CN106566807A - Concentration gradient rhIL-2 dependent iNKT cell amplification method and application thereof - Google Patents

Concentration gradient rhIL-2 dependent iNKT cell amplification method and application thereof Download PDF

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CN106566807A
CN106566807A CN201610921173.6A CN201610921173A CN106566807A CN 106566807 A CN106566807 A CN 106566807A CN 201610921173 A CN201610921173 A CN 201610921173A CN 106566807 A CN106566807 A CN 106566807A
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cell
inkt
rhil
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concentration
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闾军
孙文峰
陈辉
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Beijing Gene Qiming Biological Science And Technology Co Ltd
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Beijing Gene Qiming Biological Science And Technology Co Ltd
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Abstract

The invention discloses a concentration gradient rhIL-2 dependent iNKT cell amplification method and an application thereof. The method comprises the following steps: (1) extracting peripheral blood mononuclear cells PBMC; (2) adding [alpha]-GalCer, which is 100ng/ml in final concentration, to the PBMC cells which are extracted in the step (1), and conducting cultivation for 48h; and (3) gradually increasing an rhIL-2 concentration in a culture medium in the cells which are cultivated in the step (2) in a gradient mode. According to the cell amplification method provided by the invention, when the iNKT cells are collected on the 21st day of cultivation, the quantity of the iNKT cells is increased by 100,000 times, including more than 90% of CD4-iNKT cells which have effects of up-regulating immune reaction and directly killing tumors. The iNKT cells, which are activated and amplified by virtue of the method provided by the invention, are higher in amplification efficiency, and the method can selectively amplify the Th1-like iNKT cells and enhance functions of in vitro amplification iNKT cell immunity as well as tumor immunity surveillance and killing.

Description

INKT methods for cell expansion and its application that a kind of Concentraton gradient rhIL-2 is relied on
Technical field
The invention belongs to tumor cell immunotherapy field, more particularly it relates to have Tumor cytotoxicity The methods and applications of Th1 sample iNKT cells rapid, high volume amplification.
Background technology
Constant natural killer T (invariant nature killer T, iNKT) cell is the naturally occurring mediation of a class The immunocyte of innate immunity and acquired immunity, is the subgroup of uniqueness in T lymphocytes, with class NK (nature Killer) cell sample adjusts the innate immune function of immunoreation to stimulating rapid generation cytokine and chemotactic factor, The reaction of raw specificity is originated in by φt cell receptor (TCR) antagonism.Different from the TCR of classical T lymphocytes, iNKT cells tool The TCR α chains for having highly conserved TCR α chains, the mankind are made up of 28 parts of V α 24-J α, can be with various glycolipid class antigen-reactives.This Outward, different from the I classes or II classes of traditional T cell identification antigen presenting cell (antigen presenting cell, APC) Major histocompatibility complex molecule (major histocompatibility complex, MHCI or II) offers antigen Peptide, the sugared lipid antigen that the non-classical MHC I quasi-molecule CDld in iNKT cell energy specific recognition APC surface offer.After activation INKT cells secrete cytokine profiles, directly or indirectly participate in different immunne response.The iNKT of different subgroups or hypotype is thin Born of the same parents have different immunoloregulation functions.
INKT cells have the function of induction immunostimulant and immunosurveillance simultaneously.Under antigenic stimulus, iNKT cells can To secrete a series of cytokines, including：Interferon-γ (interferon-gamma, IFN-γ), interleukin (IL) -2,4, 10,13,17,21,22 and grain-monosystem colony stimulating factor (granulocyte-macrophage colony- Stimulating factor, GM-CSF) and tumor necrosis factor (tumor necrosis factor-alpha, TNF-α).
INKT cells pass through the inducing dendritic shape cell of CD1d-TCR complex and CD40-CD40L interaction specificitys (Dendritic Cells, DC) is ripe, and secretes IL-12.It is more that IL-12 stimulates NK, NKT cell and other T cells to produce IFN-γ.Both cytokines significantly have activated downstream effect cell mass such as NK cells, CD8 together+T cell and gamma delta T are thin Born of the same parents.The activation of iNKT cells further promotes DC to raise the expression of the costimulatory moleculeses such as CD70, CD80 and CD86.And DC expression CD70 is CD8+T cell starts necessary to adaptive immunity.The IL-2 inducing memory CD4 of the iNKT cells secretion of activation+T Helper cell proliferation.CD4+T cell is divided into different t helper cell subgroups in the presence of different cytokines.INKT cells Th1/ proinflammatory (IFN-γ, TNF-α) or Th2/ anti-inflammatories (IL-4,10,13) cytokine can be secreted, this cascade strengthens Effect simultaneously excite the natural immunity and acquired immunity, induced killer MHC positive tumor cell.MHC feminine genders are swollen Oncocyte, iNKT cells show NK cell sample MHC independence cell cytotoxic activities Jing after the activation of the glycolipid antigens such as α-GalCer, By direct killings such as abduction delivering perforin/granzyme FasL/Fas or TNF related apoptosis induction ligands (TRAIL). To CD1d+Tumor cell (such as acute T Lymphocytic leukemias, monocytic leukemia and other hematologic malignancies), With Direct Recognition and effective target cell lethal effect can be showed.
It is often accompanied by serious iNKT cell quantities to reduce and functional disorder in tumor patient body.Due to oneself expression phosphoric acid sheath Ammonia alcohol receptor (S1P1R) and various chemokine receptors (C-X-C chemokine receptor) turn iNKT cell selectives Tumor locus are moved on to, is circulated in causing colon cancer, head and neck cancer, breast carcinoma, renal cell carcinoma, melanoma and Peripheral Blood of Patients with Hepatocellular Carcinoma Property iNKT cell quantities substantially reduce, but cell produce IFN-γ ability do not damage.Although body-internal-circulation iNKT cells Quantity is reduced, but Jing α-GalCer stimulate and can breed rapidly iNKT cells in vitro, and the iNKT cells of in-vitro multiplication are same Sample shows preferable cellulotoxic effect killing tumor cell.It is various in recent years based on human body iNKT cellular immunotherapy tumors Scheme is developed, but the iNKT cells of clinical selectivity injection Activated in Vitro, in tumour patient body, remission rate is relatively low, immunity Reaction individual variation is larger, and antitumous effect is preferable not to the utmost.The mankind have CD4+And CD4-INKT cells, according to cytokine-expressing It is divided into Th1 sample iNKT cells and Th2 sample iNKT cells.Wherein, Th1 samples iNKT cells produce IFN-γ, and its function is that mediation is straight Connect tumor-killing and the function of immunoreation and immunosurveillance is raised in tumor microenvironment.The phenotype mark of Th1 sample iNKT cells Will is great expression CD8 α, and considerably less CD8 α β, there is also CD4-CD8-。CD8+INKT cells compare CD4+Or CD4-CD8- INKT cells secrete more IFN-γs, and cytotoxicity is also higher.Due to CD4 in human peripheral iNKT cells+INKT cells are accounted for Relatively high (～90%), traditional amplification method does not have selectivity to Th1 sample iNKT cell amplifications, and the iNKT cells after amplification are each Hypotype ratio is unstable, causes feedback larger to immunoreation individual variation after tumour patient.Therefore, current technological difficulties master Concentrate in the amplification of the sufficient amount of feature iNKT cell with tumor-killing and mediated immunity supervisory function bit.
There is proliferation time using the iNKT cells that fixed concentration rhIL-2 joint α-GalCer are obtained in conventional amplification method After after long (～4 weeks), amplification, iNKT cell proportions are than relatively low (～20%), growth multiple limited (average 100 times), amplification INKT cell CD4+Accounting higher (～40%), the problems such as internal killing ability is low, it is impossible to meet clinical demand.The present invention is adopted The mode increased with rhIL-2 Concentraton gradient, offers α-GalCer using DC, combines rhIL-15, rhIL-7, CD3 monoclonal anti Body is by iNKT cells by CD4+It is converted into CD4-.Culture 21 days can be by 100,000 times of iNKT cell amplifications, wherein CD8+INKT cells are accounted for Than ＞ 50%, CD4+INKT cells accounting ＜ 10%.Solve the problems, such as iNKT quantity and purity, and selective amplification Th1 samples INKT cells, can fully meet the demand of clinical treatment.
The content of the invention
The invention provides a kind of amplification Activiation method of iNKT cells of Concentraton gradient rhIL-2 dependences and its application.This The method of invention is for existing iNKT amplification in vitros culture efficiency is relatively low, ask to Th1 samples, Th2 sample iNKT cell non-selectivities etc. Topic, propose it is a kind of more effectively, and specific amplification Th1 sample iNKT lymphocytes method.
In the iNKT methods for cell expansion that a kind of Concentraton gradient rhIL-2 that the present invention is provided is relied on, cultivate the 0th day, add 100ng/mlα-GalCer；Cultivate the 0-11 days, increase rhIL-2 concentration in the way of gradient.
The present invention provide preferred version be：The time of fixed interval, rhIL-2 concentration are in equal difference or wait than increasing；Interval Time preferred 1-72 hours, the preferred 10-400IU/ml of rhIL-2 concentrations.
Technical scheme of the invention preferred is：Cultivate the ripe DC for adding same donor inducing culture on the 7th day；DC is adding It is initially charged 100ng/ml α-GalCer within two hours before entering iNKT cells to be incubated；Cultivate the 14th day and use Anti-iNKT MicroBeads human are sorted, and are sorted during the iNKT cells for obtaining add the coated culture bottle of OKT3 monoclonal antibodies and are trained Support；CD28 antibody, rhIL-15 and rhIL-7 are added in culture medium；OKT3 MAb concentrations be 1-5 μ g/ml, CD28 antibody Concentration is 1-5 μ g/ml；RhIL-15, rhIL-7 concentration is 1-10ng/ml.
The further preferred technical scheme of the present invention is as follows：
(1) extract PERIPHERAL BLOOD MONONUCLEAR CELL PBMC；
(2) final concentration of 100ng/ml α-GalCer are added to cultivate in the PBMC cells extracted to step (1) 48 hours；
(3) gradually increase rhIL-2 concentration in culture medium in the way of gradient in step (2) cultured cells.
Further, in step (3), rhIL-2 concentration is increased with Fixed Time Interval, and the Fixed Time Interval is 1-72 hours.
Further, the rhIL-2 concentration be 10-400IU/ml in wait compare or equal difference mode increase；The 11st of culture the It when, rhIL-2 concentration increases to 400IU/ml.
Further, culture adds mature dendritic cell, ripe dendron shape on the 7th day in step (3) cell culture medium Cell is 1 with the quantity ratio of iNKT cells:10.
Further, mature dendritic cell was initially charged 100ng/ml α-GalCer for two hours before iNKT cells are added It is incubated.
Further, culture is sorted on the 14th day using Anti-iNKT MicroBeads human, what sorting was obtained INKT cells are cultivated in adding the coated culture bottle of CD3 monoclonal antibodies.
Further, CD3 MAb concentrations are 1-5 μ g/ml, preferably 2 μ g/ml.
Further, CD28 antibody, rhIL-15 and rhIL-7 are added during culture；CD28 antibody concentration is 1-5 μ g/ml, excellent Elect 3 μ g/ml as；RhIL-15 concentration is 1-10ng/ml, preferably 5ng/ml；RhIL-7 concentration is 1-10ng/ml, preferably 5ng/ml。
Methods described harvests iNKT cells on the 21st day in culture, and iNKT cell number improves 100,000 times, wherein more than 90% is CD4-INKT cells, with the effect for raising immunoreation with kill tumor.
The present invention solves the problems, such as the quantity and purity of iNKT cell expansion ex vivo cultures in prior art, cultivates 21 days Can be by 100,000 times of iNKT cell amplifications, and selective amplification Th1 sample iNKT cells, wherein CD8+INKT cells accounting ＞ 50%, CD4+INKT cells accounting ＜ 10%, can fully meet the demand of clinical treatment.Gradually increase rhIL-2 concentration, can be in culture Seedling selection expands iNKT cells, in this way, iNKT cell proportions in culture can be improved to 6% left at the 7th day The right side, iNKT cell number improve 60 times.The coated ripe DC of α-GalCer were added at the 7th day, orientable induction Th1 sample iNKT are thin Born of the same parents expand, and promote CD4+INKT cells are to CD4-Conversion.In this way, can be at the 14th day by iNKT cell proportions in culture Improve to 16% or so, iNKT cell number improves 1000 times.The pure iNKT cells that the 14th day Jing magnetic bead sorting is obtained are cultivated, according to The T cell characteristic that iNKT cells have, cultivates in adding the coated culture bottle of CD3 monoclonal antibodies, and adds costimulatory moleculeses CD28 antibody, can be such that the iNKT cells of purification expand in a large number, add rhIL-15 and rhIL-7 that Th1 sample iNKT cells can be maintained to increase Grow.INKT cells are harvested within 21st day in culture, iNKT cell number improves 100,000 times, wherein more than 90% is CD4-INKT cells, With rise immunoreation and direct cytotoxicity.Present invention amplification and the iNKT cell amplifications for activating are in hgher efficiency, selectivity Ground amplification Th1 sample iNKT cells, strengthen the function that amplification in vitro iNKT cellular immunization strengthens and tumour immunity is monitored.
Description of the drawings
Fig. 1 .rhIL-2 process iNKT cellular processes.
Fig. 2. different rhIL-2 E-tests process iNKT cell Flow cytometry figures.
Fig. 3 .iNKT cell expansion ex vivo cell quantity linear graphs.
Fig. 4 .iNKT cell killings tumor cell is tested.
Fig. 5 .iNKT cell secretion of gamma-IFN capacity experimentals.
Specific embodiment
Embodiments of the invention are described below in detail, the example of illustrated embodiment is shown in the drawings.In following embodiments Experimental technique, unreceipted particular technique or condition person, then according to conventional laboratory conditions such as《ATCC cell culture handbooks》Middle institute Condition is stated, and when Reagent Company's description has been clearly stated in embodiment, then condition proposed to specifications is carried out.Institute With reagent or the unreceipted production firm person of instrument, be can pass through city available from conventional products.
Reagent used in the embodiment of the present invention：
AIM V cell culture mediums, ThermoFisher companies
Lymphoprep separating liquids (instant), Axis-Shield companies
CD3 monoclonal antibodies, CD28 monoclonal antibodies, Biolegend companies
IL-2, IL-7, IL-15, Peprotech company
α-GalCer, Enzo Life Sciences companies
Anti-iNKT MicroBeads human, Miltenyi Biotec company
Fixation/Permeabilization Solution Kit with BD GolgiPlugTM, BD companies
FITC anti-human CD3, PE anti-human TCR V α 24-J α 18, PerCP/Cy5.5anti-human CD8, PE/Cy7anti-human CD4, FITC anti-human IFN-γs, Biolegend companies
1 PERIPHERAL BLOOD MONONUCLEAR CELL of embodiment (PBMC) is extracted
(1) early morning adopt 20ml anticoagulant venous blood.
(2) the addition 15ml Lymphoprep separating liquids in 50ml centrifuge tubes.
(3) 0.9% physiological saline solution gentle inversion of equal-volume three times is slowly added in anticoagulant venous blood, is fully mixed, Blood after being diluted.
(4) blood after dilution is slowly added into into Lymphoprep separating liquids top layer using 2ml aseptic droppers, is all moved into After be sure not to rock or overturn.
(5) centrifuge tube trim, with horizontal centrifuge (swing bucket rotor) 1700rpm, ace/brake:4/0, room temperature centrifugation 30min。
(6) suction out the unnecessary blood plasma in the superiors part.Buffy coat is gently suctioned out with 2ml aseptic straws, is moved into new In 50ml centrifuge tubes, the buffy coat in all pipes is sucked in same 50ml centrifuge tubes.Add 0.9% sterile physiological salt Water is fully mixed to 50ml.
(7) centrifuge tube trim, with horizontal centrifuge (swing bucket rotor) 1700rpm, ace/brake:9/9 centrifugation, room temperature from Heart 10min.
(8) test tube is taken out, discards liquid completely.
(9) the AIM V culture medium of incubation is taken out from 37 DEG C of incubators, 2ml is taken and cell precipitation is fully mixed, note light Soft operation, it is to avoid produce bubble.Remaining culture medium is subsequently added, cell is mixed.
(10) cell suspension for mixing is moved into into 75cm2Tissue Culture Flask, gently rocks mixing, is positioned over 37 DEG C of incubators Middle culture.
2 iNKT cells of embodiment are expanded for the first time
(1) PBMC extracts the final concentration of 100ng/ml α-GalCer of same day addition and cultivates 48 hours.
(2) gradually increase the concentration of rhIL-2 in culture medium, from cell separation to culture the 11st day, rhIL-2 concentration is in Increase Deng ratio or equal difference mode.
(3) at the 11st day of culture, rhIL-2 concentration increases to 400IU/ml.
By shown in Figure 1A, from the beginning of cell separation, every 48 hours (remove the 10th day to the 11st day and be spaced 24 hours), RhIL-2 concentration is in wait to increase than mode.
By shown in Figure 1B, from the beginning of cell separation, every 72 hours (remove the 10th day to the 11st day and be spaced 48 hours), RhIL-2 concentration increases in equal difference mode.
The inducing culture of 3 dendritic cell of embodiment
(1) PBMC for obtaining density－gradient centrifuga－tion method is resuspended with AIM V culture medium, in adding Tissue Culture Dish, 37 DEG C 5%CO2Culture.
Culture dish was as a child taken out by (2) 3, was gently rocked, and suctioned out suspension cell.
(3) addition rhIL-4 containing 500IU/ml, the AIM V culture medium of 500IU/ml rhGM-CSF in culture dish.
(4) respectively after incubation the 3rd day, the 5th day half amount change liquid, add fresh rhIL-4 containing 500IU/ml, 500IU/ml The AIM V culture medium of rhGM-CSF.
(5) culture adds final concentration of 10ng/ml rhTNF- α on the 6th day.
Harvest mature dendritic cell within (6) the 7th days.
4 iNKT cells of embodiment stimulate again
(1) in 2 cell of embodiment and 3 cell culture of embodiment the 7th day, during embodiment 3 is collected in centrifugation, ripe dendron shape is thin Born of the same parents, it is resuspended with AIM V culture medium, add final concentration 100ng/ml α-GalCer to cultivate 2 hours.
(2) centrifugation removes free α-GalCer, carries out cell counting.With the first amplified production of iNKT cells:DC=10: 1 ratio by step (1) cultivate obtain DC add embodiment 2 described in and the first amplified production of iNKT cells in.
The isolated PBMC of Jing venous blood carries out flow cytometry in the 0th day, the 7th day, the 14th day respectively in culture respectively Detection.As seen from Figure 2, iNKT cells (CD3+6B11+) ratio gradually increases, and CD4 in iNKT cells+Cell proportion by Gradually reduce, CD4-CD8-Cell, CD8+Cell proportion is gradually stepped up.By shown in Fig. 2A, in example 2 from rhIL-2 concentration Increase than mode in waiting, in the 14th day iNKT cells ＞ 15%, CD8+Cell ＞ 50%, CD4+Cell ＜ 10%.By Fig. 2 B institutes Show, increased in equal difference mode from rhIL-2 concentration in embodiment 2, in the 14th day iNKT cells ＞ 4%, CD8+Cell ＞ 40%, CD4+Cell ＜ 10%.In sum, a preferred embodiment of the invention is rhIL-2 concentration in equal difference mode Increase, a further preferred embodiment is in wait to increase than mode for rhIL-2 concentration.
The iNKT cells of 5 purification of embodiment are expanded in a large number
(1) the coated Tissue Culture Flask of CD3 monoclonal antibodies should be made at the 13rd day：Prepare 2 μ g/ml CD3 monoclonal antis Body running liquid (0.9% physiological saline solution).Prepare two brand-new 75cm2Big bottle, adds what 5ml had diluted in each big bottle Antibody working solution.Bottleneck is tightened, body is jiggled, makes CD3 monoclonal antibodies diluent fully infiltrate bottom of bottle.Sealed with sealed membrane The good culture bottle mouth of pipe, keeps flat into 4 DEG C of Refrigerator stores.
(2) cultivate the 14th day, iNKT cell sortings are carried out using Anti-iNKT MicroBeads human, collect thin Born of the same parents.
(3) use and contain 100IU/ml IL-2,3 μ g/ml CD28 antibody, 5ng/mlrhIL-7,5ng/mlrhIL-15's INKT cells are fully mixed by AIM V culture medium, note gentle manipulation, it is to avoid produce bubble, prepare cell concentration be not less than 5 × 106The cell suspension of individual/ml, mixes cell.
(4) the coated culture bottle of CD3 monoclonal antibodies is taken out from 4 DEG C of refrigerators, discard liquid, add 0.9% nothings of 10ml Culture bottle of bacterium brine；The cell suspension for mixing is moved into into 75cm2Tissue Culture Flask, gently rocks mixing, puts It is placed in 37 DEG C of incubators and cultivates.
(5) incubation observation of cell state and culture medium color, can supplement appropriate culture medium according to growing state.
(6) cell can be transferred to new culture bottle after CD3 antibody stimulates 3 days, and supplements the culture medium of corresponding volume.
(7) cultivate the 21st day harvesting.
INKT cell expansion ex vivo cell quantity linear graph as seen from Figure 3.PBMC is separated from peripheral blood and is cultivated just The 0th day iNKT cell number that begin is 1 × 104Individual (accounting for PBMC total cellular score 0.1% or so), through culture in 21 days available 1 × 109 Individual iNKT cells.
6 iNKT cell killings tumor cell of liver of embodiment is tested
Effector lymphocyte：The iNKT cells of a large amount of amplifications that embodiment 5 is obtained
Target cell：Hepatoblastoma cell line HepG2, people's differentiated hepatoma cell line Huh7, high transfer hepatoma carcinoma cell It is LM3
Detection iNKT cells kill capacity experimental to tumor cell of liver and use lactic acid dehydrogenase (Lactic Dehydrogenase, LDH) method for releasing.Concrete operation method is as follows:
(1) target cell concentration is adjusted to 1 × 105Individual/ml.
(2) target cell is transferred in 96 orifice plates according to 100 μ l/ holes, three multiple holes of per group of setting.
(3) target cell Spontaneous release hole (negative control) is set.It is not added with effector lymphocyte and only adds 100 μ l culture fluid.
(4) maximum release aperture (positive control) is set.It is not added with effector lymphocyte and only adds 100 μ l10%NP40.
(5) addition 100 μ l of effector lymphocyte, effector lymphocyte in each experimental port：Target cell=0:1；1:1；3:1；10:1； 30:1；50:1.Each hole adds final concentration of 100ng/ml α-GalCer.
(6) 37 DEG C of 5%CO2Culture, cell conditioned medium was collected in 6 hours carries out lactic acid dehydrogenase (LDH) detection.
(7) culture fluid supernatant, detection LDH numerical computations activity are drawn.Killing activity (%)=[(experimental group A values-always certainly A values are discharged so)/(maximum release group A value-total Spontaneous release A values)] × 100%.
Killing experiments are grouped into:1st, DC joints iNKT groups of cells；2nd, iNKT cells independent role group；3rd, PBMC matched groups； 4th, phosphate buffer (PBS) blank control group.
Shown by Fig. 4 results, iNKT cells offer α-GalCer as antigen presenting cell in DC, amplification in vitro can be made INKT cell acute activations, to different hepatoma cell lines can SL, and in the iNKT of nonantigenic presenting cells Cell independent role group due to lacking the stimulation of strong activation signal, when effector lymphocyte's number is less, to target cell killing activity It is weaker, it was demonstrated that the effectiveness of amplification in vitro iNKT cell functions.
Detection iNKT cell IFN-γ secretion abilities use flow cytometry.Concrete operation method is as follows：
(1) according to killing experiments target cell (HepG2 cells are selected in this experiment) concentration and cell number inoculation；
(2) according to effector lymphocyte：Target cell=50:1 Inoculating efficiency cell.Each hole add final concentration of 100ng/ml α- GalCer。
(3) 2 μ l BD GolgiPlug are added per holeTM。
(4) 37 DEG C of 5%CO2Culture, collected cell respectively in 6,12 hours, carries out padding.
(5) rupture of membranes and intracellular dye are carried out according to Fixation/Permeabilization Solution Kit description Color.
(6) fixed cell, carries out Flow cytometry and analysis.
INKT cell IFN-γ secretion capacity experimentals are grouped into:1st, DC joints iNKT groups of cells；2nd, iNKT cells are individually made With group；3rd, PBMC matched groups；4th, phosphate buffer (PBS) blank control group.
Shown by Fig. 5 results, when DC is activated as antigen presenting cell, the ability of secretion of gamma-IFN is obvious for iNKT cells Higher than other groups, it was demonstrated that the effectiveness of the iNKT cell functions of In-vitro specificity amplification, further illustrate using institute of the present invention The iNKT cells for stating method amplification are mainly Th1 sample iNKT cells, with powerful tumor-killing and immune enhancing function.
The content of the publication listed in all this specification is included in this specification.Additionally, people in the art Member is it is appreciated that in the case of without departing substantially from the technical scope and flesh and blood described in claims, carry out to the present invention Various different modifications and change are possible.Present invention additionally comprises above-mentioned these modifications and change.

Claims (12)

1. a kind of Concentraton gradient rhIL-2 is relied on iNKT methods for cell expansion and its application, it is characterised in that culture the 0th day, Add 100ng/ml α-GalCer；Cultivate the 0-11 days, increase rhIL-2 concentration in the way of gradient.
2. method according to claim 1, it is characterised in that the time of fixed interval, rhIL-2 concentration are in equal difference or wait Than increasing；Interlude is 1-72 hours, and rhIL-2 concentration is 10-400IU/ml.
3. the method according to claim 1-2, it is characterised in that culture add within the 7th day same donor inducing culture into Ripe DC；DC before iNKT cells are added was initially charged 100ng/ml α-GalCer for two hours and is incubated；Cultivate use in the 14th day Anti-iNKT MicroBeads human are sorted, and are sorted the iNKT cells for obtaining and are added OKT3 monoclonal antibodies coated Cultivate in culture bottle；CD28 antibody, rhIL-15 and rhIL-7 are added in culture medium；OKT3 MAb concentrations are 1-5 μ g/ Ml, CD28 antibody concentration is 1-5 μ g/ml；RhIL-15, rhIL-7 concentration is 1-10ng/ml.
4. method according to claim 1, it is characterised in that step is：
(1) extract PERIPHERAL BLOOD MONONUCLEAR CELL PBMC；
(2) final concentration of 100ng/ml α-GalCer are added to cultivate in the PBMC cells extracted to step (1) 48 hours；
(3) gradually increase rhIL-2 concentration in culture medium in the way of gradient in step (2) cultured cells.
5. method according to claim 4, it is characterised in that methods described is further comprising the steps of：
(4) the 7th day for cultivating, in step (3) cell culture medium, add same donor inducing culture ripe dendritic cell, Mature dendritic cell is 1 with the quantity ratio of iNKT cells:10；
The preparation method of the mature dendritic cell is：The PBMC that density－gradient centrifuga－tion method is obtained is resuspended with AIM V culture medium, In adding Tissue Culture Dish, 37 DEG C of 5%CO2Culture；Culture dish was taken out in 3 hours, rocked, suction out suspension cell；To culture dish Middle addition rhIL-4 containing 500IU/ml, the AIM V culture medium of 500IU/ml rhGM-CSF；Respectively after incubation the 3rd day, the 5th Its half amount changes liquid, adds fresh rhIL-4 containing 500IU/ml, the AIM V culture medium of 500IU/ml rhGM-CSF；Cultivate the 6th day Add final concentration of 10ng/ml rhTNF- α；Harvest mature dendritic cell within 7th day.
6. method according to claim 5, it is characterised in that mature dendritic cell is adding step (3) cell culture Before base, with AIM V culture medium it is resuspended and add final concentration 100ng/ml α-GalCer be incubated 2 hours.
7. method according to claim 4, it is characterised in that methods described is also comprised the steps of：
(5) cultivating the 14th day, iNKT cell sortings being carried out using Anti-iNKT MicroBeads human, sorting is obtained into thin Born of the same parents are cultivated in the coated culture bottle of CD3 antibody, add CD28 antibody, rhIL-15 and rhIL-7 to cultivate in 37 DEG C in culture medium Cultivate in case.
8. method according to claim 7, it is characterised in that CD3 antibody concentration is 1-5 μ g/m, preferably 2 μ g/ml.
9. method according to claim 7, it is characterised in that add CD28 antibody concentration to be 1-5 in step (5) culture medium μ g/ml, preferably 3 μ g/ml；RhIL-15 concentration is 1-10ng/ml, preferably 5ng/ml；RhIL-7 concentration is 1- 10ng/ml, preferably 5ng/ml.
10. method according to claim 7, it is characterised in that cell is transferred to after stimulating 3 days by step (5) CD3 antibody New culture bottle, and the culture medium of corresponding volume is supplemented, cultivate the 21st day harvesting.
11. methods according to any claim in claim 1-10, it is characterised in that methods described is in culture the 21st It harvests iNKT cells, and iNKT cell number improves 100,000 times, wherein more than 90% is CD4-INKT cells, it is anti-with immunity is raised Should be with the effect for killing tumor.
The iNKT lymphocytes that 12. methods according to any one of claim 1-11 are prepared are preparing antitumor drug In purposes.
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CN113462646A (en) * 2021-06-30 2021-10-01 徐州医科大学 Simple and effective method for induced amplification of iNKT cells and application
CN117511868A (en) * 2023-12-06 2024-02-06 承德合润生物科技有限公司 Method for realizing quick amplification of iNKT cells

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AU2019300782B2 (en) * 2018-07-10 2023-12-21 Immunitybio, Inc. Generating CIK NKT cells from cord blood

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113462646A (en) * 2021-06-30 2021-10-01 徐州医科大学 Simple and effective method for induced amplification of iNKT cells and application
CN117511868A (en) * 2023-12-06 2024-02-06 承德合润生物科技有限公司 Method for realizing quick amplification of iNKT cells

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