CN108118030A - A kind of Dendritic Cells culture medium and cultural method - Google Patents
A kind of Dendritic Cells culture medium and cultural method Download PDFInfo
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Abstract
The invention discloses a kind of Dendritic Cells culture medium and cultural methods, including minimal medium and adding ingredient, wherein, minimal medium is made of glucose injection, normal saline solution, amino acid injection and micro-element injection, and adding ingredient includes the component of following final concentration：100 200ng/mL of GM CSF, 2 4mmol/L of glutamine, 0.2 0.5mg/L of astragalus polyose, 0.2 0.5mg/L of notoginseng polysaccharide, 1 3v/v% of chick embryo extract, 1 3v/v% of ginger juice, 1 3v/v% of kiwi-fruit juice etc..Culture medium provided by the invention, definite ingredients without animal-based protein, will not generate security risk, while also without adding in substance of high cost, it is easy to spread；Each component mutually acts synergistically in culture medium simultaneously, and using the medium culture Dendritic Cells, amplification rate is fast, and vigor is high.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of Dendritic Cells culture medium and cultural method.
Background technology
Treated autologous cell refers to the body cell applied to the self of people, allogeneic or xenogenesis (non-human), through grasping in vitro
The therapy of (or implantation) human body is fed back after work.This manipulation in vitro include cell passage in vitro, amplification, screening and
Drug or other can change the processing of cell behaviors, the body cell after manipulation in vitro can be used for the treatment of disease,
It can also be used for the diagnosis and prevention of disease.Treated autologous cell has a variety of different types, including feeding back Activation In Vitro in vivo
Mononuclearcell such as Tumor-infiltrating lymphocytes, tumor infiltrating lymphocyte, monocyte, macrophage or external
Killing cell of sensitization etc..Domestic immune cell therapy more application in treatment tumour and viral infectious diseases such as hepatitis,
AIDS etc..The important method that more mature autoimmune cell treatment technology is tumor biotherapy is applied in clinical at present
One of, it is effective activation and the amplification separated immunocompetence from autologous peripheral blood under the action of panimmunity active factors
Cell (mononuclearcell) is fed back into patient's body, direct killing or induction immune effector cell killing tumor cell or virus
Infection cell or the immune function for adjusting and enhancing body.Autoimmune cell treatment technology includes killing for cytokine induction
Hinder cellular immunotherapy, Treatment of Dendritic cell, DC-CIK cellular immunotherapies, natural killer cells immunization therapy etc..
Dendritic Cells (Dendritic Cells, DC) is U.S. Rockkflle universities Steinman and Cohn in 1973
It finds when observing spleen cell, gains the name when having dendron sample or pseudo- Microfilament when it is ripe.DC is thin from Hematopoietic Stem
Born of the same parents are widely distributed in lymphoid tissue and non-lymphoid tissue, are the most strong professional antigen presenting cells of the function being currently known,
The high expression MHC- class Ⅱmolecules of film surface, energy effective stimulus primary tape T cell activation, HLA-II antigen are thin much stronger than macrophage
Born of the same parents and B cell, therefore, DC are the main initiators of immune response, and key effect is also played in tumour immunity.Both at home and abroad
Scholar using various methods by DC processing, it is amplifying activated in vitro, and give corresponding antigenic stimulus so that DC vaccines are made,
However it is fed back to patient's body and carries out immunization therapy.
However the quantity of DC in vivo is few, it is difficult to meet the needs of clinical treatment research.But with the development of technology,
People also have investigated the culture medium of amplification in vitro DC, but such culture medium some contains animal sources substance, so as to generate one
A little security risks, although what is had is free of animal derived components, the people's source protein or recombinant protein used is expensive, unfavorable
In large-scale promotion.
The content of the invention
For above-mentioned deficiency of the prior art, the present invention provides a kind of Dendritic Cells culture medium and cultural method,
The medium component is clear and definite, non-animal derived ingredient, at low cost, easy to spread.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is：
A kind of Dendritic Cells culture medium, including minimal medium and adding ingredient, wherein, minimal medium is by glucose
Parenteral solution, normal saline solution, amino acid injection and micro-element injection are 4-6 by volume：3-5：1.5-2：1-
1.5 mixing, adding ingredient include the component of following final concentration：GM-CSF100-200ng/mL, glutamine 2-4mmol/L, Huang
Astragalus polysaccharides 0.2-0.5mg/L, notoginseng polysaccharide 0.2-0.5mg/L, chick embryo extract 1-3v/v%, ginger juice 1-3v/v%, macaque
Peach juice 1-3v/v%, IL-415-30ng/mL, TNF-α 800-12000UI/mL, ironic citrate 300-500mg/L and vitamin
C3-6mg/L。
Further, minimal medium is by glucose injection, normal saline solution, amino acid injection and micro member
Plain parenteral solution is 5 by volume：4：1.5：1 mixing.
Further, adding ingredient includes the component of following final concentration：GM-CSF 150ng/mL, glutamine 3mmol/
L, astragalus polyose 0.3mg/L, notoginseng polysaccharide 0.3mg/L, chick embryo extract 2v/v%, ginger juice 2v/v%, kiwi-fruit juice 2v/
V%, IL-420ng/mL, TNF-α 1000UI/mL, ironic citrate 400mg/L and vitamin C 5mg/L.
Astragalus polyose is prepared by the following method to obtain：The waste residue for extracting astragalus root total saponin is taken, adds 3-5 times of weight of waste residue
Water, boil 40-60min, filter, obtain the first filtrate and first precipitation, to first precipitation in plus 2-4 times of weight water, boil 30-
50min, filtering obtain the second filtrate and the second precipitation, then add in 2-3 times of water into second of precipitation again, boil 20-30min, mistake
Filter, obtains third time filtrate and third time precipitates, and merges filtrate three times, and the 1/4-1/5 of the total filtrate of spin concentration to original volume is added in
Absolute ethyl alcohol, it is stirring while adding, it places, then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain Radix Astragali
Polysaccharide.
Notoginseng polysaccharide is prepared by the following method to obtain：The waste residue for extracting arasaponin is taken, adds 3-5 times of weight of waste residue
Water, boil 40-60min, filter, obtain the first filtrate and first precipitation, to first precipitation in plus 2-4 times of weight water, boil 30-
50min, filtering obtain the second filtrate and the second precipitation, then add in 2-3 times of water into second of precipitation again, boil 20-30min, mistake
Filter, obtains third time filtrate and third time precipitates, and merges filtrate three times, and the 1/4-1/5 of the total filtrate of spin concentration to original volume is added in
Absolute ethyl alcohol, it is stirring while adding, it places, then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain Radix Notoginseng
Polysaccharide.
Chick embryo extract is prepared by the following method to obtain：Corn in chicken embryo is removed, is then homogenized, then in equal volume
DMEM dilution, 2-3h is stirred under the conditions of 20-25 DEG C, 30-80r/min, 48-96h is frozen under the conditions of being then transferred to -80 DEG C
After thaw, centrifuge, collect supernatant, chick embryo extract obtained after filtered degerming.
Using the method for above-mentioned medium culture Dendritic Cells, including：Peripheral blood is separated using density-gradient centrifugation method
Mononuclearcell with above-mentioned culture medium is mixed, is subsequently placed in 37 DEG C, 5%CO by middle mononuclearcell2Under the conditions of cultivated,
Obtain immature dendritic cell.
The present invention provides Dendritic Cells culture medium and cultural methods, have the advantages that：
Culture medium provided by the invention, definite ingredients without animal-based protein, will not generate security risk, while
Substance of high cost is not added in, it is easy to spread；Each component mutually acts synergistically in culture medium simultaneously, using the medium culture
Dendritic Cells, amplification rate is fast, and vigor is high.
Specific embodiment
Embodiment 1
A kind of Dendritic Cells culture medium, including minimal medium and adding ingredient, wherein, minimal medium is by glucose
Parenteral solution, normal saline solution, amino acid injection and micro-element injection are 4 by volume：3：1.5：1 mixing, adds
Addition point includes the component of following final concentration：GM-CSF 100ng/mL, glutamine 2mmol/L, astragalus polyose 0.2mg/L, three
Seven polysaccharide 0.2mg/L, chick embryo extract 1v/v%, ginger juice 1v/v%, kiwi-fruit juice 1v/v%, IL-415ng/mL, TNF-α
800UI/mL, ironic citrate 300mg/L and vitamin C 3mg/L.
Wherein, astragalus polyose is prepared by the following method to obtain：The waste residue for extracting astragalus root total saponin is taken, adds 3 times of waste residue
The water of weight boils 40min, filtering, obtains the first filtrate and the first precipitation, and the water of 2 times of weight is added into the first precipitation, boils 30min,
Filtering obtains the second filtrate and the second precipitation, then adds in 2 times of water into second of precipitation again, boil 20min, filter, obtains third time
Filtrate and third time precipitate, and merge filtrate three times, and the total filtrate of spin concentration to the 1/4 of original volume adds in absolute ethyl alcohol, side edged
Stirring is placed, and then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain astragalus polyose.
Notoginseng polysaccharide is prepared by the following method to obtain：The waste residue for extracting arasaponin is taken, adds 3 times of weight of waste residue
Water boils 40min, filtering, obtains the first filtrate and the first precipitation, and the water of 2 times of weight is added into the first precipitation, boils 30min, is filtered,
The second filtrate and the second precipitation are obtained, 2 times of water is then added in into second of precipitation again, boils 20min, filtering obtains third time filtrate
It is precipitated with third time, merges filtrate three times, the total filtrate of spin concentration to the 1/4 of original volume adds in absolute ethyl alcohol, and side edged stirs
It mixes, places, then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain notoginseng polysaccharide.
Chick embryo extract is prepared by the following method to obtain：Corn in chicken embryo is removed, is then homogenized, then in equal volume
DMEM dilution, 3h is stirred under the conditions of 20-25 DEG C, 30r/min, is thawed after 48h is frozen under the conditions of being then transferred to -80 DEG C,
Centrifugation, collects supernatant, chick embryo extract is obtained after filtered degerming.
Ginger juice is to clean ginger, dry, and is then squeezed, and by gained liquid by 0.22 μm of filter membrane, obtains ginger
Juice；Kiwi-fruit juice is to remove the peel Kiwi berry, is squeezed the juice, and then crosses 0.22 μm of filter membrane, obtains kiwi-fruit juice.
Using the method for above-mentioned medium culture Dendritic Cells, including：Peripheral blood is separated using density-gradient centrifugation method
Mononuclearcell with above-mentioned culture medium is mixed, is subsequently placed in 37 DEG C, 5%CO by middle mononuclearcell2Under the conditions of cultivated,
Obtain immature dendritic cell.
Embodiment 2
A kind of Dendritic Cells culture medium, including minimal medium and adding ingredient, wherein, minimal medium is by glucose
Parenteral solution, normal saline solution, amino acid injection and micro-element injection are 6 by volume：5：2：1.5 mixing, add
Addition point includes the component of following final concentration：GM-CSF 200ng/mL, glutamine 4mmol/L, astragalus polyose 0.5mg/L, three
Seven polysaccharide 0.5mg/L, chick embryo extract 3v/v%, ginger juice 3v/v%, kiwi-fruit juice 3v/v%, IL-430ng/mL, TNF-α
1200UI/mL, ironic citrate 500mg/L and vitamin C 6mg/L.
Wherein, astragalus polyose is prepared by the following method to obtain：The waste residue for extracting astragalus root total saponin is taken, adds 5 times of waste residue
The water of weight boils 60min, filtering, obtains the first filtrate and the first precipitation, and the water of 4 times of weight is added into the first precipitation, boils 50min,
Filtering obtains the second filtrate and the second precipitation, then adds in 3 times of water into second of precipitation again, boil 30min, filter, obtains third time
Filtrate and third time precipitate, and merge filtrate three times, and the total filtrate of spin concentration to the 1/5 of original volume adds in absolute ethyl alcohol, side edged
Stirring is placed, and then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain astragalus polyose.
Notoginseng polysaccharide is prepared by the following method to obtain：The waste residue for extracting arasaponin is taken, adds 5 times of weight of waste residue
Water boils 60min, filtering, obtains the first filtrate and the first precipitation, and the water of 4 times of weight is added into the first precipitation, boils 50min, is filtered,
The second filtrate and the second precipitation are obtained, 3 times of water is then added in into second of precipitation again, boils 30min, filtering obtains third time filtrate
It is precipitated with third time, merges filtrate three times, the total filtrate of spin concentration to the 1/5 of original volume adds in absolute ethyl alcohol, and side edged stirs
It mixes, places, then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain notoginseng polysaccharide.
Chick embryo extract is prepared by the following method to obtain：Corn in chicken embryo is removed, is then homogenized, then in equal volume
DMEM dilution, 2h is stirred under the conditions of 20-25 DEG C, 80r/min, is thawed after 96h is frozen under the conditions of being then transferred to -80 DEG C,
Centrifugation, collects supernatant, chick embryo extract is obtained after filtered degerming.
Ginger juice is to clean ginger, dry, and is then squeezed, and by gained liquid by 0.22 μm of filter membrane, obtains ginger
Juice；Kiwi-fruit juice is to remove the peel Kiwi berry, is squeezed the juice, and then crosses 0.22 μm of filter membrane, obtains kiwi-fruit juice.
Using the method for above-mentioned medium culture Dendritic Cells, including：Peripheral blood is separated using density-gradient centrifugation method
Mononuclearcell with above-mentioned culture medium is mixed, is subsequently placed in 37 DEG C, 5%CO by middle mononuclearcell2Under the conditions of cultivated,
Obtain immature dendritic cell.
Embodiment 3
A kind of Dendritic Cells culture medium, including minimal medium and adding ingredient, wherein, minimal medium is by glucose
Parenteral solution, normal saline solution, amino acid injection and micro-element injection are 4.5 by volume：3.5：1.5：1 is mixed
It closes, adding ingredient includes the component of following final concentration：GM-CSF120ng/mL, glutamine 2.5mmol/L, astragalus polyose
0.3mg/L, notoginseng polysaccharide 0.3mg/L, chick embryo extract 1.5v/v%, ginger juice 1.5v/v%, kiwi-fruit juice 1.5v/v%,
IL-420ng/mL, TNF-α 1000UI/mL, ironic citrate 350mg/L and vitamin C 4mg/L.
Wherein, astragalus polyose is prepared by the following method to obtain：The waste residue for extracting astragalus root total saponin is taken, adds 4 times of waste residue
The water of weight boils 40min, filtering, obtains the first filtrate and the first precipitation, and the water of 3 times of weight is added into the first precipitation, boils 40min,
Filtering obtains the second filtrate and the second precipitation, then adds in 3 times of water into second of precipitation again, boil 30min, filter, obtains third time
Filtrate and third time precipitate, and merge filtrate three times, and the total filtrate of spin concentration to the 1/5 of original volume adds in absolute ethyl alcohol, side edged
Stirring is placed, and then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain astragalus polyose.
Notoginseng polysaccharide is prepared by the following method to obtain：The waste residue for extracting arasaponin is taken, adds 4 times of weight of waste residue
Water boils 40min, filtering, obtains the first filtrate and the first precipitation, and the water of 3 times of weight is added into the first precipitation, boils 40min, is filtered,
The second filtrate and the second precipitation are obtained, 3 times of water is then added in into second of precipitation again, boils 30min, filtering obtains third time filtrate
It is precipitated with third time, merges filtrate three times, the total filtrate of spin concentration to the 1/5 of original volume adds in absolute ethyl alcohol, and side edged stirs
It mixes, places, then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain notoginseng polysaccharide.
Chick embryo extract is prepared by the following method to obtain：Corn in chicken embryo is removed, is then homogenized, then in equal volume
DMEM dilution, 3h is stirred under the conditions of 20-25 DEG C, 50r/min, is thawed after 96h is frozen under the conditions of being then transferred to -80 DEG C,
Centrifugation, collects supernatant, chick embryo extract is obtained after filtered degerming.
Ginger juice is to clean ginger, dry, and is then squeezed, and by gained liquid by 0.22 μm of filter membrane, obtains ginger
Juice；Kiwi-fruit juice is to remove the peel Kiwi berry, is squeezed the juice, and then crosses 0.22 μm of filter membrane, obtains kiwi-fruit juice.
Using the method for above-mentioned medium culture Dendritic Cells, including：Peripheral blood is separated using density-gradient centrifugation method
Mononuclearcell with above-mentioned culture medium is mixed, is subsequently placed in 37 DEG C, 5%CO by middle mononuclearcell2Under the conditions of cultivated,
Obtain immature dendritic cell.
Embodiment 4
A kind of Dendritic Cells culture medium, including minimal medium and adding ingredient, wherein, minimal medium is by glucose
Parenteral solution, normal saline solution, amino acid injection and micro-element injection are 5.5 by volume：4.5：1.5：1 is mixed
It closes, adding ingredient includes the component of following final concentration：GM-CSF180ng/mL, glutamine 3.5mmol/L, astragalus polyose
0.4mg/L, notoginseng polysaccharide 0.4mg/L, chick embryo extract 2.5v/v%, ginger juice 2.5v/v%, kiwi-fruit juice 2.5v/v%,
IL-425ng/mL, TNF-α 1000UI/mL, ironic citrate 450mg/L and vitamin C 5mg/L.
Wherein, astragalus polyose is prepared by the following method to obtain：The waste residue for extracting astragalus root total saponin is taken, adds 4 times of waste residue
The water of weight boils 40min, filtering, obtains the first filtrate and the first precipitation, and the water of 3 times of weight is added into the first precipitation, boils 40min,
Filtering obtains the second filtrate and the second precipitation, then adds in 3 times of water into second of precipitation again, boil 30min, filter, obtains third time
Filtrate and third time precipitate, and merge filtrate three times, and the total filtrate of spin concentration to the 1/5 of original volume adds in absolute ethyl alcohol, side edged
Stirring is placed, and then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain astragalus polyose.
Notoginseng polysaccharide is prepared by the following method to obtain：The waste residue for extracting arasaponin is taken, adds 4 times of weight of waste residue
Water boils 40min, filtering, obtains the first filtrate and the first precipitation, and the water of 3 times of weight is added into the first precipitation, boils 40min, is filtered,
The second filtrate and the second precipitation are obtained, 3 times of water is then added in into second of precipitation again, boils 30min, filtering obtains third time filtrate
It is precipitated with third time, merges filtrate three times, the total filtrate of spin concentration to the 1/5 of original volume adds in absolute ethyl alcohol, and side edged stirs
It mixes, places, then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain notoginseng polysaccharide.
Chick embryo extract is prepared by the following method to obtain：Corn in chicken embryo is removed, is then homogenized, then in equal volume
DMEM dilution, 3h is stirred under the conditions of 20-25 DEG C, 50r/min, is thawed after 96h is frozen under the conditions of being then transferred to -80 DEG C,
Centrifugation, collects supernatant, chick embryo extract is obtained after filtered degerming.
Ginger juice is to clean ginger, dry, and is then squeezed, and by gained liquid by 0.22 μm of filter membrane, obtains ginger
Juice；Kiwi-fruit juice is to remove the peel Kiwi berry, is squeezed the juice, and then crosses 0.22 μm of filter membrane, obtains kiwi-fruit juice.
Using the method for above-mentioned medium culture Dendritic Cells, including：Peripheral blood is separated using density-gradient centrifugation method
Mononuclearcell with above-mentioned culture medium is mixed, is subsequently placed in 37 DEG C, 5%CO by middle mononuclearcell2Under the conditions of cultivated,
Obtain immature dendritic cell.
Embodiment 5
A kind of Dendritic Cells culture medium, including minimal medium and adding ingredient, wherein, minimal medium is by glucose
Parenteral solution, normal saline solution, amino acid injection and micro-element injection are 5 by volume：4：1.5：1 mixing, adds
Addition point includes the component of following final concentration：GM-CSF 150ng/mL, glutamine 3mmol/L, astragalus polyose 0.3mg/L, three
Seven polysaccharide 0.3mg/L, chick embryo extract 2v/v%, ginger juice 2v/v%, kiwi-fruit juice 2v/v%, IL-420ng/mL, TNF-α
1000UI/mL, ironic citrate 400mg/L and vitamin C 5mg/L.
Wherein, astragalus polyose is prepared by the following method to obtain：The waste residue for extracting astragalus root total saponin is taken, adds 4 times of waste residue
The water of weight boils 40min, filtering, obtains the first filtrate and the first precipitation, and the water of 3 times of weight is added into the first precipitation, boils 40min,
Filtering obtains the second filtrate and the second precipitation, then adds in 3 times of water into second of precipitation again, boil 30min, filter, obtains third time
Filtrate and third time precipitate, and merge filtrate three times, and the total filtrate of spin concentration to the 1/5 of original volume adds in absolute ethyl alcohol, side edged
Stirring is placed, and then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain astragalus polyose.
Notoginseng polysaccharide is prepared by the following method to obtain：The waste residue for extracting arasaponin is taken, adds 4 times of weight of waste residue
Water boils 40min, filtering, obtains the first filtrate and the first precipitation, and the water of 3 times of weight is added into the first precipitation, boils 40min, is filtered,
The second filtrate and the second precipitation are obtained, 3 times of water is then added in into second of precipitation again, boils 30min, filtering obtains third time filtrate
It is precipitated with third time, merges filtrate three times, the total filtrate of spin concentration to the 1/5 of original volume adds in absolute ethyl alcohol, and side edged stirs
It mixes, places, then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain notoginseng polysaccharide.
Chick embryo extract is prepared by the following method to obtain：Corn in chicken embryo is removed, is then homogenized, then in equal volume
DMEM dilution, 3h is stirred under the conditions of 20-25 DEG C, 50r/min, is thawed after 96h is frozen under the conditions of being then transferred to -80 DEG C,
Centrifugation, collects supernatant, chick embryo extract is obtained after filtered degerming.
Ginger juice is to clean ginger, dry, and is then squeezed, and by gained liquid by 0.22 μm of filter membrane, obtains ginger
Juice；Kiwi-fruit juice is to remove the peel Kiwi berry, is squeezed the juice, and then crosses 0.22 μm of filter membrane, obtains kiwi-fruit juice.
Using the method for above-mentioned medium culture Dendritic Cells, including：Peripheral blood is separated using density-gradient centrifugation method
Mononuclearcell with above-mentioned culture medium is mixed, is subsequently placed in 37 DEG C, 5%CO by middle mononuclearcell2Under the conditions of cultivated,
Obtain immature dendritic cell.
Comparative example 1
1 difference from Example 5 of comparative example is, ginger juice and kiwi-fruit juice are lacked in culture medium, other and implementation
Example 5 is identical.
Comparative example 2
2 difference from Example 5 of comparative example is, astragalus polyose, notoginseng polysaccharide and chicken embryo extraction are lacked in culture medium
Object, other are same as Example 5.
Comparative example 3
3 difference from Example 5 of comparative example is, astragalus polyose, notoginseng polysaccharide, chicken embryo extraction are lacked in culture medium
Object, ginger juice and kiwi-fruit juice, other are same as Example 5.
Dendritic Cells is cultivated using embodiment 1-5 and comparative example 1-3 culture mediums respectively, embodiment 1-5 is cultivated
The Dendritic Cells quantity arrived is substantially many more than comparative example 1-3 quantity, especially embodiment 5, culture gained cell quantity
Maximum, and 3 gained cell quantity of comparative example is minimum, while cell viability obtained by embodiment 1-5 is best, comparative example 1-3 cells are lived
Power is substantially poorer than embodiment 1-5, and especially 3 vigor of comparative example is worst.
Claims (7)
1. a kind of Dendritic Cells culture medium, which is characterized in that including minimal medium and adding ingredient, wherein, it is basic to cultivate
Base is 4-6 by volume by glucose injection, normal saline solution, amino acid injection and micro-element injection：3-
5：1.5-2：1-1.5 is mixed, and adding ingredient includes the component of following final concentration：GM-CSF 100-200ng/mL, glutamine 2-
4mmol/L, astragalus polyose 0.2-0.5mg/L, notoginseng polysaccharide 0.2-0.5mg/L, chick embryo extract 1-3v/v%, ginger juice 1-
3v/v%, kiwi-fruit juice 1-3v/v%, IL-415-30ng/mL, TNF-α 800-12000UI/mL, ironic citrate 300-500mg/
L and vitamin C 3-6mg/L.
2. Dendritic Cells culture medium according to claim 1, which is characterized in that the minimal medium is noted by glucose
It is 5 by volume to penetrate liquid, normal saline solution, amino acid injection and micro-element injection：4：1.5：1 mixing.
3. Dendritic Cells culture medium according to claim 1, which is characterized in that it is dense that the adding ingredient includes following end
The component of degree：GM-CSF 150ng/mL, glutamine 3mmol/L, astragalus polyose 0.3mg/L, notoginseng polysaccharide 0.3mg/L, chicken embryo
Extract 2v/v%, ginger juice 2v/v%, kiwi-fruit juice 2v/v%, IL-420ng/mL, TNF-α 1000UI/mL, ironic citrate
400mg/L and vitamin C 5mg/L.
4. the Dendritic Cells culture medium according to claim 1 or 3, which is characterized in that the astragalus polyose passes through following
Method is prepared：The waste residue for extracting astragalus root total saponin is taken, adds the water of 3-5 times of weight of waste residue, boils 40-60min, filters, obtains
First filtrate and the first precipitation into the first precipitation plus the water of 2-4 times of weight, are boiled 30-50min, are filtered, obtain the second filtrate and the
Two precipitations, 2-3 times of water is then added in into second of precipitation, boils 20-30min again, is filtered, and obtains third time filtrate and third time is sunk
It forming sediment, merges filtrate three times, the 1/4-1/5 of the total filtrate of spin concentration to original volume adds in absolute ethyl alcohol, stirring while adding, places,
Then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain astragalus polyose.
5. the Dendritic Cells culture medium according to claim 1 or 3, which is characterized in that the notoginseng polysaccharide passes through following
Method is prepared：The waste residue for extracting arasaponin is taken, adds the water of 3-5 times of weight of waste residue, boils 40-60min, filters, obtains
First filtrate and the first precipitation into the first precipitation plus the water of 2-4 times of weight, are boiled 30-50min, are filtered, obtain the second filtrate and the
Two precipitations, 2-3 times of water is then added in into second of precipitation, boils 20-30min again, is filtered, and obtains third time filtrate and third time is sunk
It forming sediment, merges filtrate three times, the 1/4-1/5 of the total filtrate of spin concentration to original volume adds in absolute ethyl alcohol, stirring while adding, places,
Then filtering must precipitate, then wash precipitation with absolute ethyl alcohol, finally dry, obtain notoginseng polysaccharide.
6. the Dendritic Cells culture medium according to claim 1 or 3, which is characterized in that the chick embryo extract by with
Lower section method is prepared：Corn in chicken embryo is removed, is then homogenized, then with isometric DMEM dilutions, at 20-25 DEG C, 30-
2-3h is stirred under the conditions of 80r/min, is thawed after 48-96h is frozen under the conditions of being then transferred to -80 DEG C, centrifuges, collects supernatant,
Chick embryo extract is obtained after filtered degerming.
7. using the method for any one of the claim 1-6 medium culture Dendritic Cells, which is characterized in that including：It adopts
Mononuclearcell in peripheral blood is separated with density-gradient centrifugation method, mononuclearcell with above-mentioned culture medium is mixed, is subsequently placed in
37 DEG C, 5%CO2Under the conditions of cultivated, obtain immature dendritic cell.
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