CN112080473A - Primary cell strain of osteosarcoma lung metastasis range and culture method and application thereof - Google Patents

Primary cell strain of osteosarcoma lung metastasis range and culture method and application thereof Download PDF

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CN112080473A
CN112080473A CN202010974517.6A CN202010974517A CN112080473A CN 112080473 A CN112080473 A CN 112080473A CN 202010974517 A CN202010974517 A CN 202010974517A CN 112080473 A CN112080473 A CN 112080473A
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尹军强
沈靖南
赵巨鹏
樊泽培
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First Affiliated Hospital of Sun Yat Sen University
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Abstract

The invention provides a primary cell strain of a human osteosarcoma lung metastasis focus, a culture method and application thereof. The primary cell strain of the human osteosarcoma lung metastasis is named ZOSL-1, and the preservation number is CCTCC No: C202088. The primary cell strain of the human osteosarcoma lung metastasis stove is obtained by screening purified tumor cells by combining a pancreatin differential time digestion method, a differential time adherence method and a serum-free culture medium stimulation method, can be used for lung metastasis mechanism research of osteosarcoma and antitumor drug research, and has good medical value.

Description

Primary cell strain of osteosarcoma lung metastasis range and culture method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to primary cells of human osteosarcoma lung metastasis and a culture method and application thereof.
Background
Osteosarcoma (OS), also called osteogenic sarcoma, is commonly found in adolescents and children. Lung metastasis is the leading cause of death in osteosarcoma patients. To obtain a more effective treatment regime, an appropriate preclinical model is of critical importance.
The cell strain is one of common preclinical models and has the advantages of convenience and rapidness. However, in the in vitro culture process, the micro-environment for the in vivo growth of the tumor is lacked, and the in vitro culture environment is adapted to be mutated after repeated passage, so that the initial character of the tumor cannot be reflected. The lung metastases usually occur in multiple positions of the lung, but the size of a single lung is small, so that a proper specimen is difficult to obtain and the lung metastases are difficult to separate after the acquisition. Therefore, the existing osteosarcoma cell strains are all derived from in-situ foci or remote metastasis, and there are no cell strains of lung metastasis. Lung metastasis of osteosarcoma is the major cause of high mortality in patients. Therefore, a cell line of osteosarcoma lung metastasis is urgently needed for the research of osteosarcoma lung metastasis mechanism and medicine.
Disclosure of Invention
Based on this, there is a need to provide a primary cell strain of human osteosarcoma lung metastasis.
The primary cell strain of human osteosarcoma lung metastasis is named ZOSL-1, and the preservation number is CCTCC No: C202088.
In one embodiment, the human osteosarcoma pulmonary metastasis primary cell strain is derived from pulmonary metastases of osteosarcoma volunteers.
In one embodiment, the primary cell line of human osteosarcoma lung metastases has a cobblestone-like epithelioid cell morphology with a doubling time of 39.28 ± 3.04 h.
The invention also provides an application of the primary cell strain of the human osteosarcoma lung metastasis.
The primary cell strain of the human osteosarcoma lung metastasis is applied to research of osteosarcoma lung metastasis.
The primary cell strain of the human osteosarcoma lung metastasis is applied to research of antitumor drugs.
The invention also provides a culture method of the primary cell strain of the human osteosarcoma lung metastasis.
A method for culturing primary cell strains of human osteosarcoma lung metastases comprises the following steps: obtaining an osteosarcoma lung metastasis specimen in an aseptic environment, and cleaning the osteosarcoma lung metastasis specimen by using 5% penicillin-streptomycin-amphotericin PBS solution;
cutting osteosarcoma lung metastasis focus specimen into 1-2.5mm3Size, cultured in complete medium;
after the cells are emigrated, the tumor cells are purified by a pancreatin differential time digestion method, a differential time adherence method and a serum-free medium stimulation method in sequence to obtain the primary cell strain of the human osteosarcoma lung metastasis.
In a preferred embodiment, the complete medium is 10% Fetal Bovine Serum (FBS), 5% penicillin-streptomycin, 5% glutamine, 80% basal medium (DMEM).
In a preferred embodiment, the differential pancreatic digestion method is to separate fibroblasts from tumor cells by using pancreatic enzymes to digest the cells at different times. More preferably, 0.25. + -. 0.01% trypsin is used for digestion at 37. + -. 0.5 ℃ for 3. + -.1 min and 5. + -.2 min.
In a preferred embodiment, the differential time adherence method is to separate fibroblasts from tumor cells by setting different adherence time according to different adherence speeds of the fibroblasts and the tumor cells. The differential time adherence method comprises the steps of adding a fresh culture solution into a No. 1 culture dish, and dripping cell suspension after adherence culture; after standing and adherent culture for 30min +/-10 min in an incubator, collecting a culture solution, adding the culture solution into a No. 2 culture dish, adding a fresh culture solution into a No. 1 culture dish, and putting the culture dish into the incubator; and after the static culture is carried out in the incubator for 30min +/-10 min, the culture solution in the No. 2 culture dish is collected again, and the culture dish is replaced to repeat the operation for 1-3 times. More preferably, the different attachment times are 30min ± 10min, more preferably 30min ± 5 min.
In a preferred embodiment, the serum-free medium stimulation method is to utilize the difference of the degree of dependence of fibroblasts and tumor cells on serum, inhibit the growth of fibroblasts under the premise of ensuring the survival of tumor cells by adopting serum-free culture, and separate the fibroblasts from the tumor cells.
The primary cell strain of the lung metastasis of the human osteosarcoma screened by the invention has a cobblestone-like epithelioid cell shape, the doubling time is 39.28 +/-3.04 h, and the cell strain has a plurality of cell nucleoli. It can be used for research on lung metastasis mechanism and medicine of osteosarcoma.
The method for obtaining the primary cell strain of the human osteosarcoma lung metastasis is obtained by creatively combining a pancreatin differential time digestion method, a differential time adherence method and a serum-free culture medium stimulation method after cells are emigrated, purifying tumor cells and screening.
The osteosarcoma cell ZOSL-1 (primary cell strain ZOSL-1 of osteosarcoma lung metastasis) is preserved in China Center for Type Culture Collection (CCTCC) of the preservation unit specified by the State intellectual property office in 5 months and 27 days in 2020, and the preservation number is CCTCC No: C202088.
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FIG. 1 is a morphological diagram of primary tumor cells under an electron microscope.
FIG. 2 is a graph showing the proliferation of cells in example 2.
FIG. 3 is a schematic diagram of the result of detecting apoptosis by the flow cytometry AV/PI method in example 3.
FIG. 4 is a schematic diagram of the cell cycle results of flow cytometry in example 3.
FIG. 5 is a diagram showing the results of detection of cellular alleles in example 4.
FIG. 6 is a diagram showing the results of the cell function test in example 5.
FIG. 7 is a schematic diagram of the detection of drug resistance in cells in example 6.
FIG. 8 is a graph showing the results of the subcutaneous implantation in example 7.
Fig. 9 is a schematic illustration of the results of the tibial tray implantation of example 8.
Fig. 10 is a schematic view of the pulmonary metastasis model of the tibial cavity in example 9.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Example 1: isolated culture of osteosarcoma lung metastasis cell line
Intraoperative resection of the necrotizing zone under the tumor capsule was performed on intraoperative days of osteosarcoma patients (right tibia distal osteosarcoma right calf amputation on 5 days of right tibia 4/2016, 14 postoperative adjuvant chemotherapies; posterior thoracic CT showed posterior basal segment nodules of the left lung, about 13.3 × 18.0mm in size, and thoracoscopic left lower pulmonary wedge resection on 7 days of 2/2018), intraoperative resection of the necrotizing zone of the tumor capsule (collection of specimens approved by clinical research and laboratory animal ethics committee of the first hospital affiliated to Zhongshan university (part number: trial [2019 ]: Lung)]060), the patient signs an informed consent). The patient's active specimens were washed with PBS shaking to remove blood from the tissues. Cutting the specimen to 2mm3Size, resuspend with addition of a small amount of Fetal Bovine Serum (FBS), attach the tissue pieces piece by piece to a petri dish (diameter 10cm) using ophthalmic forceps, 5% CO at 37 ℃2The cells were allowed to stand in an incubator for 4h, and complete medium (high-glucose DMEM medium, 10% FBS, 1% penicillin and streptomycin, 1% glutamine, all from Gibco) was added thereto at 37 ℃ with 5% CO2Culturing in an incubator.
After the mass of cells had migrated out, the cells were digested with 0.25% trypsin at 37 ℃ for 3min and collected and labeled as P0 passages. The collected cells were replanted and labeled as passage P1. When the cells are full to 90%, 0.25% trypsin is used for digesting for 3min and 5min at 37 ℃, and the cells with different digestion time are collected. Cells of different digestion times, culture fluid heavy suspension. Add fresh culture medium to culture dish No. 1 and add cell suspension dropwise. And (3) after standing adherent culture for 30min in an incubator, collecting the culture solution, adding the culture solution into a No. 2 culture dish, adding a fresh culture solution into a No. 1 culture dish, and putting the culture dish into the incubator. After the static culture in the incubator is carried out for 30min, the culture solution in the No. 2 culture dish is collected again, and the culture dish is replaced to repeat the operation for 2 times. The next day, serum-free medium (high-glucose DMEM medium, 1% penicillin and streptomycin, both purchased from Gibco) was changed and placed in an incubator for culture. After the cells grow to 90% again, a polygonal and spherical tumor cell is screened after the pancreatin digestion, the adherence and the standing culture are repeated for 3 times (figure 1A), and the sterilized cotton swab is used for scratching peripheral fibroblasts to obtain primary tumor cells which are amplified. Obtaining primary tumor cells with higher tumor cell ratio by P5 generation.
And (3) inverting the microscope: ZOSL-1 grows to 80% confluency in the petri dish and is observed under an inverted microscope. The results are shown in FIG. 1B, which shows a polygonal, cobblestone, epithelioid cell morphology.
Transmission electron microscopy: and when the ZOSL-1 grows to be 80% of the full culture dish, digesting the lower cells by using pancreatin, centrifuging at 3000 r/min for 15-20min to enable the cells to be precipitated and agglomerated at the tip of a centrifugal tube, removing supernatant, and slowly adding fresh 2.5% glutaraldehyde fixing solution to avoid dispersing cell clusters. Washing with PBS buffer solution for 3 times, fixing with 2.5% glutaraldehyde electron microscope stationary liquid overnight, performing conventional transmission electron microscope sample preparation, and observing with transmission electron microscope. As a result, as shown in FIG. 1C, the nucleus has a large cytoplasmic size and many nucleoli.
Example 2: cell doubling time detection
Respectively taking 2000 ZOSL-1 cells, planting the cells in a 96-well plate, adding 100 mu L of culture solution (complete culture solution), adding 10 mu L of cck-8 reagent the next day, incubating for 1h in a dark place, detecting the light absorption value at 450nm, and replacing fresh culture solution. The assay was continued for 7 days, cell growth curves were plotted, and doubling times were calculated. A blank control was set up and PBS solution was added to the peripheral wells to reduce evaporation.
The cell doubling time DT ═ t [ lg2/(lgODt-lgOD0) ] was calculated. The proliferation curve is shown in FIG. 2, and the doubling time is 39.28. + -. 3.04 h.
Example 3: cell cycle and apoptosis detection
ZOSL-1 cells (P10, full 90% growth) were collected and after washing, apoptosis was detected using the flow cytometry AV/PI method. The results are shown in FIG. 3, which shows that 86.5% of the cells had good activity.
ZOSL-1 cells (P10, full of 90%) were collected, washed, fixed with glacial ethanol, stained with iodopyridine, and examined for cell cycle by flow cytometry. The results are shown in FIG. 4, 50.77% at G1 stage, 15.37% at G2 stage and 33.85% at S stage.
Example 4: cell STR detection and preservation
The P10 generation cells are collected, sent to the Wuhan university biological preservation center (namely China center for type culture Collection) for detection and preservation, and the preservation number is CCTCC No. C202088. The result of allele detection performed by the university of wuhan biological depository is shown in table 1 and fig. 5, and the result of allele detection shows that ZOSL-1 has no cross contamination with other cells and is not matched with the existing cell line of ATCC, i.e., a new cell line.
TABLE 1 allele detection
Figure BDA0002685297070000061
Figure BDA0002685297070000071
Example 5: transwell
Take 5 x 104ZOSL-1P 10 cells (i.e., cells having a preservation number of CCTCC No: C202088) were suspended using 200. mu.L of serum-free medium, placed in a chamber, plated in a 24-well plate, and 500. mu.L of complete medium was added to the bottom. 5% CO at 37 ℃2Standing in the incubator for 24h, sucking off the liquid inside and outside the chamber, slightly wiping off the cells which are not penetrated on the inner surface of the basement membrane of the chamber by a small cotton swab, washing the inside of the chamber by distilled water,and then rubbed again with a small cotton swab. The cell was fixed in methanol for 30 min; after the cell was aspirated, 500uL of crystal violet solution was added and the staining was performed at room temperature for 30 min. Washing with ultrapure water, air drying, and performing microscopic examination.
The results are shown in FIG. 6.A, where the cells have metastatic capacity.
800 cells of ZOSL-1P 10 were seeded in a 6-well plate, 2mL of the culture medium was added, and after 10 days of culture, the cell morphology was observed by crystal violet staining.
The results are shown in FIG. 6.B, where the cells have clonogenic capacity.
And (3) planting ZOSL-1P 10 cells in a 6-hole plate, scratching along the symmetrical line of a culture dish after the cells grow full, adding a serum-free culture solution after PBS (phosphate buffer solution) is cleaned, and observing the healing conditions of scratches within 0h, 6h, 12h and 24 h.
The results are shown in fig. 6.C, the scratch healed 53% after 24h for the cells, indicating that the cells have good migration ability.
Example 6: cell drug resistance detection
ZOSL-1P 10 cells were plated in 96-well plates at 2000 cells per well, and doxorubicin (ADM), Methotrexate (MTX), and cisplatin (DDP) were added the next day. After 72h, the OD of the cells was measured by mtt method.
The results are shown in fig. 7, the calculation IC50, ADM IC50 ═ 405.8nM, MTX IC5 ═ 948.7ng/mL, and DDP IC50 ═ 1.784 μ M, have strong drug resistance, and are very suitable for being used as preclinical research models, lung metastasis mechanism research, new drug development and the like.
Example 7: subcutaneous neoplasia
150 ten thousand ZOSL-1P 10 cells/70 mu LPBS solution are injected into nude mice (5-7 weeks old) for subcutaneous feeding in SPF environment for 2 months, and specimens are collected, HE sections are prepared, and microscopic examination is performed.
As a result, as shown in FIG. 8, a large amount of necrosis occurred in the tumor tissue, and the non-necrotic part was large in nuclear cytoplasm and some cells had nuclear fission images.
Example 8: tibial cavity tumorigenesis
150 ten thousand ZOSL-1P 10 cells/20 mu LPBS solution are injected into the left tibia cavity of a nude mouse (5-7 weeks old), raised for 2 months in SPF environment, collected, transected, HE sliced, and examined under a microscope.
The results are shown in FIG. 9, wherein both cortical and cancellous bone were invaded and transformed into sarcoma tissue, and some muscles were invaded, indicating that ZOSL-1 has stronger characteristics of sarcoma. The tumor tissue has necrosis, the non-necrotic part of the cells have larger nuclear cytoplasm, and part of the cells have nuclear fission images.
Example 9: tibia cavity in-situ lung metastasis model
The virus amount calculated by adding 1mLFBS to 9mLDMEM medium and 200. mu. LENhance solution to MOI (50). times.cell count 1.3/virus titer (108), i.e., 130. mu. L m-Cherry virus solution was added to 4mL of the dilution. And (3) planting 20 ten thousand ZOSL-1 cells in a 6-well plate, adding a virus solution the next day, culturing for 24 hours, replacing a normal culture solution, and culturing for 2-3 days to observe the cell transfection condition under a fluorescence microscope. And (4) continuously amplifying. Puromycin solution (normal culture solution added with puromycin, 1:10000, 2. mu.g/mL-7. mu.g/mL, starting from the minimum concentration for screening) is prepared, and cells are collected after 48 h.
The transfected cells were expanded. 150 million cells/20. mu.LPBS solution were injected into the tibial cavity of NSG mice (NOD-Prkdcscid Il2rgtm 1/Bcgen). After 2 months of feeding, the distribution condition of the cells is detected by a living body imaging instrument, lung tissues are collected, HE slices are prepared, and microscopic examination is carried out.
The results are shown in FIG. 10, which shows that the m-Cherry stained cells are distributed in the tibia and lung tissues, the lung tissues are multinode, the nucleus quality in the node is large, and a nuclear fission image exists.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

Claims (10)

1. The human osteosarcoma lung metastasis focus primary cell strain has the preservation number of CCTCC No: C202088.
2. The human osteosarcoma pulmonary metastasis primary cell strain of claim 1, wherein the human osteosarcoma pulmonary metastasis primary cell strain is derived from pulmonary metastases of osteosarcoma volunteers.
3. The primary cell line of human osteosarcoma lung metastasis according to claim 1, wherein the primary cell line of human osteosarcoma lung metastasis has a cobblestone-like epithelioid cell morphology with a doubling time of 39.28 ± 3.04 h.
4. Use of the primary cell line of human osteosarcoma lung metastasis according to any one of claims 1 to 3 for the study of osteosarcoma lung metastasis.
5. Use of the primary cell strain of human osteosarcoma lung metastasis as claimed in any one of claims 1 to 3 in the research of antitumor drugs.
6.A method for culturing a primary cell strain of a human osteosarcoma lung metastasis, which is characterized by comprising the following steps: obtaining an osteosarcoma lung metastasis specimen in an aseptic environment, and cleaning the osteosarcoma lung metastasis specimen by using a PBS (phosphate buffer solution) solution containing penicillin-streptomycin-amphotericin;
cutting osteosarcoma lung metastasis focus specimen into 1-2.5mm3Size, cultured in complete medium;
after the cells are emigrated, the tumor cells are purified by a pancreatin differential time digestion method, a differential time adherence method and a serum-free medium stimulation method in sequence to obtain the primary cell strain of the human osteosarcoma lung metastasis.
7. The method for culturing primary cell line of human osteosarcoma lung metastasis according to claim 6, wherein the complete culture medium comprises 10% fetal bovine serum, 5% penicillin-streptomycin, 5% glutamine, and 80% basal medium.
8. The method for culturing the primary cell line of human osteosarcoma lung metastasis according to claim 6, wherein the differential pancreatic digestion comprises digestion with 0.25 ± 0.01% trypsin at 37 ± 0.5 ℃ for 3 ± 1min and 5 ± 2min to separate fibroblasts from tumor cells.
9. The method for culturing the primary cell strain of the lung metastasis of human osteosarcoma according to claim 6, wherein the differential time adherence method comprises adding a fresh culture solution to a No. 1 culture dish, and dripping a cell suspension after adherence culture; after standing and adherent culture for 30min +/-10 min in an incubator, collecting a culture solution, adding the culture solution into a No. 2 culture dish, adding a fresh culture solution into a No. 1 culture dish, and putting the culture dish into the incubator; and after the static culture is carried out in the incubator for 30min +/-10 min, the culture solution in the No. 2 culture dish is collected again, and the culture dish is replaced to repeat the operation for 1-3 times.
10. The method for culturing the primary cell strain of human osteosarcoma lung metastasis, according to claim 9, wherein the differential time adherence method comprises adding fresh culture solution to No. 1 culture dish, and dripping cell suspension after adherence culture; after standing and adherent culture for 30min in an incubator, collecting a culture solution, adding the culture solution into a No. 2 culture dish, adding a fresh culture solution into a No. 1 culture dish, and putting the culture dish into the incubator; after the static culture in the incubator is carried out for 30min, the culture solution in the No. 2 culture dish is collected again, and the culture dish is replaced to repeat the operation for 2 times.
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CN116836934B (en) * 2023-08-31 2023-11-24 北京大橡科技有限公司 Osteosarcoma organoid culture solution, culture reagent combination and culture method

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