CN104357391A - Method for simultaneously inducing and amplifying V alpha<24+>iNKT cells and CD<3->CD<56+>NK cells - Google Patents
Method for simultaneously inducing and amplifying V alpha<24+>iNKT cells and CD<3->CD<56+>NK cells Download PDFInfo
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Abstract
The invention discloses a method for simultaneously inducing and amplifying V alpha<24+>iNKT cells and CD<3->CD<56+>NK cells. The method comprises the steps as follows: PBMC (peripheral blood mononuclear cells) are separated from peripheral blood, the concentration of the PBMC is adjusted to 2*10<6>/ml by a serum-free medium containing autologous plasma; an Anti-CD<16> antibody, -GalCer, IL-2, IL-18 and IL-21 are added, and then the mixture is transferred into a culture flask for culture; an Anti-CD3 antibody is added in a cell suspension in 24 hours; a serum-free medium containing IL-2, IL-18 and IL-21 is supplemented every two days according to the cell growth condition; the cell concentration is controlled to be 1.5*10<6>/ml; and after continuous culture is performed for 14-21 days, large quantities of high-purity V alpha<24+>iNKT 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 V alpha<24+>iNKT cells and the CD<3->CD<56+>NK cells is simple, convenient and effective.
Description
Technical field
The invention belongs to immunocyte vitro culture field, relate to a kind of by human peripheral blood single nucleus cell (PBMC) induced amplification V α simultaneously
24+iNKT cell and CD
3-cD
56+the method of NK cell.
Background technology
Malignant tumour is the major disease of serious threat our people health.The traditional means for the treatment of malignant tumour comprises operative treatment, radiotherapy (abbreviation radiotherapy) and chemotherapy (abbreviation chemotherapy).Three kinds of therapies respectively have limitation: operative treatment can not remove the micrometastasis stove of tumour, are difficult to obtain radical-ability curative effect; Radiotherapy has dose-dependent toxicity, cannot effectively remove potential metastasis, in addition, some patients is insensitive to radiotherapy because producing radiation opposing, chemotherapy easily causes general immunity to suppress, and some patients effectively cannot enter tumor tissues because of chemotherapeutics or patient produces resistance to chemotherapeutics and affects the treatment.Think at present, three major causes for the treatment of malignant tumor failure are: topical therapeutic is not thorough, and thus tumour occurs that distant metastasis and tumour patient immunity system are suppressed cannot effectively identify and remove remaining tumour cell.As can be seen here, clinical cancer therapy is in the urgent need to introducing novel methods for the treatment of.Along with deepening continuously to immunology and oncology studies, people profoundly recognize that the generation of tumour and development and the immunologic function of human body are closely related, thus after operation, radiotherapy, Chemotherapy in Treating Malignant Tumor, propose the concept of adoptive cellular immunotherapy (Adoptive cellular immunotherapy, ACI).
Adoptive cellular immunotherapy is that autologous patient or allosome lymphocyte are cultivated with cytokine profiles the immunocyte that a group activating or carry out to produce after genetic modification is also increased has anti-tumor activity in vitro, feed back in patient body, direct killing tumour cell or enhancing patient autoimmune function, thus play a kind of methods for the treatment of of antitumor action.The advantage of this therapy is the residual after removing operation, radiotherapy, chemotherapy; Lower recurrence or the possibility sent out to improve curative ratio; The immunity function strengthening patient improves curative ratio; Improve the quality of life of patient.Because of the toxic side effect that it is almost equal to zero, by the good reputation titled with " green remedy ", now become continue " operation, radiotherapy, chemotherapy " three great tradition treatment after " the fourth-largest therapy ".Mainly containing for adoptive cellular immunotherapy at present: 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), iNKT cell (invariant Natural Killer T cells, iNKT) 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.
Natural killer cell (natural killer cell, NK) and iNKT cell (invariant Natural Killer T cells, iNKT) are the important composition members of natural immune system.Wherein NK cell is mainly distributed in peripheral blood, account for the 5%-10% of peripheral blood lymphocyte ratio, participate in body anti-infective, prevent the effect such as malignant change of cell and immunity moderation, in antitumor action, it identifies, kills and wounds that the mode of target cell is different from T lymphocyte, for " non-MHC is restricted ", identify in advance without the need to tumour specific antigen and get final product direct killing tumour cell, effectively identify and the cell removed with MHC-I quasi-molecule defect or tumour cell, Tumor suppression immunologic escape effect; In the process removing tumour cell, NK cell also can express multiple can with the acceptor of MHC or non-MHC ligand binding, suppress or activation signals and regulate NK cytoactive by transmitting, produce simultaneously a large amount of with kill and wound relevant cytokine, thus performance immunoregulation effect.And iNKT cell, be the cell of a group between NK cell and T cell, cell surface had both expressed φt cell receptor TCR, also expressed NK cell receptor.This cell has strict CD1d restricted, and there is the ability of a large amount of release IL-4 and IFN-, iNKT cell not only itself has effect that is antitumor, antiviral and suppression autoimmune disorder, and can also stimulate the activity of the immunocytes such as T, NK, strengthens its cellulotoxic effect.The NK cell that clinical data display induced amplification goes out, iNKT adoptive cellular immunotherapy malignant tumour have a good application prospect, and all have certain effect to lung cancer, liver cancer, ovarian cancer, esophagus cancer, colorectal carcinoma, cancer of the stomach, cervical cancer, osteocarcinoma etc.
Main to cultivate the cell of single type in immune cell therapy, for cultivating polytype immunocyte simultaneously, because the culture condition being applicable to each cell type is not identical each other, have to implement culturing step abreast to various kinds of cell type respectively simultaneously, thus cause blood sampling volume large, complicated operation, the problems such as cost is higher.Carry out adoptive cellular immunotherapy kill the low problem of tumor activity to solve single immunocyte in prior art, the effect of further raising immune cell therapy, need combined utilization 2 kinds or two or more immunocyte to treat, the present invention can well achieve this end.
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 to propose the method for a kind of two kinds of immunocytes newly amplification in vitro simultaneously, and the method in a culture systems, in vitro under culture condition, can obtain simultaneously and widely apply higher, the active strong V α of safety, purity
24+iNKT cell and CD
3-cD
56+nK cell, thus this cell is reached can be applied to clinical quality safely and effectively.
According to an aspect of the present invention, the invention provides a kind of α of induced amplification V simultaneously
24+iNKT cell and CD
3-cD
56+nK cellular processes.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-CD toward PBMC cell suspension
16antibody ,-GalCer, IL-2, IL-18 and IL-21, proceed in culturing bottle afterwards 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-CD after 24 hours
3antibody, at 37 DEG C, 5%CO
2, cultivate under 100% humidity condition;
3rd day, add the second substratum of half volume in cell suspension, described second substratum is the serum free medium containing autologous plasma, IL-2, IL-18, IL-21;
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 V α that collected by centrifugation obtains
24+iNKT cell and CD
3-cD
56+nK cell.
Contriver is surprised to find, and utilizes method of the present invention only to utilize a culture systems, in vitro under culture condition, obtains simultaneously and widely applies higher, the active strong V α of safety, purity
24+iNKT cell and CD
3-cD
56+nK cell, thus this cell is reached can be applied to clinical quality safely and effectively.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, described Anti-CD
16the final concentration of antibody is 50ng ~ 1mg/ml.According to of the present invention one concrete example, in described first substratum and described second substratum, described Anti-CD
16the final concentration of 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-GalCer is 100ng ~ 1mg/mL.According to of the present invention one concrete example, in described first substratum and described second substratum, the final concentration of described-GalCer 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-18 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-18 is 15ng/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-21 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-21 is 20ng/ml.Thus, object cell amplification is effective.
According to embodiments of the invention, the 1st day, the described Anti-CD that cell cultures added after 24 hours
3the final concentration of antibody is 10ng ~ 1mg/ml.According to of the present invention one concrete example, the 1st day, the Anti-CD that cell cultures added after 24 hours
3the final concentration of antibody is 50ng/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 described, cell reaches 5x10
9time, the V α that collected by centrifugation obtains
24+iNKT 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-1Without L-Glutamine substratum of BI company of Israel.
In addition, according to embodiments of the invention, by method of the present invention cultivate the two kinds of cells obtained, finally can be prepared as cell therapy product, for clinical tumor adoptive cellular immunotherapy.
According to concrete examples more of the present invention, the present invention design a kind of easy, effective, can in a culturing step, autologous peripheral blood mononuclear cell be induced simultaneously and a large amount of V α that increases
24+iNKT cell and CD
3-cD
56+the method of NK cell, comprises the following steps:
0th day, getting peripheral blood, isolate PBMC with lymphocyte separation medium, using the serum free medium adjustment PBMC concentration containing 5 volume % autologous plasmas to be 2 × 10 by being separated the PBMC obtained
6/ mL, makes PBMC cell suspension, then adds Anti-CD toward PBMC cell suspension
16antibody ,-GalCer (alpha-galactosylceramide), IL (interleukin-)-2, IL-18 and IL-21, proceed in T175 culturing bottle afterwards in 37 DEG C, 5%CO
2, cultivate under 100% humidity;
1st day, cell cultures added Anti-CD after 24 hours
3antibody, at 37 DEG C, 5%CO
2, cultivate under 100% humidity;
3rd day, in cell suspension, half amount added the serum free medium of the IL-21 of IL-18, the 20ng/mL of IL-2,15ng/mL containing 5 volume % autologous plasmas, 1000U/mL;
5th day, centrifugal collecting cell, with the serum free medium of IL-21 of IL-18, the 20ng/mL of IL-2, the 15ng/mL containing 5 volume % autologous plasmas, 1000U/mL, cell is resuspended and adjust cell concn to 1.5 × 10
6/ mL, is transferred in a closed cell culture apparatus,
Continue to cultivate and results: later every 2 days sampling counting cells, in the cell culture apparatus closed, supplement the serum free medium containing autologous plasma, IL-2, IL-18, IL-21 according to count results, adjustment cell density is 1.5 × 10
6/ ml, 37 DEG C, 5%CO
2, cultured continuously is after 14 ~ 21 days under 100% humidity condition, cell reaches 5x10
9time, the V α that collected by centrifugation obtains
24+iNKT cell and CD
3-cD
56+nK cell.
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, V α of the present invention
24+iNKT cell refers to and is labeled as V α by flow cytomery cell surface molecule
24+iNKT cell, the CD of indication
3-cD
56+nK cell refers to and is labeled as CD by flow cytomery cell surface molecule
3-cD
56+nK cell (i.e. natural killer cell).
IL-21 and IL-21 (interleukin-21, IL-21): IL-21 is the newcomer in IL-2 family, by the CD activated
4+t cell is secreted, and similar to IL-2, IL-15 in structure, controllable T cell, B cell proliferation, affect NK cell NK cell proliferation, differentiation and cytotoxic activity.
According to concrete examples more of the present invention, method of the present invention can induce highly purified V α 24+iNKT and CD3-CD56+NK two kinds of cells simultaneously, these two kinds of cells all induce activated receptor NKG2D (Activating receptor of the NK cell) high expression level of V α 24+iNKT cell and CD3-CD56+NK cell simultaneously, and the expression of NKG2D has marked difference before and after amplification.Wherein, the NKG2D described in the present invention is NK, T, CD
8+the Activating receptor of T cell, iNKT cell, scavenger cell etc., NKG2D participates in the identification of virus infected cell and killing and wounding tumour cell, plays an important role in immunosurveillance and immunologic cytotoxicity.
The cell proliferation of method induced amplification of the present invention is fast, and quantity is many, and active good, NKG2D activated receptor is expressed high, and effector cell's purity is high.
Accompanying drawing explanation
Fig. 1 be flow cytometry donor 1 increase front sample express CD
3-cD
56+cD
3-cD
56+nK percentage of cells figure;
Fig. 2 is that after flow cytometry donor 1 increases, CD expressed by sample
3-cD
56+cD
3-cD
56+nK percentage of cells figure;
Fig. 3 be flow cytometry donor 2 increase front sample express CD
3-cD
56+cD
3-cD
56+nK percentage of cells figure;
Fig. 4 is that after flow cytometry donor 2 increases, CD expressed by sample
3-cD
56+cD
3-cD
56+nK percentage of cells figure;
Fig. 5 be flow cytometry donor 3 increase front sample express CD
3-cD
56+cD
3-cD
56+nK percentage of cells figure;
Fig. 6 is that after flow cytometry donor 3 increases, CD expressed by sample
3-cD
56+cD
3-cD
56+nK percentage of cells figure.
Fig. 7 be flow cytometry donor 1 increase front sample express Va
24+v α
24+iNKT percentage of cells figure;
Fig. 8 is that after flow cytometry donor 1 increases, Va expressed by sample
24+v α
24+iNKT percentage of cells figure;
Fig. 9 be flow cytometry donor 2 increase front sample express Va
24+v α
24+iNKT percentage of cells figure;
Figure 10 is that after flow cytometry donor 2 increases, Va expressed by sample
24+v α
24+iNKT percentage of cells figure;
Figure 11 be flow cytometry donor 3 increase front sample express Va
24+v α 2
4+iNKT percentage of cells figure;
Figure 12 is that after flow cytometry donor 3 increases, Va expressed by sample
24+v α
24+iNKT percentage of cells figure.
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: simultaneously induce and the V α that increases
24+iNKT cell and CD
3-cD
56+nK cellular processes
1, induced amplification
0th day, get three routine healthy human peripheral bloods (every routine 100mL), with lymphocyte separation medium (trade(brand)name Lymphoprep TM, Axis-Shield company, Norway) be separated by gradient centrifugation, collect centrifuge tube upper strata buffy layer as autologous plasma, middle layer tunica albuginea layer is PBMC cell, by the PBMC cell phosphate buffered saline (PBS) (PBS collected, GIBCO, the U.S.) washed cell twice, with AIM-V serum free medium (trade(brand)name AIM-V Medium CTS TM, GIBCO, the U.S.) cell is resuspended, get 100L cell suspension trypan exclusion stain detect cytoactive and count.According to count results, respectively acquisition PBMC cell AIM-V serum free medium being adjusted PBMC concentration is 2 × 10
6/ mL, each example takes out 1mL cell suspension, centrifugal collecting cell, with 90 volume % autologous plasmas and 10 volume % dimethyl sulfoxide (DMSO) (Dimethyl sulfoxide or DMSO, the U.S.) the frozen storing liquid 1ml that is mixed with is resuspended by cell, be transferred in the cryopreservation tube of a 2mL ,-80 degrees Celsius of refrigerators are frozen, for flow cytometer showed art.Each routine remaining cell suspension adds by culture volume the Anti-CD16 antibody that final concentration is 500ng/mL, final concentration is the-GalCer of 500ng/mL, final concentration is the IL-2 of 1000U/mL, final concentration is the IL-18 of 15ng/mL and final concentration is the IL-21 of 20ng/mL, proceed in T175 culturing bottle after mixing, 37 DEG C, 5%CO
2, start under 100% humidity condition to cultivate.
1st day, cell cultures added by culture volume the Anti-CD that final concentration is 50ng/mL after 24 hours
3antibody, 37 DEG C, 5%CO2, continue under 100% humidity condition to cultivate;
3rd day, in cell suspension, half amount is added containing 5 volume % autologous plasmas, final concentration was the serum free medium of the IL-21 of IL-18,20ng/mL of IL-2,15ng/mL of 1000U/mL, 37 DEG C, 5%CO
2, continue under 100% humidity condition to cultivate;
5th day, centrifugal collecting cell, the IL-18 being 15ng/mL with the IL-2 being 1000U/mL containing 5 volume % autologous plasmas, final concentration, final concentration, final concentration were that the serum free medium of the IL-21 of 20ng/mL is resuspended and adjust cell concn to 1.5 × 10 by cell
6after/mL, be transferred in a closed cell culture apparatus G-rex 100L (Wilson company, the U.S.), 37 DEG C, 5%CO
2, continue under 100% humidity condition to cultivate.
7-14 days: every 2 days sampling counting cells, in the cell culture apparatus closed, supplement the serum free medium containing autologous plasma, IL-2, IL-18, IL-21 according to count results, adjustment cell density is 1.5 × 10
6/ mL, 37 DEG C, 5%CO
2, cultured continuously under 100% humidity condition.
15th day, the V α that collected by centrifugation obtains
24+iNKT cell and CD
3-cD
56+nK cell.
2: increase 15 days total cells, V α
24+iNKT cell and CD
3-cD
56+the amplification times of NK cell
Flow cytometry assays: sample this 5x10
5cell/pipe, PBS washes 2 times, adds flow cytometer detection antibody, and incubated at room 20 minutes, PBS washes 2 times, and cell is analyzed by flow cytometer.
Total cells expanded: the cell (i.e. step 1 gained) that amplification obtains for 15 days is counted with hematimeter with after Trypan Blue again, by current total cellular score divided by the mononuclearcell sum before cultivation, numerical value is the amplification times of cell, and particular case is in table 1.
CD
3-cD
56+nK cells expanded: counted with hematimeter again after cell Trypan Blue amplification obtained for 15 days, by CD in Flow cytometry to cell mass when being multiplied by 15 days by current total cellular score
3-cD
56+mark CD
3-cD
56+the percentage ratio of NK cell is multiplied by the mononuclearcell of recovery by CD in Flow cytometry to cell mass divided by the mononuclearcell sum before cultivation again
3-cD
56+cD in mark
3-cD
56+the percentage ratio of NK cell, numerical value is the amplification times of NK cell, and particular case is in table 1.
Va
24+iNKT cells expanded: is counted with hematimeter again after the cell Trypan Blue that amplification is obtained for 15 days, by during current total cellular score 5 days by Va in Flow cytometry to cell mass
24+the Va of mark
24+the percentage ratio of iNKT cell is multiplied by the mononuclearcell of recovery by Va in Flow cytometry to cell mass divided by the mononuclearcell sum before cultivation again
24+the Va of mark
24+the percentage ratio of iNKT cell, numerical value is Va
24+the amplification times of iNKT cell, particular case is in table 1.
The different donor of table 1 cultivates the increment multiple table of each cell after 15 days
Embodiment 2: V α before and after flow cytomery amplification
24+iNKT cell and CD
3-cD
56+the phenotype of NK cell
By (the also namely frozen 2x10 be separated to for the 0th day of step 1 gained freeze-stored cell in embodiment 1
6pBMC cell) recovery, cultivate with embodiment 1 method the cell obtained afterwards for 15 days and carry out parallel flow cytometer detection operation, express CD
3-cD
56+cD
3-cD
56+nK percentage of cells uses anti-FITC Mouse Anti-Human CD
3detect antibody 20L (BDPharmingen, the U.S.), PE Mouse Anti-Human CD
56detection antibody 20L (BD Pharmingen, the U.S.) is carried out double-tagging and is carried out double-tagging, expresses Va
24+va
24+iNKT percentage of cells uses PE Mouse Anti-Human Invariant NKT cell antibody (BD Pharmingen, the U.S.) to carry out single mark, by stream type cell analyzer (Millipore guava easyCyte 6HT-2L, the U.S.) measure, measured value is as Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, shown in Figure 10, Figure 11, Figure 12.Wherein the X-coordinate of Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 is CD
3-FITC fluorescence antibody marks, and ordinate zou is CD
56-PE fluorescence antibody marks; The X-coordinate of Fig. 7, Fig. 8, Fig. 9, Figure 10, Figure 11, Figure 12 is Va
24+iNKT-PE fluorescence antibody marks, and ordinate zou represents Relative cell number; R2 represents Va
24+iNKT cell percentage composition establishes door (determine analyzed area, establish door according to Isotype control (Isotype Control)).
Fig. 1 be flow cytometry donor 1 increase front sample express CD
3-cD
56+cD
3-cD
56+nK percentage of cells, Fig. 2 is that after flow cytometry donor 1 increases, CD expressed by sample
3-cD
56+cD
3-cD
56+nK percentage of cells; Fig. 3 be flow cytometry donor 2 increase front sample express CD
3-cD
56+cD
3-cD
56+nK percentage of cells, Fig. 4 is that after flow cytometry donor 2 increases, CD expressed by sample
3-cD
56+cD
3-cD
56+nK percentage of cells; Fig. 5 be flow cytometry donor 3 increase front sample express CD
3-cD
56+cD
3-cD
56+nK percentage of cells, Fig. 6 is that after flow cytometry donor 3 increases, CD expressed by sample
3-cD
56+cD
3-cD
56+nK percentage of cells.As can be seen from CD after this amplification of Fig. 1-6, three increment
3-cD
56+nK cell proportion is increased to 68.68% ± 6.77% after amplification by 8.09% ± 4.45% before increasing.Prove to cultivate through present method to have induced highly purified CD
3-cD
56+nK cell.
Fig. 7 be flow cytometry donor 1 increase front sample express Va
24+va
24+iNKT percentage of cells, Fig. 8 is that after flow cytometry donor 1 increases, Va expressed by sample
24+va
24+iNKT percentage of cells; Fig. 9 be flow cytometry donor 2 increase front sample express Va
24+va
24+iNKT percentage of cells, Figure 10 is that after flow cytometry donor 2 increases, Va expressed by sample
24+va
24+iNKT percentage of cells; Figure 11 be flow cytometry donor 3 increase front sample express Va
24+va
24+iNKT percentage of cells, Figure 12 is that after flow cytometry donor 3 increases, Va expressed by sample
24+va
24+iNKT percentage of cells.As can be seen from Va after this amplification of Fig. 7-12, three increment
24+iNKT cell proportion is increased to 12.25% ± 3.98% after amplification by 0.48% ± 0.08% before increasing, and proves to induce a high proportion of CD through cultivation
3-cD
56+also highly purified Va can be induced while NK cell
24+iNKT cell.
Embodiment 3: cell high expression level NKG2D after amplification
NKG2D is NK, T, CD
8+the Activating receptor of T cell, iNKT cell, scavenger cell etc., NKG2D participates in the identification of virus infected cell and killing and wounding tumour cell, plays an important role in immunosurveillance and immunologic cytotoxicity.
The present embodiment is using NKG2D as cell activation Testing index, by the PBMC cell of recovery in embodiment 2, cultivate with embodiment 1 method the cell obtained afterwards for 15 days and carry out parallel flow cytometer detection, with NKG2D antibody (BD Pharmingen, the U.S.) carry out single mark, measured by stream type cell analyzer, measured value is as table 2, as can be seen from Table 2, after sample amplification, NKG2D expresses and is increased to 80.25% ± 6.96% after amplification from 12.73% ± 3.71% before amplification.Prove that the process of cultivating induces V α
24+iNKT cell and CD
3-cD
56+the activated receptor NKG2D high expression level of NK cell, the expression of NKG2D has marked difference before and after amplification.
Table 2 difference expresses schedule of proportion for NKG2D before and after sample body amplification
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 (10)
1. a simultaneously induced amplification V α
24+iNKT cell and CD
3-cD
56+the method of NK cell, is characterized in that, step is: the 0th day, get peripheral blood, isolate PBMC with lymphocyte separation medium, and PBMC separation obtained is 2 × 10 by the first substratum adjustment PBMC concentration
6/ mL, makes PBMC cell suspension, then adds Anti-CD16 antibody ,-GalCer, IL-2, IL-18 and IL-21 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-CD after 24 hours
3antibody, at 37 DEG C, 5%CO
2, cultivate under 100% humidity condition;
3rd day, add the second substratum of half volume in cell culture fluid, described second substratum is the serum free medium containing autologous plasma, IL-2, IL-18, IL-21;
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 V α 24 that collected by centrifugation obtains
+iNKT cell and CD
3-cD
56+nK cell.
2. induced amplification V α while of according to claim 1
24+iNKT 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 5 volume %.
3. induced amplification V α 24 while of according to claim 1
+iNKT cell and CD
3-cD
56+the method of NK cell, is characterized in that, in described first substratum and described second substratum, and described Anti-CD
16the final concentration of antibody is 50ng ~ 1mg/ml, preferred 500ng/mL.
4. induced amplification V α while of according to claim 1
24+iNKT 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-GalCer is 100ng ~ 1mg/mL, preferred 500ng/mL.
5. induced amplification V α while of according to claim 1
24+iNKT 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.
6. induced amplification V α while of according to claim 1
24+iNKT 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-18 is 1ng/ml ~ 100ng/mL, preferred 15ng/mL.
7. induced amplification V α while of according to claim 1
24+iNKT 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-21 is 1ng/ml ~ 100ng/mL, preferred 20ng/mL.
8. induced amplification V α while of according to claim 1
24+iNKT cell and CD
3-cD
56+the method of NK cell, is characterized in that, the 1st day, the described Anti-CD that cell cultures added after 24 hours
3the final concentration of antibody is 10ng ~ 1mg/ml, preferred 50ng/mL.
9. induced amplification V α while of according to claim 1
24+iNKT cell and CD
3-cD
56+the method of NK cell, is characterized in that, described cell culture apparatus is G-REX100L culture apparatus.
10. induced amplification V α while of according to claim 1
24+iNKT cell and CD
3-cD
56+the method of NK cell, is characterized in that, continue in the step cultivated and gather in the crops described, cell reaches 5x10
9time, the V α that collected by centrifugation obtains
24+iNKT cell and CD
3-cD
56+nK cell.
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