CN102168067A - Inducing culture method for regulatory T cell - Google Patents
Inducing culture method for regulatory T cell Download PDFInfo
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Abstract
The invention provides an inducing culture method for regulatory T cells and relates to an inducing culture method for regulatory gamma delta T cells. In the method, cell factors are applied and deoxyribonucleic acid (DNA) demethylated medicament decitabine is combined so as to cooperatively induce the generation of the regulatory gamma delta T cells, and an enrichment process of magnetic cell sorter (MACS) positive sorting is adopted; after enrichment, detections of cell growth state and activity are carried out so as to ensure the next experience. The method has the advantages of simple and practicable inducing and enrichment processes, short period, low cost, high inducing efficiency and good repeatability; and after enrichment, cell activity is good, the quantity and activity of cells can ensure the next in vivo and in vitro studies, thereby providing a good study platform for defining the biological characteristics of the gamma delta T cells. Thus, the method has a good popularization value.
Description
Technical field
The invention belongs to immunology and epigenetics research field, relate to the cultural method that application cell factor combined DNA demethylation medicine co-induction modulability gamma delta T cells generates.
Background technology
Gamma delta T cells has particular structure and biological function, and proportion very little (1-5%) was ignored for investigators in the past always in peripheral blood and lymphoid organ.After Kunzmann etc. found that first gamma delta T cells can increase in a large number, many more many more scholars began to be devoted to the research of such cell colony.At present, the amplification in vitro of gamma delta T cells and purifying have become a kind of routine techniques; For this reason, the biological function of such cell is day by day clear and definite.Investigators have found that gamma delta T cells bringing into play very important effect at anti-infective and anti-tumor aspect, and its adoptive immunotherapy obtained widespread use clinically, and a lot of patients benefit from it.The modulability gamma delta T cells is as a kind of subgroup of gamma delta T cells, find first by Italian scholar Casetti in September, 2009, the same jaw albumen 3(FOXP3 that expresses of its immunophenotype) transcription factor with regulatory T cells, this cell has shown powerful immune suppression function, has the effect of graft versus host disease (GVH disease) after potential treatment autoimmune disorder, solid organ transplantation rejection and the hematopoietic stem cell transplantation.But at present the other biological of modulability gamma delta T cells being learned characteristic knows little about it, the research relevant with this group cell is very few especially, thus consider with the modulability gamma delta T cells in vivo few the and existing amplification method efficient of quantity on the low side can not guarantee the inside and outside study in needed enough cell quantities relevant.The modulability gamma delta T cells that can not obtain sufficient amount becomes the bottleneck of its further investigation of restriction.Therefore, the efficient of inducing that how to improve the modulability gamma delta T cells becomes a difficult problem that presses for solution at present.
Summary of the invention
The objective of the invention is to overcome in the existing research approach and induce efficient shortcoming on the low side, a kind of method for inducing and cultivating of regulatory T cells is provided, relate to the method for inducing and cultivating that a kind of modulability gamma delta T cells is provided, can provide research platform for the biological characteristics of further clear and definite modulability gamma delta T cells.The present invention realizes by following steps:
(1) people's mononuclearcell preparation: get the peripheral blood sample, anticoagulant heparin is used the human lymphocyte parting liquid and is separated the preparation peripheral blood mononuclear cell, with containing the complete RPMI RPMI-1640 of 10% foetal calf serum re-suspended cell;
(2) mononuclearcell grouping: the mononuclearcell that is obtained is divided into ordinary method at random induces group and improve one's methods and induce group: ordinary method induces group to induce with cytokine and azoles Lai phosphoric acid for single, improve one's methods and induce group to be cytokine, azoles Lai phosphoric acid associating Decitabine co-induction, two groups of cells are provided with 3 multiple holes respectively;
(3) the modulability gamma delta T cells is induced: mononuclearcell inoculation prepared in the step (1) was calculated by the 0th day the same day, two groups of cells added at the 0th day respectively with azoles Lai phosphonic acids activation gamma delta T cells, add with interleukin-2 (IL-2), the generation of interleukin-15 (IL-15) and transforming growth factor-beta 1 (TGF-β 1) cytokine induction modulability gamma delta T cells, added Decitabine in the group improving one's methods to induce on the 1st day, then two groups of cells are respectively at the 3rd, 6, carry out half amount in 9 days and change liquid, add fresh IL-2 when changing liquid at every turn, IL-15 and TGF-β 1 cytokine, concentration was the same, respectively two groups of cells was carried out the detection of modulability gamma delta T cells quantity content in the 10th day;
(4) modulability gamma delta T cells quantity content detection: collect the cell in the step (3), adopt flow cytometry to detect gamma delta T cells and modulability gamma delta T cells quantity content respectively, with FOXP3 and the two positive cells of TXi Baoshouti γ δ (TCR γ δ) phenotypic characteristic as the modulability gamma delta T cells, the TCR γ δ positive is as the phenotypic characteristic of gamma delta T cells, and calculates the quantity percentage composition that the modulability gamma delta T cells accounts for total gamma delta T cells according to following formula:
The two positive cells of modulability gamma delta T cells quantity percentage composition (%)=FOXP3 and TCR γ δ/(the two positive cells of FOXP3 and the TCR γ δ+single positive cell of the negative TCR γ of FOXP3 δ) * 100;
(5) modulability gamma delta T cells enrichment: adopt the aseptic sorting TCR of positive sorting (MACS) method of immunomagnetic beads γ δ positive cell group, be rich in the modulability gamma delta T cells in this group cell;
(6) cell state and vigor detect after the enrichment: cell adds to continue to cultivate after 2 days with IL-2, IL-15 and TGF-β 1 cytokine and expects with inverted phase contrast microscope and platform that orchid is dyeed and check cell state and vigor after the enrichment, with the saturating good brightness of cell, form is irregular and it is agglomerating as the good standard of cell growth state to assemble; When platform was expected orchid dyeing observation of cell vigor, dead cell was dyed light blue, and viable cell is refused to dye, and calculated cell viability according to following formula: living cell rate (%)=viable cell sum/(viable cell sum+dead cell sum) * 100
The present invention is by the generation of application cell factor combined DNA demethylation medicine Decitabine co-induction modulability gamma delta T cells and adopts the enrichment process of the positive sorting of MACS, and carries out cell growth state and vigor detects to guarantee to be used for next step experiment after enrichment.It has following characteristics: (1) induces with enrichment process simple, and the cycle is short, and is with low cost; (2) induce efficient height, good reproducibility; (3) cell viability is good after the enrichment, and cell quantity and vigor all can guarantee next step the interior and in vitro study of body after the enrichment, and the excellent research platform can be provided for the biological characteristics of clear and definite modulability gamma delta T cells, have excellent popularization and are worth.
Description of drawings
Fig. 1 is that list is induced the flow cytometer showed figure of modulability gamma delta T cells with the ordinary method of cytokine and azoles Lai phosphoric acid.
Fig. 2 is the flow cytometer showed figure that improves one's methods and induce the modulability gamma delta T cells with cytokine, azoles Lai phosphoric acid combined DNA methylated transferase inhibitor Decitabine (0.5 μ mol/ml).
Fig. 3 improves one's methods the cell of inducing culture at the flow cytometer showed figure after the positive sorting enrichment of MACS.
Fig. 4 improves one's methods the cell of inducing culture at the 12nd day cell growth state figure without the MACS sorting.
Fig. 5 is that the improve one's methods cell of inducing culture is cultivated 2 days cell growth state figure after the positive sorting of MACS.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
This tests used cytokine all available from U.S. PeproTech company, Flow cytometry uses the mono-clonal fluorescence antibody available from eBioscience company, azoles Lai phosphonic acids (trade(brand)name is selected Thailand) is given for Novartis company limited, Decitabine (trade(brand)name reaches jade-like stone) is for Xian-Janssen Pharmaceutical Ltd. gives, and gamma delta T cells MACS sorting test kit is available from beautiful day Ni biotech company of Germany.
This experiment has repeated 5 normal volunteer's peripheral blood samples, has all obtained similar effect.
Embodiment 1: the inducing of modulability gamma delta T cells
(1) in the 15ml centrifuge tube, adds the 7ml lymphocyte separation medium;
(2) taking heparin anti-freezing venous blood 10 ml and the abundant mixing of equivalent Hank's liquid slowly are superimposed on the laminated fluid level along tube wall with dropper, note keeping clearly interface, the centrifugal 600g of level * 20 minutes;
(3) see after centrifugal to be divided into three layers in the pipe, the upper strata is blood plasma and Hank's liquid, and lower floor is mainly red corpuscle and granulocyte, and the middle level is a lymphocyte separation medium, and the narrow band of white cloud and mist layer based on mononuclearcell arranged at the interface in last, middle level;
(4) be inserted into the cloud and mist layer with dropper, draw mononuclearcell, insert in another 15ml centrifuge tube, add the Hank's liquid of 5 times of volumes, 300g * 5 are minute centrifugal, washed cell 2 times;
(5) last centrifugal after, abandon supernatant, add and to contain 10% foetal calf serum and 1% pair of anti-complete RPMI1640 nutrient solution, re-suspended cell is adjusted cell density to 4 * 10
6/ ml;
(6) with cell inoculation in 24 orifice plates, be divided into ordinary method and induce group and improve one's methods and induce group, each group is set up 3 multiple holes respectively, every hole 1ml, each Kong Yudi added with cytokine and azoles Lai phosphonic acids in 0 day, amount and final concentration are as shown in table 1; Improve one's methods and induce each Kong Yudi of group to add in 1 day and use Decitabine, measure as shown in table 1ly, final concentration is 0.5 μ mol/L; Then carry out half amount in the 3rd, 6,9 day two groups of each Kong Jun of cell and change liquid, the 0.5ml nutrient solution is removed in every hole, adds the fresh PRMI 1640 complete culture solution 0.5ml that contain 10% foetal calf serum again, and adds cytokine, and each porocyte factor amount sees Table 2.It is 10000U/ml that various cytokines store concentration: IL-2, and IL-15 is 10 μ g/ml, and TGF-β 1 is 1 μ g/ml; It is 1mmol/ml that Decitabine stores concentration; It is 56mmol/ml that azoles Lai phosphonic acids stores concentration.
Embodiment 2: Flow cytometry modulability gamma delta T cells
(1) gathers in the crops the cell that ordinary method is induced group and improved one's methods and induce group to cultivate 10 days, every pipe 10 respectively
6, every group one pipe, with 1 * phosphate buffered saline buffer 2ml washing secondary, centrifugal 300g before at every turn toppling over * 5 minutes topples over the back and blots mouth of pipe liquid with filter paper;
(2) with 1 * phosphate buffered saline buffer, 40 μ l re-suspended cells, add anti-TCR γ δ FITC monoclonal antibody 20 μ l, abundant mixing, 4 ℃ of lucifuges were hatched 30 minutes;
(3) 1 * phosphate buffered saline buffers 2 ml washing 2 times is toppled over filter paper after each washing and is blotted mouth of pipe liquid;
(4) add fixing/rupture of membranes (Fix/Perm) damping fluid 1ml after, mixing was hatched 30 minutes for 4 ℃;
(5) add 1 * rupture of membranes washing (Perm Wash) damping fluid 2ml washing 2 times, it is soft that attention action is wanted, and topples over filter paper after each washing and blot mouth of pipe liquid;
(6) with 1 * phosphate buffered saline buffer (FACS), 40 μ l re-suspended cells, add anti-FOXP3 PE monoclonal antibody 5 μ l, abundant mixing, 4 ℃ of lucifuges were hatched 40 minutes;
(7) add with 1 * Perm Wash damping fluid 2ml washing 2 times, after toppling over, add 500 μ l, 1 * phosphate buffered saline buffer;
(8) machine testing on the flow cytometer; Use the every pipe of CELL-QUEST software and obtain 10000 cells, with the single positive phenotypic characteristic of TCR γ δ as gamma delta T cells, with FOXP3 PE and the two positive phenotypic characteristics of TCR γ δ FITC, calculate the percentage composition that the modulability gamma delta T cells accounts for total gamma delta T cells as the modulability gamma delta T cells.Detected result finds that improving one's methods of cytokine, azoles Lai phosphoric acid combined DNA methylated transferase inhibitor Decitabine induce group and single percentage ratio of inducing modulability gamma delta T cells in the group to account for total gamma delta T cells with the ordinary method of cytokine and azoles Lai phosphoric acid to be respectively 58.1% and 32.9%; The result is referring to Fig. 1, Fig. 2, Fig. 1 is that list is induced the flow cytometer showed figure of modulability gamma delta T cells with the ordinary method of cytokine and azoles Lai phosphoric acid, Fig. 2 is the flow cytometer showed figure that improves one's methods and induce the modulability gamma delta T cells of cytokine, azoles Lai phosphoric acid combined DNA methylated transferase inhibitor Decitabine (0.5 μ mol/ml), and The above results shows and adds the generation that can effectively induce the modulability gamma delta T cells with Decitabine.FL1 refers to anti-TCR γ δ FITC among Fig. 1,2, and FL2 refers to anti-FOXP3 PE; Among the figure not with the content of bracket percentage ratio phalangeal cell group in total gamma delta T cells, percentage ratio phalangeal cell group content in total cell in door in the bracket.
Embodiment 3: immunomagnetic beads cell sorting technology enrichment modulability gamma delta T cells
(1) prepares damping fluid, form by pH 7.2 1 * phosphate buffered saline buffer, 0.5% bovine albumin and 3M ethylenediamine tetraacetic acid (EDTA) (EDTA), hereinafter to be referred as damping fluid; Counting cells gets 10
7
Centrifugal 10 minutes of (2) 300 * g remove supernatant;
(3) cell is resuspended in 40 μ l damping fluids;
(4) add the anti-TCR γ of 10 μ l δ hapten antibody;
(5) 4 ° of C were hatched 10 minutes behind the mixing;
(6) add 90 μ l damping fluids and 60 μ l antihapten microballon-FITC;
(7) mixing, 4 ° of C lucifuges were hatched 15 minutes
(8) add 2 ml damping fluids, centrifugal 10 minutes of 300 * g abandons supernatant;
(9) re-suspended cell in 500 μ l damping fluids;
(10) the MS post is placed in the corresponding MACS sorter, with 0.5ml damping fluid rinse sorting post;
(11) cell suspension is added in the sorting post;
(12) allow cell flow out,, wait until to add new liquid when liquid flow is empty in the preceding sorting post at every turn with 3 * 0.5ml damping fluid flushing sorting post;
(13) the sorting post is shifted out from sorter, be placed on the aseptic centrifuge tube of 15ml;
(14) add the 0.5ml damping fluid on the sorting post, the piston that is equipped with the sorting post pushes the magnetic mark cell fast, and the cell that pushes is the cell after the enrichment;
(15) leave and take after the enrichment 10
6Cell, with total gamma delta T cells and modulability gamma delta T cells content after the Flow cytometry enrichment, the Flow cytometry method is with embodiment 2, and detected result finds that the modulability gamma delta T cells accounts for 59.3% of the interior total cell of door, referring to Fig. 3.
Embodiment 4: cell cultures and cell growth state and vigor detect after the enrichment
(1) with cell after the remaining enrichment with 1 * phosphate buffered saline buffer washed twice, add PRMI 1640 complete culture solutions that contain 10% foetal calf serum, adjust cell density to 2 * 10
6/ ml adds cytokine IL-2, IL-15 and TGF-β 1 and is respectively 32.5U/ml, 50ng/ml, 8.5ng/ml and 1.12mmol/ml to final concentration;
(2) cell is continued cultivated 2 days in containing 37 ℃ of cell culture incubators of 5%CO2;
(3) observation of cell form under inverted phase contrast microscope, the result is referring to Fig. 4,5, Fig. 4 improves one's methods to induce in the group cell growth figure under inverted microscope through cytokine and 12 days cell of Decitabine inducing culture, the agglomerating growth of visible cell, form is irregular, cell volume is bigger than general lymphocyte, and saturating good brightness is not seen fragment; Fig. 5 cultivated second day growth figure with like cell at the 10th day after the aseptic sorting of MACS, visible a little fragment among the figure, and cell begins to merge agglomerating, and saturating good brightness is not seen particle in the born of the same parents;
(4) get 10 in addition
6Cultivate back cell totally 100 μ l, be prepared into single cell suspension;
(5) cell suspension is expected blue solution with 9:1 mixing mixing with 0.4%, placed 2 minutes;
(6) with cell counting count board difference living cell counting and dead cell, and calculate viable cell content, found that viable cell accounts for 89.3% of total cell.
Claims (2)
1. the method for inducing and cultivating of a modulability gamma delta T cells is characterized in that realizing by following steps:
(1) people's mononuclearcell preparation: get the peripheral blood sample, anticoagulant heparin is used the human lymphocyte parting liquid and is separated the preparation peripheral blood mononuclear cell, with containing the complete RPMI RPMI-1640 of 10% foetal calf serum re-suspended cell;
(2) mononuclearcell grouping: the mononuclearcell that is obtained is divided into ordinary method at random induces group and improve one's methods and induce group, ordinary method induces group to induce with cytokine and azoles Lai phosphoric acid for single, improve one's methods and induce group to be cytokine, azoles Lai phosphoric acid associating Decitabine co-induction, two groups of cells are provided with 3 multiple holes respectively;
(3) the modulability gamma delta T cells is induced: mononuclearcell inoculation prepared in the step (1) was calculated by the 0th day the same day, two groups of cells added the phosphonic acids with azoles Lai respectively at the 0th day, add and use interleukin-2, interleukin-15 and transforming growth factor-beta 1 cytokine, added Decitabine in the group improving one's methods to induce on the 1st day, then two groups of cells are respectively at the 3rd, 6, carry out half amount in 9 days and change liquid, add fresh interleukin-2 when changing liquid at every turn, interleukin-15 and transforming growth factor-beta 1 cytokine were carried out the detection of modulability gamma delta T cells quantity content respectively to two groups of cells in the 10th day;
(4) modulability gamma delta T cells quantity content detection: collect the cell in the step (3), adopt flow cytometry to detect gamma delta T cells and modulability gamma delta T cells quantity content respectively, with the phenotypic characteristic of the two positive cells of jaw albumen 3 and TXi Baoshouti γ δ as the modulability gamma delta T cells, the TCR γ δ positive is as the phenotypic characteristic of gamma delta T cells, and calculates the quantity percentage composition that the modulability gamma delta T cells accounts for total gamma delta T cells according to following formula:
The two positive cells of modulability gamma delta T cells quantity percentage composition (%)=jaw albumen 3 and TCR γ δ/(the two positive cells of jaw albumen 3 and the TCR γ δ+single positive cell of jaw albumen 3 negative TCR γ δ) * 100;
(5) modulability gamma delta T cells enrichment: adopt the aseptic sorting TCR of the positive sorting method of immunomagnetic beads γ δ positive cell group, be rich in the modulability gamma delta T cells in this group cell;
(6) cell state and vigor detect after the enrichment: cell adds to continue to cultivate after 2 days with interleukin-2, interleukin-15 and transforming growth factor-beta 1 cytokine and expects with inverted phase contrast microscope and platform that orchid is dyeed and check cell state and vigor after the enrichment.
2. the method for inducing and cultivating of a kind of modulability gamma delta T cells according to claim 1 is characterized in that: step (6) with the saturating good brightness of cell, form is irregular and it is agglomerating as the good standard of cell growth state to assemble; When platform was expected orchid dyeing observation of cell vigor, dead cell was dyed light blue, and viable cell is refused to dye, and calculated cell viability according to following formula:
Living cell rate (%)=viable cell sum/(viable cell sum+dead cell sum) * 100.
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| 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 |
| CN103436493A (en) * | 2013-08-29 | 2013-12-11 | 浙江大学 | Culture method of rapamycin induced regulatory gamma delta T cells |
| CN104357389A (en) * | 2014-10-15 | 2015-02-18 | 湖南赛诺生物科技有限责任公司 | Improved expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium |
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| CN103436493B (en) * | 2013-08-29 | 2016-02-03 | 浙江大学 | The cultural method of rapamycin induction modulability gamma delta T cells |
| CN103436493A (en) * | 2013-08-29 | 2013-12-11 | 浙江大学 | Culture method of rapamycin induced regulatory gamma delta T cells |
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