CN102191215A - Human-derived serum-free culture medium and preparation method thereof - Google Patents
Human-derived serum-free culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a human serum-free culture medium and a preparation method thereof, which uses 11 raw materials of human serum albumin solution, human recombinant insulin solution, human transferrin solution, human cholesterol solution, human catalase solution, 2-mercaptoethanol solution, ascorbic acid solution, linoleic acid solution, ethanolamine solution, human vitronectin solution and L-glutamine solution for treatment, wherein the added protein and lipid are all derived from human plasma, serum or tissues, are pharmaceutical-grade or highly purified human protein or human recombinant protein and do not contain any animal-derived component, other components all accord with United states Pharmacopeia or national standard, and the human serum-free culture medium is qualified through cell culture detection and is clinically safe and reasonable. The proliferation rate of CIK cells cultured and induced by the culture medium is superior to that of a serum-containing culture medium, the percentage of the cells CD3+ CD56+ and the killing rate of K562 leukemia cells are similar to that of the serum-containing culture medium. The invention helps to improve the safety and standardization of cell therapy.
Description
Technical field
The present invention relates to the medical experiment articles for use, especially relate to a kind of humanized's serum free medium and preparation method thereof.
Background technology
From setting up tissue and cell culture technology, just always with the cell culture medium that contains animal or human's serum.At the seventies, the biologist just worries the danger that the uncertain material in animal or human's serum brings pathogenic agent in the influence, particularly animal or human's serum of culturing cell.Along with further developing of stem-cell research, now the stem cell of cultured and amplified in vitro can have been imported or transplanted in human body and carry out the cell replacement treatment, thereby be necessary very much to carry out the development of humanized's serum free medium.We have just carried out looking into new to the wide cell spectrum of humanized serum free medium in 2007 and have begun to study.
At present, the producer that does not all have the formal humanized of production serum free medium both at home and abroad.External commercially available serum free medium all is serum free mediums (being called for short the animal source serum free medium) of the composition that contains animal-origin (as bovine serum albumin etc.), of a great variety, but a kind of serum free medium is only at a certain, the cell of two kinds of specific types, and abroad all serum-free culture base products all indicate " only using for research " on the label of specification sheets, the external substratum that also has cultivator embryonic stem cell and people's adult stem cell, as StemPro-SFM, StemSpan H3000 and KNOKOUT serum substitute, and (ancestral) cell and lymphocytic serum free medium are done in cultivator hematopoiesis, as AIM-V and X-VIVO, but wherein all contain the animal source composition, do not have the pharmaceutical grade composition.Though domestic existing serum-free protein-free medium (two no substratum), it replaces animal source albumen with the plant seed protein hydrolysate, but only be used for animal cell culture, as Chinese hamster ovary cell (CHO), hybridoma etc., the inapplicable human body cell that maybe can not be used for is cultivated.Along with carrying out and clinical application of cell therapy, the development trend in the serum free medium research and development is a preparation humanized serum free medium both at home and abroad, should not contain animal source protein or other material, can be for clinical use.The animal source serum free medium should not be used for cultivating the reason that is used for the clinical treatment cell: the animal source serum free medium contains the material of separation and purification from ox or other animal serums or tissue; may contain animal pathogen; as containing bovine viral in the bovine serum, this just causes these animal viruss to cause the danger of disease.Discovered in recent years ox sponge-type encephalopathic (BSE), it is a kind of transmissible ox neurodegenerative disease that causes by bovine viral, have very long latent period or incubation time, this discovery further makes people increase the worry of using animal serum in the bio-active products production process.For this reason, development humanized serum free medium is imperative, does not still have commercial humanized's serum free medium at present both at home and abroad.
Summary of the invention
First purpose of the present invention is to provide a kind of protein that adds and lipid, all derive from the people blood plasma, serum or tissue, for pharmaceutical grade or highly purified people's source protein or human recombination protein, do not contain any animal source composition, other composition all meets the ultrapure or analytical reagent of second one of American Pharmacopeia or " national standard chemistry reagent " or Pharmacopoeia of the People's Republic of China version in 2010, and qualified through the cell cultures detection, be used for the rational humanized's serum free medium of clinical safety.
Second purpose of the present invention is to provide a kind of preparation method of humanized's serum free medium.
First purpose of the present invention is achieved in that
A kind of humanized's serum free medium, feature is: prescription contains in the treatment of final concentration human serum albumin solution 1--10mg/ml, biosynthetic human insulin's solution 4--40 μ g/ml, human transferrin solution 30--60 μ g/ml, people's cholesterol solution 20--50 μ g/ml, human hydrogen peroxidase enzyme solution 20--50 μ g/ml, 2 mercapto ethanol solution 0.5-5.0 μ g/ml, ascorbic acid solution 25--100 μ g/ml, linolic acid solution 1.5--25 μ g/ml, ethanolamine solutions 10-60 μ g/ml, people's vitronectin solution 1.0-10 μ g/ml, L-glutamine solution 10.0--100 μ g/ml.
Second purpose of the present invention is achieved in that
A kind of preparation method of humanized's serum free medium, feature is:
A. according to a conventional method the treatment that is respectively of compound concentration with human serum albumin solution 50-500mg/ml, biosynthetic human insulin's solution 500-2000 μ g/ml, human hydrogen peroxidase enzyme solution 1000-2500 μ g/ml, 2 mercapto ethanol solution: 25-250 μ g/ml, ascorbic acid solution 1250--5000 μ g/ml, ethanolamine solutions 500-3000 μ g/ml, people's vitronectin solution 50-500 μ g/ml, L-glutamine solution 500-5000 μ g/ml;
B, the saturated human transferrin solution of preparation iron:
(1), elder generation is with the FeCl of 16.22--27.30mg
3With the 1mM HCl of 10--20ml dissolving, be made into storage liquid, packing then, freezing being stored in-20 ℃ obtain storage liquid No. 1;
(2), take by weighing the human transferrin of 120--200mg, being dissolved in the 4.0--10.0ml cutter that does not add sodium bicarbonate improves among she Ge Shi (DMEM) than Ke Shi, add No. 1 storage liquid of 270--400 μ l again, stir, promptly getting concentration is the saturated human transferrin solution of iron of 1500--3000 μ g/ml;
C, preparation people cholesterol solution: the people's cholesterol powder that takes by weighing 50--100mg, people's cholesterol powder of 50--100mg is joined 50--100ml not to be added among the DMEM of sodium bicarbonate, ultrasonic oscillator vibration with 2cm diameter peptide probe, in 4 ℃ of refrigerators, vibrated 1 hour with peak swing, make it complete emulsification, successively with the membrane filtration degerming of 1.2 μ m and 0.45 μ m, promptly getting concentration is people's cholesterol solution of 1000-2500 μ g/ml, is kept at-30 ℃;
D. prepare linolic acid solution: take by weighing the 0.015-0.03g linolic acid and be dissolved in the 100ml standard ethanol, fully vibration dissolving, promptly getting concentration is the linolic acid solution of 75-1250 μ g/ml, is kept at 8 ℃;
E, will treat with human serum albumin solution, biosynthetic human insulin's solution, human transferrin solution, people's cholesterol solution, human hydrogen peroxidase enzyme solution, 2 mercapto ethanol solution, ascorbic acid solution, linolic acid solution, ethanolamine solutions, people's vitronectin solution and L-glutamine solution and mix, stir, again with Yi Sikefu improvement cutter than Ke Shi substratum (Iscove ' s modified Dulbecco ' smedia, abbreviation IMDM) is diluted to the 1L/ bottle, reach the final concentration in the prescription, promptly get humanized's serum free medium.
The application of humanized's serum free medium in inducing culture immunologically competent cell (as CIK) and dried (ancestral) cell of hematopoiesis.
(1) vitro culture of cytokine-induced killer cell (CIK):
Separate mononuclearcell (MNCs) by the Ficoll-Hypaque density gradient centrifugation from normal people, human cord blood, patient self peripheral blood or marrow, also use the direct separation of C D34 cell of fluidic cell separation system or immunomagnetic beads method (CD34 positive cell).The MNCs cell suspension being injected culturing bottle cultivate, is 2-3x 10 at cell concn
6High-cell density under, add to add humanized's serum free medium of the specific cell factor and cultivate the CIK cell.According to the clinical treatment needs, results are cultivated the cell of 5 days and 10 days respectively, expect blue counting cells with tongue respectively, mtt assay detects cell-proliferation activity, through the two positive percentage of Flow cytometry CIK cell phenotype CD3+CD56+, with CFSE mark K562 target cell 4 hours, in the ratio of effector cell (CIK) and target cell (K562) is 10: 1 mixed culture 4 hours, washed cell 3 times adds the PI dyestuff through the cytotoxicity of Flow cytometry DC/CIK cell to the K562 target cell before the last machine.Cultivate the CIK cell simultaneously respectively with basal culture medium, external similar serum free medium AIM-V and the IMDM substratum (blood serum medium is arranged) that contains 10%FCS, cultivate after 5 and 10 days, its cellular form, cell proliferation (detecting with mtt assay), CIK cell phenotype and cytotoxicity (using Flow cytometry) are detected and compare.
(1) basal culture medium on the CIK cellular form (plumpness and aspects such as diopter) and AIM-V with that blood serum medium is arranged is similar.
(2) aspect the cell cultures rate of propagation, the Cord Blood Mononuclear Cell implant concentration is 1x10
6The time, induced CIK cell proliferation 5 days with basal culture medium, OD value (mean number ± standard deviation) is 0.614 ± 0.036, and AIM-V is 0.517 ± 0.04, and it is 0.289 ± 0.021 that blood serum medium is arranged; Basal culture medium and AIM-V and have blood serum medium relatively, P value is equal＜and 0.001.Induced CIK cell proliferation 10 days with basal culture medium, the OD value is 0.894 ± 0.072, and AIMV-V is 0.701 ± 0.088, and it is 0.621 ± 0.028 that blood serum medium is arranged, and basal culture medium compares P value＜0.04 with blood serum medium is arranged.Basal culture medium is better than that blood serum medium is arranged.
(3) aspect cell phenotype, the two positive percentage of CD3+CD56+: be 29% when basal culture medium is induced 10 days; AIM-V is 34.45%; It is 30.74% that blood serum medium is arranged; At the CIK cell to aspect K562 leukemia cell's the kill rate, induce 5 days CIK cell, imitating the target ratio is under 10: 1 situations, the CIK cell is to K562 leukemia cell's kill rate (CFSE and PI dyeing, flow cytometer detects): basal culture medium is 11.35%, AIM-V is 8.83%, and it is 11.77% that blood serum medium is arranged.Basal culture medium induce the two positive cells of CD3+CD56+ and the inductive CIK of institute cell to aspect K562 leukemia cell's the kill rate to AIM-V with that blood serum medium is arranged is similar.
(2) cultivation of (ancestral) cells in vitro is done in hematopoiesis:
Be used for hematopoiesis and do propagation and the differentiation that all formula concentration of the basal culture medium of (ancestral) cell cultures all are suitable for dried (ancestral) cell of hematopoiesis, be used for the treatment of the stem cell transplantation and the gene therapy of radioactive exposure damage, immune deficiency and hematopoietic system cancer.Adding SCF and IL-6 act on the amplification and the differentiation of early stage hemopoietic stem cell (as the CD34 positive cell) in the basal culture medium, and adding TPO and GM-CSF then act on hemopoietic progenitor cell (as a megakaryoblast and the single progenitor cell of grain) amplification and break up.Separate mononuclearcell (MNCs) by the Ficoll-Hypaque density gradient centrifugation from human peripheral, marrow or Cord blood, results tunica albuginea confluent monolayer cells is counted behind the washed cell.1x10
6The MNCs cell cultures was changed liquid 1 time in per 3 days in being added with above-mentioned cytokine humanized's serum free medium, the 14th day harvested cell is used for stem-cell therapy.
11 kinds of raw materials that the present invention is used: the human serum albumin solution is used in treatment, biosynthetic human insulin's solution, human transferrin solution, people's cholesterol solution, the human hydrogen peroxidase enzyme solution, 2 mercapto ethanol solution, ascorbic acid solution, linolic acid solution, ethanolamine solutions, people's vitronectin solution and L-glutamine solution, wherein protein and lipid derive from people's blood plasma, serum or tissue, be pharmaceutical grade or highly purified people's source protein or human recombination protein, do not contain any animal source composition, other composition all meets the ultrapure or analytical reagent of second one of American Pharmacopeia or " national standard chemistry reagent " or Pharmacopoeia of the People's Republic of China version in 2010, and qualified through the cell cultures detection, it is safe and rational being used for clinical.Can be used for smelting with its cultured cells and treat human tumor and other refractory disease.
The proliferation rate of cultivation of the present invention and inductive CIK cell is better than that blood serum medium is arranged, cell CD3+CD56+ cell percentage with to K562 leukemia cell's kill rate to have blood serum medium similar.The present invention helps to improve the security and the stdn of domestic cell therapy.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1: the preparation of humanized's serum free medium
A. prepare the saturated human transferrin solution of iron:
(1), elder generation is with the FeCl of 16.22mg
3With the 1mM HCl of 10ml dissolving, be made into storage liquid, packing then, freezing being stored in-20 ℃ obtain storage liquid No. 1;
(2), take by weighing the human transferrin of 120mg, be dissolved among the 4.0ml DMEM that does not add sodium bicarbonate, add No. 1 storage liquid of 270 μ l again, stir, be the saturated human transferrin solution of iron;
B. prepare people's cholesterol solution: the people's cholesterol powder that takes by weighing 50mg, adding 50ml does not again add among the DMEM of sodium bicarbonate, ultrasonic oscillator vibration with 2cm diameter peptide probe, in 4 ℃ of refrigerators, vibrated 1 hour with peak swing, make it complete emulsification, successively, promptly get people's cholesterol solution, be kept at-30 ℃ with the membrane filtration degerming of 1.2 μ m and 0.45 μ m;
C. prepare linolic acid solution: take by weighing the 0.02g linolic acid and be dissolved in the 100ml standard ethanol, fully vibration dissolving promptly gets linolic acid solution, is kept at 8 ℃.
D. the human serum albumin solution is used in the 50mg/ml treatment, 500 μ g/ml biosynthetic human insulin solution, 1500 μ g/ml human transferrin solution, 1000 μ g/ml people cholesterol solutions, 1000 μ g/ml human hydrogen peroxidase enzyme solution, 25ug/ml 2 mercapto ethanol solution, 1250 μ g/ml ascorbic acid solutions, 75 μ g/ml linolic acid solution, 500 μ g/ml ethanolamine solutions, 50 μ g/ml people vitronectin solution and 500 μ g/mlL-glutamine solution mix, stir, be diluted to the 1L/ bottle with IMDM again, reach the final concentration in the prescription, promptly get humanized's serum free medium.
Embodiment 2: the preparation of humanized's serum free medium
A, the saturated human transferrin solution of preparation iron:
(1), elder generation is with the FeCl of 27.30mg
3With the 1mM HCl of 20ml dissolving, be made into storage liquid, packing then, freezing being stored in-20 ℃ obtain storage liquid No. 1;
(2), take by weighing the human transferrin of 200mg, be dissolved among the 10.0ml DMEM that does not add sodium bicarbonate, add No. 1 storage liquid of 400 μ l again, stir, be the saturated human transferrin solution of iron;
B, preparation people cholesterol solution: the people's cholesterol powder that takes by weighing 100mg, adding 100ml does not again add among the DMEM of sodium bicarbonate, ultrasonic oscillator vibration with 2cm diameter peptide probe, in 4 ℃ of refrigerators, vibrated 1 hour with peak swing, make it complete emulsification, successively, promptly get people's cholesterol solution, be kept at-30 ℃ with the membrane filtration degerming of 1.2 μ m and 0.45 μ m;
C. prepare linolic acid solution: take by weighing the 0.025g linolic acid and be dissolved in the 100ml standard ethanol, fully vibration dissolving promptly gets linolic acid solution, is kept at 8 ℃.
D. the human serum albumin solution is used in the 100mg/ml treatment, 800 μ g/ml biosynthetic human insulin solution, 2000 μ g/ml human transferrin solution, 1200 μ g/ml people cholesterol solutions, 1300 μ g/ml human hydrogen peroxidase enzyme solution, 30ug/ml 2 mercapto ethanol solution, the 2000/ml ascorbic acid solution, 100 μ g/ml linolic acid solution, 1000 μ g/ml ethanolamine solutions, 100 μ g/ml people vitronectin solution and 1000 μ g/mlL-glutamine solution mix, stir, be diluted to the 1L/ bottle with IMDM again, reach the final concentration in the prescription, promptly get humanized's serum free medium.
Embodiment 3: the preparation of humanized's serum free medium
A. prepare the saturated human transferrin solution of iron:
(1), elder generation is with the FeCl of 20mg
3With the 1mM HCl of 15ml dissolving, be made into storage liquid, packing then, freezing being stored in-20 ℃ obtain storage liquid No. 1;
(2), take by weighing the human transferrin of 200mg, be dissolved among the 10.0ml DMEM that does not add sodium bicarbonate, add No. 1 storage liquid of 400 μ l again, stir, be the saturated human transferrin solution of iron;
B, preparation people cholesterol solution: the people's cholesterol powder that takes by weighing 100mg, adding 100ml does not again add among the DMEM of sodium bicarbonate, ultrasonic oscillator vibration with 2cm diameter peptide probe, in 4 ℃ of refrigerators, vibrated 1 hour with peak swing, make it complete emulsification, successively, promptly get people's cholesterol solution, be kept at-30 ℃ with the membrane filtration degerming of 1.2 μ m and 0.45 μ m;
C. prepare linolic acid solution: take by weighing the 0.025g linolic acid and be dissolved in the 100ml standard ethanol, fully vibration dissolving promptly gets linolic acid solution, is kept at 8 ℃.
D, 300mg/ml treatment is mixed with human serum albumin solution, 1000 μ g/ml biosynthetic human insulins, 2800 μ g/ml human transferrin liquid, 1500 μ g/ml people cholesterol solutions, 1600 μ g/ml human hydrogen peroxidase enzymes, 80ug/ml2-mercaptoethanol, 2500/ml xitix, 90 μ g/ml linolic acid, 1200 μ g/ml thanomins, 150 μ g/ml people vitronectin and 1200 μ g/ml L-glutamines, stir, be diluted to the 1L/ bottle with IMDM again, reach the final concentration in the prescription, promptly get humanized's serum free medium.
Embodiment 4: humanized's serum free medium is used for the culturing cell factor and induces killer cell (CIK)
In humanized's serum free medium, add following cytokine (final concentration):
(1) people interferon gamma (hrIFN): the 30ng/ml that recombinates
(2) people CD3 monoclonal antibody (OKT3): 100ng/ml
(3) people's recombinant interleukin 2 (hrIL-2): 20ng/ml
(4) people's recombinant interleukin 1 (hrIL-1): 55ng/ml.
Embodiment 5: humanized's serum free medium is used to cultivate hematopoiesis and does (ancestral) cell
In humanized's serum free medium, add following cytokine (final concentration):
(1) STEM CELL FACTOR (SCF) 100ng/ml
(2) grain macrophage stimulation factor (GM-CSF) 80ng/ml
(3) interleukin 6 (IL-6) 50ng
(4) thrombopoietin (TPO) 20ng/ml.
Claims (2)
1. humanized's serum free medium is characterized in that: fill a prescription to contain in the treatment of final concentration and uses the human serum albumin solution: 1--10mg/ml, biosynthetic human insulin's solution: 4--40 μ g/ml, human transferrin solution: 30--60 μ g/ml, people's cholesterol solution: 20--50 μ g/ml, human hydrogen peroxidase enzyme solution: 20--50 μ g/ml, 2 mercapto ethanol solution: 0.05--0.5mM, ascorbic acid solution: 25--100 μ g/ml, linolic acid solution: 1.5--25 μ g/ml, ethanolamine solutions: 10-60 μ g/ml, people's vitronectin solution: 0.1--1.0 μ g/ml, L-glutamine solution: 10.0--100 μ g/ml.
2. the preparation method of humanized's serum free medium according to claim 1 is characterized in that:
A. according to a conventional method the treatment that is respectively of compound concentration with human serum albumin solution 50-500mg/ml, biosynthetic human insulin's solution 500-2000 μ g/ml, human hydrogen peroxidase enzyme solution 1000-2500 μ g/ml, 2 mercapto ethanol solution: 25-250 μ g/ml, ascorbic acid solution 1250--5000 μ g/ml, ethanolamine solutions 500-3000 μ g/ml, people's vitronectin solution 50-500 μ g/ml, L-glutamine solution 500-5000 μ g/ml;
B, the saturated human transferrin solution of preparation iron:
(1), elder generation is with the FeCl of 16.22--27.30mg
3With the 1mM HCl of 10--20ml dissolving, be made into storage liquid, packing then, freezing being stored in-20 ℃ obtain storage liquid No. 1;
(2), take by weighing the human transferrin of 120--200mg, being dissolved in the 4.0--10.0ml cutter that does not add sodium bicarbonate improves among she Ge Shi (DMEM) than Ke Shi, add No. 1 storage liquid of 270--400 μ l again, stir, promptly getting concentration is the saturated human transferrin solution of iron of 1500--3000 μ g/ml;
C, preparation people cholesterol solution: the people's cholesterol powder that takes by weighing 50--100mg, people's cholesterol powder of 50--100mg is joined 50--100ml not to be added among the DMEM of sodium bicarbonate, ultrasonic oscillator vibration with 2cm diameter peptide probe, in 4 ℃ of refrigerators, vibrated 1 hour with peak swing, make it complete emulsification, successively with the membrane filtration degerming of 1.2 μ m and 0.45 μ m, promptly getting concentration is people's cholesterol solution of 1000-2500 μ g/ml, is kept at-30 ℃;
D. prepare linolic acid solution: take by weighing the 0.015-0.03g linolic acid and be dissolved in the 100ml standard ethanol, fully vibration dissolving, promptly getting concentration is the linolic acid solution of 75-1250 μ g/ml, is kept at 8 ℃;
E, will treat with human serum albumin solution, biosynthetic human insulin's solution, human transferrin solution, people's cholesterol solution, human hydrogen peroxidase enzyme solution, 2 mercapto ethanol solution, ascorbic acid solution, linolic acid solution, ethanolamine solutions, people's vitronectin solution and L-glutamine solution and mix, stir, be diluted to the 1L/ bottle with Yi Sikefu improvement cutter than Ke Shi substratum again, reach the final concentration in the prescription, promptly get humanized's serum free medium.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102352343A (en) * | 2011-09-28 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | Lymphocyte culture medium and use method thereof |
| 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 |
| 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 |
| CN105176925A (en) * | 2015-09-26 | 2015-12-23 | 友康恒业生物科技（北京）有限公司 | Immune cell serum-free medium and preparation and application thereof |
| CN106190946A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of culture medium for expansion of stem cells |
| CN108707579A (en) * | 2018-05-28 | 2018-10-26 | 北京美迪阿姆科技发展有限公司 | The serum free medium and preparation method and cultural method of a kind of human T lymphocyte's culture |
| CN109055311A (en) * | 2018-08-21 | 2018-12-21 | 苏州米苏生物技术有限公司 | Source of people lymphocyte cultural method based on serum free medium |
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| CN102352343A (en) * | 2011-09-28 | 2012-02-15 | 上海柯莱逊生物技术有限公司 | Lymphocyte culture medium and use method thereof |
| 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 |
| CN104371972B (en) * | 2013-08-12 | 2015-11-25 | 英普乐孚生物技术(上海)有限公司 | A kind of non-animal derived property and humanized's composition T lymphocytes culture medium and preparation method thereof |
| 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 |
| CN104371973B (en) * | 2013-08-15 | 2017-12-05 | 协和华东干细胞基因工程有限公司 | A kind of serum free medium of immunocyte |
| CN105176925A (en) * | 2015-09-26 | 2015-12-23 | 友康恒业生物科技（北京）有限公司 | Immune cell serum-free medium and preparation and application thereof |
| CN105176925B (en) * | 2015-09-26 | 2018-08-28 | 友康恒业生物科技（北京）有限公司 | A kind of immunocyte serum free medium and application thereof |
| CN106190946A (en) * | 2016-07-19 | 2016-12-07 | 安徽惠恩生物科技股份有限公司 | A kind of culture medium for expansion of stem cells |
| CN108707579A (en) * | 2018-05-28 | 2018-10-26 | 北京美迪阿姆科技发展有限公司 | The serum free medium and preparation method and cultural method of a kind of human T lymphocyte's culture |
| 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 |
| CN109055311A (en) * | 2018-08-21 | 2018-12-21 | 苏州米苏生物技术有限公司 | Source of people lymphocyte cultural method based on serum free medium |
| CN109055311B (en) * | 2018-08-21 | 2022-03-15 | 苏州沃美生物有限公司 | Human lymphocyte culture method based on serum-free culture medium |
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