CN104498434A - Preparation method of large number of dendritic cells and obtained dendritic cells - Google Patents
Preparation method of large number of dendritic cells and obtained dendritic cells Download PDFInfo
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- CN104498434A CN104498434A CN201410730272.7A CN201410730272A CN104498434A CN 104498434 A CN104498434 A CN 104498434A CN 201410730272 A CN201410730272 A CN 201410730272A CN 104498434 A CN104498434 A CN 104498434A
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Abstract
The invention belongs to the technical field of cellular immunity, and discloses a preparation method of a large number of dendritic cells and the obtained dendritic cells. The preparation method provided by the invention comprises the following steps: obtaining single karyocytes; performing adherent culture on the obtained single karyocytes to obtain precursor cells of the dendritic cells; and culturing, inducing the precursor cells of the dendritic cells, detecting, and collecting the dendritic cells. According to the preparation method provided by the invention, after the single karyocytes are obtained, the precursor cells of the dendritic cells are obtained by virtue of the adherent culture, and then the dendritic cells are obtained by culturing and inducing, so that the single karyocytes is induced and differentiated into the dendritic cells, the operation is simple, the source of the single karyocytes is sufficient, and the single karyocytes can be used for large-scale preparation of the dendritic cells to ensure that the preparation method is more beneficial for popularization and application.
Description
Technical field
The invention belongs to cellular immunization technical field, a kind of especially preparation method of dendritic cell and gained dendritic cell.
Background technology
Cellular immunotherapy therapy gathers human autoimmune's cell, through vitro culture, make that its quantity thousandfold increases, target killing function strengthens, and then feed back to human body to kill blood and tissue in pathogenic agent, cancer cells, sudden change cell, break immune tolerance, to activate and the immunological competence of enhancing body, take into account the double effects for the treatment of and health care.Cellular immunotherapy has the features such as side effect is little, targeted therapy, sphere of action are more extensive, receives much concern in clinical studies.It is reported, through the cellular immunotherapy of multiple course for the treatment of, can effectively kill except patient body inner tumour cell, promote rehabilitation, improve the quality of life of patient.Dendritic cell therapy is one of cellular immunotherapy therapy of current most study.
Dendritic cell (Dendritic cells, DC) is the antigen presenting cell (Antigen presenting cell, APC) that in body, function is the strongest, and on the one hand, it can stimulate initial T cell propagation, starts immunne response; On the other hand, dendritic cell can also antigen-presenting to the restricted CD8 of MHC-I class
+cD4 restricted with MHC-II class
+t lymphocyte, inducing specific immunity reacts, and is called as " natural immunity adjuvant ", and therefore, dendritic cell has unique status in induce immune response.In recent years, the immunotherapy based on DC cell obtains paying close attention to more and more widely in clinical application, is the focus of research both at home and abroad.
Along with going deep into of research, find that sufficient DC cell becomes the technical bottleneck in the research of DC cellular immunotherapy.In existing DC cell preparation method, mostly adopt and extract Peripheral blood culture, isolate hemopoietic stem cell, cultivation under cytokine profiles exists afterwards, induction gained hemopoietic stem cell, prepare DC cell.Whole process operation is complicated, and because cell derived is few, the quantity of gained hemopoietic stem cell is also few, finally have impact on DC cell quantity and quality, cause result for the treatment of undesirable.So, be extremely necessary the preparation method that need look for new DC cell.
Summary of the invention
In view of this, goal of the invention of the present invention is the preparation method and the gained dendritic cell that provide a kind of a large amount of dendritic cell.Preparation method provided by the invention, after acquisition mononuclearcell, through cultivating, inducing, has namely prepared dendritic cell, simple to operate, and mononuclearcell source is sufficient, can be used for a large amount of preparations of dendritic cell, makes this preparation method be more conducive to promotion and application.
In order to realize goal of the invention of the present invention, the present invention adopts following technical scheme:
The invention provides a kind of preparation method of dendritic cell, it comprises the following steps:
Step 1: obtain mononuclearcell;
Step 2: get gained mononuclearcell, adherent culture, obtains dendritic cell precursor cell;
Step 3: cultivate, induce gained dendritic cell precursor cell, detect, collect dendritic cell.
In the present invention, mononuclearcell can be separated by peripheral blood and obtain, and also can be separated by marrow and obtain.Wherein, there is in peripheral blood mononuclear cell and peripheral blood the cell of single core, comprise lymphocyte and monocyte.First vitro detection lymphocyte wants separating peripheral blood mononuclear cells, separation method main is at present Ficoll-hypaque (dextran-urografic acid methylglucamine salt) density gradient centrifugation, because the proportion of each formed elements there are differences in blood, be therefore separated.Red corpuscle and granulocyte density are greater than layering liquid, are deposited at the bottom of pipe because red corpuscle runs into Ficoll and aggegation bunchiness money shape simultaneously.Thrombocyte is then suspended in because density is little in blood plasma, has liquid-tight degree is suitable with layering mononuclearcell only intensive in the interface of plasma layer and layering liquid, in tunica albuginea shape, draws this confluent monolayer cells and pass through washing the high heart resuspended.This law is separated mononuclearcell purity can reach 95%, and wherein, lymphocyte accounts for 90% ~ 95%, and cell pick-up rate can reach more than 80%, the height of cell pick-up rate and room temperature relative, can affect cell pick-up rate during more than 25 DEG C.
Preferably, in preparation method provided by the invention, the induction agent used of the induction in step 3 comprises the first induction agent and the second induction agent;
This first induction agent comprises GM-CSF, IL-4, human serum albumin and glutamine;
This second induction agent comprises TNF-α.
GM-CSF is granulocyte-macrophage colony stimutaing factor, is a kind of cytokine.Preferably, in preparation method provided by the invention, in the first induction agent, the concentration of GM-CSF is 100ng/mL ~ 200ng/mL.In some embodiments of the invention, in preparation method provided by the invention, the concentration of the GM-CSF in the first induction agent is 100ng/mL.In other embodiment of the present invention, in preparation method provided by the invention, the GM-CSF in the first induction agent is rhGM-CSF.
IL-4 is interleukin-4, is a kind of cytokine.Preferably, in preparation method provided by the invention, in the first induction agent, the concentration of IL-4 is 30ng/mL ~ 50ng/mL.In some embodiments of the invention, in preparation method provided by the invention, the concentration of the IL-4 in the first induction agent is 30ng/mL.In other embodiment of the present invention, in preparation method provided by the invention, IL-4 is rhIL-4.
Preferably, in preparation method provided by the invention, in g/mL, in the first induction agent, the concentration of human serum albumin is 2% ~ 4%.In some embodiments of the invention, in preparation method provided by the invention, in g/mL, the concentration of the human serum albumin in the first induction agent is 2%.
Preferably, in preparation method provided by the invention, in the first induction agent, the concentration of glutamine is 2mmol/L ~ 4mmol/L.In some embodiments of the invention, in preparation method provided by the invention, in the first induction agent, the concentration of glutamine is 2mmol/L.
Preferably, in preparation method provided by the invention, in the second induction agent, the concentration of TNF-α is 500UI/mL ~ 1500UI/mL.
TNF-α is a kind of tumour necrosis factor.In some embodiments of the invention, in preparation method provided by the invention, in the second induction agent, the concentration of TNF-α is 1000UI/mL.
Preferably, in preparation method provided by the invention, cultivation in step 3, induction comprise:
In dendritic cell precursor cell, add the complete culture solution containing the first induction agent, cultivate; After adding complete culture solution described in twice, then add the mixing solutions of described complete culture solution and the second induction agent; Afterwards, detect, collect dendritic cell, to obtain final product.
In some embodiments of the invention, in preparation method provided by the invention, cultivation described in step 3, induction are specially:
In dendritic cell precursor cell, add the complete culture solution containing this first induction agent, cultivate;
Within second day, the 4th day, this complete culture solution is added respectively what cultivate;
At the 6th day that cultivates, add the mixing solutions of this complete culture solution and the second induction agent;
At the 7th day that cultivates, detect, collect dendritic cell, to obtain final product.
In other embodiment of the present invention, preparation method provided by the invention specifically comprises:
Get peripheral blood, be separated and prepare mononuclearcell;
After removing red corpuscle, thrombocyte, carry out adherent culture; In attached cell, add the complete culture solution containing rhGM-CSF, rhIL-4, human serum albumin and glutamine, continue to cultivate; In culturing process, added this complete culture solution at second day, the 4th day respectively; 6th day, add the mixing solutions of this complete culture solution and the second induction agent; 7th day, detect, collect dendritic cell, to obtain final product.
In some embodiments of the invention, described collection dendritic cell is: suck supernatant after the DC cell after induction also accelerating is carried out eccentric cleaning, add 1mL cells frozen storing liquid, frozen in the cryopreservation tube of 2mL after mixing, be placed in freezing storing box, put into-80 DEG C of Refrigerator stores; Described cells frozen storing liquid comprises 20% human serum albumin, serum free medium and DMSO, and the volume ratio of described 20% human serum albumin, serum free medium and DMSO is 5:4:1.
After preparation method provided by the invention obtains mononuclearcell, by the mode of adherent culture, obtain the attached cell of being rich in dendritic cell precursor cell, cultivate again afterwards, inductor is added in culturing process, through cultivating, inducing, stimulate maturing dendritic cell, obtain dendritic cell.Whole preparation process is simple to operate, and mononuclearcell source is sufficient, can be used for a large amount of preparations of dendritic cell, makes this preparation method be more conducive to promotion and application.Experimental result confirms, the dendritic cell quantity that preparation method provided by the invention prepares is many, can have for the preparation of the pharmaceutical preparation containing dendritic cell.The dendritic cell that preparation method provided by the invention prepares can directly add physiological saline, human serum albumin is configured to cell and feeds back liquid, for venous re-transfusion; Also can be undertaken frozen by gained dendritic cell, recover in the use used again, configuration cell feeds back liquid, for venous re-transfusion.
The invention provides a kind of preparation method and gained dendritic cell of a large amount of dendritic cell.The preparation method of dendritic cell provided by the invention comprises: obtain mononuclearcell; Get gained mononuclearcell, adherent culture, obtain dendritic cell precursor cell; Cultivate, induction gained dendritic cell precursor cell, detects, collects dendritic cell.Preparation method provided by the invention is after acquisition mononuclearcell, namely dendritic cell precursor cell is obtained by adherent culture, again through cultivating, inducing, namely prepared dendritic cell, achieved and mononuclearcell is induced to differentiate into dendritic cell, simple to operate, and mononuclearcell source is sufficient, can be used for a large amount of preparations of dendritic cell, make this preparation method be more conducive to promotion and application, gained dendritic cell can be used for useful in preparing drug formulations.Experimental result confirms, preparation method provided by the invention has successfully prepared dendritic cell, and gained dendritic cell quantity is large, and ripening degree is high.
Accompanying drawing explanation
Fig. 1 shows the flow cytometer showed result picture of gained cell in embodiment 2, and wherein R1 represents cell context;
Fig. 2 shows the flow cytometric analysis results of the CD83 of gained cell in embodiment 2, and wherein N represents expression rate;
Fig. 3 shows the flow cytometric analysis results of the CD86 of gained cell in embodiment 2;
Fig. 4 shows the flow cytometric analysis results of the HLA-DR of gained cell in embodiment 2;
Fig. 5 shows the HLA-DR of gained cell in embodiment 2
+cD83
+flow cytometric analysis results;
Fig. 6 shows the HLA-DR of gained cell in embodiment 2
+cD86
+flow cytometric analysis results.
Embodiment
The invention discloses a kind of preparation method and gained dendritic cell of a large amount of dendritic cell.Those skilled in the art with reference to present disclosure, can obtain this dendritic cell, and realize its application, special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope this paper preparation method and application are changed or suitably change with combination, realize and apply the technology of the present invention.
Reagent in the preparation method of a kind of a large amount of dendritic cell provided by the invention and gained dendritic cell and raw material all can be buied by market.
In order to enable those skilled in the art understand technical scheme of the present invention better, below in conjunction with embodiment, set forth the present invention further:
The preparation of embodiment 1 dendritic cell
Experiment material:
Blood sources in tumour patient, lung cancer, the male sex, 67 years old;
It is biological that lymphocyte separation medium derives from Tianjin Hao ocean;
LONZA x-vivo15 serum free medium derives from lonza;
RhGM-CSF derives from peprotech;
It is special precious that rhIL-4 derives from Xiamen;
It is blue biological that 20% human serum albumin (in g/mL) derives from China;
Fluorescently-labeled anti-HLA-DR antibody sources is in BD;
Fluorescently-labeled anti-CD83 antibody sources is in BD;
Fluorescently-labeled anti-CD86 antibody sources is in BD;
Collocation method containing the LONZA x-vivo15 perfect medium of rhGM-CSF, rhIL-4, Gln and human serum albumin: the above-mentioned factor is added in LONZA x-vivo15 substratum by concentration, is configured to 200mL DC complete culture solution.
Experimental technique:
(1) blood cell separator enriched mononuclearcell
Get blood, use Fresenius COM.TEC blood cell separator, circulate 12 circulations, obtains the blood sample suspension that about 80mL is rich in mononuclearcell.
(2) mononuclearcell is also induced as dendritic cell through purifying
With the separating obtained blood sample suspension of 3 pipe (15mL/ pipe) lymphocyte separation medium, get rid of red corpuscle, the compositions such as thrombocyte, obtain purer mononuclearcell, concrete operations are: density gradient centrifugation, and rotating speed is 2000 revs/min, centrifugal 20 minutes, collect tunica albuginea shape confluent monolayer cells.
Get gained mononuclearcell, join 6 T75
2in cm culturing bottle (being labeled as 1-6 culturing bottle respectively), every bottle adds 15mL LONZA x-vivo15 serum free medium, is placed on 37 DEG C, 5%CO
2in incubator, hatch 0.5h; Take out after hatching, outwell suspension cell, and clean attached cell once with LONZA x-vivo15 serum free medium, make it purer, obtain dendritic cell precursor cell.
Retain attached cell respectively, totally six parts.2% (namely adding containing rhGM-CSF (final concentration is 100ng/mL), rhIL-4 (final concentration is 30ng/mL), Gln (final concentration is 2mmol/L), 20% human serum albumin concentration in every part of attached cell is, in g/mL, the final concentration of human serum albumin is 0.4%) LONZA x-vivo15 perfect medium 15mL, in gained culture system, the concentration of attached cell is about 2x10
6individual/mL; Start to cultivate, 2% (namely 2nd, within 4 days, adding above-mentioned in each bottle is containing rhGM-CSF (final concentration is 100ng/mL), rhIL-4 (final concentration is 30ng/mL) rhIL-4, Gln (final concentration is 2mmol/L), 20% human serum albumin concentration, in g/mL, the final concentration of human serum albumin is 2%) perfect medium 5mL continue cultivate; 6th day, 2% (namely adding 5mL containing final concentration 1000UI/mL TNF-alpha factor in each bottle above-mentioned is containing rhGM-CSF (final concentration is 100ng/mL), rhIL-4 (final concentration is 30ng/mL) rhIL-4, Gln (final concentration is 2mmol/L), 20% human serum albumin concentration, in g/mL, the final concentration of human serum albumin is 0.4%) perfect medium, overnight incubation; 7th day, gather in the crops DC cell after carrying out conventional sense and preserve.
Common detection methods is that immunophenotype detects, and is specially: get and facilitate ripe rear DC cell (namely cultivating the 7th day, by sampling after cell mixing), after washing 2 times, respectively get 1 × 10 with PBS
5/ mL, 1mL, add in corresponding FCM pipe respectively.Add monoclonal antibody to be detected: each 5 μ L of anti-HLA-DR antibody, anti-CD83 antibody and anti-CD86 antibody, 4 DEG C of lucifuges hatch 30 minutes, within every 10 minutes, rock 1 time, cell is fully contacted with antibody.Wash 2 times with PBS, and be resuspended in the PBS of 400 μ L, adopt flow cytometer FASCSCalibur (BD Biosciences) to detect.
Mononuclearcell in No. 1 culturing bottle is cultivated, induce the detected result of gained cell in table 1, Fig. 1-Fig. 6.
The detected result statistics of table 11 culturing bottle
| Testing index | Positive rate |
| HLA-DR + | 99.8％ |
| CD83 + | 97.8％ |
| CD86 + | 98.2％ |
| HLA-DR +CD83 + | 98.2％ |
| HLA-DR +CD86 + | 97.2％ |
From table 1 and Fig. 1-Fig. 6, detected result is known: cell high expression level dendritic cell marker, illustrates that the present embodiment have successfully been obtained a large amount of dendritic cell.
From experimental result, preparation method provided by the invention has successfully prepared dendritic cell, and this preparation method is simple, easily operates, is more conducive to a large amount of preparations of dendritic cell.
Mononuclearcell in 2-6 culturing bottle is cultivated, the detected result of induction gained cell is close with the detected result of No. 1 culturing bottle.Illustrate that preparation method provided by the invention has successfully prepared dendritic cell, this preparation method is simple, easily operates, is more conducive to a large amount of preparations of dendritic cell.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (10)
1. a preparation method for dendritic cell, is characterized in that, comprises the following steps:
Step 1: obtain mononuclearcell;
Step 2: get described mononuclearcell, adherent culture, obtains dendritic cell precursor cell;
Step 3: cultivate, induce described dendritic cell precursor cell, detects, and collects dendritic cell.
2. preparation method according to claim 1, is characterized in that, the described induction induction agent used in step 3 comprises the first induction agent and the second induction agent;
Described first induction agent comprises GM-CSF, IL-4, human serum albumin and glutamine;
Described second induction agent comprises TNF-α.
3. preparation method according to claim 2, is characterized in that, in described first induction agent, the concentration of described GM-CSF is 100ng/mL ~ 200ng/mL.
4. the preparation method according to Claims 2 or 3, is characterized in that, in described first induction agent, the concentration of described IL-4 is 30ng/mL ~ 50ng/mL.
5. the preparation method according to any one of claim 2 to 4, is characterized in that, in g/mL, in described first induction agent, the concentration of described human serum albumin is 2% ~ 4%.
6. the preparation method according to any one of claim 2 to 5, is characterized in that, in described first induction agent, the concentration of described glutamine is 2mmol/L ~ 4mmol/L.
7. the preparation method according to any one of claim 2 to 6, is characterized in that, in described second induction agent, the concentration of described TNF-α is 500UI/mL ~ 1500UI/mL.
8. preparation method according to claim 2, is characterized in that, cultivation described in step 3, induction comprise:
In described dendritic cell precursor cell, add the complete culture solution containing described first induction agent, cultivate; Add complete culture solution described in twice, then add the mixing solutions of described complete culture solution and described second induction agent; Detect, collect dendritic cell.
9. the preparation method according to any one of claim 1 to 8, is characterized in that, described collection dendritic cell is:
Suck supernatant after dendritic cell after induction also accelerating is carried out eccentric cleaning, add 1mL cells frozen storing liquid, frozen in the cryopreservation tube of 2mL after mixing, be placed in freezing storing box, put into-80 DEG C of Refrigerator stores; Described cells frozen storing liquid comprises 20% human serum albumin, serum free medium and DMSO, and the volume ratio of described 20% human serum albumin, serum free medium and DMSO is 5:4:1.
10. the as in one of claimed in any of claims 1 to 9 dendritic cell for preparing of preparation method.
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| CN105087489A (en) * | 2015-09-14 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | DC cell culture reagent and culture method thereof |
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| CN105316287A (en) * | 2015-12-08 | 2016-02-10 | 黑龙江天晴干细胞股份有限公司 | Method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cell |
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| CN105647867A (en) * | 2016-02-28 | 2016-06-08 | 深圳爱生再生医学科技有限公司 | Method for inducing dendritic cells to be mature and dendritic cells |
| CN105670994A (en) * | 2016-02-28 | 2016-06-15 | 深圳爱生再生医学科技有限公司 | DC (dendritic cell) inducer and application thereof |
| CN105861438A (en) * | 2016-05-19 | 2016-08-17 | 上海君微生物科技有限公司 | Improvement method for rapidly preparing dendritic cells |
| CN106359368A (en) * | 2016-09-30 | 2017-02-01 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryoprotectant and cryopreservation method |
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