CN114774363A - Method for obtaining tumor stem cells - Google Patents

Method for obtaining tumor stem cells Download PDF

Info

Publication number
CN114774363A
CN114774363A CN202210180810.4A CN202210180810A CN114774363A CN 114774363 A CN114774363 A CN 114774363A CN 202210180810 A CN202210180810 A CN 202210180810A CN 114774363 A CN114774363 A CN 114774363A
Authority
CN
China
Prior art keywords
cells
tumor stem
stem cells
cell
steps
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210180810.4A
Other languages
Chinese (zh)
Inventor
刘庆喜
肖海蓉
赵梦莲
任倩影
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Boya Perception Pharmaceutical Co ltd
Qilu Institute of Technology
Original Assignee
Shenzhen Boya Perception Pharmaceutical Co ltd
Qilu Institute of Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Boya Perception Pharmaceutical Co ltd, Qilu Institute of Technology filed Critical Shenzhen Boya Perception Pharmaceutical Co ltd
Priority to CN202210180810.4A priority Critical patent/CN114774363A/en
Publication of CN114774363A publication Critical patent/CN114774363A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0695—Stem cells; Progenitor cells; Precursor cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2523/00—Culture process characterised by temperature

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Cell Biology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Oncology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention provides a method for obtaining tumor stem cells, which comprises the steps of carrying out dry culture on a glioma cell line, carrying out low-temperature stimulation on cells in the dry culture process to activate and amplify initial tumor stem seed cells, then carrying out sorting by using dry related markers, removing adherent glioma cells without dry characteristics in the culture process after sorting, and carrying out repeated tumor ball culture on residual suspension cells to obtain the tumor stem cells. The tumor stem cell acquisition method can rapidly amplify cells with strong dryness, and provides a large amount of tumor stem seed cells for later sorting and culturing.

Description

Method for obtaining tumor stem cells
Technical Field
The invention belongs to the field of cell biology, and particularly relates to a method for acquiring tumor stem cells.
Background
Tumor stem cells (CSCs) are a subpopulation of cancer cells that have the ability to resemble somatic stem cells. CSCs play a pivotal role in the development, metastasis, recurrence and resistance of tumors. The vast majority of tumors are considered to originate from CSCs, which may be one of the important reasons for primary and acquired drug resistance in clinical tumor treatment, so that the research on tumor stem cells has attracted extensive attention in the basic research of tumors at home and abroad in recent years.
At present, tumor stem cells are mainly obtained from tumor tissues and tumor cell lines, and because the tumor tissues obtained from clinical sources are limited, and part of the tumor tissues need to be subjected to clinical pathological analysis, most of the tumor tissues for separating the tumor stem mainly comprise tissues beside cancer, which also restricts the obtaining of the tumor stem cells. There is also a limitation in obtaining tumor stem cells from tumor cell lines, for example, the tumor cell lines have a low proportion of dry cells, and when they are sorted by magnetic beads or flow cytometry, certain stem cells are lost, which finally results in low obtained tumor stem cells or failure in separation.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for obtaining tumor stem cells, which comprises performing dry culture before sorting tumor stem cells, activating and amplifying initial tumor stem seed cells by low-temperature stimulation, etc., performing magnetic bead/flow sorting, and removing adherent glioma cells without stem characteristics in the process of culture after sorting to obtain tumor stem cells, thereby providing a tamping technical basis for the lack of tumor stem resources at present.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a method for obtaining tumor stem cells, comprising the steps of:
1) carrying out dry culture on the glioma cell line U118MG, and carrying out low-temperature stimulation in the culture process;
2) sorting immunomagnetic beads;
3) differential adherent purification after sorting;
4) tumor stem is rapidly amplified.
Further, the low-temperature stimulation method comprises the following steps: culturing at 4-10 deg.C for 10-30 min for 1-5 times.
Further, the step 1) specifically comprises the following steps: u-118MG cells were seeded in tumor stem cell culture medium containing 1-40ng/ml bFGF, 1-20ng/ml EGF, 0.5-4% B27-50X, 0.1-0.4% streptomycin-100X in DMEM/F12 serum-free complete medium and cultured for 3-5 days.
Further, recovering the U118MG cells and passaging the U-118MG cells before the step 1).
Further, the recovery of the U118MG cells comprises the following steps: and (3) taking out the frozen U-118MG cells from the liquid nitrogen tank, quickly putting the cells into a water bath kettle at 37 ℃ for quick thawing, then sucking the cell suspension into a pre-placed centrifuge tube containing 6mL of preheated basal culture medium for centrifugation, and culturing the cells after removing supernatant.
Further, the U-118MG cell passage is carried out when the growth density of the U-118MG cell reaches more than 95% of the bottom area of the culture flask, and the method comprises the following steps: digesting the cultured cells by using pancreatin, and suspending the cells after the digestion is finished; the cell suspension was centrifuged, and the supernatant was discarded, followed by cell culture.
Further, the step 2) specifically comprises the following steps:
1) digesting and resuspending the cell structure obtained in the step 1), centrifuging the cell suspension, collecting cell sediment, and resuspending again;
2) adding an FcR blocking reagent and CD133 magnetic beads into the cell suspension, fully and uniformly mixing, and then incubating in a refrigerator;
3) after incubationWashing, centrifuging and re-suspending the cells, adding the cells into a separation column to wash off unlabelled CD133 cells so as to obtain positive CD133 cells+And left in the separation column.
Further, the identification of the tumor stem cells is also included after the step 3) and before the step 4).
Further, the identification of the tumor stem cells comprises the following steps:
1) collecting the cultured cells, digesting, adding 4% paraformaldehyde fixing solution into the cells, fixing for 15min, centrifuging, and resuspending;
2) respectively adding 2 mu L of FITC-CD133, APC-OCT4 and PE-CD44 antibodies into the cell suspension, and incubating for 1h at room temperature in a dark place;
3) PBS washing to remove unbound CD133, OCT4, CD44 antibodies;
4) and (5) carrying out flow cytometry detection.
Compared with the prior art, the tumor stem cell obtaining method has the following advantages:
the method for obtaining the tumor stem cells improves the method for extracting the tumor stem cells, dry culture is carried out on a glioma cell line, low-temperature stimulation and other means are carried out on the cells in the dry culture process so as to activate and amplify the initial tumor stem seed cells, then sorting is carried out by utilizing dry related markers, adherent glioma cells without stem characteristic are removed in the culture process after sorting, and the cells express three stem cell specific biomarkers (CD133)+、OCT4+And CD44+) And the method can rapidly expand a large number of tumor stem cells so as to meet the working requirements of conventional tumor biomedical basic research, clinical research, antitumor drug development and the like.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, illustrate embodiments of the invention and together with the description serve to explain the invention and do not constitute a limitation of the invention. In the drawings:
FIG. 1 is a tumor stem cell map.
FIG. 2 is a diagram of flow identification of tumor stem cells;
FIG. 3 is a diagram of tumor stem cells cultured according to an embodiment of the present invention;
FIG. 4 is a diagram of tumor stem cells cultured in comparative examples of the present invention.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
The method for obtaining the tumor stem cells specifically comprises the following steps:
1) recovering U118MG cells: the cancer cell culture medium and DMEM/F12 basal medium were preheated to 37 ℃ in a water bath and 6mL of preheated basal DMEM/F12 medium was added to a 15mL centrifuge tube. Taking out frozen U-118MG cells from liquid nitrogen tank, rapidly thawing in 37 deg.C water bath pan (thawing degree is suitable for small amount of ice crystal left in freezing tube), sucking cell suspension into a pre-placed centrifuge tube containing 6mL of basic culture medium, centrifuging at room temperature of 1000r/min for 5min, discarding supernatant, adding appropriate amount of culture solution with cell concentration of 1 × 105cells/mL were grown in culture flasks at 37 ℃ with 5% CO2Culturing in an incubator, and performing liquid change treatment every 2 d.
2) Passage of U-118MG cells: and the growth density of the U-118MG cells reaches more than 95% of the bottom area of the culture flask for cell passage.
The method comprises the following specific steps:
(1) the medium and PBS were pre-warmed at 37 ℃ in advance.
(2) The cell culture was aspirated and a small amount of PBS was added to rinse the cells.
(3) Removing PBS by suction, adding appropriate amount of pancreatin to cover cells, adding 5% CO at 37 deg.C2Digesting in an incubator for about 3min, adding a culture medium with 2 times of pancreatin volume to stop digestion when cells tend to be round, cell gaps become larger and a small amount of cells float, and lightly blowing and beating the cells by using a gun head to completely suspend the cells.
(4) Collecting cell suspension, and separating at room temperature 1000r/minCentrifuging for 5min, removing supernatant, and blowing with culture medium to make cell concentration 1 × 105The cells/mL are planted in culture bottles and placed at 37 ℃ in 5% CO2Culturing in an incubator.
3) Dry culture of tumor cells: cells were cultured at 1X 105Each cell/mL was inoculated in tumor stem cell culture medium containing 10ng/mL bFGF, 20ng/mL EGF, 2mL of B27-50X, 200. mu.L of penicillin-100X DMEM/F12 serum-free complete medium and cultured 3 times for 15 minutes each at 4 ℃ during the culture for 4 days.
4) And (3) sorting immunomagnetic beads: the sorting operation was performed strictly according to the Miltenyi Biotec magnetic bead sorting instructions, with the following specific steps:
(1) the U-118MG adherent cells were washed with PBS to remove dead cells, and digested with pancreatin.
(2) The cells were resuspended in a basal medium, centrifuged at 1000 rpm for 5min and the cell pellet collected.
(3) At 107The total cells were added to 60. mu.L buffer to resuspend the cell pellet.
(4) Every 10 th7mu.L of FcR blocking reagent was added to each total cell.
(5) Every 10 th7mu.L of CD133 magnetic beads were added to each total cell, mixed well and incubated in a refrigerator (2-8 ℃) for 15 min. The incubation was continued with slow rotation using a MACSmix rotator to prevent cell sedimentation.
(6) The incubated cells were washed, 1-2mL of buffer was added to wash the cells, and then centrifuged at 300g/min for 10min, and the supernatant was discarded.
(7) At 107Each cell was suspended by blowing 500. mu.L of buffer (10)7Individual cells).
(8) The separation column (500. mu.L) was placed in a separator, and the separation column was rinsed with a rinse solution.
(9) Slowly adding the incubated cells into the separation column, collecting the unlabeled cells, and adding the unlabeled cells into the separation column again.
(10) Washing the column with appropriate amount of buffer, repeating 3 times, and washing to remove unlabeled cells (CD133), to obtain positive cells(CD133+) And left in the separation column.
(11) And taking the separation column off the separation frame, and putting the separation column on a collecting pipe for elution.
5) And (3) carrying out ball-shaped culture on the sorted cells: the cells sorted by magnetic beads were divided into 1X 105Inoculating each cell/mL in a tumor stem cell culture medium, attaching more first-generation cells in spherulitic culture to the wall, collecting cells growing in spherulitic growth, carrying out pancreatin digestion, repeatedly collecting the spherulitic cells to remove the attached cells, facilitating the purification of the tumor stem cells, and obtaining the dry cells completely suspended in spherulitic growth after repeated passage.
6) And (3) identifying the tumor stem cells: (1) collecting the dry U-118MG cells, digesting with 0.05% pancreatin, terminating the culture medium, centrifuging at 1000r/min for 5min, washing with PBS, and counting.
(2) The cell suspension was centrifuged at 1000r/min for 5min and the supernatant was discarded.
(3) Adding 4% paraformaldehyde stationary liquid, fixing for 15min, washing with PBS, centrifuging at 1000r/min for 5min, and blowing and suspending with PBS to obtain suspension with density of 1 × 106Individual cells/mL of cell suspension.
(4) mu.L each of FITC-CD133, APC-OCT4, PE-CD44 antibodies (diluted 1:50 according to the recommended proportion of the antibody specification of Meitian and whirlpool) was added and incubated for 1h at room temperature in the dark.
(5) PBS was washed 3 times to remove unbound CD133, OCT4, CD44 antibody.
(6) The cells were resuspended in 500. mu.L PBS and flow cytometric assayed as shown in FIG. 2.
7) Tumor stem rapid amplification: the cells sorted by magnetic beads were divided into 1X 105The cells/mL are inoculated in a tumor stem cell culture medium, although serum is removed, the culture medium is added with a plurality of growth factors for cell growth, such as serum substitutes of bFGF, EGF and the like, and necessary nutrients such as insulin, transferrin and the like which are used for cell survival are also reserved. In the absence of serum differentiation promoters, the sorted cells are more conducive to growth of suspended spheroids.
The tumor stem cells obtained by the method are shown in figure 1, and the obtained tumor stem cells grow in a suspension manner and are aggregated to grow into spheres.
As shown in FIG. 2, the obtained tumor stem cells had CD133 by flow assay+CD44+OCT4+Three positive properties.
Comparative example
In addition to the above examples, low temperature stimulation was not performed during the dry culture of tumor cells.
The culture results are shown in FIG. 4, and it can be seen from FIG. 4 that the cell balling effect is poor, and most of the cells are in an adherent growth state, which indicates that the low temperature condition can stimulate the growth of the tumor stem cells.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (9)

1. A method for obtaining tumor stem cells, comprising: the method comprises the following steps:
1) carrying out dry culture on the glioma cell line U118MG, and carrying out low-temperature stimulation in the culture process;
2) sorting immunomagnetic beads;
3) differential adherent purification after sorting;
4) tumor stem is rapidly amplified.
2. The method for obtaining tumor stem cells according to claim 1, wherein: the low-temperature stimulation method comprises the following steps: culturing at 4-10 deg.C for 10-30 min for 1-5 times.
3. The method for obtaining tumor stem cells according to claim 1, wherein: the step 1) specifically comprises the following steps: u-118MG cells were seeded in tumor stem cell culture medium containing 1-40ng/ml bFGF, 1-20ng/ml EGF, 0.5-4% B27-50X, 0.1-0.4% streptomycin-100X in DMEM/F12 serum-free complete medium and cultured for 3-5 days.
4. The method for obtaining tumor stem cells according to claim 1, wherein: the method also comprises the steps of recovering the U118MG cells and passaging the U-118MG cells before the step 1).
5. The method for obtaining tumor stem cells according to claim 4, wherein: the recovery of the U118MG cells comprises the following steps: and (3) taking out the frozen U-118MG cells from the liquid nitrogen tank, quickly putting the cells into a water bath kettle at 37 ℃ for quick thawing, then sucking the cell suspension into a pre-placed centrifuge tube containing 6mL of preheated basal culture medium for centrifugation, and culturing the cells after removing supernatant.
6. The method for obtaining tumor stem cells according to claim 4, wherein: and (3) carrying out cell passage on the U-118MG cells until the growth density of the U-118MG cells reaches more than 95% of the bottom area of a culture flask, wherein the cell passage comprises the following steps: digesting the cultured cells by using pancreatin, and suspending the cells after the digestion is finished; the cell suspension was centrifuged, and the supernatant was discarded before cell culture.
7. The method for obtaining tumor stem cells according to claim 1, wherein: the step 2) specifically comprises the following steps:
1) digesting and resuspending the cell structure obtained in the step 1), centrifuging the cell suspension, collecting cell sediment, and resuspending again;
2) adding an FcR blocking reagent and CD133 magnetic beads into the cell suspension, fully and uniformly mixing, and then incubating in a refrigerator;
3) washing, centrifuging and re-suspending the incubated cells, adding the cells into a separation column to wash off unlabeled CD133 cells so as to obtain positive CD133 cells+Left in the separation column.
8. The method for obtaining tumor stem cells according to claim 1, wherein: the identification of the tumor stem cells is also included after the step 3) and before the step 4).
9. The method for obtaining tumor stem cells according to claim 8, wherein: the identification of the tumor stem cells comprises the following steps:
1) collecting and digesting the cultured cells, adding 4% paraformaldehyde fixing solution into the cells for fixing for 15min, and then centrifuging and resuspending;
2) respectively adding 2 mu L of FITC-CD133, APC-OCT4 and PE-CD44 antibodies into the cell suspension, and incubating for 1h at room temperature in a dark place;
3) PBS washing to remove unbound CD133, OCT4, CD44 antibodies;
4) and (5) carrying out flow cytometry detection.
CN202210180810.4A 2022-02-25 2022-02-25 Method for obtaining tumor stem cells Pending CN114774363A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210180810.4A CN114774363A (en) 2022-02-25 2022-02-25 Method for obtaining tumor stem cells

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210180810.4A CN114774363A (en) 2022-02-25 2022-02-25 Method for obtaining tumor stem cells

Publications (1)

Publication Number Publication Date
CN114774363A true CN114774363A (en) 2022-07-22

Family

ID=82422556

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210180810.4A Pending CN114774363A (en) 2022-02-25 2022-02-25 Method for obtaining tumor stem cells

Country Status (1)

Country Link
CN (1) CN114774363A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115521916A (en) * 2022-10-11 2022-12-27 中国人民解放军中部战区总医院 Culture and identification method of primary tumor stem cells of human brain glioblastoma

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120028933A1 (en) * 2010-07-28 2012-02-02 Baust John M Cell Culture Media Supplement and Method of Molecular Stress Control
CN104312976A (en) * 2014-10-14 2015-01-28 广州市搏克肿瘤研究所 Separating method of tumor stem cells
CN106282117A (en) * 2016-08-31 2017-01-04 湖南艾佳生物科技股份有限公司 The enrichment of tumor stem cell and screening technique
CN108949689A (en) * 2018-08-07 2018-12-07 武汉合研生物医药科技有限公司 A kind of method for separating and detecting of tumor stem cell

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120028933A1 (en) * 2010-07-28 2012-02-02 Baust John M Cell Culture Media Supplement and Method of Molecular Stress Control
CN104312976A (en) * 2014-10-14 2015-01-28 广州市搏克肿瘤研究所 Separating method of tumor stem cells
CN106282117A (en) * 2016-08-31 2017-01-04 湖南艾佳生物科技股份有限公司 The enrichment of tumor stem cell and screening technique
CN108949689A (en) * 2018-08-07 2018-12-07 武汉合研生物医药科技有限公司 A kind of method for separating and detecting of tumor stem cell

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LEI SHI: "Hypothermia Stimulates Glioma Stem Spheres to Spontaneously Dedifferentiate Adjacent Non-stem Glioma Cells", CELL MOL NEUROBIOL, pages 217 - 274 *
周金方: "亚低温促进胶质瘤干细胞形成", 豆丁 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115521916A (en) * 2022-10-11 2022-12-27 中国人民解放军中部战区总医院 Culture and identification method of primary tumor stem cells of human brain glioblastoma

Similar Documents

Publication Publication Date Title
CN107299082B (en) Method for separating placenta mesenchymal cells from tissues and culturing into mesenchymal stem cells
CN114292816B (en) Lung cancer organoid culture solution, and culture reagent combination and culture method thereof
CN108611322B (en) Breast cancer circulating tumor cell line CTC-3, culture medium, and establishment method and application of CTC-3
CN104312976B (en) Separating method of tumor stem cells
CN112662627B (en) Culture solution for differentiating pluripotent stem cells into natural killer cells and differentiation method
CN114317443A (en) Breast cancer organoid culture solution, and culture reagent combination and culture method thereof
CN104630144A (en) Method for separating and culturing umbilical cord blood mesenchymal stem cells
US20220162560A1 (en) Three-dimensional culture method for large-scale preparation of stem cells
CN114774363A (en) Method for obtaining tumor stem cells
CN110804585A (en) Separation method of umbilical cord mesenchymal stem cells
CN102344909B (en) Method for separating human spermatogonial stem cells
CN108034634B (en) Method for separating endometrial mesenchymal stem cells from menstrual blood
WO2022213704A1 (en) High-migration mesenchymal stem cell, and preparation method therefor and application thereof
CN109609452A (en) A kind of efficient macrophages in vitro preparation method
CN102154203A (en) Method for directionally inducing insulin-secreting cells by endometrial stem cells
CN111411082B (en) Culture medium for culturing CD90posi cells and culture method thereof
CN112159790B (en) Method for purifying pluripotent vascular progenitor cells from perinatal tissue
CN104099296B (en) A kind of preparation method of rabbit umbilical cord mesenchymal stem cells
CN106957814B (en) Culture medium for amniotic mesenchymal stem cells and method for culturing amniotic mesenchymal stem cells
CN111254116A (en) Method for enriching and flow-sorting human ovarian cancer stem cells
CN108285890A (en) A kind of preparation method of endothelial progenitor cells
CN116836933B (en) Liver and gall cancer organoid culture solution, culture reagent combination and culture method
CN110527666A (en) A kind of sub-sieve enrichment method of cancer stem cell
CN116410929A (en) Lung cancer organoid culture solution, culture reagent combination and culture method
CN116376821B (en) Method for improving expression quantity of umbilical cord mesenchymal stem cell exosomes

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination