CN110938600A

CN110938600A - Primary tumor cell screening culture medium and method for constructing tumor cell line by using same

Info

Publication number: CN110938600A
Application number: CN201911110725.5A
Authority: CN
Inventors: 王珂; 于敏
Original assignee: TIANJIN TUMOUR HOSPITAL
Current assignee: TIANJIN TUMOUR HOSPITAL
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2020-03-31

Abstract

The invention provides a primary tumor cell screening culture medium, which consists of the following components in percentage by volume: 5 to 20 percent of acellular ascites, 3 to 15 percent of fetal calf serum and the balance of a mammalian cell culture medium. The screened culture medium component provided by the invention can selectively kill interstitial cells and has high selectivity on the growth of tumor cells. Meanwhile, the proliferation speed of the tumor cells can be improved, and the culture period is shortened. The tumor cell line construction method provided by the invention can purify the cancer cell line to more than 99%, and greatly improves the success rate of in vitro construction of the tumor cell line.

Description

Primary tumor cell screening culture medium and method for constructing tumor cell line by using same

Technical Field

The invention relates to the field of cell biology, in particular to a culture medium and a method for constructing a tumor cell line by using the culture medium.

Background

Cell lines are now widely used as cancer models, reflecting the clinical problem of the disease. These applications include mechanisms of drug resistance, new drug screening and drug development, analysis of proto-oncogene/suppressor gene expression, modeling of adhesion and transformation, and control studies of proliferation by peptide growth factors, cytokines, and hormones.

However, in the prior art, the success rate of establishing tumor cell lines from clinical specimens is only less than 30%. The reason for the failure is often attributed to the lack of proliferation of epithelial cells, which is often accompanied by hyperproliferation of mesenchymal cells. In the case of the human ovarian cancer cell line, it is difficult to establish an ovarian cancer cell line that can survive for a long period of time, and the difficulties faced by those skilled in the art are mainly the excessive proliferation of fibroblasts in culture, difficulty in determining culture conditions, difficulty in serial passages and difficulty in identification after establishing the cell line.

These problems have largely limited the research on cancer, and there is an urgent need to find a method to improve the success rate of tumor cell line construction.

Disclosure of Invention

In order to solve the above technical problems, an aspect of the present invention is to provide a primary tumor cell screening medium, which can effectively reduce mesothelial cell contamination, and separate and purify high-purity tumor cells.

The technical scheme provided by the invention is as follows:

a primary tumor cell screening medium, which consists of the following components in percentage by volume: 5 to 20 percent of acellular ascites, 3 to 15 percent of fetal calf serum and the balance of a mammalian cell culture medium.

In the prior art, the success rate of establishing tumor cell lines with clinical specimens is only less than 30%. The reason for the failure is often attributed to the lack of proliferation of epithelial cells, which is often accompanied by hyperproliferation of mesenchymal cells. Patients often contain large numbers of tumor cells in their ascites, and thus the success rate of establishing cell lines is much higher than that of establishing them with solid tumor tissues.

The difficulty in establishing a long-lived cell line is mainly the hyperproliferation of fibroblasts in culture, the difficulty in determining culture conditions, the difficulty in serial passage and the identification after establishment of the cell line, which is faced by the person skilled in the art. The present inventors have systematically developed a relatively simplified and easy-to-perform cell culture method in response to these difficult problems, successfully established epithelial tumor cell lines from ascites samples of cancer patients, and were essential for more efficient screening of potential carcinogens or anticancer drugs, development of new therapeutic approaches, and development of anti-tumor vaccines.

Preferably, in one embodiment of the present invention, the primary tumor cell screening medium further comprises an antibiotic. The antibiotic may be any suitable antibiotic, preferably penicillin and streptomycin.

The above-mentioned acellular ascites (CFA) may be ascites produced by any cancer patient, preferably ascites produced by an ovarian cancer patient. Meanwhile, the above-mentioned acellular ascites (CFA) can be prepared by any suitable decellularization method. Preferably, in one embodiment of the present invention, the method for preparing the cell-free ascites comprises: collecting ascites-free cells producing blood fiber net, centrifuging at 3000rpm for 10min to remove cells, filtering with 0.2 μm sterile filter membrane, and freezing at-20 deg.C for use.

The mammalian cell culture medium can be any suitable mammalian cell culture medium, such as RPMI1640, DMEM, MEM, and the like. Preferably, in one embodiment of the present invention, the mammalian cell culture medium is a DMEM medium.

Preferably, in an embodiment of the present invention, the primary tumor cell screening medium consists of the following components in percentage by volume: 15% of acellular ascites, 5% of fetal calf serum, 1% of double antibody and the balance of DMEM medium.

The screening culture medium can well inhibit the proliferation of mesothelial cells and indirectly damage the mesothelial cells, so that the mesothelial cells are shrunk and easily fall off, and the mesothelial cells are not damaged. Meanwhile, the proliferation speed of the tumor cells can be improved, and the culture period is shortened.

Another aspect of the invention provides the use of the above medium for the construction or culture of a tumor cell line.

In another aspect, the invention provides an application of the culture medium in the construction of ovarian cancer tumor cell lines.

In another aspect, the invention provides the use of the above culture medium for removing contamination of mesenchymal cells during cell culture.

Another aspect of the present invention provides a method for constructing a tumor cell line, comprising the steps of taking ascites and/or pleural effusion of a cancer patient, removing erythrocytes, continuously culturing the remaining cells in a complete culture medium, changing the complete culture medium into a screening culture medium when the confluency is not less than 70%, and culturing until all the cells are epithelial tumor cells;

wherein the screening culture medium is the culture medium.

Preferably, in one embodiment of the present invention, the culture medium comprises the following components by volume percent: 15% of acellular ascites, 5% of fetal calf serum, 1% of double antibody and the balance of DMEM medium.

Preferably, the cancer patient is a cancer patient who produces ascites and/or pleural effusion, and more preferably, the cancer patient is an ovarian cancer patient.

Preferably, the cancer patient produces a tumor content in the ascites and/or pleural fluid of not less than 20%.

In the construction method of the present invention, the ascites and/or pleural fluid of the cancer patient may be taken as a tissue of the cancer patient, and preferably a tissue having a high proportion of mesenchymal cells. For example, tissue containing more than 20% stromal cells.

In the method of the present invention, preferably, after removing the red blood cells, the cells are cultured in a complete medium until the mesenchymal cells in the cell suspension adhere to each other, and then the epithelial cell clot is blown up and transferred to another culture flask for further culture.

Preferably, the cells are cultured in a complete medium for 1 to 2 hours.

In the method of the present invention, preferably, after the complete medium is replaced with the screening medium, a step of further removing the stromal cells may be included, specifically, the screening medium is removed, the cell pellet is washed, then cell digest is added, when the stromal cells shrink and become round but the epithelial cells do not shrink yet, the culture flask is tapped and tapped to detach the stromal cells, after the stromal cells are removed, the culture is continued in the screening medium until all the epithelial tumor cells are removed.

In another aspect of the present invention, there is provided a tumor cell line, wherein the tumor cell line is prepared by the above method.

Preferably, in one embodiment of the present invention, the tumor cell line is an ovarian cancer cell line.

The invention has the beneficial effects that:

the screened culture medium component provided by the invention can selectively kill interstitial cells and has high selectivity on the growth of tumor cells. Meanwhile, the proliferation speed of the tumor cells can be improved, and the culture period is shortened. The tumor cell line construction method provided by the invention can purify the cancer cell line to more than 99%, and greatly improves the success rate of constructing the tumor cell line.

Drawings

FIG. 1 is a cell morphology diagram of ovarian cancer epithelial cells at different culture stages, wherein A is mixed cells after one week of complete medium culture, B is damaged interstitial cells after one week of selective medium culture, and C is purified ovarian cancer epithelial cells after interstitial cells are removed;

FIG. 2 is a graph showing the results of flow cytometry analysis to identify the phenotype of cell lines constructed by the methods of the present invention;

FIG. 3 is a graph showing the results of immunohistochemical characterization of cell line phenotypes constructed by the methods of the present invention.

Detailed Description

The invention discloses a culture medium, which can be realized by appropriately improving process parameters by a person skilled in the art with reference to the content in the specification. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art.

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Example 1: preparation of the culture Medium

The screening medium consists of the following components in percentage by volume: 15% of acellular ascites, 5% of fetal calf serum, 1% of double antibody and the balance of DMEM medium.

Mixing the above materials under aseptic condition, and storing at low temperature.

Example 2: preparation of the culture Medium

The screening medium consists of the following components in percentage by volume: 5% of acellular ascites, 8% of fetal calf serum, 1% of double antibody and the balance of RPMI1640 culture medium.

Example 3: preparation of the culture Medium

The screening medium consists of the following components in percentage by volume: 20% of acellular ascites, 6% of fetal calf serum, 1% of double antibody and the balance of MEM culture medium.

Example 4: construction of cell lines

The following procedure was carried out using the screening medium prepared in example 1, the formulation of the complete medium being: 10% FBS + DMEM medium.

1. Collection of ovarian ascites samples

Liquid samples obtained from surgery, all reagents and instruments must be sterile during collection and transport and transported to the laboratory for processing within 6h after being isolated. The sample should be marked with the word ascites, peritoneal fluid or peritoneal rinse. And records the relevant information in a table. The patient name is recommended not to be recorded with the hospitalized number as the ID number, while the information on the table is transferred to the laboratory record book to ensure that the relevant clinical information is consistent with any tumor cell lines established from the sample.

2. Primary pretreatment before inoculation of ovarian cancer cells

1) Under aseptic operation, transferring the ascites to a 50mL centrifuge tube with an aseptic conical bottom, and centrifuging at 1000rpm and 4 ℃ for 7 min;

2) discarding the supernatant;

3) adding 2mL of erythrocyte lysate (ACK), mixing uniformly and cracking;

4) centrifuging at 1000rpm and 4 deg.C for 7min, and removing supernatant;

5) repeating the above lysis step if there are still a large number of red blood cells;

6) washing the cell pellet 2 times with 10mL PBS buffer;

7) the resulting cell pellet was resuspended in 5mL of medium, which was the final cell suspension.

3. Removal of stromal cells and primary cell seeding

1) Approximately counting the viable cells in the final cell suspension obtained above with a hemocytometer and a viability dye, such as trypan blue;

2) adjusting the final cell concentration to 2X 10⁵Adding 7-10mL of cell suspension into a T25 culture flask per mL of culture medium;

3) culturing for 4 days in complete culture medium containing 1% PS, changing culture medium if the degree of confluence is 70%, and changing culture medium, and continuing culturing to 70% if the degree of confluence is less than 70%, and then performing subsequent operation;

4) using screening medium at 37 deg.C and 5% CO₂The incubator continues to culture until all cells are epithelial cells.

Example 5: construction of cell lines

1. Collection of ovarian ascites samples

2. Primary pretreatment before inoculation of ovarian cancer cells

2) discarding the supernatant;

3) adding 2mL of erythrocyte lysate (ACK), mixing uniformly and cracking;

4) centrifuging at 1000rpm and 4 deg.C for 7min, and removing supernatant;

6) washing the cell pellet 2 times with 10mL PBS buffer;

3. Removal of stromal cells and primary cell seeding

3) the cell culture flask was placed at 37 ℃ in 5% CO₂Culturing in incubator for 2 hr, observing under microscope, adhering interstitial cells in cell suspension, blowing off epithelial clot, transferring to new T25 culture bottle, placing at 37 deg.C and 5% CO₂Culturing in an incubator, wherein interstitial cells are removed for the first time;

4) culturing for 5 days in complete culture medium containing 1% PS, changing the culture solution, adding screening culture medium, culturing overnight if the confluency is 80%, and continuing to culture the culture solution to 80% confluency if the confluency is less than 80%, and performing subsequent operation;

5) washing the cell pellet 2 times with 10mL PBS buffer;

6) adding 1mL of trypsin/EDTA (1:1) solution at 37 ℃, and gently mixing to immerse the cells in the solution;

7) observing the time node that the shrinkage of the mesothelial cells becomes round but the epithelial cancer cells are not shrunk under a microscope, tapping and beating the culture bottle to help the mesothelial cells to be separated, and removing a large amount of interstitial cells for the second time;

8) washing the cell pellet 3 times with 10mL PBS buffer;

9) using screening medium at 37 deg.C and 5% CO₂The incubator continues to culture until all cells are epithelial cells.

The human ovarian adenocarcinoma primary drug-resistant strain W038 is constructed by the method, and the cell morphology of ovarian carcinoma epithelial cells at different culture stages is shown in figure 1. As can be seen from the figure, A in FIG. 1 is a control group, and the ascites mixed cells after one week of complete medium culture, regardless of mesothelial cells or cancer cells, are spread and grow, and are firmly attached to the wall, so that the purification and separation of the cancer cells are difficult. While B in FIG. 1 is the method in the working examples, it can be seen that the method can selectively culture tumor cells well.

The difficulty in culturing ascites-derived tumor cells lies in the proliferation and removal of mesothelial cells, and the culture method and the screening culture medium of the embodiment of the invention can well solve the problem, effectively reduce mesothelial cell pollution, separate and purify tumor cells with high purity, and the results are shown in fig. 1C and fig. 2.

Example 6: subculturing and freezing of cell lines

1. Liquid changing device

Inoculating the primary culture, changing the culture medium with fresh screening culture medium every 3-4 days, sucking away the old culture medium with sterile pasteur pipette, and adding new complete culture medium with new pipette.

2. Avoid cross contamination

To reduce or avoid the possibility of contamination of newly cultured cell lines with cells from other sources, the following precautions are taken:

if the conditions exist, other established cell lines are not cultured in a laboratory, if the conditions do not allow, the newly established primary culture cell line is cultured in an independent super clean bench and an independent incubator as far as possible, the used culture medium is independently filled in a sterile container and is independently marked, each culture operation is carried out in the cell culture super clean bench, a sterile new suction pipe or a gun head is replaced for each operation, the cell line with the same passage number is placed in the same layer of the incubator, and finally the established cell line is carefully maintained.

3. Subculturing

Cell populations derived from different tumors were grown at different times and pooled to 90% using 37 ℃ trypsin/EDTA solution at 1: 1.

The cell passage method comprises 1: 3-1: 4 passage and 1 time of 5-7 days.

4. Changing culture medium

After the first vial break, the cell phenotype was identified by Flow Cytometry (FCM), and supernatants were collected to check for mycoplasma status. And replacing the culture medium into a growth culture medium after bottle separation, and separating bottles at a ratio of 1:3 when the growth is 90% converged.

5. Freezing and storing

And when the seeds grow to the sufficient quantity required by the subsequent experiment and the reserved seeds, the frozen stock solution is frozen and stored by liquid nitrogen.

The components of the frozen stock solution are as follows: serum solutions containing 10% DMSO.

6. Resuscitation

Taking out the freezing tube containing cells from liquid nitrogen, rapidly rewarming in 37 deg.C water bath, wiping the freezing tube with 75% ethanol, transferring the cell suspension into 15mL centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, suspending the cells with 10% serum-containing complete culture medium, adjusting ideal cell concentration, 37 deg.C, 5% CO₂Culturing in an incubator.

Example 7: identification of cell lines

The following methods were performed using the cell lines constructed in examples 4 and 5 of the present invention, and the daughter cells of the cell lines constructed in examples 4 and 5.

Characterization of cellular phenotypes by FCM

1) Washing cells to be detected by using PBS buffer solution, and collecting the cells by using pancreatin digestion;

2) centrifuging at 1000rpm for 5min, and discarding the supernatant;

3) fixing with 4% paraformaldehyde solution for 15min, centrifuging, and removing supernatant;

4) washing with PBS buffer solution for 2 times;

5) punching in 0.3% TritonX-100 PBS for 15mins at room temperature;

6) washing with PBS buffer solution for 2 times;

7) cell counting, per tube flow tube 10⁵(ii) individual cells;

8) FITC-CK7 is added into the experimental group 1:500, FITC-CDX-2 is added into the homotypic antibody control group 1:500, and incubation and dyeing are carried out for 30min at room temperature in a dark place;

9) washing with PBS buffer solution for 1 time;

10) the results were obtained by on-line detection on a BD FACSCAnto II flow cytometer and analyzed by FlowJo 7.6 software.

The results are shown in fig. 2, and indicate that successfully cultured W038 cells FCM in the present invention proved to be purified epithelial cancer cells, and 100% of the cells expressed the epithelial ovarian tumor marker CK 7.

2. Immunohistochemical identification of cell phenotype

In the examination of the conventional direct smear method for pleuroperitoneal fluid, the detection rate of cancer cells is low, diagnosis is easy to leak, and the paraffin-embedded section method for the pleural and peritoneal effusion sediment is adopted, so that the number of collected cells is increased, the section quality is high, meanwhile, the cells can be repeatedly cut as required, and the tumor can be marked by combining immunohistochemistry, thereby improving the detection rate of the pleural and peritoneal effusion tumor cells.

Centrifuging with 52A type medical low-speed centrifuge with a centrifugation radius of 40cm for 15min at a centrifugation speed of 2500r/min for 300mL of ascites, discarding the supernatant, filtering and wrapping the precipitate with test paper, and fixing Caroy liquid for 2h, wherein if ascites tumor cells and fibrin are abundant, the ascites tumor cells and fibrin are coagulated into blocks and can be directly wrapped and fixed. Dehydrating the treated precipitate in gradient ethanol, removing water from xylene, soaking in low-melting-point paraffin wax at 54 deg.C for 2 times (each for 1 hr), embedding in paraffin wax, continuously slicing with 4-6um, cutting into 10 sections, mounting the slices on several glass slides, HE staining, and observing with optical lens. Immunohistochemistry, cytokeratin CK, etc., immunohistochemical diagnostic criteria: the positive cells are those with brownish yellow particles in cytoplasm and/or cell membrane.

The results of the organization are shown in FIG. 3: PAX-8(+), WT-1(-), ER (-), PR (-), CDX-2(-), CK7(+), P53 (90% +).

STR identification

Comparing the cells with other tumor cells by gene fingerprint technology, the cell line is confirmed to be human, come from the resources of the patient and not be polluted by other cell lines in the culture room.

1) Material detection processing and detecting method

DNA is extracted from a proper amount of test material by using Chelex100, a MicroreaderTM21 ID System STR composite amplification kit is adopted for amplification, and STR locus and sex gene Amelogenin are detected on an ABI 3130xl type genetic analyzer. Genotyping of each STR site was performed using Gene Mapper v3.2 software (ABI). The cell has clear DNA amplified spectrum and good parting result.

2) Test results

The positive and negative control results are correct in the experiment.

The results of genotyping the STR and Amelogenin loci of this cell line are shown in Table 1.

3) Test conclusion

① the STR typing of the cell DNA shows that there is no three-allele phenomenon at each locus, no human cell cross contamination is found in the cell ② the cell DNA typing does not find 100% match of the cell typing in ATCC and DSMZ cell banks.

TABLE 1 genotyping results for STR and Amelogenin sites of W038 cells

4) Mycoplasma detection

Mu.l of the cell pellet was subjected to nested PCR amplification using the Mycoplasma Detection Set amplification kit (M & C Gene technology), and the band of interest was detected by 2.0% agarose gel electrophoresis, and the fragment size of the PCR product was determined for the Mycoplasma species shown in Table 2. In the experiment, the negative control (NTC) has no band, and the positive control (P) has a band, so that the result of the experiment is accurate and reliable. No mycoplasma was detected in sample W038.

TABLE 2 Mycoplasma species corresponding to fragment size of PCR product

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A primary tumor cell screening medium is characterized by comprising the following components in percentage by volume: 5 to 20 percent of acellular ascites, 3 to 15 percent of fetal calf serum and the balance of a mammalian cell culture medium.

2. The primary tumor cell screening medium of claim 1, further comprising an antibiotic.

3. The primary tumor cell screening medium of claim 2, consisting of the following components in volume percent: 15% of acellular ascites, 5% of fetal calf serum, 1% of double antibody and the balance of DMEM medium.

4. Use of a primary tumor cell screening medium according to any one of claims 1 to 3 for constructing or culturing tumor cell lines.

5. Use of the primary tumor cell screening medium of any one of claims 1-3 for constructing ovarian cancer tumor cell lines.

6. Use of a primary tumor cell screening medium according to any of claims 1 to 3 for the removal of stromal cell contamination during cell culture.

7. A method for constructing tumor cell line is characterized in that ascites and/or pleural fluid of cancer patients are taken, red blood cells are removed, the rest cells are continuously cultured in a complete culture medium, when the confluency is not less than 70 percent, the complete culture medium is replaced by a screening culture medium, and the cells are cultured until all epithelial tumor cells are obtained;

wherein the screening culture medium is the primary tumor cell screening culture medium of any one of claims 1 to 3.

8. The method according to claim 7, wherein the cancer patient is a cancer patient producing ascites and/or pleural fluid, preferably an ovarian cancer patient.

9. The method of claim 7, wherein after removing the red blood cells, the cells are cultured in a complete medium until the mesenchymal cells in the cell suspension adhere, and then the epithelial cell clot is blown up and transferred to another culture flask for further culture.

10. The method according to claim 9, further comprising the following steps after the complete medium is replaced with the selection medium: removing the screening culture medium, washing the cell precipitate, adding cell digestive juice, tapping and beating the culture bottle to separate the interstitial cells when the interstitial cells shrink and become round but the epithelial cells do not shrink, removing the interstitial cells, and continuously culturing in the screening culture medium until all the interstitial cells are epithelial tumor cells.

11. A tumor cell line, wherein the tumor cell line is prepared by the method according to any one of claims 7 to 10, and the tumor cell line is preferably an ovarian cancer cell line.