CN111004782A

CN111004782A - Primary human intestinal cancer cell and culture method and application thereof

Info

Publication number: CN111004782A
Application number: CN201911286397.4A
Authority: CN
Inventors: 赵九娥; 贾玉霞; 智慧芳; 倪君君
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2019-12-13
Filing date: 2019-12-13
Publication date: 2020-04-14
Anticipated expiration: 2039-12-13
Also published as: CN111004782B

Abstract

The invention discloses a primary human intestinal cancer cell, a culture method and application thereof, wherein the primary human intestinal cancer cell is named as human intestinal cancer cell HCOC-751, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO. 18530. 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 human intestinal cancer cell and culture method and application thereof

Technical Field

The invention relates to the field of cell biology, in particular to a primary human intestinal cancer cell and a culture method and application thereof.

Background

The primary tumor cell culture refers to a process of obtaining tumor cells by surgical excision of tumor tissues from a tumor patient in vivo and performing short-term culture in vitro. 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. Therefore, the purification and culture of the primary human intestinal cancer cells have important significance for establishing cell strains, drug sensitivity detection experiments, personalized treatment and research on pathogenesis and treatment mechanism of intestinal cancer.

At present, in the purification and culture process of primary tumor cells, the tumor cells can be collected by simply relying on an enzyme digestion method. But has the disadvantage of poor cell status or death.

Disclosure of Invention

The invention provides a primary human intestinal cancer cell, a culture method and application thereof, and the cultured cell has a better state.

In order to achieve the purpose, the invention is realized by the following technical scheme:

in a first aspect, the invention provides a primary human intestinal cancer cell named human intestinal cancer cell HCOC-751, which has been deposited in China general Microbiological Culture Collection Center (CGMCC) at 16.10.2019 at the national institute of Microbiological Culture Collection, Ministry of sciences, Ministry of China, Ministry of microbiology, Ministry of Japan, and the accession number is CGMCC No. 3, wherein the accession number is 100101, and the accession number is CGMCC No. 18530.

The human intestinal cancer primary cell HCOC-751 provided by the invention has at least the following characteristics:

the characteristics are as follows: the primary cell is isolated and cultured from intestinal cancer tissues of Chinese people, and the cell is not transferred with any foreign gene.

And (2) the characteristics: observing the human intestinal cancer primary cell HCOC-751 under a microscope, finding out epithelial source cells which are different in size and shape, lose polarity, disorganized in arrangement, free of contact inhibition, mutually extruded and stacked or mosaic.

And (3) characteristics: human intestinal cancer primary cell HCOC-751 is identified as CK (cytokeratin) strongly expressed and CR (Calretinin) is not expressed by immunofluorescence.

And (4) the characteristics are as follows: human intestinal cancer primary cell HCOC-751 was characterized by karyotype analysis as a cancer cell heterotypic karyotype. The karyotype analysis shows that the number of chromosomes of the cell is 46, the cell is a tumor cell of a diploid karyotype, has the phenomena of translocation, inversion and the like, and accords with the genetic characteristics of human malignant tumors.

And (5) characteristics: the primary human intestinal cancer cell HCOC-751 is identified by STR genotyping, is a single human source and has no cross-contamination.

And (6) characteristics: the primary cell HCOC-751 of the human intestinal cancer can be used for the pathogenesis research, the drug sensitivity research and detection of human cancer-related diseases and the research of anti-tumor drugs.

In a second aspect, the present invention provides a method of culturing primary cells of human intestinal cancer, comprising:

taking fresh human intestinal cancer tissues, and carrying out primary isolated culture by using a culture medium containing 10% serum;

when the confluence degree of the cells obtained by primary separation and culture reaches 70-90%, removing a culture medium containing 10% serum, performing cell digestion treatment, collecting human intestinal cancer cells while digesting, and transferring the human intestinal cancer cells collected each time to a centrifuge tube containing the culture medium containing 10% serum;

centrifuging the collected human intestinal cancer cells, removing supernatant, carrying out heavy suspension by using a serum-free culture medium, inoculating the cells into a cell culture bottle for culture, collecting nonadherent human intestinal cancer cells after fibroblasts adhere to the wall, transferring the collected nonadherent human intestinal cancer cells into a new cell culture bottle for continuous culture, and repeatedly adhering the walls in the way until all the fibroblasts are eliminated to obtain purified human intestinal cancer primary cells.

In one embodiment of the present invention, after obtaining the purified primary human intestinal cancer cells, the method further comprises:

culturing the obtained purified primary human intestinal cancer cells by using a serum-free culture medium;

removing the serum-free culture medium when the confluence degree of the purified primary human intestinal cancer cells reaches 70-90%, performing cell digestion treatment, collecting the primary human intestinal cancer cells while digesting, and transferring the primary human intestinal cancer cells collected each time to a centrifuge tube containing a culture medium containing 10% serum;

and centrifuging the collected human intestinal cancer primary cells, removing supernatant, resuspending the cells in a serum-free culture medium, and inoculating the cells in a cell culture flask for culture to obtain the passaged human intestinal cancer primary cells.

Preferably, the serum-free medium comprises: DF culture medium, the additives are 1-5mM glutamine, 5-30mg/L insulin, 5-20mg/L transferrin, 5-20 μ g/L sodium selenite, 5-20 μ g/L EGF, 10-100nM hydrocortisone, 1-5mg/ml BSA, 0.1-0.5mg/ml penicillin, 0.1-0.5mg/ml kanamycin sulfate, 0.25-0.5 μ g/ml amphotericin B and 1-5 μ g/ml vancomycin.

Preferably, the cell culture conditions comprise: placing at 37 ℃ and 5% CO₂Culturing in an incubator.

Preferably, the cancer cell doubling time of the human intestinal cancer primary cells is 24-72 h.

In a third aspect, the present invention provides the use of the primary human intestinal cancer cell or the primary human intestinal cancer cell cultured by any one of the methods described above in the study of the pathogenesis of cancer-related diseases.

In a fourth aspect, the invention provides the application of the human intestinal cancer primary cell or the human intestinal cancer primary cell cultured by any one of the methods in drug sensitivity research and detection.

In a fifth aspect, the invention provides an application of the human intestinal cancer primary cell or the human intestinal cancer primary cell cultured by any one of the methods in screening or preparing an anti-tumor drug.

Preservation information

The human intestinal cancer cell HCOC-751 related by the invention is preserved in China General Microbiological culture Collection Center (CGMCC) at 16.10.2019, and the preservation address is No. 3 of Xilu 1 of Beijing province facing the sunny region in Beijing, the Microbiological research institute of Chinese academy of sciences, the postal code is 100101, and the preservation number is CGMCC NO. 18530.

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 schematic diagram of a growth curve of a primary human intestinal cancer cell according to an embodiment of the present invention;

FIG. 3 is a photograph of immunofluorescence assay of primary cells of human intestinal cancer 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

The embodiment of the invention provides a primary human intestinal cancer cell named as human intestinal cancer cell HCOC-751, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO. 18530.

Example 2

Example 2 is used to illustrate morphological observations of primary cells of human intestinal cancer.

Morphological observation results as shown in FIG. 1 were obtained by observing human intestinal cancer primary cell HCOC-751 under an inverted microscope.

Referring to fig. 1, it can be seen that primary human intestinal cancer cell HCOC-751 is in the form of epithelioid cell, and in the cancer cell mass, the cell size and shape are different, the polarity is lost, the arrangement is disordered, the cancer cell proliferation is fast, the cell proliferation is not inhibited by contact, and the cells are mutually pressed and stacked or embedded.

Example 3

Example 3 is used to illustrate the growth curve assay of primary human intestinal cancer cells.

Step 3.1: human intestinal cancer primary cells HCOC-751 confluency reached 70-90%, serum-free medium was removed, cells were washed at least twice with PBS (0.01M, pH7.4) to remove old serum-free medium and cells in poor exfoliated state, digested with 1-2ml EDTA-Trypsin, and the digestion was stopped with DF10 medium.

Step 3.2: the cells were cultured at 5X 10⁴The cells were inoculated in a 96-well plate at 200. mu.l/well.

Step 3.3: the measurement was started after 24 hours, and the absorbance was measured every 24 hours thereafter, and the average value was calculated.

Step 3.4: during the determination, the gel is stained with neutral red with the 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 then the absorbance value is determined at 550nm for 8 days. In this step, the data related to the growth curve shown in table 1 can be obtained, and further the schematic diagram of the growth curve shown in fig. 2 can be obtained.

TABLE 1

Time (sky)	Relative OD value (550nm)	Error of the measurement
			1	0.257	0.0556
2	0.55	0.1089
			3	0.931	0.2461
4	0.985	0.1381
			5	1.318	0.3309
6	1.627	0.2949
			7	1.901	0.2403
8	1.9	0.1351

Note that the relative OD values in table 1 are average values.

As is clear from Table 1 in conjunction with FIG. 1, the primary human intestinal cancer cell HCOC-751 grew well during this period, and the in vitro culture had stable cell growth characteristics.

Example 4

Example 4 is used to illustrate immunofluorescence identification of primary cells of human intestinal cancer.

Step 4.1: human intestinal cancer primary cells HCOC-751 reached a confluency of about 70-90%, serum-free medium was removed, the cells were washed at least twice with PBS (0.01M, pH7.4) to remove old serum-free medium and cells in poor exfoliated state, digested with 1-2ml of EDTA-Trypsin, and inoculated into 48-well plates.

Step 4.2: when the cell confluence reaches 70-90%, the serum-free medium is removed, washed 3 times with PBS, and removed after 30min of fixation by adding 500. mu.l/well of ice methanol.

Step 4.3: add 200. mu.l/well 2.5% BSA blocking solution, block for 60min at room temperature and aspirate the blocking solution.

Step 4.4: add 150. mu.l/well of primary antibody (CK 181: 800 dilution, CR 1:1000 dilution); after incubation at 37 ℃ for 2h, aspirate.

Step 4.5: 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.

Step 4.6: 150 μ l/well of PBS was added, and photographed by fluorescence, objective lens × eyepiece (20 × 10).

In this example, the immunofluorescence assay results shown in FIG. 3 were obtained.

In detail, CK is a common marker of epithelial cells and tumors thereof, and only CK is expressed in epithelial cells and tumor cells derived therefrom, but CR is not expressed. Referring to FIG. 3, it was found by immunofluorescence assay that HCOC-751CK is strongly expressed and CR is not expressed, indicating that HCOC-751 is derived from epithelial cells.

Example 5

Example 5 is used to illustrate the karyotyping of primary human intestinal cancer cells.

Step 5.1: cell culture

When the confluence of HCOC-751 of primary human intestinal cancer cells reaches 70-90%, serum-free medium is removed, the cells are washed at least twice with PBS (0.01M, pH7.4) to remove old serum-free medium and cells in a poor exfoliated state, the cells are digested with 1-2ml of EDTA-Trypsin, the tumor cells are collected while digesting under a microscope, and the digested tumor cells are terminated with DF10 medium. After all digestion, the supernatant was removed by centrifugation at 1000rpm for 5 min.

Step 5.2: colchicine treatment

Adding colchicine 20-25 μ l with concentration of 300 μ g/ml, and treating in 37 deg.C incubator for 25 min.

Step 5.3: 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.

Step 5.4: 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.

Step 5.5: centrifugation

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

Step 5.6: 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.

Step 5.7: 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.

Step 5.8: 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.

Step 5.9: dyeing process

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

Step 5.10: 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.

In this step, the karyotype analysis and identification result shown in FIG. 4 can be obtained.

Referring to FIG. 4, it can be seen that HCOC-751, a primary cell of human intestinal cancer, was characterized by karyotype analysis as a heterotypic cancer cell karyotype, which is different from the karyotype of normal human cells. The karyotype analysis shows that the number of chromosomes of the cell is 46, the cell is a tumor cell of a diploid karyotype, has the phenomena of translocation, inversion and the like, and accords with the genetic characteristics of human malignant tumors.

Example 6

Example 6 is used to illustrate STR identification of primary human intestinal cancer cells.

Step 6.1: human intestinal cancer primary cell HCOC-751 (1X 10) growing adherently⁶One), the cells were washed twice with PBS (0.01M, pH7.4), digested with 1-2ml EDTA-Trypsin for 5-60min, and the digestion was stopped with 10ml DF10 medium.

Step 6.2: centrifugation was carried out at 10000rpm for 1min, the supernatant was discarded, 200. mu.l of buffer GA (cell/tissue genomic DNA extraction kit DP304, Tiangen Co.) was added, and shaking was carried out until suspension was completed.

Step 6.3: add 20. mu.l of protease K solution and mix well.

Step 6.4: 200. mu.l of buffer GB (cell/tissue genomic DNA extraction kit DP304, Tiangen Co.) was added, mixed well by inversion, left at 70 ℃ for 10min, and centrifuged briefly.

Step 6.5: adding 200 μ l of anhydrous ethanol, shaking thoroughly, mixing for 15s, and centrifuging briefly.

Step 6.6: the resulting solution and flocculent precipitate were both added to an adsorption column (cell/tissue genomic DNA extraction kit DP304, Tiangen Co., Ltd.), centrifuged at 12000rpm for 30s, and the waste liquid was removed.

Step 6.7: to the adsorption column was added 500. mu.l of buffer GD (cell/tissue genomic DNA extraction kit DP304, Tiangen Co.), centrifuged at 12000rpm for 30s, and the waste liquid was removed.

Step 6.8: to the adsorption column, 600. mu.l of a rinsing solution PW (cell/tissue genomic DNA extraction kit DP304, Tiangen Co., Ltd.) was added, centrifuged at 12000rpm for 30 seconds, and the waste liquid was removed.

Step 6.9: 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 2-5 min, centrifuging at 12000rpm for 2min, and collecting the extracted DNA solution into the centrifuge tube.

Step 6.10: the DNA multiplex amplification of 21 loci (20 STR loci and 1 individuality locus) was performed using ABI 9700PCR amplification apparatus (ABI Co.).

Step 6.11: detection of the amplified fragments was performed using a ABI 3130XL type genetic analyzer.

Step 6.12: the sample data was analyzed using GenoMapper3.2 software for automated genotyping.

In this step, STR typing results can be shown in fig. 5, where 21 loci are detected, expressed as "locus/allele length": D19S433/14.2/16.2, D5S818/10/13, D21S11/32.2/33.2, D18S51/15/15, D6S1043/13/13, AMEL/X/X, D3S1358/14/16, D13S317/10/12, D7S820/11/11, D16S539/9/12, CSFIPO/12/12, PentaD/9/11, D2S441/10/14, vWA/14/19, D8S1179/13/14, TPOX/8/11, PentaE/15/15, TH01/6/9, D12S391/17/21, D2S 8/23/24, FGA/23/24.

Referring to FIG. 5, STR genotyping identified human intestinal cancer primary cells HCOC-751 as single human source, and no cross-contaminating cells.

Example 7

Example 7 is intended to illustrate the use of primary human intestinal cancer cells in the study of the pathogenesis of cancer-related diseases.

In this example, the pathogenesis of human intestinal cancer-related diseases was studied using human intestinal cancer primary cell HCOC-751.

Example 8

Example 8 is used to illustrate the use of primary human intestinal cancer cells in drug sensitivity studies and assays.

In this example, the CD-DST method was used to study the application of HCOC-751, a primary cell of human intestinal cancer, in drug sensitivity studies and assays.

Step 8.1 collagen gel drop culture

Primary human intestinal cancer cells HCOC-751 were mixed well with gel-forming solution (i: ii: iii: 8:1:1), and the cell suspension was inoculated into 6-well plates at 30 μ l/gel drop and 3 gel drops/well, and at the same time, 3 gel drops were inoculated into another 6-well plate as 0-time control group. Adding 3mL DF10 culture medium after the gel drops solidify for 1-2h, and adding 5% CO at 37 deg.C₂The culture was carried out overnight in an incubator.

Step 8.2 anti-cancer drug contact and washing

After 24h of culture, adding anti-cancer drugs, setting a negative Control hole (Control) without drugs and a positive Control hole treated by clinical common drugs for intestinal cancer, and simultaneously dyeing and fixing the 0-time group. After the corresponding time of culture, the cells were washed 2 times with DF medium for 15min each, and then cultured for 5 additional days with serum-free medium, wherein the medium was changed once on day 3.

Step 8.3 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. Then, the glue droplets were subjected to scanning analysis using a Primage image analysis system.

In this step, the results of drug sensitivity detection shown in table 2 were obtained.

TABLE 2

Name of drug	Cell survival rate
		Fluorouracil (continuous intravenous infusion) (5-FU)	78.68％
Fluorouracil and Folic acid formyl (5-FU/LV)	52.63％
		Oxaliplatin + tegafol capsule (SOX)	41.81％
Irinotecan + fluorouracil (FOLFIRI)	44.9％
		Capecitabine + oxaliplatin (XELOX)	50.57％

Example 9

Example 9 is used to illustrate the application of human intestinal cancer primary cells in screening or preparing anti-tumor drugs.

In this example, human intestinal cancer primary cell HCOC-751 was used to screen or prepare antitumor drugs.

By combining the above examples 2 to 9, it is understood that the human intestinal cancer primary cell HCOC-751 has at least the following characteristics:

And (2) the characteristics: observing primary cells of human intestinal cancer under a microscope, and finding out epithelial source cells which are different in size and shape, lose polarity, are disorganized in arrangement, are not inhibited by contact, are mutually extruded and are stacked or inlaid.

And (3) characteristics: the CK is strongly expressed and CR is not expressed by the identification of immunofluorescence.

And (4) the characteristics are as follows: the cancer cell is identified as abnormal karyotype by karyotype analysis. The karyotype analysis shows that the number of chromosomes of the cell is 46, the cell is a tumor cell of a diploid karyotype, has the phenomena of translocation, inversion and the like, and accords with the genetic characteristics of human malignant tumors.

And (5) characteristics: STR genotyping identifies cells that are of single human origin and have no cross contamination.

And (6) characteristics: can be used for the pathogenesis research, drug sensitivity research and detection of human cancer-related diseases and the research of anti-tumor drugs.

Example 10

Example 10 is intended to illustrate a method for culturing primary cells of human intestinal cancer.

Step 10.1: fresh human intestinal cancer tissues are taken and subjected to primary isolation culture by using a culture medium containing 10% serum.

Step 10.2: when the confluence degree of the cells cultured by primary separation reaches 70-90%, removing the culture medium containing 10% serum, performing cell digestion treatment, collecting human intestinal cancer cells while digesting, and transferring the human intestinal cancer cells collected each time to a centrifuge tube containing the culture medium containing 10% serum.

Step 10.3: centrifuging the collected human intestinal cancer cells, removing supernatant, carrying out heavy suspension by using a serum-free culture medium, inoculating the cells into a cell culture bottle for culture, collecting nonadherent human intestinal cancer cells after fibroblasts adhere to the wall, transferring the collected nonadherent human intestinal cancer cells into a new cell culture bottle for continuous culture, and repeatedly adhering the walls in the way until all the fibroblasts are eliminated to obtain purified human intestinal cancer primary cells.

In the present example, in step 10.3, it is preferred to culture the cells in serum-free medium, rather than in 10% serum-containing medium.

In step 10.3, after the fibroblasts adhere to the wall, the nonadherent human intestinal cancer cells can be directly sucked and transferred into a new cell culture bottle, the culture is continued by using a serum-free culture medium, the fibroblasts preferentially adhere to the wall in the process of continuous culture, after the fibroblasts adhere to the wall, the nonadherent human intestinal cancer cells are directly sucked again and transferred into the new cell culture bottle, and the process is circulated so as to realize repeated adherence until all the fibroblasts are removed.

Considering that the purified human intestinal cancer primary cells can be usually further subcultured, and since the human intestinal cancer primary cells are purified, serum-free medium is used for subculturing. Therefore, when the serum-free culture medium is used for culturing the cells in the step 10.3, the cells are attached to the wall repeatedly until the primary human intestinal cancer cells are removed of all fibroblasts, and the cells can be directly transferred to a new culture bottle for subculture without centrifugation, so that the damage influence on the primary human intestinal cancer cells caused by the centrifugation operation can be avoided.

In addition, when the cells are attached to the wall repeatedly until all fibroblasts are eliminated, if the primary cells of the human intestinal cancer are also partially attached to the wall, in order to collect the attached primary cells of the human intestinal cancer, the primary cells of the human intestinal cancer can be directly digested by EDTA-Trypsin after the serum-free culture medium is removed.

However, if the serum-containing medium is used to culture the cells in step 10.3, the cells attached to the wall repeatedly until all fibroblasts are eliminated need to be centrifuged to remove the serum-containing medium, and then the cells can be transferred to a new culture bottle for subculture, but the centrifugation operation is easily harmful to the primary human intestinal cancer cells.

In addition, when the cells adhere to the wall repeatedly until all fibroblasts are eliminated, if the primary human intestinal cancer cells also adhere to the wall partially, in order to collect the adherent primary human intestinal cancer cells, the serum-containing culture medium is removed, cleaning treatment is also needed, and then EDTA-Trypsin digestion treatment can be used. This is because the serum-containing medium usually remains on the surface of the primary cells of human intestinal cancer after the serum-containing medium is removed, and the presence of the serum-containing medium affects the digestion. Furthermore, when a serum-containing medium is used, the number of cell culture steps is increased because a washing step is also performed, as compared with the case where a serum-free medium is used, thereby increasing the possibility of cell contamination.

In the embodiment of the invention, by collecting while digesting, the digested human intestinal cancer cells can be collected in time and digestion is stopped, so that the condition that the cells are in a bad state or die due to over digestion is avoided. In addition, based on a differential adherence method, fibroblasts can be well removed by repeated adherence. Therefore, based on the culture method, the intestinal cancer cells can be quickly and stably separated and purified, so that purified primary human intestinal cancer cells are obtained by culture, the cell purification and removal efficiency is improved, the purification period is shortened, few pollutants are generated, the yield and purity of tumor cells are large, the purified and cultured cells are good in state, the culture success rate is high, and the defect that the molecular biological experiment result of the existing human intestinal cancer cell strain is greatly different from the clinical individual result can be overcome.

When the number of the purified human intestinal cancer primary cells meets the biological preservation requirement, one part of the purified human intestinal cancer primary cells can be directly taken for biological preservation, and the rest part can be used for cell characteristic analysis and cell application research and can be sealed and preserved by liquid nitrogen for later use.

For example, 1 × 10 may be used⁶The purified primary human intestinal cancer cells are resuspended in 1-2ml of cell freezing medium (90% fetal bovine serum and 10% DMSO, v/v), and then stored in liquid nitrogen for later use. Wherein DMSO (dimethyl sulfoxide) is dimethyl sulfoxide.

Next, the specific implementation steps of the primary isolation culture and the purification culture will be described.

In the above culture method, primary isolation culture is first required. In the embodiment of the present invention, the specific procedures of the primary isolation culture may be as follows (a1) - (a 6):

(a1) cleaning fresh intestinal cancer tissues by using cleaning fluid to remove impurities;

(a2) chopping the intestinal cancer tissue treated in the step (a1) into paste;

(a3) digesting the minced tumor tissue in the step (a2) by using cell dispersing enzyme, terminating the reaction after the digestion is finished, and centrifuging to take a precipitate;

(a4) adding a cell digestive juice into the cell sediment obtained in the step (a3), and terminating the reaction after digestion is finished;

(a5) filtering the cell fluid obtained in the step (a4) by a nylon membrane, and centrifuging to obtain a precipitate;

(a6) resuspending the cells from step (a5) with DF10 medium into cell culture flasks at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator.

Wherein, the DF10 culture medium is a DF culture medium containing 10% FBS (fetal bovine serum).

In the above culture method, purification culture can be performed after primary isolation culture is completed. In the examples of the present invention, the specific procedures of the purification culture may be as follows (b1) to (b 3):

(b1) removing DF10 culture medium when the confluence degree of the cells cultured by primary separation reaches 70-90%, and digesting with 1-2ml of EDTA-Trypsin for 5-60 min;

(b2) observing under a microscope, collecting human intestinal cancer cells while digesting, and transferring the human intestinal cancer cells collected each time to a centrifuge tube with DF10 culture medium;

(b3) centrifuging at 1000rpm for 5min to remove supernatant, resuspending in serum-free medium, 37 deg.C, and 5% CO₂And (3) placing the cells in a cell culture box for 10min-2h, collecting nonadherent human intestinal cancer cells after the fibroblasts adhere to the walls, transferring the cells into a new cell culture bottle for continuous culture, and repeatedly adhering the walls for 2-5 times to completely remove the fibroblasts to obtain purified primary human intestinal cancer cells.

Wherein EDTA (ethylenediaminetetraacetic acid) is ethylenediamine tetraacetic acid, and Trypsin is Trypsin.

In the embodiment of the present invention, preferably, the serum-free medium includes: DF culture medium, the additives are 1-5mM glutamine, 5-30mg/L insulin, 5-20mg/L transferrin, 5-20 μ g/L sodium selenite, 5-20 μ g/L EGF, 10-100nM hydrocortisone, 1-5mg/ml BSA, 0.1-0.5mg/ml penicillin, 0.1-0.5mg/ml kanamycin sulfate, 0.25-0.5 μ g/ml amphotericin B and 1-5 μ g/ml vancomycin.

For example, the concentration of glutamine can be 1, 2, 3, 4, or 5; the concentration of insulin can be 5, 10, 15, 20, 25 or 30; the concentration of transferrin can be 5, 10, 15 or 20; the concentration of sodium selenite can be 5, 10, 15 or 20; the concentration value of the EGF can be 5, 10, 15 or 20; the concentration of hydrocortisone can be 10, 30, 50, 70 or 100; the concentration of BSA can be 1, 2, 3, 4 or 5; the concentration of penicillin can be 0.1, 0.2, 0.3, 0.4 or 0.5; the concentration value of the kanamycin sulfate can be 0.1, 0.2, 0.3, 0.4 or 0.5; the concentration of amphotericin B can be 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5; the concentration of vancomycin can be 1, 2, 3, 3.5, 4, 4.5 or 5.

Therefore, in the embodiment of the invention, the serum-free medium specifically comprises 3.5-5 mug/ml vancomycin.

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

In detail, egf (epidemal Growth factor) is an epidermal Growth factor; BSA (albumin from bone serum) is bovine serum albumin.

The serum-free culture medium can effectively promote the growth of primary cells of human intestinal cancer and inhibit impurity cells such as fibroblasts and the like. The antibiotic is used for inhibiting the proliferation of microorganisms and avoiding the pollution of tumor cells.

When the number of the purified human intestinal cancer primary cells is small, the purified human intestinal cancer primary cells may be subcultured to culture the human intestinal cancer primary cells of one or more generations thereafter. One part of any cultured primary human intestinal cancer cells can be used for biological preservation, and the rest part can be used for cell characteristic analysis and cell application research and can be sealed and stored by liquid nitrogen for later use. Thus, the following example 11 may be present.

Example 11

Example 11 is intended to illustrate a method for culturing primary cells of human intestinal cancer.

In the present example, the culture method was substantially the same as the culture method provided in example 10 above, except that: after obtaining purified primary cells of human intestinal cancer in step 10.3, further comprising:

step 11.1: and culturing the obtained purified primary human intestinal cancer cells by using a serum-free culture medium.

Step 11.2: when the confluence degree of the purified primary human intestinal cancer cells reaches 70-90%, removing the serum-free culture medium, performing cell digestion treatment, collecting the primary human intestinal cancer cells while digesting, and transferring the primary human intestinal cancer cells collected each time to a centrifuge tube containing a culture medium containing 10% serum.

Step 11.3: and centrifuging the collected human intestinal cancer primary cells, removing supernatant, resuspending the cells in a serum-free culture medium, and inoculating the cells in a cell culture flask for culture to obtain the passaged human intestinal cancer primary cells.

In the embodiment of the invention, because the primary human intestinal cancer cells are purified, a serum-free culture medium can be selected for cell subculture. The purified primary human intestinal cancer cell can be a primary human intestinal cancer cell obtained after primary purification, and can also be any generation of primary human intestinal cancer cell after passage of the primary human intestinal cancer cell.

Next, the specific implementation procedure of subculture will be described.

In the present example, the specific procedures for subculture may be as follows (c1) to (c 4):

(c1) culturing the purified primary human intestinal cancer cells in a serum-free culture medium;

(c2) when the confluence degree of primary cells of the human intestinal cancer reaches 70-90%, washing the cells by PBS, and then digesting by 1-2ml of EDTA-Trypsin for 5-60 min;

(c3) collecting primary human intestinal cancer cells while digesting, and transferring the primary human intestinal cancer cells collected each time to a centrifuge tube with DF10 culture medium;

(c4) centrifuging at 1000rpm for 5min to remove supernatant, resuspending cells with serum-free medium, inoculating in culture flask, and culturing at 37 deg.C under 5% CO₂Culturing in an incubator to obtain the primary human intestinal cancer cells after passage.

In the embodiment of the invention, preferably, the cancer cell doubling time of the primary human intestinal cancer cells is 24-72 h. During this period, the cells are actively growing and the in vitro culture has stable cell growth characteristics.

Based on the above, since all primary cells were isolated and cultured from the same intestinal cancer tissue, the primary human intestinal cancer cells cultured by the cell culture method of any of the above examples, which are different only in cell generation number, can be used as a biological preservation, and their cell characteristics are substantially identical to those of the primary human intestinal cancer cells HCOC-751 of the above examples.

The present invention will be further illustrated by the following examples in conjunction with the foregoing, but is not limited thereto.

Example 12

Example 12 illustrates isolation and culture of primary human intestinal cancer cells.

Step 12.1: the part of intestinal cancer tissue excised at the time of surgery of the patient is collected with informed consent of the patient or patient guardian.

Step 12.2: transferring intestinal cancer tissue into cell culture dish, washing with physiological saline containing 0.1-0.5mg/ml penicillin, 0.1-0.5mg/ml kanamycin sulfate, 0.25-0.5 μ g/ml amphotericin B, and 1-5 μ g/ml vancomycin for 5-8 times to remove non-cancer tissue impurities such as fat and mucosa.

In the step, the effects of sterilization, bacteriostasis and pollution prevention can be achieved through repeated washing.

Step 12.3: the intestinal cancer tissue treated in step 12.2 is transferred to a new petri dish and the tissue mass is treated with scissors and a blade into a sludge shape.

In the step, the crushed sludge is processed, so that subsequent digestion and separation can be facilitated.

Step 12.4: transferring the crushed mud-shaped tumor tissue in the step 12.3 into a 50ml centrifuge tube, and centrifuging at 1500rpm for 5 min; removing supernatant, adding 9ml DF medium for resuspension, adding 1ml cell dispersing enzyme (containing 1-10mg/ml collagenase I and 0.2-2mg/ml hyaluronidase), at 37 deg.C and 5% CO₂Digesting in the incubator for 30min-2h at low speed by oscillation; after the enzyme reaction, 20ml of DF10 medium was added to stop the reaction, the mixture was blown up and mixed, centrifuged at 1500rpm for 5min, and the supernatant was removed.

In this step, resuspension with DF medium without additives is beneficial for better digestion.

Step 12.5: adding 5ml of cell digestive juice (EGTA-Trypsin) solution into the cell sediment obtained in the step 12.4, and uniformly mixing; standing at room temperature for 5 min; 20ml DF10 medium was added to stop the reaction, and the mixture was blown off and mixed.

Step 12.6: the cells of step 12.5 were filtered through a 300 μm nylon membrane, and the filtered cell sap was collected in a 50ml centrifuge tube, centrifuged at 1500rpm for 5min, and the supernatant was removed.

In this step, the filtering operation may remove non-tumor tissue, such as adipose tissue.

Step 12.7: the cells in step 12.6 were resuspended in 10ml DF10 medium containing antibiotics to 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator. This initial culture can last for 24-72 h.

Example 13

Example 13 is used to illustrate the purification of primary human intestinal cancer cells.

Step 13.1: when the confluency of cells reached 70-90% in step 12.7, DF10 medium was removed and digested with 1-2ml EDTA-Trypsin.

Step 13.2: under the microscope, the human intestinal cancer cells were collected while digesting, and digestion of each digested human intestinal cancer cell was terminated with DF10 medium.

Step 13.3: centrifuging at 1000rpm for 5min, removing supernatant, resuspending in serum-free medium, 37 deg.C, and 5% CO₂And (3) placing the cells in a cell culture box for 10min-2h, collecting the non-adherent intestinal cancer primary cells after the fibroblasts adhere to the walls, transferring the cells into a new cell culture bottle for continuous culture, and repeatedly adhering the walls for 2-5 times to achieve the effect of completely removing the fibroblasts, thereby obtaining the purified human intestinal cancer primary cells.

Step 13.4: and (3) culturing the human intestinal cancer primary cells obtained in the step 13.3 by using a serum-free culture medium.

The primary human intestinal cancer cells cultured in the step can be used for biological preservation, cell characteristic analysis, cell application, liquid nitrogen cryopreservation and the like.

Step 13.5: if necessary, 1 × 10⁶Resuspending primary cells of individual intestinal cancer in 1-2ml of cell freezing medium (90% fetal calf serum and 10% DMSO, v/v)) And storing in liquid nitrogen for later use.

Example 14

Example 14 is used to illustrate the subculture of primary human intestinal cancer cells.

Step 14.1: when the confluency of the cultured primary human intestinal cancer cells reaches 70-90%, the cells are washed twice with 1 XPBS (0.01M, pH7.4), and then digested with 1-2ml of EDTA-Trypsin for 5-60 min.

When the step is executed for the first time, the cultured human intestinal cancer primary cells can be the cells in the step 13.4, and when the step is not executed for the first time, the cultured human intestinal cancer primary cells can be the cells in the step 14.3, and the circulation is repeated, so that the subculture of the purified human intestinal cancer primary cells is completed.

Step 14.2: and (4) observing under a microscope, collecting the primary human intestinal cancer cells while digesting, and stopping digesting the human intestinal cancer cells digested each time by using DF10 culture medium until the primary human intestinal cancer cells are completely digested.

Step 14.3: centrifuging at 1000rpm for 5min, resuspending in serum-free medium, 5% CO at 37 deg.C₂Culturing in a cell culture box.

Aiming at the human intestinal cancer primary cells cultured in the step 13.4, the human intestinal cancer primary cells cultured in the step are different from the primary cells in terms of cell generation number and basically consistent in cell characteristics, so that the human intestinal cancer primary cells can be used for biological preservation, cell characteristic analysis, liquid nitrogen freezing storage and the like.

Step 14.4: if necessary, 1 × 10⁶Each tumor cell was resuspended in 1-2ml of cell freezing medium (90% fetal bovine serum and 10% DMSO, v/v) and stored in liquid nitrogen for use.

Step 14.5: and (4) based on the human intestinal cancer primary cells cultured in the step 14.3, performing the step 14.1 to the step 14.3 again, and circulating in this way to subculture the human intestinal cancer primary cells for 58 days of continuous subculture.

The experiment shows that when the primary human intestinal cancer cells are continuously subcultured for 58 days, the cells can still keep the normal growth in a proliferation state.

Example 15

Example 15 is used to illustrate the characterization of primary human intestinal cancer cells.

In the present example, the cell characterization analysis procedures of examples 2 to 6 were performed using the primary purified human intestinal cancer cells cultured in step 13.4 and using the primary human intestinal cancer cells cultured in step 14.3 and subcultured to any passage, respectively.

Experiments show that the cell characteristics of the primary human intestinal cancer cells, whether the primary purified human intestinal cancer cells or the primary human intestinal cancer cells subcultured to any generation, are consistent with the human intestinal cancer primary cells HCOC-751.

Example 16

Example 16 is used to illustrate the use of primary cells of human intestinal cancer.

In the embodiment of the invention, the primarily purified human intestinal cancer primary cells cultured in the step 13.4 and the human intestinal cancer primary cells cultured in the step 14.3 and subcultured to any generation are used for the pathogenesis research, drug sensitivity research and detection of human cancer-related diseases and the research of anti-tumor drugs.

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

1. The primary human intestinal cancer cell is named as human intestinal cancer cell HCOC-751, is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC NO. 18530.

2. A method for culturing primary cells of human intestinal cancer, comprising:

3. The method of claim 2,

after the obtaining of the purified primary human intestinal cancer cells, further comprising:

4. The method according to claim 2 or 3,

the serum-free culture medium comprises: DF culture medium, the additives are 1-5mM glutamine, 5-30mg/L insulin, 5-20mg/L transferrin, 5-20 μ g/L sodium selenite, 5-20 μ g/L EGF, 10-100nM hydrocortisone, 1-5mg/ml BSA, 0.1-0.5mg/ml penicillin, 0.1-0.5mg/ml kanamycin sulfate, 0.25-0.5 μ g/ml amphotericin B and 1-5 μ g/ml vancomycin.

5. The method of claim 2,

the cell culture conditions include: placing at 37 ℃ and 5% CO₂Culturing in an incubator.

6. The method according to any one of claims 2 to 5,

the doubling time of the cancer cells of the human intestinal cancer primary cells is 24-72 h.

7. Use of the human intestinal cancer primary cell of claim 1, or cultured by the method of any one of claims 2 to 6, for studying the pathogenesis of cancer-related diseases.

8. Use of the human intestinal cancer primary cell of claim 1, or cultured by the method of any one of claims 2 to 6, in drug sensitivity studies and assays.

9. Use of the human intestinal cancer primary cell of claim 1 or the human intestinal cancer primary cell cultured by the method of any one of claims 2 to 6 for screening or preparing an antitumor drug.