CN110904024A - Method for removing dead cells in suspension cell culture - Google Patents
Method for removing dead cells in suspension cell culture Download PDFInfo
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- CN110904024A CN110904024A CN201911107953.7A CN201911107953A CN110904024A CN 110904024 A CN110904024 A CN 110904024A CN 201911107953 A CN201911107953 A CN 201911107953A CN 110904024 A CN110904024 A CN 110904024A
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- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000000725 suspension Substances 0.000 title claims abstract description 13
- 238000004113 cell culture Methods 0.000 title claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims abstract description 75
- 238000000926 separation method Methods 0.000 claims abstract description 10
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 5
- 238000005119 centrifugation Methods 0.000 claims abstract description 3
- 239000006285 cell suspension Substances 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 5
- 229920001917 Ficoll Polymers 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 3
- 210000002751 lymph Anatomy 0.000 claims description 3
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 claims description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 claims description 2
- 239000012930 cell culture fluid Substances 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims description 2
- 238000007664 blowing Methods 0.000 claims 1
- 238000000432 density-gradient centrifugation Methods 0.000 claims 1
- 239000012530 fluid Substances 0.000 claims 1
- 210000000170 cell membrane Anatomy 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 230000006907 apoptotic process Effects 0.000 abstract description 3
- 206010003694 Atrophy Diseases 0.000 abstract description 2
- 230000037444 atrophy Effects 0.000 abstract description 2
- 230000030833 cell death Effects 0.000 abstract description 2
- 239000012634 fragment Substances 0.000 abstract description 2
- 230000003834 intracellular effect Effects 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 230000035699 permeability Effects 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 2
- 230000003698 anagen phase Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0081—Purging biological preparations of unwanted cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
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- Molecular Biology (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention mainly relates to a method for removing dead cells in suspension cell culture, and the cells with higher activity are obtained. This patent adopts the gradient centrifugation method to separate dead cell and dead cell piece. The principle of isolating dead cells is: after cell death, cell membrane permeability increases, intracellular water is lost and atrophy, and finally the cell membrane is ruptured. During this process of apoptosis, cells become more dense, and dead cells and fragments of dead cells become denser than normal living cells. The cell mixture is added to a lymphocyte separation medium having a density between that of dead cells and that of live cells, and then centrifuged to separate the dead cells from the live cells. Compared with the traditional centrifugal method, the method for removing dead cells in suspension cells is more thorough in separation, and meanwhile, the damage to living cells is reduced.
Description
Technical Field
The invention mainly relates to living cell purification of suspension cell culture, and belongs to the field of cell culture.
Background
During the discontinuous culture of suspension cells, the whole cell culture cycle can be divided into four stages according to the growth and division conditions of the whole cells: incubation period, logarithmic growth phase, stationary phase, decline period. When the cells enter the logarithmic growth phase, the cells are most actively divided, the cell activity is also the highest, the cell metabolites are increased along with the increase of the division times, the cell density is increased, and a small part of cells show the signs of apoptosis at the later stage of the logarithmic growth phase. After the stable period, the cell density reaches the maximum, and if the fresh culture medium is replaced by supplementing nutrition in time, the number of apoptotic cells is increased, namely, the number of dead cells is increased. Too many dead cells can affect the overall cellular activity and inhibit the growth metabolism of normal cells. Therefore, how to remove dead cells in suspension cell culture is a necessary experimental operation in the suspension cell culture process.
Currently, no commercial product specifically directed to the removal of dead cells has emerged. Most of the traditional methods for removing dead cells adopt a low-speed centrifugation method, which is not thorough in removing the dead cells on one hand, and also can separate and lose some living cells on the other hand. The patent refers to the field of 'separation of cells from blood vessels'. The principle of isolating dead cells is: after cell death, cell membrane permeability increases, intracellular water is lost and atrophy, and finally the cell membrane is ruptured. During this process of apoptosis, cells become more dense, and dead cells and fragments of dead cells become denser than normal living cells. The cell mixture is added to a lymphocyte separation medium having a density between that of dead cells and that of live cells, and then centrifuged to separate the dead cells from the live cells. Compared with the traditional centrifugal method, the method for removing dead cells in suspension cells is more thorough in separation, and meanwhile, the damage to living cells is reduced.
Disclosure of Invention
The invention relates to a method for removing dead cells in suspension cell culture, which aims to separate and remove dead cells and dead cell debris in suspension culture cells to obtain cells with higher activity.
In order to achieve the above object, the present invention proposes the following solutions:
preparing reagents and consumables for use includes: 250ml centrifuge bottles, 50ml centrifuge tubes and ficoll human lymphocyte separation medium.
All cell culture fluid required to separate dead cells was collected in a centrifuge flask, centrifuged at 2300rpm (or 800 g) for 10min, and the supernatant was decanted.
Adding appropriate amount of DPBS solution into the centrifugal flask, and mixing to make the cell concentration at 1-5 x 107Between/ml.
Several 50ml centrifuge tubes were prepared, and 15ml of ficoll human lymphocyte splitting medium was added to each tube.
The cell suspension with the adjusted cell concentration is slowly added into a centrifuge tube filled with the lymph separation liquid, and 30ml of the cell suspension is added into each tube.
After the tube was trimmed, the tube was gently placed in a centrifuge at 2300rpm (or 800 g) for 20 min.
After centrifugation, the upper 30ml of solution in each tube was slowly pipetted into the centrifuge flask, leaving the remaining portion (containing dead cells) to waste.
The cell suspension in the centrifugal bottle is blown and evenly mixed, centrifuged at 2300rpm (or 800 g) for 10min, and the supernatant is poured off.
And (4) the cell sediment in the centrifugal bottle is the separated living cells.
And (3) resuspending and uniformly mixing the cells in the centrifugal bottle by using a culture bottle, and taking 2ml of suspension to detect the proportion of living cells by a trypan blue staining method.
Through trypan blue staining detection, the proportion of the living cells in the finally obtained cell suspension is 99.5%.
Claims (2)
1. A method for removing dead cells in suspension cell culture is characterized in that a density gradient centrifugation method is adopted to remove dead cells and dead cell debris, and a used separation reagent is ficoll human lymph separation liquid.
2. A method for removing dead cells in suspension cell culture is characterized in that the operation process comprises the following steps: collecting all cell culture fluid needing to separate dead cells into a centrifuge bottle, centrifuging at 2300rpm (or 800 g) for 10min, and pouring off supernatant; adding appropriate amount of DPBS solution into the centrifugal flask, and mixing to make the cell concentration at 1-5 x 107Between/ml; preparing a plurality of 50ml centrifuge tubes, and adding 15ml of ficoll human lymphocyte splitting fluid into each tube; slowly adding the cell suspension with the adjusted cell concentration into a centrifuge tube filled with a lymph separation liquid, and adding 30ml of cell suspension into each tube; after the centrifugal tube is balanced, the centrifugal tube is gently placed into a centrifugal machine to be centrifuged at 2300rpm (or 800 g) for 20 min; after centrifugation, the upper 30ml of solution in each tube was slowly aspirated into a centrifuge bottle, leaving the remaining portion (Containing dead cells) are discarded; blowing and uniformly mixing the cell suspension in the centrifugal bottle, centrifuging at 2300rpm (or 800 g) for 10min, and pouring off the supernatant; and (4) the cell sediment in the centrifugal bottle is the separated living cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911107953.7A CN110904024A (en) | 2019-11-13 | 2019-11-13 | Method for removing dead cells in suspension cell culture |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911107953.7A CN110904024A (en) | 2019-11-13 | 2019-11-13 | Method for removing dead cells in suspension cell culture |
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| CN110904024A true CN110904024A (en) | 2020-03-24 |
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| CN201911107953.7A Pending CN110904024A (en) | 2019-11-13 | 2019-11-13 | Method for removing dead cells in suspension cell culture |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925977A (en) * | 2020-09-24 | 2020-11-13 | 北京健为医学检验实验室有限公司 | Method for removing dead cells in cell suspension |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4806476A (en) * | 1983-09-08 | 1989-02-21 | Lovelace Medical Foundation | Efficient cell fusion process |
| US5827642A (en) * | 1994-08-31 | 1998-10-27 | Fred Hutchinson Cancer Research Center | Rapid expansion method ("REM") for in vitro propagation of T lymphocytes |
| CN1823160A (en) * | 2003-07-11 | 2006-08-23 | 布拉斯蒂康生物科技研究有限责任公司 | Monocyte-derived autologous self-tolerance-inducing cells and their use in pharmaceutical preparations |
| CN101031641A (en) * | 2004-08-19 | 2007-09-05 | 伯哈德·莫瑟 | Preparation of antigen-presenting human γδT cells and its use in immunotherapy |
| CN101374942A (en) * | 2006-01-31 | 2009-02-25 | 阿斯比奥制药株式会社 | Methods for purification of stem cells and embryonic derived cardiomyocytes and putative cardiomyocytes |
| CN107533051A (en) * | 2015-03-27 | 2018-01-02 | 南加利福尼亚大学 | New target drones of the HLA G as CAR T cell immunotherapies |
| CN109554345A (en) * | 2018-11-21 | 2019-04-02 | 新格元(南京)生物科技有限公司 | A kind of digestive juice and its method that Tissues of Human Adenocarcinoma of Pancreas is separated into single living cell |
| CN110229784A (en) * | 2019-05-27 | 2019-09-13 | 立沃生物科技（深圳）有限公司 | A method of it removing the separating liquid of dead liver cell and removes dead liver cell using it |
-
2019
- 2019-11-13 CN CN201911107953.7A patent/CN110904024A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4806476A (en) * | 1983-09-08 | 1989-02-21 | Lovelace Medical Foundation | Efficient cell fusion process |
| US5827642A (en) * | 1994-08-31 | 1998-10-27 | Fred Hutchinson Cancer Research Center | Rapid expansion method ("REM") for in vitro propagation of T lymphocytes |
| CN1823160A (en) * | 2003-07-11 | 2006-08-23 | 布拉斯蒂康生物科技研究有限责任公司 | Monocyte-derived autologous self-tolerance-inducing cells and their use in pharmaceutical preparations |
| CN101031641A (en) * | 2004-08-19 | 2007-09-05 | 伯哈德·莫瑟 | Preparation of antigen-presenting human γδT cells and its use in immunotherapy |
| CN101374942A (en) * | 2006-01-31 | 2009-02-25 | 阿斯比奥制药株式会社 | Methods for purification of stem cells and embryonic derived cardiomyocytes and putative cardiomyocytes |
| CN107533051A (en) * | 2015-03-27 | 2018-01-02 | 南加利福尼亚大学 | New target drones of the HLA G as CAR T cell immunotherapies |
| CN109554345A (en) * | 2018-11-21 | 2019-04-02 | 新格元(南京)生物科技有限公司 | A kind of digestive juice and its method that Tissues of Human Adenocarcinoma of Pancreas is separated into single living cell |
| CN110229784A (en) * | 2019-05-27 | 2019-09-13 | 立沃生物科技（深圳）有限公司 | A method of it removing the separating liquid of dead liver cell and removes dead liver cell using it |
Non-Patent Citations (5)
| Title |
|---|
| H TSE、R W DUTTON: "Separation of helper and suppressor T lymphocytes on a ficoll velocity sedimentation gradient", 《J EXP MED》 * |
| W F DAVIDSON、C R PARISH: "A procedure for removing red cells and dead cells from lymphoid cell suspensions", 《J IMMUNOL METHODS》 * |
| 宋建勋等: "运用短期外周血超抗原反应T细胞系研究超抗原诱导的T细胞凋亡", 《第三军医大学学报》 * |
| 李海宁等: "中性粒细胞碱性磷酸酶对中性粒细胞功能影响的体外研究", 《中华血液学杂志》 * |
| 马新博等: "《医学免疫学实验与学习指导》", 31 October 2015, 第四军医大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111925977A (en) * | 2020-09-24 | 2020-11-13 | 北京健为医学检验实验室有限公司 | Method for removing dead cells in cell suspension |
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Inventor after: Chen Jie Inventor before: Fang Songgang Inventor before: Chen Jie |