CN107142245A - A kind of CIK cell cultural method - Google Patents
A kind of CIK cell cultural method Download PDFInfo
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- CN107142245A CN107142245A CN201710401454.3A CN201710401454A CN107142245A CN 107142245 A CN107142245 A CN 107142245A CN 201710401454 A CN201710401454 A CN 201710401454A CN 107142245 A CN107142245 A CN 107142245A
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Abstract
The present invention relates to biological technical field, more particularly to a kind of CIK cell cultural method, comprise the following steps：（1）PMNC is resuspended in CIK nutrient solutions, cell suspension is obtained；（2）Cell suspension is added in the blake bottle being coated with advance with CD 3-resisting monoclonal antibody and retronectin, 37 DEG C, 5%CO are placed in afterwards2Cultivated in saturated humidity incubator；（3）3rd day supplement CIK nutrient solution of culture；（4）The 6th day of culture, has added after CIK nutrient solutions, has been enlarged culture；（5）9th day supplement CIK nutrient solution of culture；（6）Cultivate to the 13rd 14 day, harvesting.The present invention is remarkably improved the proliferation rate of immunocyte, especially CD3+CD56+The proliferation rate of effector cell, improves its killing activity.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of CIK cell cultural method.
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,
One of important means as tumor biotherapy.
Cytokine induced kill cell（Cytokine induced killer cell, CIK）, it is by human peripheral
Mononuclearcell（Peripheral blood mononuclear cells, PBMCs）Pierced through cytokine profiles co-incubation
The a group foreign cell obtained after swashing, its main effects cell is CD3+ CD56+Double positive cells, with T lymphocytes
Powerful anti-tumor activity and non-principal histocompatibility complex（MHC）It is restricted the characteristics of kill knurl.CIK cell has propagation speed
Degree is fast, it is high to kill tumor activity, it is wide, same to multidrug resistant tumour cell sensitive, thin to normal marrow hemopoiesis precursor to kill knurl spectrum
The features such as cellular toxicity is small, is that the tumor cell killing activity having now been found that is strong, a kind of preferable effect for being adapted to clinical practice is thin
Born of the same parents, but this effector cell is extremely rare in normal peripheral blood, therefore CIK cell is considered as that current antitumor cell is adopted
The preferred option of immunization therapy.
But the problem of there is activity and poor multiplication capacity mostly in the CIK cell of existing CIK cell method culture.
The content of the invention
The purpose of the present invention is that there is provided a kind of simple and effective CIK cell culture for above-mentioned deficiency of the prior art
Method, can significantly improve the activity and multiplication capacity of immunocyte, particularly CD3+CD56+The acquisition quantity of effector cell, is improved
Its killing activity.
The purpose of the present invention is achieved through the following technical solutions：
A kind of CIK cell cultural method, comprises the following steps：
（1）PMNC is resuspended in CIK nutrient solutions, cell suspension is obtained；
（2）Cell suspension is added in the blake bottle being coated with advance with AntiCD3 McAb monoclonal antibody and retronectin,
37 DEG C, 5%CO are placed in afterwards2Cultivated in saturated humidity incubator；Adjustment cell density is 7 × 10 before culture5-2×106/mL；
（3）3rd day supplement CIK nutrient solution of culture；
（4）The 6th day of culture, has added after CIK nutrient solutions, the culture liquid mixture in blake bottle is fallen to be dispensed into different trainings
Support in container, it is 7 × 10 to be enlarged adjustment cell density before culture, culture5-1.5×106/mL；
（5）9th day supplement CIK nutrient solution of culture；
（6）Cultivate to the 12-14 days, harvesting, it is preferable that culture was harvested to the 13rd day.
Preferably, the step（2）In, final concentration of 6 the μ g/mL, retronectin of anti-CD49d McAb end before culture
Concentration is 11 μ g/mL.
Preferably, the CIK nutrient solutions include basal medium and the additive made an addition in basal medium, by dense eventually
Degree meter, the additive includes the component of following content：Poly-D-lysine 20-30 μ g/mL, BCG polysaccharide nucleic acid 5-15mg/
ML, sodium selenite 0.03-0.04 μm of ol/mL, 4- hydroxyethyl piperazineethanesulfonic acid 5-8 μ g/mL, reduced glutathione 2-4 μ g/
ML, adenosine deaminase 8-12 μ g/mL, IFN-γ sustained-release micro-spheres 2-3mg/mL, IL-2 sustained-release micro-spheres 1.5-2mg/mL, autologous blood
Starch 4-6mL/L.
Preferably, the preparation method of the IL-2 sustained-release micro-spheres comprises the following steps：
In A, the aqueous solution by IL-2, polyethylene glycol addition in advance added with mannitol and zinc sulfate, after stirring, mixed
IL-2 concentration is 1.2-2.5wt% in compound A, the mixture A, and the concentration of polyethylene glycol is 7-12 wt%；
B, PLGA-polyethylene glycol added in dichloromethane and dissolved, after stirring, obtain mixture B,
The concentration of PLGA-polyethylene glycol is 12-18wt% in the mixture B；
C, mixture A is added in mixture B, after stirring, obtains mixture C, the mixture A and mixture B
Volume ratio be 1:1-2；
D, polyvinyl alcohol, chitosan and sodium chloride is added to the water, after stirring, obtains mixture D；In the mixture D
The concentration of polyvinyl alcohol is 1-2 wt %, and the concentration of chitosan is 0.5-1 wt %, and the concentration of sodium chloride is 1-2 wt %；
E, will mixture C add mixture D in, stirring carry out solvent volatilization, it is scrubbed, centrifuge, vacuum freeze drying, system
Obtain IL-2 sustained-release micro-spheres.
Preferably, the particle diameter of the IL-2 sustained-release micro-spheres is 60-110 μm.The IFN-γ sustained-release micro-spheres of the present invention make sustained release
The envelop rate of microballoon is more than 85%.
Preferably, in the mixture A, the concentration of the mannitol is 2-4 wt%, and carbonic acid zinc concentration is 1-3
Wt%.
Preferably, the polyvinyl alcohol is 1 by mass ratio:0.5-2 atactic polyvinyl alcohol and a rule polyethylene composition.
Preferably, the degree of polymerization of the atactic polyvinyl alcohol be 600-1000, it is described between rule polyethylene the degree of polymerization be
1800-2500。
Preferably, the preparation method of the IFN-γ sustained-release micro-spheres comprises the following steps：
S1, IFN-γ, heparin, polyethylene glycol added into the aqueous solution added with glucan and pentaerythrite zinc salt in advance, stirring
After uniform, the concentration for obtaining IFN-γ in mixture I, the mixture A is 2-5wt%, and the concentration of polyethylene glycol is 5-
9wt%, the concentration of heparin is 0.5-1.5wt%, it is preferable that the concentration of the glucan is 3-5 wt%, pentaerythrite zinc salt
Concentration be 2-4 wt%；
S2, will polylactic acid-polyglycol add dichloromethane in dissolve, after stirring, obtain mixture II, the mixing
The concentration of polylactic acid-polyglycol is 13-20wt% in thing II；
S3, mixture I is added in mixture II, after stirring, obtains mixture III, the mixture I and mixing
The volume ratio of thing II is 1:1.5-2.5；
S4, polyvinyl alcohol, chitosan and sodium alginate be added to the water, after stirring, obtain mixture IV；The mixture
The concentration of polyvinyl alcohol is 1-2 wt % in IV, and the concentration of chitosan is 0.5-1 wt %, and the concentration of sodium alginate is 2-4 wt
%；Preferably, the polyvinyl alcohol is 1 by mass ratio:0.5-2 atactic polyvinyl alcohol and a rule polyethylene composition；It is described random
The degree of polymerization of polyvinyl alcohol be 600-1000, it is described between rule polyethylene the degree of polymerization be 1800-2500；
S5, mixture III added in mixture IV, stirring carries out solvent volatilization, it is scrubbed, centrifuge, vacuum refrigeration is done
It is dry, IFN-γ sustained-release micro-spheres are made.
Preferably, the particle diameter of the IFN-γ sustained-release micro-spheres is 40-80 μm.The IFN-γ sustained-release micro-spheres of the present invention make sustained release
The envelop rate of microballoon is more than 90%.
The present invention rule polyvinyl alcohol, atactic polyvinyl alcohol and chitosan by between are combined, and as the raw material of gel, are improved solidifying
The thermal property and mechanical performance of glue, while providing the space contacted between the growing space of abundance and sertoli cell knot for cell
Structure, the cell of acquisition it is active stronger.Polyvinyl alcohol, chitosan and sodium chloride are engaged, and can be strengthened between polyvinyl alcohol molecule
With the crosslinking of strand, optimize the performance of gel, shortening prepares the cycle of polyvinyl alcohol, and raising prepares the efficiency of sustained-release micro-spheres,
So as to be more beneficial for CIK growth, increase CIK amplification times, it is to avoid IL-2 and IFN-γ concentration reduce too fast, concentration difference
It is different big, the stability of influence cell proliferation rate and cell quality.
The IL-2 sustained-release micro-spheres and IFN-γ sustained-release micro-spheres of the present invention have the advantages that long-acting slow-release, had no toxic side effect.
Preferably, the basal medium is DMEM culture mediums.
The beneficial effects of the present invention are：
(1) CIK cell cultural method technique of the invention is easy and effective, it is possible to increase the activity and propagation energy of immunocyte
Power, and then the CIK cells shows for obtaining culture go out big amplification times, strong killing activity and high cell surface antigen
Fungi, bacterium, mycoplasma are not detected after content, culture in nutrient solution, endotoxin is less than 0.5EU/mL, microorganism detection index symbol
Close and require.
(2) each composition of CIK nutrient solutions of the invention is mutually coordinated, collective effect, needed for providing growth and proliferation of cell
Sufficient nutrition and good environment, significantly improve the amplification rate of immunocyte, in CIK nutrient solutions add IL-2 sustained-release micro-spheres
With IFN-γ sustained-release micro-spheres, using its slow release characteristic make growth factor in the medium can continued smooth, release, can prevent training
Support IFN-γ factor concentration change in base and bring infringement to cell soon, operating efficiency can be improved, immunocyte is steadily expanded, with
BCG polysaccharide nucleic acid and other components are engaged, and can not only reduce the toxic side effect that heavy dose of cell factor is brought, and
CIK amplification rate, especially CD3 can be improved+And CD56+CIK cell, improves its killing activity；CIK nutrient solutions addition people is certainly
Body blood plasma, it is to avoid the risk for the infectious disease that external source serum is present, improves clinical safety.
Embodiment
The invention will be further described with the following Examples.
Embodiment 1
In the present embodiment, a kind of CIK cell cultural method comprises the following steps：
（1）PMNC is resuspended in CIK nutrient solutions, cell suspension is obtained；
（2）Cell suspension is added to the T175 cultures being coated with advance with AntiCD3 McAb monoclonal antibody and retronectin
In bottle, 37 DEG C, 5%CO are placed in afterwards2In saturated humidity incubator；Adjustment cell density is 7 × 10 before culture5-2×106/mL；
（3）The 3rd day mL of supplement CIK nutrient solutions 60 of culture；
（4）The 6th day of culture, has added after CIK nutrient solutions, original nutrient solution in former T175 blake bottles is poured into new blake bottle
In, former T175 blake bottles pour into new blake bottle after cell on wall is blown and beaten completely with nutrient solution, add CIK nutrient solutions to total
Volume is 400mL, and above-mentioned nutrient solution is dispensed into four culture bags, and it is 500mL that CIK nutrient solutions to cumulative volume is added respectively,
It is 7 × 10 to be enlarged adjustment cell density before culture, culture5-2×106/mL；
（5）The 9th day mL of supplement CIK nutrient solutions 70 of culture；
（6）Cultivate to the 13rd day, harvesting；
Preferably, the step（2）In, final concentration of 6 the μ g/mL, retronectin of anti-CD49d McAb final concentration before culture
For 11 μ g/mL.
Preferably, the CIK nutrient solutions include DMEM culture mediums and make an addition to the additive in DMEM culture mediums, by dense eventually
Degree meter, the additive includes the component of following content：The μ g/mL of poly-D-lysine 25, BCG polysaccharide nucleic acid 10mg/mL, Asia
The μ g/mL of 0.035 μm of ol/mL, 4- hydroxyethyl piperazineethanesulfonic acid of sodium selenate 6, the μ g/mL of reduced glutathione 3, the μ of adenosine deaminase 9
G/mL, IFN-γ sustained-release micro-spheres 2.5mg/mL, IL-2 sustained-release micro-spheres 1.75mg/mL, autologous plasma 5mL/L.
Preferably, the preparation method of the IL-2 sustained-release micro-spheres comprises the following steps：
In A, the aqueous solution by IL-2, polyethylene glycol addition in advance added with mannitol and zinc sulfate, after stirring, mixed
IL-2 concentration is 2wt% in compound A, the mixture A, and the concentration of polyethylene glycol is 9wt%；The concentration of the mannitol is
3wt%, carbonic acid zinc concentration is 2wt%.
B, PLGA-polyethylene glycol added in dichloromethane and dissolved, after stirring, mixed
The concentration of PLGA-polyethylene glycol is 16wt% in thing B, the mixture B；
C, mixture A is added in mixture B, after stirring, obtains mixture C, the mixture A and mixture B
Volume ratio be 1:1.5；
D, polyvinyl alcohol, chitosan and sodium chloride is added to the water, after stirring, obtains mixture D；In the mixture D
The concentration of polyvinyl alcohol is 1.5 wt %, and the concentration of chitosan is 0.7 wt %, and the concentration of sodium chloride is 1.5 wt %；
E, will mixture C add mixture D in, stirring carry out solvent volatilization, it is scrubbed, centrifuge, vacuum freeze drying, system
Obtain the IL-2 sustained-release micro-spheres that particle diameter is 60-110 μm, it is preferable that the volume ratio of mixture C and mixture D is 1:2.
Preferably, in the mixture A, it is preferable that the polyvinyl alcohol is 1 by mass ratio:1 atactic polyvinyl alcohol and
Between rule polyethylene composition, the degree of polymerization of the atactic polyvinyl alcohol is 600-1000, it is described between the degree of polymerization of rule polyethylene be
1800-2500。
Preferably, the preparation method of the IFN-γ sustained-release micro-spheres comprises the following steps：
S1, IFN-γ, heparin, polyethylene glycol added into the aqueous solution added with glucan and pentaerythrite zinc salt in advance, stirring
After uniform, the concentration for obtaining IFN-γ in mixture I, the mixture A is 3wt%, and the concentration of polyethylene glycol is 6wt%, liver
The concentration of element is 1wt%, and the concentration of glucan is 4wt%, and the concentration of pentaerythrite zinc salt is 3wt%；
S2, will polylactic acid-polyglycol add dichloromethane in dissolve, after stirring, obtain mixture II, the mixing
The concentration of polylactic acid-polyglycol is 16wt% in thing II；
S3, mixture I is added in mixture II, after stirring, obtains mixture III, the mixture I and mixing
The volume ratio of thing II is 1:2；
S4, polyvinyl alcohol, chitosan and sodium alginate be added to the water, after stirring, obtain mixture IV；The mixture
The concentration of polyvinyl alcohol is 1.5wt % in IV, and the concentration of chitosan is 0.6 wt %, and the concentration of sodium alginate is 3wt %；It is preferred that
Ground, the polyvinyl alcohol is 1 by mass ratio:1 atactic polyvinyl alcohol and a rule polyethylene composition；The atactic polyvinyl alcohol
The degree of polymerization is 600-1000, it is described between rule polyethylene the degree of polymerization be 1800-2500；
S5, mixture III added in mixture IV, stirring carries out solvent volatilization, it is scrubbed, centrifuge, vacuum refrigeration is done
It is dry, the IFN-γ sustained-release micro-spheres that particle diameter is 40-80 μm are made.
Embodiment 2
In the present embodiment, a kind of CIK cell cultural method comprises the following steps：
（1）PMNC is resuspended in CIK nutrient solutions, cell suspension is obtained；
（2）Cell suspension is added to the T175 cultures being coated with advance with AntiCD3 McAb monoclonal antibody and retronectin
In bottle, 37 DEG C, 5%CO are placed in afterwards2In saturated humidity incubator；Adjustment cell density is 7 × 10 before culture5-1.5×106/
mL；
（3）The 3rd day mL of supplement CIK nutrient solutions 60 of culture；
（4）The 6th day of culture, has added after CIK nutrient solutions, original nutrient solution in former T175 blake bottles is poured into new blake bottle
In, former T175 blake bottles pour into new blake bottle after cell on wall is blown and beaten completely with nutrient solution, add CIK nutrient solutions to total
Volume is 400mL, and above-mentioned nutrient solution is dispensed into four culture bags, and it is 500mL that CIK nutrient solutions to cumulative volume is added respectively,
It is 7 × 10 to be enlarged adjustment cell density before culture, culture5-2×106/mL；
（5）The 9th day mL of supplement CIK nutrient solutions 70 of culture；；
（6）Cultivate to the 13rd day, harvesting；
Preferably, the step（2）In, final concentration of 6 the μ g/mL, retronectin of anti-CD49d McAb final concentration before culture
For 11 μ g/mL.
Preferably, the CIK nutrient solutions include basal medium and the additive made an addition in basal medium, by dense eventually
Degree meter, the additive includes the component of following content：The μ g/mL of poly-D-lysine 20, BCG polysaccharide nucleic acid 15mg/mL, Asia
The μ g/mL of 0.03 μm of ol/mL, 4- hydroxyethyl piperazineethanesulfonic acid of sodium selenate 8, the μ g/mL of reduced glutathione 2, the μ of adenosine deaminase 12
G/mL, IFN-γ sustained-release micro-spheres 2mg/mL, IL-2 sustained-release micro-spheres 2mg/mL, autologous plasma 4mL/L.
Preferably, the preparation method of the IL-2 sustained-release micro-spheres comprises the following steps：
In A, the aqueous solution by IL-2, polyethylene glycol addition in advance added with mannitol and zinc sulfate, after stirring, mixed
IL-2 concentration is 1.2wt% in compound A, the mixture A, and the concentration of polyethylene glycol is 12 wt%；
B, PLGA-polyethylene glycol added in dichloromethane and dissolved, after stirring, obtain mixture B,
The concentration of PLGA-polyethylene glycol is 12wt% in the mixture B；
C, mixture A is added in mixture B, after stirring, obtains mixture C, the mixture A and mixture B
Volume ratio be 1:1；
D, polyvinyl alcohol, chitosan and sodium chloride is added to the water, after stirring, obtains mixture D；In the mixture D
The concentration of polyvinyl alcohol is 1 wt %, and the concentration of chitosan is 1 wt %, and the concentration of sodium chloride is 1wt %；
E, will mixture C add mixture D in, stirring carry out solvent volatilization, it is scrubbed, centrifuge, vacuum freeze drying, system
Obtain the IL-2 sustained-release micro-spheres that particle diameter is 60-110 μm.
Preferably, in the mixture A, the concentration of the mannitol is 2wt%, and carbonic acid zinc concentration is 3 wt%.
Preferably, the polyvinyl alcohol is 1 by mass ratio:0.5 atactic polyvinyl alcohol and a rule polyethylene composition, it is described
The degree of polymerization of atactic polyvinyl alcohol be 600-1000, it is described between rule polyethylene the degree of polymerization be 1800-2500.
Preferably, the preparation method of the IFN-γ sustained-release micro-spheres comprises the following steps：
S1, IFN-γ, heparin, polyethylene glycol added into the aqueous solution added with glucan and pentaerythrite zinc salt in advance, stirring
After uniform, the concentration for obtaining IFN-γ in mixture I, the mixture A is 2wt%, and the concentration of polyethylene glycol is 9wt%, liver
The concentration of element is 0.5wt%, it is preferable that the concentration of the glucan is 5 wt%, and the concentration of pentaerythrite zinc salt is 2 wt%；
S2, will polylactic acid-polyglycol add dichloromethane in dissolve, after stirring, obtain mixture II, the mixing
The concentration of polylactic acid-polyglycol is 13wt% in thing II；
S3, mixture I is added in mixture II, after stirring, obtains mixture III, the mixture I and mixing
The volume ratio of thing II is 1:1.5；
S4, polyvinyl alcohol, chitosan and sodium alginate be added to the water, after stirring, obtain mixture IV；The mixture
The concentration of polyvinyl alcohol is 1 wt % in IV, and the concentration of chitosan is 1 wt %, and the concentration of sodium alginate is 2 wt %；It is preferred that
Ground, the polyvinyl alcohol is 1 by mass ratio:2 atactic polyvinyl alcohol and a rule polyethylene composition；The atactic polyvinyl alcohol
The degree of polymerization be 600-1000, it is described between rule polyethylene the degree of polymerization be 1800-2500；
S5, mixture III added in mixture IV, stirring carries out solvent volatilization, it is scrubbed, centrifuge, vacuum refrigeration is done
It is dry, the IFN-γ sustained-release micro-spheres that particle diameter is 40-80 μm are made.
Embodiment 3
In the present embodiment, a kind of CIK cell cultural method comprises the following steps：
（1）PMNC is resuspended in CIK nutrient solutions, cell suspension is obtained；
（2）Cell suspension is added to the T175 cultures being coated with advance with AntiCD3 McAb monoclonal antibody and retronectin
In bottle, 37 DEG C, 5%CO are placed in afterwards2In saturated humidity incubator；Adjustment cell density is 7 × 10 before culture5-1.5×106/
mL；
（3）The 3rd day mL of supplement CIK nutrient solutions 60 of culture；
（4）The 6th day of culture, has added after CIK nutrient solutions, original nutrient solution in former T175 blake bottles is poured into new blake bottle
In, former T175 blake bottles pour into new blake bottle after cell on wall is blown and beaten completely with nutrient solution, add CIK nutrient solutions to total
Volume is 400mL, and above-mentioned nutrient solution is dispensed into four culture bags, and it is 500mL that CIK nutrient solutions to cumulative volume is added respectively,
It is 7 × 10 to be enlarged adjustment cell density before culture, culture5-2×106/mL；
（5）The 9th day mL of supplement CIK nutrient solutions 70 of culture；；
（6）Cultivate to the 13rd day, harvesting；
Preferably, the step（2）In, final concentration of 6 the μ g/mL, retronectin of anti-CD49d McAb final concentration before culture
For 11 μ g/mL.
Preferably, the CIK nutrient solutions include DMEM culture mediums and the additive made an addition in basal medium, by dense eventually
Degree meter, the additive includes the component of following content：The μ g/mL of poly-D-lysine 30, BCG polysaccharide nucleic acid 5mg/mL, sub- selenium
The sour μ g/mL of 0.04 μm of ol/mL, 4- hydroxyethyl piperazineethanesulfonic acid of sodium 5, the μ g/mL of reduced glutathione 4, the μ g/ of adenosine deaminase 8
ML, IFN-γ sustained-release micro-spheres 3mg/mL, IL-2 sustained-release micro-spheres 1.5mg/mL, autologous plasma 6mL/L.
Preferably, the preparation method of the IL-2 sustained-release micro-spheres comprises the following steps：
In A, the aqueous solution by IL-2, polyethylene glycol addition in advance added with mannitol and zinc sulfate, after stirring, mixed
IL-2 concentration is 2.5wt% in compound A, the mixture A, and the concentration of polyethylene glycol is 7wt%；
B, PLGA-polyethylene glycol added in dichloromethane and dissolved, after stirring, obtain mixture B,
The concentration of PLGA-polyethylene glycol is 18wt% in the mixture B；
C, mixture A is added in mixture B, after stirring, obtains mixture C, the mixture A and mixture B
Volume ratio be 1: 2；
D, polyvinyl alcohol, chitosan and sodium chloride is added to the water, after stirring, obtains mixture D；In the mixture D
The concentration of polyvinyl alcohol is 2 wt %, and the concentration of chitosan is 0.5wt %, and the concentration of sodium chloride is 2 wt %；
E, will mixture C add mixture D in, stirring carry out solvent volatilization, it is scrubbed, centrifuge, vacuum freeze drying, system
Obtain the IL-2 sustained-release micro-spheres that particle diameter is 60-110 μm.
Preferably, in the mixture A, the concentration of the mannitol is 4 wt%, and carbonic acid zinc concentration is 1wt%.
Preferably, the polyvinyl alcohol is 1 by mass ratio:2 atactic polyvinyl alcohol and a rule polyethylene composition, it is described
The degree of polymerization of atactic polyvinyl alcohol be 600-1000, it is described between rule polyethylene the degree of polymerization be 1800-2500.
Preferably, the preparation method of the IFN-γ sustained-release micro-spheres comprises the following steps：
S1, IFN-γ, heparin, polyethylene glycol added into the aqueous solution added with glucan and pentaerythrite zinc salt in advance, stirring
After uniform, the concentration for obtaining IFN-γ in mixture I, the mixture A is 5wt%, and the concentration of polyethylene glycol is 5wt%, liver
The concentration of element is 1.5wt%, and the concentration of glucan is 3wt%, and the concentration of pentaerythrite zinc salt is 4 wt%；
S2, will polylactic acid-polyglycol add dichloromethane in dissolve, after stirring, obtain mixture II, the mixing
The concentration of polylactic acid-polyglycol is 20wt% in thing II；
S3, mixture I is added in mixture II, after stirring, obtains mixture III, the mixture I and mixing
The volume ratio of thing II is 1: 2.5；
S4, polyvinyl alcohol, chitosan and sodium alginate be added to the water, after stirring, obtain mixture IV；The mixture
The concentration of polyvinyl alcohol is 2 wt % in IV, and the concentration of chitosan is 0.5 wt %, and the concentration of sodium alginate is 4 wt %；It is preferred that
Ground, the polyvinyl alcohol is 1 by mass ratio:0.5 atactic polyvinyl alcohol and a rule polyethylene composition；The atactic polyvinyl alcohol
The degree of polymerization be 600-1000, it is described between rule polyethylene the degree of polymerization be 1800-2500；
S5, mixture III added in mixture IV, stirring carries out solvent volatilization, it is scrubbed, centrifuge, vacuum refrigeration is done
It is dry, the IFN-γ sustained-release micro-spheres that particle diameter is 40-80 μm are made.
Comparative example
The cultural method that comparative example is used, comprises the following steps：
（1）PMNC is resuspended in control group nutrient solution, cell suspension is obtained；The control group nutrient solution does not add
Plus BCG polysaccharide nucleic acid, IL-2 sustained-release micro-spheres and IFN-γ sustained-release micro-spheres, it is dense added with the IL-2 that concentration is 300IU/mL
Spend the IFN-γ for 300IU/mL；
（2）Cell suspension is added to the T175 cultures being coated with advance with AntiCD3 McAb monoclonal antibody and retronectin
In bottle, 37 DEG C, 5%CO are placed in afterwards2In saturated humidity incubator；Adjustment cell density is 7 × 10 before culture5-1.5×106/
mL；Final concentration of 6 the μ g/mL, retronectin of anti-CD49d McAb final concentration of 11 μ g/mL before culture,
（3）The 3rd day mL of supplement control group nutrient solution nutrient solution 60 of culture；
（4）The 6th day of culture, has added after CIK nutrient solutions, original nutrient solution in former T175 blake bottles is poured into new blake bottle
In, former T175 blake bottles pour into new blake bottle after cell on wall is blown and beaten completely with control group nutrient solution, add control group
Nutrient solution to cumulative volume is 400mL, and above-mentioned nutrient solution is dispensed into four culture bags, and control group nutrient solution is added respectively to total
Volume is 500mL, and it is 7 × 10 to be enlarged adjustment cell density before culture, culture5-2×106/mL；
（5）The 9th day mL of supplement control group nutrient solution 70 of culture；
（6）Cultivate to the 13rd day, harvesting；
The embodiment 1-3 of table 1 and the CIK cells of comparative example culture statistics and qualification result
| Project | Embodiment 1 | Embodiment 2 | Embodiment 3 | Comparative example |
| Proliferation times | 689 | 672 | 679 | 470 |
| Cell viability % | 98.0 | 97.6 | 98.2 | 92.1 |
| CD3+CD56+Effector cell's ratio % | 41.9 | 38.3 | 39.1 | 28.6 |
As seen from the above table, the CIK cell cultural method that the present invention is provided can improve CIK amplification rate, especially CD3+ CD56+The acquisition quantity of effector cell, so as to improve the killing activity of CIK cell.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than to present invention guarantor
The limitation of scope is protected, although being explained with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent substitution, without departing from the reality of technical solution of the present invention
Matter and scope.
Claims (10)
1. a kind of CIK cell cultural method, it is characterised in that：Comprise the following steps：
（1）PMNC is resuspended in CIK nutrient solutions, cell suspension is obtained；
（2）Cell suspension is added in the blake bottle being coated with advance with AntiCD3 McAb monoclonal antibody and retronectin,
37 DEG C, 5%CO are placed in afterwards2Cultivated in saturated humidity incubator；Adjustment cell density is 7 × 10 before culture5-2×106/mL；
（3）3rd day supplement CIK nutrient solution of culture；
（4）The 6th day of culture, has added after CIK nutrient solutions, the culture liquid mixture in blake bottle is fallen to be dispensed into different trainings
Support in container, it is 7 × 10 to be enlarged adjustment cell density before culture, culture5-1.5×106/mL；
（5）9th day supplement CIK nutrient solution of culture；
（6）Cultivate to the 12-14 days, harvesting.
2. a kind of CIK cell cultural method according to claim 1, it is characterised in that：The CIK nutrient solutions include basis
Culture medium and the additive made an addition in basal medium, by final concentration, the additive includes the component of following content：It is many
Polylysine 20-30 μ g/mL, BCG polysaccharide nucleic acid 5-15mg/mL, 0.03-0.04 μm of ol/mL, 4- ethoxy of sodium selenite
Piperazine ethanesulfonic acid 5-8 μ g/mL, reduced glutathione 2-4 μ g/mL, adenosine deaminase 8-12 μ g/mL, IFN-γ sustained-release micro-spheres
20-30mg/mL, IL-2 sustained-release micro-spheres 1.5-2mg/mL, autologous plasma 4-6mL/L.
3. a kind of CIK cell cultural method according to claim 2, it is characterised in that：The system of the IL-2 sustained-release micro-spheres
Preparation Method comprises the following steps：
In A, the aqueous solution by IL-2, polyethylene glycol addition in advance added with mannitol and zinc sulfate, after stirring, mixed
IL-2 concentration is 1.2-2.5wt% in compound A, the mixture A, and the concentration of polyethylene glycol is 7-12 wt%；
B, polyglycolic acid-PLA-polyethylene glycol added in dichloromethane and dissolved, after stirring, obtain mixture
The concentration of polyglycolic acid-PLA-polyethylene glycol is 12-18wt% in B, the mixture B；
C, mixture A is added in mixture B, after stirring, obtains mixture C, the mixture A and mixture B
Volume ratio be 1:1-2；
D, polyvinyl alcohol, chitosan and sodium chloride is added to the water, after stirring, obtains mixture D；In the mixture D
The concentration of polyvinyl alcohol is 1-2 wt %, and the concentration of chitosan is 0.5-1 wt %, and the concentration of sodium chloride is 1-2 wt %；
E, will mixture C add mixture D in, stirring carry out solvent volatilization, it is scrubbed, centrifuge, vacuum freeze drying, system
Obtain IL-2 sustained-release micro-spheres.
4. a kind of CIK cell cultural method according to claim 3, it is characterised in that：The grain of the IL-2 sustained-release micro-spheres
Footpath is 60-110 μm.
5. a kind of CIK cell cultural method according to claim 3, it is characterised in that：It is described sweet in the mixture A
The concentration for revealing alcohol is 2-4 wt%, and carbonic acid zinc concentration is 1-3 wt%.
6. a kind of CIK cell cultural method according to claim 3, it is characterised in that：The polyvinyl alcohol is by mass ratio
For 1:0.5-2 atactic polyvinyl alcohol and a rule polyethylene composition.
7. a kind of CIK cell cultural method according to claim 6, it is characterised in that：The atactic polyvinyl alcohol it is poly-
It is right be 600-1000, it is described between rule polyethylene the degree of polymerization be 1800-2500.
8. a kind of CIK cell cultural method according to claim 2, it is characterised in that：The IFN-γ sustained-release micro-spheres
Preparation method comprises the following steps：
S1, IFN-γ, heparin, polyethylene glycol added into the aqueous solution added with glucan and pentaerythrite zinc salt in advance, stirring
After uniform, the concentration for obtaining IFN-γ in mixture I, the mixture A is 2-5wt%, and the concentration of polyethylene glycol is 5-
9wt%, the concentration of heparin is 0.5-1.5wt%；
S2, PLA-polyethylene glycol added in dichloromethane and dissolved, after stirring, obtain mixture II, the mixing
The concentration of polylactic acid-polyglycol is 13-20wt% in thing II；
S3, mixture I is added in mixture II, after stirring, obtains mixture III, the mixture I and mixing
The volume ratio of thing II is 1:1.5-2.5；
S4, polyvinyl alcohol, chitosan and sodium alginate be added to the water, after stirring, obtain mixture IV；The mixture
The concentration of polyvinyl alcohol is 1-2 wt % in IV, and the concentration of chitosan is 0.5-1 wt %, and the concentration of sodium alginate is 2-4 wt
%；
S5, mixture III added in mixture IV, stirring carries out solvent volatilization, it is scrubbed, centrifuge, vacuum refrigeration is done
It is dry, IFN-γ sustained-release micro-spheres are made.
9. a kind of CIK cell cultural method according to claim 8, it is characterised in that：The IFN-γ sustained-release micro-spheres
Particle diameter is 40-80 μm.
10. a kind of CIK cell cultural method according to claim 1, it is characterised in that：The basal medium is DMEM
Culture medium.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988155A (en) * | 2017-12-06 | 2018-05-04 | 暨赛再生医学科技有限公司 | A kind of preparation method of CIK cell |
| CN111172110A (en) * | 2019-12-20 | 2020-05-19 | 中科细胞科技(广州)有限公司 | Culture method of umbilical cord blood CIK cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103690933A (en) * | 2014-01-08 | 2014-04-02 | 昆明制药集团股份有限公司 | Stem cell factor sustained-release microspheres and preparation method thereof |
| CN104248628A (en) * | 2013-06-25 | 2014-12-31 | 深圳翰宇药业股份有限公司 | Lixisenatide controlled-release microspheres and preparation method thereof |
| CN104818248A (en) * | 2015-03-25 | 2015-08-05 | 苏州佰通生物科技有限公司 | Immunocyte culture medium, and culture method and application of immunocytes |
| CN106591231A (en) * | 2016-12-02 | 2017-04-26 | 中国计量大学 | Bacilli calmette-giierin polysaccharide nucleic acid for promoting proliferation and differentiation of CIK (cytokine-induced killer) cells, CIK cell culture medium, CIK cell culture method, and application of Bacilli calmette-giierin polysaccharide nucleic acid |
-
2017
- 2017-05-31 CN CN201710401454.3A patent/CN107142245A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104248628A (en) * | 2013-06-25 | 2014-12-31 | 深圳翰宇药业股份有限公司 | Lixisenatide controlled-release microspheres and preparation method thereof |
| CN103690933A (en) * | 2014-01-08 | 2014-04-02 | 昆明制药集团股份有限公司 | Stem cell factor sustained-release microspheres and preparation method thereof |
| CN104818248A (en) * | 2015-03-25 | 2015-08-05 | 苏州佰通生物科技有限公司 | Immunocyte culture medium, and culture method and application of immunocytes |
| CN106591231A (en) * | 2016-12-02 | 2017-04-26 | 中国计量大学 | Bacilli calmette-giierin polysaccharide nucleic acid for promoting proliferation and differentiation of CIK (cytokine-induced killer) cells, CIK cell culture medium, CIK cell culture method, and application of Bacilli calmette-giierin polysaccharide nucleic acid |
Non-Patent Citations (3)
| Title |
|---|
| 乔飞鸿等: "卡介菌多糖核酸体外抗病毒活性试验", 《中国农业大学学报》 * |
| 任玮等: "重组人纤维蛋白片段诱导CIK的增殖及对肺癌耐顺铂细胞的杀伤作用", 《生物工程学报》 * |
| 黄招琴等: "卡介菌多糖核酸对CIK细胞活性的影响", 《湖南师范大学自然科学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107988155A (en) * | 2017-12-06 | 2018-05-04 | 暨赛再生医学科技有限公司 | A kind of preparation method of CIK cell |
| CN107988155B (en) * | 2017-12-06 | 2019-01-11 | 暨赛再生医学科技有限公司 | A kind of preparation method of CIK cell |
| CN111172110A (en) * | 2019-12-20 | 2020-05-19 | 中科细胞科技(广州)有限公司 | Culture method of umbilical cord blood CIK cells |
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