CN109593713A - A kind of cultural method of autologous peripheral blood lymphocyte - Google Patents
A kind of cultural method of autologous peripheral blood lymphocyte Download PDFInfo
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- CN109593713A CN109593713A CN201811639114.5A CN201811639114A CN109593713A CN 109593713 A CN109593713 A CN 109593713A CN 201811639114 A CN201811639114 A CN 201811639114A CN 109593713 A CN109593713 A CN 109593713A
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 210000005105 peripheral blood lymphocyte Anatomy 0.000 title claims abstract description 16
- 210000004027 cell Anatomy 0.000 claims abstract description 30
- 210000004698 lymphocyte Anatomy 0.000 claims abstract description 14
- 239000012930 cell culture fluid Substances 0.000 claims abstract description 11
- 210000005259 peripheral blood Anatomy 0.000 claims abstract description 10
- 239000011886 peripheral blood Substances 0.000 claims abstract description 10
- 108010074328 Interferon-gamma Proteins 0.000 claims abstract description 9
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 9
- 230000002093 peripheral effect Effects 0.000 claims abstract description 9
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 8
- 102000008070 Interferon-gamma Human genes 0.000 claims abstract description 7
- 239000006143 cell culture medium Substances 0.000 claims abstract description 7
- 229960003130 interferon gamma Drugs 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims description 24
- 238000011534 incubation Methods 0.000 claims description 10
- 238000000926 separation method Methods 0.000 claims description 8
- 210000004369 blood Anatomy 0.000 claims description 5
- 239000008280 blood Substances 0.000 claims description 5
- 210000002966 serum Anatomy 0.000 claims description 5
- 102000004125 Interleukin-1alpha Human genes 0.000 claims description 4
- 108010082786 Interleukin-1alpha Proteins 0.000 claims description 4
- 239000000654 additive Substances 0.000 claims description 4
- 230000000996 additive effect Effects 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 4
- 230000001464 adherent effect Effects 0.000 claims description 3
- 230000010100 anticoagulation Effects 0.000 claims description 3
- 244000309466 calf Species 0.000 claims description 3
- 229920000669 heparin Polymers 0.000 claims description 3
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims description 3
- 229960001008 heparin sodium Drugs 0.000 claims description 3
- 239000002609 medium Substances 0.000 claims description 3
- 210000001616 monocyte Anatomy 0.000 claims description 3
- 239000012679 serum free medium Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 238000005406 washing Methods 0.000 claims description 2
- 210000004405 cytokine-induced killer cell Anatomy 0.000 abstract description 8
- 238000009169 immunotherapy Methods 0.000 abstract description 6
- 230000003321 amplification Effects 0.000 abstract description 4
- 230000001413 cellular effect Effects 0.000 abstract description 4
- 230000000694 effects Effects 0.000 abstract description 4
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 4
- 230000000259 anti-tumor effect Effects 0.000 abstract description 3
- 206010028980 Neoplasm Diseases 0.000 description 4
- 102000004127 Cytokines Human genes 0.000 description 3
- 108090000695 Cytokines Proteins 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000000087 hemolymph Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 239000003726 plant lectin Substances 0.000 description 1
- 239000000047 product 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
- C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/24—Interferons [IFN]
-
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Abstract
The invention discloses a kind of cultural methods of autologous peripheral blood lymphocyte, specifically includes the following steps: (1) is added in appropriate serum-free cell culture medium from separating peripheral blood mononuclear cells in peripheral blood, and the interferon-γ that concentration is 1000-2000U/ml is added, it is placed in incubator stationary culture 3 days；(2) mononuclearcell cultivated in step (1) is transferred in the culture bottle containing 3 monoclonal antibody of cell surface molecule and 16 monoclonal antibody of cell surface molecule, and the interferon-2 that concentration is 1000-2000U/ml is added, stationary culture 3 days；(3) cell culture fluid that 500-1000mL is added into step (2), is added the interferon-2 of 1000-2000U/ml, and stationary culture 3 days；(4) cell culture fluid for continuing to add 500-1000mL into step (3), is placed in incubator stationary culture 3 days, repeats this step 2-3 time, obtained autologous peripheral lymphocyte.The present invention can cultivate the CIK cell of high amplification rate and high activity, and then be applied in antitumor adoptive cellular immunotherapy well.
Description
Technical field
Invention is related to culture in vitro of lymphocytes technical field, specially a kind of culture side of autologous peripheral blood lymphocyte
Method.
Background technique
Adoptive immunotherapy is that sensitized lymphocyte (having specific immunity) or its product are defeated by cellular immunity function
The low person's (such as tumour patient) of energy, makes its obtain anti-tumor immunity, it is a kind of immunotherapy for treating tumour, and
CIK is that human peripheral blood single nucleus cell is used to cytokine profiles (such as CD 3-resisting monoclonal antibody, IL-2 and IFN-γ in vitro
Deng) a group foreign cell that obtains afterwards for a period of time of co-incubation, the applications well for CIK cell is considered as that a new generation is anti-
The preferred option of tumour adoptive cellular immunotherapy
But at present for the Vitro Culture Techniques of CIK cell there are amplification efficiencies lower, ability lethal for cancer cell
It is poor, mainly due to cytokine profiles (type and dosage of such as CD 3-resisting monoclonal antibody, IL-2 and IFN-γ, even
Influencing each other between cytokine profiles, and then CIK cell activity obtained is influenced, so that CIK cell
It can not be applied to well in antitumor adoptive cellular immunotherapy, for this purpose, we have proposed a kind of autologous peripheral hemolymphs
The cultural method of cell.
Summary of the invention
The cultural method for being designed to provide a kind of autologous peripheral blood lymphocyte of invention, to solve above-mentioned background technique
The problem of middle proposition.
To achieve the above object, invention provides the following technical solutions: a kind of cultural method of autologous peripheral blood lymphocyte,
Specifically includes the following steps:
(1) firstly, from peripheral blood separating peripheral blood mononuclear cells, then, according to density 1 × 106Appropriate nothing is added
In serum cell culture fluid, and the interferon-γ that concentration is 1000-2000U/ml is added, is placed in incubator stationary culture 3 days,
Obtain first culture solution；
(2) firstly, 3 monoclonal antibody of cell surface molecule and 16 monoclonal antibody of cell surface molecule are wrapped in advance
The mononuclearcell cultivated in step (1) is then transferred to containing 3 monoclonal antibody of cell surface molecule and cell surface point by quilt
In the culture bottle of sub 16 monoclonal antibodies, equimultiple adds serum-free cell culture medium, and it is 1000-2000U/ml's that concentration, which is added,
Interferon-2 is placed in incubator stationary culture 3 days, obtains second incubation liquid；
(3) it firstly, second incubation liquid obtained by step (2) to be added to the cell culture fluid of 500-1000mL, is then added
The interferon-2 of 1000-2000U/ml is placed in incubator stationary culture 3 days, and culture solution three times is made；
(4) firstly, being placed in training for culture solution continues to add the cell culture fluid of 500-1000mL three times obtained by step (3)
It supports case stationary culture 3 days, repeats this step 2-3 times, autologous peripheral lymphocyte is made.
Preferably, in the step (1) peripheral blood separation method are as follows: firstly, every 50mL peripheral blood of acquisition is added
After mixing with fresh anticoagulation 10mL with the Hanks liquid 1:1 mixed liquor mixed after 5IU heparin sodium, being placed in revolving speed is 1800r/min
Low speed desk centrifuge be centrifuged 15min, then, collect plasma layer and lymphocyte separation medium interface mononuclearcell,
Finally, adding Hank ' s liquid of the 0.2mL containing 20% calf serum to mix again by every milliliter of blood preparation after washed liquid washs 2 times
Outstanding 37 DEG C of cell, 5%CO2Supernatant is removed after cultivating 2h in incubator, obtains adherent monocyte.
Preferably, the condition of stationary culture is 37 DEG C of temperature, relative humidity 100%, CO in step (1)-(4)2Content
For 5% CO2Culture in incubator.
Preferably, the serum-free medium in the step (1) is added with following components: 10% autologous plasma, interferon-
γ, 1000-2000U/ml, phytolectin P, 500~1000ng, interleukin 1 α, 100~200U.
Preferably, 3 monoclonal antibody additive amount of cell surface molecule is 50-100ng, cell surface in the step (2)
16 monoclonal antibody additive amount of molecule is 50-100ng.
Preferably, in the step (4) after autologous peripheral lymphocyte obtained centrifugation after washing removes culture solution, weight
It is suspended from and saves in liquid.
Compared with prior art, advantageous effect of the invention is:
1, the cultural method of the autologous peripheral blood lymphocyte extracts peripheral blood mononuclear cells and from peripheral blood to pass
Based on the CIK cell cultural method of system, the type and dosage of addition cell factor are improved, and then be made with high amplification rate
And the CIK cell of high activity, and then be applied in antitumor adoptive cellular immunotherapy well；
2, the cultural method of the autologous peripheral blood lymphocyte uses serum-free medium in incubation, and animal is discharged
Other disturbing factors such as foreign aid's virus that serum carries, will not impact CIK cell in vitro amplification procedure, guarantee CIK
The quality of cell；
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment one: invention provides a kind of technical solution: a kind of method of separation and Extraction peripheral blood mononuclear cells, specifically
The following steps are included: firstly, every 50mL peripheral blood of acquisition is added after 5IU heparin sodium and fresh anticoagulation 10mL and Hanks
After the mixed liquor mixing that liquid 1:1 is mixed, it is placed in the low speed desk centrifuge that revolving speed is 1800r/min and is centrifuged 15min, then, receive
Collect the mononuclearcell of plasma layer and lymphocyte separation medium interface, finally, after washed liquid washs 2 times, by every milliliter of blood
Liquid sample adds Hank ' s liquid of the 0.2mL containing 20% calf serum 37 DEG C of suspension cell, 5%CO again2After cultivating 2h in incubator
Supernatant is removed, adherent monocyte is obtained.
Embodiment two:
Invention provides a kind of technical solution: a kind of cultural method of autologous peripheral blood lymphocyte specifically includes following step
It is rapid:
(1) firstly, according to the method for one of embodiment one separation and Extraction peripheral blood mononuclear cells outside 100mL
Separating peripheral blood mononuclear cells in all blood, then, according to density 1 × 106It is added solidifying containing 10% autologous plasma, plant in right amount
In the serum-free cell culture medium for collecting element P, 500ng, interleukin 1 α, 100U, and the interferon-γ that concentration is is added, sets
In 37 DEG C of temperature, relative humidity 100%, CO2The CO that content is 5%2Culture culture 3 days, are cultivated for the first time in incubator
Liquid；
(2) firstly, by 3 monoclonal antibody of 50ng cell surface molecule and 16 monoclonal antibody of 50ng cell surface molecule into
Row is coated in advance, and the mononuclearcell cultivated in step (1) is then transferred to 3 monoclonal antibody of cell surface molecule containing 50ng
In the culture bottle of 16 monoclonal antibody of 50ng cell surface molecule, equimultiple adds serum-free cell culture medium, and concentration is added
For the interferon-2 of 1000U/ml, it is placed in 37 DEG C of temperature, relative humidity 100%, CO2The CO that content is 5%2It is quiet in incubator
Culture culture 3 days is set, second incubation liquid is obtained；
(3) firstly, second incubation liquid obtained by step (2) to be added to the cell culture fluid of 500mL, 1000U/ml is then added
Interferon-2, be placed in 37 DEG C of temperature, relative humidity 100%, CO2The CO that content is 5%2Stationary culture 3 is cultivated in incubator
It, is made culture solution three times；
(4) firstly, being placed in temperature for culture solution continues to add the cell culture fluid of 500mL three times obtained by step (3)
37 DEG C, relative humidity 100%, CO2The CO that content is 5%2Stationary culture 3 days in incubator repeat this step 3 time, are made certainly
Body periphery lymphocyte；
After (5) 2 weeks, after being washed removal culture solution after autologous peripheral lymphocyte obtained in step (4) centrifugation, weight
It is suspended from and saves in liquid.
Embodiment two:
Invention provides a kind of technical solution: a kind of cultural method of autologous peripheral blood lymphocyte specifically includes following step
It is rapid:
(1) firstly, according to the method for one of embodiment one separation and Extraction peripheral blood mononuclear cells outside 100mL
Separating peripheral blood mononuclear cells in all blood, then, according to density 1 × 106It is added solidifying containing 10% autologous plasma, plant in right amount
In the serum-free cell culture medium for collecting element P, 1000ng, interleukin 1 α, 200U, and the interferon-γ that concentration is is added, sets
In 37 DEG C of temperature, relative humidity 100%, CO2The CO that content is 5%2Culture culture 3 days, are cultivated for the first time in incubator
Liquid；
(2) firstly, by 3 monoclonal antibody of 100ng cell surface molecule and 16 monoclonal antibody of 100ng cell surface molecule
It is coated in advance, the mononuclearcell cultivated in step (1) is then transferred to 3 monoclonal of cell surface molecule containing 100ng
In antibody and the culture bottle of 16 monoclonal antibody of 100ng cell surface molecule, equimultiple adds serum-free cell culture medium, and is added
Concentration is the interferon-2 of 2000U/ml, is placed in 37 DEG C of temperature, relative humidity 100%, CO2The CO that content is 5%2Incubator
Interior stationary culture culture 3 days, obtains second incubation liquid；
(3) firstly, second incubation liquid obtained by step (2) to be added to the cell culture fluid of 1000mL, 2000U/ is then added
The interferon-2 of ml is placed in 37 DEG C of temperature, relative humidity 100%, CO2The CO that content is 5%2Culture stands training in incubator
It supports 3 days, culture solution three times is made；
(4) firstly, being placed in temperature for culture solution continues to add the cell culture fluid of 1000mL three times obtained by step (3)
Spend 37 DEG C, relative humidity 100%, CO2The CO that content is 5%2Stationary culture 3 days in incubator repeat this step 3 time, are made
Autologous peripheral lymphocyte；
After (5) 2 weeks, after being washed removal culture solution after autologous peripheral lymphocyte obtained in step (4) centrifugation, weight
It is suspended from and saves in liquid.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (6)
1. a kind of cultural method of autologous peripheral blood lymphocyte, it is characterised in that: specifically includes the following steps:
(1) firstly, from peripheral blood separating peripheral blood mononuclear cells, then, according to density 1 × 106It is thin that appropriate serum-free is added
In born of the same parents' culture solution, and the interferon-γ that concentration is 1000-2000U/ml is added, is placed in incubator stationary culture 3 days, obtains just
Secondary culture solution；
(2) firstly, 3 monoclonal antibody of cell surface molecule and 16 monoclonal antibody of cell surface molecule are coated in advance,
Then the mononuclearcell cultivated in step (1) is transferred to containing 3 monoclonal antibody of cell surface molecule and cell surface molecule
In the culture bottle of 16 monoclonal antibodies, equimultiple adds serum-free cell culture medium, and it is the dry of 1000-2000U/ml that concentration, which is added,
Element -2 is disturbed, incubator stationary culture is placed in 3 days, obtains second incubation liquid；
(3) firstly, second incubation liquid obtained by step (2) to be added to the cell culture fluid of 500-1000mL, 1000- is then added
The interferon-2 of 2000U/ml is placed in incubator stationary culture 3 days, and culture solution three times is made；
(4) firstly, being placed in incubator for culture solution continues to add the cell culture fluid of 500-1000mL three times obtained by step (3)
It stationary culture 3 days, repeats this step 2-3 times, autologous peripheral lymphocyte is made.
2. a kind of cultural method of autologous peripheral blood lymphocyte as described in claim 1, it is characterised in that: the step
(1) separation method of peripheral blood in are as follows: firstly, every 50mL peripheral blood of acquisition is added after 5IU heparin sodium and fresh anticoagulation
After 10mL is mixed with the Hanks liquid 1:1 mixed liquor mixed, it is placed in the low speed desk centrifuge that revolving speed is 1800r/min and is centrifuged
Then 15min collects the mononuclearcell of plasma layer and lymphocyte separation medium interface, finally, washed liquid washs 2 times
Afterwards, add Hank ' s liquid of the 0.2mL containing 20% calf serum 37 DEG C of suspension cell, 5%CO again by every milliliter of blood preparation2It is incubated for
Supernatant is removed after cultivating 2h in case, obtains adherent monocyte.
3. a kind of cultural method of autologous peripheral blood lymphocyte as described in claim 1, it is characterised in that: step (1)-
(4) condition of stationary culture is 37 DEG C of temperature, relative humidity 100%, CO in2The CO that content is 5%2Culture in incubator.
4. a kind of cultural method of autologous peripheral blood lymphocyte as described in claim 1, it is characterised in that: the step
(1) serum-free medium in is added with following components: 10% autologous plasma, interferon-γ, and 1000-2000U/ml, plant are solidifying
Collect element P, 500~1000ng, interleukin 1 α, 100~200U.
5. a kind of cultural method of autologous peripheral blood lymphocyte as claimed in claim 2, it is characterised in that: the step
(2) 3 monoclonal antibody additive amount of cell surface molecule is 50-100ng in, and 16 monoclonal antibody additive amount of cell surface molecule is
50-100ng。
6. a kind of cultural method of autologous peripheral blood lymphocyte as claimed in claim 4, it is characterised in that: the step
(4) it is resuspended in and saves in liquid after washing removes culture solution after autologous peripheral lymphocyte obtained centrifugation in.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811639114.5A CN109593713A (en) | 2018-12-29 | 2018-12-29 | A kind of cultural method of autologous peripheral blood lymphocyte |
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| CN201811639114.5A CN109593713A (en) | 2018-12-29 | 2018-12-29 | A kind of cultural method of autologous peripheral blood lymphocyte |
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|---|---|---|---|---|
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| CN106434554A (en) * | 2016-08-31 | 2017-02-22 | 北京同立海源生物科技有限公司 | Preparation method of NK cells |
-
2018
- 2018-12-29 CN CN201811639114.5A patent/CN109593713A/en active Pending
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|---|---|---|---|---|
| US20020115214A1 (en) * | 1988-11-23 | 2002-08-22 | Carl H. June | Methods for selectively stimulating proliferation of t cells |
| US20030235908A1 (en) * | 2000-02-24 | 2003-12-25 | Xcyte Therapies, Inc. | Activation and expansion of cells |
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| Title |
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