CN104498434B - A kind of preparation method of a large amount of BMDCs, gained BMDC - Google Patents
A kind of preparation method of a large amount of BMDCs, gained BMDC Download PDFInfo
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Abstract
The invention belongs to cellular immunity technical field, discloses a kind of largely preparation method of BMDC and gained BMDC.Preparation method provided by the invention includes：Obtain mononuclearcell；Gained mononuclearcell is taken, adhere-wall culture, obtains dendritic cell precursor cell；Culture, induction gained dendritic cell precursor cell, detection, collects BMDC.Preparation method provided by the invention is after mononuclearcell is obtained, dendritic cell precursor cell is obtained by adhere-wall culture, again by culture, induction, BMDC is prepared, realize and mononuclearcell is induced to differentiate into BMDC, it is simple to operate, and mononuclearcell source is sufficient, available for a large amount of preparations of BMDC, the preparation method is set to be more conducive to promotion and application.
Description
Technical field
The invention belongs to cellular immunity technical field, the preparation method and gained dendron shape of especially a kind of BMDC are thin
Born of the same parents.
Background technology
Cellular immunotherapy therapy is collection human autoimmune's cell, by vitro culture, makes the thousands of multiplications of its quantity
The enhancing of more, target killing function, human body is then fed back to again to kill pathogen in blood and tissue, cancer cell, mutation
Cell, the immunocompetence of break immune tolerance, activation and enhancing body, takes into account the double effects treated with health care.Cellular immunity
Treatment has the characteristics that Small side effects, targeted therapy, sphere of action are more extensive, receives much concern in clinical studies.It is reported that
By the cellular immunotherapy of multiple courses for the treatment of, it can effectively kill except patient's body tumour cell, promote rehabilitation, improve the life of patient
Bioplasm amount.BMDC therapy is one of cellular immunotherapy therapy of current most study.
BMDC (Dendritic cells, DC) is the most strong antigen presenting cell (Antigen of in vivo functionality
Presenting cell, APC), on the one hand, it can stimulate initial T cell to breed, and start immune response；On the other hand,
BMDC can also present antigen and give MHC-I classes restricted CD8+With the restricted CD4 of MHC-II classes+T lymphocytes, induction
Specific immune response, it is referred to as " innate immunity adjuvant ", therefore, BMDC has unique in immune response is induced
Status.In recent years, the immunization therapy based on DC cells obtains more and more extensive concern in clinical practice, is domestic and international
The focus of research.
With going deep into for research, finding the DC cells of abundance becomes the technical bottleneck in the research of DC cellular immunotherapies.
In existing DC cells preparation method, mostly using Peripheral blood culture is extracted, candidate stem cell is isolated, afterwards in various kinds of cell
Cultivated in the presence of the factor, induce gained candidate stem cell, prepare DC cells.Whole process complex operation, and it is because thin
Born of the same parents source is few, the quantity of gained candidate stem cell is also few, finally have impact on DC cell quantities and quality, causes therapeutic effect to be paid no attention to
Think.So, it is quite necessary to the preparation method of new DC cells need to be looked for.
The content of the invention
In view of this, goal of the invention of the invention is preparation method and the gained tree for providing a kind of a large amount of BMDCs
Prominent shape cell.Preparation method provided by the invention by cultivating, inducing, that is, prepares after mononuclearcell is obtained
BMDC, it is simple to operate, and mononuclearcell source is sufficient, available for a large amount of preparations of BMDC, makes the preparation
Method is more conducive to promotion and application.
In order to realize the goal of the invention of the present invention, the present invention adopts the following technical scheme that：
The invention provides a kind of preparation method of BMDC, it comprises the following steps：
Step 1：Obtain mononuclearcell；
Step 2：Gained mononuclearcell is taken, adhere-wall culture, obtains dendritic cell precursor cell；
Step 3：Culture, induction gained dendritic cell precursor cell, detection, collect BMDC.
In the present invention, mononuclearcell can be separated by peripheral blood and obtained, and can also be separated and obtained by marrow.Wherein, periphery
Blood mononuclear cell is the cell for having in peripheral blood single core, including lymphocyte and monocyte.Vitro detection lymph is thin
Born of the same parents first have to separating peripheral blood mononuclear cells, and separation method main at present is Ficoll-hypaque (glucans-general shadow
Portugal's amine) density-gradient centrifugation method, because the proportion of each visible component has differences in blood, therefore separated.Red blood cell and
Granulocyte density is more than layering liquid, at the same because red blood cell runs into Ficoll and aggegation bunchiness money shape and be deposited on ttom of pipe.Blood platelet
Then it is suspended in because density is small in blood plasma, it is intensive in plasma layer and layering liquid has the mononuclearcell suitable with being layered liquid-tight degree only
Interface in, in tunica albuginea shape, draw the confluent monolayer cells and pass the scrubbed high heart and be resuspended.This law separation mononuclearcell purity is reachable
95%, wherein, lymphocyte accounts for 90%~95%, and cell pick-up rate is up to more than 80%, the height of cell pick-up rate and room
Temperature is relevant, can influence cell pick-up rate during more than 25 DEG C.
Preferably, in preparation method provided by the invention, induction agent used in the induction in step 3, which includes the first induction, to be tried
Agent and the second induction agent；
First induction agent includes GM-CSF, IL-4, human serum albumin and glutamine；
Second induction agent includes TNF-α.
GM-CSF is granulocyte-macrophage colony stimutaing factor, is a kind of cell factor.Preferably, the present invention provides
Preparation method in, GM-CSF concentration is 100ng/mL~200ng/mL in the first induction agent.In some realities of the present invention
Apply in example, in preparation method provided by the invention, the concentration of the GM-CSF in the first induction agent is 100ng/mL.In the present invention
Other embodiment in, 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 cell factor.Preferably, in preparation method provided by the invention, first lures
The concentration for leading IL-4 in reagent is 30ng/mL~50ng/mL.In some embodiments of the invention, preparation provided by the invention
In method, the concentration of the IL-4 in the first induction agent is 30ng/mL.In the other embodiment of the present invention, the present invention
In the preparation method of offer, IL-4 rhIL-4.
Preferably, in preparation method provided by the invention, in terms of g/mL, the concentration of human serum albumin in the first induction agent
For 2%~4%.In some embodiments of the invention, in preparation method provided by the invention, in terms of g/mL, the first induction examination
The concentration of human serum albumin in 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, glutamy
The concentration of amine is 2mmol/L.
Preferably, in preparation method provided by the invention, in the second induction agent, the concentration of TNF-α for 500UI/mL~
1500UI/mL。
TNF-α is a kind of TNF.In some embodiments of the invention, preparation method provided by the invention
In, in the second induction agent, the concentration of TNF-α is 1000UI/mL.
Preferably, in preparation method provided by the invention, cultivated in step 3, induction includes：
The complete culture solution containing the first induction agent, culture are added into dendritic cell precursor cell；Adding twice
The complete culture solution and then the mixed solution for adding the complete culture solution and the second induction agent；Afterwards, examined
Survey, collect BMDC, produce.
In some embodiments of the invention, in preparation method provided by the invention, cultivated described in step 3, induce tool
Body is：
The complete culture solution containing first induction agent, culture are added into dendritic cell precursor cell；
The complete culture solution is added respectively within second day, the 4th day in culture；
At the 6th day of culture, the mixed solution of the complete culture solution and the second induction agent is added；
At the 7th day of culture, detected, collect BMDC, produce.
In the other embodiment of the present invention, preparation method provided by the invention specifically includes：
Peripheral blood is taken, separation prepares mononuclearcell；
After removing red blood cell, blood platelet, adhere-wall culture is carried out；Into attached cell add containing rhGM-CSF, rhIL-4,
The complete culture solution of human serum albumin and glutamine, continue to cultivate；In incubation, mended respectively second day, the 4th day
Add the complete culture solution；6th day, add the mixed solution of the complete culture solution and the second induction agent；7th day, examined
Survey, collect BMDC, produce.
In some embodiments of the invention, the collection BMDC is：The DC cells induced and after accelerating are entered
Supernatant is sucked after row eccentric cleaning, 1mL cells frozen storing liquids is added, is frozen after mixing in 2mL cryopreservation tube, be placed in freezing storing box
In, it is put into -80 DEG C of refrigerators and preserves；The cells frozen storing liquid includes 20% human serum albumin, serum free medium and DMSO, described
The volume ratio of 20% human serum albumin, serum free medium and DMSO is 5:4:1.
After preparation method provided by the invention obtains mononuclearcell, by way of adhere-wall culture, obtain rich in tree
The attached cell of prominent shape cell-progenitor cells, is cultivated, derivant is added in incubation again afterwards, by cultivating, inducing,
Maturing dendritic cell is stimulated, obtains BMDC.Whole preparation process is simple to operate, and mononuclearcell source is sufficient,
Available for a large amount of preparations of BMDC, the preparation method is set to be more conducive to promotion and application.Experimental result confirms that the present invention carries
The BMDC quantity that the preparation method of confession prepares is more, can have for preparing the pharmaceutical preparation containing BMDC.
The BMDC that preparation method provided by the invention prepares can be directly added into physiological saline, human serum albumin is configured to carefully
Born of the same parents feed back liquid, for venous re-transfusion；Also gained BMDC can be frozen, is recovered, matched somebody with somebody again in using of using
Put cell and feed back liquid, for venous re-transfusion.
The invention provides a kind of preparation method of a large amount of BMDCs and gained BMDC.It is provided by the invention
The preparation method of BMDC includes：Obtain mononuclearcell；Gained mononuclearcell is taken, adhere-wall culture, obtains dendron shape
Cell-progenitor cells；Culture, induction gained dendritic cell precursor cell, detection, collects BMDC.It is provided by the invention
Preparation method obtains dendritic cell precursor cell after mononuclearcell is obtained, by adhere-wall culture, then pass through and cultivate,
Induction, that is, prepared BMDC, realized mononuclearcell being induced to differentiate into BMDC, simple to operate,
And mononuclearcell source is sufficient, available for a large amount of preparations of BMDC, the preparation method is more conducive to promotion and application,
Gained BMDC can be used for preparing pharmaceutical preparation.Experimental result confirms that preparation method provided by the invention, which is successfully prepared, to be obtained
BMDC was obtained, gained BMDC quantity is big, and maturity is high.
Brief description of the drawings
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 of a large amount of BMDCs and gained BMDC.People in the art
Member may be referred to present disclosure, obtains the BMDC, realizes its application, it is accordingly required in particular to, it is noted that all similar replaces
Change and change apparent to those skilled in the art, they are considered as being included in the present invention.The present invention's
Method is described by preferred embodiment, and related personnel can substantially not depart from present invention, spirit and model
Enclose it is interior this paper preparation method and application are modified or suitably changed with combining, to realize and using the technology of the present invention.
A kind of preparation method of a large amount of BMDCs provided by the invention and reagent and original in gained BMDC
Material can be bought by market.
In order that those skilled in the art better understood when technical scheme, with reference to implementation
Example, is expanded on further the present invention：
The preparation of the BMDC of embodiment 1
Experiment material：
Blood sources are in tumor patient, lung cancer, male, 67 years old；
Lymphocyte separation medium is from Tianjin Hao oceans biology；
LONZA x-vivo15 serum free mediums derive from lonza；
RhGM-CSF derives from peprotech；
RhIL-4 is special precious from Xiamen；
20% human serum albumin (in terms of g/mL) is from the blue biology of China；
The anti-HLA-DR antibody sources of fluorescence labeling are in BD；
The anti-CD83 antibody sources of fluorescence labeling are in BD；
The anti-CD86 antibody sources of fluorescence labeling are in BD；
The configuration side of LONZA x-vivo15 complete mediums containing rhGM-CSF, rhIL-4, Gln and human serum albumin
Method：The above-mentioned factor is added in LONZA x-vivo15 culture mediums by concentration, is configured to 200mL DC complete culture solutions.
Experimental method：
(1) blood cell separator enriched mononuclearcell
Blood is taken, using Fresenius COM.TEC blood cell separators, 12 circulations is circulated, obtains about 80mL and be rich in
The blood sample suspension of mononuclearcell.
(2) mononuclearcell is by purifying and inducing as BMDC
With the 3 pipe separating obtained blood sample suspensions of (15mL/ pipes) lymphocyte separation medium, the composition such as red blood cell, blood platelet is excluded,
Purer mononuclearcell is obtained, concrete operations are：Density gradient centrifugation, rotating speed are 2000 revs/min, centrifuge 20 points
Clock, collect tunica albuginea shape confluent monolayer cells.
Gained mononuclearcell is taken, is added to 6 T752In cm blake bottles (being respectively labeled as 1-6 blake bottles), every bottle
15mL LONZA x-vivo15 serum free mediums are added, are placed on 37 DEG C, 5%CO2In incubator, 0.5h is incubated；Incubation finishes
After take out, outwell suspension cell, and attached cell is cleaned once with LONZA x-vivo15 serum free mediums, make it purer
Only, dendritic cell precursor cell is obtained.
Retain attached cell respectively, totally six parts.Into every part of attached cell, each addition is (final concentration of containing rhGM-CSF
100ng/mL), rhIL-4 (final concentration of 30ng/mL), Gln (final concentration of 2mmol/L), 20% human serum albumin concentration are
2% (that is, in terms of g/mL, the final concentration of LONZA x-vivo15 complete medium 15mL 0.4%) of human serum albumin, gained
In cultivating system, the concentration of attached cell is about 2x106Individual/mL；Start to cultivate, added in the 2nd, 4 day each bottle and above-mentioned contain rhGM-
CSF (final concentration of 100ng/mL), rhIL-4 (final concentration of 30ng/mL) rhIL-4, Gln (final concentration of 2mmol/L), 20%
Human serum albumin concentration is that 2% (that is, in terms of g/mL, the final concentration of complete medium 5mL 2%) of human serum albumin continues to train
Support；6th day, the 5mL that the TNF-α factors of 1000UI/mL containing final concentration are added in each bottle was above-mentioned (final concentration of containing rhGM-CSF
100ng/mL), rhIL-4 (final concentration of 30ng/mL) rhIL-4, Gln (final concentration of 2mmol/L), 20% human serum albumin are dense
Spend and (that is, in terms of g/mL, the final concentration of complete medium 0.4%) of human serum albumin, be incubated overnight for 2%；7th day, enter
DC cells are harvested after row conventional detection and are preserved.
Common detection methods detect for immunophenotype, are specially：Take and facilitate ripe rear DC cells (to cultivate the 7th day, by cell
Sampled after mixing), after washing 2 times with PBS, respectively take 1 × 105/ mL, 1mL, it is separately added into corresponding FCM pipes.Add list to be detected
Clonal antibody：Anti- HLA-DR antibody, anti-CD83 antibody and each 5 μ L of anti-CD86 antibody, 4 DEG C of lucifuges are incubated 30 minutes, every 10 minutes
Rock 1 time, cell is fully contacted with antibody.Washed 2 times, and be resuspended in 400 μ L PBS with PBS, using flow cytometer
FASCSCalibur (BD Biosciences) is detected.
Mononuclearcell culture, the testing result of induction gained cell in No. 1 blake bottle are shown in Table 1, Fig. 1-Fig. 6.
The testing result statistics of No. 11 blake bottles of table
| 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 it was found from testing result：The high expression dendritic cell marker of cell, illustrates the present embodiment
It has successfully been obtained substantial amounts of BMDC.
It was found from experimental result, preparation method provided by the invention has successfully prepared BMDC, the preparation side
Method is simple, and easily operation, is more beneficial for a large amount of preparations of BMDC.
Detection of the testing result of mononuclearcell culture, induction gained cell in 2-6 blake bottles with No. 1 blake bottle
As a result it is close.Illustrate that preparation method provided by the invention has successfully prepared BMDC, the preparation method is simple, easily
Operation, is more beneficial for a large amount of preparations of BMDC.
The explanation of above example is only intended to help the method and its core concept for understanding the present invention.It should be pointed out that pair
For those skilled in the art, under the premise without departing from the principles of the invention, the present invention can also be carried out
Some improvement and modification, these are improved and modification is also fallen into the protection domain of the claims in the present invention.
Claims (3)
1. a kind of preparation method of BMDC, it is characterised in that comprise the following steps：
Step 1：Obtain mononuclearcell；
Step 2：The mononuclearcell is taken, adhere-wall culture, obtains dendritic cell precursor cell；
Step 3：Cultivate, induce the dendritic cell precursor cell, detect, collect BMDC, described in step 3 lures
Leading induction agent used includes the first induction agent and the second induction agent；
First induction agent is made up of GM-CSF, IL-4, human serum albumin and glutamine, and the concentration of the GM-CSF is
100ng/mL~200ng/mL, the IL-4 concentration are 30ng/mL~50ng/mL, in terms of g/mL, the human serum albumin
Concentration is 2%~4%, and the concentration of the glutamine is 2mmol/L~4mmol/L；
Second induction agent is made up of TNF-α, and the concentration of the TNF-α is 500UI/mL~1500UI/mL.
2. preparation method according to claim 1, it is characterised in that cultivated described in step 3, induction includes：
The complete culture solution containing first induction agent, culture are added into the dendritic cell precursor cell；Add two
The secondary complete culture solution, then add the mixed solution of the complete culture solution and second induction agent；Detection, collect tree
Prominent shape cell.
3. preparation method according to claim 1 or 2, it is characterised in that it is described collection BMDC be：
Supernatant is sucked after the BMDC induced and after accelerating is carried out into eccentric cleaning, 1mL cells frozen storing liquids are added, after mixing
Freeze in 2mL cryopreservation tube, be placed in freezing storing box, be put into -80 DEG C of refrigerators and preserve；The cells frozen storing liquid includes 20% people's blood
Albumin, serum free medium and DMSO, the volume ratio of 20% human serum albumin, serum free medium and DMSO is 5:4:
1。
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| CN105087489B (en) * | 2015-09-14 | 2018-09-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of DC cell culture reagents and its cultural method |
| 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 |
| 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 |
| CN105647866A (en) * | 2016-02-28 | 2016-06-08 | 深圳爱生再生医学科技有限公司 | DC (dendritic cell) induction activating agent 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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