CN107988157A - A kind of Dendritic Cells Induced culture medium and its application - Google Patents
A kind of Dendritic Cells Induced culture medium and its application Download PDFInfo
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Abstract
The present invention relates to technical field of cell culture, in particular to a kind of Dendritic Cells Induced culture medium and its application.The culture medium adds following component in terms of culture volume in 1640 culture mediums of RPMI：The volume fraction of hyclone is 8%~12%, 5~10ng/ml of hrGM CSF, 4 5~10ng/ml of IL, 3 100~160ng/ml of IL, 60~120ng/ml of SCF, 60~100ng/ml of hrBDNF, 5,442 120~200ng/ml of CYM.The culture medium is used as key regulator using hrBDNF and CYM 5442 so that Human cord blood mononuclear cells induction can be shorten to 10 days or so as the DC times, and the DC of gained has outstanding bioactivity.
Description
Technical field
The present invention relates to technical field of cell culture, in particular to a kind of Dendritic Cells Induced culture medium and its
Using.
Background technology
Dendritic Cells (Dendritic cell, DC) has belonged to the leucocyte category of derived stem cell, is that primary immune is anti-
Most important antigen presenting cell in answering, specific activation T n cells.The immune function of tumor patient Dendritic Cells is not
Entirely, tumor-cell antigen processes function downward, low expression or does not express costimulatory molecules, is it is produced the main of immunologic escape
Reason.Dendritic cells treatment cultivates inductive formation DC in vitro by using the autologous monocyte of patient, and then load is corresponding
Tumour antigen, the dendritic cells of load tumour antigen are made, then stimulate after these dendritic cells are injected in vivo internal swollen
Knurl killer lymphocyte is bred, and is played long-term oncological surveillance effect and tumor-killing effect, is achieveed the purpose that tumors destroyed.Should
It is one of Main way of oncobiology treatment with the immunization therapy centered on antigen presenting cell-Dendritic Cells.
The acquisition of Dendritic Cells is the prerequisite studied it, and still, Dendritic Cells and its precursor are in body
Interior content is few, and external evoked peripheral blood mononuclear cells carries out biological therapy to obtain sufficient amount of Dendritic Cells,
On Specimen origin and still have difficulty in technology.
Bleeding of the umbilicus then abundance, is passed into disuse always.Numerous studies in recent years find, making in per unit volume bleeding of the umbilicus
Hemocytoblast is more more than marrow and peripheral hematopoietic stem cells amount, more original.Studies have reported that peripheral blood, cord blood mononuclear cells with
GM-CSF, IL-4, TNF α can be divided into functional Dendritic Cells after co-culturing.But DC facilitates ripe in vitro in the prior art
Scheme is domestic mainly to use the TNF-a factors still without unified standard, but the DC maturity of this method activation is low, it is impossible to point
Proinflammatory factor IL-12 is secreted, the DC functional defects for causing culture to obtain, and incubation time is longer.Largely limit DC epidemic diseases
The development of the clinical practice work of seedling.Therefore, it is necessary to it is further perfect to the progress of existing DC cell culture protocols, effectively to carry
Effect of the high DC vaccines in induction anti-inflammatory or anti-tumor immune response.
In view of this, it is special to propose the present invention.
The content of the invention
The first object of the present invention is to provide a kind of Dendritic Cells (Dendritic cell, DC) inducing culture,
Brain-derived neurotrophic factor (hrBDNF) is recombinated as key regulator using S1P1 receptor stimulating agents CYM-5442 and people, and
Adapt to adjust original Dendritic Cells Induced culture medium, DC induced maturations and clinical practice can be applied to well.
The second object of the present invention is to provide a kind of Dendritic Cells Induced culture medium in induction people's bleeding of the umbilicus list
A nucleus to DC break up in application, the Dendritic Cells Induced culture medium can induce Human cord blood mononuclear cells to
DC differentiation and maturations, not only the Fiber differentiation time is shorter, and obtained DC can secrete more proinflammatory factor IL-12.
In order to realize the above-mentioned purpose of the present invention, spy uses following technical scheme：
A kind of Dendritic Cells Induced culture medium, in terms of culture volume, added in RPMI-1640 culture mediums it is following into
Point：
The volume fraction of hyclone is 8%~12%, 5~10ng/ml of hrGM-CSF, IL-45~10ng/ml, IL-3
100~160ng/ml, 60~120ng/ml of SCF, 60~100ng/ml of hrBDNF, 120~200ng/ml of CYM-5442.
Brain-derived neurotrophic factor BDNF (Brain derived neurotrophic factor) is in inflammation and allergy
Play the role of key among reaction.Before some researches show that, BDNF and NGF can stimulate DC to be activated in inflammatory reaction,
And promote IL6 or IL10 release (Clinical and Experimental Allergy, 37,1701-1708,2007,
O.Noga&M.Peiser et.al)；But researchs of the BDNF in DC differentiation and maturations are induced is also seldom.
CYM-5442 is the activator of S1P1 (1 phosphate acceptors of Sphingosine-1-phosphate sphingols), there is research
Show that CYM-5442 can enter DC and adjust DC and play immune function, also there are indications that the table of S1P1 acceptors in ripe DC
It can increase up to amount, but same this area has no researchs of the CYM-5442 in DC differentiation and maturations are induced.
The BDNF and CYM-5442 of debita spissitudo are added in Dendritic Cells Induced culture environment by the present invention, can be significantly
Accelerate the induced maturation speed of Dendritic Cells, and the two cooperation has synergistic function.In the prior art, hrGM-CSF,
IL-4, IL-3 and SCF are more common DC inducing cultures component, but IL-4, IL-3 are used cooperatively to less, sheet
Application, which finds IL-4, IL-3 and hrBDNF and CYM-5442 being used cooperatively, has more preferably inducing effect.
The invention further relates to Dendritic Cells Induced culture medium as described above to induce Human cord blood mononuclear cells to tree
Application in prominent shape cell differentiation.
Compared with prior art, beneficial effects of the present invention are：
(1) the present invention provides a kind of Dendritic Cells Induced culture medium, adjusted using hrBDNF and CYM-5442 as key
The factor is controlled, the Dendritic Cells Induced culture of finished product is constructed on the basis of existing Dendritic Cells Induced culture medium
Base, the culture medium are particularly suitable for correlative study and clinical practice of the Human cord blood mononuclear cells to differentiation of dendritic cells；
(2) in order to strengthen the effect of the coordinated between each component, with the Dendritic Cells more preferably expressed, the present invention
Optimization defines the proportioning between each component of Dendritic Cells Induced culture medium；
(3) Dendritic Cells Induced culture medium provided by the present invention is used, Human cord blood mononuclear cells induction becomes DC
Time can shorten to 10 days or so, and the secretory volume of IL-12p70 and IL-10 is significantly increased compared with the prior art.
Embodiment
The present invention relates to a kind of Dendritic Cells Induced culture medium, in terms of culture volume, in RPMI-1640 culture mediums
Add following component：
The volume fraction of hyclone is 8%~12%, 5~10ng/ml of hrGM-CSF, IL-45~10ng/ml, IL-3
100~160ng/ml, 60~120ng/ml of SCF, 60~100ng/ml of hrBDNF, 120~200ng/ml of CYM-5442.
In order to make to reach more preferable culture effect, it is preferable that hyclone is superfine hyclone.
In order to make Dendritic Cells Induced culture medium preferably play inducing action, with the more obvious dendron shape of acquired character
Cell, it is preferred that in terms of culture volume, the volume fraction of hyclone is 9%~11%, 7~8ng/ml of hrGM-CSF,
7~8ng/ml of IL-4,120~140ng/ml of IL-3, SCF80~100ng/ml；
It is furthermore preferred that in terms of culture volume, the volume fraction of hyclone is 10%, hrGM-CSF7.5ng/ml, IL-
4 7.5ng/ml、IL-3 130ng/ml、SCF 90ng/ml。
Preferably, Dendritic Cells Induced culture medium as described above, in terms of culture volume, 70~90ng/ of hrBDNF
140~180ng/ml of ml, CYM-5442.
Preferably, Dendritic Cells Induced culture medium as described above, in terms of culture volume, 75~85ng/ of hrBDNF
150~170ng/ml of ml, CYM-5442.
Preferably, Dendritic Cells Induced culture medium as described above, in terms of culture volume, hrBDNF 80ng/ml,
CYM-5442 160ng/ml。
In addition, cell is contaminated in incubation in order to prevent, it is preferred that Dendritic Cells Induced as described above
Culture medium, in terms of culture volume, the culture medium further includes：
90~110U/ml of penicillin, 90~110U/ml of streptomysin；More preferably penicillin 100U/ml, streptomysin 100U/
ml。
According to an aspect of the present invention, the invention further relates to Dendritic Cells Induced culture medium as described above in induction people
Application of the cord blood mononuclear cells into differentiation of dendritic cells.
Preferably, application as described above, the time of cell Fiber differentiation is 8~12 days, more preferably 10 days.
Preferably, application as described above, the Human cord blood mononuclear cells are with 1 × 104The density inoculation in/hole is trained
Support.
Preferably, application as described above, replaces 1 subculture in every 2~3 days in incubation.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person, the condition suggested according to normal condition or manufacturer carry out.Reagents or instruments used without specified manufacturer, is
Can be with conventional products that are commercially available.
Embodiment
(1) main agents
1. granulocyte-macrophage colony-stimulating factor (rh GM-CSF)：R&D Systems,Minneapolis,MN,USA.
2. interleukin 3 (IL-3)：Kylin roc Bioceuticals Inc..
3. interleukin 4 (IL-4)：R&D Systems,Minneapolis,MN,USA.
4. stem cell factor (SCF)：Kylin roc Bioceuticals Inc..
5.hrBDNF：PeproTech companies.
6.CYM-5442：Sigma-Aldrich companies.
7. hyclone:Hyclone companies.
8.hrNGF-β:Sigma-Aldrich companies.
9.EPO：Sigma-Aldrich companies.
(2) key instruments
1. carbon dioxide constant incubator：3336 types, Forma Scienfic companies of the U.S.
2. superclean bench：SW-CT-1B types, Suzhou.
3. low temperature refrigerator：MDF-382E types, Japanese SANYO companies.
(3) experimental methods
1. the preparation of cord blood mononuclear cells.
It is prepared by cord blood mononuclear cells：During baby due, Cord blood, anticoagulant heparin, every time blood sampling are taken using 18G syringe needles
15-35 milliliters.Bleeding of the umbilicus room temperature is 2500 revs/min lower, centrifuges 10 minutes, removes upper plasma, Ficoll- is suspended in after PBS dilutions
On Paque lymphocyte separation mediums (d=1.077), 1500 revs/min at room temperature, centrifugation 20 minutes, collect positioned at separating medium it
Between cord blood mononuclear cells, after PBS washings, prepare mononuclearcell suspension, adjust concentration of cell suspension 5 × 106/ml。
2. the culture of cord blood cells：1×106The culture of/ml cord blood mononuclear cells is in floor space 25m2Plastic culture bottle,
Factor-containing rh GM-CSF, IL-3, SCF and EPO nutrient solution in, 37 DEG C, 5% carbon dioxide, under the conditions of saturated humidity,
Fresh medium was replaced at interval of 7 days, adds cell factor.
3. dendritic cells in vitro induced amplification cultivating system：
Experimental group：RPMI-1640 9ml, hyclone 1ml, hrGM-CSF 7.5ng/ml, IL-47.5ng/ml, IL-3
130ng/ml, SCF 90ng/ml, hrBDNF 80ng/ml, CYM-5442 160ng/ml, penicillin 100U/ml, streptomysin
100U/ml。
Control group 1：RPMI-1640 9ml, hyclone 1ml, hrGM-CSF 7.5ng/ml, IL-47.5ng/ml, IL-3
130ng/ml, SCF 90ng/ml, hrNGF 80ng/ml, CYM-5442 160ng/ml, penicillin 100U/ml, streptomysin
100U/ml。
Control group 2：RPMI-1640 9ml, hyclone 1ml, hrGM-CSF 7.5ng/ml, IL-47.5ng/ml, IL-3
130ng/ml, SCF 90ng/ml, hrBDNF 240ng/ml, penicillin 100U/ml, streptomysin 100U/ml.
Control group 3：RPMI-1640 9ml, hyclone 1ml, hrGM-CSF 7.5ng/ml, IL-3130ng/ml, SCF
90ng/ml, penicillin 100U/ml, streptomysin 100U/ml.
Control group 4：RPMI-1640 9ml, hyclone 1ml, hrGM-CSF 7.5ng/ml, EPO0.2IU/ml, IL-3
130ng/ml, SCF 90ng/ml, hrBDNF 80ng/ml, CYM-5442 160ng/ml, penicillin 100U/ml, streptomysin
100U/ml。
Blank control group：Without the cord blood mononuclear cells of dendritic cells in vitro inducing culture Fiber differentiation.
Human cord blood mononuclear cells prepared by upper step are with 1 × 104The density in/hole is inoculated in 37 in 6 porocyte culture plates
DEG C, 5%CO2Cultivated.
Replace 1 differential medium within 2-3 days, observe the morphological change of cell, cell training during culture under the microscope
Cell is collected after supporting 10 days.
Further to verify the cultural method of Dendritic Cells provided by the invention and its Dendritic Cells obtained tool
There is significant beneficial effect, following verified is set.
1. the facs analysis of cell phenotype
Culture 10 days after, respectively collect each group cell, add PBS solution suspension cell, adjustment cell concentration most 1 ×
105/ ml, takes 100 μ 1 to add centrifuge tube, and it is dense eventually to be then respectively adding fluorescent labeled antibody (anti-CD83, CD1a and CD11c) adjustment
Spend for 5 μ g/ml, put in 4 DEG C of refrigerators, lucifuge reaction 15min, addition PBS solution is washed 2 times, made with the homotype Ig of fluorescent marker
For control, upper machine (FACS calibur) detects cell fluorescence intensity, Cell quest software analysis.
1 cell phenotype FACS statistical results of table
CD1a is the more special representative of Dendritic Cells, and CD83 is in the mark of ripe DC, CD11c Dendritic Cells
A kind of integral protein of height expression.As can be known from the above table, compared with control group 1~4, what culture medium provided by the present invention was induced
The quantity of CD83+, CD1a+ and CD11c+ positive cell is significantly improved in DC；And when shortage BDNF (control groups in culture medium
1) or during CYM-5442 (control group 2), ripe DC quantity is remarkably decreased, this explanation BDNF and CYM-5442 can synergistic activation
DC, has significant synergies.In addition, the explanation of control group 1 is with the functionally similar NGF of BDNF and there is no with CYM-5442's
The effect of synergistic activation DC.When the IL-4 in culture medium is replaced with EPO, ripe DC quantity has also declined, and illustrates IL-
The performance of the synergistic effect of 4 couples of BDNF and CYM-5442, which also has, to be had a certain impact.
Supernatant IL-12p70 and IL-10 content before and after 2.ELISA methods detection Fiber differentiation
The supernatant for collecting the Dendritic Cells of each group respectively carries out ELISA detections, detects IL-12p70 and IL- in supernatant
10 secretion situation, detection data see the table below：
The secretion level (x ± s, ng/ml) of table 2IL-12p70 and IL-10
From the data in table 2, it can be seen that in experimental group, the level of the IL-12p70 and IL-10 of Dendritic Cells secretion are much larger than each
Control group, and IL-12p70 can directly act on CD8+T cells makes its propagation, strengthen cell immunoreceptor and form permanent note
Property T lymphocytes, the IL-12 for being capable of secreting high levels is one of high mark of Dendritic Cells bioactivity：It is it follows that logical
Cross the bioactivity that the Dendritic Cells bioactivity that the present invention is obtained is better than the Dendritic Cells that control group is obtained.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, but it will be understood by those of ordinary skill in the art that：Its
It can still modify to the technical solution described in foregoing embodiments, either to which part or all technical characteristic
Carry out equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention skill
The scope of art scheme.
Claims (10)
1. a kind of Dendritic Cells Induced culture medium, it is characterised in that in terms of culture volume, in RPMI-1640 culture mediums
Add following component：
The volume fraction of hyclone is 8%~12%, 5~10ng/ml of hrGM-CSF, 5~10ng/ml of IL-4, IL-3
100~160ng/ml, 60~120ng/ml of SCF, 60~100ng/ml of hrBDNF, 120~200ng/ml of CYM-5442.
2. Dendritic Cells Induced culture medium according to claim 1, it is characterised in that in terms of culture volume, tire ox
The volume fraction of serum is 9%~11%, 7~8ng/ml of hrGM-CSF, 7~8ng/ml of IL-4,120~140ng/ of IL-3
80~100ng/ml of ml, SCF.
3. Dendritic Cells Induced culture medium according to claim 1, it is characterised in that in terms of culture volume,
70~90ng/ml of hrBDNF, 140~180ng/ml of CYM-5442.
4. Dendritic Cells Induced culture medium according to claim 3, it is characterised in that in terms of culture volume,
75~85ng/ml of hrBDNF, 150~170ng/ml of CYM-5442.
5. Dendritic Cells Induced culture medium according to claim 4, it is characterised in that in terms of culture volume,
hrBDNF 80ng/ml、CYM-5442 160ng/ml。
6. according to Claims 1 to 5 any one of them Dendritic Cells Induced culture medium, it is characterised in that with medium body
Product meter, the culture medium further include：
90~110U/ml of penicillin, 90~110U/ml of streptomysin；Preferably penicillin 100U/ml, streptomysin 100U/ml.
7. claim 1~6 any one of them Dendritic Cells Induced culture medium is inducing Human cord blood mononuclear cells to tree
Application in prominent shape cell differentiation.
8. application according to claim 7, it is characterised in that the time of cell Fiber differentiation is 8~12 days, is preferably 10
My god.
9. application according to claim 7, it is characterised in that the Human cord blood mononuclear cells are with 1 × 104The density in/hole
Inoculation is cultivated.
10. application according to claim 9, it is characterised in that replace 1 subculture in every 2~3 days in incubation.
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|---|---|---|---|---|
| WO2008001379A3 (en) * | 2006-06-28 | 2009-04-30 | Yeda Res & Dev | Activated myeloid cells for promoting tissue repair and detecting damaged tissue |
| US8247227B2 (en) * | 2006-08-28 | 2012-08-21 | The Cleveland Clinic Foundation | Dendritic cell precursors |
| CN102690783A (en) * | 2012-06-07 | 2012-09-26 | 广西医科大学 | Artocarpus lingnanensis lectin capable of inducing dentritic cells to mature and proliferate in vitro |
| 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 |
-
2017
- 2017-11-28 CN CN201711214789.0A patent/CN107988157B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008001379A3 (en) * | 2006-06-28 | 2009-04-30 | Yeda Res & Dev | Activated myeloid cells for promoting tissue repair and detecting damaged tissue |
| US8247227B2 (en) * | 2006-08-28 | 2012-08-21 | The Cleveland Clinic Foundation | Dendritic cell precursors |
| CN102690783A (en) * | 2012-06-07 | 2012-09-26 | 广西医科大学 | Artocarpus lingnanensis lectin capable of inducing dentritic cells to mature and proliferate in vitro |
| 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 |
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