CN107794242A - A kind of CIK cell culture medium - Google Patents
A kind of CIK cell culture medium Download PDFInfo
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Abstract
The invention discloses a kind of CIK cell culture medium, including basal medium and the additive made an addition in basal medium, by final concentration, the additive includes the component of following content：The 35ng/ml of CD3 excitated types monoclonal antibody 20, the 2.5mg/ml of 0.1 2 sustained-release micro-spheres of 0.2mmol/L, IL of human serum albumin 1.2, the 40mg/ml of IFN γ sustained-release micro-spheres 30, μ g/ml of soluble polysaccharide 60 80, μ g/ml of LBP-X 60 80, μ g/ml of lentinan 80 100, the μ g/ml of vitamin C 28 and the μ g/ml of ginsenoside 5 10.The culture medium each component is mutually coordinated, collective effect, and the nutrition of abundance and good environment can be provided for CIK cell growth and breeding, it is possible to increase the activity and multiplication capacity of immunocyte, and then the CIK cell for obtaining culture shows higher amplification times.
Description
Technical field
A kind of technical field of cell culture of the present invention, and in particular to CIK cell culture medium.
Background technology
Adoptive cellular immunotherapy is that there is the powerful immunocyte for killing tumor activity to feed back to patient's external evoked
A kind of Biotherapy method, it not only has the ability of remaining cancer cell in contact element, while can strengthen the immunity of host, because
This, adoptive cellular immunotherapy is as one of important means of tumor biotherapy.Cytokine induced kill cell
(Cytokine induced killer cell, CIK), is by human peripheral blood single nucleus cell (Peripheralblood
Mononuclear cell, PBMC) a group foreign cell that is obtained after the stimulation of cytokine profiles co-incubation, its is main
Effector cell is CD3+CD56+Double positive cells, with the powerful anti-tumor activity of T lymphocytes and non-principal histocompatbility
Compound (MHC) restricted the characteristics of killing knurl, therefore, CIK cell are considered as current antitumor cell adoptive immunotherapy
Preferred option.
CIK cell can largely be bred by external evoked, and conventional CIK preparation methods are using culture medium to separation
PMNC is cultivated, and is added Porcine HGF and carried out stimulation induction, finally obtains a number of CIK
Cell.But the problem of activity and poor multiplication capacity mostly be present using the CIK cell of existing medium culture, it is final to obtain
CD3+CD56+CIK cell quantity is not ideal enough.
The content of the invention
For above-mentioned deficiency of the prior art, the invention provides a kind of CIK cell culture medium, can effectively solve existing
Medium culture CIK cell mostly exist activity and multiplication capacity it is poor the problem of.
To achieve the above object, the technical solution adopted for the present invention to solve the technical problems is：
A kind of CIK cell culture medium, including basal medium and the additive that makes an addition in basal medium, by final concentration
Meter, additive include the component of following content：CD3 excitated type monoclonal antibodies 20-35ng/ml, human serum albumin 0.1-0.2mmol/L,
IL-2 sustained-release micro-spheres 1.2-2.5mg/ml, IFN-γ sustained-release micro-spheres 30-40mg/ml, soluble polysaccharide 60-80 μ g/ml, LBP-X
60-80 μ g/ml, lentinan 80-100 μ g/ml, the μ of Catergen -8 g/ml and ginsenoside 5-10 μ g/ml.
Further, a kind of CIK cell culture medium, including basal medium and the addition that makes an addition in basal medium
Agent, by final concentration, additive includes the component of following content：CD3 excitated type monoclonal antibodies 28ng/ml, human serum albumin
0.15mmol/L, IL-2 sustained-release micro-spheres 2.2mg/ml, IFN-γ sustained-release micro-spheres 35mg/ml, μ g/ml of soluble polysaccharide 70, matrimony vine are more
68 μ g/ml of sugar, μ g/ml of lentinan 90, the μ g/ml of vitamin C 5 and the μ g/ml of ginsenoside 8.
Further, IL-2 sustained-release micro-spheres are prepared by the following method to obtain：
(1) mannitol and zinc sulfate are separately added into water, mixed, 8-10wt% Osmitrol and 8- is made
10wt% zinc sulfate solution, both are 3-4 by volume again：1-2 is mixed；
(2) IL-2 and polyethylene glycol being added in step (1) resulting solution and mixed so that IL-2 concentration is 2-3wt%,
The concentration of polyethylene glycol is 8-10wt%；
(3) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 20-25wt% in compound；
(4) it is 1-2 by volume by step (2) gains and step (4) gains：1.5-3 mixing；
(5) polyvinyl alcohol, chitosan and sodium chloride are added to the water, stirred, polyvinyl alcohol, chitosan and sodium chloride
Concentration be respectively 1-2wt%, 0.6-1.2wt% and 1-2wt%, then add step (4) gains, stir 1-1.5h, use
Water washing, it is then centrifuged for separating, last vacuum freeze drying, IL-2 sustained-release micro-spheres is made.
Further, the particle diameter of IL-2 sustained-release micro-spheres is 60-80 μm.
Further, IFN-γ sustained-release micro-spheres are prepared by the following method to obtain：
(1) IFN-γ and polyethylene glycol are separately added into advance added with glucan, zinc sulfate and pentaerythrite zinc salt
In the aqueous solution, stir；Wherein, the final concentration of IFN-γ, polyethylene glycol, glucan, zinc sulfate and pentaerythrite zinc salt point
Wei not 3-6wt%, 5-10wt%, 5-6wt%, 1-3wt% and 2-4wt%；
(2) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 20-25wt% in compound；
(3) it is 1 by volume by step (1) gains and step (2) gains：2-3 is mixed；
(4) polyvinyl alcohol, chitosan and sodium alginate are added to the water, stirred, polyvinyl alcohol, chitosan and marine alga
The concentration of sour sodium is respectively 1-2wt%, 0.6-1.2wt% and 1-2wt%, then adds step (3) gains, stirs 1-
1.5h, it is washed with water, is then centrifuged for separating, last vacuum freeze drying, the second cell factor sustained release spheroid is made.
Further, the particle diameter of IFN-γ sustained-release micro-spheres is 60-80 μm.
Further, basal medium is DMEM culture mediums.
A kind of CIK cell culture medium provided by the invention, has the advantages that：
The CIK cell culture medium each component of the present invention is mutually coordinated, collective effect, can be that CIK cell growth and breeding carries
Nutrition and good environment for abundance, it is possible to increase the activity and multiplication capacity of immunocyte, and then the CIK for obtaining culture
Cells show goes out higher amplification times, stronger killing activity and more cell surface antigen content, cultivates wild Oryza species
In do not detect fungi, bacterium, mycoplasma, microorganism detection index meets the requirements.
Embodiment
Embodiment 1
A kind of CIK cell culture medium, including basal medium and the additive made an addition in basal medium, basis culture
Base is DMEM culture mediums, and by final concentration, additive includes the component of following content：CD3 excitated type monoclonal antibodies 20ng/ml, people's blood
Albumin 0.1mmol/L, IL-2 sustained-release micro-spheres 1.2mg/ml, IFN-γ sustained-release micro-spheres 30mg/ml, μ g/ml of soluble polysaccharide 60, Chinese holly
μ g/ml of Qi polysaccharide 60, μ g/ml of lentinan 80, the μ g/ml of vitamin C 2 and the μ g/ml of ginsenoside 5.
Wherein, IL-2 sustained-release micro-spheres are prepared by the following method to obtain：
(1) mannitol and zinc sulfate are separately added into water, mixed, 8wt% Osmitrol and 8wt% is made
Zinc sulfate solution, both are 3 by volume again：1 mixing；
(2) IL-2 and polyethylene glycol are added in step (1) resulting solution and mixed so that IL-2 concentration is 2wt%, is gathered
The concentration of ethylene glycol is 8wt%；
(3) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 20wt% in compound；
(4) it is 1 by volume by step (2) gains and step (4) gains：1.5 mixing；
(5) polyvinyl alcohol, chitosan and sodium chloride are added to the water, stirred, polyvinyl alcohol, chitosan and sodium chloride
Concentration be respectively 1wt%, 0.6wt% and 1wt%, then add step (4) gains, stir 1h, be washed with water, Ran Houli
The heart separates, last vacuum freeze drying, and IL-2 sustained-release micro-spheres are made, and its particle diameter is 60-80 μm.
IFN-γ sustained-release micro-spheres are prepared by the following method to obtain：
(1) IFN-γ and polyethylene glycol are separately added into advance added with glucan, zinc sulfate and pentaerythrite zinc salt
In the aqueous solution, stir；Wherein, the final concentration of IFN-γ, polyethylene glycol, glucan, zinc sulfate and pentaerythrite zinc salt point
Wei not 3wt%, 5wt%, 5wt%, 1wt% and 2wt%；
(2) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 20wt% in compound；
(3) it is 1 by volume by step (1) gains and step (2) gains：2 mixing；
(4) polyvinyl alcohol, chitosan and sodium alginate are added to the water, stirred, polyvinyl alcohol, chitosan and marine alga
The concentration of sour sodium is respectively 1wt%, 0.6wt% and 1wt%, then adds step (3) gains, stirs 1h, is washed with water, so
After centrifuge, last vacuum freeze drying, be made the second cell factor sustained release spheroid, its particle diameter be 60-80 μm.
Embodiment 2
A kind of CIK cell culture medium, including basal medium and the additive made an addition in basal medium, basis culture
Base is DMEM culture mediums, and by final concentration, additive includes the component of following content：CD3 excitated type monoclonal antibodies 35ng/ml, people's blood
Albumin 0.2mmol/L, IL-2 sustained-release micro-spheres 2.5mg/ml, IFN-γ sustained-release micro-spheres 40mg/ml, μ g/ml of soluble polysaccharide 80, Chinese holly
μ g/ml of Qi polysaccharide 80, μ g/ml of lentinan 100, the μ g/ml of vitamin C 8 and the μ g/ml of ginsenoside 10.
Wherein, IL-2 sustained-release micro-spheres are prepared by the following method to obtain：
(1) mannitol and zinc sulfate are separately added into water, mixed, 10wt% Osmitrol and 10wt% is made
Zinc sulfate solution, both again by volume be 2：1 mixing；
(2) IL-2 and polyethylene glycol are added in step (1) resulting solution and mixed so that IL-2 concentration is 3wt%, is gathered
The concentration of ethylene glycol is 10wt%；
(3) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 25wt% in compound；
(4) it is 2 by volume by step (2) gains and step (4) gains：3 mixing；
(5) polyvinyl alcohol, chitosan and sodium chloride are added to the water, stirred, polyvinyl alcohol, chitosan and sodium chloride
Concentration be respectively 2wt%, 1.2wt% and 2wt%, then add step (4) gains, stir 1.5h, be washed with water, then
Centrifuge, last vacuum freeze drying, IL-2 sustained-release micro-spheres are made, its particle diameter is 60-80 μm.
IFN-γ sustained-release micro-spheres are prepared by the following method to obtain：
(1) IFN-γ and polyethylene glycol are separately added into advance added with glucan, zinc sulfate and pentaerythrite zinc salt
In the aqueous solution, stir；Wherein, the final concentration of IFN-γ, polyethylene glycol, glucan, zinc sulfate and pentaerythrite zinc salt point
Wei not 6wt%, 10wt%, 6wt%, 1wt% and 4wt%；
(2) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 25wt% in compound；
(3) it is 1 by volume by step (1) gains and step (2) gains：3 mixing；
(4) polyvinyl alcohol, chitosan and sodium alginate are added to the water, stirred, polyvinyl alcohol, chitosan and marine alga
The concentration of sour sodium is respectively 2wt%, 1.2wt% and 2wt%, then adds step (3) gains, stirs 1.5h, is washed with water,
It is then centrifuged for separating, last vacuum freeze drying, the second cell factor sustained release spheroid is made, its particle diameter is 60-80 μm.
Embodiment 3
A kind of CIK cell culture medium, including basal medium and the additive made an addition in basal medium, basis culture
Base is DMEM culture mediums, and by final concentration, additive includes the component of following content：CD3 excitated type monoclonal antibodies 25ng/ml, people's blood
Albumin 0.13mmol/L, IL-2 sustained-release micro-spheres 1.5mg/ml, IFN-γ sustained-release micro-spheres 32mg/ml, μ g/ml of soluble polysaccharide 65,
μ g/ml of LBP-X 65, μ g/ml of lentinan 85, the μ g/ml of vitamin C 4 and the μ g/ml of ginsenoside 6.
Wherein, IL-2 sustained-release micro-spheres are prepared by the following method to obtain：
(1) mannitol and zinc sulfate are separately added into water, mixed, 8wt% Osmitrol and 10wt% is made
Zinc sulfate solution, both again by volume be 4：1 mixing；
(2) IL-2 and polyethylene glycol are added in step (1) resulting solution and mixed so that IL-2 concentration is 3wt%, is gathered
The concentration of ethylene glycol is 8wt%；
(3) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 22wt% in compound；
(4) it is 1.5 by volume by step (2) gains and step (4) gains：2 mixing；
(5) polyvinyl alcohol, chitosan and sodium chloride are added to the water, stirred, polyvinyl alcohol, chitosan and sodium chloride
Concentration be respectively 1.2wt%, 0.8wt% and 1.2wt%, then add step (4) gains, stir 1.5h, be washed with water,
It is then centrifuged for separating, last vacuum freeze drying, IL-2 sustained-release micro-spheres is made, its particle diameter is 60-80 μm.
IFN-γ sustained-release micro-spheres are prepared by the following method to obtain：
(1) IFN-γ and polyethylene glycol are separately added into advance added with glucan, zinc sulfate and pentaerythrite zinc salt
In the aqueous solution, stir；Wherein, the final concentration of IFN-γ, polyethylene glycol, glucan, zinc sulfate and pentaerythrite zinc salt point
Wei not 4wt%, 6wt%, 5.5wt%, 1.5wt% and 2.5wt%；
(2) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 22wt% in compound；
(3) it is 1 by volume by step (1) gains and step (2) gains：3 mixing；
(4) polyvinyl alcohol, chitosan and sodium alginate are added to the water, stirred, polyvinyl alcohol, chitosan and marine alga
The concentration of sour sodium is respectively 1.2wt%, 0.8wt% and 1.2wt%, then adds step (3) gains, stirs 1.5h, uses water
Washing, is then centrifuged for separating, last vacuum freeze drying, and the second cell factor sustained release spheroid is made, and its particle diameter is 60-80 μm.
Embodiment 4
A kind of CIK cell culture medium, including basal medium and the additive made an addition in basal medium, basis culture
Base is DMEM culture mediums, and by final concentration, additive includes the component of following content：CD3 excitated type monoclonal antibodies 32ng/ml, people's blood
Albumin 0.18mmol/L, IL-2 sustained-release micro-spheres 2.2mg/ml, IFN-γ sustained-release micro-spheres 38mg/ml, μ g/ml of soluble polysaccharide 75,
μ g/ml of LBP-X 75, μ g/ml of lentinan 95, the μ g/ml of vitamin C 6 and the μ g/ml of ginsenoside 8.
Wherein, IL-2 sustained-release micro-spheres are prepared by the following method to obtain：
(1) mannitol and zinc sulfate are separately added into water, mixed, 10wt% Osmitrol and 10wt% is made
Zinc sulfate solution, both again by volume be 3：1 mixing；
(2) IL-2 and polyethylene glycol being added in step (1) resulting solution and mixed so that IL-2 concentration is 2.8wt%,
The concentration of polyethylene glycol is 9.2wt%；
(3) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 23wt% in compound；
(4) it is 2 by volume by step (2) gains and step (4) gains：3 mixing；
(5) polyvinyl alcohol, chitosan and sodium chloride are added to the water, stirred, polyvinyl alcohol, chitosan and sodium chloride
Concentration be respectively 1.8wt%, 1.0wt% and 1.8wt%, then add step (4) gains, stir 1.5h, be washed with water,
It is then centrifuged for separating, last vacuum freeze drying, IL-2 sustained-release micro-spheres is made, its particle diameter is 60-80 μm.
IFN-γ sustained-release micro-spheres are prepared by the following method to obtain：
(1) IFN-γ and polyethylene glycol are separately added into advance added with glucan, zinc sulfate and pentaerythrite zinc salt
In the aqueous solution, stir；Wherein, the final concentration of IFN-γ, polyethylene glycol, glucan, zinc sulfate and pentaerythrite zinc salt point
Wei not 5wt%, 8wt%, 8wt%, 2.2wt% and 3.5wt%；
(2) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 23wt% in compound；
(3) it is 1 by volume by step (1) gains and step (2) gains：3 mixing；
(4) polyvinyl alcohol, chitosan and sodium alginate are added to the water, stirred, polyvinyl alcohol, chitosan and marine alga
The concentration of sour sodium is respectively 1.8wt%, 1.0wt% and 1.8wt%, then adds step (3) gains, stirs 1.5h, uses water
Washing, is then centrifuged for separating, last vacuum freeze drying, and the second cell factor sustained release spheroid is made, and its particle diameter is 60-80 μm.
Embodiment 5
A kind of CIK cell culture medium, including basal medium and the additive made an addition in basal medium, basis culture
Base is DMEM culture mediums, and by final concentration, additive includes the component of following content：CD3 excitated type monoclonal antibodies 28ng/ml, people's blood
Albumin 0.15mmol/L, IL-2 sustained-release micro-spheres 2.2mg/ml, IFN-γ sustained-release micro-spheres 35mg/ml, μ g/ml of soluble polysaccharide 70,
μ g/ml of LBP-X 68, μ g/ml of lentinan 90, the μ g/ml of vitamin C 5 and the μ g/ml of ginsenoside 8.
Wherein, IL-2 sustained-release micro-spheres are prepared by the following method to obtain：
(1) mannitol and zinc sulfate are separately added into water, mixed, 10wt% Osmitrol and 10wt% is made
Zinc sulfate solution, both again by volume be 3：1 mixing；
(2) IL-2 and polyethylene glycol being added in step (1) resulting solution and mixed so that IL-2 concentration is 2.5wt%,
The concentration of polyethylene glycol is 9wt%；
(3) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 23wt% in compound；
(4) it is 2 by volume by step (2) gains and step (4) gains：3 mixing；
(5) polyvinyl alcohol, chitosan and sodium chloride are added to the water, stirred, polyvinyl alcohol, chitosan and sodium chloride
Concentration be respectively 1.5wt%, 1.0wt% and 1.5wt%, then add step (4) gains, stir 1.5h, be washed with water,
It is then centrifuged for separating, last vacuum freeze drying, IL-2 sustained-release micro-spheres is made, its particle diameter is 60-80 μm.
IFN-γ sustained-release micro-spheres are prepared by the following method to obtain：
(1) IFN-γ and polyethylene glycol are separately added into advance added with glucan, zinc sulfate and pentaerythrite zinc salt
In the aqueous solution, stir；Wherein, the final concentration of IFN-γ, polyethylene glycol, glucan, zinc sulfate and pentaerythrite zinc salt point
Wei not 5wt%, 6wt%, 5wt%, 2wt% and 3wt%；
(2) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, this is mixed
Polyglycolic-polylactic acid concentration is 23wt% in compound；
(3) it is 1 by volume by step (1) gains and step (2) gains：3 mixing；
(4) polyvinyl alcohol, chitosan and sodium alginate are added to the water, stirred, polyvinyl alcohol, chitosan and marine alga
The concentration of sour sodium is respectively 1.5wt%, 1.0wt% and 1.5wt%, then adds step (3) gains, stirs 1.5h, uses water
Washing, is then centrifuged for separating, last vacuum freeze drying, and the second cell factor sustained release spheroid is made, and its particle diameter is 60-80 μm.
Comparative example 1
The difference from Example 5 of comparative example 1 is, soluble polysaccharide and LBP-X are free of in additive, remaining and reality
It is identical to apply example 5.
Comparative example 2
The difference from Example 5 of comparative example 2 is, vitamin C and ginsenoside are free of in additive, remaining and implementation
Example 5 is identical.
Comparative example 3
The difference from Example 5 of comparative example 3 is, in additive without soluble polysaccharide, LBP-X, lentinan,
Vitamin C and ginsenoside, remaining is same as Example 5.
Experimental example
The medium culture CIK cell provided using embodiment 1-5 and comparative example 1-3, after then carrying out Trypan Blue,
Cell count is carried out with cell counting count board, and calculates cell number and Cell viability, it the results are shown in Table 1：
As shown in Table 1, the medium culture CIK cell that embodiment 1-5 is provided, its cell number are far above comparative example, explanation
Embodiment 1-5 amplification times are higher than comparative example, and the motility rate of cell is also apparently higher than comparative example, CD3+CD56+Effector cell's obtains
Obtain quantity also to greatly increase, hence it is evident that higher than comparative example, especially embodiment 5, best results.
Claims (7)
1. a kind of CIK cell culture medium, it is characterised in that including basal medium and the addition made an addition in basal medium
Agent, by final concentration, the additive includes the component of following content：CD3 excitated type monoclonal antibodies 20-35ng/ml, human serum albumin
0.1-0.2mmol/L, IL-2 sustained-release micro-spheres 1.2-2.5mg/ml, IFN-γ sustained-release micro-spheres 30-40mg/ml, soluble polysaccharide 60-80
μ g/ml, LBP-X 60-80 μ g/ml, lentinan 80-100 μ g/ml, vitamin C 2-8 μ g/ml and ginsenoside 5-10 μ
g/ml。
2. CIK cell culture medium according to claim 1, it is characterised in that including basal medium and make an addition to basis
Additive in culture medium, by final concentration, the additive includes the component of following content：CD3 excitated type monoclonal antibodies 28ng/
Ml, human serum albumin 0.15mmol/L, IL-2 sustained-release micro-spheres 2.2mg/ml, IFN-γ sustained-release micro-spheres 35mg/ml, soluble polysaccharide 70
μ g/ml, μ g/ml of LBP-X 68, μ g/ml of lentinan 90, the μ g/ml of vitamin C 5 and the μ g/ml of ginsenoside 8.
3. CIK cell culture medium according to claim 1 or 2, it is characterised in that the IL-2 sustained-release micro-spheres pass through following
Method is prepared：
(1) mannitol and zinc sulfate are separately added into water, mixed, 8-10wt% Osmitrol and 8-10wt% is made
Zinc sulfate solution, both are 3-4 by volume again：1-2 is mixed；
(2) IL-2 and polyethylene glycol are added in step (1) resulting solution and mixed so that IL-2 concentration is 2-3wt%, poly- second
The concentration of glycol is 8-10wt%；
(3) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, the mixture
Middle polyglycolic-polylactic acid concentration is 20-25wt%；
(4) it is 1-2 by volume by step (2) gains and step (4) gains：1.5-3 mixing；
(5) polyvinyl alcohol, chitosan and sodium chloride are added to the water, stirred, polyvinyl alcohol, chitosan and sodium chloride it is dense
Degree is respectively 1-2wt%, 0.6-1.2wt% and 1-2wt%, then adds step (4) gains, stirs 1-1.5h, is washed with water
Wash, be then centrifuged for separating, last vacuum freeze drying, IL-2 sustained-release micro-spheres are made.
4. CIK cell culture medium according to claim 5, it is characterised in that the particle diameter of the IL-2 sustained-release micro-spheres is 60-
80μm。
5. CIK cell culture medium according to claim 1 or 2, it is characterised in that the IFN-γ sustained-release micro-spheres by with
Lower section method is prepared：
(1) IFN-γ and polyethylene glycol are separately added into advance added with the water-soluble of glucan, zinc sulfate and pentaerythrite zinc salt
In liquid, stir；Wherein, the final concentration of IFN-γ, polyethylene glycol, glucan, zinc sulfate and pentaerythrite zinc salt is respectively
3-6wt%, 5-10wt%, 5-6wt%, 1-3wt% and 2-4wt%；
(2) polyglycolic-polylactic acid is added in dichloromethane solution, ultrasonic disperse 2-3min, obtains mixture, the mixture
Middle polyglycolic-polylactic acid concentration is 20-25wt%；
(3) it is 1 by volume by step (1) gains and step (2) gains：2-3 is mixed；
(4) polyvinyl alcohol, chitosan and sodium alginate are added to the water, stirred, polyvinyl alcohol, chitosan and sodium alginate
Concentration be respectively 1-2wt%, 0.6-1.2wt% and 1-2wt%, then add step (3) gains, stir 1-1.5h, use
Water washing, it is then centrifuged for separating, last vacuum freeze drying, the second cell factor sustained release spheroid is made.
6. CIK cell culture medium according to claim 7, it is characterised in that the particle diameter of the IFN-γ sustained-release micro-spheres is
60-80μm。
7. CIK cell culture medium according to claim 1 or 2, it is characterised in that the basal medium is cultivated for DMEM
Base.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108410795A (en) * | 2018-04-12 | 2018-08-17 | 安庆医药高等专科学校 | The formula and production method and culture dish of a kind of culture medium |
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| WO2005070418A1 (en) * | 2004-01-21 | 2005-08-04 | M N L Pharma Limited | Immunomodulatory alkaloids |
| CN105087483A (en) * | 2015-07-24 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Immune cell culture medium and culture method, as well as application of ginsenoside |
| CN105087486A (en) * | 2015-08-17 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture |
| CN107119013A (en) * | 2017-04-14 | 2017-09-01 | 南华生物医药股份有限公司 | A kind of preparation method of enhanced CIK cell and the application of soluble polysaccharide and LBP-X |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005070418A1 (en) * | 2004-01-21 | 2005-08-04 | M N L Pharma Limited | Immunomodulatory alkaloids |
| CN105087483A (en) * | 2015-07-24 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | Immune cell culture medium and culture method, as well as application of ginsenoside |
| CN105087486A (en) * | 2015-08-17 | 2015-11-25 | 广州赛莱拉干细胞科技股份有限公司 | CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture |
| CN107119013A (en) * | 2017-04-14 | 2017-09-01 | 南华生物医药股份有限公司 | A kind of preparation method of enhanced CIK cell and the application of soluble polysaccharide and LBP-X |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108410795A (en) * | 2018-04-12 | 2018-08-17 | 安庆医药高等专科学校 | The formula and production method and culture dish of a kind of culture medium |
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