CN102168067B - Inducing culture method for regulatory T cell - Google Patents
Inducing culture method for regulatory T cell Download PDFInfo
- Publication number
- CN102168067B CN102168067B CN201110035687A CN201110035687A CN102168067B CN 102168067 B CN102168067 B CN 102168067B CN 201110035687 A CN201110035687 A CN 201110035687A CN 201110035687 A CN201110035687 A CN 201110035687A CN 102168067 B CN102168067 B CN 102168067B
- Authority
- CN
- China
- Prior art keywords
- cells
- cell
- gamma delta
- modulability
- enrichment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
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, more 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) transcription factor of expressing of its immunophenotype with regulatory T cells; This cell has shown powerful immune suppression function, has the effect of graft versus host disease after potential treatment autoimmune disorder, solid organ transplantation rejection and the HSCT.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 research platform be provided for the biological characteristics of further clear and definite modulability gamma delta T cells.The present invention realizes through 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 PMNC, with containing the complete RPMI RPMI-1640 of 10% foetal calf serum re-suspended cell;
(2) The mononuclear cells Grouping: mononuclear cells obtained by conventional methods were divided into groups and improved method of induction induced group: conventional methods induce cytokine alone group and azole Lai acid induction, improved methods of inducing cytokine group , azole Lai phosphate combined synergistically induced decitabine, two cell groups were set three wells;
(3) regulation of γδ? T cell induction: Step (1) prepared in mononuclear cells were inoculated on day 0, day by calculating, two cells on day 0 were added with activated acid Lai yl γδ? T cells, plus interleukin -2 (IL-2),? interleukin -15 (IL-15) and transforming growth factor-β1 (TGF-β1) cytokines induced regulatory γδ? T cells generated on day 1 in improved methods induce Join the group decitabine, then two cells were carried out in the first half of 3,6,9 days medium was changed every time the medium was changed by adding fresh IL-2, IL-15 and TGF-β1 cytokine concentration with the former , respectively, in the first 10 days of the two groups of regulatory cells γδ? T cells to test the content;
(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 cell pass through good brightness, 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 through the generation of application cell factor combined DNA demethylation medicine NSC 127716 co-induction modulability gamma delta T cells and adopts the enrichment process of the positive sorting of MACS, and after enrichment, carries out cell growth state and vigor detects to guarantee to be used for next step experiment.It has following characteristics: (1) induces with enrichment process simple, and the cycle is short, and is with low cost; (2) induce efficient high, 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
Figure 1 is a single cytokines and azole with a conventional method Lai acid induced regulatory γδ? T cells in flow cytometric analysis of FIG.
Figure 2 is a cytokine, azole Lai Joint phosphate DNA methylation inhibitor decitabine (0.5μmol/ml) improved method of induced regulatory γδ? T cells analyzed by flow chart.
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 combines accompanying drawing and embodiment to be further described.
Cytokines used in this experiment were purchased from American PeproTech companies fluorescent flow cytometry using monoclonal antibody was purchased from eBioscience Corporation, azole Lai acid (trade name Zometa) Novartis Pharmaceutical Co., Ltd. gifts, decitabine (Goods Ming Dake) Janssen pharmaceutical Co., Ltd. for the gift, γδ? T cell MACS sorting kit was purchased from Germany Miltenyi biotech.
This experiment repetition 5 normal volunteer's peripheral blood samples, 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, has one to be the main narrow band of white cloud and mist layer with the mononuclearcell 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, adjustment cell density to 4 * 10
6/ ml;
(6) The cells were seeded in 24-well plates were divided into groups and conventional methods improved method of induction induction group, each group was set up three holes, each hole 1ml, each well was added on day 0 of cytokines and azole Lai phosphine acid, the amount and the final concentration shown in Table 1; improved method for inducing group on day 1 of each hole plus land decitabine, the amount shown in Table 1, the final concentration of 0.5μmol / L; Then in section 3,6, 9 days for two cells in each well were half the amount fluid, removing 0.5ml culture medium per well, then add fresh containing 10% FBS PRMI? 1640 complete medium 0.5ml, and added cytokines, the amount of cytokine to each well Table 2.Storage density of various cytokines: IL-2 is 10000U/ml, IL-15 was 10μg/ml, TGF-β1 to 1μg/ml; storage decitabine concentration was 1mmol/ml; yl Lai acid concentration of 56mmol storage / ml.
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 * 5 minute before at every turn toppling over are toppled over the back and are blotted 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.Test results show that cytokines, oxazole acid combined Lai DNA methylation inhibitor decitabine improved method of inducing cytokine alone group and azole Lai and phosphoric acid group, a conventional method of regulatory induced γδ? T cells in the total γδ? T cell percentages were 58.1% and 32.9%; results of Figure 1, Figure 2, Figure 1 is a single cytokines and azole with a conventional method Lai acid induced regulatory γδ? T cells, flow cytometry, and Fig. 2 is a cytokine, an oxazole acid combined Lai DNA methylation inhibitor decitabine (0.5μmol/ml) improved method of induced regulatory γδ? T cells analyzed by flow diagram, the results show that the land plus docetaxel Bin can effectively induce regulatory γδ? T cell formation.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 crowd in total gamma delta T cells, percentage ratio phalangeal cell crowd 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% BAS and 3M YD 30 (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) let 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) on the sorting post, add the 0.5ml damping fluid, the piston that is equipped with the sorting post pushes the magnetic mark cell come fast, and pushing the cell that comes 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 contain 10% foetal calf serum, adjustment 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 to cultivate 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, and Fig. 4 improves one's methods to induce in the group through cytokine and the cell growth figure of 12 days cell of NSC 127716 inducing culture under inverted microscope, the agglomerating growth of visible cell; Form is irregular; Cell volume is bigger than general lymphocyte, passes through good brightness, does not see 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, passes through good brightness, does not see 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, the result finds 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 through 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 PMNC, with containing the complete RPMI RPMI-1640 of 10% foetal calf serum re-suspended cell;
(2) mononuclearcell divides into groups: 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 rice phosphoric acid for single; Improve one's methods and induce group to be cytokine, azoles rice phosphoric acid associating NSC 127716 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 with azoles rice phosphonic acids at the 0th day respectively; Add with interleukin-2, interleukin-15 and transforming growth factor-beta 1 cytokine; Added NSC 127716 in the group improving one's methods to induce on the 1st day; Then two groups of cells carried out half amount respectively at the 3rd, 6,9 day and change liquid, added fresh interleukin-2, interleukin-15 and transforming growth factor-beta 1 cytokine when changing liquid at every turn, respectively two groups of cells were 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 jaw albumen 3 and the phenotypic characteristic of the two positive cells of 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:
Modulability gamma delta T cells quantity percentage composition (%)=jaw albumen 3 and the two positive cells of TCR γ δ/(jaw albumen 3 and the two positive cells of 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 cell pass through good brightness, 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%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110035687A CN102168067B (en) | 2011-02-11 | 2011-02-11 | Inducing culture method for regulatory T cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110035687A CN102168067B (en) | 2011-02-11 | 2011-02-11 | Inducing culture method for regulatory T cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102168067A CN102168067A (en) | 2011-08-31 |
| CN102168067B true CN102168067B (en) | 2012-08-29 |
Family
ID=44489384
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110035687A Active CN102168067B (en) | 2011-02-11 | 2011-02-11 | Inducing culture method for regulatory T cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102168067B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555666A (en) * | 2013-07-17 | 2014-02-05 | 浙江大学 | Culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells |
Families Citing this family (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013174404A1 (en) * | 2012-05-23 | 2013-11-28 | Ganymed Pharmaceuticals Ag | Combination therapy involving antibodies against claudin 18.2 for treatment of cancer |
| 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 |
| CN103436493B (en) * | 2013-08-29 | 2016-02-03 | 浙江大学 | The cultural method of rapamycin induction modulability gamma delta T cells |
| CN104357389B (en) * | 2014-10-15 | 2017-04-26 | 湖南赛诺生物科技股份有限公司 | Expansion culture medium for regulatory T cells of human cord blood origin and application method of expansion culture medium |
| 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 |
| CN105925526B (en) * | 2015-09-11 | 2019-11-15 | 中国人民解放军总医院 | A kind of active method of enhancing CIK cell and CIK cell and its preparation method and application |
| CN108546678A (en) * | 2018-04-19 | 2018-09-18 | 王文娟 | A kind of in-vitro separation amplification method of human peripheral Treg cells and its application |
| CN110699318A (en) * | 2018-07-09 | 2020-01-17 | 广西慧宝源健康产业有限公司 | T cell culture medium and culture method |
| CN108949685B (en) * | 2018-08-02 | 2022-03-29 | 吉林大学第一医院 | Method for in vitro induction and expansion of gamma delta T cells with high killing activity |
| CN114216836A (en) * | 2021-12-02 | 2022-03-22 | 星汉德生物医药(大连)有限公司 | Flow method for specifically detecting proportion of TCR positive T cells in product |
| CN115678846B (en) * | 2022-09-01 | 2023-06-16 | 广东龄值生物科技有限公司 | Tumor specific gamma delta T cell and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101031641A (en) * | 2004-08-19 | 2007-09-05 | 伯哈德·莫瑟 | Preparation of antigen-presenting human gamma delta t cells and use in immunotherapy |
| WO2007117602A2 (en) * | 2006-04-07 | 2007-10-18 | Biogen Idec Ma Inc. | Isolation and use of human regulatory t cells |
-
2011
- 2011-02-11 CN CN201110035687A patent/CN102168067B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101031641A (en) * | 2004-08-19 | 2007-09-05 | 伯哈德·莫瑟 | Preparation of antigen-presenting human gamma delta t cells and use in immunotherapy |
| WO2007117602A2 (en) * | 2006-04-07 | 2007-10-18 | Biogen Idec Ma Inc. | Isolation and use of human regulatory t cells |
Non-Patent Citations (1)
| Title |
|---|
| 杨静琳.小鼠脾脏来源的调节性表型γδT细胞的存在和体外诱导.《中国免疫学杂志》.2008,第24卷488-491. * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555666A (en) * | 2013-07-17 | 2014-02-05 | 浙江大学 | Culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102168067A (en) | 2011-08-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102168067B (en) | Inducing culture method for regulatory T cell | |
| CN105754941B (en) | In-vitro induced amplification culture method for peripheral blood NK cells | |
| CN108508196B (en) | Kit and method for detecting human regulatory T cell subtype | |
| CN103330720B (en) | Mixing stem cell injection and preparation method thereof | |
| CN102268405B (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| CN107022524A (en) | A kind of method of amplification NK cells a large amount of from PMNC | |
| CN109666640A (en) | The method of the external pure culture of natural killer cells | |
| CN106011061A (en) | In-vitro large-scale amplification method of natural killer cells | |
| CN103436493B (en) | The cultural method of rapamycin induction modulability gamma delta T cells | |
| CN103555666A (en) | Culture method for increasing amplification efficiency and activity of Vgamma9Vdelta2T cells | |
| CN104845934A (en) | Mass preparation method for cord blood CD34+ hematopoietic stem cell-derived dendritic cells | |
| CN102741695A (en) | 3D ADCC NK FACS assay | |
| CN112251406A (en) | Exosome sorting method for NK cell activation stage | |
| CN111394310A (en) | Enhanced NK cell for strong immunoregulation and strong killing of tumor cells and virus infected cells, and preparation method and kit thereof | |
| CN108004209A (en) | A kind of bleeding of the umbilicus regulatory T cells amplification in vitro method | |
| CN111944754A (en) | Natural killer cell culture method | |
| CN106190972A (en) | A kind of induced amplification cultural method of peripheral blood gamma delta T cells | |
| CN105483082A (en) | Method for obtaining leukocytes by adopting LRS and application of obtained leukocytes in basic experiment | |
| CN106635769A (en) | Device and method for separating cells | |
| CN103484428A (en) | Applications of CAPE (Caffeic Acid Phenylethyl Ester) in culturing hematopoietic stem/progenitor cells in vitro | |
| CN109486758A (en) | A kind of external efficient amplification reagent of peripheral blood NK cell and operating instruction | |
| CN104195107B (en) | Purposes of the microcapsule bubble in induction stem cell macronucleus differentiation | |
| Cui et al. | Targeting myeloma–osteoclast interaction with Vγ9Vδ2 T cells | |
| CN104862276B (en) | A kind of method that dimensional culture stimulates induction generation Activated platelets with biochemistry | |
| CN203333672U (en) | Cell treatment kit for obtaining stem cells from hematopoietic organs and blood specimens |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |