CN102154205A - Method for preparing high-purity and high-cytotoxin-activity gamma delta T cells - Google Patents
Method for preparing high-purity and high-cytotoxin-activity gamma delta T cells Download PDFInfo
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Abstract
The invention discloses a method for preparing high-purity and high-cytotoxin-activity gamma delta T cells and belongs to the field of in-vitro cell culture. The method comprises: collecting and separating peripheral blood mononuclear cells (PBMC) of a patient, screening gamma delta T positive cells by Mini MACS, activating by a phospho-antigen (E)-4-hydroxy-3-methylbut-2-enyl-pyrophophate (HDMAPP) (3 mu M), adding a gene recombinant human cell factor interleukin (IL)-2 (100IU/ml) and IL-21 (10ng/ml), placing at 37 DEG C, incubating in a culture box containing 5 of percent CO2, supplying a IL-2-containing culture medium after 4 days to continue culturing for 7 to 14 days, and thus, obtaining the high-purity and high-cytotoxin-activity gamma delta T cells. In the invention, a magnetic activated cell sorting technique is used to obtain the high-purity gamma delta T-Cell-Receptor (TCR) positive cells, and the IL-2 and IL-21 are used together to stimulate the cultured gamma delta T cells to strengthen the antitumor function of the cultured gamma delta T cells; and the problems of low purity and low cytotoxin activity of gamma delta T cells amplified in vitro in the prior art are solved.
Description
Technical field
The invention belongs to cell in vitro and cultivate the field, specifically is the preparation method of the gamma delta T cells of a kind of high purity, high cytotoxic activity.
Background of invention
Gamma delta T cells is the specifc immunity cell type between the acquired immunity and the natural immunity, has the antigen-specific recognition function and not have MHC restricted.The gamma delta T cells that is used for antitumor research at present mainly is meant V γ 9V δ 2T cell, accounts for the 50-95% of peripheral blood gamma delta T cells.
The V γ 9V δ 2T cell of human peripheral is discerned non-peptide class phosphorylation antigen by cell surface TCR, need not the restriction of MHC molecule and offer in the antigen recognition process.The non-peptide class phosphorylation antigen that is usually used in activating V γ 9V δ 2T cell at present has: isoprene tetra-sodium (IPP), nitrogenous Diphosphonate (N-BPs) and alkylamine.Wherein IPP is the intermediate product of mevalonic acid path, can directly cause the activation of V γ 9V δ 2T cell.N-BPs and alkylamine are rate-limiting enzyme--the farnesyl diphosphate synthases by inhibition mevalonic acid path, and the interior IPP of born of the same parents is accumulated, thus indirect activation V γ 9V δ 2T cell.Because IPP extensively exists in tumour cell, V γ 9V δ 2T cell is stimulated IPP adapt to, and be weak reaction or reactionless.So using more efficiently phosphoric acid antigen is to need one of technical barrier that solves at present.
Current research shows, the product hydroxyl first isopentenyl diphosphate (HMBPP) of 2-methyl-erythritol-4-phosphoric acid approach is the strongest V γ 9V δ 2T cell activator of present function, its stimulation V γ 9V δ 2T ability of cell proliferation is 10000 times of IPP, (trade(brand)name: Picostim) structural formula and HMBPP are basic identical, confirm to have equally the function of powerful stimulation V γ 9V δ 2T cell through experiment in vitro for the synthetic phosphorylation antigen HDMAPP of institute of France Innate Pharma company.
Phosphorylation antigen and IL-2 coupling can be at external selective amplification V γ 9V δ 2T cells, and obtained confirmation in clinical trial.Utilize setting up of this method amplification in vitro liver cancer, colorectal cancer, neoplastic hematologic disorder patient from body V γ 9V δ δ 2T cell technology.These V γ 9V δ 2T cell great majority are CD27
-CD45RA
+Phenotype is expressed pore-forming protein, granzyme A and B and secretion of gamma-IFN, and presents many cells clone's diversity.
Carried out at present the I phase clinical study of V γ 9V δ 2T cell therapy late tumor, people such as Bennouna discover that to 10 example kidneys in late period 60% (6/10) patient disease is stable, reach for 25.7 weeks steady time.People such as Dieli utilize V γ 9V δ 2T cell agonist and IL-2 to treat 18 routine hormonal resistance prostate cancers, and the result has 3 example parts to alleviate, and 5 examples are stable.Although V γ 9V δ 2T cell therapy is being obtained challenging result aspect feasibility, treatment tolerance and the preliminary curative effect, but this therapy does not obtain optimum curative effect as yet, and its major cause is that the interior tumor killing activity of the body of V γ 9V δ 2T cell is still waiting to improve.Therefore, it is very necessary to seek to strengthen the new tool of antitumor cell cytotoxic activity of V γ 9V δ 2T cell.
IL-21 is a good cell factor, and the IL-21R wide expression is in the lymphocytic cell surface that comprises the activatory gamma delta T cells.Cytokine couplings such as IL-21 and IL-2, IL-15 can be worked in coordination with stimulates original and memory CD8
+T cell and NKT cell proliferation, IL-21 can stimulate the short-term propagation of gamma delta T cells equally, and strengthens NK and CD8 by raising pore-forming protein and granzyme
+The anti-tumor function of T cell, so we select IL-21 to stimulate V γ 9V δ 2T cell.This invention confirms that IL-21 can promote the division of phosphorylation antigen activatory V γ 9V δ 2T cell, temporal induction V γ 9V δ 2T cell proliferation to a certain extent.The common V γ 9V δ 2T cell anti-tumor function that stimulates of IL-21 and IL-2 significantly strengthens, and shows as CD56 and some lysosome particles and expresses increase.
Because V γ 9V δ 2T cell content in human peripheral is atomic, single cell purity that stimulates two weeks of amplification to obtain with phosphoric acid antigen HDMAPP still is lower than 70%, can't provide a kind of highly purified cell preparation for clinical, therefore we invent a kind of immunological magnetic bead sorting technology enrichment V γ 9V δ 2T cell of using earlier, unite the method for IL-2 and IL-21 amplification high purity V γ 9V δ 2T cell then with HDMAPP.
Summary of the invention
The invention provides the preparation method of the gamma delta T cells of a kind of high purity, high cytotoxic activity, can improve gamma delta T cells purity, and CD56
+, pore-forming protein, granzyme expression rate.Overcome the existing method for preparing gamma delta T cells and have the technical problem that purity is low, cytotoxic activity is low.
The technical solution adopted in the present invention is: a kind of method for preparing V γ 9V δ 2T cell, comprise the steps: to gather and separate peripheral blood mononuclear cells, and obtain gamma delta T cells through Mini MACS sorting earlier, activate through HDMAPP again, add IL-2 and IL-21 simultaneously, place 37 ℃, 5%CO
2Incubator in hatch, replenish the substratum contain IL-2 after 4 days and continue to cultivate 7-14 days, can obtain the V γ 9V δ 2T cell of high purity, high cytotoxic activity.
According to the present invention, described substratum is GT-551 human lymphocyte substratum or RPMI1640, and adds 10% self blood plasma.Described mononuclearcell is to adopt Ficoll density gradient centrifugation separated and collected, and cleans with physiological saline, obtains behind the low-speed centrifugal.Described gamma delta T cells is meant V γ 9V δ 2T cell, adopts the indirect immunomagnetic beads forward of Mini MACS sorting method to obtain.The final concentration of described HDMAPP is 3 μ M, and the final concentration of cytokine IL-2 is 100IU/ml, and the final concentration of IL-21 is 10ng/ml.According to the concentration difference of mononuclearcell, cell continued incubation time about 7-14 days.
Raw material that the present invention is used and reagent except that specifying, all commercially available getting.
The invention has the beneficial effects as follows: utilize the immunological magnetic bead sorting technology to obtain highly purified gamma delta T CR positive cell, coupling IL-2 and IL-21 stimulate the gamma delta T cells of cultivation jointly then, and its anti-tumor function is strengthened.Solved the low and low problem of cytotoxic activity of gamma delta T cells purity of prior art amplification in vitro.
Embodiment
Specify the present invention with embodiment below, but the present invention is not limited.The experimental technique of all unreceipted actual conditionses among the following embodiment, the operation instructions that being the method for observing a usual practice and producer provides is carried out.
Embodiment 1: the preparation of gamma delta T cells
1) preparation of peripheral blood mononuclear cell (PBMC)
Gather patient's anticoagulation with blood cell separator, centrifugal 10 minutes of 1500rpm/min, draw upper plasma, 56 ℃ of deactivations are centrifugal standby after 30 minutes, and with physiological saline two-fold dilution hemocyte precipitation, proportion is 1.077 human lymphocyte parting liquid and dilutes blood in 1: 1.5 the ratio adding centrifuge tube, centrifugal 15 minutes of 2000rpm/min, the careful leukocytic cream that extracts cleans twice with physiological saline, obtains peripheral blood mononuclear cell (PBMC) behind the low-speed centrifugal.
2) the indirect immunomagnetic beads forward of Mini MACS sorting gamma delta T cells
With the resuspended peripheral blood mononuclear cell of RPMI1640 that contains 10% (V/V) self blood plasma, and adjust cell concn to 10
8/ ml adds the anti-TCR gamma/delta of 10 μ l Hapten antibody, hatches 1 hour for 4 ℃ behind the mixing, uses twice of MACS damping fluid centrifuge washing; With MACS damping fluid re-suspended cell to 10
8/ ml adds behind the anti-Hapten magnetic bead of 20 μ l 4 ℃ and hatched 40 minutes, treats sorting with 500 μ l MACS damping fluid re-suspended cells behind the centrifuge washing.The MS post is placed on the Mini MACS, with 500 μ lMACS damping fluids flushings MS post, 500 μ l cell suspensions of above-mentioned preparation are added to allow its spontaneous current mistake in the MS post, with 500 μ lMACS damping fluids flushing pillar three times, what flow through the MS post is gamma/delta
-The T cell.Take off the MS post and add the 1ml damping fluid attached parietal cell is washed from the post upper punch fast, collection be gamma delta T cells.
3) efficient gamma delta T cells induces amplification
The gamma delta T cells of above-mentioned acquisition is placed the RPMI1640 nutrient solution that contains HDMAPP (3 μ M), put into 225cm
2Culturing bottle in, add cytokine IL-2 (final concentration is 100IU/ml) and IL-21 (final concentration is 10ng/ml) simultaneously, place 37 ℃, 5%CO
2Incubator in hatch per 24 hours observation of cell forms and growing state, and counting cells.Replenish the substratum that contains IL-2 in every 2-3 days and continue to cultivate 7-14 days, can obtain the gamma delta T cells of high purity, high cytotoxic activity.
Embodiment 2
1. the gamma delta T cells morphological observation of Pei Yanging
Cell cultures 24h is visible gamma delta T cells adherent growth, the about 3-6 of each a colony cell, and colony begins to become big behind the 48h, cultivates visible big colony and the single adherent growth cell of minute quantity of 10d, and individual cells is bar shuttle shape.Cultivate the capable auspicious Ji's Albert'stain Albert of cell smear of 10d, find that the most cells volume is big, be oval or irregular shape, it is oval or circular that nucleus mostly is greatly, and chromatin is loose, and nuclear membrane is irregular, and projection is arranged, and visible 1 the little kernel of each cell is mazarine; Kytoplasm is abundant, dyes grey blue, and form is irregular, and pseudopodium is arranged, and there is the light phenomenon of dying at nearly nuclear place, and also has to be irregular shape.
Transmission electron microscope sees that cell is rounded, there is the microvillus of more elongated protrusion on the surface, sees many group golgi apparatuses in the kytoplasm, more rich rough surfaced endoplasmic reticulum, plastosome is tiny, the electron density height is also shown in high electron density particle and fat and drips, and the nucleus ellipse has depression, how tiny euchromatin is, be evenly distributed, heterochromatin is few, and kernel is obvious.Get the cell 100 μ l that cultivated 10 days, add 100 μ l, 0.4% trypan blue staining fluid, viable cell is not colored, and dead cell is dyed blueness, the microscopically counting.The cell viability of the present invention's preparation is greater than 90%.
2. gamma delta T cells purity of Pei Yanging and immunophenotype detect
Get respectively and cultivate 0,4,7,10 day concentration about 10
6The cell 100 μ l of/ml add respectively and detect with mouse-anti people CD3-FITC, V δ 2-FITC, CD56-PE antibody 10 μ l, and the room temperature lucifuge was hatched 30 minutes, used the PBS washed twice, detected the cell preparation of the present invention's preparation, CD3 through flow cytometer
+The T high enrichment reaches 99.5% ± 0.3%, CD3
+V δ 2
+The T cell increases 93.8% ± 4%, CD3 rapidly from 1.3% ± 0.6%
+V δ 2
+The T cell reached peak value on the 7th day in cultivation, and it is fixed to keep steady up to the 10th Tian Bao.
3. the mensuration of the gamma delta T cells cytotoxic activity of Pei Yanging
The renal adenocarcinoma 786-0 cell strain of taking the logarithm vegetative period is as target cell, and target cell suspension concentration is 1 * 10
5/ ml, 5 * 10
4/ ml, 2.5 * 10
4/ ml, every hole is got 100 μ l and is laid in 96 well culture plates, cultivates 24 hours, and the cell concn of getting the present invention's preparation is adjusted to 1 * 10
6/ ml, add in 96 orifice plates, effect target ratio was respectively 10: 1,20: 1 and 40: 1, each concentration is established 3 multiple holes and is established blank respectively, cultivating after 48 hours every hole, to add concentration be the MTT20 μ l of 5mg/ml, continues to cultivate after 4 hours and abandon supernatant, and every hole adds DMSO 100 μ l, read absorbance in the 570nm place, calculate kill rate.
The result shows that V γ 9V δ 2T cell that the present invention prepares is higher than single during with IL-2 5 times to the killing activity of 786-0 cell strain, confirms that IL-2 and IL-21 coupling have synergy to the anti-tumour effect that strengthens V γ 9V δ 2T cell.
Claims (6)
1. the preparation method of the gamma delta T cells of a high purity, high cytotoxic activity is characterized in that preparation process is as follows:
1) gathers patient's peripheral blood, separate and the collection peripheral blood mononuclear cell;
2) with anti-TCR gamma/delta microballon test kit Mini MACS sorting gamma delta T cells;
3) with the positive cell of the resuspended above-mentioned acquisition of substratum that contains 10% self blood plasma, after phosphoric acid antigen HDMAPP activation, add cytokine IL-2 and IL-21 and cultivated 3 days;
4) add the fresh culture that contains IL-2 according to the every 2-3 of cell growth condition days and continue to cultivate 7-14 days, obtain the gamma delta T cells of high amplification times.
2. the preparation method of the gamma delta T cells of high purity as claimed in claim 1, high cytotoxic activity is characterized in that: mononuclearcell is to adopt Ficoll density gradient centrifugation separated and collected, obtains behind the low-speed centrifugal.
3. the preparation method of the gamma delta T cells of high purity as claimed in claim 1, high cytotoxic activity is characterized in that: gamma delta T cells is to adopt the indirect immunomagnetic beads forward of Mini MACS sorting method to obtain.
4. the preparation method of the gamma delta T cells of high purity as claimed in claim 1, high cytotoxic activity, it is characterized in that: described substratum is meant GT-T551 human lymphocyte nutrient solution or RPMI1640, the final concentration of HDMAPP is 3 μ M, the final concentration of IL-2 is 100IU/ml, and the final concentration of IL-21 is 10ng/ml.
5. the preparation method of the gamma delta T cells of high purity as claimed in claim 1, high cytotoxic activity is characterized in that: cell places cell culture bags or culturing bottle to cultivate, and culture environment is 37 ℃, 5%CO
2, described cell concn reaches 3-5 * 10
8/ ml time-division flask culture.
6. the gamma delta T cells that obtains of aforesaid method can be applied to treat in the medicine of kinds of tumors and virus disease.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994448A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of gamma-delta-T cells |
| CN105886468A (en) * | 2015-05-22 | 2016-08-24 | 北京康爱瑞浩生物科技股份有限公司空港分公司 | Method for efficiently inducing [gamma][delta]T cells and application of [gamma][delta]T cells |
| CN106190972A (en) * | 2015-04-30 | 2016-12-07 | 广州复大医疗股份有限公司 | A kind of induced amplification cultural method of peripheral blood gamma delta T cells |
| CN109517793A (en) * | 2018-11-30 | 2019-03-26 | 广州长峰生物技术有限公司 | A kind of method for building up of NK cell and gamma delta T cells co-cultivation |
| CN113105550A (en) * | 2014-04-10 | 2021-07-13 | 拉法医疗有限公司 | Immunoglobulins that bind to human V gamma 9V delta 2T cell receptors |
| CN113710257A (en) * | 2019-02-24 | 2021-11-26 | 盖米达细胞有限公司 | Methods of γ δ T cell homing and retention, optionally using natural killer cells, for generating cell compositions for therapy |
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| CN1506456A (en) * | 2002-12-12 | 2004-06-23 | 中国医学科学院基础医学研究所 | External amplification process of gamma delta T-lymphocyte |
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2011
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| WO1995025745A1 (en) * | 1994-03-24 | 1995-09-28 | Gradimir Misevic | Fucose containing proteoglycan or acid glycan and their pharmace utical use |
| CN1506456A (en) * | 2002-12-12 | 2004-06-23 | 中国医学科学院基础医学研究所 | External amplification process of gamma delta T-lymphocyte |
Non-Patent Citations (2)
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994448A (en) * | 2012-12-13 | 2013-03-27 | 上海柯莱逊生物技术有限公司 | Method for in-vitro amplification of gamma-delta-T cells |
| CN113105550A (en) * | 2014-04-10 | 2021-07-13 | 拉法医疗有限公司 | Immunoglobulins that bind to human V gamma 9V delta 2T cell receptors |
| CN106190972A (en) * | 2015-04-30 | 2016-12-07 | 广州复大医疗股份有限公司 | A kind of induced amplification cultural method of peripheral blood gamma delta T cells |
| CN105886468A (en) * | 2015-05-22 | 2016-08-24 | 北京康爱瑞浩生物科技股份有限公司空港分公司 | Method for efficiently inducing [gamma][delta]T cells and application of [gamma][delta]T cells |
| CN105886468B (en) * | 2015-05-22 | 2019-09-06 | 北京康爱瑞浩生物科技股份有限公司空港分公司 | A kind of method and application of efficient induction gamma delta T cells |
| CN109517793A (en) * | 2018-11-30 | 2019-03-26 | 广州长峰生物技术有限公司 | A kind of method for building up of NK cell and gamma delta T cells co-cultivation |
| CN109517793B (en) * | 2018-11-30 | 2022-05-10 | 广州长峰生物技术有限公司 | Establishment method for NK cell and gamma delta T cell co-culture |
| CN113710257A (en) * | 2019-02-24 | 2021-11-26 | 盖米达细胞有限公司 | Methods of γ δ T cell homing and retention, optionally using natural killer cells, for generating cell compositions for therapy |
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Application publication date: 20110817 |