CN114015655A

CN114015655A - HBRCA-959 cell line and culture method and application thereof

Info

Publication number: CN114015655A
Application number: CN202111513775.5A
Authority: CN
Inventors: 贾永娟; 周坤; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2022-02-08
Anticipated expiration: 2041-12-09
Also published as: CN114015655B

Abstract

The invention relates to a cell HBRCA-959 and a culture method and application thereof. The primary human breast cancer cell is named as human breast cancer cell HBRCA-959 and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 23022.

Description

HBRCA-959 cell line and culture method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to an HBRCA-959 cell line.

Background

The breast cancer is a phenomenon that mammary epithelial cells generate uncontrolled proliferation under the action of various carcinogenic factors. The early stage of the disease often shows symptoms of breast lumps, nipple discharge, axillary lymphadenectasis and the like, and the later stage of the disease can generate distant metastasis due to cancer cells to generate multi-organ lesion, thereby directly threatening the life of a patient.

Since breast cancer seriously harms human health, in order to research pathogenesis and treatment method thereof, commercial cell lines are used as materials. However, after long-term culture, the biological characteristics and genetics of the cells are easily changed, which is not favorable for the research of pathogenesis and treatment method.

However, in the prior art, the research and inhibition of cancer mechanism mostly adopt identification of related molecular markers of immune system or universal cancer markers, such as the composition for detecting breast cancer stem cells provided in chinese patent with publication number CN112898428A, which includes a first antibody nucleic acid conjugate specifically binding to cell surface CD44 protein, a second antibody nucleic acid conjugate specifically binding to cell surface CD24 protein, a DNA single-chain T chain and a DNA single-chain F chain, wherein the T chain is complementarily bound to the nucleic acid chain of the first antibody nucleic acid conjugate, and the F chain is complementarily bound to the nucleic acid chain of the second antibody nucleic acid conjugate, which together form a programmed DNA loop, erase the signal marker on the surface of the breast cancer cell of CD44 positive CD24 positive phenotype, and retain the signal probe labeled to the breast cancer stem cells. CD24 protein is a mucin-like adhesion molecule, and a large number of researches show that CD24 can be seen in tumors, inflammations and autoimmune diseases and plays different roles in different diseases, so that the detection of the markers of CD24 and CD44 used in the patents has high possibility of having non-specific disease detection errors, and causing misleading problems in secondary diagnosis such as tissue section and the like.

The primary tumor cell culture refers to a process of obtaining tumor cells by surgical excision of tumor tissues from a tumor patient body and performing short-term culture in vitro. The primary cultured tumor cells have the biological characteristics which are not changed greatly because the tissues are just isolated, still retain the original genetic characteristics, have the characteristics similar to those of the tumor cells in vivo, are suitable for the research of related experiments on biochemical molecules, drug sensitivity and mechanisms, and have more convincing data. Therefore, the culture of primary cells of human breast cancer is particularly necessary.

The invention separates a human breast cancer primary cell from the disease tissue of a clinical patient and obtains a specific culture method thereof, thereby providing a specific model for the subsequent breast cancer diagnosis and pharmacological research.

Furthermore, on the one hand, due to the differences in understanding to the person skilled in the art; on the other hand, since the applicant has studied a great deal of literature and patents when making the present invention, but the disclosure is not limited thereto and the details and contents thereof are not listed in detail, it is by no means the present invention has these prior art features, but the present invention has all the features of the prior art, and the applicant reserves the right to increase the related prior art in the background.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a cell HBRCA-959 which is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 23022. A primary human breast cancer cell isolated from a clinical lesion tissue is named as a human breast cancer cell HBRCA-959, and is deposited in China General Microbiological Culture Collection Center (CGMCC) at 24 days 08 and 24 months 2021, wherein the deposition address is No. 3 of the national institute of microbiology, Ministry of sciences, North China, national institute of microbiology, the postal code is 100101, and the deposition number is CGMCC NO. 23022.

The invention provides a culture medium. The culture medium is a serum-free culture medium for culturing primary cells of human breast cancer. The culture medium comprises: an additive, an antibiotic that inhibits the growth of microorganisms, and a DF medium, wherein the additive comprises a cell growth inducer, a cell growth nutrient, and an ATP competitive inhibitor.

According to a preferred embodiment, the medium for culturing primary cells of human breast cancer comprises: additives, antibiotics and DF media. Wherein the additives comprise glutamine, insulin, transferrin, sodium selenite, epidermal growth factor, bovine serum albumin BSA, Y-27632 and Wnt3 a. The antibiotics include penicillin, kanamycin sulfate, amphotericin B, cefmetazole, vancomycin and other antibiotics for inhibiting the growth of microorganisms.

In detail, Y-27632 is an ATP-competitive ROCK-I and ROCK-II inhibitor.

Preferably, the medium for culturing human breast cancer primary cells comprises: 1-10mM glutamine, 3-50mg/L insulin, 3-30mg/L transferrin, 5-50 μ g/L sodium selenite, 5-50 μ g/L LEGF, 1-5mg/mL BSA, 5-20 μ M Y-27632, 50-500 ng/mL Wnt3a, 0-2000 μ g/mL penicillin, 0-3000 μ g/mL kanamycin sulfate, 0-10 μ g/mL amphotericin B, 0-500 μ g/mL cefmetazole, and 0-50 μ g/mL vancomycin.

According to a preferred embodiment, since the cell HBRCA-959 is derived from human breast cancer tissues and has morphological characteristics and physiological and biochemical properties of breast cancer, the cell HBRCA-959 can be applied to the development of a drug for inhibiting or treating breast cancer.

A method of culturing cells comprising the steps of:

providing a cell HBRCA-959;

the cells were placed in the above-mentioned medium, and HBRCA-959 cells were cultured under conditions allowing the cells to proliferate.

According to a preferred embodiment, a method of culturing primary cells of human breast cancer comprises:

s1: preserving the collected fresh human breast cancer tissue with a sample preservation solution;

the sample preservation solution comprises fetal calf serum, antibiotics and a DF culture medium, wherein the antibiotics comprise penicillin, kanamycin sulfate and amphotericin B;

s2: washing the breast cancer tissue preserved in S1 with a washing solution;

wherein the cleaning solution comprises physiological saline or PBS buffer solution containing antibiotics;

s3: taking the breast cancer tissue cleaned and treated by S2, and carrying out primary isolated culture by using a serum-containing culture medium;

wherein the serum-containing culture medium comprises fetal calf serum, antibiotics and DF culture medium;

s4: and (3) taking the cells obtained by S3 primary separation culture, and purifying and subculturing the cells by using any one of the provided serum-free culture media to obtain the primary human breast cancer cells.

In detail, the DF medium is DME/F-121: 1 medium (HyClone, USA) which is common in the art.

Preferably, the serum-containing medium comprises: penicillin 50-2000. mu.g/mL, kanamycin sulfate 20-3000. mu.g/mL, amphotericin B0.5-10. mu.g/mL.

Preferably, the cleaning liquid comprises: penicillin 0.1-1mg/mL, kanamycin sulfate 0.1-1mg/mL, amphotericin B1-10 μ g/mL.

Modern research suggests that breast cancer is not a single disease but rather a heterogeneous collection of diseases characterized by a diversity of histologies, genomic alterations, gene expression, metastatic behavior, and therapeutic responses. For a model constructed by the human breast cancer primary cells, the positive influence of the human breast cancer primary cell secretory protein on diseases also has value on the mechanism research of breast cancer. Similarly, the use of the cells in protein production can provide a basis for the subsequent protein marker and protein level targeted drug research of breast cancer. Thus, the use of human breast cancer primary cells, or the HBRCA-959 cell line, for the production of proteins is also worth exploring.

Similarly, in the field of molecular medicine, the screening of biomarkers and biomarker compositions is an effective means for early diagnosis of diseases. The cell HBRCA-959 is separated from human breast cancer tissues and has specificity on breast cancer identification. Based on this, the present invention designs a marker. Markers include cell HBRCA-959, used for screening for breast cancer.

Further, the present invention designs a kit comprising a cell HBRCA-959, an expression vector for expressing a gene contained in the cell and a cell culture medium for culturing the cell. The screening is carried out by means of RT-PCR or cell culture, and the cells are further taken as targets for selecting a range, thereby providing the possibility of multi-target drug screening for patients.

The invention provides a human breast cancer primary cell or cell HBRCA-959, a human breast cancer primary cell or cell HBRCA-959 obtained by culturing a serum-free culture medium for culturing the human breast cancer primary cell or cell HBRCA-959, or a human breast cancer primary cell or cell HBRCA-959 obtained by culturing by using the culture method, and application of the human breast cancer primary cell or cell HBRCA-959 in drug sensitivity research and detection.

The invention provides an application of a human breast cancer primary cell or cell HBRCA-959, a human breast cancer primary cell or cell HBRCA-959 obtained by culturing a serum-free culture medium for culturing the human breast cancer primary cell or cell HBRCA-959, or a human breast cancer primary cell or cell HBRCA-959 obtained by culturing by using the culture method in the construction of a breast cancer animal model.

The invention provides an application of a human breast cancer primary cell or cell HBRCA-959, a human breast cancer primary cell or cell HBRCA-959 obtained by culturing a serum-free culture medium for culturing the human breast cancer primary cell or cell HBRCA-959, or a human breast cancer primary cell or cell HBRCA-959 obtained by culturing by using the culture method in the development of a medicament for inhibiting breast cancer.

The invention provides a human breast cancer primary cell or cell HBRCA-959, a human breast cancer primary cell or cell HBRCA-959 obtained by culturing a serum-free culture medium for culturing the human breast cancer primary cell or cell HBRCA-959, or a human breast cancer primary cell or cell HBRCA-959 obtained by culturing by the culture method, and application of the human breast cancer primary cell or cell HBRCA-959 in-vitro research of breast cancer.

It should be noted that the application of the human breast cancer primary cells provided by the invention is for the purpose of diagnosis and treatment of non-diseases.

The human breast cancer cell HBRCA-959 provided by the invention at least has the following characteristics:

the method is characterized in that: the primary separation culture is from the breast cancer tissue of Chinese, and the cell is not modified by any gene engineering and gene modification.

And (2) feature: the human breast cancer cell HBRCA-959 observed under a microscope is in an epithelial cell form, has high tumor cell proliferation speed, unequal cell sizes and forms, is irregular or polygonal, is disorganized in arrangement, has no contact inhibition, has less clear cell boundary, and has the morphological characteristics of the breast cancer cell.

And (3) feature: the multiplication time of a cell population of the human breast cancer cell HBRCA-959 is 130 hours, the cell proliferation speed is high, the cell growth is active, the cell activity state is good, the cell culture stability is high, and the in vitro culture has stable cell growth characteristics.

And (4) feature: human breast cancer cells HBRCA-959 were immunofluorescently identified as strongly expressed tumor cell markers CK18 (cytokeratin 18) and CK8 (cytokeratin 8) of epithelial origin.

And (5) feature: the human breast cancer cell HBRCA-959 is identified as the abnormal karyotype of the tumor cell through karyotype analysis, the chromosome structure and the number of the cell are abnormal, the chromosome number of the cell is 52, the cell belongs to the hyperploid karyotype, and the genetic characteristic of the human malignant tumor is met.

And (6) feature: the human breast cancer cell HBRCA-959 is identified by STR (Short Tandem Repeat) genotyping, is a cell which is a single human source and has no cross contamination, and is a newly obtained breast cancer primary cell.

And (7) feature: the human breast cancer cell HBRCA-959 can be used for drug sensitivity research and detection, breast cancer animal model construction, breast cancer inhibition drug development and breast cancer in-vitro research.

Drawings

FIG. 1 is a cell morphology of human breast cancer cell HBRCA-959 provided by an embodiment of the present invention;

FIG. 2 is a graph showing the growth of HBRCA-959 cells of human breast cancer according to an embodiment of the present invention;

FIG. 3 is a diagram showing the result of immunofluorescence identification of human breast cancer cell HBRCA-959 according to an embodiment of the present invention;

FIG. 4 is a chromosome karyotype analysis chart of human breast cancer cells HBRCA-959 provided by an embodiment of the present invention;

FIG. 5 is a STR genotyping graph of human breast cancer cell HBRCA-959 provided in one embodiment of the present invention.

Detailed Description

The following detailed description is made with reference to the accompanying drawings.

It should be noted that due to the specificity of breast cancer tissues, the lack of effective available breast cancer specimens, the technical difficulty in establishing breast cancer primary cell cultures and other factors, the existing in vitro breast cancer research model is seriously lacked, thereby greatly limiting the research on the cytology level of breast cancer. Based on the above, the embodiment of the invention provides a human breast cancer primary cell, a culture method and an application thereof, and can provide good experimental materials for relevant researches such as a molecular generation mechanism and a clinical treatment approach of breast cancer. The following is a description of the human breast cancer primary cells, and a culture method and application thereof, respectively.

Example 1

The embodiment of the invention provides a primary human breast cancer cell which is named as a human breast cancer cell HBRCA-959 and is preserved in the China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC NO. 23022.

The human breast cancer cell HBRCA-959 is primarily separated and cultured from the breast cancer tissue of Chinese people, and the cell is not modified and modified by any gene engineering.

Next, the biological properties of HBRCA-959, which is a human breast cancer cell, will be described.

1.1 morphological Observation of cells

The human breast cancer cell HBRCA-959 is observed under an inverted microscope, is in an epithelial cell form, has high tumor cell proliferation speed, unequal cell sizes and forms, is irregular or polygonal, is disorganized in arrangement, has no contact inhibition, has less clear cell boundary, and has the morphological characteristics of breast cancer cells. The morphological observation results are shown in fig. 1.

1.2 measurement of growth Curve and doubling time

1.2.1 growth Curve determination procedure

(1) When the confluence of human breast cancer cells HBRCA-959 reached 70-90%, the serum-free medium was removed, the cells were washed at least twice with PBS (0.01M, pH7.4) to remove old serum-free medium and cells with poor float, digested with 1-2mL of EDTA-Trypsin (Trypsin-EDTA digest), and the digestion was stopped with DF10 medium (DF medium and 10% FBS).

Wherein, EDTA (ethylene Diamine Tetraacetic acid) is ethylenediamine Tetraacetic acid, and Trypsin is Trypsin.

(2) The cells were cultured at 2.5X 10⁵Perml were plated into 36 wells of 12 48-well plates, and 500. mu.l of cell suspension was added per well.

(3) Discarding the culture medium in 3 holes every 24 hours from the inoculation time, adding EDTA-Trypsin for digestion treatment, suspending the cells, counting the average value of the cell density in 3 holes for 3 times per hole, taking the average value, and continuously culturing the cells in the rest holes until the 12 th day; the medium was changed every 2 days.

(4) The measurement was continued for 12 days, and the growth curve data shown in the following Table 1 were obtained.

1.2.2 growth Curve measurements

Table 1 shows the growth curve data obtained for 12 consecutive days of the assay.

TABLE 1

Cultivation time (sky)	Mean cell density
		1	11.3×10⁴Per mL
2	11.73×10⁴Per mL
		3	13.29×10⁴Per mL
4	17.01×10⁴Per mL
		5	19.02×10⁴Per mL
6	20.05×10⁴Per mL
		7	21.85×10⁴Per mL
8	23.10×10⁴Per mL
		9	26.53×10⁴ Per mL
10	30.20×10⁴ Per mL
		11	34.80×10⁴ Per mL
12	41.87×10⁴Per mL

Based on the cell growth curve data in table 1, a schematic diagram of the cell growth curve as shown in fig. 2 was obtained. In FIG. 2, the abscissa represents the incubation time (days), and the ordinate represents the number of cells (. times.10)⁴one/mL).

Referring to the growth curve data in table 1 and the schematic diagram of the growth curve shown in fig. 2, it can be seen that human breast cancer cells HBRCA-959 grow well for 12 consecutive days, with a 10-day logarithmic growth phase.

1.2.3 cell population doubling time

The population doubling time of tumor cells was calculated as: DT × [ lg2/(lgNt-lgNo) ]. Where DT is doubling time, t is time interval, No is initial cell number, and Nt is terminal cell number.

The cell population doubling time of the human breast cancer cell HBRCA-959 was calculated to be-130 h.

In conclusion, the human breast cancer cell HBRCA-959 has the advantages of high cell proliferation speed, active cell growth, good cell activity state, high cell culture stability and stable cell growth characteristic in vitro culture.

1.3 immunofluorescence identification

1.3.1 immunofluorescence identification procedure

(1) The human breast cancer cell HBRCA-959 confluence degree reaches about 70-90%, a serum-free culture medium is removed, cells are washed at least twice by PBS (0.01M, PH7.4) to remove old serum-free culture medium and floating cells with poor state, and the cells are digested by 1-2mL EDTA-Trypsin and inoculated to a 48-pore plate coated with collagen;

(2) when the confluence degree of the cells reaches 70-90%, removing the serum-free culture medium, washing with PBS for 3 times, adding 500 mu L/hole of glacial methanol, and removing after fixing for 30 min;

(3) adding 300 μ L/hole 2.5% BSA blocking solution, blocking at room temperature for 60min, and sucking out the blocking solution;

(4) add 300. mu.L/well of primary antibody (Keratin 18Mouse mAb, 1:800 dilution; Keratin 8Rabbit mAb 1:200 dilution), incubate overnight at 4 ℃ and aspirate;

(5) adding 300 mu L/hole of fluorescent secondary antibody (Anti-Mouse IgG (second antibody) -FITC antibody produced in rabbitt, diluted 1: 100; Cy3 labeled goat Anti-rabbit IgG, diluted 1: 500), incubating for 1h at room temperature in the dark, sucking out the secondary antibody, and washing for 3 times with PBS;

(6) adding 2 drops of DAPI stock solution (ab104139) into each well, and dyeing for 5min at room temperature in a dark place;

(7) the DAPI was aspirated and washed 3 times with PBS;

(8) add 300. mu.L/well PBS, take pictures by fluorescence, objective lens Xocular lens (10X 10), and obtain the immunofluorescence identification results.

1.3.2 immunofluorescence identification results

CytoKeratin (CK) is a common marker for epithelial cells, and CK is expressed in epithelial cells and tumor cells of epithelial origin.

The immunofluorescence assay results are shown in FIG. 3. Referring to FIG. 3, it was found that DAPI stained the tumor cell nucleus and that the human breast cancer cells HBRCA-959CK18 and CK8 were strongly expressed, indicating that the human breast cancer cells HBRCA-959 are derived from epithelial cells.

1.4 karyotyping

1.4.1 karyotype analysis and identification procedure

(1) Cell culture

When the confluency of the human breast cancer cells HBRCA-959 reaches 70-90%, removing the serum-free medium culture, washing the cells with PBS (0.01M, PH7.4) at least twice to remove the old serum-free medium and the cells with poor shedding state, digesting with 1-2mL EDTA-Trypsin, observing under a microscope, collecting the digested cells while digesting, and terminating the digestion of the collected cells with DF10 medium until all the cells are digested. Centrifuge at 1000rpm for 5min and remove supernatant.

(2) Colchicine treatment

Adding colchicine 20-50 μ L with concentration of 300 μ g/mL into the cell precipitate, and treating in 37 deg.C incubator for 25 min.

(3) Hypotonic treatment

After the colchicine treatment was completed, the mixture was centrifuged (1300rpm, 10min) and the supernatant was discarded. Then adding 0.075mol/L KCl solution in 37 ℃ water bath to obtain a hypotonic solution of 10mL, blowing the hypotonic solution into a cell suspension by using a suction pipe, and placing the cell suspension in 37 ℃ water bath for treatment for 30-60 min.

(4) Pre-fixing

After the hypotonic treatment, 1mL of fixative was added to each tube and the water bath was continued at 37 ℃ for 5 min.

(5) Centrifugation

Centrifuge at 1300rpm for 10min, and discard the supernatant.

(6) Fixing

Adding 6-8mL of fixative into a centrifuge tube, immediately gently blowing and beating into single cell suspension by using a pipette, fixing in a water bath at 37 ℃ for 30min, centrifuging at 1300rpm for 10min, and removing supernatant.

(7) Second fixing

Adding 6-8mL of fixative into a centrifuge tube, immediately gently blowing and beating into single cell suspension by using a pipette, fixing in a water bath at 37 ℃ for 15min, centrifuging at 1300rpm for 10min, and removing supernatant.

(8) Tabletting

Adding about 0.2mL of new stationary liquid into a centrifuge tube, slightly blowing and beating the new stationary liquid into cell suspension by using a suction tube, taking out borneol from a freezing chamber of a refrigerator, adjusting the borneol to be proper suspension, dripping 1-2 drops of suspension into each slice, and baking the slices for 3 hours at 75 ℃.

(9) Dyeing process

Staining with 6% Giemsa dye for 10min, then clamping the slide out with forceps, washing the two sides gently with tap water, drying at room temperature, and performing microscopic examination.

(10) Microscopic examination

After the slides were dried, they were examined under a microscope. And (3) firstly, searching for a good split phase by using a low power microscope, and then observing by using a high power oil microscope to obtain a chromosome karyotype analysis and identification result.

1.4.2 karyotype analysis and identification results

The results of karyotyping are shown in FIG. 4. Referring to fig. 4, the human breast cancer cell HBRCA-959 was identified as a tumor cell allotype by karyotyping analysis, the chromosome structure and number of the cell were abnormal, the number of chromosomes of the cell was 52, and the cell belongs to the hyperploid karyotype, and meets the genetic characteristics of the human malignant tumor.

1.5STR identification

1.5.1STR identification procedure

(1) Human breast cancer cells grown adherently HBRCA-959(1 × 10)⁶One) the cells were washed twice with PBS (0.01M, pH7.4), digested sequentially with 1-2mL EDTA-Trypsin for 3-30min, and 10mL DF10 medium was used to stop the digestion reaction.

(2) Centrifugation was carried out at 10000rpm for 1min, the supernatant was discarded, and 200. mu.L of buffer GA (cell/tissue genomic DNA extraction kit DP304, Tiangen Co.) was added thereto and shaken to thoroughly suspend the mixture.

(3) Add 20. mu.L of protease K solution and mix well.

(4) 200. mu.L of buffer GB (cell/tissue genomic DNA extraction kit DP304, Tiangen Co.) was added, mixed well by inversion, left at 70 ℃ for 10min, and centrifuged briefly.

(5) Adding 200 μ L of anhydrous ethanol, shaking thoroughly, mixing for 15s, and centrifuging briefly.

(6) The resulting solution and flocculent precipitate were both added to an adsorption column (cell/tissue genomic DNA extraction kit DP304, Tiangen Co., Ltd.), centrifuged at 12000rpm for 30s, and the waste liquid was removed.

(7) To the adsorption column was added 500. mu.L of buffer GD (cell/tissue genomic DNA extraction kit DP304, Tiangen Co., Ltd.), centrifuged at 12000rpm for 30s, and the waste liquid was removed.

(8) To the adsorption column, 600. mu.L of a rinsing solution PW (cell/tissue genomic DNA extraction kit DP304, Tiangen Co., Ltd.) was added, centrifuged at 12000rpm for 30 seconds, and the waste liquid was removed.

(9) Transferring the adsorption column into another centrifuge tube, adding 50-200 μ L of elution buffer TE (cell/tissue genome DNA extraction kit DP304, Tiangen corporation) dropwise into the middle part of the adsorption membrane, standing at room temperature for 2-5min, centrifuging at 12000rpm for 2min, and collecting the extracted DNA solution into the centrifuge tube.

(10) The amplification is carried out by adopting a golden eye TM20A STR multiplex amplification kit, and 21 known STR loci and sex genes Amelogenin are continuously detected and analyzed on an ABI3100 type genetic analyzer.

(11) And analyzing the sample data by using GeneMapper3.2 software, and carrying out automatic genotyping to obtain an STR typing result.

1.5.2STR identification results

The STR typing results are shown in fig. 5. Referring to fig. 5, 21 loci were examined, expressed as "locus/allele length": D19S433/13.2/14/14.2, D5S818/11/13, D21S11/29/30, D18S51/13/20, D6S1043/18/20, AMEL/X/X, D3S1358/15/15, D13S317/10/11, D7S820/10/11, D16S539/13/13, CSFIPO/10/11, PentaD/9/12, D2S441/10/10, vWA/14/18, D8S1179/10/13, TPOX/11/11, PentaE/12/18, TH01/7/7, D12S391/23/24, D2S1338/23/23, FGA/23/24.

From the above, the STR genotyping identification can lead to the following conclusions:

(1) the HBRCA-959 of the human breast cancer cell only has a three-allele phenomenon at a D19S433 gene locus, and no human cell cross contamination is found in the cell.

(2) Comparing the STR data of the human breast cancer cell HBRCA-959 with those of STR databases of ATCC (american type culture collection center) and DSMZ (national culture collection center in germany), it was found that no cell matching 100% of the cell typing of the human breast cancer cell HBRCA-959 was found in the ATCC and DSMZ cell banks.

(3) Comparing the STR data of human breast cancer cell HBRCA-959 with those in STR databases of ATCC and DSMZ, it can be seen that the STR data match rate of the HROC278Met T2M 2(CVCL _1U90) cell is relatively highest, namely 75%.

In conclusion, the STR genotyping identification of the human breast cancer cell HBRCA-959 proves that the cell is a single human source and has no cross contamination, and is a newly obtained breast cancer primary cell.

1.6 cell uses

1.6.1 application in drug sensitivity research and detection

1.6.1.1 drug susceptibility testing procedure (CD-DST method)

(1) Collagen gel drop culture

The human breast cancer cell HBRCA-959 and the gel composition (I: II: III: 8:1:1) are mixed uniformly, the cell suspension is inoculated into a 6-well plate according to the ratio of 30 mu L/gel drop and 3 gel drops/well, and simultaneously, 3 gel drops are inoculated into another 6-well plate to be used as a 0-time control group. Adding 3mL DF culture medium containing 10% fetal calf serum after the gel drops are solidified after 1-2h, and treating with 5% CO at 37 deg.C₂The culture was carried out overnight in an incubator.

(2) Anti-cancer drug exposure and cleaning

And (2) adding an anti-cancer drug after culturing the primary human breast cancer cells obtained in the step (1) for 24 hours, setting a negative Control hole (Control) without the drug and a positive Control hole treated by a clinical breast cancer common drug (note that the drug can be generally selected according to the main clinical manifestations of a cell source patient), and dyeing and fixing the 0-time group. After the corresponding time of culture, the cells were washed 2 times with DF medium for 15min each, and then cultured for 5 additional days with serum-free medium, wherein the medium was changed once on day 3.

(3) Dye fixation and scanning

And (3) performing staining fixation on the 8 th day, staining the cells for 2h by neutral red with the final concentration of 50 mug/mL, washing the cells for 3 times and 5min each time by PBS, fixing the cells for 45min by neutral formalin, washing the cells for 15min by distilled water, and performing ventilation drying to obtain the tumor cells with activity. And then scanning and analyzing the glue drops by using a Primage image analysis system to obtain a drug sensitive detection result.

1.6.1.2 result of drug sensitivity detection

The results of drug sensitivity detection (CD-DST method) of HBRCA-959 in human breast cancer cells are shown in Table 2 below.

TABLE 2

Name of drug	Cell survival rate (%)
		Carboplatin + paclitaxel (CBDCA + PAC)	94.75
Cisplatin + Noveba (CDDP + VNR)	74.21
		Capecitabine + docetaxel (CAP + DOC)	88.26
Epirubicin + docetaxel (EPI + DOC)	95.38
		Cisplatin + Gemcitabine (CDDP + GEM)	90.43

Wherein, if the cell survival rate is less than 50%, it can indicate that the individual tumor cell has higher sensitivity (i.e. high sensitivity) to the corresponding drug; if the cell survival rate is 50-60%, it can indicate that the sensitivity of individual tumor cells to the corresponding drug is in the critical range of high sensitivity and low sensitivity (i.e. critical); if the cell survival rate is greater than 60%, it can indicate that the individual tumor cells have low sensitivity (i.e., drug resistance) to the corresponding drug.

Referring to table 2, it can be seen that human breast cancer cell HBRCA-959 is low-sensitive to five medication regimens of carboplatin + paclitaxel (CBDCA + PAC), cisplatin + navelbine (CDDP + VNR), capecitabine + docetaxel (CAP + DOC), epirubicin + docetaxel (EPI + DOC), cisplatin + gemcitabine (CDDP + GEM), and the like, and thus can be used for effectiveness evaluation of the 5-combination clinical breast cancer common medication. Through the drug sensitivity detection experiment, clinical medication guidance can be more pertinently carried out on related breast cancer patients (such as breast cancer patients of human breast cancer cells HBRCA-959, other breast cancer patients with the same or similar clinical manifestations or drug sensitivity characteristics or breast cancer metastasis lesion patients and the like), and the drug sensitivity detection method has important significance on personalized treatment of the breast cancer patients.

1.6.2 use in constructing animal models of Breast cancer

Human breast cancer cells in logarithmic growth phase, HBRCA-959, were inoculated into mammals (e.g., nude mice) at a prescribed dose and cultured for several days to construct an animal model of breast cancer. The breast cancer animal model can be used for development research of breast cancer, research of transfer mechanism, curative effect evaluation of radiotherapy and chemotherapy drug clinical treatment and the like.

1.6.3 application in developing medicine for inhibiting breast cancer

The human breast cancer cell HBRCA-959 is cultured from breast cancer tissue which is just separated from the body, has small change of biological characteristics, retains the original genetic characteristics, and can be applied to screening or evaluating drugs for treating breast cancer.

Different chemotherapeutic drugs are added into a culture medium of HBRCA-959 of human breast cancer cells, and the changes of cell growth, proliferation, death, period and the like are observed to obtain a primary effective candidate drug. And then the candidate drug is used in a breast cancer animal model, the in vivo effect of the drug is detected, the general condition, survival time and tumor size change of the animal and the change condition of the animal body after the drug effect are observed, and therefore the curative effect evaluation and the mechanism analysis are carried out on the candidate drug.

1.6.4 used for in vitro study of breast cancer

The human breast cancer cell HBRCA-959 is cultured from breast cancer tissue which is just separated, has small biological characteristic change, reserves the original genetic characteristic, can be used as a cell material for in vitro research of human breast cancer pathogenesis and individualized treatment, and further provides a foundation for establishment of a human breast cancer pathogenesis model and preparation, screening, evaluation and the like of anti-human breast cancer drugs.

In summary, the human breast cancer cell HBRCA-959 provided by the embodiments of the present invention has at least the following characteristics:

Example 2

This embodiment is a further improvement of embodiment 1, and repeated contents are not described again.

This example illustrates a method for culturing primary human breast cancer cells, comprising the following steps 1-4. The human breast cancer cell provided in example 1, HBRCA-959, was cultured using this culture method.

Step 1, preserving the collected fresh human breast cancer tissue by using a sample preservation solution. Wherein, the sample preservation solution comprises fetal bovine serum, antibiotics and DF culture medium.

And 2, cleaning the breast cancer tissues preserved in the step 1 by using a cleaning solution. Wherein the cleaning solution comprises physiological saline containing the antibiotic.

And 3, taking the breast cancer tissue cleaned and treated in the step 2, and performing primary isolated culture by using a serum-containing culture medium. Wherein the serum-containing medium comprises fetal bovine serum, the antibiotic and DF medium.

Specifically, step 3 may include the following step 3.1) -step 3.3).

And 3.1) treating the cleaned breast cancer tissue by using cell dispersing enzyme.

Specifically, step 3.1) may include the following step 3.1.1) -step 3.1.4).

Step 3.1.1), chopping the cleaned breast cancer tissue into a minced shape.

Wherein, the breast cancer is chopped into a paste shape, which is convenient for the breast cancer tissue to digest into tumor cells with proper size.

Step 3.1.2), the smashed breast cancer tissues are centrifuged for 5-10min at 800-.

Wherein, the supernatant is removed by centrifugation, and impurities such as adipose tissue, mucosal tissue, connective tissue, and necrotic tissue in breast cancer tissue can be removed.

Step 3.1.3), adding cell dispersing enzyme in sequence, placing at 37 deg.C and 5% CO₂Digesting in the incubator at low speed under oscillation for 20min-2h to disperse breast cancer tissues and prevent cell agglomeration, adding DF10 culture medium to stop reaction, and blowing to disperse and mix uniformly.

Wherein, the cell dispersing enzyme can be: contains 1-10mg/ml collagenase I and 0.2-2mg/ml hyaluronidase.

Step 3.1.4), centrifuging for 5-10min at 1500rpm of 800-.

And 3.2) digesting by using cell digestive juice.

In particular, step 3.2) may comprise: adding cell digestive juice into the cells obtained after the digestion treatment of the cell dispersing enzyme, blowing, uniformly mixing, digesting for 2-20min at room temperature, adding DF10 culture medium to terminate the reaction, blowing, uniformly mixing, centrifuging and removing supernatant to obtain the cells digested by the cell digestive juice.

Wherein the cell digestive juice may be EGTA-Trypsin.

And 3.3) culturing the cells obtained after the digestion treatment by using a serum-containing culture medium to obtain primary isolated and cultured cells.

Specifically, step 3.3) may include the following step 3.3.1) -step 3.3.3).

Step 3.3.1), filtering the cells obtained after the digestion treatment through a 200-400 mu m nylon membrane, and collecting the filtered cells. Among them, filtration using a nylon membrane can filter non-tumor tissues such as adipose tissues.

Step 3.3.2), centrifuging for 5-10min at 800-₂Culturing in an incubator. For example, culturing for 24-48h to make the cells grow adherently.

Step 3.3.3), when the cultured cells grow completely adherent to the wall, the serum-containing culture medium is replaced by a serum-free culture medium and placed in37℃、5％CO₂And continuously culturing in the incubator to obtain the cells obtained by primary separation culture.

And 4, taking the cells obtained by the primary separation and culture in the step 3, and purifying and subculturing the cells by using a serum-free culture medium to obtain the primary human breast cancer cells. Wherein the serum-free medium comprises additives, the antibiotic and a DF medium.

In the step, when the confluency of the cultured cells reaches 70-90%, the cultured cells are purified by combining a pancreatin differential time digestion method, a differential time adherence method and a repeated adherence method to obtain the primary cells of the human breast cancer.

Based on this, step 4 may include the following steps 4.1) -4.4).

And 4.1) removing serum-free culture medium in the cultured cells, and digesting with EDTA-Trypsin for 3-30 min.

And 4.2) observing under a microscope, discarding fibroblasts digested first by EDTA-Trypsin, then digesting while collecting cells subjected to digestion treatment, and stopping digestion of the collected cells by using a DF10 culture medium until all the cells are digested.

Step 4.3), centrifuging for 5-10min at 1500rpm of 800-₂And (3) allowing fibroblasts to adhere to the wall in the incubator for 5-10min, collecting non-adherent human breast cancer primary cells, transferring the non-adherent human breast cancer primary cells into a new cell culture bottle for continuous culture, and repeatedly adhering the wall for 2-6 times to obtain purified human breast cancer primary cells. Thus, the fibroblast can be completely eliminated, and the cell state is poor or death caused by over digestion can be avoided.

And 4.4) subculturing the purified human breast cancer primary cells.

In this example, the antibiotics include penicillin, kanamycin sulfate, amphotericin B. During the preservation, the cleaning of breast cancer tissues and the culture of tumor cells, the presence of antibiotics can play a role in bacteriostasis and sterilization so as to protect primary cells from being polluted by microorganisms, and the antibiotics can kill gram-positive bacteria, gram-negative bacteria, mycoplasma and fungi.

In this embodiment, the supplement includes glutamine, insulin, transferrin, sodium selenite, epidermal growth factor, bovine serum albumin, Y-27632, and Wnt3 a.

Preferably, the cleaning liquid includes: penicillin 0.1-1mg/mL, kanamycin sulfate 0.1-1mg/mL, amphotericin B1-10 μ g/mL.

For example, in the wash solution, the concentration of penicillin may be 0.1, 0.3, 0.5, 0.7, 0.8, or 1mg/mL, the concentration of kanamycin sulfate may be 0.1, 0.3, 0.5, 0.7, 0.8, or 1mg/mL, and the concentration of amphotericin B may be 1, 3, 5, 8, or 10. mu.g/mL.

For example, in serum-containing media, penicillin may be present at a concentration of 50, 200, 300, 500, 1000, or 2000. mu.g/mL, kanamycin sulfate may be present at a concentration of 20, 200, 500, 1000, 2000, or 3000. mu.g/mL, and amphotericin B may be present at a concentration of 0.5, 2, 4, 6, 8, or 10. mu.g/mL.

Preferably, the serum-free medium comprises: 1-10mM glutamine, 3-50mg/L insulin, 3-30mg/L transferrin, 5-50 μ g/L sodium selenite, 5-50 μ g/L EGF, 1-5mg/mL BSA, 5-20 μ M Y-27632, 50-500 ng/mL Wnt3a, 0-2000 μ g/mL penicillin, 0-3000 μ g/mL kanamycin sulfate, 0-10 μ g/mL amphotericin B, 0-500 μ g/mL cefmetazole, and 0-50 μ g/mL vancomycin. The serum-free culture medium can effectively promote the growth of primary cells of human breast cancer and inhibit impurity cells such as fibroblasts and the like.

For example, in serum-free medium, glutamine can be at a concentration of 1, 3, 5, 8, or 10mM, insulin can be at a concentration of 5, 10, 15, 30, 40, or 50mg/L, transferrin can be at a concentration of 3, 10, 20, or 30mg/L, sodium selenite can be at a concentration of 5, 25, 35, or 50 μ g/L, EGF can be at a concentration of 5, 20, 35, or 50 μ g/L, BSA can be at a concentration of 1, 2, 3, 4, or 5mg/mL, Y-27632 can be at a concentration of 5, 10, 15, or 20 μ M, Wnt3a can be at a concentration of 50, 100, 200, 300, 400, or 500/mL, penicillin can be at a concentration of 0, 50, 200, 300, 500, 1000, or 2000 μ g/mL, kanamycin can be at a concentration of 0, 20, 200, 500, 1000, 2000, or 3000 μ g/mL, kanamycin, and amphotericin B can be at a concentration of 0.5 ng/L, 2. 4, 6, 8 or 10 mug/mL, the concentration of cefmetazole can be 0, 100, 300 or 500 mug/mL, and the concentration of vancomycin can be 0, 10, 30 or 50 mug/mL.

Next, the specific implementation of the above-mentioned step 1 to step 4 will be described with reference to the following specific culture method.

For step 1:

in step 1, the collected fresh human breast cancer tissue is preserved with a sample preservation solution, which is specifically realized in the following steps 1.1-1.2:

step 1.1, collecting the part of breast cancer tissue excised by the patient during surgery under the condition that the patient or the patient guardian agrees with the patient.

And 1.2, preserving the breast cancer tissues collected in the step 1.1 by using a sample preserving fluid.

For step 2:

in step 2, the breast cancer tissue preserved in step 1 is washed with a washing solution, which is specifically realized by the following steps 2.1-2.2:

and 2.1, transferring the breast cancer tissues preserved in the step 1.2 into a cell culture dish, washing for 6 times by using a cleaning solution, and removing non-cancer tissue impurities such as fat, mucosa, red blood cells and the like.

And 2.2, transferring the breast cancer tissue cleaned and treated in the step 2.1 into a new culture dish.

For step 3:

in step 3, the breast cancer tissue cleaned and treated in step 2 is taken and subjected to primary isolation culture by using a serum-containing culture medium, and the specific implementation is shown in the following steps 3.1 to 3.6:

and 3.1, cutting the breast cancer tissues cleaned and treated in the step 2.2 into small pieces by using scissors and a blade, and chopping the small pieces into a smashed mud shape.

Step 3.2, transferring the breast cancer tissue crushed into mud in the step 3.1 into a 50mL centrifuge tube, centrifuging at 1300rpm for 6min, and transferringRemoving supernatant, adding 9mL DF medium sequentially for resuspension, adding 1mL cell dispersing enzyme containing 10mg/mL collagenase I and 2mg/mL hyaluronidase sequentially, and adding 5% CO at 37 deg.C₂Digesting for 1h in the incubator by low speed oscillation to disperse breast cancer tissues and prevent cell agglomeration. After the cell dispersing enzyme reaction is finished, 20mL of DF10 culture medium is added to stop the reaction, the mixture is blown to be mixed evenly and is centrifuged for 5min at 1000rpm, the supernatant is removed, and the cell sediment is taken.

And 3.3, adding 4mL of EGTA-Trypsin solution into the cell sediment obtained in the step 3.2, uniformly mixing, standing at room temperature for 5min, sequentially adding 20mL of DF10 culture medium to terminate the reaction, blowing, dispersing and uniformly mixing to obtain the cell digested by the cell digestive juice.

In the step, the EGTA-Trypsin solution is cell digestive juice, and the digestion time can be 3-5 min.

And 3.4, filtering the cells obtained in the step 3.3 through a 300-micron nylon membrane, collecting the filtered cell sap into a 50mL centrifuge tube, centrifuging at 1000rpm for 5min, removing the supernatant, and taking cell precipitates.

Step 3.5, resuspending the cell pellet obtained in step 3.4 with 10mL of serum-containing medium to 25cm²The culture bottles were sequentially placed at 37 ℃ and 5% CO₂The culture was carried out in an incubator overnight (24 h).

Step 3.6, when the cells cultured in the step 3.5 grow completely adherent to the wall, the serum-containing culture medium is changed to a serum-free culture medium and is placed at 37 ℃ and 5% CO₂And continuing culturing in the incubator.

Wherein, the cells obtained by the culture in the step 3.6 are the cells obtained by primary separation culture.

For step 4:

in step 4, the cells isolated and cultured in the step 3 are purified and subcultured with a serum-free medium to obtain primary human breast cancer cells, which is specifically realized in the following steps 4.1 to 4.5 (step 4.1 to step 4.3 are purification and culture processes, and step 4.4 to step 4.6 are subculture processes):

step 4.1, when the confluency of the cells cultured in step 3.6 reaches 70-90%, the serum-free medium is removed and digested with 1-2mL EDTA-Trypsin.

And 4.2, observing under a microscope, discarding the fibroblasts digested by the EDTA-Trypsin when the cells are separated from the culture bottle, and then digesting while collecting the digested cells. The cells collected at each time were transferred to a centrifuge tube containing DF10 medium to terminate the digestion in time until all cells were digested.

In this step, by terminating digestion in time, poor cell state or death due to over-digestion can be avoided.

Step 4.3, centrifugation at 1000rpm for 5min, removal of supernatant, resuspension in serum-free medium and 5% CO at 37 ℃₂And (3) placing the cells in a cell culture box for 0.5h, collecting the cells which are not attached to the wall after the fibroblasts are attached to the wall, transferring the cells into a new culture bottle for continuous culture, and repeatedly attaching to the wall for 5 times to completely remove the fibroblasts to obtain the purified primary human breast cancer cells.

Based on the content, the culture method adopts a combined mode of a pancreatin differential time digestion method, a differential time adherence method and a repeated adherence method to purify the cells, can avoid poor cell state or death caused by over digestion in the purification process, obtains the human breast cancer primary cells with good state and without fibroblast and other impurity cells, and improves the success rate of culturing the human breast cancer primary cells.

And 4.4, culturing the human breast cancer primary cells obtained in the step 4.3 by using a serum-free culture medium, washing the cells at least twice by using 1-fold concentration (1 x) phosphate buffer saline solution PBS (0.01M, PH7.4) when the confluence degree of the cells reaches 70-90% so as to remove old serum-free culture medium and cells with poor shedding state, and then digesting the cells for 3-30min by using 1-2mL of EDTA-Trypsin.

And 4.5, observing under a microscope, collecting the digested cells while digesting, and stopping digestion of the collected cells by using DF10 culture medium in time until all cells are digested.

Step 4.6, centrifuge at 1000rpm for 6min, remove supernatant, resuspend with serum-free medium, inoculate in new culture flask, in order at 37 deg.C, 5% CO₂Cell culture boxAnd culturing to obtain the primary human breast cancer cells after passage.

Thereafter, the above steps 4.4-4.6 are repeated as needed, except that when step 4.4 is not performed for the first time, the human breast cancer primary cells cultured in step 4.4 are no longer the human breast cancer primary cells obtained in step 4.3, but are the human breast cancer primary cells obtained in the previous step 4.6 (i.e., the previous generation of human breast cancer primary cells).

It should be noted that, since the primary isolation culture is from the same breast cancer tissue, the cells obtained in step 4.3 and each step 4.6 are human breast cancer primary cells, and have the same cell characteristics, and the difference is only in the difference of cell generation number. Thus, any generation of human breast cancer primary cells cultured by the culture method provided by the embodiment has the same cell characteristics as the human breast cancer cells HBRCA-959.

Based on the above, for the primary cells of human breast cancer obtained in step 4.3 and each step 4.6, (1 × 10) can be used as necessary⁶Individual) human breast cancer primary cells were resuspended in 1-2mL of cell cryopreservation solution (90% fetal bovine serum and 10% DMSO, v/v) and stored in liquid nitrogen for future use. Wherein DMSO (dimethyl sulfoxide) is dimethyl sulfoxide.

The serum-free medium used in the above culture method includes: DF medium, 5mM glutamine, 25mg/L insulin, 10mg/L transferrin, 20. mu.g/L sodium selenite, 30. mu.g/L EGF, 1.5mg/mL BSA, 10. mu. M Y-27632, 100ng/mL Wnt3a, 500. mu.g/mL penicillin, 500. mu.g/mL kanamycin sulfate, 5. mu.g/mL amphotericin B, 100. mu.g/mL cefmetazole, 10. mu.g/mL vancomycin.

The serum-containing culture medium used in the above culture method comprises: DF medium, 10% FBS (total bone serum), 1000. mu.g/mL penicillin, 1500. mu.g/mL kanamycin sulfate, 50. mu.g/mL amphotericin B.

DF Medium is DME/F-121: 1 medium.

The washing solution used in the above culture method comprises: physiological saline, 0.5mg/mL penicillin, 0.5mg/mL kanamycin sulfate, 5. mu.g/mL amphotericin B.

It should be noted that the method for culturing primary human breast cancer cells provided in this embodiment may be used for culturing not only primary breast cancer tumor cells, but also primary breast cancer tumor cells of metastatic lesions of breast cancer.

Example 3

This embodiment is a further improvement on embodiment 1 and/or embodiment 2, and repeated contents are not described again.

This example illustrates the use of human breast cancer primary cells for non-disease diagnostic and therapeutic purposes. The human breast cancer primary cell may be the human breast cancer cell HBRCA-959 provided in example 1, the human breast cancer primary cell cultured by the culture method provided in example 2, or the human breast cancer primary cell cultured by the serum-free culture medium provided in example 3.

The application of the human breast cancer primary cells provided by the embodiment of the invention can be particularly used for drug sensitivity research and detection, breast cancer animal model construction, breast cancer inhibition drug development, breast cancer in-vitro research and the like.

It should be noted that the above-mentioned embodiments are exemplary, and that those skilled in the art, having benefit of the present disclosure, may devise various arrangements that are within the scope of the present disclosure and that fall within the scope of the invention. It should be understood by those skilled in the art that the present specification and figures are illustrative only and are not limiting upon the claims. The scope of the invention is defined by the claims and their equivalents. The present description contains several inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally", each indicating that the respective paragraph discloses a separate concept, the applicant reserves the right to submit divisional applications according to each inventive concept. Throughout this document, the features referred to as "preferably" are only an optional feature and should not be understood as necessarily requiring that such applicant reserves the right to disclaim or delete the associated preferred feature at any time.

Claims

1. The HBRCA-959 cell line is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO. 23022.

2. The cell line of claim 1, wherein the cells have the morphological characteristics of breast cancer cells.

3. A culture medium for culturing the cell of claim 1 or 2, comprising: an additive, an antibiotic that inhibits the growth of a microorganism, and a DF medium, wherein the additive comprises a cell growth inducer, a cell growth nutrient, and an ATP competitive inhibitor.

4. The culture medium of claim 3, wherein the cell growth inducing hormone and cell growth nutrient comprise glutamine, insulin, transferrin, sodium selenite, EGF, BSA, and Wnt3 a.

5. The cell line of claim 1 or 2, wherein the cell is capable of being used in the development of a medicament for inhibiting or treating breast cancer.

6. A method of culturing cells comprising

Providing a cell as claimed in claim 1 or 2;

placing the cells in the medium of any one of claims 4 or 3, and culturing the cells under conditions that allow the cells to proliferate.

7. Use of a cell according to any of claims 1 or 2 for the manufacture of a protein.

8. A marker and a kit comprising the cell according to any one of claims 1 or 2, an expression vector for expressing a gene contained in the cell, and a cell culture medium for culturing the cell.

9. Use of a cell line according to claim 1 or 2, wherein the cells, including the cells according to claim 1 or 2 or the cells HBRCA-959 cultured in the medium according to claim 4 or 3, or the cells HBRCA-959 cultured by the culture method according to claim 6, for the in vitro study of breast cancer.

10. A method for isolating primary breast cancer cells, wherein the primary cells are isolated and cultured from human breast cancer tissue, and the method for culturing the cells is the method as claimed in claim 6.