CN112831472B

CN112831472B - Primary human thymoma cell, culture method and application thereof, and serum-free culture medium

Info

Publication number: CN112831472B
Application number: CN202110174485.6A
Authority: CN
Inventors: 倪君君; 智慧芳; 李春凤
Original assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Current assignee: Beijing Harmony Health Medical Diagnostics Co ltd
Priority date: 2021-02-07
Filing date: 2021-02-07
Publication date: 2022-08-12
Anticipated expiration: 2041-02-07
Also published as: CN112831472A

Abstract

The present disclosure provides a human thymoma primary cell, a culture method, an application and a serum-free culture medium thereof, wherein the human thymoma primary cell is named as human thymoma cell HThy-943, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, and has a preservation number of CGMCC NO. 21020.

Description

Primary human thymoma cell, culture method and application thereof, and serum-free culture medium

Technical Field

The invention relates to the technical field of biology, in particular to a human thymoma primary cell, a culture method and application thereof, and a serum-free culture medium.

Background

It is well known that the thymus develops many types of tumors, including thymoma, germ cell tumors, neurogenic tumors, lymphoblastic leukemia/lymphoma and others. Thymoma is an epithelial thymic tumor, the most common pre-mediastinal tumor, and is not primarily gender-prone, accounting for about 20-30% of all mediastinal tumors occurring in adults. They are generally considered to have an inert growth pattern, but are malignant because of the potential for local infiltration, pleural dissemination, and even systemic metastases of the tumor. In addition, 20-40% of patients with thymoma have paraneoplastic syndromes, including Myasthenia Gravis (MG), pure red cell aplastic hyperthyroidism, endocrinopathies, and other connective tissue diseases. Studies have shown that about 10-30% of MG patients develop thymic tumors. According to different reports, 15-60% of patients with thymoma develop MG. There are also studies discussing different clinical characteristics of thymomas with and without MG. These studies have focused primarily on the presence of MG and its effect on the prognosis of patients with thymoma.

Because thymoma is a serious health hazard to humans, commercial cell lines are used as materials for the study of the pathogenesis and therapeutic methods of thymoma. However, after long-term culture, the biological characteristics and genetics of the cells are easily changed, which is not favorable for the research of pathogenesis and treatment method.

The primary tumor cell culture refers to a process of obtaining tumor cells by surgical excision of tumor tissues from a tumor patient body and performing short-term culture in vitro. The primary cultured tumor cells have the biological characteristics that the tissue is just isolated, the biological characteristics are not changed greatly, the primary genetic characteristics are still kept, 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. Therefore, the culture of primary cells of the human thymoma is particularly necessary.

Disclosure of Invention

The embodiment of the invention provides a primary human thymoma cell, a culture method and application thereof, and a serum-free culture medium.

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 thymoma cell named human thymoma cell HThy-943, which has been deposited in China General Microbiological Culture Collection Center (CGMCC) at 12 months and 3 days 2020, and the deposit address is Beijing university institute No. 1, Ministry of Japan and Japan, institute of microbiology, institute of China academy of sciences, Ministry of Japan, No. 3, Ministry of Japan, and the deposit number is CGMCC No. 21020.

In a second aspect, the invention provides a serum-free medium for culturing primary cells of human thymoma, comprising: additives, antibiotics and DF media;

wherein the Supplement comprises glutamine, insulin, transferrin, sodium selenite, epidermal growth factor, bovine serum albumin, Y-27632 and N-2 Supplement;

the antibiotics comprise penicillin, kanamycin sulfate and amphotericin B.

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

Preferably, the serum-free medium comprises: 1-5mM glutamine, 5-30mg/L insulin, 5-20mg/L transferrin, 5-20 μ g/L sodium selenite, 5-20 μ g/L EGF, 1-5mg/mL BSA, 5-20 μ M Y-27632, 0.5-5% N-2Supplement, 10-60 μ g/mL penicillin, 10-60 μ g/mL kanamycin sulfate, 0.15-0.5 μ g/mL amphotericin B.

In a third aspect, the present invention provides a method for culturing primary cells of human thymoma, comprising:

step 1, preserving the collected fresh human thymoma tissues by using a sample preserving fluid;

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

step 2, cleaning the thymoma tissues preserved in the step 1 by using a cleaning solution;

wherein the cleaning solution comprises physiological saline containing the antibiotic;

step 3, taking the thymus tumor tissue cleaned and treated in the step 2, and carrying out primary isolated culture by using a culture medium containing serum;

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

and 4, taking the cells obtained by the primary separation and culture in the step 3, and purifying and subculturing the cells by using any serum-free culture medium provided in the second aspect to obtain the primary human thymoma cells.

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

Preferably, the serum-containing medium comprises: penicillin 10-60. mu.g/mL, kanamycin sulfate 10-60. mu.g/mL, amphotericin B0.15-0.5. mu.g/mL.

Preferably, the cleaning liquid includes: penicillin 0.1-0.8mg/mL, kanamycin sulfate 0.1-0.8mg/mL, amphotericin B1.5-5. mu.g/mL.

In a fourth aspect, the present invention provides the human thymoma primary cell described in the first aspect, the human thymoma primary cell cultured by the serum-free medium described in the second aspect, or the human thymoma primary cell cultured by the culture method described in the third aspect, and applications of the human thymoma primary cell in drug sensitivity research and detection.

In a fifth aspect, the present invention provides the use of the human thymoma primary cell of the first aspect, the human thymoma primary cell cultured by the serum-free medium of the second aspect, or the human thymoma primary cell cultured by the culture method of the third aspect, in constructing a thymoma animal model.

In a sixth aspect, the present invention provides the use of the primary human thymoma cell of the first aspect, the primary human thymoma cell cultured in the serum-free medium of the second aspect, or the primary human thymoma cell cultured by the culture method of the third aspect in the development of a thymoma inhibitory drug.

In a seventh aspect, the present invention provides the use of the primary human thymoma cell of the first aspect, the primary human thymoma cell cultured in the serum-free medium of the second aspect, or the primary human thymoma cell cultured by the culture method of the third aspect in vitro research of thymoma.

The primary cells of the human thymoma provided by the invention are applied for the purposes of diagnosis and treatment of non-diseases.

The human thymoma cell HThy-943 provided by the invention has at least the following characteristics:

the method is characterized in that: the primary culture is isolated from the thymus tumor tissue of Chinese, and the cell has no foreign gene transferred.

And (2) characteristic: the human thymoma cell HThy-943 is in an epithelial cell shape, is high in tumor cell proliferation speed, is irregular in cell size and shape, is irregular or polygonal, loses polarity, is disorderly arranged, is not inhibited by contact, is mutually extruded, is stacked or inlaid, is less clear in cell boundary, and has the morphological characteristics of the thymoma cell.

And (3) feature: the multiplication time of the cell population of the human thymoma cell HThy-943 is 32-84 hours, the cell proliferation speed is high, the cell growth is active, the cell activity state is good, the cell culture stability is high, and the in vitro culture has stable cell growth characteristics.

And (4) characteristic: human thymoma cell HThy-943 was immunofluorescent-identified as strongly expressed as epithelial-derived tumor cell marker CK (cytokeratin).

And (5) feature: the human thymoma cell HThy-943 is identified as a tumor cell heterotypic karyotype through karyotype analysis, the chromosome structure and the number of the cell are abnormal, the chromosome number of the cell is 74, the cell belongs to a super-triploid karyotype, and the cell accords with the genetic characteristic of human malignant tumor.

And (6) feature: human thymoma cell HThy-943 is identified by STR (Short Tandem Repeat) genotyping, is a cell which is a single human source and has no cross contamination, and is a newly obtained thymoma primary cell.

And (7) feature: the human thymoma cell HThy-943 can be used for drug sensitivity research and detection, construction of a thymoma animal model, development of a thymoma inhibiting drug and in-vitro research of thymoma.

Cell preservation information

The human thymoma cell HThy-943 related by the invention has been preserved in China General Microbiological Culture Collection Center (CGMCC) at 12 months and 3 days in 2020, the preservation address is No. 3 of Beijing West Luo No. 1 of the sunward area, the institute of microbiology of the Chinese academy of sciences, the postal code is 100101, and the preservation number is CGMCC NO. 21020.

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 cellular morphology of human thymoma cell HThy-943 provided by an embodiment of the present invention;

FIG. 2 is a graph showing the growth of human thymoma cell HThy-943 according to an embodiment of the present invention;

FIG. 3 is a graph showing the immunofluorescence identification of human thymoma cell HThy-943 according to an embodiment of the present invention;

FIG. 4 is a chromosome karyotype analysis chart of human thymoma cell HThy-943 according to an embodiment of the present invention;

FIG. 5 is a STR genotyping graph of human thymoma cell HThy-943 provided by an embodiment of the 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.

It should be noted that due to the specificity of the thymoma tissue, the lack of a useful thymoma specimen, the technical difficulty in establishing primary thymoma cell culture and other factors, the existing thymoma in vitro research model is seriously lacked, thereby greatly limiting the research on the thymoma cytology level. Based on the above, the embodiment of the invention provides a human thymoma primary cell, a culture method, an application and a serum-free culture medium thereof, and can provide good experimental materials for relevant researches such as biochemical molecules, generation mechanisms and clinical treatment approaches of thymoma. The following description is directed to the human thymoma primary cells, their culture method, application and serum-free medium, respectively.

Example 1

The embodiment of the invention provides a human thymoma primary cell named as human thymoma cell HThy-943, which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 21020.

The human thymoma cell HThy-943 is isolated and cultured from the thymus tumor tissue of Chinese, and no foreign gene is introduced into the cell.

Next, the biological properties of human thymoma cell HThy-943 will be described.

1.1 morphological Observation of cells

The human thymoma cell HThy-943 is observed under an inverted microscope, is in an epithelial cell form, has the characteristics of rapid tumor cell proliferation, unequal cell sizes and forms, irregular or polygonal shapes, no polarity loss, disordered arrangement, no contact inhibition, mutual extrusion, stacking or mosaic shapes and unclear cell boundaries, and has the morphological characteristics of thymoma cells. The morphological observation results are shown in fig. 1.

1.2 measurement of growth Curve and doubling time

1.2.1 growth Curve determination procedure

(1) When the confluence of human thymoma cells HThy-943 reached 70-90%, the serum-free medium was removed, the cells were washed at least twice with PBS (0.01M, pH7.4) to remove old serum-free medium and floating cells, digested with 1-2mL of EDTA-Trypsin (Trypsin-EDTA digest), and the digestion was stopped with DF10 medium (DF medium and 10% FBS).

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

(2) Cells were cultured at 2.5X 10 ⁵ Perml were seeded into 30 wells of 10 24-well plates, and 1ml of cell suspension was added per well.

(3) Discarding the culture medium in 3 holes every 24 hours from the inoculation time, adding EDTA-Trypsin for digestion treatment, suspending the cells, counting the average value of the cell density in 3 holes for 3 times per hole, taking the average value, and continuously culturing the cells in the rest holes until the 10 th day; the medium was changed every 2 days.

(4) The measurement was continued for 10 days, and the growth curve data shown in the following Table 1 were obtained.

1.2.2 growth Curve measurements

Table 1 shows the growth curve data obtained for 10 consecutive days of the assay.

TABLE 1

Cultivation time (sky)	Mean cell density
		1	2.9×10 ⁴ Per mL
2	5.2×10 ⁴ Per mL
		3	6.8×10 ⁴ Per mL
4	8.1×10 ⁴ Per mL
		5	8.9×10 ⁴ Per mL
6	10.5×10 ⁴ Per mL
		7	11.2×10 ⁴ Per mL
8	13.4×10 ⁴ Per mL
		9	14.7×10 ⁴ Per mL
10	14.1×10 ⁴ Per 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 represents the culture time (days) and the ordinate represents the number of cells.

Referring to the growth curve data in table 1 and the schematic diagram of the growth curve shown in fig. 2, it can be seen that human thymoma cell HThy-943 grew well for 10 consecutive days, with a logarithmic growth phase of 8 days.

1.2.3 cell population doubling time

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

The doubling time of the cell population of the human thymoma cell HThy-943 is calculated to be 32-84 h.

In conclusion, the human thymoma cell HThy-943 has the advantages of high cell proliferation speed, active cell growth, good cell activity state, high cell culture stability and stable cell growth characteristic in vitro culture.

1.3 immunofluorescence identification

1.3.1 immunofluorescence identification procedure

(1) Human thymoma cells HThy-943 reached around 70-90% confluence, 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 floating cells, digested with 1-2mL EDTA-Trypsin, and seeded into 48-well plates.

(2) When the cell confluence reaches 70-90%, the serum-free medium is removed, washed 3 times with PBS, and then removed after 30min of fixation by adding 500. mu.L/well of ice methanol.

(3) Adding 200 μ L/well 2.5% BSA blocking solution, blocking at room temperature for 60min, and sucking out the blocking solution.

(4) 150 μ L/well of primary antibody (Keratin 18(DC10) Mouse mAb, 1:800 dilution; Anti-Calretinin antibody produced in rabbite, 1:1000 dilution) was added, incubated overnight at 4 ℃ and aspirated.

(5) mu.L/well of a fluorescent secondary antibody (Anti-Mouse IgG (second antibody) -FITC antibody produced in rabbitt, diluted 1: 100; Cy 3-labeled goat Anti-rabbit IgG, diluted 1: 500) was added, incubated at room temperature for 1h in the absence of light, aspirated off the secondary antibody, and washed 3 times with PBS.

(6) 2 drops of DAPI stock (ab104139) were added to each well and stained for 5min at room temperature in the dark.

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

(8) 150 μ L/well of PBS was added, and the immunofluorescence assay was performed by taking a photograph of the sample using an objective lens Xeyepiece (20X 10).

1.3.2 immunofluorescence identification results

CytoKeratin (CK) is a common marker of epithelial cells, and CK is expressed in epithelial cells and tumor cells of epithelial origin, but not Calretinin (CR).

The immunofluorescence assay results are shown in FIG. 3. Referring to fig. 3, it was found that DAPI stained tumor cell nuclei, and HThy-943 CK was strongly expressed and CR was not expressed, indicating that HThy-943 originated from epithelial cells.

1.4 karyotyping

1.4.1 karyotype analysis and identification procedure

(1) Cell culture

When the human thymoma cell HThy-943 confluency reaches 70-90%, removing the serum-free medium culture, washing the cells with PBS (0.01M, pH7.4) at least twice to remove the old serum-free medium and the cells with poor sloughing state, digesting with 1-2mL EDTA-Trypsin, observing under a microscope, collecting the digested cells while digesting, and terminating the digestion of the collected cells with DF10 medium until all the cells are digested. Centrifuge at 1000rpm for 5min and remove supernatant.

(2) Colchicine treatment

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

(3) Hypotonic treatment

After the colchicine treatment was completed, the mixture was centrifuged (1300rpm, 10min) and the supernatant was discarded. Then adding 0.075mol/L KCl solution in 37 ℃ water bath to obtain a hypotonic solution of 10mL, blowing the hypotonic solution into a cell suspension by using a suction pipe, and placing the cell suspension in 37 ℃ water bath for treatment for 30-35 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) Centrifugation

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

(6) Fixing

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

(7) Second fixing

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

(8) Tabletting

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

(9) Dyeing process

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

(10) Microscopic examination

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

1.4.2 karyotype analysis and identification results

The results of karyotyping are shown in FIG. 4. Referring to fig. 4, the human thymoma cell HThy-943 was identified by karyotype analysis as a tumor cell heterotypic karyotype, the chromosome structure and number of the cell were abnormal, and the cell was a tumor cell with a hyper-triploid karyotype, the number of chromosomes was 74, which is different from the karyotype of normal cells of human body and meets the genetic characteristics of human malignant tumor.

1.5STR identification

1.5.1STR identification procedure

(1) Human thymoma cell HThy-943(1 × 10) growing adherently ⁶ One) the cells were washed twice with PBS (0.01M, pH7.4), digested sequentially with 1-2mL EDTA-Trypsin for 3-30min, and 10mL DF10 medium was used to stop the digestion reaction.

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

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

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

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

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

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

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

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

(10) Using a golden eye ^TM Amplifying the 20A STR composite amplification kit, and continuously detecting and analyzing 21 known STR loci and sex genes Amelogenin on an ABI 3100 type genetic analyzer.

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

1.5.2STR identification results

The STR typing results are shown in fig. 5. Referring to fig. 5, 21 loci were examined, expressed as "locus/allele length": D19S433/13/15, D5S818/12/12, D21S11/30/32, D18S51/13/15, D6S1043/11/15, AMEL/X/Y, D3S1358/15/15, D13S317/10/10, D7S820/13/13, D16S539/9/12, CSFIPO/10/10, PentaD/11/12, D2S441/10/11, vWA/14/17/18, D8S1179/14/17, TPOX/8/9, PentaE/11/14, TH01/7/7, D12S391/20/20, D2S1338/23/25, FGA/24/24.

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

(1) human thymoma cell HThy-943 only has three-allelic gene phenomenon at vWA locus, and no human cell cross contamination is found in the cell.

(2) Comparing the STR data of human thymoma cell HThy-943 with those of STR databases of ATCC (american type culture collection center) and DSMZ (national culture collection center in germany), it was found that no cell matching 100% of the cell type of human thymoma cell HThy-943 was found in both ATCC and DSMZ cell banks.

(3) Comparing the STR data of human thymoma cell HThy-943 with those of STR databases of ATCC and DSMZ, it is known that the STR data match rate of MDA-MB-468(HTB-132) cell is relatively highest, 70%.

In conclusion, the STR genotyping identification of the human thymoma cell HThy-943 proves that the human thymoma cell is a single human-derived cell without cross contamination and is a newly obtained thymoma primary cell.

1.6 cell uses

1.6.1 application in drug sensitivity research and detection

1.6.1.1 drug susceptibility testing procedure (CD-DST method)

(1) Collagen gel drop culture

Mixing human thymoma cell HThy-943 with gel constituent (I: II: III: 8:1:1), and making into fine gel drops (30 μ L/gel drop and 3 gel drops/hole)The cell suspension was inoculated in 6-well plates, while 3 gel drops were inoculated in another 6-well plate as a 0-time control. 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.C ₂ The culture was carried out overnight in an incubator.

(2) Anti-cancer drug exposure and cleaning

After culturing the primary human thymoma cells obtained in the step (1) for 24 hours, adding an anti-cancer drug, and setting a negative Control hole (Control) without a drug and a positive Control hole treated by a clinical thymoma common drug (note that the drug can be selected according to the main clinical manifestation of a patient from which the cells are derived), 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.

(3) Dye fixation and scanning

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

1.6.1.2 result of drug sensitivity detection

The results of drug sensitivity detection (CD-DST method) of human thymoma cell HThy-943 are shown in Table 2 below.

TABLE 2

Name of drug	Cell survival rate
		Carboplatin (CBDCA)	64.41％
Cisplatin (CDDP)	72.97％
		Adriamycin (ADR)	96.03％
Epirubicin (EPI)	85.40％
		Etoposide (VP-16)	80.28％
Taxol (PAC)	83.54％
		Novier book (VNR)	91.12％
Gemcitabine (GEM)	90.59％
		Fluorouracil (static push) (5-FU)	83.86％
Docetaxel (DOC)	79.72％

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

Referring to Table 2, human thymoma cells HThy-943 were all hyposensitive to carboplatin (CBDCA), Cisplatin (CDDP), doxorubicin (ADR), Epirubicin (EPI), etoposide (VP-16), Paclitaxel (PAC), navelbine (VNR), Gemcitabine (GEM), fluorouracil (immortal) (5-FU), and Docetaxel (DOC), and thus were used for efficacy evaluation of the 10 clinical thymoma common drugs. Through the drug sensitivity detection experiment, clinical medication guidance can be more pertinently carried out on related thymoma patients (such as human thymoma cell HThy-943 thymoma patients, other thymoma patients with the same or similar clinical manifestations or drug sensitivity characteristics or thymoma metastasis lesion patients and the like), and the drug sensitivity detection method has important significance on personalized treatment of the thymoma patients.

1.6.2 use in constructing animal models of thymoma

Human thymoma cells HThy-943 in logarithmic growth phase are inoculated into mammals (e.g., nude mice) at a prescribed dose, and cultured for several days to construct a thymoma animal model. The thymoma animal model can be used for development research of thymoma, 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 thymoma inhibiting drugs

The human thymoma cell HThy-943 is cultured from a thymoma tissue just isolated from the body, has small change of biological characteristics, retains the original genetic characteristics, and can be applied to screening or evaluating medicaments for treating the thymoma.

Different chemotherapeutic drugs are added into a culture medium of the human thymoma cell HThy-943, and the changes of cell growth, proliferation, death, period and the like are observed to obtain a primary effective candidate drug. And then the candidate drug is used in a thymoma animal model, the in-vivo effect of the drug is detected, the general condition, survival time and tumor size change of animals are observed, and the change condition of animal organisms after the drug effect is observed, so that the curative effect evaluation and the mechanism analysis of the candidate drug are carried out.

1.6.4 used for extrasomatic research of thymoma

The human thymoma cell HThy-943 is cultured from a thymoma tissue just isolated from the body, has small biological property change, retains the original genetic property, can be used as a cell material for in vitro research of human thymoma pathogenesis and individualized treatment, and further provides a foundation for establishment of a human thymoma pathogenesis model and preparation, screening, evaluation and the like of an anti-human thymoma drug.

In summary, the human thymoma cell HThy-943 provided by the embodiments of the present invention has at least the following characteristics:

the method is characterized in that: the primary culture is isolated from the thymalfoma tissue of Chinese, and the cells are not introduced with any foreign genes.

And (2) feature: the human thymoma cell HThy-943 is in an epithelial cell shape, is high in tumor cell proliferation speed, is irregular in cell size and shape, is irregular or polygonal, loses polarity, is disorderly arranged, is not inhibited by contact, is mutually extruded, is stacked or inlaid, is less clear in cell boundary, and has the morphological characteristics of the thymoma cell.

And (4) feature: human thymoma cell HThy-943 was immunofluorescent-identified as strongly expressed as epithelial-derived tumor cell marker CK (cytokeratin).

And (5) feature: the human thymoma cell HThy-943 is identified as a tumor cell heterotypic karyotype through karyotype analysis, the chromosome structure and the number of the cell are abnormal, the chromosome number of the cell is 74, the cell belongs to a super-triploid karyotype, and the genetic characteristic of the human malignant tumor is met.

And (7) characteristic: the human thymoma cell HThy-943 can be used for drug sensitivity research and detection, construction of a thymoma animal model, development of a thymoma inhibiting drug and in-vitro research of thymoma. The use is for non-disease diagnostic and therapeutic purposes.

Example 2

This example illustrates a method for culturing primary human thymoma cells, comprising the following steps 1-4. The human thymoma cell HThy-943 provided in example 1 was cultured by this culture method.

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

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

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

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

Step 3.1), the cleaned thymoma tissue is treated by cell dispersing enzyme.

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

Step 3.1.1), chopping the cleaned thymoma tissues into a crushed mud shape.

Wherein, the thymus gland tumor tissue can be conveniently digested into tumor cells with proper size by being chopped into a crushed mud shape.

Step 3.1.2), the muddy thymoma tissue is centrifuged for 5-10min at 800-.

Wherein the supernatant is removed by centrifugation to remove impurities in the thymoma tissue, such as adipose tissue, mucosal tissue, connective tissue, and necrotic tissue.

Step 3.1.3), adding cell dispersing enzyme in sequence, placing at 37 deg.C and 5% CO ₂ Digesting in the culture box at low speed for 20min-2h under shaking to disperse thymoma tissue and prevent cell agglomeration, adding DF10 culture medium to stop reaction, and blowing to disperse and mix uniformly.

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

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

And 3.2) digesting by using cell digestive juice.

In particular, step 3.2) may comprise: adding cell digestive juice into the cells obtained after the digestion treatment of the cell dispersing enzyme, blowing, uniformly mixing, digesting for 2-7min at room temperature, adding DF10 culture medium to stop the reaction, blowing, uniformly mixing, centrifuging and removing supernatant to obtain the cells digested by the cell digestive juice.

Wherein the cell digestive juice may be EGTA-Trypsin.

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

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

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

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

Step 3.3.3), when the cultured cells grow completely adherent to the wall, the serum-containing culture medium is replaced by a serum-free culture medium, and the cells are placed at 37 ℃ and 5% CO ₂ And continuously culturing in the incubator to obtain the cells obtained by primary separation culture.

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

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

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

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

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

Step 4.3), centrifuging for 5-10min at 800-1500rpm in sequence, removing the supernatant, resuspending the cell pellet with serum-free medium, and placing the resuspended cells at 37 ℃ and 5% CO ₂ And (3) allowing fibroblasts to adhere to the wall in the incubator for 5-100min, collecting unadhered human thymoma primary cells, transferring the unadhered human thymoma primary cells into a new cell culture bottle for continuous culture, and repeatedly adhering the walls for 2-6 times to obtain purified human thymoma primary cells. Thus, the fibroblast can be completely eliminated, and the cell state is poor or death caused by over digestion can be avoided.

And 4.4) carrying out subculture on the purified primary human thymoma cells.

In this example, the antibiotics include penicillin, kanamycin sulfate, amphotericin B. During the preservation, cleaning and tumor cell culture of thymalfoma tissue, the antibiotic has bacteriostasis and bactericidal effect to protect primary cell from being polluted by microbe and kill gram-positive bacteria, gram-negative bacteria, mycoplasma and fungi.

In this embodiment, the Supplement includes glutamine, insulin, transferrin, sodium selenite, epidermal growth factor, bovine serum albumin, Y-27632, and N-2 Supplement.

For example, in the wash solution, the concentration of penicillin may be 0.1, 0.3, 0.5, 0.7, or 0.8mg/mL, the concentration of kanamycin sulfate may be 0.1, 0.3, 0.5, 0.7, or 0.8mg/mL, and the concentration of amphotericin B may be 1.5, 2.5, 3.5, 4.5, or 5. mu.g/mL.

For example, in serum-containing media, penicillin can be present at a concentration of 10, 20, 30, 40, 50, or 60. mu.g/mL, kanamycin sulfate can be present at a concentration of 10, 20, 30, 40, 50, or 60. mu.g/mL, and amphotericin B can be present at a concentration of 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, or 0.5. mu.g/mL.

Preferably, the serum-free medium comprises: 1-5mM glutamine, 5-30mg/L insulin, 5-20mg/L transferrin, 5-20 μ g/L sodium selenite, 5-20 μ g/L EGF, 1-5mg/mL BSA, 5-20 μ M Y-27632, 0.5-5% N-2Supplement, 10-60 μ g/mL penicillin, 10-60 μ g/mL kanamycin sulfate, 0.15-0.5 μ g/mL amphotericin B. The serum-free culture medium can effectively promote the growth of primary cells of the human thymoma and inhibit foreign cells such as fibroblasts and the like.

For example, in serum-free medium, glutamine can be at a concentration of 1, 2, 3, 4, or 5mM, insulin can be at a concentration of 5, 10, 15, 20, 25, or 30mg/L, transferrin can be at a concentration of 5, 10, 15, or 20mg/L, sodium selenite can be at a concentration of 5, 11, 15, or 20 μ g/L, EGF can be at a concentration of 5, 10, 15, or 20 μ g/L, BSA can be at a concentration of 1, 2, 3, 4, or 5mg/mL, Y-27632 can be at a concentration of 5, 10, 15, or 20 μ M, N-2Supplement can be at a volume percent of 0.5, 1, 2, 3, 4, or 5%, penicillin can be at a concentration of 10, 20, 30, 40, 50, or 60 μ g/mL, kanamycin sulfate can be at a concentration of 10, 20, 30, 40, 50, or 60 μ g/mL, amphotericin B can be at a concentration of 0.15, 30, or 5 μ g/mL, 0.2, 0.25, 0.3, 0.35, 0.4 or 0.5. mu.g/mL.

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

For step 1:

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

step 1.1, collecting the part of thymoma tissue excised by the patient at the time of surgery under the informed consent of the patient or patient guardian.

And step 1.2, preserving the thymoma tissues collected in the step 1.1 by using a sample preservation solution.

For step 2:

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

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

Step 2.2, the thymus tumor tissue washed in step 2.1 is transferred to a new culture dish.

For step 3:

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

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

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

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

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

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

Step 3.5, resuspending the cell pellet obtained in step 3.4 with 10mL of serum-containing medium to 25cm ² The culture bottles were sequentially placed at 37 ℃ and 5% CO ₂ The culture was carried out in an incubator overnight (24 h).

Step 3.6, when the cells cultured in the step 3.5 grow completely adherent to the wall, the serum-containing culture medium is changed to a serum-free culture medium and is placed at 37 ℃ and 5% CO ₂ And continuing culturing in the incubator.

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

For step 4:

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

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

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

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

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

Based on the content, the culture method adopts a combined mode of a pancreatin differential time digestion method, a differential time adherence method and a repeated adherence method to purify the cells, so that the cells are prevented from being in poor states or dead due to over digestion in the purification process, the primary human thymoma cells in good states and without fibroblast and other foreign cells are obtained, and the success rate of culturing the primary human thymoma cells is improved.

Step 4.4, culturing the primary human thymoma cells obtained in step 4.3 in a serum-free medium, washing the cells at least twice with 1-fold concentration (1 ×) Phosphate Buffered Saline (PBS) (0.01M, PH7.4) when the confluency of the cells reaches 70-90% to remove old serum-free medium and cells with poor sloughing status, and then digesting with 1-2mL EDTA-Trypsin for 3-30 min.

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

Step 4.6, centrifuge at 1000rpm for 5min, remove supernatant, resuspend with serum-free medium, inoculate in new culture flask, in order at 37 deg.C, 5% CO ₂ And culturing in a cell culture box to obtain the primary human thymoma cells after passage.

Thereafter, steps 4.4-4.6 above are repeated as necessary, except that when step 4.4 is not performed for the first time, the human thymoma primary cells cultured in step 4.4 are not the human thymoma primary cells obtained in step 4.3, but the human thymoma primary cells obtained in step 4.6 above (i.e., the previous generation of human thymoma primary cells).

It should be noted that, since the primary cells were isolated and cultured from the same thymoma tissue, the cells obtained in step 4.3 and each step 4.6 were human thymoma primary cells, and all had the same cell characteristics, except that the cell generation number was different. Thus, any generation of primary human thymoma cells cultured by the culture method provided in this example has the same cell characteristics as the human thymoma cells HThy-943.

Based on the above, for the primary human thymoma cells obtained in step 4.3 and each step 4.6, (1 × 10) is added as necessary ⁶ Individual) human thymoma 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 future use. Wherein DMSO (dimethyl sulfoxide) is dimethyl sulfoxide.

The serum-free medium used in the above culture method includes: DF medium, 3mM glutamine, 20mg/L insulin, 10mg/L transferrin, 11. mu.g/L sodium selenite, 10. mu.g/L EGF, 3mg/mLBSA, 10. mu. M Y-27632, 1% N-2Supplement, 50. mu.g/mL penicillin, 50. mu.g/mL kanamycin sulfate, 0.25. mu.g/mL amphotericin B.

The serum-containing culture medium used in the above culture method comprises: DF medium, 10% FBS (total bone serum), 50. mu.g/mL penicillin, 50. mu.g/mL kanamycin sulfate, 0.25. mu.g/mL amphotericin B.

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

The washing solution used in the above culture method comprises: physiological saline, 0.5mg/mL penicillin, 0.5mg/mL kanamycin sulfate, 2.5. mu.g/mL amphotericin B.

It should be noted that the method for culturing primary human thymoma cells provided in this embodiment can be used for culturing not only primary thymoma tumor cells, but also primary thymoma metastasis primary tumor cells.

Example 3

This example provides serum-free media for culturing primary human thymoma cells. The serum-free medium may be any of the serum-free media used in example 2 above, and the human thymoma cell HThy-943 provided in example 1 can be cultured using the serum-free medium.

Example 4

This example illustrates the use of primary human thymoma cells for non-disease diagnostic and therapeutic purposes. The human thymoma primary cell may be the human thymoma cell HThy-943 provided in example 1, the human thymoma primary cell cultured by the culture method provided in example 2, and the human thymoma primary cell cultured by the serum-free culture medium provided in example 3.

The application of the primary human thymoma cell provided by the embodiment of the invention can be particularly used for drug sensitivity research and detection, construction of a thymoma animal model, development of a thymoma inhibiting drug, in-vitro thymoma research and the like.

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 thymoma cell is named as human thymoma cell HThy-943, is preserved in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC number 21020.

2. Use of the human thymoma primary cell of claim 1 in the construction of a thymoma animal model.

3. Use of the primary human thymoma cell of claim 1 in the preparation of a thymoma-inhibiting medicament.