CN102352343A - Lymphocyte culture medium and use method thereof - Google Patents
Lymphocyte culture medium and use method thereof Download PDFInfo
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- CN102352343A CN102352343A CN2011102981520A CN201110298152A CN102352343A CN 102352343 A CN102352343 A CN 102352343A CN 2011102981520 A CN2011102981520 A CN 2011102981520A CN 201110298152 A CN201110298152 A CN 201110298152A CN 102352343 A CN102352343 A CN 102352343A
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Abstract
The invention relates to a lymphocyte culture medium, which belongs to the technical field of in-vitro cell culture and comprises a basic culture medium and serum substitutes, wherein the serum substitutes comprise recombinant human blood albumin, recombinant human transferrin and recombinant human insulin and are used for solving the problems that most of the existing lymphocyte culture medium adopts animal or human serum or ingredients of the animal or human serum as additives, the disease spreading risk is increased, and simultaneously, the quality in different batches cannot be unified.
Description
Technical field
The invention belongs to cell in vitro culture technique field, relate to a kind of lymphocytes culture medium, in addition, the present invention also provides a kind of cultural method of lymphocytes culture medium.
Background technology
Effectively the cellular immunization treatment be unable to do without at external a large amount of cultivations, amplification and activated lymphocyte.This wherein the maximum reagent of usage quantity be exactly substratum.Substratum is that lymphocytic growth provides basic environment; Be included as the lymphocyte survival suitable osmotic pressure is provided; PH value; For lymphocytic growth provides all required nutritional factor; Comprising inorganic salt, amino acid, functional albumen, VITAMIN etc., also is the discharging place of the various meta-bolitess of lymphocyte.Traditional substratum is in order to provide these nutritional factor; The serum and the component thereof that mostly adopt the animal or human be as additive, and more common have new-born calf serum, foetal calf serum, people AB serum, bovine serum albumin, human serum albumin, a human transferrin etc.Using the advantage of animal serum is that enough amount supplies are arranged; Low price; But the shortcoming that is to use animal serum is that the potential risk that disseminates infection is arranged, and because foreign protein causes hypersensitive generation, and the well lymphocytic growth of backer of animal serum.And the benefit of end user AB serum is can well the lymphocytic growth of backer, but people AB serum source difficulty, and supply is limited, and has the risk that disseminates infection.
Existing commercial serum-free lymphocytes culture medium mostly is added with humanized's components such as human serum albumin, human transferrin.Because these components derive from people's serum, though the detection technique and the disinfection technology of various pathogenic agent are arranged, but still keep away unavoidable pathophorous risk; Owing to derive from different donors, cause a batch differences, be unfavorable for the control of quality.
Summary of the invention
Technical problem to be solved by this invention is that serum and component thereof that existing lymphocytes culture medium adopts the animal or human mostly are as additive; Increased pathophorous risk; The while different batches; Quality can't be unified problem; And this problem of being directed against provides a kind of lymphocytes culture medium; It is suitable with the serum free medium that contains humanized's composition to experiment showed, that use lymphocytes culture medium according to the invention is used for the lymphocytic effect of cultivator.
The present invention solves the problems of the technologies described above through following technical scheme:
A kind of lymphocytes culture medium comprises basic medium and serum substitute, it is characterized in that said serum substitute comprises recombinant human serum albumin, recombinant human Transferrins,iron complexes and recombinant human insulin.
The main effect of human transferrin is to intracellular transport iron (Fe
3+), nearly all cell growth all be unable to do without the demand to iron, and Transferrins,iron complexes is mainly synthetic by liver in vivo.Human serum albumin is the maximum a kind of protein of content in the human plasma; Mainly play and keep oncotic pressure; Store and effects such as transportation lipid, hormone and trace element; Mainly synthetic in vivo by liver; The main effect of Regular Insulin is to promote the picked-up of cell to glucose, in vivo by pancreatic secretion.
The composition of lymphocytes culture medium according to the invention does not all adopt animal or human's serum, thereby has avoided pathophorous risk, and in addition, the used basic medium of the present invention is RPMI 1640 substratum of animal origin-free.Preferably, this substratum is RPMI 1640 substratum that do not contain glutamine.
Recombinant human serum albumin is to use yeast, bacterium etc. to produce the use of no any animal derived components in the production process for carrier among the present invention.Its working concentration is 0.1mg/L to 40mg/L, and preferably, its working concentration is 0.5mg/L to 10mg/L, more preferably is 2mg/L.
The recombinant human Transferrins,iron complexes is to use yeast, bacterium etc. to produce the use of no any animal derived components in the production process for carrier among the present invention.Its working concentration is 5mg/L to 200mg/L, and preferably, its working concentration is 10mg/L to 100mg/L, more preferably is 50mg/L.
Among the present invention the recombinant human insulin to be to use yeast be the Regular Insulin of the pharmaceutical grade produced of carrier, the use of no any animal derived components in the production process.Its working concentration is 1mg/L to 100mg/L, and preferably, its working concentration is 5mg/L to 50mg/L, more preferably is 10mg/L.
Certainly, as lymphocytes culture medium, the present invention has also added lipid, trace compound and VITAMIN to serum substitute.Can synthesize in cell though the needed lipid of culturing cell is most of; But few part lipid belongs to cell can not the synthetic lipid; Need replenish by the external world; The lipid that has added for example according to the invention just belongs to the cell cultures needs; But can not the synthetic lipid, this lipoids comprises linolic acid, linolenic acid, cholesterol etc.Wherein, the cholesterol source is chemosynthesis, and linolic acid and linolenic acid source are plant.In practice, for increasing the solubleness of lipid, can add solubility promoter Tween 80.
And trace element is the active centre of many enzymes in the cell like iron, zinc, selenium, copper etc.; Also need add to suitable form in the substratum; Trace compound according to the invention comprises iron nitrate, Sodium Selenite, copper sulfate and zinc sulfate, so that iron, zinc, selenium and copper to be provided to cell.
The present invention has also added vitamin-E in serum substitute, to satisfy the cell cultures needs.
In order to make the good effect of lymphocytes culture medium performance according to the invention, the present invention has done like delimit lipid, trace compound and VITAMIN:
The concentration of cholesterol is 0.5mg/L-5mg/L in the said lymphocytes culture medium; Linoleic concentration is 0.05mg/L-0.5mg/L in the said lymphocytes culture medium; Linolenic concentration is 0.05mg/L-0.5mg/L in the said lymphocytes culture medium.
Iron nitrate content is 0.001mg/L to 1mg/L, is preferably 0.01mg/L to 0.5mg/L, more preferably 0.1mg/L.
Sodium Selenite content is 0.0001mg/L to 0.1mg/L, is preferably 0.001mg/L to 0.05mg/L, more preferably 0.01mg/L.
Copper sulfate content is 0.1mg/L to 0.00001mg/L, is preferably 0.01mg/L to 0.0001mg/L, more preferably 0.001mg/L.
Zinc sulfate content is 10mg/L to 0.01mg/L, is preferably 1mg/L to 0.1mg/L, more preferably 0.5mg/L.
The concentration of vitamin-E is 0.01mg/L-10mg/L in the said lymphocytes culture medium.
Said lymphocytes culture medium comprises that also concentration is the thanomin of 0.1mg/L-10mg/L, is preferably 1mg/L to 2mg/L, more preferably 1.5mg/L;
Said lymphocytes culture medium comprises that also concentration is the ascorbic acid phosphoric acid esters of 0.1mg/L-10mg/L, is preferably 0.5mg/L to 5mg/L, more preferably 2.5mg/L;
Said lymphocytes culture medium comprises that also concentration is the gentamicin of 1IU/ml-100IU/ml, is preferably 10IU/ml to 40IU/ml, more preferably 10IU/ml;
Said lymphocytes culture medium comprises that also concentration is sodium hydrogencarbonate or the 5.958g/L HEPES of 2.0g/L, and as the damping fluid of regulating pH value, the pH value that makes lymphocytes culture medium is between the 6.8-7.2;
Said lymphocytes culture medium also comprises 2% autoserum.
The present invention also provides a kind of method of lymphocytes culture medium, and this method comprises the step of using the said lymphocytes culture medium of forward direction to add glutamine.
Description of drawings
Fig. 1 kills the ratio of outflow comparison diagram by the CIK cell of said A, B, C and four groups of culture medium culturing of D among the embodiment;
Fig. 2 be among the embodiment by said A, B, C and four groups of substratum of D through embodiment medium size lymphocyte proliferation experiment and the described method culturing cell of survival rate and the survival rate comparison diagram that obtains;
Fig. 3 be among the embodiment by said A, B, C and four groups of substratum of D through embodiment medium size lymphocyte proliferation experiment and the described method culturing cell of survival rate and the propagation multiple comparison diagram that obtains.
Embodiment
Embodiment
A kind of lymphocytes culture medium, this lymphocytes culture medium is compared with traditional humanized's component and serum substratum that contains for the non-animal derived property substratum of the human lymphocyte that can efficiently increase, and lymphocytic amplification ability is with tumor activity is suitable extremely.
The collocation method of lymphocytes culture medium
In every 1L RPMI 1640 substratum, add recombinant human serum albumin 2mg; Recombinant human Transferrins,iron complexes 50mg; Recombinant human insulin 10mg, cholesterol 2mg, linolic acid 0.2mg; Linolenic acid 0.2mg; Vitamin-E 1mg, Tween 802mg, iron nitrate 0.1mg; Sodium Selenite 0.01mg; Copper sulfate 0.001mg, zinc sulfate 0.5mg, thanomin 1.5mg; Ascorbic acid phosphoric acid esters 2.5mg; Gentamicin 10IU stirs and makes abundant dissolving, uses 0.1mol/L hydrochloric acid and 0.1mol/L sodium hydroxide to regulate pH value as between the 6.8-7.2; 0.1um the membrane filtration degerming, packing.
Face with preceding adding glutamine 300mg.
Lymphocyte proliferation assay and survival rate
Use lymphocyte separation medium density gradient centrifugation to separate peripheral blood mononuclear cell, the frozen mononuclearcell of perhaps recovering.Add and contain centrifugal 10 minutes of 5% heat-inactivated fetal bovine serum (FBS) physiological saline 300g, remove supernatant.Counting cells is made into 1*10 with corresponding substratum
6/ ml cell suspension.Be divided into following several groups: A:RPMI 1640+10%FBS; B: be to use humanized's albumin and Transferrins,iron complexes to substitute recombinant human albumin and recombinant human Transferrins,iron complexes respectively with unique difference of lymphocytes culture medium; C: lymphocytes culture medium, D: lymphocytes culture medium+2% autoserum.Add the 2ml substratum by every hole and insert 6 orifice plates, 3 every group multiple holes.Add 1000IU/ml IFN-r on the 1st day, and cultivated adding 50ng/ml CD3 monoclonal antibody (OKT3) after 24 hours, 300IU/ml IL-2,100IU/ml IL-1a.Per 3 days later on countings are added and are contained the 300IU/mlIL-2 nutrient solution to 1*10
6/ ml.Cultivated 15 days, cell proliferation multiple and survival rate are calculated in the blue dyeing of platform phenol, see Fig. 2 and Fig. 3.
The lymphocytotoxicity experiment
Getting the CIK cell of cultivating 15 days and be the effector cell, is target cell with the K562 cell, is inoculated into than 10: 1 in 96 orifice plates by imitating target, and 37 spend 5%CO
2Cultivated 24 hours.Survey absorbance with mtt assay, calculate and kill ratio of outflow.A:RPMI 1640+10%FBS; B: other component is identical, uses the lymphocytes culture medium of alternative recombinant human albumin of humanized's albumin and Transferrins,iron complexes and recombinant human Transferrins,iron complexes, C: lymphocytes culture medium; D: lymphocytes culture medium+2% autoserum, see Fig. 1.
Kill ratio of outflow %=1-(test holes-effector cell hole)/target cell hole
Claims (17)
1. a lymphocytes culture medium comprises basic medium and serum substitute, it is characterized in that said serum substitute comprises recombinant human serum albumin, recombinant human Transferrins,iron complexes and recombinant human insulin.
2. a kind of lymphocytes culture medium according to claim 1, the concentration that it is characterized in that recombinant human serum albumin in the said lymphocytes culture medium is 0.1mg/L-40mg/L.
3. a kind of lymphocytes culture medium according to claim 1, the concentration that it is characterized in that recombinant human Transferrins,iron complexes in the said lymphocytes culture medium is 5mg/L-200mg/L.
4. a kind of lymphocytes culture medium according to claim 1, the concentration that it is characterized in that Recombulin in the said lymphocytes culture medium is 1mg/L-100mg/L.
5. a kind of lymphocytes culture medium according to claim 1 is characterized in that serum substitute also comprises lipid, trace compound and VITAMIN, and said lipid comprises cholesterol, linolic acid, linolenic acid.
6. a kind of lymphocytes culture medium according to claim 5, the concentration that it is characterized in that cholesterol in the said lymphocytes culture medium is 0.5mg/L-5mg/L; Linoleic concentration is 0.05mg/L-0.5mg/L in the said lymphocytes culture medium; Linolenic concentration is 0.05mg/L-0.5mg/L in the said lymphocytes culture medium.
7. a kind of lymphocytes culture medium according to claim 6 is characterized in that said lymphocytes culture medium also comprises Tween80.
8. a kind of lymphocytes culture medium according to claim 6 is characterized in that said VITAMIN is a vitamin-E, and the concentration of vitamin-E is 0.01mg/L-10mg/L in the said lymphocytes culture medium.
9. a kind of lymphocytes culture medium according to claim 6 is characterized in that said trace compound comprises iron nitrate, Sodium Selenite, copper sulfate and zinc sulfate.
10. a kind of lymphocytes culture medium according to claim 9, the concentration that it is characterized in that iron nitrate in the said lymphocytes culture medium is 0.001mg/L-1mg/L; The concentration of Sodium Selenite is 0.0001mg/L-0.1mg/L in the said lymphocytes culture medium; The concentration of copper sulfate is 0.00001mg/L-0.1mg/L in the said lymphocytes culture medium; The sulfuric acid zinc concentration is 0.01mg/L-10mg/L in the said lymphocytes culture medium.
11. a kind of lymphocytes culture medium according to claim 1 is characterized in that said lymphocytes culture medium comprises that also concentration is the thanomin of 0.1mg/L-10mg/L.
12. a kind of lymphocytes culture medium according to claim 1 is characterized in that said lymphocytes culture medium comprises that also concentration is the ascorbic acid phosphoric acid esters of 0.1mg/L-10mg/L.
13. a kind of lymphocytes culture medium according to claim 1 is characterized in that said lymphocytes culture medium comprises that also concentration is the gentamicin of 1IU/ml-100IU/ml.
14. a kind of lymphocytes culture medium according to claim 1 is characterized in that said lymphocytes culture medium comprises that also concentration is sodium hydrogencarbonate or the 5.958g/L HEPES of 2.0g/L.
15. a kind of lymphocytes culture medium according to claim 1 is characterized in that said basic medium is RPMI 1640 substratum.
16. a kind of lymphocytes culture medium according to claim 1 is characterized in that said lymphocytes culture medium also comprises 2% autoserum.
17. the method for use of the said lymphocytes culture medium of claim 1 comprises the step of using the said lymphocytes culture medium of forward direction to add glutamine.
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| CN104371973A (en) * | 2013-08-15 | 2015-02-25 | 协和华东干细胞基因工程有限公司 | Serum-free medium of immune cells |
| CN104371972A (en) * | 2013-08-12 | 2015-02-25 | 英普乐孚生物技术(上海)有限公司 | T lymphocyte culture medium without any animal original or human original component and preparation method thereof |
| CN104450614A (en) * | 2014-12-05 | 2015-03-25 | 上海安集协康生物技术有限公司 | Animal protein-free immune cell serum-free medium and using method thereof |
| CN104830766A (en) * | 2015-05-22 | 2015-08-12 | 广州和能生物科技有限公司 | Serum-free human peripheral blood lymphocyte culture medium |
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| CN108707579B (en) * | 2018-05-28 | 2022-08-09 | 北京美迪阿姆科技发展有限公司 | Serum-free medium for human T lymphocyte culture, preparation method and culture method |
| CN108977409A (en) * | 2018-08-21 | 2018-12-11 | 苏州米苏生物技术有限公司 | Source of people lymphocytes culture medium, preparation method and application |
| CN108865996A (en) * | 2018-08-21 | 2018-11-23 | 苏州米苏生物技术有限公司 | Serum-free T lymphocyte culture medium, preparation method and application |
| CN108865996B (en) * | 2018-08-21 | 2022-06-24 | 苏州沃美生物有限公司 | Serum-free T lymphocyte culture medium, preparation method and application thereof |
| CN108977409B (en) * | 2018-08-21 | 2022-06-28 | 苏州沃美生物有限公司 | Culture medium for human-derived lymphocytes, preparation method and application thereof |
| CN110583622A (en) * | 2019-08-30 | 2019-12-20 | 依科赛生物科技（太仓）有限公司 | T cell serum-free freezing medium and use method thereof |
| CN110669730A (en) * | 2019-10-23 | 2020-01-10 | 杭州宝荣科技有限公司 | Human peripheral blood lymphocyte culture medium |
| CN110669730B (en) * | 2019-10-23 | 2022-03-29 | 杭州杰毅麦特医疗器械有限公司 | Human peripheral blood lymphocyte culture medium |
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