CN104357390A - Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells - Google Patents
Method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells Download PDFInfo
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Abstract
The invention discloses a method for simultaneous and efficient amplification of CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells. The method comprises the steps as follows: the concentration of separated PBMC (peripheral blood mononuclear cells) is adjusted by a serum-free medium containing autologous plasma, an Anti-CD16 antibody, IL-2 and IL-15 are added, and then the mixture is transferred into a T175 culture flask for culture; an Anti-CD3 antibody and an Anti-CD137 antibody are added; a serum-free medium containing the autologous plasma, IL-2 and IL-15 is supplemented every two days according to the cell growth condition; the cell concentration is controlled to be about 1.5*10<6>/ml; and after culture is performed for 14-21 days, large quantities of high-purity CD<3+>CD<56+>CIK cells and CD<3->CD<56+>NK cells can be obtained simultaneously, and the total cell quantity can reach an effective value of the cell quantity required for adoptive cellular immunotherapy clinically for tumor. The method for simultaneous and efficient amplification of the CD<3+>CD<56+>CIK cells and the CD<3->CD<56+>NK cells is simple, convenient, effective and high in cell killing activity.
Description
Technical field
The invention belongs to immunocyte vitro culture field, relate to a kind of by peripheral blood mononuclear cell (PBMC) efficient amplification CD simultaneously
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell.
Background technology
Malignant tumour is the disease of serious harm human health, and its mortality ratio is in ascendant trend year by year.The traditional treatment means of tumour mainly comprise operation, chemotherapy and radiotherapy.But traditional treatment means kills and wounds shortage specificity to tumour cell, also has damage, along with obvious toxic side effects to normal histocyte.In recent years, along with the development of immunology and Protocols in Molecular Biology, the 4th kind of pattern, i.e. biotherapy of oncotherapy of being born.Biotherapy can more specific killing off tumor cells, normal tissue cell not damaged, and the immunity system by activating patient self produces recurrence and the transfer of immunological memory effect and then prophylaxis of tumours, is the useful supplement of operation, chemotherapy, radiotherapy.
In the biotherapy of tumour, a kind of important method is exactly adoptive cellular immunotherapy (Adoptive cellular immunotherapy, ACI), namely by being separated patient's autoimmunization cell, the external induced activation through cytokine profiles or by genetic modification after increase, obtain the immunocyte in a large number with powerful anti-tumor activity and feed back to by direct killing tumour cell in patient body, or by the immune response activating body killing tumor cell indirectly.Cellular immunotherapy is passable, the risk of effective reduction Malignant Tumor Recurrence and transfer, comprehensive amplification body's immunity is to inhibiting tumor cell, to remove after traditional treatment free cancer cell in MRD and body, the remarkable quality of life improving patient, the lifetime of effective prolongation patient, and without any side effects and treatment misery, be united and applied in clinical anticancer excellent with traditional treatment and more and more receive publicity.
The immunocyte applied in biotherapy mainly contains: Tumor-infiltrating lymphocytes (lymPhokine activated killer, LAK), tumor infiltrating lymphocyte (tumor infiltrating lymphocytes, TIL), cytokine induced kill cell (cytokine induced killer cell, and natural killer cell (natural killer cell CIK), NK), NKT cell (Natural Killer T cells, NKT) etc., high with its tumor cell killing activity, kill and wound the advantages such as spectrum is wide, important clinical value is demonstrated in the complex therapy of tumour.
CIK cell (cytokine induced kill cell) is with CD
3+cD
56+two positive cell is the cell that a group of main effects cell has non-specific killing ability.Because its main effects cell expresses CD3 and CD56 two kinds of membrane protein molecules simultaneously, therefore be also called NK cell sample T lymphocyte, with the anti-tumor activity that T lymphocyte is powerful, and the non-MHC of NK cell is restricted kills knurl advantage.Kill that tumor activity is high, to kill knurl spectrum wide because CIK cell has, normal tissue toxicity is low, the external feature such as can highly to increase, and is widely used adoptive immunotherapy cell clinically.
NK cell (natural killer cell), it is the class non-specific immunity cell belonging to lymphocyte linage, it need not contact antigen in advance and can kill and wound by the host cell of virus infection, it is the Major Members of human immune system, not only resist virus infection to play an important role, and the NK cell after activation can synthesize and secrete cytokine profiles, closely related with the antitumor of body and immunoloregulation function, can extensively identify, rapid dissolving, kill and wound, destroy cancer cells, to metastases and recurrence arch-criminal---tumor stem cell has significant lethal effect.
Because CIK, NK cell has powerful anti-tumor activity, in recent years, along with the progress of immunocyte technology of preparing and the cognition of people to immune cell therapy and the raising of acceptance, CIK and NK cell is all widely used in clinical cancer therapy, but be all increased respectively by external CIK, NK immunocyte to patient, blood sampling volume is large, and high in cost of production factor becomes the bottleneck of various kinds of cell immunotherapy development.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is a kind of method proposing new immunocyte amplification in vitro, and the method can utilize a culture systems, obtains simultaneously widely apply higher, the active strong CD of safety, purity under condition of in vitro culture
3+cD
56+cIK cell and CD
3-cD
56+nK cell.
According to an aspect of the present invention, the invention provides a kind of efficient amplification CD simultaneously
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell.According to the embodiment of the present invention, steps of the method are:
0th day, get peripheral blood, isolate PBMC with lymphocyte separation medium, PBMC separation obtained is 2 × 10 by the first substratum adjustment PBMC concentration
6/ ml, makes PBMC cell suspension, then adds Anti-CD16 antibody, IL-2 and IL-15 toward PBMC cell suspension, proceeds to afterwards in culturing bottle in 37 DEG C, 5%CO
2, start under 100% humidity condition to cultivate, described first substratum is the serum free medium containing autologous plasma;
1st day, cell cultures added Anti-CD3 antibody after 24 hours, Anti-CD137 antibody, at 37 DEG C, 5%CO
2, cultivate under 100% humidity condition;
3rd day, in cell suspension, half amount added the second substratum, and described second substratum is the serum free medium containing autologous plasma, IL-2, IL-15;
5th day, centrifugal collecting cell, and with described second substratum, cell is resuspended and adjust cell concn to 1.5 × 10
6/ ml, is transferred in cell culture apparatus;
Continue to cultivate and results: later every 2 days sampling counting cells, supplement described second substratum according to count results in described cell culture apparatus, adjustment cell density is 1.5 × 10
6/ ml, 37 DEG C, 5%CO
2, cultured continuously after 14 ~ 21 days under 100% humidity condition, the CD that collected by centrifugation obtains
3+cD
56+cIK cell and CD
3-cD
56+nK cell.
Contriver is surprised to find, method of the present invention is utilized only to utilize a culture systems, under condition of in vitro culture, higher, the active strong CD of safety, purity can be widely applied from autologous peripheral blood mononuclear cell (PBMC) efficient amplification acquisition simultaneously in a culturing step
3+cD
56+cIK cell and CD
3-cD
56+nK cell.In addition, according to embodiments of the invention, method of the present invention is simple to operation, and cost is lower, is suitable for wide popularization and application.
Wherein, it should be noted that, term " autologous plasma " used in this application refers to, the blood plasma of the donor identical with described derived from peripheral blood.
According to embodiments of the invention, in described first substratum and described second substratum, the concentration of autologous plasma is 1%-20% volume.According to of the present invention one concrete example, in described first substratum and described second substratum, the concentration of autologous plasma is 1 volume %.Thus, object cell amplification is effective.
According to embodiments of the invention, in described first substratum and described second substratum, the final concentration of described Anti-CD16 antibody is 50ng ~ 1mg/ml.According to of the present invention one concrete example, in described first substratum and described second substratum, the final concentration of described Anti-CD16 antibody is 500ng/ml.Thus, object cell amplification is effective.
According to embodiments of the invention, in described first substratum and described second substratum, the final concentration of described IL-2 is 1000 ~ 6000U/mL.According to of the present invention one concrete example, in described first substratum and described second substratum, the final concentration of described IL-2 is 1000U/mL.Thus, object cell amplification is effective.
According to embodiments of the invention, in described first substratum and described second substratum, the final concentration of described IL-15 is 1ng/ml ~ 100ng/ml.According to of the present invention one concrete example, in described first substratum and described second substratum, the final concentration of described IL-15 is 20ng/ml.Thus, object cell amplification is effective.
According to embodiments of the invention, the 1st day, the final concentration of the described Anti-CD3 antibody that cell cultures added after 24 hours was 10ng ~ 1mg/ml.According to of the present invention one concrete example, the 1st day, the final concentration of the Anti-CD3 antibody that cell cultures added after 24 hours was 50ng/ml.Thus, object cell amplification is effective.
According to embodiments of the invention, the 1st day, the final concentration of the described Anti-CD137 antibody that cell cultures added after 24 hours was 10ng ~ 1mg/ml.According to of the present invention one concrete example, the 1st day, the final concentration of the Anti-CD137 antibody that cell cultures added after 24 hours was 500ng/ml.Thus, object cell amplification is effective.
According to embodiments of the invention, described cell culture apparatus is G-REX 100L culture apparatus.Wherein, it should be noted that, described cell culture apparatus is closed state in culturing process.
According to embodiments of the invention, continue in the step cultivated and gather in the crops, when the total cell count of culture system is not less than 5x10 described
9time, the CD that collected by centrifugation obtains
3+cD
56+cIK cell and CD
3-cD
56+nK cell.Object cell is obtained thereby, it is possible to effectively collect.
According to some embodiments of the present invention, the X-VIVO15 of GCT551 and LONZA that described serum-free culture can be AIM-V serum free medium, Japan cures precious day, at least one of the BIOTARGET-1 Without L-Glutamine substratum of BI company of Israel.
In addition, according to embodiments of the invention, the two kinds of cells obtained by method of the present invention, may be used for preparing cell therapy product, and then for clinical tumor adoptive cellular immunotherapy.
Efficient amplification CD while of the present invention
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, owing to just can induce and amplify 2 kinds of effector cells in a culture system simultaneously, need to implement culturing step abreast to 2 kinds of effector cells relative to traditional method simultaneously, its cost reduces, and method is simple, easy handling, amplification efficiency is high, be easy to promote, relative to the CIK cell that widely used ordinary method is cultivated, the killing activity of cell mass and the secretion of cytokine are all improved largely.
Wherein, it should be noted that, " the 0th day " of the present invention, refer to the same day of sampling, within the 1st day, refer to the first day after sampling, the 3rd day, the 5th day ... by that analogy.
In addition, " CD of the present invention
3+cD
56+cIK cell " refer to and be labeled as CD by flow cytomery cell surface molecule
3+cD
56+cIK cell (i.e. cytokine induced kill cell is also called NK cell sample T lymphocyte), " CD
3-cD
56+nK cell " refer to and be labeled as CD by flow cytomery cell surface molecule
3-cD
56+nK cell (i.e. natural killer cell).
According to concrete examples more of the present invention, method of the present invention is easy, effective, can in a culturing step from autologous peripheral blood mononuclear cell (PBMC) simultaneously efficient amplification obtain CD
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, and compared with the cultural method of routine, the present invention at least tool has the following advantages:
1, have employed Novel cell culture device G-rex 100L in step, the ventilation of its special bottom improves ordinary method Tissue Culture Flask or cell culture bags culturing process causes the problems such as cellular respiration difficulty due to the continuous increase of culture volume, significantly improves cell amplification efficiency.
2, in step only by Anti-CD3, Anti-CD16, Anti-CD137 tri-kinds of Antibody Combination, achieve CD in a culture systems
3+cD
56+cIK cell and CD
3-cD
56+activate while NK cell.CD
3+cD
56+cIK main cell is differentiated by the T cell of induced activation, and the first signal that Anti-CD3 antibody provides T cell activation as antigenic stimulation carrys out activated T cell; CD
3-cD
56+nK cell, neither B cell neither T cell, but CD
3-cD
56+nK cell can express IgGFc acceptor Fc γ R III (CD16) of low affinity, CD
3-cD
56+nK cell lacks CD3 molecule, but it can express the ζ chain molecule of CD3, and forms IgGFc low-affinity receptor to activate NK cell together with CD16; CD137/CD137L is then one of TNFR/TNF superfamily member, and interacting that the costimulatory signal that jointly mediates is same with anti-CD3 and anti-CD16 antibody can the propagation of irritation cell.Method of the present invention, stimulates T cell and NK cell high-throughput activatable propagation by the synergy that three kinds of monoclonal antibodies are mutual, thus makes to obtain the CD met clinical needs in a culture systems simultaneously
3+cD
56+cIK cell and CD
3-cD
56+nK cell quantity.
3, just achieve CD only by IL-2 and IL-15 two kinds of cytokines in step
3+cD
56+cIK cell and CD
3-cD
56+the amplification of NK cell, the IL-2R α chain that IL-2 can stimulate NK cell expressing a large amount of, thus NK cell is bred in a large number, and under IL-2 stimulates NK cell expressing adhesion molecule, make CD
3-cD
56+particle in NK kytoplasm increases and promotes the expression of serine easterase mRNA, thus improves CD
3-cD
56+the cytotoxic activity of NK cell; IL-15 is a kind of multi-functional cytokine, is the decisive factor that hemopoietic forebody cell is grown to NK cell directional, has promotion CD
3-cD
56+nK hyperplasia, raises NK cytotoxic activity, promotes that the various cytokine of NK emiocytosis participates in immunomodulatory and chemotaxis, by the mutual synergy between two kinds of factors, not only can promote CD
3-cD
56+the amplification of NK cell, can also increase substantially CD
3+cD
56+the cytotoxic activity of CIK cell.
4, combination of cytokines induced amplification CD is simultaneously utilized
3+cD
56+cIK cell and CD
3-cD
56+nK cell technology, can improve the CD in traditional C IK colony
3+cD
56+cIK cell and CD
3-cD
56+nK cell content, works in coordination with and strengthens the antitumor curative effect of immunocyte.
5, the cell that obtains of the inventive method, has the ability of killing activity and secrete cytokines, and cell cultures whole process adopts serum free medium, ensures, outside cell quantity and activity, to add the security of clinical application.
Accompanying drawing explanation
Fig. 1 is conventional group and the cultivated days of experimental group two kinds of different methods gained cells and the graph of a relation of cell proliferation multiple;
In Fig. 2, left figure is that stream type cell analyzer detection conventional group cell expresses CD respectively
3+cD
56+cIK cell and expression CD
3-cD
56+nK percentage of cells figure, right figure are that experimental group cell expresses CD respectively
3+cD
56+cIK cell and expression CD
3-cD
56+nK percentage of cells figure;
In Fig. 3, left figure is the percentage of cells figure of secrete cytokines IFN-γ after stream type cell analyzer test experience group cell and target cell effect, and right figure is the percentage of cells figure of experimental group cell Autocrine cytokine IFN-γ;
In Fig. 4, left figure is the percentage of cells figure that stream type cell analyzer detects secrete cytokines IFN-γ after conventional group cell and target cell effect, and right figure is the percentage of cells figure of experimental group cell Autocrine cytokine IFN-γ;
The percentage of cells figure of secrete cytokines CD107a after left figure stream type cell analyzer test experience group cell and target cell effect in Fig. 5, right figure is the percentage of cells figure of experimental group cell Autocrine CD107a;
In Fig. 6, left figure is the percentage of cells figure that stream type cell analyzer detects secrete cytokines CD107a after conventional group cell and target cell effect, and right figure is the percentage of cells figure of experimental group cell Autocrine CD107a.
Embodiment
Be described below in detail embodiments of the invention, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has element that is identical or similar functions from start to finish.Be exemplary below by the embodiment be described with reference to the drawings, be intended to for explaining the present invention, and can not limitation of the present invention be interpreted as.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1:
One, from peripheral blood PBMC cell efficient amplification CD simultaneously
3+cD
56+cIK cell and CD
3-cD
56+the method (hereinafter referred to as experimental group) of NK cell.
0th day:
1 gathers healthy human peripheral blood (100ml), with lymphocyte separation medium (Axis-Shield company, Norway) by gradient centrifugation separating peripheral blood mononuclear cells, collects the yellow blood plasma in separator tube upper strata as autologous plasma simultaneously.
2 use phosphate buffered saline (PBS) (PBS) (GIBCO company, the U.S.), washed cell twice, refuse dye method detect cytoactive and count with trypan blue (GIBCO company, the U.S.).
3, by separating obtained peripheral blood lymphocytes 1.17x108 cell suspension (GIBCO company, U.S.) in containing the AIM-V serum free medium of culture volume 1% autologous plasma, make cell initial inoculation concentration be 2.5X106/ml, altogether 47ml.
Step 3 gained peripheral blood lymphocytes (2.5X106/ml, altogether 47ml) is seeded to a 175cm by 4
2in Tissue Culture Flask (Greiner Bio-one Germany).
Anti-CD16 antibody is that 500ng/ml is added in the cell suspension of step 4 with final concentration by 5, and at 37 DEG C, 5%CO2, cultivates 24 hours under 100% humidity.
1st day:
Anti-CD3 antibody, Anti-CD137 antibody were added in cell suspension after 24 hours by 6 cell cultures, made Anti-CD3 antibody final concentration 50ng/ml, Anti-CD137 antibody final concentration 500ng/ml, at 37 DEG C, and 5%CO
2, continue under 100% humidity to cultivate.
2nd day:
By platform, 7 expect that orchid refuses dye method counting cells and statistics motility rate every sampling in 2 days, according to cell counting situation, add and contain
The AIM-V serum free medium of culture volume 1% autologous plasma, 1000U/ml IL-2 and 20ng/ml IL-15, adjustment cell density is 1.5 × 106/ml, 37 DEG C, 5%CO
2, continue under saturated humidity to cultivate.
5th day:
8 centrifugal collecting cells, cell is resuspended and adjust cell density to 1.5 × 106/ml containing the AIM-V serum free medium of culture volume 1% autologous plasma, 1000U/ml IL-2 and 20ng/ml IL-15, proceed in G-rex 100L (wilson company, the U.S.) cell culture apparatus
6-14 days:
Every 2 days sampling counting cells after 9, contain the AIM-V serum free medium of 1 volume % autologous plasma, 1000U/ml IL-2 and 20ng/ml IL-15 toward supplemented medium in the cell culture apparatus closed according to count results, adjustment cell density is 1.5 × 106/ml, 37 DEG C, 5%CO
2, continue to be cultured to the 14th day under saturated humidity.
Two, conventional amplification CIK cultural method (hereinafter referred to as conventional group) from peripheral blood PBMC cell
It is 1 × 10 that the peripheral blood mononuclear cell AIM-V serum free medium of collection is adjusted concentration
6u/ml, adds IFN-γ on the 0th day, makes IFN-γ final concentration in the medium be 1000U/ml, subsequently nutrient solution is placed in 37 DEG C, CO
2concentration is 5%, and humidity is cultivate 24 hours in the constant incubator of 100%;
Within 1st day, add IL-1 α, IL-2 and Anti-CD
3antibody, makes IL-1 α, IL-2 and Anti-CD
3the final concentration of antibody in AIM-V serum free medium is respectively 300U/ml, 1000U/ml and 500U/ml, continues at 37 DEG C, CO
2concentration is 5%, and humidity is cultivate in the constant incubator of 100%;
Later every 2 days sampling counting cells, add the fresh AIM-V serum free medium containing IL-2 according to count results, make IL-2 be 1000U/ml at the new final concentration added in substratum, adjustment cell concn remains on 1.5 × 10
6/ ml, proceeds to cell culture bags and is cultured to the 14th day after volume of culture is greater than 200ml.
Three, result test
Collected at the 14th day and be used for every test by experimental group described in the present embodiment and conventional prescription method institute cultured cells.
The CD that this two kinds of preparation methods obtain is compared from the following aspects
3+cD
56+cIK cell and CD
3-cD
56+the otherness of NK cell, the conventional group of CD being the method provided with the present embodiment step 2 and preparing
3+cD
56+cIK cell and CD
3-cD
56+nK cell, experimental group is CD prepared by the method provided with the present embodiment step one
3+cD
56+cIK cell and CD
3-cD
56+nK cell.
1: the mensuration of cell proliferation multiple
Count with blood counting chamber after being dyeed by the cell trypan blue obtained (GIBCO, the U.S.), by current total cellular score divided by the mononuclearcell number before cultivation, numerical value is the amplification times of cell again.Can the proliferative conditions of dynamic observation of cell by this method, particular case is shown in Fig. 1, and as can be seen from Figure 1, experimental group is suitable with conventional group cell proliferation rate, and total cellular score can meet clinical application.
2: flow cytometer detection two groups of cells (express CD
3+cD
56+cIK cell and expression CD
3-cD
56+nK cell) phenotype analytical
Two groups of cells respectively get 5 × 10 on the 14th day what cultivate
5cell is in 1.5ml centrifuge tube, and centrifugal collecting cell, PBS washs 2 times, adds 100 μ l PBS re-suspended cells, then adds FITC Mouse Anti-Human CD
3detect antibody 20 μ l (BD Pharmingen, the U.S.), PE Mouse Anti-Human CD
56detect antibody 20 μ l (BD Pharmingen, the U.S.) carry out double-tagging, 20 minutes are hatched in room temperature dark place, PBS washes 2 times, wash away Excess antibody, finally use 500ul PBS that cell is resuspended, by stream type cell analyzer (Millipore guava easyCyte 6HT-2L, the U.S.) measure, the results are shown in Figure 2.The result display of Fig. 2, the CD of experimental group
3+cD
56+cIK cell and CD
3-cD
56+nK cell percentages example is all greatly improved than conventional group.
3: the expression level of cytokine IFN-γ in flow cytometer detection two groups of cells
In the K562 tumour cell (China typical culture collection center, Wuhan) of taking the logarithm vegetative period to aseptic 15ml centrifuge tube, 1500rpm, centrifugal 5 minutes, abandon supernatant, cell precipitation is resuspended with 1640 substratum containing culture volume 10%FBS, and adjustment cell concn is 5 × 10
6/ ml, inoculating cell is in 96 hole flat undersides, and 100 μ l/ holes, inoculate 2 holes, be placed in 37 DEG C, CO
2concentration is 5%, humidity be in the constant incubator of 100% cultivate 24 hours stand-by as target cell;
Collecting the cultivation experimental group of 14 days and conventional group culturing cell respectively, is 5 × 10 with the 1640 substratum adjustment cell densities containing culture volume 10%FBS
6/ ml, action effect cell.100 μ l are respectively got, 5 × 10 after pipettor piping and druming mixing cell
5cell adds to 5 × 10 of 100 μ l in aforementioned 96 orifice plates
5in target cell, make effect target than being 1:1 (effect target ratio: the ratio of effector cell's quantity and target cell numbers, below herewith), BFA 1 μ l (Protein Transport Inhibitor (Containing Brcfcldin A) is added by every ml cell suspension, BD Pharmingen, U.S.), and set two groups of cells not reacting with tumour cell as effector cell's blank, 37 DEG C, 5%CO
2cell mixing 1x10 is respectively got after hatching 4 hours altogether
6, PBS washes 2 times, often pipe adds PE Mouse Anti-Human CD56 detection antibody 20 μ l (BD Pharmingen, the U.S.), incubated at room 30min, be fixed agent 500 μ l (fixation/permeabilization Kit, BD Pharmingen, the U.S.) incubated at room 15min, PBS washes, add rupture of membranes agent 500 μ l (fixation/permeabilization Kit, BD Pharmingen, the U.S.) room temperature 5Min, add FITC Mouse Anti-HumanIFN-γ and detect antibody 20 μ l (BD Pharmingen, the U.S.), incubated at room 15min, PBS washes 2 times, finally use 500 μ lPBS that cell is resuspended, CD is measured by stream type cell analyzer
56+in cell, the expression level of IFN-γ, the results are shown in Figure 3, Fig. 4.
Final cell expresses the spontaneous expression rate of IFN-γ positive rate (the %)-IFN-γ (%) that IFN-γ percentage ratio method of calculation=add target cell stimulates
According to final express cell IFN-γ percentage ratio method of calculation, as can be seen from Figure 3, CD in experimental group cell
56+the percentage ratio that effector cell expresses IFN-γ is 34.18%, and CD in conventional group in Fig. 4
56+the percentage ratio that effector cell expresses IFN-γ is only 1.28%, and the cell count of experimental group cell expressing IFN-γ is 26.7 times of conventional group, and illustrative experiment group cell cytokine IFN-γ expression level is higher.
4: retting conditions (the expressing CD107a) ability of flow cytometer detection two groups of cells
Lysosomal associated membrane albumen-1 (LAMP-1 or CD107a) is a kind of albumen of high-glycosylation, accounts for 50% of lysosomal membrane protein53.CD107a molecule is expressed on normal NK cells and CIK surface hardly, but when NK and CIK main effects cell killing target cell, toxic granulations will arrive serosal surface and and cell membrane fusion, granular contents is caused to discharge, finally cause the death of target cell, along with degranulated generation, CD107a molecule is transported to surface of cell membrane, and the up-regulated of CD107a molecule and the secretion of pore-forming protein consistent.Therefore, the CD of CD107a molecule positive expression
3+cD
56+cIK cell and CD
3-cD
56+nK cell can represent the effector cell with killing activity.
In the K562 tumour cell (China typical culture collection center, Wuhan) of taking the logarithm vegetative period to aseptic 15ml centrifuge tube, 1500rpm, centrifugal 5 minutes, abandon supernatant, cell precipitation is resuspended with 1640 substratum containing culture volume 10%FBS, and adjustment cell concn is 5 × 10
6/ ml, inoculating cell is in 96 hole flat undersides, and 100 μ l/ holes, inoculate 2 holes, be placed in 37 DEG C, CO
2concentration is 5%, humidity be in the constant incubator of 100% cultivate 24 hours stand-by as target cell;
Collecting the cultivation experimental group of 14 days and conventional group culturing cell respectively, is 5 × 10 with the 1640 substratum adjustment cell densities containing culture volume 10%FBS
6/ ml, action effect cell.100 μ l are respectively got, 5 × 10 after pipettor piping and druming mixing cell
5cell adds to 5 × 10 of 100 μ l in aforementioned 96 orifice plates
5in target cell, make effect target than being 1:1, monensin 0.67 μ l (Protein Transport inhibitor (containing Monensin) is added by every ml cell suspension, BD Pharmingen, the U.S.), and set two groups of cells not reacting with tumour cell as effector cell's blank, and 37 DEG C, 5%CO
2after hatching 1.5 hours altogether, respectively get cell mixing 1x10
6, PBS washes 2 times, and often pipe adds APC Mouse Anti-Human CD56 detection antibody 20 μ l, PE Mouse Anti-Human CD107a detects antibody 20 μ l (BD Pharmingen, the U.S.), incubated at room 30 min, PBS washes 2 times, measures CD by stream type cell analyzer
56+the expression level of CD107a in cell, the results are shown in Figure 5 and 6.
Final cell expresses the spontaneous expression rate of CD107a positive rate (the %)-CD107a (%) that CD107a percentage ratio method of calculation=add target cell stimulates
CD107a percentage ratio method of calculation are expressed according to final cell, as can be seen from Figure 5, CD56 in experimental group cell
+the percentage ratio that effector cell expresses CD107a is 22.45%, and CD in conventional group in Fig. 6
56+the percentage ratio that effector cell expresses CD107a is only 6.32%, and the cell count of experimental group cell expressing CD107a is 3.55 times of conventional group, and the lethal effect of illustrative experiment group cells against tumor cells is more powerful.
Although illustrate and describe embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention when not departing from principle of the present invention and aim, revising, replacing and modification.
Claims (9)
1. a simultaneously efficient amplification CD
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, step is:
0th day, get peripheral blood, isolate PBMC with lymphocyte separation medium, PBMC separation obtained is 2 × 10 by the first substratum adjustment PBMC concentration
6/ ml, makes PBMC cell suspension, then adds Anti-CD16 antibody, IL-2 and IL-15 toward PBMC cell suspension, proceeds to afterwards in culturing bottle in 37 DEG C, 5%CO
2, start under 100% humidity condition to cultivate, described first substratum is the serum free medium containing autologous plasma;
1st day, cell cultures added Anti-CD3 antibody after 24 hours, Anti-CD137 antibody, at 37 DEG C, 5%CO
2, cultivate under 100% humidity condition;
3rd day, in cell suspension, half amount added the second substratum, and described second substratum is the serum free medium containing autologous plasma, IL-2, IL-15;
5th day, centrifugal collecting cell, and with described second substratum, cell is resuspended and adjust cell concn to 1.5 × 10
6/ ml, is transferred in cell culture apparatus;
Continue to cultivate and results: later every 2 days sampling counting cells, supplement described second substratum according to count results in described cell culture apparatus, adjustment cell density is 1.5 × 10
6/ ml, 37 DEG C, 5%CO
2, cultured continuously after 14 ~ 21 days under 100% humidity condition, the CD that collected by centrifugation obtains
3+cD
56+cIK cell and CD
3-cD
56+nK cell.
2. efficient amplification CD while of according to claim 1
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, in described first substratum and described second substratum, the concentration of autologous plasma is 1%-20% volume, preferably 1 volume %.
3. efficient amplification CD while of according to claim 1
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, in described first substratum and described second substratum, the final concentration of described Anti-CD16 antibody is 50ng ~ 1mg/ml, preferred 500ng/ml.
4. efficient amplification CD while of according to claim 1
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, in described first substratum and described second substratum, the final concentration of described IL-2 is 1000 ~ 6000U/mL, preferred 1000U/mL.
5. efficient amplification CD while of according to claim 1
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, in described first substratum and described second substratum, the final concentration of described IL-15 is 1ng/ml ~ 100ng/ml, preferred 20ng/ml.
6. efficient amplification CD while of according to claim 1
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, the 1st day, and the final concentration of the described Anti-CD3 antibody that cell cultures added after 24 hours is 10ng ~ 1mg/ml, preferred 50ng/ml.
7. efficient amplification CD while of according to claim 1
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, the 1st day, and the final concentration of the described Anti-CD137 antibody that cell cultures added after 24 hours is 10ng ~-1mg/ml, preferred 500ng/ml.
8. efficient amplification CD while of according to claim 1
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, described cell culture apparatus is G-REX100L culture apparatus.
9. efficient amplification CD while of according to claim 1
3+cD
56+cIK cell and CD
3-cD
56+the method of NK cell, is characterized in that, continues in the step cultivated and gather in the crops, when the total cell count of culture system is not less than 5x10 described
9time, the CD that collected by centrifugation obtains
3+cD
56+cIK cell and CD
3-cD
56+nK cell.
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