CN110885788B - Primary cells of human intestinal cancer, application and culture method - Google Patents

Primary cells of human intestinal cancer, application and culture method Download PDF

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CN110885788B
CN110885788B CN201911286394.0A CN201911286394A CN110885788B CN 110885788 B CN110885788 B CN 110885788B CN 201911286394 A CN201911286394 A CN 201911286394A CN 110885788 B CN110885788 B CN 110885788B
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intestinal cancer
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human intestinal
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CN110885788A (en
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李春凤
赵九娥
智慧芳
倪君君
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Beijing Harmony Health Medical Diagnostics Co ltd
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Abstract

The invention provides a primary human intestinal cancer cell, an application and a culture method, wherein the primary human intestinal cancer cell is named as HCOC-691 with the preservation number of CGMCC NO. 18528. The preservation address is as follows: china, beijing. The human intestinal cancer primary cell can be applied to the research of pathogenesis of cancer-related diseases, the research and the detection of drug sensitivity and the research of anti-tumor drugs. Compared with the cell state of the primary tumor cells cultured in the existing purification culture process, the cell state of the primary human intestinal cancer cells is better.

Description

Primary cells of human intestinal cancer, application and culture method
Technical Field
The invention relates to the technical field of biology, in particular to a primary cell of human intestinal cancer, application and a culture method.
Background
Intestinal cancer is a common digestive tract disease, generally, the biological characteristics of primarily cultured tumor cells are not changed greatly due to the fact that tissues are just isolated, the original genetic characteristics are still kept, the gene retention amount is over 90 percent, the intestinal cancer cell is suitable for biochemical molecular experiments, drug sensitivity experiments and mechanism research related experiments, and the data of the intestinal cancer cell is more convincing. The establishment of the primary human intestinal cancer cells has important significance for the establishment of cell strains, drug sensitivity detection experiments, personalized treatment and research on the pathogenesis and treatment mechanism of intestinal cancer.
At present, the acquisition of primary human intestinal cancer cells is usually carried out by purely enzyme digestion. However, this method may cause poor cell status or death due to over-digestion, which in turn leads to the failure of primary cell culture for human intestinal cancer.
Disclosure of Invention
The embodiment of the invention provides a primary human intestinal cancer cell, an application and a culture method, which can culture the primary human intestinal cancer cell in a good state.
For this reason, the above object of the present invention is achieved by the following technical solutions:
in a first aspect, the invention provides a primary human intestinal cancer cell, which is named as human intestinal cancer cell HCOC-691 with the collection number of CGMCC NO. 18528.
Preferably, the first and second electrodes are formed of a metal,
the karyotype of the primary human intestinal cancer cell HCOC-691 is as follows: the number of chromosomes was 49, a hyperdiploid karyotype.
In a second aspect, the present invention provides a use of the primary human intestinal cancer cell of the first aspect in the study of the pathogenesis of cancer-related diseases.
In a third aspect, the invention also provides an application of the human intestinal cancer primary cell in the first aspect in research of anti-tumor drugs.
In a fourth aspect, the invention also provides an application of the human intestinal cancer primary cell in drug sensitivity research and detection.
In a fifth aspect, the present invention provides a method for culturing primary cells of human intestinal cancer, comprising:
washing intestinal cancer tissues by using a washing solution, wherein the washing solution comprises: physiological saline containing antibiotics;
digesting the cleaned intestinal cancer tissue by using cell dispersing enzyme;
digesting the cells obtained after the cell dispersing enzyme digestion treatment by using a cell digestion solution;
culturing the cells obtained after the digestion treatment of the cell digestive juice by using a serum-containing culture medium and a serum-free culture medium, wherein the serum-containing culture medium comprises: DF medium, 10% Fetal Bovine Serum (FBS) and the antibiotic, the serum-free medium comprising: DF media, supplements and said antibiotics;
when the confluency of the cultured cells reaches 70-90%, purifying the cultured cells by combining a pancreatic enzyme differential digestion method, a differential adherence method and a repeated adherence method to obtain primary cells of human intestinal cancer.
In particular, the cell dispersing enzyme may be: contains 1-10mg/ml collagenase I and 0.2-2mg/ml hyaluronidase.
With respect to the concentration of collagenase I, 1-10mg/ml refers to any concentration within the range of 1mg/ml to 10mg/ml, such as 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml, 3mg/ml, 3.3mg/ml, 3.5mg/ml, 4mg/ml, 4.5mg/ml, 5mg/ml, 5.5mg/ml, 6mg/ml, 6.6mg/ml, 7mg/ml, 7.5mg/ml, 8mg/ml, 8.4mg/ml, 8.8mg/ml, 9mg/ml, 9.5mg/ml and 10 mg/ml.
0.2-2mg/ml means any concentration in the range of 0.2mg/ml to 2mg/ml with respect to the concentration of hyaluronidase, such as 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, 0.5mg/ml, 0.6mg/ml, 0.7mg/ml, 0.8mg/ml, 0.9mg/ml, 1mg/ml, 1.1mg/ml, 1.2mg/ml, 1.3mg/ml, 1.4mg/ml, 1.5mg/ml, 1.6mg/ml, 1.7mg/ml, 1.8mg/ml and 2 mg/ml.
Preferably, the first and second electrodes are formed of a metal,
the additive, comprising: glutamine, insulin, transferrin, sodium selenite, epidermal growth factor EGF, hydrocortisone and bovine serum albumin BSA.
Preferably, the first and second electrodes are formed of a metal,
the antibiotic comprises: penicillin, kanamycin sulfate, amphotericin B, and vancomycin.
Specifically, in the serum-free medium supplement, the concentration of glutamine is 1-5mM, the concentration of insulin is 5-30mg/L, the concentration of transferrin is 5-20mg/L, the concentration of sodium selenite is 5-20 μ g/L, EGF, the concentration of hydrocortisone is 10-100nM, and the concentration of BSA is 1-5 mg/ml.
Specifically, the concentrations of penicillin, kanamycin sulfate, amphotericin B, and vancomycin in the serum-free medium, the serum-containing medium, and the antibiotics in the washing solution are 0.1 to 0.5mg/ml, and 1 to 5. mu.g/ml, respectively.
Specifically, the concentration of penicillin in the cleaning solution is 0.1-0.5mg/ml, the concentration of kanamycin sulfate is 0.1-0.5mg/ml, the concentration of amphotericin B is 0.25-0.5 mu g/ml, and the concentration of vancomycin is 1-5 mu g/ml.
With respect to the concentration of glutamine, 1-5mM means any concentration in the range of 1mM to 5mM, for example, 1mM, 1.3mM, 1.8mM, 2mM, 2.2mM, 2.7mM, 3mM, 3.3mM, 3.8mM, 4mM, 4.3mM, 4.8mM, and 5 mM.
With respect to the concentration of insulin, 5-30mg/L refers to any concentration within the range of 5mg/L to 30mg/L, such as 5mg/L, 5.3mg/L, 5.8mg/L, 6mg/L, 6.3mg/L, 6.8mg/L, 7mg/L, 9mg/L, 11mg/L, 13mg/L, 14mg/L, 15mg/L, 17mg/L, 19mg/L, 21mg/L, 22mg/L, 23mg/L, 25mg/L, 26mg/L, 29mg/L, and 30 mg/L.
With respect to the concentration of transferrin, 5-20mg/L refers to any concentration in the range of 5mg/L to 20mg/L, such as 5mg/L, 5.3mg/L, 5.7mg/L, 6mg/L, 6.4mg/L, 8mg/L, 8.7mg/L, 10mg/L, 11.4mg/L, 12mg/L, 13mg/L, 15mg/L, 17mg/L, and 20 mg/L.
With respect to the concentration of sodium selenite, 5-20. mu.g/L refers to any concentration in the range of 5. mu.g/L to 20. mu.g/L, such as 5. mu.g/L, 5.5. mu.g/L, 6. mu.g/L, 6.5. mu.g/L, 7. mu.g/L, 9. mu.g/L, 11. mu.g/L, 13. mu.g/L, 15. mu.g/L, 17. mu.g/L and 20. mu.g/L.
With respect to the concentration of EGF, 5-20. mu.g/L means any concentration in the range of 5. mu.g/L to 20. mu.g/L, such as 5. mu.g/L, 5.8. mu.g/L, 6. mu.g/L, 7. mu.g/L, 9. mu.g/L, 11. mu.g/L, 13. mu.g/L, 15. mu.g/L, 17. mu.g/L and 20. mu.g/L.
With respect to the concentration of hydrocortisone, 10-100nM refers to any concentration in the range of 10nM to 100nM, e.g., 10nM, 20nM, 30nM, 40nM, 50nM, 60nM, 70nM, 80nM, 90nM and 100 nM.
With respect to the concentration of BSA, 1-5mg/ml means any concentration within the range of 1mg/ml to 5mg/ml, such as 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml, 3mg/ml, 3.5mg/ml, 4mg/ml, 4.5mg/ml and 5 mg/ml.
With respect to the concentration of penicillin, 0.1-0.5mg/ml refers to any concentration in the range of 0.1mg/ml to 0.5mg/ml, such as 0.1mg/ml, 0.15mg/ml, 0.25mg/ml, 0.35mg/ml, 0.45mg/ml and 0.5 mg/ml.
As for the concentration of kanamycin sulfate, 0.1-0.5mg/ml means any concentration within the range of 0.1mg/ml to 0.5mg/ml, for example, 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml and 0.5 mg/ml.
With respect to the concentration of amphotericin B, 0.25 to 0.5. mu.g/ml means any concentration in the range of 0.25. mu.g/ml to 0.5. mu.g/ml, such as 0.25. mu.g/ml, 0.3. mu.g/ml, 0.35. mu.g/ml, 0.4. mu.g/ml, 0.45. mu.g/ml and 0.5. mu.g/ml.
With respect to the concentration of vancomycin, 1-5. mu.g/ml refers to any concentration ranging from 1. mu.g/ml to 5. mu.g/ml, such as 1. mu.g/ml, 1.5. mu.g/ml, 2. mu.g/ml, 3. mu.g/ml, 3.5. mu.g/ml, 4. mu.g/ml and 5. mu.g/ml.
Preferably, the first and second electrodes are formed of a metal,
the method for culturing the cells obtained after the digestion treatment of the cell digestive juice by using the serum-containing culture medium and the serum-free culture medium comprises the following steps:
filtering the cells obtained after the cell digestive juice digestion treatment through a 200-plus 300 mu m nylon membrane, and collecting the filtered cells;
centrifuging at 1000-2Culturing in an incubator;
when the cultured cells grow completely adherent, the serum-containing culture medium is replaced by a serum-free culture medium, and the cells are placed at 37 ℃ and 5% CO2And continuing culturing in the incubator.
Specifically, when tumor cells are cultured, it is necessary to filter non-tumor tissue, such as adipose tissue, using a nylon membrane, then centrifuge the filtered cells, collect the precipitated cells, and re-suspend the cells in a serum-containing medium in a cell culture flask at 37 ℃ and 5% CO2Culturing for 24-72h in an incubator to ensure that the cells grow completely adherent.
Preferably, the first and second electrodes are formed of a metal,
the method for purifying the cultured cells by combining a pancreatin differential time digestion method, a differential time wall attaching method and a repeated wall attaching method to obtain primary cells of the human intestinal cancer comprises the following steps:
removing the serum-free medium from the cultured cells and treating with EDTA-Trypsin digestion;
observing under a microscope, discarding fibroblasts digested first by EDTA-Trypsin, then collecting cells after digestion while digesting, and stopping digestion of the collected cells by using DF10 culture medium until all cells are digested;
sequentially centrifuging at 2000rpm of 1000-2Culturing in incubator for 10-120min to make fibroblast adhere to wall;
collecting the primary cells of the human intestinal cancer which are not attached to the wall, continuously culturing, and repeatedly attaching to the wall for 2-5 times to obtain the primary cells of the human intestinal cancer.
Specifically, in the process of purifying cultured cells, the serum-free culture medium is removed firstly, then EDTA-Trypsin (i.e., Trypsin-EDTA digestive solution) is added, the cells are observed under a microscope to shrink and become round, fibroblasts digested firstly by EDTA-Trypsin are discarded, then the cells after digestion treatment are collected while digestion is carried out, the collected cells are stopped digesting by using DF10 culture medium until all the cells are digested completely, then the supernatant is centrifuged, and the serum-free culture medium is used for resuspension in sequence, and 5% CO is carried out at 37 ℃2Placing in a cell culture box for 10-120min, collecting primary cells of human intestinal cancer which are not attached to the wall after the fibroblasts are attached to the wall, transferring the primary cells into a new cell culture bottle for continuous culture, and repeatedly attaching to the wall for 2-5 times, so that the effect of completely removing the fibroblasts can be achieved, and meanwhile, the phenomenon that the cells are poor in state or die due to over digestion can be avoided.
It is understood that the amount of EDTA-Trypsin is slightly larger than that of the cultured cells. Typically 25cm2The cell culture bottle adopts 1-2ml of EDTA-Trypsin.
Specifically, DF10 media included: DF medium and 10% FBS.
Specifically, the digestion time of EDTA-Trypsin is 5-60 min.
Preferably, the first and second electrodes are formed of a metal,
the method for digesting the cleaned intestinal cancer tissue by using the cell dispersing enzyme comprises the following steps:
chopping the cleaned intestinal cancer tissues into a crushed mud shape;
centrifuging the muddy intestinal cancer tissue at 1000-;
adding cell dispersing enzyme in sequence, and standing at 37 deg.C and 5% CO2Digesting in the incubator for 0.5-2h by low speed oscillation, adding DF10 culture medium to stop reaction, blowing and mixing uniformly;
centrifugation was carried out at 1000-2000rpm for 3-8min in sequence, and the supernatant was removed to obtain the cells after digestion with the cell dispersing enzyme.
Specifically, in order to facilitate the subsequent digestion of the intestinal cancer tissue into tumor cells of a suitable size, the intestinal cancer tissue needs to be chopped into a minced state before being digested, and then the supernatant is removed by centrifugation to remove impurities in the intestinal cancer tissue, such as adipose tissue, membrane tissue, connective tissue, and necrotic tissue. And adding a DF culture medium for resuspension, and then dispersing the intestinal cancer tissues by using cell dispersing enzyme under the condition of low-speed oscillation to prevent cell agglomeration. Stopping digestion by using DF10 culture medium after digestion, blowing to disperse and mix evenly, centrifuging to remove supernatant, and reserving precipitated cells.
Preferably, the first and second electrodes are formed of a metal,
the method for digesting the cells obtained after the cell dispersing enzyme digestion treatment by using the cell digestive juice comprises the following steps:
and adding a cell digestive solution into the cells obtained after the digestion treatment of the cell dispersing enzyme, blowing, uniformly mixing, standing at room temperature for 3-8min, adding the DF10 culture medium to stop the reaction, blowing, uniformly mixing, and obtaining the cells digested by the cell digestive solution.
Specifically, after dispersed cells are obtained, adding a cell digestive juice, terminating the reaction by using a DF10 culture medium, blowing, uniformly mixing, centrifuging and removing a supernatant to obtain the cells after the cell digestive juice is digested.
Specifically, the cell digest may be EGTA-Trypsin.
The embodiment of the invention provides a primary cell of human intestinal cancer, an application and a culture method, wherein a cleaning solution is used for cleaning and pre-treating intestinal cancer tissues to remove impurities, the cleaned intestinal cancer tissues are digested into tumor cells with proper size by cell dispersing enzyme and cell digestive fluid in sequence on the premise of not damaging cell membranes, and then a serum-containing culture medium and a serum-free culture medium are used for culture. The cleaning solution, the culture medium containing serum and the serum-free culture medium all contain antibiotics, so that the antibacterial and bactericidal effects can be achieved when intestinal cancer tissues are cleaned and tumor cells are cultured, and the influence of pollution on normal cell culture is prevented. When the confluence degree of the cells reaches 70-90%, a pancreatic enzyme differential digestion method, a differential adherence method and a repeated adherence method are combined for purification, so that the condition of the cells is poor or the cells die due to over digestion in the purification process is avoided, the human intestinal cancer primary cells with good conditions and without fibroblast and other foreign cells are obtained, and the success rate of culturing the human intestinal cancer primary cells is improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a cell morphology of a primary human intestinal cancer cell provided by an embodiment of the present invention;
FIG. 2 is a graph showing the growth of primary human intestinal cancer cells according to an embodiment of the present invention;
FIG. 3 is a graph showing the immunofluorescence assay results of primary human intestinal cancer cells according to an embodiment of the present invention;
FIG. 4 is a chromosome karyotype analysis chart of primary human intestinal cancer cells provided by an embodiment of the present invention;
FIG. 5 is a STR genotyping graph of primary human intestinal cancer cells, according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer and more complete, the technical solutions in the embodiments of the present invention will be described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention, and based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without creative efforts belong to the scope of the present invention.
Example 1: isolation culture of primary cells of human intestinal cancer
(1) Collecting partial intestinal cancer tissues excised during surgery of a patient under the condition that the patient or a patient guardian agrees;
(2) transferring the intestinal cancer tissues collected in the step (1) into a cell culture dish, washing for 8 times by using a cleaning solution (physiological saline containing 0.4mg/ml penicillin, 0.4mg/ml kanamycin sulfate, 0.4 mu g/ml amphotericin B and 4 mu g/ml vancomycin) to remove non-cancer tissue impurities such as fat, mucous membranes and the like;
(3) transferring the intestinal cancer tissues cleaned and treated in the step (2) into a new culture dish, cutting the intestinal cancer tissues cleaned and treated into small pieces by using scissors and a blade, and chopping the intestinal cancer tissues into a minced shape;
(4) transferring minced intestinal cancer tissue into 50ml centrifuge tube, centrifuging at 2000rpm for 8min, removing supernatant, sequentially adding 9ml DF culture medium for resuspension, sequentially adding 1ml cell dispersing enzyme containing 5mg/ml collagenase I and 1mg/ml hyaluronidase, and treating at 37 deg.C and 5% CO2Digesting in an incubator for 1h by low-speed oscillation; after the cell dispersing enzyme reaction is finished, adding 20ml of DF10 culture medium to stop the reaction, blowing and uniformly mixing; centrifuging at 1000rpm for 5min, removing supernatant, and collecting cell precipitate;
(5) adding 5ml of EGTA-Trypsin solution into the cell sediment obtained in the step (4), uniformly mixing, standing for 5min at room temperature, sequentially adding 20ml of DF10 culture medium to terminate the reaction, blowing and uniformly mixing to obtain cells after cell digestive juice digestion;
(6) filtering the cells obtained in the step (5) through a 200-micron nylon membrane, collecting the filtered cell sap into a 50ml centrifuge tube, centrifuging at 1500rpm for 5min, removing supernatant, and taking cell precipitates;
(7) resuspending the cell pellet from step (6) 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).
(8) When the cultured cells grow completely adherent, the serum-containing culture medium is replaced by a serum-free culture medium, and the cells are continuously cultured in a 5% CO2 culture box at 37 ℃.
Example 2: purification of primary human intestinal cancer cells
(1) When the confluency of the cells cultured in step (8) of example 1 reached 70-90%, the serum-free medium was removed and digested with 1-2ml of EDTA-Trypsin.
(2) Observing under a microscope until the cells are separated from the culture bottle, discarding fibroblasts digested by EDTA-Trypsin, collecting the cells after digestion while digesting, and stopping digestion of the collected cells by using DF10 culture medium until all the cells are digested.
(3) Centrifuging at 1500rpm for 6min, removing supernatant, resuspending with serum-free medium, and culturing at 37 deg.C with 5% CO2Placing in a cell culture box for 1.5h, collecting nonadherent cells after fibroblasts adhere to the wall, transferring into a new culture bottle for continuous culture, and repeatedly adhering to the wall for 3 times to completely remove the fibroblasts, thereby obtaining the primary human intestinal cancer cell HCOC-691.
In particular, it is contemplated that the purified human intestinal cancer primary cells can generally be further subcultured, and since the human intestinal cancer primary cells are purified, serum-free media can be used for subculturing. Therefore, in example 2, when the cells were cultured using the serum-free medium, the primary human intestinal cancer cells that were attached to the cell walls repeatedly until all fibroblasts were removed were subcultured without centrifugation, and the influence of the centrifugation on the primary human intestinal cancer cells was avoided.
Example 3: morphological observation
It can be found by observation under an inverted microscope that the primary cells of human intestinal cancer cultured after the above examples 1 and 2 have epithelial-like cell morphology, and the cell size and morphology in the cancer cell mass are different, lose polarity, are disorganized, and have rapid proliferation, no contact inhibition, and are mutually squeezed to form a stack or a mosaic. The results are shown in detail in FIG. 1.
Example 4: subculturing of primary human intestinal cancer cells
(1) The primary human intestinal cancer cells obtained in step (3) of example 2 were washed at least twice with 1-fold concentration (1X) of phosphate buffered saline PBS (0.01M, pH 7.4) when the confluency of the cells reached 70-90% to remove old serum-free medium and cells in poor exfoliated state, and then digested with 1-2ml of EDTA-Trypsin for 5-60 min.
(2) The cells after digestion were collected while observing under a microscope, and the collected cells were terminated with DF10 medium until all cells were digested.
(3) Sequentially centrifuging at 1000-2000rpm for 3-8min, removing supernatant, re-suspending with serum-free medium, inoculating into new culture flask, sequentially culturing at 37 deg.C and 5% CO2Culturing in a cell culture box.
(4) If necessary, (1X 10)6Individual) human intestinal 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.
Example 5: growth curve determination of primary human intestinal cancer cells
(1) The primary human intestinal cancer cells obtained in step (3) of example 2 were taken, and when the confluency of the cells reached 70-90%, the serum-free medium was removed, the cells were washed at least twice with PBS (0.01M, pH 7.4) to remove old serum-free medium and cells in a poor floating state, digested with 1-2ml of EDTA-Trypsin, and the digestion was stopped with DF 10.
(2) The cells were cultured at 5X 104The cells were inoculated in a 96-well plate at 200. mu.l/well.
(3) The measurement was started after 24 hours, and the absorbance was measured every 24 hours thereafter, and the average value was calculated.
(4) During the determination, the gel is stained with neutral red with a final concentration of 100 mug/ml for 2h, washed with PBS for 3 times, added with neutral red eluent (water: ethanol: acetic acid: 49: 50: 1), shaken and mixed for 10min, and measured for absorbance at 550nm for 8 days.
Table 1 below shows the growth curve data of human intestinal cancer primary cells HCOC-691.
TABLE 1
Time (sky) Relative OD value (average) (550nm) Error of the measurement
1 0.1200 0.0100
2 0.2500 0.0900
3 0.2800 0.0700
4 0.5600 0.2000
5 0.6200 0.1400
6 0.7900 0.2000
7 0.9600 0.2400
8 0.8900 0.1800
From Table 1 above, it can be seen that the human intestinal cancer primary cells HCOC-691 grew well for 8 consecutive days. The growth curve of primary human intestinal cancer cells HCOC-691 is shown in FIG. 2. In FIG. 2, the abscissa represents the cultivation time in (days) and the ordinate represents the OD value.
Specifically, the doubling time of the cancer cells of the human intestinal cancer primary cell HCOC-691 is 24-72 h. The cells grow actively, and the in vitro culture has stable cell growth characteristics.
Example 6: immunofluorescence identification of primary cells of human intestinal cancer
(1) Taking the human intestinal cancer primary cells obtained in the step (3) of the example 2, removing the serum-free culture medium when the confluence degree of the human intestinal cancer primary cells reaches about 70-90%, washing the cells at least twice by using PBS (0.01M, PH 7.4) to remove old serum-free culture medium and floating cells with poor state, digesting by using 1-2ml of EDTA-Trypsin, and inoculating the cells to a 48-well plate.
(2) When the cell confluence reaches 70% -90%, the serum-free medium is removed, PBS is washed for 3 times, 500 mul/hole of glacial methanol is added, and the cell is removed after being fixed for 30 min.
(3) Add 200. mu.l/well 2.5% BSA blocking solution, block for 60min at room temperature and aspirate the blocking solution.
(4) Add 150. mu.l/well of primary antibody (CK 181: 800 dilution, CR 1:1000 dilution); after incubation at 37 ℃ for 2h, aspirate.
(5) 150 μ l/well of fluorescent secondary antibody (Anti-Mouse-IgG-FITC 1:100 dilution; Cy 3-labeled goat Anti-rabbit IgG 1: 500 dilution) was added, incubated at room temperature for 1h in the dark, aspirated secondary antibody, and washed 3 times with PBS.
(6) 150 μ l/well of PBS was added, and photographed by fluorescence, objective lens × eyepiece (20 × 10).
Specifically, Cytokeratin (CK) is a common marker of epithelial cells, and only CK, but not Calretinin (CR), is expressed in epithelial cells and tumor cells derived from the epithelial cells. Immunofluorescence identification shows that the human intestinal cancer primary cell CK is strongly expressed, CR is not expressed, which indicates that the human intestinal cancer primary cell is derived from epithelial cells, and specific results are shown in figure 3.
Example 7: chromosome karyotype analysis and identification of primary cells of human intestinal cancer
(1) Cell culture: taking the human intestinal cancer primary cells obtained in the step (3) of example 2, removing the serum-free medium culture when the confluence degree of the cells reaches 70-90%, washing the cells at least twice by PBS (0.01M, PH 7.4) to remove old serum-free medium and cells with poor shedding state, digesting by 1-2ml of EDTA-Trypsin, observing under a microscope, collecting the digested cells while digesting, and stopping the digestion of the collected cells by DF10 medium until all the cells are digested. Centrifuge at 1000rpm for 5min and remove supernatant.
(2) Colchicine treatment: adding colchicine 20-25 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 deg.C water bath to obtain a hypotonic solution of 10ml, blowing with a pipette to obtain cell suspension, and treating in 37 deg.C water bath for 30-35 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) Centrifuging: 1300rpm, 10min, discard supernatant.
(6) Fixing: adding 6-8ml of fixing solution into a centrifuge tube, immediately and 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) And (3) second fixing: adding 6-8ml of fixing solution into a centrifuge tube, immediately and 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 0.2ml of new fixative into a centrifuge tube, slightly blowing with a suction tube to obtain cell suspension, taking out Borneolum Syntheticum from a freezing chamber of a refrigerator, adjusting to appropriate suspension, adding 1-2 drops of suspension dropwise into each tablet, and baking at 75 deg.C for 3 hr.
(9) Dyeing: 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. The good split phase was first looked for with a low power microscope and then observed with a high power oil lens.
The primary cells of the human intestinal cancer are identified to be heterotypic karyotypes through chromosome karyotype analysis, namely tumor cells of the hyperploid karyotype, the number of chromosomes is 49, the karyotype of the human intestinal cancer is different from that of normal cells of a human body, and the genetic characteristics of human malignant tumors are met. The specific result is shown in figure 4.
Example 8: short tandem repeat STR identification of primary cells of human intestinal cancer
(1) Taking the human intestinal cancer primary cells obtained in the step (3) of the example 2, and carrying out adherent growth on the human intestinal cancer primary cells (1X 10)6Respectively) washing the cells twice with PBS (0.01M, pH 7.4), sequentially digesting with 1-2ml of EDTA-Trypsin for 5-60min, and terminating the digestion reaction with 10ml of DF 10;
(2) centrifuging at 10000rpm for 1min, discarding the supernatant, adding 200 μ l buffer GA (cell/tissue genome DNA extraction kit DP304, Tiangen corporation), and shaking to completely suspend;
(3) adding 20 mul of protease K solution, and mixing uniformly;
(4) adding 200 μ l buffer solution GB (cell/tissue genome DNA extraction kit DP304, Tiangen corporation), fully reversing, mixing, standing at 70 deg.C for 10min, and centrifuging briefly;
(5) adding 200 μ l of anhydrous ethanol, shaking thoroughly, mixing for 15s, and centrifuging briefly;
(6) adding the obtained solution and flocculent precipitate into an adsorption column (cell/tissue genome DNA extraction kit DP304, Tiangen corporation), centrifuging at 12000rpm for 30s, and removing waste liquid;
(7) adding 500 μ l buffer GD (cell/tissue genome DNA extraction kit DP304, Tiangen corporation) into the adsorption column, centrifuging at 12000rpm for 30s, and removing waste liquid;
(8) adding 600 μ l of rinsing solution PW (cell/tissue genome DNA extraction kit DP304, Tiangen Co., Ltd.) into the adsorption column, centrifuging at 12000rpm for 30s, and removing waste liquid;
(9) transferring the adsorption column into another centrifuge tube, dripping 50-200 μ l of elution buffer TE (cell/tissue genome DNA extraction kit DP304, Tiangen corporation) 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) performing DNA composite amplification of 21 loci (20 STR loci and 1 individualized locus) by using an ABI 9700PCR (ABI company);
(11) detecting the amplified fragments by using ABI 3130XL type genetic analyzer;
(12) sample data were analyzed using the GenoMapper3.2 software for automated genotyping, STR typing results are shown in FIG. 5, and 21 loci were examined, expressed as "locus/allele length": D19S433/12.2/13, D5S818/11/11, D21S11/31/31, D18S51/12/14, D6S1043/18/19, AMEL/X/Y, D3S1358/16/17/18, D13S317/8/10, D7S820/10/12, D16S539/11/15, CSFIPO/10/13, PentaD/9/12, D2S441/15/17, vWA/14/16, D8S1179/14/17, TPOX/8/11, PentaE/13/22, TH01/7/9.3, D12S391/18/20, D2S1338/18/23, FGA/22/25.
In conclusion, STR identification is carried out on the purified human intestinal cancer primary cells, and the result proves that the cells are of single human source and have no cross contamination.
Example 9: primary cell drug sensitivity detection of human intestinal cancer (CD-DST method)
(1) Collagen gel drop culture
Primary human intestinal cancer cells obtained in step (3) of example 2 were mixed with a gel-forming solution (i: ii: iii: 8:1:1) uniformly, and the cell suspension was seeded into 6-well plates and 3 gel drops into another 6-well plate at 30 μ l/gel drop/well to serve 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.C2Overnight in the incubatorAnd (5) culturing.
(2) Anti-cancer drug exposure and cleaning
And (2) after culturing the primary human intestinal cancer cells obtained in the step (1) for 24 hours, adding an anti-cancer drug, and dyeing and fixing the 0-time group while arranging a negative Control hole (Control) without the drug and a positive Control hole treated by clinical intestinal cancer common drugs. 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 mu g/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. The drops were then scanned for analysis using the Primage image analysis system.
The results of drug sensitivity test (CD-DST method) of HCOC-691 primary cells of human intestinal cancer are shown in the following Table 2:
TABLE 2
Name of drug Cell survival rate
Fluorouracil and Folic acid formyl (5-FU/LV) 29.32％
Tiji ao capsule (TS-1) 23.78％
Capecitabine (CAP) 16.12％
Irinotecan (CPT-11) 37.27％
Oxaliplatin (L-OHP) 27.29％
The primary human intestinal cancer cells can be used for evaluating the drug effectiveness of fluorouracil + leucovorin (5-FU/LV), tegafur capsule (TS-1), Capecitabine (CAP), irinotecan (CPT-11) and oxaliplatin (L-OHP).
The primary human intestinal cancer cell is named as human intestinal cancer cell HCOC-691, and is preserved in China general microbiological culture Collection center (CGMCC) in 2019, 10 months and 16 days; the preservation address is as follows: china, Beijing, institute of sciences, China; the preservation number is CGMCC NO. 18528.
It should be noted that:
the serum-free medium in the above examples was: DF Medium (i.e., DME/F-121: 1 medium) supplemented with 3mM glutamine, 3mg/L insulin, 3mg/L transferrin, 10. mu.g/L sodium selenite, 10. mu.g/L EGF, 50nM hydrocortisone, 3mg/ml BSA, 0.3mg/ml penicillin, 0.3mg/ml kanamycin sulfate, 0.4. mu.g/ml amphotericin B, and 3. mu.g/ml vancomycin.
The serum-containing medium in the above examples was: DF medium, 10% FBS, 0.3mg/ml penicillin, 0.3mg/ml kanamycin sulfate, 0.4. mu.g/ml amphotericin B and 3. mu.g/ml vancomycin.
The DF10 medium in the above examples was a mixture of DF medium and 10% FBS.
CCTCC identification experiments prove that the following conclusion is reached.
1. Only one-third of the alleles were found in the D3S1358 locus of the COC-691 cell line, indicating that there was no cross-contamination of the human-derived cell line.
2. STR data for COC-691 cell lines in the ATCC and DSMZ databases, the profiles of which did not match the current data exactly.
3. The highest match between STR data of COC-691 cell line and D283(HTB-185) cell line in ATCC database was 79%.
The embodiments of the invention have at least the following beneficial effects:
1. the primary human intestinal cancer cells provided by the invention are isolated and cultured from intestinal cancer tissues of Chinese people, and the cells are not introduced with any foreign genes and are identified as cancer cell heterotypic karyotypes through karyotype analysis. STR genotyping identifies cells that are of single human origin and have no cross contamination.
2. The primary human intestinal cancer cells provided by the invention are epithelial cells which are different in size and form, lose polarity, disorganized in arrangement, free of contact inhibition, mutually extruded and stacked or inlaid when observed under a microscope.
3. The primary human intestinal cancer cell provided by the invention can be used for the research on pathogenesis of human cancer-related diseases, the research and detection on drug sensitivity, the research on anti-tumor drugs and the like.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
Finally, it is to be noted that: the above description is only a preferred embodiment of the present invention, and is only used to illustrate the technical solutions of the present invention, and not to limit the protection scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall fall within the protection scope of the present invention.

Claims (3)

1. The primary human intestinal cancer cell is named as human intestinal cancer primary cell HCOC-691 with the preservation number of CGMCC NO. 18528.
2. Use of the primary human intestinal cancer cell of claim 1 in a drug sensitivity assay.
3. Use of the primary human intestinal cancer cell of claim 1 in the preparation of an anti-tumor medicament.
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