer. The separation column was washed with 5mL of separation buffer and placed on a magnetic rack. The cell suspension was passed through a cell sorting column, which was washed three times with 3mL of sorting buffer, and CD326 negative cells were eluted. The separation column was removed from the magnetic rack and washed once with 5mL of separation buffer, eluting CD326 positive cells.

Note that: it is ready for use.

6. The sorted CD326 positive cells were centrifuged at 800g for 10 minutes at room temperature, and the supernatant was discarded. The inoculation and adherent culture was performed with DMEM medium containing 10% serum at a density of 10 ⁵/3.5 cm cell culture dishes.

EXAMPLE 9 passage of primary tumor cells of urinary System tumor

1. The cells of the primary tumor of the urinary system which are cultivated by adherence are cleaned by sterile PBS solution after the culture medium is removed.

2. 0.05% Trypsin digests cells at room temperature for 30-300s, during which time the cell state is observed until most of the cells are digested into spheres.

3. The digestion reaction was stopped with 10 volumes of DMEM medium containing 10% serum, and 800g of the cell suspension was collected and centrifuged at room temperature for 10 minutes, and the supernatant was discarded.

4. The supernatant was discarded after centrifugation at 800g for 10 minutes at room temperature.

5. Cell pellet was resuspended in DMEM medium containing 10% serum and cell counted. Inoculation and adherent culture (with the medium of example 5 plus 10% FBS) was performed at a density of 10 ⁵/3.5 cm cell culture dishes.

Through the purification procedure of example 8 and the subculture of example 9, high purity urinary system tumor primary tumor cells can be obtained (fig. 4 is a comparison of a clear field pattern of a bladder cancer primary tumor cell with an immunofluorescent staining pattern).

EXAMPLE 10 cryopreservation of primary tumor cells of urinary System

After the urinary system tumor primary tumor cells purified and cultured in example 10 are subjected to passage expansion for 2-3 times, the urinary system tumor primary tumor cells can be frozen:

5. Cell pellet was resuspended at a density of 10 ⁶/mL with cell cryopreservation solution (see example 1), 1mL of cell suspension per tube in a 2mL cryopreservation tube, and the gradient-cooled cassette was frozen overnight and transferred to liquid nitrogen for long term storage.

EXAMPLE 11 resuscitation of primary cells of urinary System tumor

The primary tumor cells of the urinary system tumor stored in the liquid nitrogen can be recovered:

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

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

3. Cells in the cryopreservation tube were transferred to a 15ml centrifuge tube, supplemented with ten volumes of DMEM medium containing 10% serum, thoroughly mixed, centrifuged at 800g for 10 min at room temperature, and the supernatant discarded.

4. Cell pellet was resuspended in DMEM medium containing 10% serum and cell counted. The inoculation and adherent culture were performed at a density of 10 ⁵/3.5 cm cell culture dishes.

Example 12 comparative urinary tumor micro-tumor Structure Forming Capacity of different Primary Medium

The procedure for primary culture of all samples in this example was identical (see above), with only the medium formulation being different. The various primary media tested are shown in table 25. Wherein scheme B is the formulation employed in the present invention, see in particular Table 9.

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Note that: in scheme A, the final concentration of human recombinant protein EGF is 50ng/mL; the final concentration of the human recombinant protein bFGF is 20ng/mL; the final concentration of the human recombinant protein HGF is 20ng/mL; the final concentration of the human recombinant protein MSP is 20ng/mL; the final concentration of N-acetyl-L-cysteine was 1mM; the final concentration of Y-27632 was 10. Mu.M; dihydrotestosterone at a final concentration of 10nM; prostaglandin E2 was added at a final concentration of 10. Mu.M. In scheme B, the final concentration of the human recombinant protein EGF is 50ng/mL; the final concentration of the human recombinant protein bFGF is 20ng/mL; the final concentration of the human recombinant protein HGF is 20ng/mL; the final concentration of the human recombinant protein MSP is 20ng/mL; the final concentration of the human recombinant protein IL-2 is 20ng/mL; the final concentration of the human recombinant protein IL-15 is 20ng/mL; the final concentration of the N-acetyl-L-cysteine is 1mM; the final concentration of Y-27632 was 10. Mu.M, the final concentration of Dihydrotestosterone nM, and the final concentration of Prostaglandin E2 was 10. Mu.M. In scheme C, the final concentration of the human recombinant protein EGF is 50ng/mL; the final concentration of the human recombinant protein bFGF is 20ng/mL; the final concentration of the human recombinant protein MSP is 20ng/mL; the final concentration of the human recombinant protein IL-2 is 20ng/mL; the final concentration of the human recombinant protein IL-15 is 20ng/mL; the final concentration of the human recombinant protein HGF is 20ng/mL; the final concentration of Forskolin is 5 mu M; the final concentration of Y-27632 was 10. Mu.M.

Three primary cell culture media protocols 10 samples were treated for kidney cancer, bladder cancer and ureter cancer, respectively, and sample treatment and culture procedures were performed as described in examples 3, 4 and 5, and the cell mass structure was observed in the culture system after 3 days of culture, as shown in table 26.

It can be seen that the urinary system tumor microtumor model can be cultured using both media of regimen A, B, whereas the regimen C media is not suitable for the formation of urinary system tumor microtumor models. The success rate of the urinary system tumor micro-tumor model formed in the culture medium of the scheme B is higher, the number and the volume of the obtained micro-tumors are larger, and the method has the advantage compared with the culture medium of the scheme A.

Example 13 comparison of the genetic background of urinary tumor microtumor models formed by different Primary cell culture Medium

The DNA extraction procedure mentioned in the examples below was performed using the Tiangen blood/tissue/cell genome extraction kit (DP 304).

The library construction procedure mentioned in the examples below was performed using the NEB DNA sequencing library construction kit (E7645).

The high throughput sequencing referred to in the examples below refers to the Illumina HiSeq X-ten sequencing platform.

The invention obtains an example of bladder cancer primary surgery focus sample in the embodiment 12, and a bladder cancer micro-tumor model obtained by culturing two culture mediums according to the sample use scheme A, B, and the DNA extraction, the whole genome sequencing and the copy number variation analysis method are as follows:

1. 10mg of bladder cancer original focus tissue block and 10 ⁵ cell clusters of the bladder cancer microtumor model obtained by in vitro culture in examples 3, 4 and 5 are respectively subjected to DNA extraction, library establishment and whole genome high throughput sequencing (WGS), and the sequencing depth is 30×.

2. Copy number variation analysis (CNV) was performed on both sets of sequencing results of the primary lesion and bladder cancer microtumor models, respectively, and the copy number variation between the primary bladder cancer tumor tissue and bladder cancer microtumor model was compared, as shown in fig. 5. The results show that the urinary system tumor micro-tumor model obtained by culturing the two culture mediums can well retain the copy number variation characteristics of the urinary system tumor primary focus. There was no significant difference between the two media in maintaining the genetic background of the primary foci.

Example 14 comparison of urinary tumor microtumor model cell composition formed by different Primary cell culture Medium

The invention obtains a urinary system tumor micro-tumor model obtained by culturing two culture mediums of scheme A, B in example 12 and a urinary system tumor primary disease focus sample (two examples of kidney cancer, bladder cancer and ureteral membrana cancer), and respectively carries out multiple immunofluorescence staining to identify the cell composition (the method is shown in example 7). The results are shown in Table 27.

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It can be seen that both culture media a and B can well retain tumor cells in the original lesions, and that the formed urinary system tumor microtumor model is composed of tumor cells with higher purity. The culture medium B can maintain the activity of tumor cells in the original focus, can well preserve fibroblasts and T cells existing in tissues, and has greater advantages in the aspect of maintaining cell diversity compared with the culture medium A.

Therefore, the culture medium (Table 9) of the micro-tumor model of the urinary system tumor can protect various cell types in the original focus to the greatest extent, promote the spontaneous formation of a multi-cell structure under the suspension condition, and construct an in-vitro micro-tumor model extremely close to the characteristics of the original focus.

Example 15 comparison of success Rate of different sample dissociation liquid cultures

The procedure of the primary culture of all samples in this example was completely identical (see above), and only the sample dissociation liquid formulation was different. The various sample dissociation solutions tested are shown in table 28 wherein scheme D is the formulation employed in the present invention, and in particular in table 3.

The sample dissociation solution is prepared at present.

Samples (4 cases of kidney cancer, bladder cancer and ureter cancer) with the weight of the solid tumor tissue mass of the urinary system tumor exceeding 100mg were selected, and the samples were divided into four parts in average, and the sample treatment and culture operations were performed by the methods described in examples 3, 4 and 5, respectively, using the above four sample dissociation liquids. The observation of the formation of cell mass structure in the culture system after 3 days of culture is shown in tables 29 and 30:

It can be seen that the sample dissociation liquid formula has a certain influence on the formation capacity of the urinary system tumor micro-tumor model and the size of the micro-tumor cell mass. The sample dissociation solution (Table 3) used in the invention can gently dissociate cells in tissues, maintain cell activity to the greatest extent, and improve the formation efficiency and quality of a micro tumor model.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.

Claims

1. A culture medium for culturing a microtumor model of a urinary system tumor, characterized by: the culture medium consists of an antibacterial and antifungal agent triple antibody HEPES, glutaMax, a human recombinant protein EGF, a human recombinant protein bFGF, a human recombinant protein HGF, a human recombinant protein MSP, a human recombinant protein IL-2, a human recombinant protein IL-15, N-acetyl-L-cysteine, an N-2 additive, Y-27632, dihydrotestosterone, prostaglandin E2, ITS-X and a basic culture medium;

The antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B;

In the medium, the final concentration of HEPES is 8-12mM; the final concentration of the GlutaMax is 0.8-1.2% by volume; the final concentration of the human recombinant protein EGF is 10-100ng/mL; the final concentration of the human recombinant protein bFGF is 10-50ng/mL; the final concentration of the human recombinant protein HGF is 5-25ng/mL; the final concentration of the human recombinant protein MSP is 5-25ng/mL; the final concentration of the human recombinant protein IL-2 is 10-100ng/mL; the final concentration of the human recombinant protein IL-15 is 10-100ng/mL; the final concentration of the N-acetyl-L-cysteine is 0.5-2mM; the final concentration of the N-2 additive is 1% by volume; the final concentration of the Y-27632 is 5-20 mu M; the final concentration of the dihydrotestosterone is 1-10nM; the final concentration of prostaglandin E2 is 1-10 mu M; the final concentration of ITS-X was 1% by volume.

2. The medium of claim 1, wherein: the basal culture medium is ADVANCED DMEM/F12 culture medium; and/or

In the culture medium, the final concentration of penicillin in the antibacterial and antifungal agent three antibodies is 100-200U/mL; the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL; the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL.

3. A kit for culturing a microtumor model of a urinary system tumor, consisting of the medium of claim 1 and all or part of: sample dissociation liquid, sample preservation liquid, sample cleaning liquid, cell digestion liquid, digestion termination liquid and cell frozen stock liquid;

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

The sample preservation solution consists of fetal bovine serum, an antibacterial antifungal agent, HEPES and HBSS; the antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B; in the sample preservation solution, the final concentration of the fetal bovine serum is 1-5% by volume; the final concentration of penicillin in the antibacterial antifungal agent three antibodies is 100-200U/mL; the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL; the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL; the final concentration of HEPES is 8-12 mM; the balance is HBSS;

the sample cleaning liquid consists of an antibacterial antifungal agent three antibody and PBS; the antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B; in the sample cleaning solution, the final concentration of penicillin in the antibacterial and antifungal agent three antibodies is 100-200U/mL; the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL; the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL; the balance being PBS;

The cell digestive juice comprises the following components: every 10mL of the cell digestive juice contains 4-6mL of Ackutase, EDTA with the final concentration of 5mM, 1.5-2.5mL TrypLE Express and the balance of PBS;

The digestion stopping solution consists of fetal bovine serum, an antibacterial antifungal agent, a third antibody and a DMEM culture medium; the antibacterial and antifungal agent three antibodies are penicillin, streptomycin and amphotericin B; in the digestion stopping solution, the final concentration of the fetal bovine serum is 8-12% by volume; the final concentration of penicillin in the antibacterial antifungal agent three antibodies is 100-200U/mL; the final concentration of streptomycin in the antibacterial and antifungal agent is 100-200 mug/mL; the final concentration of amphotericin B in the antibacterial antifungal agent is 200-250ng/mL; the balance is DMEM culture medium;

The cell freezing solution consists of ADVANCED DMEM/F12 culture medium, DMSO and 1% methyl cellulose solution; wherein the volume ratio of the ADVANCED DMEM/F12 culture medium, the DMSO and the 1% methyl cellulose solution is 20:2:0.8-1.2; the 1% methyl cellulose solution is a 10g/L methyl cellulose aqueous solution.

4. Use of the medium of claim 1 or 2 or the kit of claim 3 in a micro-tumor model for culturing a urinary system tumor.

5. The use according to claim 4, characterized in that: the urinary system tumor is kidney cancer, bladder cancer or ureter cancer.

6. A method of culturing a microtumor model of a urinary system tumor, comprising the steps of:

(a1) Dissociating solid tumor tissue of a urinary system tumor with the sample dissociation solution of claim 3;

(a2) And (3) culturing the cells dissociated in the step (a 1) in a suspension manner by using the culture medium to form cell clusters, thus obtaining the micro-tumor model of the urinary system tumor.

7. The method according to claim 6, wherein: in step (a 1), the solid tumor tissue of the urinary system tumor is dissociated with the sample dissociation liquid according to a method comprising the steps of: according to the dosage of not more than 0.5mg tissue of 1mL of the sample dissociation liquid, carrying out sample dissociation on the sheared solid tumor tissue of the urinary system tumor by using the sample dissociation liquid at 37 ℃ for 30 minutes to 2 hours; and/or

In step (a 2), the dissociated cells of (a 1) are suspension-cultured with the medium according to a method comprising the steps of: using a cell culture container with a low adsorption surface, and culturing dissociated cells of (a 1) in a suspension manner by using the culture medium at 37 ℃ under the condition of 5% CO ₂; and/or

Prior to step (a 1), further comprising the step of subjecting the solid tumor tissue of the urinary system tumor to a dissociation pretreatment as follows: washing the surface of a solid tumor tissue sample of the urinary system tumor by using 70-75% ethanol by volume percent; washing a solid tumor tissue sample of the urinary system tumor with the sample washing solution of claim 3 and then with sterile PBS solution; and/or

In step (a 1), the dissociation treatment of the solid tumor tissue of the urinary system tumor by using the sample dissociation solution further comprises the following steps: terminating the dissociation reaction with the digestion stop liquid of claim 3, and collecting the cell suspension; filtering the cell suspension to remove tissue fragments and adherent cells; after centrifugation, cells were resuspended in sterile PBS; re-centrifugation followed by resuspension of the cell pellet with the medium of claim 1.

8. The method according to claim 7, wherein: the dissociation pretreatment is performed for a period of time of not more than 12 hours in vitro on the solid tumor tissue sample of the urinary system tumor, and the sample is stored in the sample storage solution according to claim 3 until the dissociation pretreatment is performed.

9. The method according to claim 6, wherein: the urinary system tumor is kidney cancer, bladder cancer or ureter cancer.

10. A method for obtaining solid tumor primary cells of a urinary system tumor, which is to isolate solid tumor primary cells of a urinary system tumor from a micro tumor model of a urinary system tumor obtained by the method according to any one of claims 6 to 9.