CN114015655B

CN114015655B - HBRCA-959 cell line and culture method and application thereof

Info

Publication number: CN114015655B
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: 2023-09-05
Anticipated expiration: 2041-12-09
Also published as: CN114015655A

Abstract

The invention relates to a cell HBRCA-959, a culture method and application thereof. The primary cell of human breast cancer 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 field of biotechnology, in particular to an HBRCA-959 cell line.

Background

Breast cancer is a phenomenon that breast epithelial cells undergo proliferation control under the action of various cancerogenic factors. Early stage of the disease is often manifested by symptoms such as breast lump, nipple discharge, axillary lymphadenectasis, and the like, and late stage of the disease can cause distant metastasis due to cancer cells, and the disease can cause multi-organ lesions, thereby directly threatening the life of patients.

Since breast cancer is a serious hazard to human health, commercial cell lines are often used as materials for studying pathogenesis and treatment methods. However, after long-term culture, the biological characteristics and genetics of the strain are easy to change, which is unfavorable for researching pathogenesis and treatment methods.

However, in the prior art, the research and inhibition of cancer mechanism mostly adopts related molecular markers of immune system to identify or commonly use cancer markers, such as a composition for detecting breast cancer stem cells provided in chinese patent publication No. CN112898428A, including a first antibody nucleic acid conjugate that specifically binds to cell surface CD44 protein, a second antibody nucleic acid conjugate that specifically binds to cell surface CD24 protein, a DNA single-chain T-chain and a DNA single-chain F-chain, the T-chain being complementarily bound to the nucleic acid strand of the first antibody nucleic acid conjugate, the F-chain being complementarily bound to the nucleic acid strand of the second antibody nucleic acid conjugate, together forming a programmed DNA loop, erasing the signal markers on the surface of breast cancer cells of CD44 positive CD24 positive phenotype, and retaining the signal probes labeled to breast cancer stem cells. The CD24 protein is a mucin-like adhesion molecule, and a great deal of researches show that CD24 can be seen in tumor, inflammation and autoimmune diseases and can play different roles in different diseases, so that the detection of the markers of CD24 and CD44 used in the above patent is very likely to have non-specific disease detection errors, and the misleading problem of the direction of secondary diagnosis such as tissue section and the like is caused.

Primary tumor cell culture refers to a process in which tumor cells are obtained by surgical excision of tumor tissue from a tumor patient and cultured in vitro for a short period of time. The biological characteristics of the primary cultured tumor cells are not greatly changed due to the fact that the tissues are just isolated, original genetic characteristics are still reserved, the characteristics of the primary cultured tumor cells are similar to those of the primary cultured tumor cells in vivo, the primary cultured tumor cells are suitable for related experiments of biochemical molecules, drug sensitivity and mechanism exploration, and the data of the primary cultured tumor cells are more convincing. It can be seen that the culture of primary cells of human breast cancer is particularly necessary.

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

Furthermore, there are differences in one aspect due to understanding to those skilled in the art; on the other hand, since the applicant has studied a lot of documents and patents while making the present invention, the text is not limited to details and contents of all but it is by no means the present invention does not have these prior art features, but the present invention has all the prior art features, and the applicant remains in the background art to which the right of the related prior art is added.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a cell HBRCA-959 which is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.23022. A primary human breast cancer cell isolated from clinical pathological tissues is named as a human breast cancer cell HBRCA-959, and is preserved in China general microbiological culture Collection center (China General Microbiological Culture Collection Center, CGMCC) for 24 th month in 2021, wherein the preservation address is Beijing Chaoyang area Beichen Xiyu No. 1, 3, the national institute of microbiology, postal code 100101, and the preservation 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: additives, antibiotics to inhibit microbial growth, and DF medium, wherein the additives include cell growth inducers, cell growth nutrients, and ATP competitive inhibitors.

According to a preferred embodiment, the medium for culturing human breast cancer primary cells comprises: additives, antibiotics and DF medium. Wherein the additives include glutamine, insulin, transferrin, sodium selenite, epidermal growth factor, bovine serum albumin BSA, Y-27632 and Wnt 3a. Antibiotics include antibiotics inhibiting the growth of microorganisms such as penicillin, kanamycin sulfate, amphotericin B, cefmetazole, vancomycin, and the like.

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 μg/L sodium selenite, 5-50 μg/LEGF, 1-5mg/mL BSA, 5-20 μ M Y-27632, 50-500 ng/mL Wnt 3a, 0-2000 μg/mL penicillin, 0-3000 μg/mL kanamycin sulfate, 0-10 μg/mL amphotericin B, 0-500 μg/mL cefmetazole, 0-50 μg/mL vancomycin.

According to a preferred embodiment, since the cell HBRCA-959 is derived from human breast cancer tissue and has morphological characteristics and physiological and biochemical properties of breast cancer, the cell HBRCA-959 can be used in the development of a medicament 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 medium and the HBRCA-959 cells were cultured under conditions that allow proliferation of the cells.

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 bovine serum, antibiotics and DF culture medium, wherein the antibiotics comprise penicillin, kanamycin sulfate and amphotericin B;

S2: cleaning the breast cancer tissue preserved in the step S1 by using a cleaning solution;

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

s3: taking breast cancer tissues subjected to S2 cleaning treatment, and carrying out primary separation culture by using a serum-containing culture medium;

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

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

In detail, DF medium is DME/F-12:1 medium (HyClone, U.S.A.) common in the art.

Preferably, the serum-containing medium comprises: 50-2000. Mu.g/mL penicillin, 20-3000. Mu.g/mL kanamycin sulfate, 0.5-10. Mu.g/mL amphotericin B.

Preferably, the cleaning liquid comprises: 0.1-1mg/mL penicillin, 0.1-1mg/mL kanamycin sulfate, 1-10 μg/mL amphotericin B.

Modern research suggests that breast cancer is not a single disease, but rather is a heterogeneous collection of diseases characterized by histological, genomic changes, gene expression, metastatic behaviour, and therapeutic response diversity. For models constructed with human breast cancer primary cells, the positive effects of human breast cancer primary cell secreted proteins on disease are also valuable for the mechanistic studies of breast cancer. Similarly, the use of cells in the production of proteins can provide basis for subsequent protein markers for breast cancer and protein level targeted drug studies. Thus, the use of the human breast cancer primary cell, or 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 disease. The cell HBRCA-959 is isolated from human breast cancer tissue and has specificity for recognizing breast cancer. Based on this, the present invention is directed to a marker. Markers include cellular HBRCA-959 for screening for breast cancer.

Further, the present invention relates to 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. Screening is carried out by means of RT-PCR or cell culture, and the cells are further used as target selection range, so that the possibility of multi-target drug screening is provided 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 an application of the human breast cancer primary cell or cell HBRCA-959 obtained by culturing the culture method in drug sensitivity research and detection.

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 an application of the human breast cancer primary cell or cell HBRCA-959 obtained by culturing the culture method in constructing a breast cancer animal model.

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 an application of the human breast cancer primary cell or cell HBRCA-959 obtained by culturing the culture method in developing 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 an application of the human breast cancer primary cell or cell HBRCA-959 obtained by culturing the culture method in-vitro research of breast cancer.

The application of the primary human breast cancer cells provided by the invention is the purpose of diagnosis and treatment of non-diseases.

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

feature 1: the primary isolated and cultured breast cancer tissue from Chinese, the cell has not undergone any genetic engineering and genetic modification.

Feature 2: the human breast cancer cell HBRCA-959 is observed under a microscope, is in an epithelial-like cell form, has a relatively high proliferation speed of tumor cells, has different cell sizes and forms, is in an irregular shape or a polygonal shape, is arranged in a disorder, has no contact inhibition, has an underscore cell boundary, and has the morphological characteristics of breast cancer cells.

Feature 3: the multiplication time of the 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.

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

Feature 5: the human breast cancer cell HBRCA-959 is identified as tumor cell heterokaryon type by karyotype analysis, the chromosome structure and the number of the cells are abnormal, the chromosome number of the cells is 52, the human breast cancer cell HBRCA-959 belongs to the super-diploid karyotype, and accords with the genetic characteristics of human malignant tumor.

Feature 6: human breast cancer cell HBRCA-959 is identified by STR (Short Tandem Repeat ) genotyping, is a single human-derived cell without cross contamination, and is a newly obtained primary breast cancer cell.

Feature 7: the human breast cancer cell HBRCA-959 can be used for drug sensitivity research and detection, building a breast cancer animal model, developing a breast cancer inhibiting drug and in-vitro research of breast cancer.

Drawings

FIG. 1 is a cell morphology diagram of human breast cancer cells HBRCA-959 according to one embodiment of the present invention;

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

FIG. 3 is a graph showing the result of immunofluorescence assay of human breast cancer cells HBRCA-959 according to an embodiment of the present invention;

FIG. 4 is a graph of a chromosome karyotype analysis of human breast cancer cells HBRCA-959 according to one embodiment of the present invention;

FIG. 5 is a STR genotyping chart of human breast cancer cells HBRCA-959 according to one embodiment of the present invention.

Detailed Description

The following detailed description refers to the accompanying drawings.

It should be noted that, due to the specificity of breast cancer tissue, the lack of effective and available breast cancer specimens, the technical difficulty of establishing primary cell culture of breast cancer, and other factors, the existing in vitro research model of breast cancer is seriously lacking, so that the research of the cytology level of breast cancer is greatly limited. Based on the above, the embodiment of the invention provides a primary cell of human breast cancer, and a culture method and application thereof, which can provide good experimental materials for related researches such as molecular generation mechanism and clinical treatment path of breast cancer. The primary human breast cancer cells, and the culture method and application thereof are described below.

Example 1

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

The human breast cancer cell HBRCA-959 is primarily isolated and cultured from breast cancer tissues of Chinese, and the cell is not subjected to any genetic engineering and modification.

The biological properties of human breast cancer cells HBRCA-959 are described below.

1.1 morphological observations of cells

The human breast cancer cell HBRCA-959 is observed under an inverted microscope, is in an epithelial-like cell form, has a relatively high proliferation speed of tumor cells, is in an irregular or polygonal shape and is in disorder arrangement, non-contact inhibition and undershot in cell limit, and has morphological characteristics of breast cancer cells. The morphological observation results are shown in FIG. 1.

1.2 determination of growth curve and doubling time

1.2.1 growth Curve determination procedure

(1) When the cell confluence of human breast cancer cells HBRCA-959 reached 70-90%, serum-free medium was removed, cells were washed with PBS (0.01M, pH 7.4) at least twice to remove old serum-free medium and floating poorly conditioned cells, treated with 1-2mL EDTA-Trypsin (Trypsin-EDTA digest) and stopped with DF10 medium (DF medium and 10% FBS).

Wherein EDTA (Ethylene Diamine Tetraacetic Acid) is ethylenediamine tetraacetic acid and Trypsin is Trypsin.

(2) Cells were grown at 2.5X10 ⁵ Per ml of the cells were inoculated into 36 wells of 12 48-well plates, and 500. Mu.l of the cell suspension was added to each well.

(3) Discarding the culture medium in 3 holes every 24 hours from the inoculation time, adding EDTA-Trypsin for digestion treatment, suspending cells, counting the average value of the cell density in 3 holes, counting 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 growth curve data shown in table 1 below were obtained.

1.2.2 growth Curve measurement results

Table 1 shows growth curve data obtained from continuous measurement for 12 days.

TABLE 1

Cultivation time (Tian)	Average cell density
		1	11.3×10 ⁴ personal/mL
2	11.73×10 ⁴ personal/mL
		3	13.29×10 ⁴ personal/mL
4	17.01×10 ⁴ personal/mL
		5	19.02×10 ⁴ personal/mL
6	20.05×10 ⁴ personal/mL
		7	21.85×10 ⁴ personal/mL
8	23.10×10 ⁴ personal/mL
		9	26.53×10 ⁴ personal/mL
10	30.20×10 ⁴ personal/mL
		11	34.80×10 ⁴ personal/mL
12	41.87×10 ⁴ personal/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. The abscissa of FIG. 2 shows the incubation time (days), and the ordinate shows the cell number (. Times.10) ⁴ and/mL).

Referring to the growth curve data in Table 1 and the growth curve diagram 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 calculation formula of the population doubling time of tumor cells is as follows: dt=tx [ lg 2/(lgNt-lgNo) ]. Wherein DT is doubling time, t is time interval, no is initial cell number, and Nt is final cell number.

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

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 characteristics in vitro culture.

1.3 immunofluorescence assay

1.3.1 immunofluorescence identification step

(1) Human breast cancer cells HBRCA-959 reached a confluence of about 70-90%, serum-free medium was removed, cells were washed with PBS (0.01M, pH 7.4) at least twice to remove old serum-free medium and floating poorly conditioned cells, digested with 1-2mL EDTA-Trypsin, and inoculated into collagen-coated 48-well plates;

(2) When the cell confluence reaches 70-90%, removing serum-free culture medium, washing 3 times with PBS, adding 500 μl/hole of ice methanol, fixing for 30min, and removing;

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

(4) Adding 300 μl/well primary antibody (dilution of Keratin 18Mouse mAb,1:800; dilution of Keratin 8Rabbit mAb 1:200 release), and aspiration after overnight incubation at 4deg.C;

(5) Adding 300 μl/well of fluorescent secondary antibody (secondary antibody-Mouse IgG (white molecular) -FITC antibody produced in rabbit,1:100 dilution; cy 3-labeled goat Anti-rabbit IgG,1:500 dilution), incubating at room temperature in the dark for 1h, sucking out the secondary antibody, and washing with PBS for 3 times;

(6) 2 drops of DAPI stock solution (ab 104139) are added into each hole, and the mixture is dyed for 5min at room temperature in a dark place;

(7) Aspiration of DAPI, PBS wash 3 times;

(8) 300. Mu.L/well PBS was added, and the immunofluorescence was obtained by taking a photograph, and then using an objective lens X ocular lens (10X 10).

1.3.2 immunofluorescence assay results

CytoKeratin (CK) is a common marker of epithelial cells, expressing CK 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 by immunofluorescence assay that DAPI stained tumor nuclei, and that human breast cancer cells HBRCA-959CK18 and CK8 were strongly expressed, indicating that human breast cancer cells HBRCA-959 were derived from epithelial cells.

1.4 identification by chromosome karyotyping

1.4.1 chromosome karyotype analysis and identification step

(1) Cell culture

When the human breast cancer cells HBRCA-959 reached a confluence of 70-90%, the serum-free medium culture was removed, the cells were washed with PBS (0.01M, PH7.4) at least twice to remove old serum-free medium and shed poorly conditioned cells, treated with 1-2mL EDTA-Trypsin digestion, observed under a microscope, the digested cells were collected while digestion was performed, and the collected cells were terminated with DF10 medium until all cells were digested. Centrifuge at 1000rpm for 5min and remove supernatant.

(2) Colchicine treatment

Colchicine 20-50 μl was added to the cell pellet at a concentration of 300 μg/mL and treated in an incubator at 37 ℃ for 25min.

(3) Hypotonic treatment

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

(4) Prefixed

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

(5) Centrifuging

Centrifugation at 1300rpm for 10min, and discarding supernatant.

(6) Fixing

6-8mL of fixative solution is added into a centrifuge tube, a single cell suspension is gently blown by a suction tube, the mixture is fixed in a water bath at 37 ℃ for 30min, and after centrifugation at 1300rpm for 10min, the supernatant is discarded.

(7) Second fixing

6-8mL of fixative solution is added into a centrifuge tube, a single cell suspension is gently blown by a suction tube, the mixture is fixed in a water bath at 37 ℃ for 15min, and after centrifugation at 1300rpm for 10min, the supernatant is discarded.

(8) Tabletting

Adding new fixing solution about 0.2mL into the centrifuge tube, gently blowing into cell suspension with a suction tube, taking out Borneolum Syntheticum from the freezing chamber of the refrigerator, adjusting to suitable suspension, dropwise adding 1-2 drops of suspension, and baking at 75deg.C for 3 hr.

(9) Dyeing

The slide was stained with 6% Giemsa for 10min, then clamped with forceps, gently rinsed on both sides with tap water, dried at room temperature and visualized.

(10) Microscopic examination

After the slides are dried, they are examined under a microscope. And searching for a good split phase by using a low-power mirror, and observing by using a high-power oil mirror to obtain a chromosome karyotype analysis and identification result.

1.4.2 chromosome karyotype analysis and identification results

The results of the karyotyping analysis and identification are shown in FIG. 4. Referring to FIG. 4, the human breast cancer cell HBRCA-959 is identified by karyotype analysis as tumor cell heterokaryotype, the chromosome structure and number of the cell are abnormal, the chromosome number of the cell is 52, the cell belongs to the super diploid karyotype, and the genetic characteristics of human malignant tumor are met.

1.5STR identification

1.5.1STR identification procedure

(1) Human breast cancer cells HBRCA-959 (1×10) grown by adherence ⁶ And then twice washing the cells with PBS (0.01M, PH7.4), sequentially digesting with 1-2mL EDTA-Trypsin for 3-30min, and stopping the digestion with 10mL DF10 medium.

(2) Centrifuge at 10000rpm for 1min, discard supernatant, add 200 μl buffer GA (cell/tissue genomic DNA extraction kit DP304, tengen) and shake to complete suspension.

(3) mu.L of protease K solution was added and mixed well.

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

(5) 200 mu L of absolute ethyl alcohol is added, fully and uniformly mixed for 15s by shaking, and the mixture is centrifuged briefly.

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

(7) To the adsorption column, 500. Mu.L of buffer GD (cell/tissue genomic DNA extraction kit DP304, tiangen) was added, and the mixture was centrifuged at 12000rpm for 30s to remove the waste liquid.

(8) 600. Mu.L of a rinse PW (cell/tissue genomic DNA extraction kit DP304, tiangen) was added to the column, and the mixture was centrifuged at 12000rpm for 30s to remove the waste liquid.

(9) Transferring the adsorption column into another centrifuge tube, dripping 50-200 μl of elution buffer TE (cell/tissue genome DNA extraction kit DP304, tiangen Co.) 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 Goldeney TM20A STR composite amplification kit is adopted for amplification, and 21 known STR sites and sex genes Amelogenin are continuously detected and analyzed on an ABI3100 genetic analyzer.

(11) Sample data were analyzed using genemap 3.2 software for automatic genotyping to obtain STR typing results.

1.5.2STR identification results

The STR typing results are shown in FIG. 5. Referring to fig. 5, 21 loci are detected, 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, CSFIPO/10/11, pentaD/9/12, D2S441/10, vWA/14/18, D8S1179/10/13, TPOX/11, pentaE/12/18, TH01/7/7, D12S391/23/24, D2S1338/23, FGA/23/24.

From the above, the following conclusions can be drawn through STR genotyping identification:

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

(2) Comparing the STR data of human breast cancer cells HBRCA-959 with those of ATCC (American type culture Collection) and DSMZ (national center for culture Collection of Germany), it is found that no cells 100% matching the cell type of human breast cancer cells HBRCA-959 are found in both ATCC and DSMZ cell banks.

(3) Comparing the STR data of human breast cancer cells HBRCA-959 with those of ATCC and DSMZ STR databases, it was found that the matching rate of the STR data with HROC278Met T2M 2 (cvcl_1u90) cells was 75% to the highest.

In conclusion, STR genotyping identification is carried out on human breast cancer cells HBRCA-959, and the result proves that the human breast cancer cells are single cells of human origin and have no cross contamination, and are a newly obtained breast cancer primary cell.

1.6 cell uses

1.6.1 use in drug sensitivity studies and detection

1.6.1.1 step of detecting drug sensitivity (CD-DST method)

(1) Drop culture of collagen gel

Human breast cancer cells HBRCA-959 were mixed with gel constituting liquid (I: II: III=8:1:1) uniformly, and cell suspensions were inoculated in 6 well plates according to 30. Mu.L/drop, 3 drops/well, and 3 drops were inoculated in another 6 well plate at the same time as a 0-time control group. After 1-2h, adding 3mL DF culture medium containing 10% foetal calf serum after gel drop coagulation, and adding 5% CO at 37deg.C ₂ Culturing in an incubator overnight.

(2) Anticancer drug contact and cleaning

After the primary cells of the human breast cancer in the step (1) are cultured for 24 hours, anticancer drugs are added, negative Control holes (Control) without drugs are arranged, and positive Control holes treated by common drugs for clinical breast cancer (it is to be noted that the drug selection can be usually carried out according to the main clinical manifestation of a cell-derived patient) are arranged, and meanwhile, the 0-time group is fixed by staining. After the corresponding incubation time, the medium was washed 2 times with DF medium for 15min each, and then incubated with serum-free medium for 5 days, with the medium being changed once on day 3.

(3) Dyeing fixation and scanning

On day 8, staining and fixing, neutral red staining with final concentration of 50 μg/mL for 2h, PBS washing the cells for 3 times, 5min each time, neutral formalin fixing for 45min, distilled water washing for 15min, and air drying to obtain active tumor cells. And then scanning and analyzing the adhesive drop by using a Primage image analysis system to obtain a drug sensitivity detection result.

1.6.1.2 drug sensitivity test results

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

TABLE 2

Drug name	Cell viability (%)
		Carboplatin+paclitaxel (CBDCA+PAC)	94.75
Cisplatin + Norvitamin (CDDP + VNR)	74.21
		Capecitabine+docetaxel (CAP+DOC)	88.26
Epirubicin+docetaxel (EPI+DOC)	95.38
		Cisplatin + gemcitabine (CDDP + GEM)	90.43

If the cell survival rate is less than 50%, it can be shown that the sensitivity of the individual tumor cells to the corresponding drugs is higher (i.e. high sensitivity); if the cell survival rate is 50-60%, it can be shown that the sensitivity of individual tumor cells to the corresponding drugs is in the critical range (i.e. critical) of high sensitivity and low sensitivity; if the cell viability is greater than 60%, it may be indicated that the individual tumor cells are less sensitive (i.e., resistant) to the corresponding drug.

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

1.6.2 use in constructing animal models of breast cancer

Human breast cancer cells HBRCA-959 in the logarithmic growth phase were inoculated in a mammal (e.g., nude mice) at prescribed doses and cultured for several days to construct a breast cancer animal model. 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 use in the development of medicaments for inhibiting breast cancer

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

By adding different therapeutic drugs into the human breast cancer cell HBRCA-959 culture medium, the changes of cell growth proliferation, death, period and the like are observed, and preliminary effective candidate drugs are obtained. And then the candidate medicine is used for a breast cancer animal model, the in-vivo action of the medicine is detected, the general condition, the survival time and the tumor size change of animals and the organism change condition of the animals after the action of the medicine are observed, and therefore the curative effect evaluation and the mechanism analysis are carried out on the candidate medicine.

1.6.4 for in vitro study of breast cancer

The human breast cancer cell HBRCA-959 is cultured from the breast cancer tissue which is just isolated, has little change of biological characteristics, retains original genetic characteristics, can be used as a cell material for in vitro research of human breast cancer pathogenesis and personalized treatment, and further provides a basis for establishment of a human breast cancer pathogenesis model, preparation, screening, evaluation and the like of anti-human breast cancer medicaments.

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

Example 2

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

This example is useful for illustrating a method of culturing primary cells of human breast cancer, comprising the following steps 1-4. The human breast cancer cells HBRCA-959 provided in example 1 were obtained by culturing 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 tissue stored in the step 1 by using a cleaning solution. Wherein the cleaning solution comprises physiological saline containing the antibiotic.

And step 3, taking the breast cancer tissue after the cleaning treatment in the step 2, and carrying out 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 steps 3.1) -3.3).

Step 3.1), treating the washed breast cancer tissue with a cell-dispersing enzyme.

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

Step 3.1.1), chopping the washed breast cancer tissue into a mashed state.

Wherein, the breast cancer tissue can be conveniently digested into tumor cells with proper size by being chopped into mud.

Step 3.1.2), centrifuging the crushed muddy breast cancer tissue at 800-1500rpm for 5-10min, removing the supernatant, and adding DF medium into the cell sediment after removing the supernatant for resuspension.

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

Step 3.1.3), cell-dispersing enzyme was added sequentially, and the mixture was placed at 37℃with 5% CO ₂ Low-speed shaking digestion is carried out for 20min-2h in an incubator to disperse breast cancer tissues and prevent cell agglomeration, then DF10 culture medium is added to stop reaction, and the mixture is blown off and mixed uniformly.

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

Step 3.1.4), centrifuging at 800-1500rpm for 5-10min, and removing supernatant to obtain cell after cell dispersion enzyme digestion.

Step 3.2), performing digestion treatment by using cell digestion liquid.

Specifically, step 3.2) may comprise: adding cell digestive juice into cells obtained after cell dispersion enzyme digestion treatment, blowing off and mixing uniformly, digesting for 2-20min at room temperature, adding DF10 culture medium to stop reaction, blowing off and mixing uniformly, centrifuging to remove supernatant, and obtaining cells digested by the cell digestive juice.

Wherein the cell digestive juice can be EGTA-Trypsin.

Step 3.3), culturing the digested cells in a serum-containing medium to obtain primary isolated cultured cells.

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

Step 3.3.1), the cells obtained after the digestion treatment were filtered through a nylon membrane of 200 to 400 μm, and the filtered cells were collected. Among them, filtration with nylon membrane can filter out non-tumor tissues such as adipose tissues.

Step 3.3.2), centrifuging at 800-1500rpm for 5-10min, removing supernatant, resuspending the supernatant-removed cell pellet with serum-containing medium, and placing the resuspended cells at 37deg.C, 5% CO ₂ Culturing in an incubator. Such as for example, 24-48 hours to allow for complete adherent growth of the cells.

Step 3.3.3), after the cultured cells are grown completely by adherence, the serum-containing medium is changed to serum-free medium, and the cells are placed at 37 ℃ and 5% CO ₂ Culturing is continued in an incubator to obtain primary isolated cultured cells.

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

In the step, when the confluence of the cultured cells reaches 70-90%, the cultured cells can be purified by adopting a mode of combining a pancreatin differential time digestion method, a differential time adherence method and a repeated adherence method, so as to obtain the primary cells of the human breast cancer.

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

Step 4.1), removing serum-free medium from the cultured cells, and subjecting the cells to EDTA-Trypsin digestion for 3-30min.

Step 4.2), observing under a microscope, discarding the fibroblast digested by EDTA-Trypsin, then collecting the digested cell while digesting, and stopping the digestion of the collected cell by using DF10 culture medium until all the cells are digested.

Step 4.3), sequentially centrifuging at 800-1500rpm for 5-10min, removing supernatant, and resuspending the supernatant-removed cell pellet with serum-free medium, and placing the resuspended cells at 37deg.C, 5% CO ₂ And (3) placing in an incubator for 5-10min, attaching fibroblasts to the wall, collecting non-attached human breast cancer primary cells, transferring the non-attached human breast cancer primary cells into a new cell culture flask for continuous culture, and repeatedly attaching the fibroblasts to the wall for 2-6 times to obtain purified human breast cancer primary cells. Thus, the effect of completely removing the fibroblasts can be achieved, and meanwhile, poor cell state or death caused by over digestion can be avoided.

Step 4.4), subculturing the purified primary cells of human breast cancer.

In this example, the antibiotics include penicillin, kanamycin sulfate, amphotericin B. The antibiotic can play a role in bacteriostasis and sterilization during the preservation and cleaning of breast cancer tissues and the culture of tumor cells, so as to protect primary cells from microbial contamination, and has killing effect on gram-positive bacteria, gram-negative bacteria, mycoplasma and fungi.

In this example, the additives include glutamine, insulin, transferrin, sodium selenite, epidermal growth factor, bovine serum albumin, Y-27632, and Wnt 3a.

Preferably, the cleaning solution includes: 0.1-1mg/mL penicillin, 0.1-1mg/mL kanamycin sulfate, 1-10 μg/mL amphotericin B.

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

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

Preferably, the serum-free medium comprises: 1-10mM glutamine, 3-50mg/L insulin, 3-30mg/L transferrin, 5-50 μg/L sodium selenite, 5-50 μg/L EGF, 1-5mg/mL BSA, 5-20 μ M Y-27632, 50-500 ng/mL Wnt 3a, 0-2000 μg/mL penicillin, 0-3000 μg/mL kanamycin sulfate, 0-10 μg/mL amphotericin B, 0-500 μg/mL cefmetazole, 0-50 μg/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.

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

The specific implementation of the above steps 1 to 4 will be described below with reference to the following specific cultivation method.

For step 1:

in step 1, the collected fresh human breast cancer tissue is preserved by a sample preservation solution, and the specific implementation is shown in the following steps 1.1-1.2:

step 1.1, collecting partial breast cancer tissues excised during operation of a patient under the condition of informed consent of the patient or patient guardian.

Step 1.2, preserving the breast cancer tissue collected in step 1.1 by using a sample preservation solution.

For step 2:

in step 2, the breast cancer tissue preserved in step 1 is washed with a washing liquid, and the specific implementation is shown in the following steps 2.1-2.2:

and 2.1, transferring the breast cancer tissue stored in the step 1.2 into a cell culture dish, and washing the breast cancer tissue with a washing liquid for 6 times to remove non-cancer tissue impurities such as fat, mucous membrane, red blood cells and the like.

Step 2.2, transferring the breast cancer tissue after the cleaning treatment in step 2.1 to a new culture dish.

For step 3:

in step 3, the breast cancer tissue after the cleaning treatment in step 2 is taken and subjected to primary separation culture by a serum-containing culture medium, and the specific implementation is shown in the following steps 3.1-3.6:

and 3.1, shearing the breast cancer tissue after the cleaning treatment in the step 2.2 into small pieces by using scissors and a blade, and chopping the small pieces into mud.

Step 3.2, transferring the mashed breast cancer tissue of step 3.1 into a 50mL centrifuge tube, centrifuging at 1300rpm for 6min, removing supernatant, sequentially adding 9mL DF culture medium for resuspension, sequentially adding 1mL cell dispersion enzyme containing 10mg/mL collagenase I and 2mg/mL hyaluronidase, and concentrating at 37deg.C and 5% CO ₂ The cells were prevented from clumping by shaking and digesting for 1 hour at low speed in an incubator to disperse breast cancer tissue. After the cell dispersion enzyme reaction was completed, 20mL of DF10 medium was added to terminate the reaction, the mixture was blown off and centrifuged at 1000rpm for 5min, and the supernatant was removed to obtain a cell pellet.

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 off and uniformly mixing to obtain the cell digested with the cell digestive juice.

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

Step 3.4, filtering the cells obtained in the step 3.3 through a 300 mu m nylon membrane, collecting the filtered cell fluid into a 50mL centrifuge tube, centrifuging at 1000rpm for 5min, removing the supernatant, and taking a cell precipitate.

Step 3.5, the cell pellet obtained in step 3.4 was inoculated by re-suspension to 25cm with 10mL of serum-containing medium ² Sequentially placing at 37deg.C and 5% CO in culture flask of (C) ₂ Culturing in an incubator overnight (24 h).

Step 3.6, after the cells cultured in step 3.5 are grown completely, the serum-containing medium is changed to serum-free medium and placed at 37℃in 5% CO ₂ Culturing is continued in the incubator.

Wherein, the cells obtained by culturing in the step 3.6 are primary isolated and cultured cells.

For step 4:

in step 4, the primary isolated and cultured cells in step 3 are taken, and purified and subcultured with serum-free medium to obtain primary cells of human breast cancer, wherein the specific implementation is as follows in steps 4.1-4.5 (step 4.1-step 4.3 is a purification culture process, and step 4.4-step 4.6 is a subculture process):

step 4.1, when the confluence of the cells cultured in step 3.6 reached 70-90%, the serum-free medium was removed and treated with 1-2mL EDTA-Trypsin digestion.

And 4.2, observing under a microscope, discarding the fibroblast digested by EDTA-Trypsin when the cell is separated from the culture flask, and collecting the digested cell while digesting. Cells collected each time were transferred to centrifuge tubes with DF10 medium placed in order to terminate digestion in time until all cells were digested.

In this step, by terminating digestion in time, poor cell status or death due to excessive digestion can be avoided.

Step 4.3, centrifugation at 1000rpm for 5min, removal of supernatant, resuspension with serum-free medium, and 5% CO at 37 ℃ ₂ Placing 0.5 in a cell incubatorh, after the fibroblast is attached, collecting the non-attached cells, transferring the cells into a new culture flask for continuous culture, and repeatedly attaching the cells for 5 times to completely remove the fibroblast, thereby obtaining the purified human breast cancer primary cells.

Based on the above, the method for culturing the human breast cancer primary cells adopts the combination of the pancreatin differential time digestion method, the differential time adherence method and the repeated adherence method to purify the cells, so that the condition of the cells is poor or the cells die due to excessive digestion in the purification process can be avoided, the human breast cancer primary cells which are good in condition and free of fibroblast and other impurity cells can be obtained, and the success rate of culturing the human breast cancer primary cells can be improved.

Step 4.4, culturing the primary cells of the human breast cancer obtained in the step 4.3 with a serum-free medium, washing the cells at least twice with 1-fold concentration (1×) phosphate buffer solution PBS (0.01M, pH 7.4) when the cell confluency reaches 70-90%, removing old serum-free medium and cells in poor state of shedding, and digesting with 1-2mL EDTA-Trypsin for 3-30min.

And 4.5, observing under a microscope, and collecting the digested cells while digesting, wherein the collected cells are timely stopped from being digested by DF10 culture medium until all the cells are digested.

Step 4.6, centrifuging at 1000rpm for 6min, removing supernatant, re-suspending with serum-free medium, inoculating into new culture flask, sequentially culturing at 37deg.C and 5% CO ₂ Culturing in a cell incubator to obtain the primary cells of the breast cancer after passage.

Thereafter, the above steps 4.4 to 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 human breast cancer primary cells of the previous generation).

Since the primary isolated culture is from the same breast cancer tissue, the cells obtained in step 4.3 and step 4.6 are both primary human breast cancer cells, and have the same cell characteristics, and the difference is only that the cell algebra is different. Thus, any of the primary human breast cancer cells cultured by the culture method provided in this example had 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 of step 4.6, (1X 10) may be used as necessary ⁶ Personal) human breast cancer primary cells were resuspended in 1-2mL of cell cryopreservation (90% fetal bovine serum and 10% DMSO, v/v) and stored in liquid nitrogen for later 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 μg/L sodium selenite, 30 μg/L EGF, 1.5mg/mL BSA, 10 μg M Y-27632, 100ng/mL Wnt3a, 500 μg/mL penicillin, 500 μg/mL kanamycin sulfate, 5 μg/mL amphotericin B, 100 μg/mL cefmetazole, 10 μg/mL vancomycin.

The serum-containing medium used in the above culture method includes: DF medium, 10% FBS (fetal bovine serum ), 1000. Mu.g/mL penicillin, 1500. Mu.g/mL kanamycin sulfate, 50. Mu.g/mL amphotericin B.

DF medium, namely DME/F-12:1 medium.

The cleaning solution used in the culture method comprises the following components: physiological saline, 0.5mg/mL penicillin, 0.5mg/mL kanamycin sulfate, 5 μg/mL amphotericin B.

It should be noted that, the method for culturing primary breast cancer cells provided in this embodiment can be used for culturing primary breast cancer tumor cells, and also for culturing primary breast cancer metastasis tumor cells.

Example 3

This embodiment is a further improvement of embodiment 1 and/or embodiment 2, and the repeated description is omitted.

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

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

It should be noted that the above-described embodiments are exemplary, and that a person skilled in the art, in light of the present disclosure, may devise various solutions that fall within the scope of the present disclosure and fall within the scope of the present disclosure. It should be understood by those skilled in the art that the present description and drawings are illustrative and not limiting to the claims. The scope of the invention is defined by the claims and their equivalents. The description of the invention encompasses multiple inventive concepts, such as "preferably," "according to a preferred embodiment," or "optionally," all means that the corresponding paragraph discloses a separate concept, and that the applicant reserves the right to filed a divisional application according to each inventive concept. Throughout this document, the word "preferably" is used in a generic sense to mean only one alternative, and not to be construed as necessarily required, so that the applicant reserves the right to forego or delete the relevant preferred feature at any time.

Claims

1. An HBRCA-959 cell line, which is characterized in that the HBRCA-959 cell line is preserved in the China general microbiological culture Collection center (ccm) with the preservation number of CGMCC No. 23022.

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

3. The cell line according to claim 1 or 2, wherein the cell line is used for the development of a medicament for inhibiting or treating breast cancer.

4. Use of a cell line according to claim 1 or 2, in an in vitro study of breast cancer in humans.

5. The use according to claim 4, wherein the in vitro study of breast cancer is an animal model study of breast cancer.