CN113502266A - Culture method for CIK-containing cells in peripheral blood - Google Patents

Culture method for CIK-containing cells in peripheral blood Download PDF

Info

Publication number
CN113502266A
CN113502266A CN202110784404.4A CN202110784404A CN113502266A CN 113502266 A CN113502266 A CN 113502266A CN 202110784404 A CN202110784404 A CN 202110784404A CN 113502266 A CN113502266 A CN 113502266A
Authority
CN
China
Prior art keywords
cells
culture
medium
peripheral blood
culturing
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
CN202110784404.4A
Other languages
Chinese (zh)
Inventor
占震锋
李庆静
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.)
Shanghai Nanbinjiang Cell Biotechnology Co ltd
Original Assignee
Shanghai Nanbinjiang Cell Biotechnology Co ltd
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 Shanghai Nanbinjiang Cell Biotechnology Co ltd filed Critical Shanghai Nanbinjiang Cell Biotechnology Co ltd
Priority to CN202110784404.4A priority Critical patent/CN113502266A/en
Publication of CN113502266A publication Critical patent/CN113502266A/en
Pending legal-status Critical Current

Links

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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • 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
    • C12N5/0634—Cells from the blood or the immune system
    • 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
    • C12N2500/05—Inorganic 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2301—Interleukin-1 (IL-1)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2321—Interleukin-21 (IL-21)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]
    • 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
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • 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
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/51—B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • 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
    • C12N2501/998—Proteins not provided for elsewhere
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses a culture method for CI K cells contained in peripheral blood, which belongs to the technical field of biology, and the culture method for CI K cells contained in peripheral blood comprises the following steps: step 1, extracting peripheral blood and culturing mononuclear cells; and 2, culturing the CI K cells. In addition, the CD28 monoclonal antibody is added in the culture process of the CI K cells, the activation of the T cells can be increased by utilizing CD28, the generation of the T cells I L-2 and the proliferation of the antigen-specific polyclonal T cells are promoted, the proliferation multiple of the CI K cells can be obviously increased, and the proliferation capacity of the CI K cells is improved; i L-1 alpha, I L-15 and I L-21 stimulating factors are also added to induce the expression of perforin, granzyme B and CD107a in the cultured CIK cells, so that the cytotoxic activity of the CI K cells is improved.

Description

Culture method for CIK-containing cells in peripheral blood
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture method of CIK (cytokine induced killer) cells contained in peripheral blood.
Background
CIK (Cytokine-Induced Killer Cells) Chinese is all called as Cytokine-Induced Killer Cells, and is a group of heterogeneous Cells obtained by co-culturing human peripheral blood mononuclear Cells with a plurality of cytokines for a period of time in vitro. It has both strong anti-tumor activity of T lymphocytes and non-MHC (major histocompatibility antigen) restricted tumor killing ability of NK cells (natural killer cells). The CIK cell has the characteristics of high tumor killing activity, wide tumor killing spectrum, low toxicity to normal tissues, high in-vitro amplification and the like, and is an adoptive immunotherapy cell widely used clinically at present.
At present, the common activation method of the CIK cells is to activate the peripheral blood mononuclear cells obtained by separation by using factors such as CD28 monoclonal antibody, interleukin-1 (IL-1 α), interleukin-2 (IL-2) and gamma-interferon (IFN- γ), and further to amplify the cells by using a static and intermittent liquid changing method in a long-term culture method, however, the common phenomena of low activation efficiency, long culture period and limitation of cell growth due to nutrient depletion or toxic byproduct accumulation exist, and the cell amplification effect is difficult to meet clinical requirements.
Disclosure of Invention
The invention aims to provide a method for culturing CIK cells contained in peripheral blood, which is used for solving the technical problems of poor reproductive capacity and low cytotoxic activity of the CIK cells cultured by the conventional CIK cell culture method.
The purpose of the invention can be realized by the following technical scheme:
a culture method of CIK cells contained in peripheral blood comprises the following steps:
step 1, peripheral blood extraction and mononuclear cell culture: collecting peripheral blood of a patient or a volunteer in a sterile environment, adding heparin sodium, and centrifuging once by using a lymph separation liquid to separate mononuclear cells; sucking the mononuclear cell layer by using a capillary, washing by using physiological saline, centrifuging for the second time, purifying, and repeating for 2-3 times; then suspending the cells in a medium A and adjusting the cell density to 4X 106L, and inoculating the culture medium A to 30cm2Then the flask was placed at 37 ℃ and 5% CO2Culturing for 24h in an incubator at the humidity of 97-100%, collecting cells, centrifuging for three times, and removing supernatant to obtain cultured mononuclear cells;
step 2, culturing the CIK cells: suspending the cultured mononuclear cells obtained in step 1 with an activation medium B and adjusting the cell density to 1.8X 106L, and inoculating the activated medium B to 180cm2The CD28 antibody-coated culture flask was placed at 37 ℃ and 5% CO2Culturing at 97-100% humidity in incubator for 24 hr, sequentially adding IL-15 and IL-21, culturing, replacing amplification culture medium C every 1-2 days for half a day, and maintaining cell density at 1.8 × 106mL, cultured to the required amount.
Further, in the step 1, the centrifugation conditions of the first centrifugation are that the centrifugal force is 260Xg, the centrifugation time is 18min, the temperature is 13-18 ℃, the centrifugation conditions of the second centrifugation are that the centrifugal force is 300Xg, the centrifugation time is 23min, the temperature is 20-23 ℃, the centrifugation conditions of the third centrifugation are that the centrifugal force is 250Xg, the centrifugation time is 17min, and the temperature is 17-22 ℃.
Further, in the step 1, the culture medium A is formed by mixing culture medium dry powder, L-glutamine, basic fibroblast growth factor, vitamin C, ALK inhibitor, GSK-3 inhibitor, insulin, human plasma albumin and ammonium bicarbonate, wherein the concentration of the L-glutamine is 0.5-1.5g/L, the concentration of the basic fibroblast growth factor is 0.2-0.6mg/L, the concentration of the vitamin C is 12-18mg/L, ALK inhibitor is 1-5ug/L, GSK-3 inhibitor is 1-5ug/L, the concentration of the insulin is 6-9mg/L, the concentration of the human plasma albumin is 1-5g/L, and the concentration of the ammonium bicarbonate is 5-15 g/L.
Further, in step 2, the activated medium B is formed by adding IL-2, IL-1. alpha. and IFN-. gamma.to the medium A, and the concentration of IL-2 is 5X 104-1×105U/L, IL-1. alpha. concentration 5X 104-1×105U/L, IFN-gamma concentration of 2X 105-2×106U/L, the culture bottle coated with the CD28 antibody is formed by coating the bottom of the culture bottle with a coating solution and incubating for 70min at 14 ℃, wherein the coating solution is prepared by dissolving CD28 monoclonal antibody and IFN-gamma in PBS buffer solution, and the concentration of CD28 is 1 × 105-1×106U/L, IFN-gamma concentration of 2X 105-2×106U/L。
Furthermore, the concentrations of IL-15 and IL-21 in the culture solution after the addition in the step 2 are respectively 16-19 mug/L and 7-9 mug/L.
Further, the activation medium C in step 2 is formed by adding IL-2 to the medium A, and the concentration of IL-2 is 2X 105-1×106U/L。
The invention has the beneficial effects that:
1. in the invention, the CD28 monoclonal antibody is added in the culture process of the CIK cells, the activation of the T cells can be increased by utilizing CD28, the generation of the IL-2 of the T cells and the proliferation of the antigen-specific polyclonal T cells are promoted, in addition, a large amount of activated and increased effector T cells are stimulated by the CD28 antibody, the proliferation multiple of the CIK cells can be obviously increased, and the reproductive capacity of the CIK cells is improved;
2. IL-1 alpha, IL-15 and IL-21 stimulating factors are added in the culture process of the CIK cells, so that the expression of perforin, granzyme B and CD107a in the cultured CIK cells is induced, and the cytotoxic activity of the CIK cells is improved.
Therefore, the culture method of CIK cells contained in peripheral blood provided by the invention can solve the technical problems of poor reproductive capacity and low cytotoxic activity of the CIK cells cultured by the conventional CIK cell culture method.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
a culture method of CIK cells contained in peripheral blood comprises the following steps:
step 1, peripheral blood extraction and mononuclear cell culture: collecting peripheral blood of a patient or a volunteer in a sterile environment, adding heparin sodium, and centrifuging with a lymph separation solution at 15 deg.C under a centrifugal force of 260Xg for 18min to separate mononuclear cells; sucking the mononuclear cell layer by capillary, washing with normal saline, centrifuging at 20 deg.C and 300Xg for 23min, and repeating for 2 times; then suspending the cells in a medium A and adjusting the cell density to 4X 106L, and inoculating the culture medium A to 30cm2Then the flask was placed at 37 ℃ and 5% CO2Culturing for 24h at 97% humidity in an incubator, collecting cells, centrifuging for 17min at 17 ℃ and 250Xg centrifugal force, and removing supernatant to obtain cultured mononuclear cells;
step 2, culturing of CIK cellsCulturing: suspending the cultured mononuclear cells obtained in step 1 with an activation medium B and adjusting the cell density to 1.8X 106L, and inoculating the activated medium B to 180cm2The CD28 antibody-coated culture flask was placed at 37 ℃ and 5% CO2Culturing at 97% humidity in incubator for 24 hr, sequentially adding IL-15 and IL-21, culturing, replacing amplification culture medium C every 1.5 days for half, and maintaining cell density at 1.8 × 106The culture flask coated with the CD28 antibody is formed by coating the bottom of the culture flask with a coating solution and incubating for 70min at 14 ℃.
The raw materials used in the above steps are shown in table 1.
TABLE 1
Figure BDA0003158573940000041
Figure BDA0003158573940000051
Example 2:
a culture method of CIK cells contained in peripheral blood comprises the following steps:
step 1, peripheral blood extraction and mononuclear cell culture: collecting peripheral blood of a patient or a volunteer in a sterile environment, adding heparin sodium, and centrifuging with a lymph separation solution at 16 deg.C under a centrifugal force of 260Xg for 18min to separate mononuclear cells; sucking the mononuclear cell layer by capillary, washing with normal saline, centrifuging at 20 deg.C and 300Xg for 23min, and repeating for 2 times; then suspending the cells in a medium A and adjusting the cell density to 4X 106L, and inoculating the culture medium A to 30cm2Then the flask was placed at 37 ℃ and 5% CO2Culturing at 98% humidity in an incubator for 24h, collecting cells, centrifuging at 19 deg.C under 250Xg for 17min, and removing supernatant to obtain cultured mononuclear cells;
step 2, culturing the CIK cells: step 1 is to obtainThe cultured mononuclear cells of (4) are suspended in an activation medium B and adjusted to a cell density of 1.8X 106L, and inoculating the activated medium B to 180cm2The CD28 antibody-coated culture flask was placed at 37 ℃ and 5% CO2Culturing at 98% humidity in incubator, sequentially adding IL-15 and IL-21 after 24 hr, culturing, replacing half of the culture medium C every 2 days, and maintaining cell density at 1.8 × 106The culture flask coated with the CD28 antibody is formed by coating the bottom of the culture flask with a coating solution and incubating for 70min at 14 ℃.
The raw materials used in the above steps are shown in table 2.
TABLE 2
Figure BDA0003158573940000061
Example 3:
a culture method of CIK cells contained in peripheral blood comprises the following steps:
step 1, peripheral blood extraction and mononuclear cell culture: collecting peripheral blood of a patient or a volunteer in a sterile environment, adding heparin sodium, and centrifuging with a lymph separation solution at 18 deg.C under a centrifugal force of 260Xg for 18min to separate mononuclear cells; sucking the mononuclear cell layer by capillary, washing with normal saline, centrifuging at 23 deg.C and 300Xg for 23min, and repeating for 2 times; then suspending the cells in a medium A and adjusting the cell density to 4X 106L, and inoculating the culture medium A to 30cm2Then the flask was placed at 37 ℃ and 5% CO2Culturing for 24h at 100% humidity in an incubator, collecting cells, centrifuging for 17min at 22 ℃ and 250Xg centrifugal force, and removing supernatant to obtain cultured mononuclear cells;
step 2, culturing the CIK cells: suspending the cultured mononuclear cells obtained in step 1 with an activation medium B and adjusting the cell density to 1.8X 106L, and inoculating the activated medium B to 180cm2The CD28 antibody-coated culture flask was placed at 37 ℃ and 5%CO2Culturing at 100% humidity in incubator for 24 hr, sequentially adding IL-15 and IL-21, culturing, replacing half of the culture medium C every 2 days, and maintaining cell density at 1.8 × 106The culture flask coated with the CD28 antibody is formed by coating the bottom of the culture flask with a coating solution and incubating for 70min at 14 ℃.
The raw materials used in the above steps are shown in table 3.
TABLE 3
Figure BDA0003158573940000071
Comparative example 1:
the comparative example was cultured by the conventional culture method, comprising the following steps:
centrifuging peripheral blood 70mL at 2000rpm/min for 20min, separating and collecting plasma, inactivating at 56 deg.C for 30min, centrifuging at 1500rpm/min for 10min, and collecting plasma from body for preparing activation culture medium;
diluting blood with sterile PBS (1.5 times of plasma volume) to obtain mononuclear cells, and separating Peripheral Blood Mononuclear Cells (PBMCs) with Ficoll-Paque;
cell density was adjusted to 1X 10 using RPMI1640 medium containing 10% volume fetal bovine serum6/mL, adding IFN-gamma, and the final concentration is 2000U/mL; after 24 hours, the CD3 monoclonal antibody was added to a final concentration of CD3 of 50. mu.g/mL and human interleukin-2 (IL-2) at a final concentration of 500U/mL IL-2 and 50000U/mL IL-1 α, and the samples were counted at intervals of 48 hours at 4X 105Density passage at/mL supplemented with IL-2, IL-1 α and IFN- γ.
Example 4:
first, CIK cell proliferation fold
Cell counting is carried out on a full-automatic five-classification blood analyzer, CIK cells obtained by the culture methods of the above examples 1-3 and the comparative example 1 are counted respectively on the 3 rd day, the 5 th day, the 10 th day and the 15 th day of culture, and the amplification factor is calculated, and the calculation formula is as follows:
cell growth factor is the number of cells after culture/the number of cells seeded before culture.
Cell proliferation multiple comparison statistical table
Figure BDA0003158573940000081
As shown in table 4:
TABLE 4
3d 5d 10d 15d
Example 1 4.3±0.3 22.7±3.2 231.7±17.2 1317.1±71.1
Example 2 5.1±0.2 23.1±3.1 232.8±16.9 1321.3±72.3
Example 3 4.7±0.3 22.5±3.3 234.5±17.4 1319.1±71.9
Comparative example 1 2.7±0.4 11.5±2.4 132.4±13.9 986.7±43.2
It can be seen from table 4 that the fold increase during the culture was much higher in examples 1 to 3 than in comparative example 1, with significant difference.
II, determination of cell killing activity of CIK
The cells cultured in examples 1-3 and comparative example 1 for 9 days were collected, washed 3 times with PBS and prepared to 2X 106The cell suspension of/L is effector cells. A549, MFC-7 and HME1 cells in logarithmic growth phase are prepared into 2x 105Taking the/L cell suspension as a target cell, equivalently mixing 0.5mL of cell suspension for effective target cells respectively, incubating the mixture in an incubator with 37 ℃, 5% CO2 and saturated humidity for 6h, gently mixing the mixture, suspending the cells, centrifuging the suspended cells for 10min at 252Xg, collecting supernatant, using a lactate dehydrogenase kit to operate according to the requirements of a reagent specification, measuring the absorbance value (A) under the wavelength of 340nm of a full-automatic biochemical analyzer, setting 5 multitubes for each detection sample, and taking an average value. The killing activity was calculated according to the following formula:
killing activity (%) - (A)Experimental group-AEffector cell natural release group)/(AMaximum release group of target cells-ATarget cell natural release group)]×100％。
The killing activity was measured and the data are shown in Table 5.
TABLE 5
A549 MFC-7 HME1
Example 1 killing activity (%) 80.91 57.77 72.31
Example 2 killing activity (%) 79.37 58.34 71.79
Example 3 killing activity (%) 80.63 57.89 71.81
Comparative example 1 killing activity (%) 64.37 48.91 48.79
As can be seen from Table 5, the killing activity of the CIK cells obtained in examples 1-3 on A549, MFC-7 and HME1 cells is significantly higher than that of the CIK cells obtained in comparative example 1 on A549, MFC-7 and HME1 cells.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is merely exemplary and illustrative of the principles of the present invention and various modifications, additions and substitutions of the specific embodiments described herein may be made by those skilled in the art without departing from the principles of the present invention or exceeding the scope of the claims set forth herein.

Claims (7)

1. A method for culturing CIK-containing cells in peripheral blood, which comprises the following steps:
step 1, peripheral blood extraction and mononuclear cell culture: collecting peripheral blood in a sterile environment, adding heparin sodium, and performing primary centrifugal separation by using a lymph separation liquid to obtain mononuclear cells; sucking the mononuclear cell layer, washing with physiological saline, centrifuging for the second time, and purifying; then suspending the cells in a medium A and adjusting the cell density to 4X 106L, inoculating the culture medium A into a culture bottle, culturing for 24h, collecting cells, centrifuging for three times, and removing supernatant to obtain cultured mononuclear cells;
step 2, culturing the CIK cells: suspending the cultured mononuclear cells obtained in step 1 with an activation medium B and adjusting the cell density to 1.8X 106L, inoculating the activated culture medium B into a culture flask coated with CD28 antibody, culturing for 24 hr, sequentially adding IL-15 and IL-21, culturing, replacing half of the culture medium C every 1-2 days, and maintaining cell density at 1.8 × 106mL, cultured to the required amount.
2. The method for culturing CIK cells in peripheral blood according to claim 1, wherein the centrifugation conditions of the first centrifugation in step 1 are 260Xg and 18min at a temperature of 13-18 ℃, the centrifugation conditions of the second centrifugation are 300Xg and 23min at a temperature of 20-23 ℃, and the centrifugation conditions of the third centrifugation are 250Xg and 17min at a temperature of 17-22 ℃.
3. The method for culturing CIK-containing cells in peripheral blood according to claim 1, wherein the culture medium A in step 1 is formed by mixing culture medium dry powder, L-glutamine, basic fibroblast growth factor, vitamin C, ALK inhibitor, GSK-3 inhibitor, insulin, human plasma albumin and ammonium bicarbonate.
4. The method according to claim 1, wherein the activated medium B in step 2 is formed by adding IL-2, IL-1 α and IFN- γ to the medium A, and the concentration of IL-2 is 5X 104-1×105U/L, IL-1. alpha. concentration 5X 104-1×105U/L, IFN-gamma concentration of 2X 105-2×106U/L。
5. The method of claim 1, wherein the culture flask coated with the CD28 antibody in step 2 is prepared by coating the bottom of the culture flask with a coating solution prepared by dissolving the CD28 monoclonal antibody and IFN-gamma in PBS buffer solution, and incubating at 14 ℃ for 70min, wherein the concentration of CD28 is 1X 105-1×106U/L, IFN-gamma concentration of 2X 105-2×106U/L。
6. The method according to claim 1, wherein the concentrations of IL-15 and IL-21 added in step 2 in the culture medium are 16-19 μ g/L and 7-9 μ g/L, respectively.
7. The method of claim 1, wherein the activated medium C in step 2 is formed by adding IL-2 to the medium A, and the concentration of IL-2 is 2X 105-1×106U/L。
CN202110784404.4A 2021-07-12 2021-07-12 Culture method for CIK-containing cells in peripheral blood Pending CN113502266A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110784404.4A CN113502266A (en) 2021-07-12 2021-07-12 Culture method for CIK-containing cells in peripheral blood

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110784404.4A CN113502266A (en) 2021-07-12 2021-07-12 Culture method for CIK-containing cells in peripheral blood

Publications (1)

Publication Number Publication Date
CN113502266A true CN113502266A (en) 2021-10-15

Family

ID=78012422

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110784404.4A Pending CN113502266A (en) 2021-07-12 2021-07-12 Culture method for CIK-containing cells in peripheral blood

Country Status (1)

Country Link
CN (1) CN113502266A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115651905A (en) * 2022-11-16 2023-01-31 南京鼓楼医院 Staged culture method for in-vitro amplification of human CIK cells and application thereof
CN118147067A (en) * 2024-05-11 2024-06-07 北京岷德生物科技有限公司 Culture solution for improving capability of CIK cells to kill tumor cells, method and application

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102925410A (en) * 2012-11-19 2013-02-13 昆明理工大学附属医院 Method for preparing CIK cell by using heparin anticoagulant plasma
WO2015081741A1 (en) * 2013-12-04 2015-06-11 深圳市合一康生物科技有限公司 Method and kit for preparing immunological cells
CN105018423A (en) * 2015-05-27 2015-11-04 贵州北科泛特尔生物科技有限公司 CIK cell culturing method
CN105087487A (en) * 2015-09-23 2015-11-25 协和干细胞基因工程有限公司 Efficient CIK amplifying method
CN111849890A (en) * 2020-07-30 2020-10-30 昆明医科大学 Separation and culture method of peripheral blood mononuclear cells

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102925410A (en) * 2012-11-19 2013-02-13 昆明理工大学附属医院 Method for preparing CIK cell by using heparin anticoagulant plasma
WO2015081741A1 (en) * 2013-12-04 2015-06-11 深圳市合一康生物科技有限公司 Method and kit for preparing immunological cells
CN105018423A (en) * 2015-05-27 2015-11-04 贵州北科泛特尔生物科技有限公司 CIK cell culturing method
CN105087487A (en) * 2015-09-23 2015-11-25 协和干细胞基因工程有限公司 Efficient CIK amplifying method
CN111849890A (en) * 2020-07-30 2020-10-30 昆明医科大学 Separation and culture method of peripheral blood mononuclear cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
刘军权等: "不同刺激因子对人CIK细胞功能影响", 《中国实验血液学杂志J》 *
刘军权等: "多种活化因子共刺激对人外周血T淋巴细胞体外增殖和功能的影响", 《第十届徐州科技论坛论文集》 *
孙志亚等: "不同组合CD3、CD28 抗体对CIK 细胞诱导培养的探索研究", 《重庆医学》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115651905A (en) * 2022-11-16 2023-01-31 南京鼓楼医院 Staged culture method for in-vitro amplification of human CIK cells and application thereof
CN118147067A (en) * 2024-05-11 2024-06-07 北京岷德生物科技有限公司 Culture solution for improving capability of CIK cells to kill tumor cells, method and application

Similar Documents

Publication Publication Date Title
CN1878860B (en) Method of isolating and culturing mesenchymal stem cell derived from umbilical cord blood
CN113502266A (en) Culture method for CIK-containing cells in peripheral blood
CN103167876A (en) Improved hematopoietic stem and progenitor cell therapy
CN107475192B (en) Lymphocyte population using memory stem T cell as main component and in-vitro efficient amplification method thereof
CN110643574A (en) Preparation method of feeder cells for rapid culture of tumor infiltrating lymphocytes
CN111944754A (en) Natural killer cell culture method
EP1687415B1 (en) Method of isolating and culturing mesenchymal stem cell derived from cryopreserved umbilical cord blood
CN115044555B (en) Culture medium for in-vitro amplification and dryness maintenance of human umbilical blood hematopoietic stem cells
CN116333986A (en) Culture method for exosome activated NK cells
CN112608896A (en) NK cell culture method and application thereof
CN111548994B (en) Cell culture medium and method for culturing NK cells by using same
CN111172110B (en) Culture method of umbilical cord blood CIK cells
CN115247148B (en) Culture medium for culturing immune cells and cell culture method
CN104109653A (en) Method of large-scale amplification of human peripheral blood DNT cell by utilization of animal-serum-free culture system
CN110862962A (en) Method for culturing and amplifying NK cells in vitro by using gallic acid
CN112300992B (en) NK cell culture solution and multistage activated NK cell culture method
CN107090434B (en) Blood anticoagulation and preservation method for improving CIK cell culture effect
CN108004213B (en) Method and kit for rapid amplification of CIK cells
CN113564118A (en) CIK cell culture solution and culture method for enhancing CD3+ CD56+ of CIK cells
CN115404215A (en) Method for efficiently activating and amplifying NK immune cells in vitro
CN110872575A (en) In-vitro amplification method of iNKT cells
CN113293130A (en) Culture method of tumor specific T cells
CN110564684A (en) Method for in vitro stabilizing, large-amount amplification and high-cytotoxic-activity NK cells and application
CN1554748A (en) Method for preparing megakaryocytic preparation by amplifying macronucleus ancestral cell and mature megacaryocyte and use
CN111004780B (en) Purification and separation culture method for improving killing activity of immune cells

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
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20211015