阅读说明：本技术 一种结直肠癌实体瘤组织样本解离液 (Dissociation liquid for colorectal cancer solid tumor tissue sample ) 是由 张函槊 尹申意 于 2018-06-13 设计创作，主要内容包括：本发明公开了一种结直肠癌实体瘤组织样本解离液。本发明提供的样本解离液由胶原酶I、胶原酶II、胶原酶IV和PBS组成；其中,所述胶原酶I在所述样本解离液中的终浓度为150-250U/mL；所述胶原酶II在所述样本解离液中的终浓度为150-250U/mL；所述胶原酶IV在所述样本解离液中的终浓度为50-150U/mL；余量均为PBS。本发明用温和细胞解离试剂处理结直肠癌实体瘤组织,最大程度的保证了组织中癌细胞的活力。利用本发明方法得到的结直肠癌原代细胞培养物可以用于多种细胞水平的体外实验、二代测序、构建动物模型、构建细胞系等等。可以预见,本发明的培养方法和本发明所提供的样本解离液在结直肠癌的研究和临床诊断治疗领域具有广泛的应用前景。(The invention discloses a colorectal cancer solid tumor tissue sample dissociation liquid. The sample dissociation liquid provided by the invention consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase II in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase IV in the dissociation liquid of the sample is 50-150U/mL; the balance being PBS. The invention uses mild cell dissociation reagent to process colorectal cancer solid tumor tissue, which guarantees the activity of cancer cell in the tissue to the maximum extent. The colorectal cancer primary cell culture obtained by the method can be used for in-vitro experiments, next generation sequencing, animal model construction, cell line construction and the like of various cell levels. The culture method and the sample dissociation liquid provided by the invention have wide application prospects in the fields of research and clinical diagnosis and treatment of colorectal cancer.)

1. A colorectal cancer solid tumor tissue sample dissociation liquid is characterized in that: the sample dissociation liquid consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase II in the sample dissociation liquid is 150-250U/mL; the final concentration of the collagenase IV in the dissociation liquid of the sample is 50-150U/mL; the balance being PBS.

2. The sample dissociation liquid according to claim 1, wherein: the sample dissociation solution is a solution obtained by mixing the collagenase I, the collagenase II, the collagenase IV, and the PBS.

3. The sample dissociation liquid according to claim 1, wherein: each component in the sample dissociation liquid exists independently.

4. The sample dissociation liquid according to claim 3, wherein: said collagenase I, said collagenase II and said collagenase IV are present in the form of a mother liquor;

specifically, the mother liquor is 10 or 20 times of the mother liquor;

the 10 × collagenase I solution consists of the collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL; the balance being PBS;

a 10 × collagenase II stock solution consists of the collagenase II and PBS; wherein the final concentration of collagenase II is 2000U/mL; the balance being PBS;

20 × collagenase IV stock consists of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance being PBS.

5. A kit for dissociating colorectal cancer solid tumor primary cells from colorectal cancer solid tumor tissue, comprising the sample dissociation solution of any one of claims 1 to 4 and the digestion stop solution of claim 9.

6. Use of the sample dissociation liquid of any one of claims 1-4 or the kit of parts of claim 5 for dissociating colorectal cancer solid tumor primary cells from colorectal cancer solid tumor tissue.

7. A method of dissociating colorectal cancer solid tumor primary cells from colorectal cancer solid tumor tissue, comprising the steps of: treating the minced colorectal cancer solid tumor tissue with the sample dissociation liquid preheated at 37 ℃ according to the dosage of 0.1-0.3mL of the sample dissociation liquid according to any one of claims 1-4 per mg of tissue, and dissociating the sample at 37 ℃ for 15 minutes to 3 hours.

8. The method of claim 7, wherein: after the sample dissociation liquid is used for dissociation treatment of the colorectal cancer solid tumor tissue, the method further comprises the following steps: terminating the dissociation reaction by using a digestion termination solution, and collecting cell suspension; the cell suspension was filtered to remove tissue debris and adherent cells.

9. The method of claim 8, wherein: the digestion stop solution consists of fetal calf serum, an antibacterial antifungal agent triantion and a DMEM medium; wherein the final concentration of the fetal calf serum in the digestion stop solution is 8-12% (volume percentage); the final concentration of penicillin in the three antibiotics of the antibacterial and antifungal agents in the digestion stop solution is 100-200U/mL; the final concentration of streptomycin in the three-antibody of the antibacterial antifungal agent in the digestion stop solution is 100-200 mu g/mL; the final concentration of amphotericin B in the antibacterial antifungal agent triantion in the digestion stop solution is 250-500 ng/mL; the balance is DMEM medium.

10. A sample dissociation liquid or kit of reagents or use or method according to any one of claims 1 to 9, wherein: the colorectal cancer is primary colorectal cancer, the pathological stage is stage II, stage III or stage IV, and colorectal cancer or colorectal cancer metastasis focus of various pathological types.

Technical Field

The invention relates to the technical field of biology, in particular to a method for culturing primary cells of colorectal cancer solid tumors.

Background

Colorectal cancer is one of the most common health malignancies that severely threaten humans. The incidence rate of colorectal cancer in China is 9.24%, and the colorectal cancer accounts for the fourth place in all malignant tumors. The mortality rate of colorectal cancer is 11.77%, and the colorectal cancer accounts for the fifth place in all malignant tumors. With the development of economy, improvement of living standard and change of life style, the incidence rate of colorectal cancer will be on an increasing trend. In addition, the risk of recurrence and metastasis of colorectal cancer is high, and more than 50% of colorectal cancer patients have different degrees of recurrence and metastasis within months to years after radical treatment.

Although there is a great deal of investment in research and medical institutions throughout the world in studying the etiology and development of colorectal cancer, human beings are still poorly aware of this disease. Colorectal cancer is a complex disease, the occurrence and development of which are dynamic processes involving the interaction of a plurality of signal molecules, a complex molecular regulation network is formed, and the colorectal cancer is also influenced by external environmental factors. The etiology, occurrence and development process of colorectal cancer are highly variable among individuals and cannot be determined in a whole. Therefore, the trend of taking the primary cell culture of colorectal cancer solid tumor as a model to carry out individual accurate research is the colorectal cancer research field and even the colorectal cancer diagnosis and treatment field.

How to efficiently dissociate primary tumor cells from a tumor tissue sample and ensure the activity of the tumor cells is an important link influencing the separation and culture effects of the primary tumor cells.

Disclosure of Invention

In order to effectively solve the technical problems, the invention provides a novel primary cell culture technology of colorectal cancer solid tumor and a matched reagent.

In a first aspect, the invention claims a dissociation liquid for a colorectal cancer solid tumor tissue sample.

The sample dissociation liquid provided by the invention consists of collagenase I, collagenase II, collagenase IV and PBS; wherein the final concentration of the collagenase I in the dissociation solution of the sample is 150-250U/mL (such as 200U/mL); the final concentration of the collagenase II in the dissociation solution of the sample is 150-250U/mL (such as 200U/mL); the final concentration of the collagenase IV in the dissociation solution of the sample is 50-150U/mL (such as 100U/mL); the balance being PBS.

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

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

Further, the sample dissociation liquid may be present in two forms:

the sample dissociation solution is a solution obtained by mixing the collagenase I, the collagenase II, the collagenase IV, and the PBS. The sample dissociation liquid needs to be prepared immediately.

Secondly, each component in the sample dissociation liquid exists independently and is prepared according to a formula when in use.

Further, collagenase I, collagenase II and collagenase IV can exist in the form of stock solution (mother liquor) (can be stored for a long time at the temperature of 20 ℃); specifically, the amount of the stock solution (mother liquor) may be 10 times or 20 times.

The 10 × collagenase I stock consists of the collagenase I and PBS; wherein the final concentration of collagenase I is 2000U/mL.

A 10 × collagenase II stock solution consists of the collagenase II and PBS; wherein the final concentration of collagenase II is 2000U/mL; the balance being PBS.

20 × collagenase IV stock consists of the collagenase IV and PBS; wherein the final concentration of collagenase IV is 2000U/mL; the balance being PBS.

The enzyme activities of collagenase I, collagenase II and collagenase IV are defined above.

In a second aspect, the invention claims a kit for dissociating colorectal cancer solid tumor primary cells from colorectal cancer solid tumor tissue.

The kit for dissociating colorectal cancer solid tumor primary cells from colorectal cancer solid tumor tissue provided by the invention comprises the sample dissociation solution and the digestion stop solution.

In a third aspect, the present invention claims the use of the sample dissociation liquid or the kit as described above for dissociating colorectal cancer solid tumor primary cells from colorectal cancer solid tumor tissue.

In a fourth aspect, the invention claims a method of dissociating colorectal cancer solid tumor primary cells from colorectal cancer solid tumor tissue.

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

Further, after the sample dissociation liquid is used for dissociation treatment of the colorectal cancer solid tumor tissue, the method also comprises the following steps: terminating the dissociation reaction with 8-15 (e.g., 10) times the volume of the digestion stop solution, and collecting the cell suspension; filtering the cell suspension with a 100 μm or 40 μm sterile cell strainer to remove tissue debris and adherent cells; 800-1000g (e.g., 800g) of the suspension is centrifuged at room temperature for 10-15 minutes (e.g., 10 minutes), and the supernatant is discarded; then resuspend the cells in 3-5mL (e.g., 5mL) sterile PBS; then 800-1000g (e.g., 800g) are centrifuged at room temperature for 10-15 minutes (e.g., 10 minutes), and the supernatant is discarded.

Wherein the digestion stop solution consists of fetal calf serum, antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) and DMEM culture medium; wherein the final concentration of the fetal calf serum in the digestion stop solution is 8-12% (such as 10%,% represents volume percentage content); the final concentration of penicillin in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the digestion stop solution is 100-200U/mL (such as 100U/mL); the final concentration of streptomycin in the antibacterial antifungal agent triantibody (penicillin-streptomycin-amphotericin B) in the digestion stop solution is 100-200 [ mu ] g/mL (such as 100 [ mu ] g/mL); the final concentration of amphotericin B in the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) in the digestion stop solution is 250-500ng/mL (such as 250 ng/mL); the balance is DMEM 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 ingredientsAn antifungal agent.

In a specific embodiment of the invention, the brand of fetal bovine serum is Gibco # 16000-; the brand code of the antibacterial antifungal agent triantion (penicillin-streptomycin-amphotericin B) is Gibco # 15240062; the DMEM medium is sold under the brand name Gibco # 11965-092.

In the first to fourth aspects, the colorectal cancer may be specifically primary colorectal cancer, pathologically staged at stage II, III or IV, colorectal cancer of various pathotypes or colorectal cancer metastasis, a sample with a surgical specimen weight of more than 20 mg.

In the present invention, all of the above PBS's may be 1 XPBS, pH 7.3-7.5. The concrete 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 colorectal cancer solid tumor from fresh colorectal cancer solid tumor tissue and a matched reagent, the invention adopts a mild cell dissociation reagent to treat the colorectal cancer solid tumor tissue, the activity of cancer cells in the tissue is ensured to the maximum extent, and by utilizing the sample dissociation liquid provided by the invention and combining the method of the invention, the following beneficial effects can be achieved:

1. the dosage of the tissue sample is less, and only 20mg of colorectal cancer operation sample is needed;

2. the method can be used for culturing primary tumor cells of colorectal cancer primary tumors and can also be used for culturing primary tumor cells of colorectal cancer metastasis focuses;

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

4. the culture stability is high, and the success rate of in vitro culture of the qualified colorectal cancer operation specimen by using the method is up to 70 percent;

5. the purity of the cells is high, the ratio of cancer cells in the primary colorectal cancer cell culture obtained by the method can reach 70-95%, and the interference of mixed cells is less.

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

Drawings

FIG. 1 shows a single cell obtained after treatment of colorectal cancer tissue. The scale is 100 μm, 100 times magnification.

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

FIG. 3 is a graph showing the staining of colorectal cancer cell mass sections HE obtained after primary culture of colorectal cancer tissue. The scale is 100 μm, 200 times magnification.

FIG. 4 is an immunofluorescence staining pattern of cancer cell masses obtained after primary culture of colorectal cancer tissue. The scale is 50 μm, 200 times magnification

FIG. 5 shows that copy number variation analysis (CNV) performed on the basis of sequencing results shows that the copy number variation of primary colorectal cancer cell cultures (P1, P2, P3, P4, P5) is highly consistent with that of primary colorectal cancer Tumor tissue (Tumor).

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.