CN108220235A - A kind of Activated in Vitro expands people's natural kill（NK）The method of cell and its special cultivating system - Google Patents

A kind of Activated in Vitro expands people's natural kill（NK）The method of cell and its special cultivating system Download PDF

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CN108220235A
CN108220235A CN201710000748.5A CN201710000748A CN108220235A CN 108220235 A CN108220235 A CN 108220235A CN 201710000748 A CN201710000748 A CN 201710000748A CN 108220235 A CN108220235 A CN 108220235A
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郭子宽
王恒湘
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Cellbang (beijing) Biotechnology Co Ltd
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Abstract

Method and its special cultivating system the invention discloses a kind of Activated in Vitro amplification people's natural kill (NK) cell.The cultivating system includes cell activator and NK cell amplification culture mediums cell activator is used for the activation of NK cells, and NK cell amplification culture mediums are used for the amplification of NK cells.The method that the Activated in Vitro expands people's natural kill (NK) cell, it is using mononuclearcell as initiator cell, cell activator is added in culture vessel and carries out bed board, then it is inoculated with the mononuclearcell suspension made of the NK cell amplification culture mediums containing autologous plasma to culture vessel, amplification is cultivated under normal condition, you can obtain people's natural kill (NK) cell of high-purity.The cultivating system and Activated in Vitro amplification method of the present inventor's natural kill (NK) cell will play a significant role in the fields such as the basic research of immunotherapy of tumors, immunocyte, have application prospect.

Description

A kind of method of Activated in Vitro amplification people's natural kill (NK) cell and its special culture System
Technical field
The invention belongs to the cultural method and cultivating system of cell in cell and tissue culture field, more particularly to one kind The method of Activated in Vitro amplification people's natural kill (NK) cell and its special cultivating system.
Background technology
Natural killer cells (natural killer cells, abbreviation NK cell) refers to be present in peripheral blood, marrow, leaching Fawn on and other tissues in large granular lymphocyte, surface characteristics molecule be CD56, do not express CD3 (T lymphocytes One species specific antigen).
NK cells have unique immunoregulation capability and cytotoxicity, can be without sensitization (prior Sensitization it is quick to remove malignant cell and virus infected cell in the case of).Meanwhile NK cells do not have own cells Detailed cytotoxicity, this is mainly due to internal normal cells to express itself MHC-I class molecule, can be with NK cell surfaces Inhibitory receptor-killer cell immunoglobulin-like receptors (killer cell immunoglobulin-like Receptors, KIR) it combines, so as to inhibit NK cell activity, this phenomenon is referred to as the theory (self-missing that itself misses the target hypothesis).However, tumour cell and the usual low expression of the metainfective cell of virus or MHC-I class molecules are not expressed, this It cannot inhibit NK cell killing activities after a little cell contact NK cells, the NK cells so as to be activated are killed.
The cytotoxicity of NK cells is powerful, and a NK cell can be by discharging one individual of perforin and granzyme killing Product is more than the tumour cell of NK cell several times.Since NK cells have powerful tumor cytotoxicity activity, people think always NK cells are the important cells of immunosurveillance.2000, lancet (Lancet) magazine delivered the article of a Japanese scholars, They occupy the follow-up in 11 years of China Association for Promoting Democracy's row to 3625, it is found that the higher cancer incidence of NK cell activity is lower, and reaches statistics Learn significant difference (ImaiK, et al.Natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence:an 11-year follow-up study of a general population.Lancet(2000)356:1795–9)。
However, due to the presence of number of mechanisms, cancer patient has serious NK cell dysfunctions, in tumor microenvironment In (Platonova S, et al.Profound coordinated alterations of intra- even more so tumoral NK cell phenotype and function in lung carcinoma.Cancer Res 2011；71 (16):5412–22).Based on this, researchers devise the clinical trial protocol using NK cell therapy recurrent intractable tumours. But using self NK cell therapy malignant tumours, effect is not yet come to a conclusion.Some researches show that extract transfer melanoma Or the peripheral blood of clear-cell carcinoma patient, it detaches, purify and expand NK cells, the NK cells of activation are thin with very strong Vitro Tumor Born of the same parents kill ability, and the time-to-live is more than 1 week or even still had a large amount of NK to be transfused after 48 days NK cells in vivo after venoclysis Cell survival.However, reach 10 in each dosage of NK cells10When a above, 8 patients have therapeutic effect without 1 (Parkhurst MR,et al.Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression.Clin Cancer Res.2011；17(19):6287-97).To myeloma patient's autologous NK cells Functional analysis is carried out with allosome NK cells, it is found that autologous NK cells are weak to the toxic action of myeloma cell, allosome NK cells are to bone Strong (Szmania S, the et al.Ex Vivo Expanded Natural Killer Cells of the toxic action of myeloma cells Demonstrate Robust Proliferation In Vivo In High-Risk Relapsed Multiple Myeloma Patients.J Immunother.2015；38(1):24-36).Clinical examination is carried out to the NK cells of lung cancer patient Test research, result of study show to be transfused NK cells be it is safe, still, curative effect and historical control indifference (Yang YJ et al.A trial of autologous ex vivo-expanded NK cell-enriched lymphocytes with docetaxel in patients with advanced non-small cell lung cancer as second-or third-line treatment:phase IIa study.Anticancer Res.2013；33(5):2115-22).To leaching The NK cells of bar knurl and breast cancer patients carry out clinical experimental study, and result of study is also indicated that is treated using autologous NK cells, effect Fruit can not show a candle to expected (Burns LJ, et al.IL-2-based immunotherapy after autologous transplantation for lymphoma and breast cancer induces immune activation and cytokine release:a phase I/II trial.Bone Marrow Transplantation 2003；32(1): 177-186).The reason in part for above-mentioned phenomenon occur is, the inhibition receptors of NK cell surfaces can with tumor cell surface from Body HLA molecules combine, and so as to inhibit the cytotoxic activity of NK cells, and reduce ADCC effects (Szmania S, et al.Ex Vivo Expanded Natural Killer Cells Demonstrate Robust Proliferation In Vivo In High-Risk Relapsed Multiple Myeloma Patients.J Immunother.2015；38(1):24- 36).What is more important, in the therapeutic process of autologous NK cells, to achieve the purpose that internal cell amplification, often in infusion NK The IL-2 of large dosage is used after cell.However, under IL-2 effects, while NK cells expand, there is immunosuppressive action Regulatory T-cell (T regulatory cells, Tregs) ratio significantly increases (Littwitz-Salomon E et al.Activated regulatory T cells suppress effector NK cell responses by an IL- 2-mediated mechanism during an acute retroviral infection.Retrovirology.2015； 12(1):66；Skrombolas D et al.Challenges and developing solutions for increasing the benefits of IL-2treatment in tumor therapy.Expert Rev Clin Immunol.2014；10(2):207-17；Kerdiles Y,et al.T cell regulation of natural killer cells.J Exp Med.2013Jun 3；210(6):1065-8).NK cells to the cell that regulatory T-cell generates because Son is very sensitive, these factors include IL-10 and TGF-beta.Therefore, the increase of regulatory T-cell is seriously affected or even is inhibited The cytotoxicity of NK cells.This is also to treat tumour using IL-2 merely, in most cases the main reason for weak curative effect. Therefore, for the cellular immunotherapy of tumour, allosome NK cells may be better choice.
The complete clinical evidence of allosome NK cells control tumor development is published in 2002《Science》Magazine (Ruggeri L,et al.Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants.Science.2002；295(5562):2097-2100).By grinding Study carefully the relation of the NK cell KIR congruences between the effect of Allogeneic Hematopoietic Stem Cell Transplantation treatment leukaemia and donor-recipient Analysis, author have found that in appreciable 92 patients KIR is not harmonious patient (34), graft rejection incidence for 0, II degree with Upper GVHD incidences are 0, and acute myeloid leukemia (AML) patient is recurred for 5 years without 1.However, 58 patients being harmonious in KIR In, more than 10%, patient's AML recurrence rate is 75% for rate of rejection and II poison Yi Shang GVHD incidences.In addition, two groups of ALL diseases People's prognosis indifference.Result of study proves that alloreactive NK cell (allo-reactive NK cells, Allo-NK) can be with GVHD and graft rejection effect are reduced, and its anti-AML leukaemia cell's effect is the pass that hematopoietic stem cell transplantation cures leukaemia Key.Meanwhile more than result of study shows that curing certain tumours using Allo-NK cells has possibility.
From after Ruggeri etc. has found that NK cells heterologous lowers leukemia relapse phenomenon, from University of Minnesota Miller et al. first reported the research using Haploidentical Stem NK cell therapies AML, and result of study finds infusion NK cells It is safe, moreover, in 19 prognosis mala patients AML, 5 can reach complete incidence graph, illustrate with allosome NK cell therapies AML has validity (Miller JS et al.Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer.Blood.2005；105(8):3051-7).
After Haploidentical Hematopoietic Stem Cell Transplantation 2 weeks and 3 weeks, infusion donor NK cells mean dose up to 2.0 × 108/ kg weight does not find any serious cell infusion reaction, is compared, finds with the historical control through identical pretreatment GVHD incidences, hematopoiesis Implantation Time and transplant related mortality are without any difference, and still, NK cell infusions are that a prediction is white The independent factor of blood disease recurrence can significantly lower recurrence rate (Choi I, the et al.Donor-Derived of leukaemia Natural Killer Cells Infused after Human Leukocyte Antigen-Haploidentical Hematopoietic Cell Transplantation:A Dose-Escalation Study.BBMT 2014；20:696- 704)。
So, in hematopoietic stem cell transplantation, infusion Activated in Vitro expands the NK cells of (activation) or out of donor body The NK cells of fresh separated, which kind of cell can reach preferable therapeutic effectIn a prospective clinical trial is studied, Someone to receive the high risk leukemia patient of Haploidentical Stem transplanting as research object, patient after candidate stem cell is transplanted the 2nd, after 40 and 100 days, receive the NK cell infusions of donor source purifying.Result of study shows compared with historical control, is transfused donor The NK cells of source purifying are to graft rejection or recurrence without any advantage, and therefore, author proposes the NK of infusion Activated in Vitro amplification Cell may have preferable therapeutic effect (Stern M, et al.Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT:a prospective phase II study in two centers.BMT 2013；48:433-8).Some researches show that the not activated NK cells of infusion are to receptor Peripheral blood cells and cell factor have no effect；However, infusion IL-2 activation NK cells after, Evaluation of Cytokines in Peripheral Blood and Chemotactic factor (CF), such as IFN-gamma, IL-6, MIP-1b quickly increase, and peripheral blood NK cell quantity drastically declines, after 24 hours Restore normal.Result of study shows that the traveling locus of peripheral blood cells can be changed in the NK cells for being transfused Activated in Vitro amplification, promotes Process (Brehm C, the et al.IL-2 Stimulated but that infusion external source and host NK cell-penetratings pass through endothelial tissue Not Unstimulated NK Cells Induce Selective Disappearance of Peripheral Blood Cells:Concomitant Results to a Phase I/II Study.PLoS ONE 2011；6(11):e27351). To the progress functional analysis of peripheral blood in gastric cancer patients NK cells, it was also found that compared with static NK cells, the NK of Activated in Vitro amplification Cell has stronger Vitro Cytotoxicity, and can be obviously prolonged life span (the Mimura K, et of tumor-bearing mice al.Therapeutic potential of highly cytotoxic natural killer cells for gastric cancer.Int.J.Cancer.2014；135:1390-8).In addition, gamma/delta T cells and alpha/ with activation Beta T cells are compared, and the NK cells of Activated in Vitro amplification have stronger anti-tumor activity (Deng X, et al.Ex vivo-expanded natural killer cells kill cancer cells more effectively than ex vivo-expanded γ δ T cells or α β T cells.International Immunopharmacology 2014；22:486-91).
In conclusion NK cells are immune surveillance cells important in vivo, the allosome NK cells of Activated in Vitro amplification are returned It is defeated internal, there is certain meaning for treating certain tumor types.Cell therapy must reach certain quantity, moreover, carefully Born of the same parents' product reaches certain purity, can ensure validity and the safety for the treatment of.If remaining is larger amount of different in cell products Body T lymphocytes, by have cause graft versus host disease(GVH disease) occur risk (He F, Warlick E, Miller JS, et al.Lymphodepleting chemotherapy with donor lymphocyte infusion post- allogeneic HCT for hematological malignancies is associated with severe,but therapy-responsive aGvHD.Bone Marrow Transplant.2016；51(8):1107-12).
To realize effective amplification of NK cells, and ensure that the clinical practice safety of NK cells amplification product and batch are steady Qualitative, people often using serum free medium, add the autologous plasma of low concentration to promote NK cell Proliferations and maintain NK cells Activity.But the amplification system that different researchers use is different, operability, repeatability and the amplification of NK cells of experiment Quality and quantity it is also different.In the clinical test carried out earliest, people are often using in the method for immunomagnetic beads separation blood CD3-/CD56+ cells (NK cells) utilize serum free medium+autologous plasma+IL-2 or other cell factors, amplification later The NK cells of purifying.The NK cell purities that this method obtains are high, still, due to the supporting role of no other immunocytes, NK cells expandeds are not high.For example, after Miller etc. is using immunomagnetic beads screening CD3-/CD5- cells, using fluidic cell Instrument screens CD56+ cells, with DMEM/F12 (2:1) as basic culture medium, addition β mercaptoethanols, ethanol amine, vitamin C and Sodium selenite, using autologous plasma as nutritional supplement, the quantity of NK cells has only expanded 30 times of (Pierson after culture 35 days BA,McGlave PB,Hu WS,Miller JS.Natural killer cell proliferation is dependent on human serum and markedly increased utilizing an enriched supplemented basal medium.J Hematother.1995；4(3):149-58).More it should be noted that, immunomagnetic beads method purifying NK is thin Born of the same parents, cost is very high, and needs special instrument and equipment (Clini-MACS), is unfavorable for the clinical expansion of NK cell therapies.Cause This, this method with magnetic bead or flow cytometer screening NK cells, clinical value is little, gradually by biological therapy Researcher abandons.
Just because of this, at present, most researchers use mononuclearcell as initiator cell culture NK cells.NK cells Ratio wherein is about 10%, and NK cell proportions increase after culture in 2-3 weeks.Most researchers use K562 cells as taste Layer is supported, specific activation simultaneously expands NK cells.K562 cells are a kind of leukaemia cells, from a chronic myelogenous leukemia urgency The patient of property Transformation of Leukemia.K562 cells are not only frequently as the killing target cell of activated NK, and research also indicates that, NK cells It can be activated under K562 cytositimulations, especially transfect the K562 cells of film mating type IL-15 and 41BBL, NK cells can be stimulated Fast breeding (Shah NN, Baird K, Delbrook CP et al.Acute GVHD in patients receiving IL-15/4-1BBL activated NK cells following T-cell-depleted stem cell transplantation.Blood.2015；125(5):784-92；Kamiya T,Chang YH,Campana D.Expanded and Activated Natural Killer Cells for Immunotherapy of Hepatocellular Carcinoma.Cancer Immunol Res.2016；4(7):574-81).But tumour cell chemicotherapy act on or it is quiet Under state, largely (Mineo M, Garfield SH, the Taverna S, et of the excretion body containing tumour information nucleic acid can be discharged al.Exosomes released by K562 chronic myeloid leukemia cells promote angiogenesis in a Src-dependent fashion.Angiogenesis.2012；15(1):33-45), these are swollen Knurl information can be transferred to cell (the Ratajczak MZ, Ratajczak of surrounding by the form of horizontal transfer J.Horizontal transfer of RNA and proteins between cells by extracellular microvesicles:14 years later.Clin Transl Med.2016；5(1):7).What is more important has research Normal hematopoetic cells vicious transformation (Zhu X, You Y, Li Q, et can be made by showing the excretion body of K562 cell deriveds al.BCR-ABL1–positive microvesicles transform normal hematopoietic transplants through genomic instability:implications for donor cell leukemia.Leukemia 2014；28:1666-75).Therefore, using K562 cells as trophoderm, the safety especially long-term safety of clinical practice Property, it is the worth the problem of of discussing.
Also there is researcher by the use of mononuclearcell as initiator cell, cultivate Human peripheral blood NK cells.In this art, With the anti-CD3antibody of low dosage and the antihuman CD 52 antibody bed board of saturated dose, cause NK cells in mononuclearcell group Activation, the NKGM-1 culture mediums amplification produced later with Japan Kohjin Bio companies, NK cell numbers can be the of culture Expand 800-1000 times within 14 days.But NK cell proportions are average in the cell mass obtained only accounts for about 60%, Just because of this, It is 1 in effect target ratio:In the case of 1, cell finished product averagely only has 60% (36.3%-87.3%) to the killing rate of K562 cells (Masuyama J,Murakami T,Iwamoto S,Fujita S.Ex vivo expansion of natural killer cells from human peripheral blood mononuclear cells co-stimulated with anti- CD3and anti-CD52 monoclonal antibodies.Cytotherapy.2016；18(1):80-90).
Therefore, activation and amplification that new cultivating system carries out NK cells are explored, with ensure in acquisition cell mass NK it is thin Born of the same parents are at high proportion and NK cells against tumor cells have stronger killing activity, so as to ensure NK cell therapy clinical practices Validity, be a project for still needing to conscientiously study.
Invention content
First purpose of the present invention is to provide a kind of culture for Activated in Vitro amplification people's natural kill (NK) cell System.
Cultivating system provided by the present invention for Activated in Vitro amplification people's natural kill (NK) cell swashs including cell Agent living and NK cell amplification culture mediums, the cell activator include：(1) anti-human CD16 monoclonal antibodies, (2) macrophage swash Lipopeptid -2 (macrophage activating lipopeptide-2, MALP-2) and (3) living is dissolved anti-human CD16 monoclonals and is resisted The buffer solution of body and macrophage activation lipopeptid -2 (MALP-2)；The NK cell amplification culture mediums include：1) basal medium, 2) free serum culture based additive, 3) cyclooxygenase 2 (COX-2) inhibitor and 4) cell factor.
Wherein：In cell activator, a concentration of 0.5-5mg/L of use of the anti-human CD16 monoclonal antibodies (recommends A concentration of 1-2mg/L, preferred concentration 1mg/L)；A concentration of 1- of use of the macrophage activation lipopeptid -2 (MALP-2) 100mg/L (recommended density 5-25mg/L, preferred concentration 10mg/L)；It is described to dissolve anti-human CD16 monoclonal antibodies and macrophage The buffer solution of cell-stimulating lipopeptid -2 (MALP-2) is phosphate buffer, Tris-CL-NaCL solution or it is other without EDTA or The buffer solution of other chelating agents, the pH value of buffer solution are 7.0-7.5 (recommendation pH value be 7.1-7.3, preferable ph 7.2).
Three kinds of components in the cell activator can separate storage (depositing in respectively in different containers) or mixing is deposited It puts.
In NK cell amplification culture mediums：The basal medium be selected from RPMI1640 culture mediums, IMDM culture mediums, The combination (preferably RPMI1640 culture mediums) of one kind or two of which or three kinds of basal mediums in DMEM/F12 culture mediums；Institute It states free serum culture based additive and includes NaHCO3, human albumin, actrapid monotard, human transferrin, sodium selenite, ethanol amine, Lipid mixtures (such as mixing of linoleic acid, leukotrienes, oleic acid, cholesterol), biotin, Sodium Pyruvate, hydroxyethyl piperazine second Thiosulfonic acid, L-Glutamine, polyamine mixture (such as mixing of putrescine, cadaverine, spermidine, spermine), hydrocortisone amber A variety of in sour sodium, essential amino acid (such as lysine, tryptophan) and nonessential amino acid (such as glutamic acid, alanine) or All；Wherein albumin, insulin, transferrins, selenium, lipid mixtures, essential amino acid and nonessential amino acid are required Ingredient, biotin, Sodium Pyruvate, hydroxyethyl piperazine second thiosulfonic acid, L-Glutamine and polyamine mixture is promote cell Proliferation Additive, hydrocortisone sodium succinate is to cultivate the Cord Blood-Derived NK cells specialist additive that is；The cyclooxygenase 2 (COX-2) inhibitor is (excellent for one kind in Indomethacin (indomethacin), NS-398 and celecoxib (celecoxib) Indomethacin is selected as, a concentration of 0.5 μM -50 μM of Indomethacin, recommended density is 1-20 μM, and preferred concentration is 5 μM)；It is described Cell factor is including at least two in IL-2, IL-15, IL-12 and IL-18, it is preferable to use IL-2 and IL-15 cell factor groups It closes, a concentration of the 2 × 10 of IL-25-3×106U/L (200U/mL-3000U/mL), preferably 1 × 106U/L (1000U/mL), A concentration of 10-100mg/L (10-100ng/mL) of IL-15, preferably 50mg/L (50ng/mL).
Commercialized combination culture medium, such as EX VIVO series of products, NKGM-1, AIM-V, it is added in culture medium The NK cells such as insulin, transferrins, selenium amplification needed nutrient matter, is also suitable for the amplification of NK cells.
Using RPMI1640 culture mediums as basic culture medium, every liter of (/L) NK cell expands general NK cell amplification culture mediums Increase culture medium to contain：RPMI1640 pulvis 10.39g, NaHCO32g, human albumin 0.5-5g, Insulin-Transferrin-selenium mix Close object 1-10mL, ethanol amine 1-10 × 10-4M, lipid mixtures 1-10mL, biotin 1-20mg, Sodium Pyruvate 55-220mg, hydroxyl Ethyl piperazidine second thiosulfonic acid 1-20mM, L-Glutamine 146-730mg, polyamine mixture 1-10mL, RPMI1640 amino acid are molten Liquid 1-20mL, 0.5-10 μM of Indomethacin, IL-2 2 × 105-3×106U,IL-15 10-100mg。
The formula of Human peripheral blood NK cells amplification culture medium is preferably：Every liter of (/L) culture medium 10.39g containing RPMI1640, NaHCO32.04g, human albumin 2g, Insulin-Transferrin-selenium mixture 10mL, ethanol amine 6 × 10-4M, lipid mixtures 1mL, biotin 10mg, Sodium Pyruvate 110mg, hydroxyethyl piperazine second thiosulfonic acid 2mM, L-Glutamine 292mg, polyamine mixture 1mL, RPMI1640 amino acid solution 20mL, 5 μM of Indomethacin, IL-2 1 × 106U,IL-15 50mg。
The formula of people's Cord Blood Natural Killer Cells: Impact amplification culture medium is preferably：Every liter of (/L) culture medium 10.39g containing RPMI1640, NaHCO32.04g, human albumin 2g, Insulin-Transferrin-selenium mixture 10mL, ethanol amine 6 × 10-4M, lipid mixtures 1mL, biotin 10mg, Sodium Pyruvate 110mg, hydroxyethyl piperazine second thiosulfonic acid 2mM, L-Glutamine 292mg, polyamine mixture 1mL, RPMI1640 amino acid solution 20mL, injection hydrocortisone sodium succinate 1 × 10-6M, 5 μM of Indomethacin, IL-2 1 ×106U,IL-15 50mg。
Second object of the present invention is to provide a kind of method of Activated in Vitro amplification people's natural kill (NK) cell.
The method of Activated in Vitro amplification people's natural kill (NK) cell provided by the present invention, uses aforementioned culture body System is using isolated mononuclearcell as initiator cell, first adds in culture vessel the cell activator and carry out Then bed board is hanged to culture vessel inoculation mononuclearcell made of the NK cell amplification culture mediums containing autologous plasma Liquid cultivates cell, obtains people's natural kill (NK) cell of activated amplification.
In this method：The NK cell deriveds are in the mononuclearcell of people's marrow, peripheral blood or umbilical vein blood (bleeding of the umbilicus)；
Three kinds of components in the cell activator can be used separately or be used in mixed way；Three kinds of components are separated into bed board method For：With isopropanol dissolving macrophage activation lipopeptid -2 (MALP-2), it is dense to macrophage activation lipopeptid -2 that buffer solution is added dropwise It spends for 1-100mg/L (recommend 1-2mg/L, preferably 1mg/L), then -2 solution of macrophage activation lipopeptid is added in into culture vessel In, solution is made to be laid in bottom of culture vessel, in 37 DEG C stand more than 1 hour (preferably 1-2 hours) or 4 DEG C standing 2 hours with It is upper to use (or non-at-scene be used in 4 DEG C of storages, use in 3 weeks), then -2 solution of absorption macrophage activation lipopeptid, with buffering Liquid washing bottom of culture vessel is unadsorbed in macrophage activation peptiolipid -2 of bottom of culture vessel to remove, and adds anti-human A concentration of 0.5-5mg/L of CD16 monoclonal antibodies and buffer solution to anti-human CD16 monoclonal antibodies (recommends 5-25mg/L, preferably 10mg/L), culture vessel is placed in 37 DEG C and stands 1 hour or more by minimum additive amount to ensure solution confluent cultures container bottom (preferably 1-2 hours) or 4 DEG C of standings 2 hours or more, it is non-at-scene to be used in 4 DEG C of storages, used in 3 weeks；
It is by the method that the cell activator of three kinds of components mixing carries out bed board：NK cell activators are added in into culture vessel In, minimum additive amount stands 1 hour or more (preferably 1-2 hours) to ensure that solution is paved with entire bottom of culture vessel, in 37 DEG C Or 4 DEG C of standings 2 hours or more, it is non-at-scene to be used in 4 DEG C of storages, used in 3 weeks；
The cell density of the mononuclearcell suspension is 0.5-10 × 106/ mL, is recommended as 1-5 × 106/ mL, preferably 2 ×106/mL；The adding proportion of autologous plasma is 1-20% (V/V), preferably 10% (V/V)；
The condition of culture of the culture cell is 37 DEG C, 5%CO2, 95% air, 100% humidity, incubation time 10 My god -21 days, recommend 10-18 days, preferably 14 days；It is preferred that gradually expanding culture scale and increasing incubation time, expand culture scale Method be addition containing 1-20% (preferably 10%, V/V) autologous plasma NK cell amplification culture mediums, incubation time is according to cell Quantity and growth conditions are increased；Using people's natural kill (NK) cell of the method harvest activation amplification of conventional centrifugal.
The method of specific Activated in Vitro amplification people's natural kill (NK) cell, includes the following steps：
1) the isolated mononuclearcell from peripheral blood or bleeding of the umbilicus；
2) cell activator is added in culture vessel and carries out bed board：
By the separated cell activator of three kinds of components carry out bed board or
The cell activator that three kinds of components are mixed carries out bed board；
3) to the Human peripheral blood NK cells amplification culture medium of the culture vessel inoculation containing 1-20% (V/V) autologous plasma Or mononuclearcell suspension made of people's Cord Blood Natural Killer Cells: Impact amplification culture medium, in 37 DEG C, 5%CO2, 95% air, 100% - 21 days 10 days (recommending 10-18 days, preferably 14 days) is cultivated under damp condition, obtains the warp of high-purity (purity is up to 60-94%) Activate people's natural kill (NK) cell of amplification.
People's natural kill (NK) cell of Activated in Vitro amplification also belongs to the content of present invention in aforementioned manners.
The present invention provides a kind of cultivating systems for Activated in Vitro amplification people's natural kill (NK) cell.The culture body System includes cell activator and NK cell amplification culture mediums, and cell activator is used for the activation of NK cells, NK cell amplification cultivations Base is used for the amplification of NK cells.The cultivating system has the following advantages：
1st, specific chemical components are clear (chemically defined), avoid the difference between cultivating system batch, from And it ensure that the stability between batch；
2nd, without heterologous protein ingredient (animal protein molecule, such as cow's serum, bovine serum albumin(BSA), ox blood fat eliminating egg It is white etc.), exempt from so as to avoid human body caused by possible zoonosis in cell output aggregate clinical practice and animal protein The risk of epidemic disease reaction；
3rd, be applicable not only to the culture of the NK cells of human peripheral or derived from cord blood, be also applied for other tissues such as marrow or The culture of other animal NK cells.
The present invention also provides it is a kind of with above-mentioned cultivating system Activated in Vitro amplification people's natural kill (NK) cell method, This method has the following advantages：
1st, using the mononuclearcell of healthy human peripheral blood or umbilical vein blood as initiator cell, without pure before culture Change NK cells, you can complete the activation amplification procedure of NK cells；
2nd, trophoderm is not added in cultivating system, the NK cell purities of acquisition are high, active good：Under normal circumstances, by 2 weeks After activation amplification, NK cell proportions can be increased to more than 60% by 5%-15%, and therefore, the activation amplification procedure of NK cells is also The purification process of NK cells；
3rd, after overactivation expands, NK cell quantities can improve nearly 2000 times or more；
4th, the NK cells after overactivation expands have very strong K562 cell killing activities, and killing rate is up to 90%.
In conclusion the cultivating system and Activated in Vitro amplification method of the present inventor's natural kill (NK) cell, it will be swollen Knurl treatment and the basic research of immunocyte etc. play a significant role in fields, have a extensive future.
The present invention is described in further details with reference to specific embodiment.
Description of the drawings
Fig. 1 is the technology path that Activated in Vitro expands people's natural kill (NK) cell.Macrophage activation lipopeptid -2 and NK Cell specific antibody (anti-human CD16 monoclonal antibodies) pre- bed board, two kinds of molecule in close be incorporated into culture bottle (or culture dish or Culture bag) bottom surface.After adding in mononuclearcell, Monocytes/Macrophages and NK cells are activated, NK cell advantage pcrs, number Amount and ratio gradually rise.
Fig. 2 is the cellular morphology of human peripheral blood single nucleus cell culture 48 hours.By human peripheral blood single nucleus cell culture After 48 hours, it is seen that adherent cell colony.By colony, there are a large amount of spindle cell (Monocytes/Macrophages, arrow institute Show).Indicate size：100μm.
Fig. 3 is the form of colony that human peripheral blood single nucleus cell culture is formed after 72 hours.Scale：100μm.
Fig. 4 is before human peripheral blood single nucleus cell culture and the flow cyctometry analysis knot of 14 days NK cell proportions of culture Fruit.Human peripheral blood single nucleus cell is collected, is activated using the NK activator in embodiment, uses the described culture medium of embodiment Culture 14 days.Flow cyctometry is analyzed after antibody label.NK cell proportions are increased to 84% from 8.67% before activation amplification.
Fig. 5 is NK cells (killing cell controls) and Activated in Vitro expand (14 days) from human peripheral blood single nucleus cell NK cells (experimental cell) to the killing rate measurement results of K562 cells (leukaemia cell).Before mononuclearcell culture (ctr) and after culture 14 days, according to different effect targets than measuring killing rate of the cell to K562 leukaemia cell.Ordinate：It kills Hinder rate (%), abscissa：Imitate target ratio.
Fig. 6 is the cellular morphology of Human cord blood mononuclear cells culture 48 hours.By Human cord blood mononuclear cells, according to aforementioned After method culture 48 hours, it is seen that adherent cell colony.By colony, have a large amount of spindle cell (Monocytes/Macrophages, Shown in asterisk).Indicate size：100μm.
Fig. 7 is the form of colony that Human cord blood mononuclear cells culture is formed after 72 hours.Scale：100μm.
Fig. 8 be Human cord blood mononuclear cells culture before and culture 14 days NK cell proportions flow cyctometry analysis result. Human cord blood mononuclear cells are collected, is activated using the NK activator in embodiment, uses the described medium culture of embodiment 14 days.Flow cyctometry is analyzed after antibody label.NK cell proportions are increased to 94.5% from 7.22% before activation amplification.
Fig. 9 is NK cells (killing cell controls) and Activated in Vitro amplification (14 days) from Human cord blood mononuclear cells NK cells (experimental cell) are to the killing rate measurement result of K562 cells (leukaemia cell).Before mononuclearcell culture (ctr) And after cultivating 14 days, according to different effect targets than measuring killing rate of the cell to K562 leukaemia cell.Ordinate：Killing rate (%), abscissa：Imitate target ratio.
Specific embodiment
Monocytes/Macrophages are the special immunocytes of a group, compared with other types of immunocyte, monokaryon/macrophage Cell is subjected to a variety of stimulations, and the various cell factor of secreting function and other bioactive substances.In addition, monokaryon/macrophage Another biological property of cell is that it has very strong adherent performance.Mononuclearcell is mainly by Monocytes/Macrophages, T The compositions such as lymphocyte, bone-marrow-derived lymphocyte, NK cells, NKT cells, the adherent ability of Monocytes/Macrophages is most in these cells By force.
NK cell deriveds used in the present invention are in the mononuclearcell of people's marrow, peripheral blood or bleeding of the umbilicus.
Based on this, inventor devises following technology path, and amplification NK cells are activated by initiator cell of mononuclearcell. (1) in Initial stage of culture, the NK cells in mononuclearcell are specifically activated, the advantage activation of NK cells are formed, also that is, comparing In other cells, the reactivity of NK cells nonspecific cell factor such as IL-2 to target cell of activation is thin higher than other types Born of the same parents' such as T lymphocytes.(2) cell factor of high dose is added in, the NK cells of activation is made to enter the amplification stage, form NK cells Advantage pcr, finally making NK cells, ratio sum number amount is significantly improved while activation.
Specific technology path is as shown in Figure 1：
The first step：With the anti-human CD16 monoclonal antibodies bed board (coating) of the specific antibody of NK cells, meanwhile, it will swash Macrophage activation lipopeptid -2 (macrophage the activating lipopeptide-2, MALP- of Monocytes/Macrophages living 2) bed board, two kinds of molecule in close are incorporated into culture bottle (or culture dish or culture bag) bottom surface.
Second step：Mononuclearcell is added in, the Monocytes/Macrophages quick wall attaching in cell mass, and it is thin in local macrophage Born of the same parents are activated under the effect of lipopeptid -2, largely secrete the cell factors such as IL-12, IL-15, TNF-alpha.Meanwhile the NK in cell mass Cell is combined with the anti-human CD16 monoclonal antibodies of culture bottle bottom.At this point, a cell culture is formd in culture bottle bottom Microenvironment, in this microenvironment, NK cells and Monocytes/Macrophages close contact carry out iuntercellular dialogue (cross- Talk), the cell factor and anti-human CD16 monoclonal antibodies (NK cell specific antibodies) that NK cells are secreted in Monocytes/Macrophages Stimulation is lower to be activated, and is finally completed the activation process of NK cells.
Third walks：The NK cells of activation exogenous cytokines IL-15 and IL-2 stimulation under rapid amplifying, with culture when Between extension, the ratios of NK cells is continuously improved, and the sum of NK cells is continuously increased, so as to complete the amplification procedure of NK cells.
The present invention expands the cultivating system of people's natural kill (NK) cell for Activated in Vitro, including cell activator and NK Cell amplification culture medium, cell activator are used for the activation of NK cells, and NK cell amplification culture mediums are used for the amplification of NK cells, institute Cell activator is stated to include：(1) anti-human CD16 monoclonal antibodies, -2 (macrophage of (2) macrophage activation lipopeptid Activating lipopeptide-2, MALP-2) and (3) dissolve anti-human CD16 monoclonal antibodies and macrophage activation fat The buffer solution of peptide -2；The NK cell amplification culture mediums include：1) basal medium, 2) free serum culture based additive, 3) ring (COX-2) inhibitor of oxidizing ferment 2 and 4) cell factor.
Cell activator
In cell activator, the effect of each component and content are as follows：
(1) anti-human CD16 monoclonal antibodies：NK cells are the special lymphocytes of a group, characteristic marker for CD3-/ CD56+.According to CD56 expressions, NK cells can be divided into CD56 low expressions (CD56dim) and CD56 high expression (CD56bri) two Subgroup.CD56dimCell height expresses CD16, has the function of very strong killing tumor cell and virus infected cell, also that is, tool There is very strong cytotoxicity.CD56briCell low expression CD16, major function are the cells such as secretion gamma interferon (IFN-γ) The factor.Before cell culture, using anti-human CD16 monoclonal antibodies bed board, antibody can advantage combination CD56briCell, so as to make training Supporting the cell activated in the process has stronger killing activity.Monoclonal antibody bed board is routine techniques, anti-human CD16 monoclonals A concentration of 0.5-5mg/L of use (0.5-5 μ g/mL) of antibody, recommended density are 1-2mg/L (1-2 μ g/mL), and preferred concentration is 1mg/L(1μg/mL)。
(2) macrophage activation lipopeptid -2 (macrophage activating lipopeptide-2, MALP-2)： MALP-2 is a mycoplasma species outer membrane substance, can pass through TLR-2 (the Toll-like receptor- on Monocytes/Macrophages surface 2, Toll-like receptor 2) and TLR-6 (Toll-like receptor 6) activated mononuclear/macrophage, it is promoted to secrete IL-12, IL-15, TNF- The cell factors such as alpha.Wherein, cell factor necessary to IL-15 is NK cell developments knocks out nothing in IL-15 DNA murine bodies NK cells.TLR-2 signals and the adaptin of domain containing the TIR (TIR domain-containing in Monocytes/Macrophages Adapter protein, TIRAP) it is related, the latter is the key that activation IL-15 gene expression adaptins.MALP-2's is another A biological characteristics are high-adhesiveness.The present invention is exactly to utilize these features, before people's mononuclearcell is cultivated, MALP-2 is pre- Bed board.Monocytes/Macrophages quick wall attaching in mononuclearcell is stimulated secretion IL-12, IL-15, TNF- by MALP-2 The cell factors such as alpha, the NK cells in mononuclearcell also fall to culture bottle/ware/bag under the effect of anti-human CD16 antibody Bottom so as to receive the cytokine profiles stimulation of Monocytes/Macrophages secretion, achievees the purpose that activated NK.Macrophage is thin Born of the same parents activate a concentration of 1-100mg/L of use (1-100 μ g/mL) of lipopeptid -2 (MALP-2), and recommended density is 5-25mg/L (5-25 μ g/mL), preferred concentration is 10mg/L (10 μ g/mL).
(3) buffer solution of anti-human CD16 monoclonal antibodies and macrophage activation lipopeptid -2 (MALP-2) is dissolved：Can be Phosphate buffer or Tris-CL-NaCL solution can also use other buffer solutions without EDTA or other chelating agents.No Same buffer solution does not influence the adhesion effect of people CD16 monoclonal antibodies and macrophage activation lipopeptid -2 (MALP-2), but slow The pH value of fliud flushing influences the process that protein molecule is adhered to culture bottle/ware/bag, and therefore, the pH value of buffer solution is 7.0-7.5, Recommendation pH value be 7.1-7.3, preferable ph 7.2.Anti-human CD16 monoclonal antibodies can be directly dissolved in buffer solution, and macrophage is thin Born of the same parents activate lipopeptid -2 (MALP-2) that can first be dissolved in isopropanol, then diluted with buffer solution.
Three kinds of components in cell activator can separate storage (depositing in respectively in different containers) or mixed storage.It can It is now with the current can also prepare after store for future use.
NK cell amplification culture mediums
In NK cell amplification culture mediums, the effect of each component and content are as follows：
1) basal medium：It is trained for the amplification of NK cells, such as RPMI1640 culture mediums, IMDM culture mediums or DMEM/F12 Base etc. is supported, the combination culture NK cells of above two or three kinds of basal mediums can also be used.The basal medium is preferred For RPMI1640 culture mediums.
2) when using above-mentioned basal medium, the NK such as insulin, transferrins, selenium cells amplification institute is also added in the present invention Nutriment is needed, so as to reach preferable expanding effect.Free serum culture based additive is the nutrients needed for the amplification of NK cells Matter, including NaHCO3, human albumin, actrapid monotard, human transferrin, sodium selenite, ethanol amine, lipid mixtures, biotin, Sodium Pyruvate, hydroxyethyl piperazine second thiosulfonic acid, L-Glutamine, polyamine mixture, injection hydrocortisone sodium succinate, must Need amino acid and nonessential amino acid etc. therein a variety of or whole, wherein albumin, insulin, transferrins, selenium, lipid are mixed It is required ingredient to close the components such as object, essential amino acid and nonessential amino acid, needs to add when cultivating Cord Blood-Derived NK cells Hydrocortisone sodium succinate, biotin, Sodium Pyruvate, hydroxyethyl piperazine second thiosulfonic acid, L-Glutamine, polyamine mixture Addition can promote the proliferation of cell, obtain enough cell numbers to ensure, can add above-mentioned substance.
NaHCO3Effect be to maintain the acid-base value of solution；The effect of human albumin is transhipment nutriment to intracellular； Actrapid monotard's effect is stimulation glycometabolism, contributes to cell growth and proliferation, improves the vigor of cell and the yield of protein；People The effect of transferrins is transhipment iron plasma；The effect of sodium selenite is to provide selenium element, and with antioxidation；Ethyl alcohol The effect of amine is to provide the lipid material precursor of cell Proliferation needs；Lipid mixtures (such as linoleic acid, leukotrienes, oleic acid, courage The mixing of sterol) effect be to maintain the normal lipid metaboli of cell；The effect of biotin (biotin, biotin) is helper cell Fat, sugar and amino acid metabolism；The effect of Sodium Pyruvate is to provide carbon source for cell；The effect of Hepes is to adjust the soda acid of solution Degree；The effect of L-Glutamine is to provide cell nucleic acid and protein essential amino acid；Polyamine mixture (such as it is rotten The mixing of amine, cadaverine, spermidine, spermine) effect be polyamines can by with ligand and film egg in DNA, RNA, various cytoplasms White interaction, promotes cell Proliferation；The effect of injection hydrocortisone sodium succinate is to promote hematopoietic cell to NK cells Differentiation, is the substance added in derived from cord blood NK cell culture；Essential amino acid (such as lysine, tryptophan) is that body cannot The actively amino acid of synthesis, effect are to provide the amino acid needed for cell Proliferation；Nonessential amino acid (such as glutamic acid, the third ammonia Acid etc.) effect be to provide amino acid needed for cell Proliferation.
1) and 2) commercialized combination culture medium, such as EX VIVO series of products, NKGM-1, AIM-V in addition, contained, The NK such as insulin, transferrins, selenium cells amplification needed nutrient matter has been added in culture medium, has been also suitable for NK cells Amplification.
3) cyclooxygenase 2 (COX-2) inhibitor：It is to offset macrophage activation lipopeptid -2 (MALP-2) to stimulate list that it, which is acted on, The PGE-2 of core/macrophages secrete is to the inhibiting effect of NK cells.Existing data has confirmed, macrophage activation lipopeptid- 2 (MALP-2) can improve the activity of cyclooxygenase 2 (COX-2), increase prostaglandin E2 (PGE-2) in culture cell conditioned medium Concentration (Mitsunari M, et al.Macrophage-activating lipopeptide-2 induces cyclooxygenase-2 and prostaglandin E(2)via toll-like receptor 2 in human placental trophoblast cells.J Reprod Immunol.2006；72:46-59).PGE-2 is a kind of with very The small-molecule substance of strong inhibition NK cell activity.Therefore, to retain Monocytes/Macrophages secretion moderate stimulation NK cell activations Activity, while eliminate restraining factors therein, cox 2 inhibitor added in cultivating system.A variety of cox 2 inhibitors all have There are similar function, such as Indomethacin (indomethacin), NS-398 and celecoxib (celecoxib), preferably Yin Diindyl U.S. is pungent, a concentration of 0.5 μM -50 μM of Indomethacin, and recommended density is 1-20 μM, and preferred concentration is 5 μM.
4) cell factor is including IL-2, IL-15, IL-2+IL-15, IL-12, IL-18 etc., effect be by with cell table The receptor in face combines, and promotes cell Proliferation.These cell factors can be combined or are used alone, and it is thin can to reach effectively amplification NK The purpose of born of the same parents.Present invention preferably uses IL-2 and IL-15 (IL-2+IL-15) combination of cytokines, IL-2 a concentration of 2 × 105-3×106U/L (200U/mL-3000U/mL), preferably 1 × 106A concentration of 10- of U/L (1000U/mL), IL-15 100mg/L (10-100ng/mL), preferably 50mg/L (50ng/mL).
It is described in detail with reference to embodiments.Method therefor is conventional method unless otherwise instructed in embodiment.
Percent concentration of the present invention is that mass/mass (W/W, unit g/100g) percentage is dense unless otherwise instructed Degree, mass/volume (W/V, unit g/100mL) percent concentration or volume/volume (V/V, Unit/mL/100mL) percentage are dense Degree.
The acquirement approach of various biomaterials described in embodiment is only to provide a kind of approach for testing acquisition to reach To specifically disclosed purpose, the limitation to biological material source of the present invention should not be become.In fact, used biomaterial Source be it is extensive, it is any keep on the right side of the law the biomaterial that can be obtained with moral ethics can be according to carrying in embodiment Show and be replaced.
Embodiment is being implemented down based on the technical solution of the present invention, gives detailed embodiment and specific Operating process, embodiment will be helpful to understand the present invention, but protection scope of the present invention is not limited to following embodiments.
Embodiment 1 expands people's natural kill (NK) cell with human peripheral blood single nucleus cell Activated in Vitro
NK cell deriveds used in the present embodiment are in the mononuclearcell of human peripheral.
First, the cultivating system of Activated in Vitro amplification people's natural kill (NK) cell
For the cultivating system of Activated in Vitro amplifying NK cells of human beings, including NK cell activators and NK cell amplification cultivations Base, specific formula are as follows：
1st, the formula of human peripheral blood cell's activator：Every liter of (/L) activator contains：
(1) (0.5-5mg is equal by 1mg for anti-human CD16 monoclonal antibodies (being purchased from Beijing Tong Lihai sources Bioisystech Co., Ltd) Can, recommended density 1-2mg)；
(2) macrophage activation lipopeptid -2 (MALP-2) (is the U.S. purchased from Beijing Abace Biotechnology Co., Ltd. Creative Peptides products) 10mg (1-100mg, recommended density 5-25mg)；
(3) solvent is 0.01M Tris-CL-NaCL solution.Tris-CL-NaCL solution contains Tris alkali 1.21g/L, NaCl 9g/L adjust pH value to 7.2 (7.0-7.5, recommendation pH value are 7.1-7.3) with HCl.
Above-mentioned three kinds of reagents are purchased from Chinese Sinopharm Chemical Reagent Co., Ltd..Three kinds in the cell activator Component can separate storage (depositing in respectively in different containers) or mixed storage.
2nd, the formula of general NK cell amplification culture mediums, using RPMI1640 culture mediums as basic culture medium (according to The method of specification prepares RPMI1640 culture mediums), as shown in table 1, every liter of (/L) NK cell amplification culture mediums addition RPMI1640 pulvis 10.39g, NaHCO3In addition 2g adds human albumin 0.5-5g, Insulin-Transferrin-selenium mixture 1-10mL, ethanol amine 1-10 × 10-4M, lipid mixtures 1-10mL, biotin 1-20mg, Sodium Pyruvate 55-220mg, ethoxy Piperazine second thiosulfonic acid 1-20mM, L-Glutamine 146-730mg, polyamine mixture 1-10mL, RPMI1640 amino acid solution 1- 20mL, injection hydrocortisone sodium succinate 1-10 × 10-6M, 0.5-10 μM of Indomethacin, IL-2 2 × 105-3×106U, IL-15 10-100mg。
The formula of Human peripheral blood NK cells amplification culture medium is preferably：Every liter of (/L) culture medium contains：RPMI1640 pulvis 10.39g NaHCO32.04g, human albumin 2g, Insulin-Transferrin-selenium mixture 10mL, ethanol amine 6 × 10-4M, fat Class mixture 1mL, biotin 10mg, Sodium Pyruvate 110mg, hydroxyethyl piperazine second thiosulfonic acid 2mM, L-Glutamine 292mg are more Amine blends 1mL, RPMI1640 amino acid solution 20mL, 5 μM of Indomethacin, IL-2 1 × 106U, IL-15 50mg.
After more than component is dissolved with water for injection, by 0.22 μm of membrane filtration degerming, put 4 DEG C and be kept in dark place.
The formula of 1 Human peripheral blood NK cells amplification culture medium of table
2nd, people's natural kill (NK) cell is expanded with human peripheral blood single nucleus cell Activated in Vitro
1st, the separation of human peripheral blood single nucleus cell and the processing of autologous plasma
Heparin (being purchased from Hebi City pharmaceutical factory of Henan Province) anti-freezing is added in into healthy human peripheral blood 30mL, 500g centrifuges 10 points Clock collects blood plasma 15mL, blood plasma is taken out after twenty minutes in 56 DEG C of standings, and 1200g is centrifuged 5 minutes, removes pellet platelets, obtains It is spare to autologous plasma.
Above-mentioned blood Ficoll density gradient centrifugations are collected mononuclearcell by empirically room conventional method.Cell count, Sum is 4.2 × 107It is a.Leave and take 2 × 106A cell is analyzed for flow cyctometry, is as follows described.Remaining cell (4.0×107It is a) it adds in containing 10% (V/V) autologous plasma (2mL) and 1000U/mL containing recombinant human il-2, recombined human IL-15 The NK cell amplification culture mediums (18mL) of 50ng/mL, the cell density of mononuclearcell suspension is 2.0 × 106A/mL (0.5- 10×106/ mL, is recommended as 1-5 × 106/ mL), for cultivating.
2nd, people's natural kill (NK) cell is activated
1) culture bottle bed board
Bed board is carried out with the separated cell activator of three kinds of components：
(1) 10 μ g macrophage activations lipopeptids -2 (MALP-2) are dissolved with 3mL isopropanols, 7mL 0.01M is added dropwise For pH7.2Tris-CL-NaCL solution to a concentration of 1mg/L of macrophage activation lipopeptid -2, addition floor space is 75cm2Training It supports in bottle, 1 hour or more (preferably 1-2 hours) is stood in 37 DEG C or 4 DEG C of standings 2 hours or more can be used (if not being scene It uses, can be in 4 DEG C of storages, use in 3 weeks)；- 2 solution of macrophage activation lipopeptid is absorbed, with 0.01M pH7.2Tris-CL- NaCL solution washing culture bottle bottom 1 time does not attach to the macrophage activation lipopeptid -2 of container bottom with removal；It (2) will be anti-human 100 μ g and 10mL 0.01M pH7.2Tris-CL-NaCL solution of CD16 monoclonal antibodies is mixed to anti-human CD16 monoclonal antibodies A concentration of 10mg/L, add in culture bottle in, by culture bottle be placed in 37 DEG C stand 1 hour or more (preferably 1-2 hours) or 4 DEG C it is quiet It puts and can be used within 2 hours or more (if not being onsite application, can be in 4 DEG C of storages, use in 3 weeks).
To simplify procedures, can the cell activator that three kinds of components described in 5mL mix directly be added in floor space is 75cm2Culture bottle in, more than 1 hour (preferably 1-2 hour) or 4 DEG C stood in 37 DEG C stand be within 2 hours or more it is usable (such as It is not onsite application, can be in 4 DEG C of storages, use in 3 weeks).
2) inoculating cell
The culture bottle of bed board is taken out, with injection brine 1 time, adds in 20mL peripheral blood mononuclear cells (cell density is 2.0 × 10 to suspension6A/mL), it is placed in 37 DEG C, 5%CO2, 95% air, cultivate under 100% humidity.
3rd, people's natural kill (NK) cell is expanded
After 72 hours, the peripheral blood NK cell amplification culture medium of 10% autologous plasma (2mL) and table 1 is added in into culture bottle (18mL) continues culture 48 hours.Cell total volume is 40mL.
Later, cell suspension is transferred in cell culture bags, adds in 10% autologous plasma (4mL) and peripheral blood NK cell expands Increase culture medium (36mL), continue culture 48 hours.At this point, cell total volume is 80mL.
10% autologous plasma (7mL) and peripheral blood NK cell amplification culture medium (73mL) are added in, continues culture 48 hours.This When, cell total volume is 160mL.
Peripheral blood NK cell amplification culture medium 160mL is supplemented, is cultivated 24 hours.At this point, cell total volume is 320mL.
Peripheral blood NK cell amplification culture medium 320mL is supplemented, is cultivated 24 hours.At this point, cell total volume is 640mL.
Peripheral blood NK cell amplification culture medium 640mL is supplemented, is cultivated 24 hours.At this point, cell total volume is 1280mL.
Peripheral blood NK cell amplification culture medium 1320mL is supplemented, is cultivated 48 hours.At this point, cell total volume is 2600mL.
Peripheral blood NK cell amplification culture medium 1000mL is supplemented, is cultivated 48 hours.At this point, cell total volume is 3600mL.
Cell culture total time is 14 days.Cell total volume is 3600mL.
4th, cell is harvested
500g centrifuges 10min, harvests cell.Total number of cells are 9.28 × 109It is a that (initial cell sum is 4 × 107It is a), Cell viability is 97%, and viable count is 9 × 109A, total number of cells expand 225 times.
3rd, cellular morphology is observed
The cellular morphology of human peripheral blood single nucleus cell culture 48 hours such as Fig. 2 (mark sizes：100 μm) shown in, it is seen that Adherent cell colony by colony, there is a large amount of spindle cell (Monocytes/Macrophages, arrow are signified), prompting monokaryon/huge Phagocyte is to the stimulation of NK cells.
Form such as Fig. 3 (mark sizes of 72 hours colonies formed of human peripheral blood single nucleus cell culture：100 μm) institute Show, it is seen that cell colony volume becomes larger, and shows that cell comes into vegetative state.
4th, flow cyctometry is analyzed
It takes culture preceding and the human peripheral blood single nucleus cell of activation amplification cultivation 2 weeks (14 days), addition FITC labels resists The anti-CD56 antibody of CD3 monoclonal antibodies and PE labels, to add in idiotype control antibodies pipe as control, room temperature is protected from light 20 Minute.FACSCalibur collects cell, WinMdi2.9 software analysis results after washing.According to culture before and cultivate after NK cells (CD3-/CD56+) ratio and total cell number calculate the amplification times of NK cells.
Before human peripheral blood single nucleus cell culture and culture 2 weeks (14 days) NK cell proportions flow cyctometry analysis result As shown in figure 4, NK cell proportions are increased to 84% from 8.67% before activation amplification, 225 times of basis is expanded in total number of cells On, NK total number of cells expand 2180 times.
5th, K562 cell killings are tested
Detect human peripheral blood single nucleus cell (killing cell controls) and Activated in Vitro amplification (14 days) from people periphery The NK cells (experimental cell) of blood mononuclear cell are to the killing rate of K562 cells (leukaemia cell).It is added in 96 well culture plates The K562 cells (deriving from ATCC) of exponential phase, per hole 2 × 104A, total volume is 50 μ L.Cell is divided into：(1) target is imitated Than 1:2, NK cell per wells inoculation 1 × 104A, total volume is 50 μ L；(2) effect target is than 1:1, NK cell per well inoculation 2 × 104It is a, Total volume is 50 μ L；(3) effect target is than 10:1, NK cell per well inoculation 2 × 105A, total volume is that 50 μ L or human peripheral are single Nucleus inoculation 2 × 105A, total volume is 50 μ L；(4) 50 μ L of culture medium are compareed as target cell；(5) only add respective counts for every group The NK cells or human peripheral blood single nucleus cell of amount.Every group of 8 holes.The tetrazolium bromide of a concentration of 1mg/mL is added in after 4 hours per hole (MTT) 10 μ L, 37 DEG C are reacted 4 hours.Add in 10% neopelex, 100 μ L.It measures per hole 560nm OD values (OD560).Before culture after (ctr) and culture 14 days, according to different effect target than measuring human peripheral blood single nucleus cell to K562 The killing rate of leukaemia cell.Killing rate=(experimental group OD values-target cell control group OD values-respective numbers target cell control OD Value)/(target cell control OD values) × 100%.
Human peripheral blood single nucleus cell (killing cell controls) and Activated in Vitro expand thin from the single core of human peripheral The NK cells (experimental cell) of born of the same parents are to killing rate measurement result such as Fig. 5 (ordinates of K562 cells (leukaemia cell)：Killing rate (%), abscissa：Imitate target ratio) shown in, compared with human peripheral blood single nucleus cell, activated amplification it is thin from the single core of human peripheral The NK cells of born of the same parents significantly improve the killing ability of K562 cells, and effect target ratio is 1:Averagely killing rate is more than 90% when 1.
Embodiment 2 expands people's natural kill (NK) carefully with human umbilical vein's blood (people's bleeding of the umbilicus) mononuclearcell Activated in Vitro Born of the same parents
NK cell deriveds used in the present embodiment are in the mononuclearcell of people's bleeding of the umbilicus.
First, the cultivating system of Activated in Vitro amplification people's natural kill (NK) cell
For the cultivating system of Activated in Vitro amplifying NK cells of human beings, including NK cell activators and NK cell amplification cultivations Base, specific formula are as follows：
1st, the formula of human umbilical cord blood cell activator：It is same as Example 1.
2nd, people's Cord Blood Natural Killer Cells: Impact amplification culture medium is on the formula basis of the general NK cell amplification culture mediums of embodiment 1 On, increase injection hydrocortisone sodium succinate 1-10 × 10-6M。
2nd, people's natural kill (NK) cell is expanded with Human cord blood mononuclear cells Activated in Vitro
1st, the separation of Human cord blood mononuclear cells and the processing of autologous plasma
Use blood taking bag (being purchased from medical appliance factory of Nangeer Biological Medicine Co., Ltd., Sichuan) acquisition Healthy People navel Original anti-coagulants sodium citrate 28mL in blood 80mL, wherein blood taking bag, practical bleeding of the umbilicus amount are 52mL.500g is centrifuged 10 minutes, is received Collect the blood plasma 50mL containing anti-coagulants.Blood plasma is taken out after twenty minutes in 56 DEG C of standings, 1200g is centrifuged 5 minutes, and removal blood platelet sinks It forms sediment, obtains autologous plasma, it is spare.
Empirically room conventional method, by above-mentioned blood Ficoll density gradient centrifugations to collect mononuclearcell.Cytometer Number, sum are 1.1 × 108It is a.Leave and take 1 × 107A cell is analyzed for flow cyctometry, is as follows described.Remaining is thin Born of the same parents (1.0 × 108It is a) it adds in containing 10% autologous plasma (2mL) and recombinant human il-2 1000U/mL, the NK of IL-15 50ng/mL Cell amplification culture medium (18mL), the cell density of mononuclearcell suspension is 5.0 × 106A/mL (0.5-10 × 106/ mL is equal Can, it is recommended as 1-5 × 106/ mL), for cultivating.
2nd, people's natural kill (NK) cell is activated
1) culture bottle bed board
Bed board is carried out with the separated cell activator of three kinds of components：
(1) 30 μ g macrophage activations lipopeptids -2 (MALP-2) are dissolved with 9mL isopropanols, 21mL 0.01M is added dropwise For pH7.2Tris-CL-NaCL solution to a concentration of 1mg/L of macrophage activation lipopeptid -2, addition floor space is 250cm2Training Support bottle in, in 37 DEG C stand more than 1 hour (preferably 1-2 hours) or 4 DEG C standing 2 hours it is used above (non-at-scene to be used in 4 DEG C Storage uses in 3 weeks), -2 solution of macrophage activation lipopeptid is absorbed, is washed with 0.01M pH7.2Tris-CL-NaCL solution Culture bottle bottom 1 time does not attach to the macrophage activation lipopeptid -2 of container bottom with removal；
(2) by anti-human 300 μ g and 30mL 0.01M pH7.2 Tris-CL-NaCL solution of CD16 monoclonal antibodies mix to A concentration of 10mg/L of anti-human CD16 monoclonal antibodies is added in culture bottle, and culture bottle is placed in 37 DEG C stands 1 hour or more (preferably 1-2 hours) or 4 DEG C of standings 2 hours or more (non-at-scene to be used in 4 DEG C of storages, used in 3 weeks).
To simplify procedures, can the cell activator that three kinds of components mix in 20mL step 1 directly be added in into bottom surface Product is 250cm2Culture bottle in, in 37 DEG C stand more than 1 hour (preferably 1-2 hours) or 4 DEG C standing 2 hours it is used above (non-at-scene to be used in 4 DEG C of storages, used in 3 weeks).
2) inoculating cell
The culture bottle of bed board is taken out, with injection brine 1 time, 50mL cord blood mononuclear cells is added in and hangs (cell density is 5.0 × 10 to liquid6A/mL), it is placed in 37 DEG C, 5%CO2, 95% air, cultivate under 100% humidity.
3rd, people's natural kill (NK) cell is expanded
After 72 hours, 10% autologous plasma (5mL) and people's Cord Blood Natural Killer Cells: Impact amplification culture medium (45mL) are added in, continues to cultivate 48 hours.Cell total volume is 100mL.
Later, cell suspension is transferred in cell culture bags, adds in 10% autologous plasma (10mL) and people's Cord Blood Natural Killer Cells: Impact Amplification culture medium (90mL) continues culture 48 hours.At this point, cell total volume is 200mL.
10% autologous plasma (20mL) and people's Cord Blood Natural Killer Cells: Impact amplification culture medium (180mL) are added in, continues culture 48 hours. At this point, cell total volume is 400mL.
10% autologous plasma (10mL) and people's Cord Blood Natural Killer Cells: Impact amplification culture medium (390mL) are added in, continues culture 24 hours. At this point, cell total volume is 800mL.
People Cord Blood Natural Killer Cells: Impact amplification culture medium 800mL is supplemented, is cultivated 48 hours.At this point, cell total volume is 1600mL.
People Cord Blood Natural Killer Cells: Impact amplification culture medium 1600mL is supplemented, is cultivated 48 hours.At this point, cell total volume is 3.2L.
People Cord Blood Natural Killer Cells: Impact amplification culture medium 1800mL is supplemented, is cultivated 48 hours.At this point, cell total volume is 5L.
Cell culture total time is 14 days.Cell total volume is 5L.
4th, cell is harvested
500g centrifuges 10min, harvests cell.Total number of cells are 1.6 × 1010It is a that (initial cell sum is 1.0 × 108 It is a), cell viability 95%, viable count is 1.52 × 1010A, total number of cells expand 152 times.
3rd, cellular morphology is observed
The cellular morphology of Human cord blood mononuclear cells culture 48 hours such as Fig. 6 (mark sizes：100 μm) shown in, it is seen that patch The cell colony of wall by colony, there is a large amount of spindle cell (Monocytes/Macrophages, shown in asterisk), prompts monokaryon/macrophage Cell is to the stimulation of NK cells.
Form such as Fig. 7 (mark sizes of 72 hours colonies formed of Human cord blood mononuclear cells culture：100 μm) shown in, Visible cell colony volume becomes larger, and shows that cell is in vegetative state.
4th, flow cyctometry is analyzed
Analysis method is same as Example 1.
Before Human cord blood mononuclear cells culture and cultivate the flow cyctometry analysis result of 2 weeks (14 days) NK cell proportions such as Shown in Fig. 8, NK cell proportions are increased to 94.5% from 7.22% before activation amplification, and 152 times of basis is expanded in total number of cells On, NK total number of cells expand 1989 times.
5th, K562 cell killings are tested
Detect Human cord blood mononuclear cells (killing cell controls) and Activated in Vitro amplification (14 days) from people's bleeding of the umbilicus list The NK cells (experimental cell) of a nucleus are to the killing rate of K562 cells (leukaemia cell).Detection method and 1 phase of embodiment Together.
Human cord blood mononuclear cells (killing cell controls) and Activated in Vitro expand from Human cord blood mononuclear cells NK cells (experimental cell) are to killing rate measurement result such as Fig. 9 (ordinates of K562 cells (leukaemia cell)：Killing rate (%), abscissa：Imitate target ratio) shown in, compared with Human cord blood mononuclear cells, activated amplification from Human cord blood mononuclear cells NK cells significantly improve the killing ability of K562 cells, and effect target ratio is 1:Averagely killing rate is more than 90% when 1.
2 mutual authentication of the embodiment 1 and embodiment present invention is for Activated in Vitro amplification people's natural kill (NK) cell Cultivating system and method can realize NK using the mononuclearcell of healthy human peripheral blood or umbilical vein blood as initiator cell The activation amplification of cell, amplification quantity improve nearly 2000 times or more, and the NK cells after overactivation expands have very strong K562 cell killing activities, effect target ratio are 1:Averagely killing rate is more than 90% when 1.Can rationally it infer, the present invention is for external The cultivating system and method for activation amplification people's natural kill (NK) cell can also apply to thin to NK from other tissues such as marrow The culture of born of the same parents, it is suitable for the culture of animal NK cells.

Claims (10)

1. for the cultivating system of Activated in Vitro amplification people's natural kill (NK) cell, expanded including cell activator and NK cells Culture medium, the cell activator include：(1) anti-human CD16 monoclonal antibodies, (2) macrophage activation lipopeptid -2 (macrophage activating lipopeptide-2, MALP-2) and (3) dissolves anti-human CD16 monoclonal antibodies and macrophage The buffer solution of cell-stimulating lipopeptid -2 (MALP-2)；
The NK cell amplification culture mediums include：1) basal medium, 2) free serum culture based additive, 3) cyclooxygenase 2 (COX-2) inhibitor and 4) cell factor.
2. the cultivating system according to claim 1 for Activated in Vitro amplification people's natural kill (NK) cell, feature It is：In cell activator, a concentration of 0.5-5mg/L of use (the recommended density 1- of the anti-human CD16 monoclonal antibodies 2mg/L, preferred concentration 1mg/L)；A concentration of 1-100mg/L of use of the macrophage activation lipopeptid -2 (MALP-2) (is pushed away Recommend a concentration of 5-25mg/L, preferred concentration 10mg/L)；It is described to dissolve anti-human CD16 monoclonal antibodies and macrophage activation fat The buffer solution of peptide -2 (MALP-2) is phosphate buffer, Tris-CL-NaCL solution or other without EDTA or other chelating agents Buffer solution, the pH value of buffer solution is 7.0-7.5 (recommendation pH value be 7.1-7.3, preferable ph 7.2).
3. the cultivating system according to claim 1 or 2 for Activated in Vitro amplification people's natural kill (NK) cell, special Sign is：Three kinds of components in the cell activator can separate storage (depositing in respectively in different containers) or mixing is deposited It puts.
4. according to any cultivating systems for Activated in Vitro amplification people's natural kill (NK) cell of claim 1-3, It is characterized in that：In NK cell amplification culture mediums：
One kind in RPMI1640 culture mediums, IMDM culture mediums, DMEM/F12 culture mediums of the basal medium or wherein The combination (preferably RPMI1640 culture mediums) of two or three of basal medium；
The free serum culture based additive includes NaHCO3, human albumin, actrapid monotard, human transferrin, sodium selenite, second Hydramine, lipid mixtures (such as mixing of linoleic acid, leukotrienes, oleic acid, cholesterol), biotin, Sodium Pyruvate, ethoxy piperazine Piperazine second thiosulfonic acid, L-Glutamine, polyamine mixture (such as mixing of putrescine, cadaverine, spermidine, spermine), hydrocortisone It is more in sodium succinate, essential amino acid (such as lysine, tryptophan) and nonessential amino acid (such as glutamic acid, alanine) Kind is whole；Wherein albumin, insulin, transferrins, selenium, lipid mixtures, essential amino acid and nonessential amino acid are Required ingredient, biotin, Sodium Pyruvate, hydroxyethyl piperazine second thiosulfonic acid, L-Glutamine and polyamine mixture is promote cell The additive of proliferation, hydrocortisone sodium succinate are to cultivate the specialist additive that Cord Blood-Derived NK cells are；
Cyclooxygenase 2 (COX-2) inhibitor is Indomethacin (indomethacin), NS-398 and celecoxib (celecoxib) (preferably Indomethacin, a concentration of 0.5 μM -50 μM of Indomethacin, recommended density is 1-20 μ to one kind in M, preferred concentration are 5 μM)；
The cell factor is including at least two in IL-2, IL-15, IL-12 and IL-18, it is preferable to use IL-2 and IL-15 are thin Intracellular cytokine combines, and a concentration of the 2 × 10 of IL-25-3×106U/L (200U/mL-3000U/mL), preferably 1 × 106U/L (1000U/mL), a concentration of 10-100mg/L (10-100ng/mL) of IL-15, preferably 50mg/L (50ng/mL).
5. the cultivating system according to claim 4 for Activated in Vitro amplification people's natural kill (NK) cell, feature It is：General NK cell amplification culture mediums are using RPMI1640 culture mediums as basic culture medium, every liter of (/L) NK cells amplification Culture medium contains：RPMI1640 pulvis 10.39g, NaHCO32g, human albumin 0.5-5g, Insulin-Transferrin-selenium mixing Object 1-10mL, ethanol amine 1-10 × 10-4M, lipid mixtures 1-10mL, biotin 1-20mg, Sodium Pyruvate 55-220mg, hydroxyl second Base piperazine second thiosulfonic acid 1-20mM, L-Glutamine 146-730mg, polyamine mixture 1-10mL, RPMI1640 amino acid solution 1-20mL, 0.5-10 μM of Indomethacin, IL-2 2 × 105-3×106U,IL-15 10-100mg。
6. the cultivating system according to claim 5 for Activated in Vitro amplification people's natural kill (NK) cell, feature It is：The formula of Human peripheral blood NK cells amplification culture medium is preferably：Every liter of (/L) culture medium 164010.39g containing RPMI, NaHCO32.04g, human albumin 2g, Insulin-Transferrin-selenium mixture 10mL, ethanol amine 6 × 10-4M, lipid mixtures 1mL, biotin 10mg, Sodium Pyruvate 110mg, hydroxyethyl piperazine second thiosulfonic acid 2mM, L-Glutamine 292mg, polyamine mixture 1mL, RPMI1640 amino acid solution 20mL, 5 μM of Indomethacin, IL-2 1 × 106U,IL-15 50mg。
7. the cultivating system according to claim 5 for Activated in Vitro amplification people's natural kill (NK) cell, feature It is：The formula of people's Cord Blood Natural Killer Cells: Impact amplification culture medium is preferably：Every liter of (/L) culture medium contains RPMI164010.39g, NaHCO3 2.04g, human albumin 2g, Insulin-Transferrin-selenium mixture 10mL, ethanol amine 6 × 10-4M, lipid mixtures 1mL are raw Object element 10mg, Sodium Pyruvate 110mg, hydroxyethyl piperazine second thiosulfonic acid 2mM, L-Glutamine 292mg, polyamine mixture 1mL, RPMI1640 amino acid solution 20mL, injection hydrocortisone sodium succinate 1 × 10-6M, 5 μM of Indomethacin, IL-2 1 × 106U,IL-15 50mg。
8. a kind of method of Activated in Vitro amplification people's natural kill (NK) cell, is trained using claim 1 to 7 any one of them The system of supporting is using isolated mononuclearcell as initiator cell, first adds in the cell activator in culture vessel Bed board is carried out, is then inoculated with the mononuclearcell made of the NK cell amplification culture mediums containing autologous plasma to culture vessel Suspension cultivates cell, obtains people's natural kill (NK) cell of activated amplification.
9. the method for Activated in Vitro amplification people's natural kill (NK) cell according to claim 8, it is characterised in that：
The NK cell deriveds are in the mononuclearcell of people's marrow, peripheral blood or umbilical vein blood (bleeding of the umbilicus)；
Three kinds of components in the cell activator can be used separately or be used in mixed way；Three kinds of components are separated bed board method is： With isopropanol dissolving macrophage activation lipopeptid -2 (MALP-2), it is a concentration of to macrophage activation lipopeptid -2 that buffer solution is added dropwise 1-100mg/L (recommends 1-2mg/L, preferably 1mg/L), then -2 solution of macrophage activation lipopeptid is added in culture vessel, makes Solution is laid in bottom of culture vessel, and 1 hour or more (preferably 1-2 hours) is stood in 37 DEG C or 4 DEG C stand 2 hours or more, so - 2 solution of macrophage activation lipopeptid is absorbed afterwards, and it is unadsorbed in culture vessel bottom to remove to wash bottom of culture vessel with buffer solution Macrophage activation peptiolipid -2 in portion add anti-human CD16 monoclonal antibodies and buffer solution to anti-human CD16 monoclonal antibodies A concentration of 0.5-5mg/L (recommending 5-25mg/L, preferably 10mg/L), minimum additive amount is ensures solution confluent cultures container bottom Culture vessel is placed in 37 DEG C and stands 1 hour or more (preferably 1-2 hours) or 4 DEG C of standings 2 hours or more by portion；
It is by the method that the cell activator of three kinds of components mixing carries out bed board：NK cell activators are added in culture vessel, Minimum additive amount stands 1 hour or more (preferably 1-2 hours) or 4 to ensure that solution is paved with entire bottom of culture vessel, in 37 DEG C DEG C stand 2 hours or more；
The cell density of the mononuclearcell suspension is 0.5-10 × 106/ mL, is recommended as 1-5 × 106/ mL, preferably 2 × 106/mL；The adding proportion of autologous plasma is 1-20% (V/V), preferably 10% (V/V)；
The condition of culture of the culture cell is 37 DEG C, 5%CO2, 95% air, 100% humidity, incubation time be 10 days -21 My god, recommend 10-18 days, preferably 14 days；It is preferred that gradually expanding culture scale and increasing incubation time, expand the method for culture scale NK cell amplification culture medium of the addition containing 1-20% (preferably 10%, V/V) autologous plasma, incubation time according to cell quantity and Growth conditions are increased；Using people's natural kill (NK) cell of the method harvest activation amplification of conventional centrifugal.
10. the method for Activated in Vitro amplification people's natural kill (NK) cell according to claim 8 or claim 9, it is characterised in that： Include the following steps：
1) the isolated mononuclearcell from peripheral blood or bleeding of the umbilicus；
2) cell activator is added in culture vessel and carries out bed board：
By the separated cell activator of three kinds of components carry out bed board or
The cell activator that three kinds of components are mixed carries out bed board；
3) to culture vessel inoculation the Human peripheral blood NK cells amplification culture medium containing 1-20% (V/V) autologous plasma or institute Mononuclearcell suspension made of people's Cord Blood Natural Killer Cells: Impact amplification culture medium is stated, in 37 DEG C, 5%CO2, 95% air, 100% humidity Under the conditions of cultivate -21 days 10 days (recommend 10-18 days, preferably 14 days), obtain the activated of high-purity (purity is up to 60-94%) People's natural kill (NK) cell of amplification.
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111235107A (en) * 2020-03-26 2020-06-05 于继莲 Additive and culture medium for immune cell culture and immune cell culture method
CN111925987A (en) * 2020-09-24 2020-11-13 深圳市一五零生命科技有限公司 NK cell serum-free culture solution and NK cell culture method
CN112063653A (en) * 2020-09-09 2020-12-11 广东昭泰体内生物医药科技有限公司 Method for preparing NK (natural killer) sample cells based on electrotransfer reprogramming plasmid
CN113373113A (en) * 2021-08-13 2021-09-10 北京肿瘤医院(北京大学肿瘤医院) Method for enhancing anti-tumor effect of immune cells
CN113416701A (en) * 2021-07-28 2021-09-21 新疆西部赛澳生物科技有限责任公司 NK cell culture medium and culture method
CN113694217A (en) * 2021-08-30 2021-11-26 沈阳药科大学 Pharmaceutical composition containing IL-15 cationic liposome complex and celecoxib liposome as well as preparation method and application of pharmaceutical composition
CN113736733A (en) * 2020-05-29 2021-12-03 基亚细胞科技有限公司 Culture medium for in vitro activation of immune cells
CN113736732A (en) * 2020-05-29 2021-12-03 基亚细胞科技有限公司 Method for activating immune cells in vitro
CN113881629A (en) * 2020-07-03 2022-01-04 山东荆卫生物科技有限公司 Culture medium and culture method for efficiently amplifying NK cells in vitro
CN114032210A (en) * 2021-11-06 2022-02-11 曾东升 Culture medium and culture method for NK cell amplification
CN114891744A (en) * 2022-07-13 2022-08-12 山东省齐鲁干细胞工程有限公司 Freezing umbilical cord blood NK cell in-vitro amplification method
CN115521914A (en) * 2022-10-12 2022-12-27 西北工业大学 Human primary natural killer cell in-vitro amplification system and method
JP7335971B2 (en) 2019-03-27 2023-08-30 ソプ シン，ジ Additive composition for NK cell culture medium, NK cell culture method using the additive composition, and cosmetic composition for improving skin troubles obtained by the culture method

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104152412A (en) * 2014-08-13 2014-11-19 赛业（苏州）生物科技有限公司 Clinical applicable culture system for efficient amplification of NK cells
CN104818248A (en) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 Immunocyte culture medium, and culture method and application of immunocytes
CN104919042A (en) * 2013-01-15 2015-09-16 阿部博幸 Method for manufacturing immunocyte-containing composition, and cancer-treating composition
CN104928241A (en) * 2015-03-27 2015-09-23 北京康爱瑞浩生物科技股份有限公司 Activation method for enhanced NK (Natural Killer) cells and cell preparation method
CN105238754A (en) * 2015-11-20 2016-01-13 赵顺英 Method for in vitro culture of high-proliferation and high-mortality NK cells
CN105238752A (en) * 2015-10-22 2016-01-13 苏州科贝生物技术有限公司 Culture system and culture method for efficient amplification in vitro of autologous NK cells
CN105861435A (en) * 2016-05-11 2016-08-17 南京华奥生物医药技术有限公司 In-vitro amplification method of natural killer cells (NK)
CN106222141A (en) * 2016-10-17 2016-12-14 湖南丰晖生物科技有限公司 NK cell culture fluid and cell culture processes

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104919042A (en) * 2013-01-15 2015-09-16 阿部博幸 Method for manufacturing immunocyte-containing composition, and cancer-treating composition
CN104152412A (en) * 2014-08-13 2014-11-19 赛业（苏州）生物科技有限公司 Clinical applicable culture system for efficient amplification of NK cells
CN104818248A (en) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 Immunocyte culture medium, and culture method and application of immunocytes
CN104928241A (en) * 2015-03-27 2015-09-23 北京康爱瑞浩生物科技股份有限公司 Activation method for enhanced NK (Natural Killer) cells and cell preparation method
CN105238752A (en) * 2015-10-22 2016-01-13 苏州科贝生物技术有限公司 Culture system and culture method for efficient amplification in vitro of autologous NK cells
CN105238754A (en) * 2015-11-20 2016-01-13 赵顺英 Method for in vitro culture of high-proliferation and high-mortality NK cells
CN105861435A (en) * 2016-05-11 2016-08-17 南京华奥生物医药技术有限公司 In-vitro amplification method of natural killer cells (NK)
CN106222141A (en) * 2016-10-17 2016-12-14 湖南丰晖生物科技有限公司 NK cell culture fluid and cell culture processes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHRISTINA MÜLLER等: "The TLR‑2/TLR‑6 agonist macrophage‑activating lipopeptide‑2 augments human NK cell cytotoxicity when PGE2 production by monocytes is inhibited by a COX‑2 blocker", 《CANCER IMMUNOL IMMUNOTHER》 *
吴燕峰等: "《实用医学细胞培养技术》", 31 January 2010, 广州：中山大学出版社 *
赵杨祉等: "NK细胞与CIK细胞体外诱导扩增及其活性比较", 《吉林大学学报（医学版）》 *

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7335971B2 (en) 2019-03-27 2023-08-30 ソプ シン，ジ Additive composition for NK cell culture medium, NK cell culture method using the additive composition, and cosmetic composition for improving skin troubles obtained by the culture method
CN111235107B (en) * 2020-03-26 2021-11-09 海南南医安启生物医疗科技有限公司 Additive and culture medium for immune cell culture and immune cell culture method
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CN113736732B (en) * 2020-05-29 2023-04-25 基亚细胞科技有限公司 Method for in vitro activation of immune cells
CN113736733B (en) * 2020-05-29 2023-04-25 基亚细胞科技有限公司 Culture medium for in vitro activation of immune cells
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CN113416701A (en) * 2021-07-28 2021-09-21 新疆西部赛澳生物科技有限责任公司 NK cell culture medium and culture method
CN113373113A (en) * 2021-08-13 2021-09-10 北京肿瘤医院(北京大学肿瘤医院) Method for enhancing anti-tumor effect of immune cells
CN114250198A (en) * 2021-08-13 2022-03-29 北京肿瘤医院(北京大学肿瘤医院) Method for enhancing anti-tumor effect of immune cells
CN113694217A (en) * 2021-08-30 2021-11-26 沈阳药科大学 Pharmaceutical composition containing IL-15 cationic liposome complex and celecoxib liposome as well as preparation method and application of pharmaceutical composition
CN114032210A (en) * 2021-11-06 2022-02-11 曾东升 Culture medium and culture method for NK cell amplification
CN114891744B (en) * 2022-07-13 2022-10-25 山东省齐鲁干细胞工程有限公司 Freezing umbilical cord blood NK cell in-vitro amplification method
CN114891744A (en) * 2022-07-13 2022-08-12 山东省齐鲁干细胞工程有限公司 Freezing umbilical cord blood NK cell in-vitro amplification method
CN115521914A (en) * 2022-10-12 2022-12-27 西北工业大学 Human primary natural killer cell in-vitro amplification system and method
CN115521914B (en) * 2022-10-12 2024-04-19 西北工业大学 In-vitro amplification system and method for human primary natural killer cells

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Application publication date: 20180629