WO2017173696A1 - 脐血nk细胞的体外扩增方法及其试剂盒与应用 - Google Patents

脐血nk细胞的体外扩增方法及其试剂盒与应用 Download PDF

Info

Publication number
WO2017173696A1
WO2017173696A1 PCT/CN2016/081198 CN2016081198W WO2017173696A1 WO 2017173696 A1 WO2017173696 A1 WO 2017173696A1 CN 2016081198 W CN2016081198 W CN 2016081198W WO 2017173696 A1 WO2017173696 A1 WO 2017173696A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cord blood
medium
recombinant human
human interleukin
Prior art date
Application number
PCT/CN2016/081198
Other languages
English (en)
French (fr)
Inventor
武晓云
刘学敏
康会彦
王云虹
樊晓翔
李志刚
丁炜
武立华
李金艳
沈殿富
张慧珍
Original Assignee
北京京蒙高科干细胞技术有限公司
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 北京京蒙高科干细胞技术有限公司 filed Critical 北京京蒙高科干细胞技术有限公司
Priority to US16/090,258 priority Critical patent/US20190112577A1/en
Priority to EP16897630.6A priority patent/EP3441460B1/en
Publication of WO2017173696A1 publication Critical patent/WO2017173696A1/zh

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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12N5/0635—B lymphocytes
    • 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
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C12N2500/42—Organic phosphate, e.g. beta glycerophosphate
    • 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]
    • C12N2501/2318—Interleukin-18 (IL-18)
    • 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
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the invention belongs to the field of cell engineering technology, and particularly relates to an in vitro expansion method of umbilical cord blood NK cells and a kit for in vitro expansion of umbilical cord blood NK cells, and relates to the umbilical cord blood NK cells in preparing tumor immunotherapy drugs and tumor immunotherapy. Applications.
  • Natural killer cells are the third type of lymphocytes other than T and B cells. Their cell morphology is also different from that of T and B cells. NK cells are large particles containing ozyllium particles in the cytoplasm. Lymphocytes, which are widely present in lymphoid organs and peripheral tissues, account for about 5% to 10% of the total number of lymphocytes in normal peripheral blood, 1% to 2% in the spleen, and NK cells in lymph nodes and bone marrow. As an important part of the body's natural immunity, NK cells are important immune regulatory cells that resist infection and prevent malignant transformation of cells, and play an important role in immune surveillance and immune defense.
  • NK cells are reduced.
  • diseases such as tumors, autoimmune diseases, aging-related diseases and HIV infection
  • the proportion, number and function of NK cells are reduced.
  • the US Centers for Disease Control reported that the vitality and quantity of NK cells is an important guarantee for human health. The incidence of almost all diseases is related to the apparent lack of activity of NK cells. When people have a disease, whether chronic, sporadic or acute, the activity of NK cells is below average. Reducing NK cells to a high level of activity is a better way to treat diseases.
  • NK cells are characterized by: NK cells can directly dissolve and destroy tumor cells and virus-infected cells without pre-stimulation, by secreting perforin, serine proteases such as granzymes A and B, and chondroitin sulfate proteoglycans. Isogenic molecules degrade cell membranes, destroy target cell integrity and exert cytosolic effect; NK cell surface expression FasL (Fas ligand) and TRAIL (TNF-related opoptosis-inducing ligand) can cause target cells by binding to receptors on target cells.
  • FasL Fas ligand
  • TRAIL TNF-related opoptosis-inducing ligand
  • NK cells release a large number of cytokines such as interferon- ⁇ and tumor necrosis factor- ⁇ after activation; NK cells mediate antibody-dependent cytotoxicity by binding cell membrane Fc ⁇ RIII (CD16) to the Fc segment of the antibody.
  • cytokines such as interferon- ⁇ and tumor necrosis factor- ⁇ after activation
  • NK cells mediate antibody-dependent cytotoxicity by binding cell membrane Fc ⁇ RIII (CD16) to the Fc segment of the antibody.
  • NK cells kill tumor cells by NK cells in vivo was observed, but the survival rate and recurrence rate of patients did not improve compared with the control group, which may facilitate immune evasion of autologous NK cells during tumor development.
  • Related, and clinical trials of adoptive immunotherapy of NK cells are often extremely advanced patients who have undergone multiple chemotherapy failures. The tumor burden is heavy, the body immunity is poor, and the function of NK cells is seriously deficient.
  • NK cells of allogeneic origin are a better choice.
  • Some researchers have studied the treatment of leukemia patients by transplanting allogeneic NK cells, and found that it can delay recurrence without causing graft-versus-host disease, which has caused people's attention to allogeneic reactivity of NK cells. From the results of current clinical studies, heterologous NK cells are significantly superior to autologous NK cells in terms of complete remission rate, event-free survival, recurrence, and mortality. Derived NK cells are not associated with graft versus host response.
  • the killing ability of NK cells to tumor cells depends on the regulation of signal balance between NK cell surface activating receptors and inhibitory receptors.
  • the killer cell immunoglobulin-like receptor (KIR) composition and major histocompatibility class I (MHC) -I) The family of receptors that bind, plays an important role in regulating the activation threshold of human NK cells.
  • KIR killer cell immunoglobulin-like receptor
  • MHC major histocompatibility class I
  • screening optimal donor can greatly enhance the killing ability of NK cells to recipient tumor cells and improve the effect of adoptive immunotherapy of NK cells.
  • Current KIR-based donor selection has received much attention in improving hematopoietic stem cell/bone marrow transplantation and adoptive immunotherapy of tumors.
  • Heterologous NK infusion has become a new strategy for clinical NK cell adoptive immunotherapy.
  • Cord blood as a ready-made heterogeneous resource no ethical problems, rich sources, easier to find the best donor, and the development of T lymphocytes in cord blood is immature, mostly naive cells, acute and chronic grafts after transplantation
  • the incidence of anti-host disease is low and mild, so cord blood has become a promising source of heterologous NK cells.
  • Clinical infusion of NK cells requires sufficient cell purity and cell number, but NK cells account for a small proportion of cord blood cells, and the number is small. It needs to be isolated and cultured in vitro to obtain a large number of high-purity NK cells. Meet clinical applications. Therefore, how to obtain high-purity, high-quality NK cells becomes the key to adoptive immunotherapy of NK cells (Document 1: Klingemann H: Challenges of cancer therapy with natural killer cells. Cytotherapy 2015, 17(3): 245-249.).
  • NK cells account for about 10% to 20% of lymphocytes in cord blood, and their functions are immature. The ability to kill is low.
  • the NK cells can be sorted by flow cytometry or magnetic bead sorter, and then the number of NK cells and the ability to kill tumors can be increased by in vitro expansion.
  • Celgene Cellular Therapeutics uses cryopreserved cord blood to separate mononuclear cells from lymphocyte fractions
  • the enrichment kit (StemCell Technologies, which contains CD3, CD4, CD14, CD19, CD20, CD36, CD66b, CD123, HLA-DR, and glycophorin A antibodies) sorts CD56 + CD3 - NK cells to a purity of 71%.
  • the number of cells was 1.5 ⁇ 10 7 / part cord blood, and then passed through the starting medium (including: IMDM, 10% FBS, 35 mg / mL transferrin, 5 ⁇ g / mL insulin, 20 ⁇ M ethanolamine, 1 ⁇ g / mL unsaturated fatty acid, 1 ⁇ g /mL linoleic acid, 0.2 ⁇ g/mL palmitic acid, 2.5 ⁇ g/mL bovine serum albumin, 0.1 ⁇ g/mL phytohemagglutinin, 1% cyan-streptomycin, 200 IU/mL interleukin-2) added trophoblast cells ( Mitomycin C-treated peripheral blood mononuclear cells and K562 cells were cultured for 5-7 days at 37 ° C under 5% CO 2 and transferred to maintenance medium (IMDM, 10% FBS, 2% human AB serum).
  • NK cells start from hematopoietic stem cells, and the cord blood is rich in hematopoietic stem cells.
  • the hematopoietic stem cells are enriched by flow cytometry or magnetic bead sorter, and a certain number of mature NK cells are obtained by in vitro differentiation.
  • Glycostem Therapeutics will cryopreserved cord blood using the CliniMACS CD34 magnetic bead sorting kit to sort and enrich CD34 + hematopoietic stem cells with a purity of 67%, and the number of cells is 3.8 ⁇ 10 6 /ml cord blood in cell expansion medium I (include: Medium, 10% human serum, high-dose cytokine combination (SCF, Flt3L, TPO, IL-7, low molecular weight heparin) and low-dose factor combination (GM-CSF, G-CSF, IL-6) were cultured for 9 days.
  • cell expansion medium I include: Medium, 10% human serum, high-dose cytokine combination (SCF, Flt3L, TPO, IL-7, low molecular weight heparin) and low-dose factor combination (GM-CSF, G-CSF, IL-6) were cultured for 9 days.
  • NK cell differentiation medium low dose combination is replaced with (IL-7, SCF, IL-15 and IL-2), other than medium I
  • NK cells with a purity of >90% and an average of 2 ⁇ 10 9 /part cord blood
  • sorting technology affects the activity of NK cells, and also needs sorting instruments;
  • a first object of the present invention is to provide a simple and safe method for in vitro expansion of cord blood NK cells.
  • the method for in vitro expansion of cord blood NK cells comprises the following steps:
  • cord blood NK cells adjust the density of cord blood mononuclear cells to 0.5 to 5 ⁇ 10 6 /mL with lymphocyte culture medium, and add at least 1 to 10 ⁇ g/mL zoledronic acid and 200 to 2000 IU/mL.
  • Recombinant human interleukin-2 cultured in a 36 ° C ⁇ 40 ° C, 5% CO 2 saturated humidity environment for 1 to 5 days, collecting cell fluid;
  • Proliferation and culture of cord blood NK cells cells are obtained from the cell liquid of step 1), and the cell density is adjusted to 0.5 to 5 ⁇ 10 6 /mL with lymphocyte medium, and then 200 to 2000 IU / mL of recombinant human interleukin - 2, culture in 36 ° C ⁇ 38 ° C, 5% CO 2 saturated humidity environment, add fresh medium every 2 to 3 days and adjust the cell density to 0.5 ⁇ 5 ⁇ 10 6 / mL, culture 14 ⁇ 35 days, Cord blood NK cells were harvested.
  • the cord blood mononuclear cells used in the step 1) are isolated from fresh anticoagulated umbilical cord blood or cryopreserved to recover cord blood, and the separation process is as follows:
  • the white membrane-like mononuclear cell layer of the second layer above was aspirated and washed with PBS to obtain cord blood mononuclear cells.
  • the lymphocyte medium in the step 1) may be AIM Medium CTS TM (available from Life Technology Corporation USA) or GMP S & XFM TM -CD lymphocyte medium (Beijing Beijing Meng Transtech Stem cell Technologies Co., Ltd. product of the same name), preferably GMP S & XFM TM -CD lymphocyte culture.
  • the lymphocyte medium in the step 1) is AIM Medium CTS when TM, preferably activated culture: with AIM Medium CTS TM lymphocyte medium is adjusted to a cell density of 1 to 3 ⁇ 10 6 /mL, and then added 1 ⁇ 5 ⁇ g / mL zoledronic acid and 500 ⁇ 1500 IU / mL recombinant human interleukin-2, at 36 ° C ⁇ 38 ° C Incubated in 5% CO 2 saturated humidity for 2 to 4 days; activated culture is better: using AIM Medium CTS TM lymphocyte medium was adjusted to a cell density of 2 ⁇ 10 6 /mL, and 2 ⁇ g/mL zoledronic acid and 1000 IU/mL recombinant human interleukin-2 were added at 37 ° C in a 5% CO 2 saturated humidity environment. Cultivate for 3 days.
  • lymphocytes medium for GMP S & XFM TM -CD following activation of the culture is preferably one of:
  • A Adjust the cell density to 1-3 ⁇ 10 6 /mL with GMP S&XFM TM -CD lymphocyte medium, and add 1-5 ⁇ g/mL zoledronic acid and 500-1500 IU/mL recombinant human interleukin-2.
  • the activation culture is better: adjust the cell density to 2 ⁇ 10 6 /mL with GMP S & XFM TM -CD lymphocyte medium, and then add 2 ⁇ g / mL zoledronic acid and 1000 IU / mL recombinant human interleukin-2, cultured in 37 ° C, 5% CO 2 saturated humidity environment for 3 days;
  • NK cells preferably cultured using GMP S & XFM TM -CD lymphocyte separation medium (Beijing Ltd. Beijing Meng Transtech dry cell product of the same name, the same record number of medical devices: Jinghai mechanical apparatus sample No.
  • GMP S & XFM TM -CD lymphocyte separation medium Beijing Ltd. Beijing Meng Transtech dry cell product of the same name, the same record number of medical devices: Jinghai mechanical apparatus sample No.
  • the operation is preferably as follows: the cell with the pipette solution was transferred to a centrifuge tube, 150g, centrifuged 10min, the supernatant was discarded by GMP S & XFM TM -CD adjustment lymphocyte culture, a cell density of 0.5 ⁇ 2 ⁇ 10 6 / mL (more preferably 1 ⁇ 10 6 / mL), and then add 500 ⁇ 1500 IU / mL (more preferably 1000 IU / mL) recombinant human interleukin-2, cultured in 37 ° C, 5% CO 2 saturated humidity environment Adding fresh medium every 2 to 3 days to adjust the cell density to 0.5 to 2 ⁇ 10 6 /mL (more preferably 1 ⁇ 10 6 /mL), and adding 500 to 1500 IU / mL (more preferably 1000 IU / mL) Recombinant human interleukin-2 is cultured for 18 to 24 days (more preferably 21 days) to obtain cord blood
  • the specific activation medium used in the step 1) of the in vitro expansion method of the cord blood NK cells described above is also the subject of the invention.
  • the dedicated activation medium is one of the following combinations:
  • an AIM of 1 to 5 ⁇ g/mL zoledronic acid and 500 to 1500 IU/mL recombinant human interleukin-2 is added.
  • GMP S&XFM TM -CD Lymph with 1–10 ⁇ g/mL zoledronic acid, 200-2000 IU/mL recombinant human interleukin-2, 1-100 ng/mL recombinant human interleukin-15 and 1-100 ng/mL recombinant human interleukin-18 Cell culture medium.
  • the specific proliferation medium used in the step 2) of the in vitro expansion method of the cord blood NK cells described above is also the subject of the present invention.
  • the specific proliferation medium was added 200 ⁇ 2000IU / mL lymphocyte medium (preferably 1000IU / mL) of recombinant human interleukin-2 GMP S & XFM TM -CD.
  • cord blood NK cells harvested by the above in vitro amplification method are also within the scope of the present invention.
  • the cell purity is over 90%.
  • the use of the umbilical cord blood NK cells obtained by the method of the present invention in the preparation of tumor immunotherapy drugs or the application in tumor immunotherapy also belong to the present invention.
  • Another object of the present invention is to provide an in vitro expansion kit for cord blood NK cells.
  • the in vitro expansion kit for cord blood NK cells provided by the invention mainly comprises the following reagents:
  • Lymphocyte separation solution of umbilical cord blood NK cells Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd., the same name), or other commercially available lymphocyte separation solution;
  • Cord blood NK cell activation medium AIM Medium CTS TM (Life Technology, USA) medium or GMP S&XFM TM -CD lymphocyte medium (commercially available from Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd.), and zoledronic acid, recombinant human interleukin-2, recombinant human interleukin-15 And recombinant human interleukin-18;
  • NK cells in growth medium GMP S & XFM TM -CD lymphocyte medium (Stem Cell Tech Beijing Beijing Meng goods Ltd.) and recombinant human interleukin-2.
  • the reagent in the in vitro expansion kit of umbilical cord blood NK cells is used in the above-described method for in vitro expansion of umbilical cord blood NK cells.
  • the present invention provides an in vitro expansion method and related kit for umbilical cord blood NK cells which are simple and safe to operate. Compared with the existing methods for isolating and expanding cord blood NK cells, the present invention has the following beneficial effects:
  • cord blood mononuclear cells does not require a cell sorting step, that is, there is no need to sort NK cells or hematopoietic stem cells, which simplifies the cell preparation process and greatly reduces the preparation cost of cord blood NK cells;
  • the cell culture medium used for the preparation of NK cells is completely free of animal-derived components, has good safety, and has clear chemical composition, which increases the stability between batches;
  • the in vitro expansion method and kit for cord blood NK cells of the invention will play an important role in the preparation of tumor immunotherapy drugs and tumor immunotherapy, and has broad application prospects.
  • 1 is a conventional method for isolating and expanding cord blood NK cells
  • FIG. 2 is a flow chart of a method for in vitro expansion of cord blood NK cells according to the present invention
  • Figure 3 is a diagram showing the cell morphology of fresh umbilical cord blood and cryopreserved umbilical cord blood NK cells before and after expansion in vitro;
  • Figure 4 is a growth curve of in vitro expansion of fresh cord blood and cryopreserved cord blood NK cells
  • Figure 5 shows the results of flow cytometry detection of purity of fresh umbilical cord blood and cryopreserved cord blood NK cells before and after expansion in vitro;
  • Figure 6 is a graph showing the killing effect of tumor cells in vitro on freshly harvested cord blood and cryopreserved cord blood NK cells.
  • the present invention aims to provide a method for preparing umbilical cord blood NK cells in vitro, and to provide a dedicated activation medium and a dedicated proliferation medium used in the method.
  • the method for in vitro expansion of cord blood NK cells of the present invention may comprise the following steps:
  • Activated culture of cord blood NK cells adjust the density of cord blood mononuclear cells to 0.5 to 5 ⁇ 10 6 /mL (preferably 1 to 3 ⁇ 10 6 /mL, most preferably, using lymphocyte medium (described in detail below) 2 ⁇ 10 6 / mL), inoculate the cell suspension into the cell culture flask; add 1 ⁇ 10 ⁇ g / mL (preferably 1-5 ⁇ g / mL, most preferably 2 ⁇ g / mL) zoledronic acid and 200 ⁇ 2000 IU / mL (preferably 500-1500 IU/mL, most preferably 1000 IU/mL) recombinant human interleukin-2 (formation-specific activation medium), cultured in an environment of 36 ° C to 40 ° C, 5% CO 2 saturated humidity for 1 to 5 days;
  • the lymphocyte medium in step 1) can be AIM Medium CTS TM (purchased from Life Technology, USA) or GMP S&XFM TM -CD Lymphocyte Medium (Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd., the same name, the same medical device record number: Jinghai Machinery and Equipment 20150008) ), preferably GMP S & XFM TM -CD lymphocyte culture.
  • a detailed description of the GMP S&XFM (TM) -CD lymphocyte medium can also be found in the literature 4: Patent Application No.: 201310082166.8, CN103146648A.
  • the activation culture method in step 1) is preferably: using AIM Medium CTS TM lymphocyte medium adjusts cord blood mononuclear cell density to 0.5 to 5 ⁇ 10 6 /mL (preferably 1 to 3 ⁇ 10 6 /mL, most preferably 2 ⁇ 10 6 / mL), cell suspension Inoculate the cell culture flask and then to AIM 1 to 10 ⁇ g/mL (preferably 1 to 5 ⁇ g/mL, most preferably 2 ⁇ g/mL) of zoledronic acid and 200 to 2000 IU/mL (preferably 500 to 1500 IU/mL, most preferably 1000 IU/) is added to the Medium CTS TM lymphocyte medium.
  • Recombinant human interleukin-2 is cultured for 1 to 5 days (preferably 2 to 4 days, most preferably 3 days) in an environment of 36 ° C to 38 ° C (preferably 37 ° C) and 5% CO 2 saturated humidity.
  • GMP S&XFM TM -CD lymphocyte medium is selected, and the specific activation medium is composed of: 1 to 10 ⁇ g/mL (preferably 1 to 5 ⁇ g/mL, most preferably 2 ⁇ g/mL) zoledronic acid and 200 to 2000 IU/mL. (preferably 500 ⁇ 1500IU / mL, most preferably 1000IU / mL) of recombinant human interleukin-2 GMP S & XFM TM -CD lymphocyte culture.
  • the activation step culture method 1) is more preferably: with GMP S & XFM TM -CD lymphocyte culture density adjusted cell individual cord blood is 0.5 ⁇ 5 ⁇ 10 6 / mL ( preferably 1 ⁇ 3 ⁇ 10 6 / mL, most preferably 2 ⁇ 10 6 / mL), inoculating the cell suspension into a cell culture flask, and adding 1 to 10 ⁇ g / mL (preferably 1 to 5 ⁇ g ) to the GMP S&XFM TM -CD lymphocyte medium.
  • the culture is carried out for 1 to 5 days (preferably 2 to 4 days, most preferably 3 days) in a 5% CO 2 saturated humidity environment.
  • GMP S&XFM TM -CD lymphocyte medium is selected, and the specific activation medium is composed of: 1 to 10 ⁇ g/mL (preferably 1 to 5 ⁇ g/mL, most preferably 2 ⁇ g/mL) zoledronic acid and 200 to 2000 IU/mL.
  • recombinant human interleukin-2 (preferably 500 to 1500 IU/mL, most preferably 1000 IU/mL) recombinant human interleukin-2, 1 to 100 ng/mL (preferably 1 to 20 ng/mL, most preferably 10 ng/mL) recombinant human interleukin-15 and 1 to 100 ng/mL (preferably 1 ⁇ 20ng / mL, and most preferably 10ng / mL) of recombinant human interleukin-GMP S & XFM TM -CD -18 lymphocyte culture.
  • the activation step culture method 1) is more preferably: with GMP S & XFM TM -CD lymphocyte culture medium adjustment cord blood mononuclear cell density of 0.5 ⁇ 5 ⁇ 10 6 / mL ( preferably 1 to 3, ⁇ 10 6 /mL, most preferably 2 ⁇ 10 6 /mL), inoculate the cell suspension into the cell culture flask, and add 1 to 10 ⁇ g / mL (preferably 1 ⁇ ) to the GMP S&XFM TM -CD lymphocyte medium.
  • the culture medium is changed by using the special activation medium, for example, at 38 ° C to 40 ° C (preferably 38.5 ° C to 39.5 ° C, most preferably 39 ° C), and 5% CO 2 saturated humidity for 0.5 to 3 days (preferably 0.5 to 1). Days, most preferably 1 day), will give the optimal conditions for step 1) activation of cultured cord blood NK cells.
  • the special activation medium for example, at 38 ° C to 40 ° C (preferably 38.5 ° C to 39.5 ° C, most preferably 39 ° C), and 5% CO 2 saturated humidity for 0.5 to 3 days (preferably 0.5 to 1). Days, most preferably 1 day), will give the optimal conditions for step 1) activation of cultured cord blood NK cells.
  • NK cells in culture 2) the proliferation of NK cells in culture: a pipette Step 1) to give the cell liquid was transferred to a centrifuge tube, 150g, centrifuged 10min, the supernatant was discarded, lymphocyte culture cell density was adjusted by GMP S & XFM TM -CD is 0.5 to 5 ⁇ 10 6 /mL (preferably 0.5 to 2 ⁇ 10 6 / mL, preferably 1 ⁇ 10 6 / mL), the cell suspension is inoculated into a cell culture flask, and then added 200 to 2000 IU / mL ( Preferably, 500-1500 IU/mL, preferably 1000 IU/mL) recombinant human interleukin-2 (formation of a dedicated proliferation medium), cultured at 36 ° C to 38 ° C (preferably 37 ° C), 5% CO 2 saturated humidity, every 2 to 3 days supplemented with fresh GMP S&XFM TM -CD lymphocyte medium to adjust the cell density to 0.5 ⁇ 5
  • Step 2) proliferation of NK cell culture process is preferably as follows: the tube with the pipette cell suspension transferred into a centrifuge tube, 150g (centrifugal force unit) centrifugal 10min, supernatant was discarded, lymphocyte medium adjusting GMP S & XFM TM -CD The cell density was 1 ⁇ 10 6 /mL, the cell suspension was inoculated into the cell culture flask, and 1000 IU/mL recombinant human interleukin-2 was added to the cell culture flask, and cultured at 37 ° C, 5% CO 2 saturated humidity environment. Fresh medium was added every 2 to 3 days, and the cell density was adjusted to 1 ⁇ 10 6 /mL, and cultured for 21 days, and umbilical cord blood NK cells were harvested.
  • the cord blood mononuclear cells used in the step 1) are separated from the cord blood, and the separation method is as follows:
  • step 1.2 using commercially available other lymphocyte separation liquid according to its instructions) centrifuge for 20 ⁇ 40min (preferably 30min);
  • the centrifuge tube is divided into four layers with clear interface from top to bottom. From top to bottom, it is: pale yellow plasma layer, white membrane-like mononuclear cell layer, transparent separation layer and red In the red blood cell layer, the white membrane-like mononuclear cell layer was carefully aspirated into another centrifuge tube and washed with PBS to obtain cord blood mononuclear cells.
  • the present invention also provides an in vitro amplification kit for umbilical cord blood NK cells, which mainly comprises the following reagents:
  • Lymphocyte separation solution of cord blood mononuclear cells Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd., the same name, the same medical device record number: Jinghai Machinery, 20150002, sample density separation solution), or other commercially available lymphocytes Separation solution
  • Cord blood NK cell activation medium AIM Medium CTS TM (purchased from Life Technology, USA) medium or GMP S&XFM TM -CD lymphocyte medium (Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd., the same name, the same medical device record number: Jinghai Machinery, 20150008) Medium); and other agents as described in one of the above), including zoledronic acid, recombinant human interleukin-2, recombinant human interleukin-15, and recombinant human interleukin-18;
  • Cord blood NK cell proliferation medium GMP S&XFM TM -CD lymphocyte medium (Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd. with the same name, the same medical device record number: Jinghai Machinery and Equipment 20150008) And the reagent described above recombinant human interleukin-2.
  • the percentage concentration is mass/mass (W/W, unit g/100g) percentage concentration, mass/volume (W/V, unit g/100mL) percentage concentration or volume/volume (V/V, unless otherwise specified). Percent concentration in units of mL/100 mL).
  • Example 1 In vitro expansion of cord blood NK cells and detection
  • the method for in vitro expansion of cord blood NK cells of the present invention comprises the following steps:
  • the centrifuge tube is divided into four layers with clear interface from top to bottom. From top to bottom, it is: pale yellow plasma layer, white film NK cell layer, transparent separation liquid layer and red red blood cells. Layer, carefully absorb the white membrane-like mononuclear cell layer into another centrifuge tube, and wash it with PBS for 2 to 3 times to obtain mononuclear cells.
  • AIM Medium CTS TM lymphocyte culture medium purchased from Life Technology, Inc.
  • AIM Medium CTS TM lymphocyte culture medium purchased from Life Technology, Inc.
  • 2 ⁇ 10 6 th /mL 0.5 ⁇ 5 ⁇ 10 6 / mL can, more 1 ⁇ 3 ⁇ 10 6 cells / mL.
  • Below 0.5 ⁇ 10 6 /mL or higher than 5 ⁇ 10 6 /mL, which is not conducive to the activation of NK cells) inoculate a single nuclear cell suspension into a cell culture flask, and then to AIM Add 2 ⁇ g/mL to Medium CTS TM lymphocyte medium (1 ⁇ 10 ⁇ g/mL, preferably 1 ⁇ 5 ⁇ g/mL.
  • NK cell activation below 1 ⁇ g/mL, affect NK cell activation; above 10 ⁇ g/mL, activate NK) Little effect) Zoledronic acid (Zhaitai, Novartis) and 1000IU/mL (200-2000IU/mL, preferably 500-1500IU/mL. Below 200IU/mL, affecting the activation of NK cells; higher than 2000IU/mL, the cells are easy to die.
  • Recombinant human interleukin-2 (Delusheng, Beijing Sihuan Bio) at 37 ° C (36 ° C ⁇ 38 ° C can be, below 36 ° C or above 38 ° C, the cells are not normal Growth), 5% CO 2 saturated humidity environment for 3 days (1 to 5 days, preferably 2 to 4 days. Less than 1 day, NK cell activation is lower; higher than 5 days, the effect on NK activation Not big).
  • NK cells less than 0.5 ⁇ 10 6 /mL or more than 5 ⁇ 10 6 /mL, are not conducive to the proliferation of NK cells), inoculated into the cell culture flask, and then add 1000IU / mL (200 ⁇ 2000IU / mL can be, better 500 ⁇ 1500IU/mL. Below 200IU/mL, affecting the proliferation of NK cells; above 2000IU/mL, the cells are easy to die.
  • Recombinant human interleukin-2, 37 ° C (36 ° C ⁇ 38 ° C can be, below 36 ° C or high) At 38 ° C, the cells can not grow normally), cultured in a 5% CO 2 saturated humidity environment, supplemented with fresh lymphocyte medium every 2 to 3 days to adjust the cell density to 0.5 ⁇ 5 ⁇ 10 6 / mL (preferably 1 ⁇ 10 6 / mL, preferably 0.5 ⁇ 2 ⁇ 10 6 / mL.
  • Cell density is less than 0.5 ⁇ 10 6 / mL, the cells will proliferate slowly; when the cells are higher than 5 ⁇ 10 6 / mL, the cells are easy fast Inhibition of contact, affecting cell proliferation), and adding 1000 IU / mL (200 ⁇ 2000 IU / mL can be, preferably 500 ⁇ 1500 IU / mL) recombinant human interleukin-2, culture 14 ⁇ 35 days (better 18 ⁇ 24 days) Most preferably 21 days), cord blood NK cells are obtained.
  • 21 days was preferably used as a harvest point of umbilical cord blood NK cells.
  • Umbilical cord blood NK cells isolated from fresh anticoagulated umbilical cord blood or cryopreserved cord blood were activated and expanded in vitro (obtained by the above method), observed under a microscope, and the cell morphology was obtained by amplification.
  • the shape of the circle and the size gradually become uniform and irregular cell morphology, and the cell body and the nucleus volume increase (the cells expanded for 21 days are shown in Figure 3); the number of cells is from the original 4.36 ⁇ 10 8 /mL (fresh umbilical cord blood), 3.25 ⁇ 10 8 cells/mL (freeze-recovered cord blood), proliferated to 5.08 ⁇ 10 10 cells/mL (fresh cord blood), 3.94 ⁇ 10 10 cells/mL for 21 days of expansion culture.
  • Fig. 4 also shows that the expanded culture is a rapid proliferative phase from 14 days to 21 days, and then to a slow fading phase at 35 days, so that umbilical cord blood NK cells can be harvested in a period of 14 to 35 days depending on the situation.
  • RESULTS The purity of NK cells expanded and cultured for 21 days increased from the initial 1.23% (fresh cord blood), 0.79% (freeze-recovered cord blood) to 94.58% (fresh cord blood), and 94.37% (freeze recovery). Cord blood), as shown in Figure 5.
  • A549 cells human lung adenocarcinoma cells, derived from ATCC
  • A549 cells human lung adenocarcinoma cells, derived from ATCC
  • Killing rate (%) [1- (experimental group OD value - OD value of individual effector cells) OD OD value of individual target cells] ⁇ 100%
  • Example 3 10 ml of frozen resuscitation cord blood (sample 3) was taken in the same manner as in Example 1.
  • the density of mononuclear cells was adjusted to 2 ⁇ 10 6 using GMP S&XFM TM -CD lymphocyte medium (same name commercial product of Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd., with the medical device record number: Jinghai Machinery Co., Ltd. 20150008).
  • the cell suspension is inoculated into the cell culture flask, and then into the GMP S & XFM TM -CD lymphocyte medium
  • Recombinant human interleukin-2 (Delusheng, Beijing Sihuan Bio), cultured at 37 ° C (36 ° C ⁇ 38 ° C), 5% CO 2 saturated humidity environment for 1 to 5 days (better 2 ⁇ 4 days, preferably 3 days. Below 1 day, NK cell activation is lower; after 5 days, it has little effect on NK activation).
  • Example 3 10 ml of frozen resuscitation cord blood (sample 3) was taken in the same manner as in Example 1.
  • GMP S&XFM TM -CD lymphocyte medium the same name of Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd., with the medical device record number: Jinghai Machinery and Equipment 20150008, /mL (0.5 ⁇ 5 ⁇ 10 6 / mL can be, preferably 1 ⁇ 3 ⁇ 10 6 / mL), the cell suspension is inoculated into the cell culture flask, and then into the GMP S & XFM TM -CD lymphocyte medium
  • Recombinant human interleukin-2 (Delusheng, Beijing Sihuan Bio)
  • 10ng/mL (1 ⁇ 100ng/ml, preferably 1-20ng
  • NK Below 1ng/ml, NK
  • the activation has no effect. Above 100 ng/mL, it has little effect on NK activity.
  • Recombinant human interleukin-15 (purchased from Peprotech) and 10 ng/mL (1 to 100 ng/ml, preferably 1-20 ng/ml) None. Below 1 ng/ml, it has no effect on NK activation. Above 100 ng/mL, it has little effect on NK activity.
  • Recombinant human interleukin-18 (purchased from Peprotech) at 37 ° C (36 ° C ⁇ 38 °C available), 5% CO 2 and saturated humidity environment Incubated for 3 days (days 1 to 5 can, more 2 to 4 days is less than one day, low NK cell activation;. Than 5 days, has little effect on the activation of NK).
  • Example 4 in vitro expansion of cord blood NK cells (activation culture protocol 4)
  • Example 3 10 ml of frozen resuscitation cord blood (sample 3) was taken in the same manner as in Example 1.
  • the same activation medium as that of the activated culture scheme 3 is used, and the culture condition is changed to 39 ° C ⁇ 0.5 ° C (38 ° C to 40 ° C can be used to increase the culture temperature, on the one hand, it is beneficial to accelerate the activation of umbilical cord blood NK cells, and the activation efficiency is improved. On the other hand, it can increase the expansion ratio, purity and biological activity of NK in umbilical cord blood.
  • NK cells cannot adapt and die, and culture in 5% CO 2 saturated humidity for 1 day (0.5 to 3 days) Yes, below 0.5 days, the activation of NK is lower, and after 3 days, NK cells will die).
  • Umbilical cord blood NK cells expanded in vitro from 10 ml of cryopreserved resuscitation cord blood were subjected to the following assays using the method described in Example 1 (Example 1 - Example 4 using sample 3 as the starting blood source) Parallel operation):
  • the invention is from umbilical cord blood mononuclear cells (at this time, the proportion of NK cells in mononuclear cells is relatively low, see Table 2, the ratio of NK cells in 0 days is only 9.56%), and can be obtained through two steps of activation and amplification. High purity NK cells.
  • Table 1-1 shows the total number of cells (including expanded NK cells and other cells) counted before and after amplification.
  • the 0-day cord blood mononuclear cells are the cells before activation and expansion of the present invention (including a small amount of NK cells and other cells), which is equivalent to a control; 0-day cord blood NK cells refer to NK cells therein.
  • the 21-day cord blood NK cells are the total number of cells after 21 days of mononuclear cell expansion. Therefore, the purity of NK is very high, and most of them are NK cells, so the cells after 21 days of expansion are called NK cells (accurately speaking Mononuclear cells dominated by NK cells).
  • Example 4 Example 3> Implementation Example 2> Example 1.
  • Example 1 Example 2
  • Example 3 Example 4 0 day cord blood NK cell purity 9.56% 9.56% 9.56% 9.56% 21-day cord blood NK cell purity 90.69% 92.46% 93.75% 95.39%
  • CIK cell culture reference literature Document 6: Adoptive immunotherapy with cytokine-induced killer cells generated with a new good Manufacturing practice-grade protocol.Cytotherapy, 2012; Early Online: 1–10.DOI: 10.3109/14653249.2012.681038) Obtained.
  • Example 3 Under the fluorescence microscope, the umbilical cord blood NK cells harvested in vitro for 21 days in each of the examples showed strong killing ability against A549 tumor cells, and the amplification of the present invention was compared with the positive control CIK cells. NK cells have greater activity.
  • the amount of cord blood per serving is 100ml (usually the amount of cord blood is between 50ml and 120ml).
  • Example 5 In vitro amplification kit for umbilical cord blood NK cells
  • the kit for in vitro expansion of cord blood NK cells of the invention mainly comprises the following reagents:
  • Lymphocyte separation solution of umbilical cord blood NK cells Lymphocyte separation solution (Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd., the same name product, the same medical device record number: Jinghai Machinery, 20150002) Liquid), or other commercially available lymphocyte separation solutions;
  • Cord blood NK cell activation medium AIM Medium CTS TM (purchased from Life Technology, USA) medium or GMP S&XFM TM -CD lymphocyte medium (Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd.)
  • umbilical cord blood NK cell proliferation medium GMP S&XFM TM -CD lymphocyte medium (Beijing Jingmeng High-Tech Stem Cell Technology Co., Ltd., the same name, the same medical device record number: Jinghai Machinery, 20150008) Recombinant human interleukin-2.
  • This kit can be used by referring to the methods of Examples 1-4.
  • the present invention has the following advantages:
  • the reagents used do not contain animal-derived ingredients and are safe;
  • High NK cell yield the purity of the obtained umbilical cord blood NK cells is over 90%, and the total number of cells can reach 10 10-11 / part of cord blood (100 ml according to the sample of Example 1);
  • the invention obtains umbilical cord blood NK cells from the isolated cord blood mononuclear cells by activated culture and proliferation culture, does not require cell sorting, does not need trophoblast cells, has simple operation, good versatility, low cost, and the reagents used do not contain animal sources. Ingredients, good safety, and high yield of harvested NK cells, the total number of cells can reach 10 10-11 / part of cord blood, the purity is more than 90%, and the killing power to tumor cells is high.
  • the invention can be applied to the preparation of a tumor immunotherapy drug.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Rheumatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供脐血NK细胞的体外扩增方法及其试剂盒与应用，脐血NK细胞的体外扩增方法包括分离的脐血单个核细胞、活化培养和增殖培养脐血NK细胞。本发明不需要细胞分选，不需要滋养层细胞，所用试剂不含动物源成分，NK细胞产量高，获得的脐血NK细胞的纯度达90％以上，细胞总数可达10 10-11个/份脐血，对肿瘤细胞的杀伤力高。

Description

[根据细则37.2由ISA制定的发明名称]　脐血NK细胞的体外扩增方法及其试剂盒与应用 技术领域
本发明属于细胞工程技术领域，特别涉及一种脐血NK细胞的体外扩增方法及脐血NK细胞体外扩增试剂盒,还涉及该脐血NK细胞在制备肿瘤免疫治疗药物及肿瘤免疫治疗中的应用。
背景技术
自然杀伤细胞(Nature killer cell，NK)是除T、B细胞之外的第三类淋巴细胞，其细胞形态也与T、B细胞不同，NK细胞是胞浆中含嗜天青颗粒的大颗粒淋巴细胞，广泛存在于淋巴器官和外周组织中，在正常外周血中约占淋巴细胞总数的5％～10％，脾中占1％～2％，淋巴结和骨髓中也有NK细胞存在。NK细胞作为人体天然免疫的重要组成部分，是机体抗御感染和防止细胞恶性转化的重要免疫调节细胞，在免疫监视、免疫防御中起着十分重要的作用。以往研究表明在某些疾病的发生过程中，如肿瘤、自身免疫性疾病、衰老相关性疾病和HIV感染等，会导致NK细胞比例、数量和功能降低。2012年美国疾病控制中心报告NK细胞的活力和数量是人体健康的重要保证，几乎所有疾病的发病都与NK细胞的活性明显不足有关。当人们患有某种疾病时，无论是慢性的、偶发性的或是急性的，NK细胞的活性都在平均水平以下。让NK细胞恢复到高水平的活性，是比较好的治疗疾病方法。与T淋巴细胞不同，NK细胞的特点是：NK细胞不需预先刺激就可直接溶解破坏肿瘤细胞和病毒感染细胞，通过分泌穿孔素、丝氨酸蛋白酶如颗粒酶A和B、硫酸软骨素蛋白聚糖等分子降解细胞膜、破坏靶细胞完整性而发挥溶细胞效应；NK细胞表面表达FasL(Fas ligand)和TRAIL(TNF-related opoptosis-inducing ligand)可通过与靶细胞上的受体结合引起靶细胞的凋亡；活化后NK细胞会释放大量的干扰素-γ、肿瘤坏死因子-α等细胞因子；NK细胞通过细胞膜FcγRIII(CD16)与抗体Fc段结合从而介导抗体依赖的细胞毒性作用。随着对NK细胞生物功能及活化机制的了解，利用NK细胞为靶点的免疫治疗对疾病的早期预防、控制和治疗具有重要意义。
近年来，免疫治疗己经成为继手术、放化疗及内分泌治疗之后的第四大治疗模式，并逐渐受到重视。过继性细胞免疫治疗法(Adoptive cellular immunotherapy)作为免疫治疗方法的一种，在临床研究方面取得了较大的进展。目前已经开展将自体NK细胞通过过继免疫治疗应用于血液肿瘤(急性髓系白血病、急性淋巴细胞白血病等)和实体瘤(脑胶质瘤、肾癌和恶性黑色素瘤)的临床研究，其安全性得到肯定，但临床试验的结果并不尽如人意，表现在将自体外周血进行NK细胞分选后回输，虽 然观察到体内NK细胞对肿瘤细胞的杀伤增强，但是患者的生存率和复发率与对照组相比并无改善，这可能与肿瘤在发展过程中容易对自体NK细胞形成免疫逃避(immune evasion)有关，而且NK细胞过继性免疫治疗的临床试验受试者往往都是经过多种化疗失败的极度晚期的患者，瘤负荷重，机体免疫力差，NK细胞的功能有严重缺陷。
要提高NK细胞的临床治疗疗效就必须改变以往的治疗策略。为了克服这些缺陷，异体来源的NK细胞是一个比较好的选择。有学者研究通过移植异体NK细胞治疗白血病患者，发现能延缓复发，且不引起移植物抗宿主病，这引起人们对NK细胞的同种异体反应性的关注。从目前的临床研究的结果来看，异源性NK细胞在完全缓解率(Complete remission rate)、无事件生存期(event-free survival)、复发和死亡率方面明显优于自体NK细胞，而且异源性NK细胞并不伴随移植物抗宿主反应。NK细胞对肿瘤细胞的杀伤能力依赖于NK细胞表面活化性受体和抑制性受体间的信号平衡调控，杀伤细胞免疫球蛋白样受体(KIR)组成与主要组织相容性I类(MHC-I)结合的受体家族，对调节人NK细胞的活化阈值起重要作用。最近越来越多的研究表明利用KIR受体/MHC-I配体的错配，筛选最优的供体能大大增强NK细胞对受体肿瘤细胞的杀伤能力，提高NK细胞过继免疫治疗的效果。当前基于KIR的供者选择对提高造血干细胞/骨髓的移植和肿瘤的过继免疫治疗方面的研究倍受关注。异源性NK输注已经成为临床上NK细胞过继免疫治疗的新策略。
脐血作为现成的异源性资源，无伦理问题，来源丰富，更容易找到最优的供者，且脐血中T淋巴细胞发育不成熟,多为幼稚细胞，移植后发生急性和慢性移植物抗宿主病的几率低、程度轻，所以脐血已经成为异源NK细胞极具前景的来源。临床输注NK细胞，需要足够的细胞纯度和细胞数量，但NK细胞在脐血细胞中所占比例较低，数量也很少，需要经过体外分离及培养扩增，获得大量高纯度的NK细胞才能满足临床应用。因而如何获得高纯度、高质量的NK细胞成为NK细胞过继免疫治疗的关键(文献1：Klingemann H:Challenges of cancer therapy with natural killer cells.Cytotherapy 2015,17(3):245-249.)。
目前分离及扩增脐血NK细胞的方法主要有两种(如图1所示)：1.NK细胞在脐血中约占淋巴细胞的10％～20％，功能不成熟，对肿瘤细胞的杀伤能力低下。可利用流式细胞仪或者磁珠分选仪分选富集NK细胞，然后通过体外扩增增加NK细胞的数量和杀伤肿瘤的能力。Celgene Cellular Therapeutics公司将冻存复苏的脐带血用淋巴细胞分离液分离单个核细胞，利用
Figure PCTCN2016081198-appb-000001
富集试剂盒(StemCell Technologies，试剂盒包含CD3、CD4、CD14、CD19、CD20、CD36、CD66b、CD123、HLA-DR和glycophorin A抗体)分选CD56+CD3-NK细胞，纯度可达71％，细胞数为 1.5×107/份脐血，然后通过起始培养基(包括：IMDM，10％FBS，35mg/mL转铁蛋白，5μg/mL胰岛素，20μM乙醇胺，1μg/mL不饱和脂肪酸，1μg/mL亚油酸，0.2μg/mL棕榈酸，2.5μg/mL牛血清白蛋白，0.1μg/mL植物凝集素，1％青-链霉素，200IU/mL白介素-2)添加滋养层细胞(丝裂霉素C处理的外周血单个核细胞和K562细胞)，37℃5％CO2条件下培养NK细胞5～7天后，转移至维持培养基(IMDM，10％FBS,2％人AB血清，1％青-链霉素，200IU/mL白介素-2)培养至21天后，每一份脐血可获得CD56+CD3-NK细胞纯度>80％，平均细胞总数为1.2×109/份脐血(文献2：Kang L,Voskinarian-Berse V,Law E,Reddin T,Bhatia M,Hariri A,Ning Y,Dong D,Maguire T,Yarmush M et al:Characterization and ex vivo Expansion of Human Placenta-Derived Natural Killer Cells for Cancer Immunotherapy.Frontiers in immunology 2013,4:101.)。2.NK细胞起始于造血干细胞，脐血中含有丰富的造血干细胞，利用流式细胞仪或磁珠分选仪分选富集造血干细胞，通过体外分化获得一定数量的成熟NK细胞。Glycostem Therapeutics公司将冻存复苏脐血利用CliniMACS CD34磁珠分选试剂盒分选富集CD34+造血干细胞纯度为67％，细胞数为3.8×106/份脐血，在细胞扩增培养基I(包括：
Figure PCTCN2016081198-appb-000002
培养基，10％人血清，高剂量细胞因子组合(SCF，Flt3L，TPO，IL-7，低分子量肝素)和低剂量因子组合(GM-CSF，G-CSF，IL-6))培养9天，然后在细胞扩增培养基II(TPO更换为IL-15，其它成分与培养基I一样)扩增至14天，最后在NK细胞分化培养基(低剂量因子组合更换为(IL-7，SCF，IL-15和IL-2)，其它与培养基I一样)分化培养至35天，获得纯度>90％，平均数量2×109/份脐血的NK细胞(文献3：Spanholtz J,Preijers F,Tordoir M,Trilsbeek C,Paardekooper J,de Witte T,Schaap N,Dolstra H:Clinical-grade generation of active NK cells from cord blood hematopoietic progenitor cells for immunotherapy using a closed-system culture process.PloS one 2011,6(6):e20740.)。
综上所述，现有技术中脐血NK细胞的制备方法存在以下缺点：
1、需要分选细胞(分选NK细胞或者造血干细胞)，分选技术影响NK细胞的活性，也需要分选仪器；
2、需要滋养层细胞，K562细胞的引入极大影响扩增NK细胞的安全性；
3、操作复杂，技术通用性差；
4、分离和扩增阶段成本高；
5、使用动物源成分添加物，安全性差；
6、NK细胞产量低。
发明内容
本发明的第一个目的是提供一种简单且安全的脐血NK细胞的体外扩增方法。
本发明所提供的脐血NK细胞的体外扩增方法，包括以下步骤：
1)活化培养脐血NK细胞：用淋巴细胞培养基调整脐血单个核细胞密度为0.5～5×106个/mL，再至少添加1～10μg/mL唑来膦酸和200～2000IU/mL重组人白介素-2，在36℃～40℃、5％CO2饱和湿度环境中培养1～5天，收集细胞液；
2)增殖培养脐血NK细胞：从步骤1)的细胞液中获取细胞，用淋巴细胞培养基调整细胞密度为0.5～5×106个/mL，再添加200～2000IU/mL重组人白介素-2，在36℃～38℃、5％CO2饱和湿度环境中培养，每隔2～3天补充新鲜培养基并调整细胞密度为0.5～5×106个/mL，培养14～35天，收获得到脐血NK细胞。
所述步骤1)中用到的脐血单个核细胞分离自新鲜抗凝脐血或冻存复苏脐带血，分离过程如下：
1.1取新鲜抗凝脐血或冻存复苏脐带血，用1～2倍体积的PBS稀释；
1.2向淋巴细胞分离液上方缓慢加入等体积的稀释脐血，保持两液面界面清晰；
1.3室温980g，离心20～30min；
1.4离心结束后，吸取上面第二层的白膜状的单个核细胞层，用PBS清洗，得到脐血单个核细胞。
所述步骤1)中的淋巴细胞培养基可为AIM
Figure PCTCN2016081198-appb-000003
Medium CTSTM(购自美国Life Technology公司)或GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司同名商品)，优选为GMP S&XFMTM-CD淋巴细胞培养基。
当所述步骤1)中的淋巴细胞培养基为AIM
Figure PCTCN2016081198-appb-000004
Medium CTSTM时，活化培养优选为：用AIM
Figure PCTCN2016081198-appb-000005
Medium CTSTM淋巴细胞培养基调整细胞密度为1～3×106个/mL，再添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2，在36℃～38℃、5％CO2饱和湿度环境中培养2～4天；活化培养更优为：用AIM
Figure PCTCN2016081198-appb-000006
Medium CTSTM淋巴细胞培养基调整细胞密度为2×106个/mL，再添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2，在37℃、5％CO2饱和湿度环境中培养3天。
当所述步骤1)中的淋巴细胞培养基为GMP S&XFMTM-CD时，活化培养优选为以下之一种：
A.用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为1～3×106个/mL，再添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2，在36℃～38℃、5％CO2饱和湿度环境中培养2～4天；活化培养更优为：用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为2×106个/mL，再添加2μg/mL唑来膦酸和1000IU/mL重组人白介素 -2，在37℃、5％CO2饱和湿度环境中培养3天；
B.用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为1～3×106个/mL，再添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2，1～100ng/mL(较好1～20ng/mL)重组人白介素-15和1～100ng/mL(较好1～20ng/mL)重组人白介素-18，在36℃～38℃、5％CO2饱和湿度环境中培养2～4天；活化培养更优为：用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为2×106个/mL，再添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2、10ng/mL重组人白介素-15和10ng/mL重组人白介素-18，在37℃、5％CO2饱和湿度环境中培养3天；
C.用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为1～3×106个/mL，再添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2，1～100ng/mL(较好1～20ng/mL)重组人白介素-15和1～100ng/mL(较好1～20ng/mL)重组人白介素-18，在38.5℃～39.5℃、5％CO2饱和湿度环境中培养0.5～1天；活化培养更优为：用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为2×106个/mL，再添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2、10ng/mL重组人白介素-15和10ng/mL重组人白介素-18，在39℃、5％CO2饱和湿度环境中培养1天。
所述步骤2)增殖培养脐血NK细胞中优选使用GMP S&XFMTM-CD淋巴细胞分离液(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150002号中的样本密度分离液)，操作优选为：用移液管将细胞液移至离心管中，150g，离心10min，弃上清，用GMP S&XFMTM-CD淋巴细胞培养基调整,细胞密度为0.5～2×106个/mL(更优选1×106个/mL)，再添加500～1500IU/mL(更优选1000IU/mL)重组人白介素-2，在37℃、5％CO2饱和湿度环境中培养，每隔2～3天补充新鲜培养基调整细胞密度为0.5～2×106个/mL(更优选1×106个/mL)，并添加500～1500IU/mL(更优选1000IU/mL)重组人白介素-2，培养18～24天(更优选21天)，得到脐血NK细胞。
以上所述脐血NK细胞的体外扩增方法步骤1)中使用的专用活化培养基也属于发明内容。专用活化培养基为以下组配之一：
添加1～10μg/mL唑来膦酸和200～2000IU/mL重组人白介素-2的AIM
Figure PCTCN2016081198-appb-000007
Medium CTSTM淋巴细胞培养基；
优选为添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2的AIM
Figure PCTCN2016081198-appb-000008
Medium CTSTM淋巴细胞培养基；
更优为添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2的AIM
Figure PCTCN2016081198-appb-000009
Medium CTSTM淋巴细胞培养基；或
添加1～10μg/mL唑来膦酸和200～2000IU/mL重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基；
优选为添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基；
更优为添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基；或
添加1～10μg/mL唑来膦酸、200～2000IU/mL重组人白介素-2、1～100ng/mL重组人白介素-15和1～100ng/mL重组人白介素-18的GMP S&XFMTM-CD淋巴细胞培养基。
优选为添加1～5μg/mL唑来膦酸、500～1500IU/mL重组人白介素-2、1～20ng/mL重组人白介素-15和1～20ng/mL重组人白介素-18的GMP S&XFMTM-CD淋巴细胞培养基。
更选为添加2μg/mL唑来膦酸、1000IU/mL重组人白介素-2、10ng/mL重组人白介素-15和10ng/mL重组人白介素-18的GMP S&XFMTM-CD淋巴细胞培养基。
以上所述脐血NK细胞的体外扩增方法步骤2)中使用的专用增殖培养基也属于本发明内容。该专用增殖培养基为添加200～2000IU/mL(优选1000IU/mL)重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基。
以上所述体外扩增方法收获的脐血NK细胞也属于本发明内容。该细胞纯度超过90％。
本发明方法得到的脐血NK细胞在制备肿瘤免疫治疗药物中的应用或在肿瘤免疫治疗中的应用也属于本发明内容。
本发明另一目的是提供一种脐血NK细胞的体外扩增试剂盒。
本发明所提供的脐血NK细胞的体外扩增试剂盒，主要包括以下试剂：
1)脐血NK细胞的淋巴细胞分离液(北京京蒙高科干细胞技术有限公司同名商品)，或市售其它淋巴细胞分离液；
2)脐血NK细胞活化培养基：AIM
Figure PCTCN2016081198-appb-000010
Medium CTSTM(美国Life Technology公司)培养基或GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司商品)，以及唑来膦酸、重组人白介素-2、重组人白介素-15和重组人白介素-18；
3)脐血NK细胞增殖培养基：GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司商品)和重组人白介素-2。
脐血NK细胞的体外扩增试剂盒中试剂按上述脐血NK细胞的体外扩增方法进行使用。
采取以上方案，本发明提供了一种操作简单且安全的脐血NK细胞的体外扩增方法以及相关试剂盒。与现有的分离及扩增脐血NK细胞的方法相比，本发明具有以下有益效果：
1)直接使用脐血单个核细胞，不需要进行细胞分选步骤，即不需要分选NK细胞或者造血干细胞，简化了细胞制备流程，大大降低了脐血NK细胞的制备成本；
2)不需要滋养层细胞，避免引入滋养层细胞给制备的脐血NK细胞带来安全风险；
3)制备NK细胞所使用的细胞培养基完全无动物源成分，安全性好，而且化学成分明确，增加了批次之间的稳定性；
4)NK细胞产量高，获得的脐血NK细胞的纯度达90％以上，细胞总数可达1010-11个/份脐血。
本发明脐血NK细胞的体外扩增方法和试剂盒将在制备肿瘤免疫治疗药物及肿瘤的免疫治疗中发挥重要作用，应用前景广阔。
附图说明
图1为现有分离及扩增脐血NK细胞的方法；
图2为本发明脐血NK细胞体外扩增方法的流程；
图3为新鲜脐血和冻存复苏脐血NK细胞体外扩增前后的细胞形态图；
图4为新鲜脐血和冻存复苏脐血NK细胞体外扩增的生长曲线；
图5为新鲜脐血和冻存复苏脐血NK细胞体外扩增前后纯度的流式细胞仪检测结果；
图6为体外扩增的新鲜脐血和冻存复苏脐血NK细胞的肿瘤靶细胞杀伤效果。
具体实施方式
针对现有技术中分离及扩增脐血NK细胞中的不足，本发明旨在提供一种脐血NK细胞的体外制备方法，并提供方法中用到的专用活化培养基和专用增殖培养基。
本发明脐血NK细胞的体外扩增方法，可包括以下步骤：
1)活化培养脐血NK细胞：用淋巴细胞培养基(下文详细介绍)调整脐血单个核细胞密度为0.5～5×106个/mL(优选1～3×106个/mL，最优选2×106个/mL)，将细胞悬液接种细胞培养瓶中；再添加1～10μg/mL(优选1～5μg/mL，最优选2μg/mL)唑来膦酸和200～2000IU/mL(优选500～1500IU/mL，最优选1000IU/mL)重组人白介素-2(形成专用活化培养基)，在36℃～40℃、5％CO2饱和湿度环境中培养1～5天；
具体的，步骤1)中的淋巴细胞培养基可为AIM
Figure PCTCN2016081198-appb-000011
Medium CTSTM(购自美国Life  Technology公司)或GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)，优选为GMP S&XFMTM-CD淋巴细胞培养基。GMP S&XFMTM-CD淋巴细胞培养基的详细说明也可参考文献4：专利申请号：201310082166.8，CN103146648A中的描述。
根据选用的淋巴细胞培养基的种类，相应得到不同的专用活化培养基。
一)选用AIM
Figure PCTCN2016081198-appb-000012
Medium CTSTM淋巴细胞培养基，专用活化培养基组成为：添加1～10μg/mL(优选1～5μg/mL，最优选2μg/mL)唑来膦酸和200～2000IU/mL(优选500～1500IU/mL，最优选1000IU/mL)重组人白介素-2的AIM
Figure PCTCN2016081198-appb-000013
Medium CTSTM淋巴细胞培养基。
利用该专用活化培养基，步骤1)中的活化培养方法优选为：用AIM
Figure PCTCN2016081198-appb-000014
Medium CTSTM淋巴细胞培养基调整脐血单个核细胞密度为0.5～5×106/mL(优选1～3×106个/mL，最优选2×106个/mL)，将细胞悬液接种细胞培养瓶中，再向AIM
Figure PCTCN2016081198-appb-000015
Medium CTSTM淋巴细胞培养基中添加1～10μg/mL(优选1～5μg/mL，最优选2μg/mL)唑来膦酸和200～2000IU/mL(优选500～1500IU/mL，最优选1000IU/mL)重组人白介素-2，在36℃～38℃(优选37℃)、5％CO2饱和湿度环境中培养1～5天(优选2～4天，最优选3天)。
二)选用GMP S&XFMTM-CD淋巴细胞培养基，专用活化培养基组成为：添加1～10μg/mL(优选1～5μg/mL，最优选2μg/mL)唑来膦酸和200～2000IU/mL(优选500～1500IU/mL，最优选1000IU/mL)重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基。
利用该专用活化培养基，步骤1)中的活化培养方法更优选为：用GMP S&XFMTM-CD淋巴细胞培养基调整脐血单个细胞密度为0.5～5×106/mL(优选1～3×106个/mL，最优选2×106个/mL)，将细胞悬液接种细胞培养瓶中，再向GMP S&XFMTM-CD淋巴细胞培养基中添加1～10μg/mL(优选1～5μg/mL，最优选2μg/mL)唑来膦酸和200～2000IU/mL(优选500～1500IU/mL，最优选1000IU/mL)重组人白介素-2，在36℃～38℃(优选37℃)、5％CO2饱和湿度环境中培养1～5天(优选2～4天，最优选3天)。
三)选用GMP S&XFMTM-CD淋巴细胞培养基，专用活化培养基组成为：添加1～10μg/mL(优选1～5μg/mL，最优选2μg/mL)唑来膦酸和200～2000IU/mL(优选500～1500IU/mL，最优选1000IU/mL)重组人白介素-2、1～100ng/mL(优选1～20ng/mL，最优选10ng/mL)重组人白介素-15和1～100ng/mL(优选1～20ng/mL，最优选10ng/mL)重组人白介素-18的GMP S&XFMTM-CD淋巴细胞培养基。
利用该专用活化培养基，步骤1)中的活化培养方法更优选为：用GMP S&XFMTM-CD淋巴细胞培养基调整脐血单个核细胞密度为0.5～5×106/mL(优选1～3×106个/mL，最优选2×106个/mL)，将细胞悬液接种细胞培养瓶中，再向GMP S&XFMTM-CD淋巴细胞培养基中添加1～10μg/mL(优选1～5μg/mL，最优选2μg/mL)唑来膦酸和200～2000IU/mL(优选500～1500IU/mL，最优选1000IU/mL)重组人白介素-2、1～100ng/mL(优选1～20ng/mL，最优选10ng/mL)重组人白介素-15和1～100ng/mL(优选1～20ng/mL，最优选10ng/mL)重组人白介素-18，在36℃～38℃(优选37℃)、5％CO2饱和湿度环境中培养1～5天(优选2～4天，最优选3天)。
利用该专用活化培养基，改变培养条件，如在38℃～40℃(优选38.5℃～39.5℃，最优选39℃)、5％CO2饱和湿度环境中培养0.5～3天(优选0.5～1天，最优选1天)，将得到步骤1)活化培养脐血NK细胞的最优条件。
2)增殖培养脐血NK细胞：用移液管将步骤1)得到的细胞液移至离心管中，150g，离心10min，弃上清，用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为0.5～5×106个/mL(较优0.5～2×106个/mL，优选1×106个/mL)，将细胞悬液接种细胞培养瓶中，再添加200～2000IU/mL(较优500～1500IU/mL，优选1000IU/mL)重组人白介素-2(形成专用增殖培养基)，在36℃～38℃(优选37℃)、5％CO2饱和湿度环境中培养，每隔2～3天补充新鲜GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为0.5～5×106个/mL(较优0.5～2×106个/mL，优选1×106个/mL)，并添加200～2000IU/mL(较优500～1500IU/mL，优选1000IU/mL)重组人白介素-2，培养14～35天(较优18～24天，优选21天)，收获得到脐血NK细胞。
步骤2)中增殖培养中专用增殖培养基组成为：添加200～2000IU/mL(较优500～1500IU/mL，优选1000IU/mL)重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基。
步骤2)增殖培养脐血NK细胞过程优选为：用移液管将细胞液移至离心管中，150g(离心力单位)，离心10min，弃上清，用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为1×106个/mL，将细胞悬液接种细胞培养瓶中，再向细胞培养瓶中添加1000IU/mL重组人白介素-2，在37℃、5％CO2饱和湿度环境中培养，每隔2～3天补充新鲜培养基并调整细胞密度为1×106个/mL，培养21天，收获得到脐血NK细胞。
上述方法中，步骤1)中用到的脐血单个核细胞分离于脐带血，分离方法如下：
1.1取新鲜抗凝脐血或冻存复苏脐带血100mL，添加100mL PBS稀释脐血；
1.2于10个50mL离心管中分别加入20mL淋巴细胞分离液(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150002号中的样本密度分离液，该分离液的详细内容也可参考文献5：专利号：ZL 201110456878.2， CN102533650A中的介绍)，将200mL稀释脐血均分分别缓慢加入分离液面上方，每个离心管加20mL稀释脐血，保持两液面界面清晰；该步骤中分离液也可以是市售其它淋巴细胞分离液。
1.3室温，400～1200g(优选980g，为一种离心力单位。如步骤1.2使用市售其它淋巴细胞分离液则按照其说明书操作)离心20～40min(优选30min)；
1.4离心结束后，离心管中由上至下分为界面清晰的四层，从上而下依次为：浅黄色的血浆层、白膜状的单个核细胞层、透明的分离液层以及红色的红细胞层，小心吸取白膜状的单个核细胞层到另一离心管中，用PBS清洗，得到脐血单个核细胞。
依据以上方法，本发明还提供了一种脐血NK细胞的体外扩增试剂盒，主要包括以下试剂：
1)脐血单个核细胞的淋巴细胞分离液(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150002号中的样本密度分离液)，或市售其它淋巴细胞分离液；
2)脐血NK细胞活化培养基：AIM
Figure PCTCN2016081198-appb-000016
Medium CTSTM(购自美国Life Technology公司)培养基或GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)；以及按上述一)至四)描述的其它试剂，包括唑来膦酸、重组人白介素-2、重组人白介素-15和重组人白介素-18；
3)脐血NK细胞增殖培养基：GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)，以及上述描述的试剂重组人白介素-2。
以下结合实施例进一步描述本发明，实施例中所用方法如无特别说明均为常规方法。
所述百分比浓度如无特别说明均为质量/质量(W/W，单位g/100g)百分比浓度、质量/体积(W/V，单位g/100mL)百分比浓度或体积/体积(V/V，单位mL/100mL)百分比浓度。
实施例中描述到的各种生物材料的取得途径仅是提供一种实验获取的途径以达到具体公开的目的，不应成为对本发明生物材料来源的限制。事实上，所用到的生物材料的来源是广泛的，任何不违反法律和道德伦理能够获取的生物材料都可以按照实施例中的提示替换使用。
实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的 操作过程，实施例将有助于理解本发明，但是本发明的保护范围不限于下述的实施例。
实施例1、体外扩增脐血NK细胞及检测
一、体外扩增脐血NK细胞(活化培养方案一)
如图1所示，本发明脐血NK细胞的体外扩增方法，包括以下步骤：
1)分离脐血单个核细胞
1.1分别取100mL的新鲜抗凝脐血(样本1)和冻存复苏脐带血(样本2，脐带血均由武警总医院妇产科提供，并经医院伦理委员会批准)，添加100mL PBS(配方：NaCl8.0g，KCl0.2g，Na2HPO41.44g，KH2PO40.24g，添加蒸馏水至1000mL，调节pH7.4)稀释脐血；
1.2于10个50mL离心管中分别加入20mL淋巴细胞分离液(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150002号中的样本密度分离液)，将稀释脐血分别缓慢加入分离液面上方，每个离心管加20mL稀释脐血，保持两液面界面清晰；
1.3室温，980g,离心30min；
1.4离心结束后，离心管中由上至下分为界面清晰的四层，从上而下依次为：浅黄色的血浆层、白膜状的NK细胞层、透明的分离液层以及红色的红细胞层，小心吸取白膜状的单个核细胞层到另一离心管中，用PBS清洗2～3次，得到单个核细胞。
2)活化培养脐血NK细胞
用AIM
Figure PCTCN2016081198-appb-000017
Medium CTSTM淋巴细胞培养基(购自美国Life Technology公司)调整单个核细胞密度为2×106个/mL(0.5～5×106/mL均可，较优1～3×106个/mL。低于0.5×106/mL或者高于5×106/mL，都不利于NK细胞的活化)，将单个核细胞悬液接种细胞培养瓶中，再向AIM
Figure PCTCN2016081198-appb-000018
Medium CTSTM淋巴细胞培养基中添加2μg/mL(1～10μg/mL均可，较优1～5μg/mL。低于1μg/mL，影响NK细胞活化；高于10μg/mL，对NK的活化影响不大)唑来膦酸(择泰，诺华制药)和1000IU/mL(200～2000IU/mL均可，较优500～1500IU/mL。低于200IU/mL，影响NK细胞的活化；高于2000IU/mL，细胞容易死亡)重组人白介素-2(德路生，北京四环生物)，在37℃(36℃～38℃均可，低于36℃或者高于38℃，细胞均无法正常生长)、5％CO2饱和湿度环境中培养3天(1～5天均可，较优2～4天。低于1天，NK细胞活化较低；高于5天后，对NK的活化影响不大)。
3)增殖培养脐血NK细胞
用移液管将细胞液移至离心管中，150g，离心10min，弃上清，用GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)调整细胞密度为1×106个/mL(0.5～5×106/mL均可，较优0.5～2×106个/mL。低于0.5×106个/mL或者高于5×106/mL时，都不利于NK细胞的增殖)，接种至细胞培养瓶中，再添加1000IU/mL(200～2000IU/mL均可，较优500～1500IU/mL。低于200IU/mL，影响NK细胞的增殖；高于2000IU/mL，细胞容易死亡)重组人白介素-2，37℃(36℃～38℃均可，低于36℃或者高于38℃时，细胞不能正常生长)、5％CO2饱和湿度环境中培养，每隔2～3天补充新鲜淋巴细胞培养基调整细胞密度为0.5～5×106个/mL(优选1×106个/mL，较优0.5～2×106个/mL。细胞密度低于0.5×106个/mL，细胞将增殖缓慢；当细胞高于5×106个/mL，细胞容易很快形成接触抑制，影响细胞的增殖)，并添加1000IU/mL(200～2000IU/mL均可，较优500～1500IU/mL)重组人白介素-2，培养14～35天(较优18～24天，最优选21天)，得到脐血NK细胞。由于细胞在21天时扩增数量较多，且杀伤活性最大，21天后细胞进入稳定期，细胞的杀伤活性下降，所以优选21天作为脐血NK细胞的收获点。
二、检测体外扩增的脐血NK细胞
1、脐血NK细胞计数
1)轻轻吹打脐血NK细胞悬液，成NK单细胞悬液；
2)取NK单细胞悬液20μl，加入20μl 0.2％台盼蓝染色液(购自SIGMA公司)中，反复抽吸轻轻混合均匀；
3)取20μl混合液加入Countstar(IC1000)细胞计数仪(购自上海睿钰生物科技有限公司)的计数板槽中；
4)静置1分钟，读数。
结果：从新鲜抗凝脐血或冻存复苏脐带血中分离的脐血NK细胞经过体外活化、扩增培养后(用上述一的方法获得)，在显微镜下观察，细胞形态由扩增前的圆形、大小各异，逐渐变为均一的不规则细胞形态，细胞胞体和胞核体积增大(扩增培养21天的细胞如图3所示)；细胞数量由最初的4.36×108个/mL(新鲜脐血)、3.25×108个/mL(冻存复苏脐血)增殖至扩增培养21天的5.08×1010个/mL(新鲜脐血)、3.94×1010个/mL(冻存复苏脐血)，如图4所示。图4还显示扩增培养14天至21天为细胞快速增殖期，之后至35天为缓慢凋零期，因此可根据情况在14～35天培养期内收获脐血NK细胞。
2、流式细胞仪检测脐血NK细胞的纯度
1)取单细胞悬液,150g离心10分钟；
2)用PBS重悬细胞沉淀，调节细胞浓度为1×106个/100μl，置流式检测管中；
3)加抗体CD56、CD3(购自BD公司)，轻轻吹打混匀，4℃避光孵育30分钟，同时设置同型对照；
4)1500g离心5分钟,弃上清；
5)加100μl PBS，轻轻吹打混匀，上机检测。
结果：一中扩增培养21天的NK细胞的纯度由最初的1.23％(新鲜脐血)、0.79％(冻存复苏脐血)提高至94.58％(新鲜脐血)、94.37％(冻存复苏脐血)，如图5所示。
3、CCK-8法测定脐血NK细胞的杀瘤活性
1)取生长良好的A549细胞(人肺腺癌细胞，来源于ATCC)为靶细胞，用0.25％胰蛋白酶消化；
2)台盼蓝染色计数，调整细胞浓度至5×104个/mL；
3)每孔100ul于96孔板中，置于37℃5％CO2孵育过夜。
4)取脐血NK细胞悬液为效应细胞，调整细胞浓度至2×l06个/mL；
5)按照效靶比为5：1、10：1、20：1加入96孔板中，每组设3复孔；
6)37℃5％CO2的孵箱中培养4h；
7)每孔加入15μl CCK-8(购自碧云天)，继续孵育2h；
8)用酶标仪检测450nm波长的OD值；
9)计算杀伤率：
杀伤率(％)＝[1-(实验组OD值-单独效应细胞OD值)÷单独靶细胞OD值]×100％
结果：荧光显微镜下观察一中体外扩增培养21天的脐血NK细胞对A549肿瘤细胞的杀伤能力大大提高，如图6所示。
上述检测结果表明，用本发明方法体外扩增的脐血NK细胞数量高、纯度高、对肿瘤细胞的杀伤活性高。
实施例2、体外扩增脐血NK细胞(活化培养方案二)
如图1所示，脐血NK细胞的体外扩增包括以下过程：
1)从冻存复苏脐带血中分离脐血单个核细胞
取10ml冻存复苏脐带血(样本3)，方法与实施例1相同。
2)活化培养脐血NK细胞(对照组为实施例1)
用GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司同名商 品，同医疗器械备案号：京海械备20150008号中的细胞培养基)调整单个核细胞密度为2×106个/mL(0.5～5×106/mL均可，较优1～3×106个/mL)，将细胞悬液接种细胞培养瓶中，再向GMP S&XFMTM-CD淋巴细胞培养基中添加2μg/mL(1～10μg/mL均可，较优1～5μg/mL)唑来膦酸(择泰，诺华制药)和1000IU/mL(200～2000IU/mL均可，较优500～1500IU/mL)重组人白介素-2(德路生，北京四环生物)，在37℃(36℃～38℃均可)、5％CO2饱和湿度环境中培养1～5天(较优2～4天，优选3天。低于1天，NK细胞活化较低；高于5天后，对NK的活化影响不大)。
相同的添加试剂对细胞活化的影响与实施例1描述的相同，不再赘述。
3)增殖培养脐血NK细胞
与实施例1相同。
实施例3、体外扩增脐血NK细胞及检测(活化培养方案三)
如图1所示，脐血NK细胞的体外扩增包括以下过程：
1)从冻存复苏脐带血中分离脐血单个核细胞
取10ml冻存复苏脐带血(样本3)，方法与实施例1相同。
2)活化培养脐血NK细胞
用GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)调整单个核细胞密度为2×106个/mL(0.5～5×106/mL均可，较优1～3×106个/mL)，将细胞悬液接种细胞培养瓶中，再向GMP S&XFMTM-CD淋巴细胞培养基中添加2μg/mL(1～10μg/mL均可，较优1～5μg/mL)唑来膦酸(择泰，诺华制药)、1000IU/mL(200～2000IU/mL均可，较优500～1500IU/mL)重组人白介素-2(德路生，北京四环生物)、10ng/mL(1～100ng/ml均可，较优1～20ng/ml均可。低于1ng/ml时，对NK的活化无作用，高于100ng/mL时，对NK活性影响不大)重组人白介素-15(购自Peprotech公司)和10ng/mL(1～100ng/ml均可，较优1～20ng/ml均可。低于1ng/ml时，对NK的活化无作用，高于100ng/mL时，对NK活性影响不大)重组人白介素-18(购自Peprotech公司)，在37℃(36℃～38℃均可)、5％CO2饱和湿度环境中培养3天(1～5天均可，较优2～4天。低于1天，NK细胞活化较低；高于5天后，对NK的活化影响不大)。
相同的添加试剂对细胞活化的影响与实施例1描述的相同，不再赘述。
3)增殖培养脐血NK细胞
与实施例1相同。
实施例4、体外扩增脐血NK细胞(活化培养方案四)
如图1所示，脐血NK细胞的体外扩增包括以下过程：
1)从冻存复苏脐带血中分离脐血单个核细胞
取10ml冻存复苏脐带血(样本3)，方法与实施例1相同。
2)活化培养脐血NK细胞
使用与活化培养方案三相同的活化培养基，培养条件变化为在39℃±0.5℃(38℃～40℃均可，提高培养温度，一方面有益于加快脐血NK细胞活化，活化效率提高，另一方面可以提高脐血NK的扩增倍数、纯度和生物活性。但高于40℃时，NK细胞不能适应，出现死亡)、5％CO2饱和湿度环境中培养1天(0.5～3天均可，低于0.5天，NK的活化较低，高于3天后，NK细胞会出现死亡)。
3)增殖培养脐血NK细胞
与实施例1相同。
对以上实施例方法得到的脐血NK细胞进行检测：
脐血NK细胞检测
用实施例1中二描述的方法对从10ml冻存复苏脐带血(样本3)体外扩增的脐血NK细胞进行以下检测(利用实施例1-实施例4方法以样本3为起始血源进行平行操作)：
1、脐血NK细胞计数
从冻存复苏脐带血中分离的脐血单个核细胞经过体外活化、扩增培养21天后，细胞扩增结果如表1-1和表1-2所示。
本发明是从脐血单个核细胞(此时NK细胞在单个核细胞所占比例较低，见表2,0天的NK细胞比例仅仅为9.56％)，经过活化和扩增两步骤，可以获得高纯度的NK细胞。表1-1显示扩增前后所计数都是细胞(包括扩增后的NK细胞和其它细胞)的总数。NK细胞的绝对数是细胞总数×NK细胞的比例，即：表1-1数据×表2＝表1-2。
0天脐血单个核细胞是本发明活化和扩增前的细胞(包括少量NK细胞和其它细胞)，相当于对照；0天脐血NK细胞是指其中的NK细胞。21天脐血NK细胞是单个核细胞扩增21天后的细胞总数，因此时NK纯度很高，绝大多数都是NK细胞，所以21天扩增后的细胞被称为NK细胞(准确说是以NK细胞为主的单个核细胞)。
该结果显示实施例1-实施例4方案均使脐血细胞总数成百倍扩增(表1-1)，其中NK细胞的绝对数上千倍扩增(表1-2)，且扩增效果实施例4＞实施例3＞实施 例2＞实施例1。
表1-1脐血细胞的总体数量及扩增倍数(10ml脐血量)
Figure PCTCN2016081198-appb-000019
表1-2脐血NK细胞的绝对数量及扩增倍数(10ml脐血量)
Figure PCTCN2016081198-appb-000020
2、流式细胞仪检测脐血NK细胞的纯度
从冻存复苏脐带血中分离的脐血单个核细胞经过体外活化、扩增培养21天后，NK细胞纯度结果如表2所示。
表2脐血NK细胞的纯度
  实施例1 实施例2 实施例3 实施例4
0天脐血NK细胞纯度 9.56％ 9.56％ 9.56％ 9.56％
21天脐血NK细胞纯度 90.69％ 92.46％ 93.75％ 95.39％
表2数据表明脐血单个核细胞中NK比例由原来的9.56％，经过各实施例的扩增，NK细胞纯度均能达到90％以上，且实施例4＞实施例3＞实施例2＞实施例1。
3、CCK-8法测定脐血NK细胞的杀瘤活性
实验以0天脐血单个核细胞(活化和扩增前的细胞)为阴性对照，以CIK细胞(一种杀伤肿瘤的免疫细胞)为阳性对照。CIK细胞的培养参照文献(文献6：Adoptive immunotherapy with cytokine-induced killer cells generated with a new good  manufacturing practice-grade protocol.Cytotherapy,2012；Early Online:1–10.DOI:10.3109/14653249.2012.681038)获得。
结果如表3所示，荧光显微镜下观察各实施例体外扩增21天收获的脐血NK细胞均显示对A549肿瘤细胞较强的杀伤能力，与阳性对照CIK细胞相比，本发明扩增的NK细胞具有更强的活性。同样效靶比下实施例4＞实施例3＞实施例2＞实施例1，表明实施例4扩增的细胞杀伤能力最强。
表3脐血NK细胞对A549肿瘤细胞的杀伤能力
Figure PCTCN2016081198-appb-000021
由以上实施例可以看出，本发明所提供的脐血NK细胞体外扩增方法操作简单且安全，与现有的分离及扩增脐血NK细胞的方法(现有技术)的比较如表4所示。
表4现有技术与本发明技术的比较
Figure PCTCN2016081198-appb-000022
注：每份脐血量为100ml(通常一份脐血的量约在50ml-120ml之间)。
实施例5、脐血NK细胞的体外扩增试剂盒
本发明脐血NK细胞的体外扩增试剂盒，主要包括以下试剂：
1)脐血NK细胞的淋巴细胞分离液：淋巴细胞分离液(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150002号中的样本密度分离 液)，或市售其它淋巴细胞分离液；
2)脐血NK细胞活化培养基：AIM
Figure PCTCN2016081198-appb-000023
Medium CTSTM(购自美国Life Technology公司)培养基或GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司
同名商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)，以及唑来膦酸、重组人白介素-2、重组人白介素-15和重组人白介素-18；
3)脐血NK细胞增殖培养基：GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司同名商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)和重组人白介素-2。
该试剂盒可参照实施例1-4的方法进行使用。
综上，本发明具有以下优点：
1)不需要细胞分选；
2)不需要滋养层细胞；
3)操作简单，通用性好；
4)成本低；
5)所用试剂不含动物源成分，安全性好；
6)NK细胞产量高：获得的脐血NK细胞的纯度达90％以上，细胞总数可达1010-11个/份脐血(按实施例1样本为100ml)；
7)对肿瘤细胞的杀伤力高。
工业应用性
本发明从分离的脐血单个核细胞通过活化培养和增殖培养获得脐血NK细胞，不需要细胞分选，不需要滋养层细胞，操作简单，通用性好，成本低，所用试剂不含动物源成分，安全性好，且收获的NK细胞产量高，细胞总数可达1010-11个/份脐血，纯度达90％以上，对肿瘤细胞的杀伤力高。本发明能应用于肿瘤免疫治疗药物的制备中。

Claims (11)

  1. 一种脐血NK细胞的体外扩增方法，包括以下步骤：
    1)活化培养脐血NK细胞：用淋巴细胞培养基调整脐血单个核细胞密度为0.5～5×106个/mL，再至少添加1～10μg/mL唑来膦酸和200～2000IU/mL重组人白介素-2，在36℃～40℃、5％ CO2饱和湿度环境中培养1～5天，收集细胞液；
    2)增殖培养脐血NK细胞：从步骤1)的细胞液中获取细胞，用淋巴细胞培养基调整细胞密度为0.5～5×106个/mL，再添加200～2000IU/mL重组人白介素-2，在36℃～38℃、5％ CO2饱和湿度环境中培养，每隔2～3天补充新鲜培养基并调整细胞密度为0.5～5×106个/mL，培养14～35天，收获得到脐血NK细胞。
  2. 根据权利要求1所述的脐血单个核细胞的体外扩增方法，其特征在于：所述步骤1)中用到的脐血单个核细胞分离自新鲜抗凝脐血或冻存复苏脐带血，分离过程如下：
    1.1取新鲜抗凝脐血或冻存复苏脐带血，用1～2倍体积的PBS稀释；
    1.2向淋巴细胞分离液上方缓慢加入等体积的稀释脐血，保持两液面界面清晰；
    1.3室温980g，离心20～30min；
    1.4离心结束后，吸取上面第二层的白膜状的单个核细胞层，用PBS清洗，得到脐血单个核细胞。
  3. 根据权利要求1或2所述的脐血NK细胞的体外扩增方法，其特征在于：所述步骤1)中的淋巴细胞培养基可为AIM
    Figure PCTCN2016081198-appb-100001
    Medium CTSTM(美国Life Technology公司商品)或GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司商品)，优选为GMP S&XFMTM-CD淋巴细胞培养基。
  4. 根据权利要求3所述的脐血NK细胞的体外扩增方法，其特征在于：所述步骤1)中的淋巴细胞培养基为
    Figure PCTCN2016081198-appb-100002
    Medium CTSTM，活化培养优选为：用
    Figure PCTCN2016081198-appb-100003
    Medium CTSTM淋巴细胞培养基调整细胞密度为1～3×106个/mL，再添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2，在36℃～38℃、5％ CO2饱和湿度环境中培养2～4天；活化培养更优为：用AIM
    Figure PCTCN2016081198-appb-100004
    Medium CTSTM淋巴细胞培养基调整细胞密度为2×106个/mL，再添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2，在37℃、5％ CO2饱和湿度环境中培养3天。
  5. 根据权利要求3所述的脐血NK细胞的体外扩增方法，其特征在于：所述步骤1)中的淋巴细胞培养基为GMP S&XFMTM-CD淋巴细胞培养基，活化培养优选为以下之一种：
    A.用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为1～3×106个/mL，再添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2，在36℃～38℃、5％ CO2饱和湿度环境中培养2～4天；活化培养更优为：用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为2×106个/mL，再添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2，在37℃、5％CO2饱和湿度环境中培养3天；
    B.用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为1～3×106个/mL，再添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2，1～100ng/mL(较好1～20ng/mL)重组人白介素-15和1～100ng/mL(较好1～20ng/mL)重组人白介素-18，在36℃～38℃、5％ CO2饱和湿度环境中培养2～4天；活化培养更优为：用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为2×106个/mL，再添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2、10ng/mL重组人白介素-15和10ng/mL重组人白介素-18，在37℃、5％ CO2饱和湿度环境中培养3天；
    C.用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为1～3×106个/mL，再添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2，1～100ng/mL(较好1～20ng/mL)重组人白介素-15和1～100ng/mL(较好1～20ng/mL)重组人白介素-18，在38.5℃～39.5℃、5％ CO2饱和湿度环境中培养0.5～1天；活化培养更优为：用GMP S&XFMTM-CD淋巴细胞培养基调整细胞密度为2×106个/mL，再添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2、10ng/mL重组人白介素-15和10ng/mL重组人白介素-18，在39℃、5％ CO2饱和湿度环境中培养1天。
  6. 根据权利要求1-5任一所述的脐血NK细胞的体外扩增方法，其特征在于：所述步骤2)中淋巴细胞培养基为GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司商品)，增殖培养脐血NK细胞优选为：用移液管将细胞液移至离心管中，150g，离心10min，弃上清，用GMP S&XFMTM-CD淋巴细胞培养基调整,细胞密度为0.5～2×106个/mL(更优选1×106个/mL)，再添加500～1500IU/mL(更优选1000IU/mL)重组人白介素-2，在37℃、5％ CO2饱和湿度环境中培养，每隔2～3天补充新鲜培养基调整细胞密度为0.5～2×106个/mL(更优选1×106个/mL)，并添加500～1500IU/mL(更优选1000IU/mL)重组人白介素-2，培养18～24天(更优选21天)，得到脐血NK细胞。
  7. 权利要求1至6任一项所述脐血NK细胞体外扩增方法中步骤1)使用的专用活化培养基，为以下组配之一：
    A组：添加1～10μg/mL唑来膦酸和200～2000IU/mL重组人白介素-2的AIM
    Figure PCTCN2016081198-appb-100005
    Medium CTSTM淋巴细胞培养基；
    优选为添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2的AIM
    Figure PCTCN2016081198-appb-100006
    Medium CTSTM淋巴细胞培养基；
    更优为添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2的AIM
    Figure PCTCN2016081198-appb-100007
    Medium CTSTM淋巴细胞培养基；或
    B组：添加1～10μg/mL唑来膦酸和200～2000IU/mL重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基；
    优选为添加1～5μg/mL唑来膦酸和500～1500IU/mL重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基；
    更优为添加2μg/mL唑来膦酸和1000IU/mL重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基；或
    C组：添加1～10μg/mL唑来膦酸、200～2000IU/mL重组人白介素-2、1～100ng/mL重组人白介素-15和1～100ng/mL重组人白介素-18的GMP S&XFMTM-CD淋巴细胞培养基；
    优选为添加1～5μg/mL唑来膦酸、500～1500IU/mL重组人白介素-2、1～20ng/mL重组人白介素-15和1～20ng/mL重组人白介素-18的GMP S&XFMTM-CD淋巴细胞培养基；
    更选为添加2μg/mL唑来膦酸、1000IU/mL重组人白介素-2、10ng/mL重组人白介素-15和10ng/mL重组人白介素-18的GMP S&XFMTM-CD淋巴细胞培养基；
  8. 权利要求1至6任一项所述脐血NK细胞体外扩增方法中步骤2)使用的专用增殖培养基，为添加200～2000IU/mL(优选1000IU/mL)重组人白介素-2的GMP S&XFMTM-CD淋巴细胞培养基。
  9. 用权利要求1-6任一所述方法体外扩增收获的纯度在90％以上的脐血NK细胞。
  10. 权利要求9所述脐血NK细胞在制备肿瘤免疫治疗药物或在肿瘤免疫治疗中 的应用。
  11. 一种脐血NK细胞的体外扩增试剂盒，主要包括以下试剂：
    1)脐血NK细胞的淋巴细胞分离液：北京京蒙高科干细胞技术有限公司商品，同医疗器械备案号：京海械备20150002号中的样本密度分离液，或市售其它淋巴细胞分离液；
    2)脐血NK细胞活化培养基：AIM
    Figure PCTCN2016081198-appb-100008
    Medium CTSTM培养基(美国Life Technology公司商品)或GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)，以及唑来膦酸、重组人白介素-2、重组人白介素-15和重组人白介素-18；
    3)脐血NK细胞增殖培养基：GMP S&XFMTM-CD淋巴细胞培养基(北京京蒙高科干细胞技术有限公司商品，同医疗器械备案号：京海械备20150008号中的细胞培养基)和重组人白介素-2。
PCT/CN2016/081198 2016-04-07 2016-05-06 脐血nk细胞的体外扩增方法及其试剂盒与应用 WO2017173696A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/090,258 US20190112577A1 (en) 2016-04-07 2016-05-06 Ex vivo expansion method for cord blood nk cell, and kit and application thereof
EP16897630.6A EP3441460B1 (en) 2016-04-07 2016-05-06 Ex vivo expansion method for cord blood nk cell, and kit and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610214351.1A CN107267454A (zh) 2016-04-07 2016-04-07 一种脐血nk细胞的体外扩增方法及其试剂盒与应用
CN201610214351.1 2016-04-07

Publications (1)

Publication Number Publication Date
WO2017173696A1 true WO2017173696A1 (zh) 2017-10-12

Family

ID=60000823

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/081198 WO2017173696A1 (zh) 2016-04-07 2016-05-06 脐血nk细胞的体外扩增方法及其试剂盒与应用

Country Status (4)

Country Link
US (1) US20190112577A1 (zh)
EP (1) EP3441460B1 (zh)
CN (1) CN107267454A (zh)
WO (1) WO2017173696A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110129267A (zh) * 2018-02-08 2019-08-16 上海细胞治疗集团有限公司 冻存人外周血单个核细胞的复苏液及方法
CN110607275A (zh) * 2019-08-20 2019-12-24 北京致仁生物科技有限公司 一种增强型自然杀伤细胞的培养方法
CN114015653A (zh) * 2021-12-15 2022-02-08 山东省齐鲁干细胞工程有限公司 一种冻存脐带血来源的nk细胞及其制备方法
CN114736858A (zh) * 2022-06-13 2022-07-12 广东先康达生物科技有限公司 一种脐带血nkt细胞的培养液及培养方法

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108165531A (zh) * 2017-12-23 2018-06-15 淮北智淮科技有限公司 一种造血干细胞用的脐带血单核细胞培养方法
CN109957543A (zh) * 2017-12-26 2019-07-02 上海尚泰生物技术有限公司 利用脐带血大量扩增脐血nk细胞的方法
JP7268039B2 (ja) 2018-02-01 2023-05-02 エヌケーマックス カンパニー リミテッド がん治療のためのナチュラルキラー細胞および組成物の製造方法
CN111073854A (zh) * 2020-01-23 2020-04-28 中科宝承生物医学科技有限公司 一种适用于脐血来源nk细胞的高效扩增工艺
CN111500535B (zh) * 2020-04-30 2023-04-14 惠和生物技术(上海)有限公司 用于体外培养人自然杀伤细胞的方法和培养基
CN111876389B (zh) * 2020-07-30 2023-09-08 广东康盾创新产业集团股份公司 一种扩增car-t细胞的方法
CN113151168B (zh) * 2021-04-21 2024-03-15 苏州科贝生物技术有限公司 一种人nk细胞培养体系及制备方法
WO2022240808A1 (en) * 2021-05-11 2022-11-17 Cytoimmune Therapeutics, Inc. Methodsand compositions for efficiently expanding cord blood nk cells
CN113293136B (zh) * 2021-05-24 2023-06-13 成都新生命霍普医学检验实验室有限公司 一种复苏细胞的方法及应用
CN113430168A (zh) * 2021-08-12 2021-09-24 山东省齐鲁干细胞工程有限公司 一种无血清培养冻存脐血nk细胞的方法及其试剂盒
CN114507640B (zh) * 2022-03-25 2023-09-08 和携科技有限公司 一种高增殖能力和高细胞毒性cik细胞的培养方法及其应用
WO2024102954A1 (en) 2022-11-10 2024-05-16 Massachusetts Institute Of Technology Activation induced clipping system (aics)
CN118048305A (zh) * 2024-04-16 2024-05-17 清泽医疗科技(广东)有限公司 一种nk细胞体外培养试剂盒及培养方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014053650A1 (en) * 2012-10-04 2014-04-10 Ab Science Use of masitinib for treatment of cancer in patient subpopulations identified using predictor factors
WO2014089169A2 (en) * 2012-12-04 2014-06-12 Oncomed Pharmaceuticals, Inc. Immunotherapy with binding agents
CN105112370A (zh) * 2015-08-25 2015-12-02 深圳市科晖瑞生物医药有限公司 一种体外刺激外周血γδT细胞高效增殖的方法及其应用
CN105462923A (zh) * 2014-12-17 2016-04-06 山东大学第二医院 一种人自然杀伤细胞体外高效扩增方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101077912B1 (ko) * 2008-10-24 2011-10-31 주식회사 메디셀 제대혈로부터 효율적인 자연살해세포의 증식 및 분화 방법
KR101679602B1 (ko) * 2009-09-11 2016-11-25 다카라 바이오 가부시키가이샤 내추럴킬러 세포의 제조방법
ES2627910T3 (es) * 2009-12-29 2017-08-01 Gamida-Cell Ltd. Métodos para potenciar la proliferación y la actividad de células destructoras naturales
WO2012009422A1 (en) * 2010-07-13 2012-01-19 Anthrogenesis Corporation Methods of generating natural killer cells
CN102533650B (zh) * 2011-12-30 2014-05-21 北京京蒙高科干细胞技术有限公司 一种细胞分离介质及细胞分离方法
US20160075996A1 (en) * 2013-03-27 2016-03-17 Biotherapy Institute Of Japan Method for producing nk cell-enriched blood preparation
CN104357391B (zh) * 2014-10-15 2017-12-26 恒瑞源正（深圳）生物科技有限公司 同时诱导扩增Vα24+iNKT细胞和CD3‑CD56+NK细胞的方法
CN104928243B (zh) * 2015-07-13 2019-01-18 山西大医院 实体瘤患者自体nk细胞分离、活化扩增及活性检测方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014053650A1 (en) * 2012-10-04 2014-04-10 Ab Science Use of masitinib for treatment of cancer in patient subpopulations identified using predictor factors
WO2014089169A2 (en) * 2012-12-04 2014-06-12 Oncomed Pharmaceuticals, Inc. Immunotherapy with binding agents
CN105462923A (zh) * 2014-12-17 2016-04-06 山东大学第二医院 一种人自然杀伤细胞体外高效扩增方法
CN105112370A (zh) * 2015-08-25 2015-12-02 深圳市科晖瑞生物医药有限公司 一种体外刺激外周血γδT细胞高效增殖的方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3441460A4 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110129267A (zh) * 2018-02-08 2019-08-16 上海细胞治疗集团有限公司 冻存人外周血单个核细胞的复苏液及方法
CN110129267B (zh) * 2018-02-08 2023-09-22 上海细胞治疗集团有限公司 冻存人外周血单个核细胞的复苏液及方法
CN110607275A (zh) * 2019-08-20 2019-12-24 北京致仁生物科技有限公司 一种增强型自然杀伤细胞的培养方法
CN110607275B (zh) * 2019-08-20 2021-06-15 北京致仁生物科技有限公司 一种增强型自然杀伤细胞的培养方法
CN114015653A (zh) * 2021-12-15 2022-02-08 山东省齐鲁干细胞工程有限公司 一种冻存脐带血来源的nk细胞及其制备方法
CN114736858A (zh) * 2022-06-13 2022-07-12 广东先康达生物科技有限公司 一种脐带血nkt细胞的培养液及培养方法
CN114736858B (zh) * 2022-06-13 2022-08-19 广东先康达生物科技有限公司 一种脐带血nkt细胞的培养液及培养方法

Also Published As

Publication number Publication date
EP3441460B1 (en) 2021-10-06
EP3441460A1 (en) 2019-02-13
CN107267454A (zh) 2017-10-20
EP3441460A4 (en) 2019-09-18
US20190112577A1 (en) 2019-04-18

Similar Documents

Publication Publication Date Title
WO2017173696A1 (zh) 脐血nk细胞的体外扩增方法及其试剂盒与应用
CN108588022B (zh) 体外培养富集人cd4+和cd8+ tcm细胞的方法
CN113151168B (zh) 一种人nk细胞培养体系及制备方法
JPH09511903A (ja) 樹状突起細胞を調製する方法、かくして得られる細胞および本方法を実施するための容器
CN104711225B (zh) Nk细胞的体外制备方法
US20210371820A1 (en) Method for amplifying antigen-specific regulatory t cells in vitro
CN111500535B (zh) 用于体外培养人自然杀伤细胞的方法和培养基
CN115558641B (zh) 高纯度效应免疫细胞群及其培养方法、试剂组合物和应用
KR20180117829A (ko) 세포밀도 조절을 이용한 자연살해세포의 대량증식방법
Condiotti et al. Ex vivo expansion of CD56+ cytotoxic cells from human umbilical cord blood
CN115651903B (zh) 高杀伤力的免疫细胞群及其培养方法、试剂组合物和应用
JPWO2020044538A1 (ja) ヒトリンパ球細胞培養用無血清培地
AU2020359872A1 (en) Engineered human immune cells, preparation method and application thereof
US20220396766A1 (en) Method for arterial endothelial-enhanced functional t cell generation
CN110607276A (zh) 高效扩增脐带血nk细胞的无血清培养方法
Blazsek et al. Large scale recovery and characterization of stromal cell-associated primitive haemopoietic progenitor cells from filter-retained human bone marrow
Frias et al. Generation of functional natural killer and dendritic cells in a human stromal-based serum-free culture system designed for cord blood expansion
CN113549595A (zh) 基于外周血的nk细胞体外培养方法
CN114402065A (zh) 低密度细胞培养
CN105106237A (zh) 一种高效杀伤肿瘤细胞生物制剂
CN108486055B (zh) 培养基及其在中央记忆型t淋巴细胞培养中的应用
CN111172110A (zh) 一种脐带血cik细胞的培养方法
CN110862962A (zh) 一种没食子酸在体外培养扩增nk细胞的方法
CN114438028B (zh) 一种体外扩增外周血nk的方法
Kjærgaard et al. Infiltration patterns of short‐and long‐term cultured A‐NK and T‐LAK cells following adoptive immunotherapy

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2016897630

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2016897630

Country of ref document: EP

Effective date: 20181107

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16897630

Country of ref document: EP

Kind code of ref document: A1