CN114908057A - Method for efficiently amplifying NK cells in vitro by taking engineered erythrocytes as trophoblast cells - Google Patents
Method for efficiently amplifying NK cells in vitro by taking engineered erythrocytes as trophoblast cells Download PDFInfo
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- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 37
- 210000003743 erythrocyte Anatomy 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000000338 in vitro Methods 0.000 title claims abstract description 26
- 210000002993 trophoblast Anatomy 0.000 title claims abstract description 13
- 210000004027 cell Anatomy 0.000 claims abstract description 20
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 claims abstract description 6
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims description 8
- 230000004936 stimulating effect Effects 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 4
- 238000012986 modification Methods 0.000 claims description 4
- 210000005259 peripheral blood Anatomy 0.000 claims description 4
- 239000011886 peripheral blood Substances 0.000 claims description 4
- 230000001502 supplementing effect Effects 0.000 claims description 4
- 210000004748 cultured cell Anatomy 0.000 claims description 3
- 210000004700 fetal blood Anatomy 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 210000005087 mononuclear cell Anatomy 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 4
- 238000002360 preparation method Methods 0.000 abstract description 3
- 230000000638 stimulation Effects 0.000 abstract description 3
- 238000001415 gene therapy Methods 0.000 abstract description 2
- 108010052285 Membrane Proteins Proteins 0.000 abstract 1
- 102000018697 Membrane Proteins Human genes 0.000 abstract 1
- 150000001299 aldehydes Chemical class 0.000 abstract 1
- 230000003321 amplification Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 102000001398 Granzyme Human genes 0.000 description 2
- 108060005986 Granzyme Proteins 0.000 description 2
- 230000001461 cytolytic effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000003248 secreting effect Effects 0.000 description 2
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- 102000005598 Chondroitin Sulfate Proteoglycans Human genes 0.000 description 1
- 108010059480 Chondroitin Sulfate Proteoglycans Proteins 0.000 description 1
- -1 IL-15 Proteins 0.000 description 1
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229930192851 perforin Natural products 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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Abstract
A method for using engineered erythrocyte as trophoblast cell to efficiently amplify NK cell in vitro belongs to the field of cell gene therapy, and comprises the steps of using low-concentration glutaraldehyde to aldehyde erythrocyte surface protein, marking a plurality of different protein combinations on the surface of erythrocyte, including IL-15,4-1BB, CD335 monoclonal antibody, CD2 monoclonal antibody and the like, using the engineered erythrocyte as trophoblast cell to repeatedly stimulate PBMC in vitro to generate a large amount of NK cells, and using the engineered erythrocyte as trophoblast cell to efficiently amplify NK cell in vitro, wherein the method has the following advantages: firstly, the use of tumor cell lines such as K562 and the like is avoided, the preparation process of NK cells can be ensured to be safer, secondly, a large number of high-killing activity NK cells can be obtained in vitro by using the repeated stimulation of the engineered erythrocytes, and the number of the obtained NK cells can be increased by more than 30% compared with a factor using method. The method for preparing the safe and sufficient NK cells can better meet the clinical use requirement.
Description
Technical Field
The invention belongs to the field of biological medicine, in particular to the field of cell gene therapy.
Background
Natural Killer Cell NK cells are mainly characterized in that tumor cells and virus infected cells can be directly dissolved and destroyed without pre-stimulation, Cell membranes are degraded by secreting perforin, serine protease such as granzyme A and B, chondroitin sulfate proteoglycan and other molecules, the integrity of target cells is destroyed to play a cytolytic effect, the cytolytic effect on tumor cells without MHC molecule expression is good, the content of NK cells in human peripheral blood is very low (5-10%), and a powerful tool is provided for basic research and clinical application research by amplifying NK cells in vitro in a large scale. The existing in-vitro large-scale amplification method of the NK cells can be divided into a 'trophoblast method' and a 'factor method', wherein the trophoblast method is mainly characterized in that IL-15 and 41-BB genes are transferred into leukemia cell line K562 cells, and the cells are irradiated and then used as trophoblast cells for culturing the NK cells. The factor method uses her-2 monoclonal antibody IL-15 and IL-12 and other methods to efficiently induce the amplification of NK cells in vitro. At present, the two methods have some problems which influence the popularization and the application of clinical large-scale NK cell cells. In the trophoblast method, the tumor cell line K562 cells are used as trophoblast cells, and the cells are subjected to transgenic treatment, so that the ability of secreting factors is retained although the treated K562 cells lose amplification activity, more verification is needed in the aspect of clinical use safety, and in addition, the state of the trophoblast cells directly has a decisive influence on the culture effect of NK cells. The factor method is simple to use, but has the problem that the factor method is influenced by individual differences of samples, and the stable and consistent yield and purity of the NK cell amplification cannot be obtained in all samples.
Disclosure of Invention
The invention provides a method for efficiently amplifying NK cells in vitro by taking engineered erythrocytes as trophoblast cells, which is used for overcoming the defects in the prior art.
The invention is realized by the following technical scheme:
an engineered erythrocyte trophoblast cell for efficiently amplifying NK cells in vitro, which is characterized by comprising the following components in percentage by weight: the engineered erythrocyte is formed by modifying proteins such as IL-15,4-1BB, CD335 monoclonal antibody, CD2 monoclonal antibody and the like on the surface of the erythrocyte, and the engineered erythrocyte trophoblast cell can be used for repeatedly stimulating PBMC to obtain a large amount of high-purity NK cells in vitro.
The protein combination of the engineered erythrocyte surface modification comprises IL-15,4-1BB, CD335 monoclonal antibody and CD2 monoclonal antibody, and is prepared by the following method:
the method comprises the following steps: shaking fresh red blood cells with 0.025% glutaraldehyde at room temperature for 45 min;
step two: after being centrifugally washed by 300g of PBS solution for 8min, the mixture is respectively reacted with 10 mu g/ml of CD335 monoclonal antibody, 10 mu g/ml of IL-15, 5 mu g/ml of CD2 monoclonal antibody and 10 mu g/ml of 4-1BB for 2 hours;
step three: washing with PBS solution for 8min at 300g to obtain engineered erythrocyte, and storing the engineered erythrocyte trophoblast cell in 2-8 deg.C environment.
The engineered erythrocyte trophoblast cell can be used for repeatedly stimulating PBMC to obtain a large amount of high-purity NK cells in vitro, and the specific use method is as follows:
the method comprises the following steps: mononuclear cells were obtained from cord and peripheral blood as engineered erythrotrophoblast cells/MNC 2: 1, bottle-laying and culturing in proportion;
step two: and (3) supplementing a fresh culture solution every other day, and culturing on the 7 th day according to the ratio of the engineered erythrocyte trophoblast cells/MNC 2: 1 to re-stimulate the cultured cells of interest;
step three: supplementing liquid every other day, and culturing for 14-15 days to obtain target cells.
The invention has the advantages that: according to the invention, the preparation process of the NK cells can be ensured to be safer by avoiding using tumor cell lines such as K562 and the like, a large number of high-killing activity NK cells can be obtained in vitro by using the engineering erythrocytes for repeated stimulation, and the prepared safe and sufficient NK cells can better meet the clinical use requirements.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic diagram of a process for preparing engineered erythrotrophoblast cells of the present invention;
FIG. 2 is a schematic diagram of the in vitro NK cell efficient amplification process of the present invention;
FIG. 3 is a flow chart of the present invention for efficiently amplifying NK cell purity in vitro;
FIG. 4 is a graph showing the multiplication factor of NK cells efficiently amplified in vitro according to the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 preparation of engineered erythrocyte trophoblast cells for efficient in vitro expansion of NK cells:
the method comprises the following steps: shaking fresh red blood cells with 0.025% glutaraldehyde at room temperature for 45 min;
step two: after being centrifugally washed by 300g of PBS solution for 8min, the mixture is respectively reacted with 10 mu g/ml of CD335 monoclonal antibody, 10 mu g/ml of IL-15, 5 mu g/ml of CD2 monoclonal antibody and 10 mu g/ml of 4-1BB for 2 hours;
step three: washing with PBS solution for 8min at 300g to obtain engineered erythrocyte, and storing the engineered erythrocyte trophoblast cell in 2-8 deg.C environment.
Example 2
The specific using method of the high-purity NK cell in vitro obtained by repeatedly stimulating PBMC with the engineered erythrocyte trophoblast cell for efficiently amplifying the NK cell in vitro is as follows:
the method comprises the following steps: mononuclear cells were obtained from cord and peripheral blood and cultured for the first day according to the engineered erythrotrophoblast cells/MNC 2: 1, bottle-laying and culturing in proportion;
step two: fresh culture medium (containing IL-2500U/ml) is supplemented every other day, and the culture is carried out on the 7 th day according to the ratio of the engineered erythrocyte trophoblast cells/MNC 2: 1 to re-stimulate the cultured cells of interest;
step three: fresh culture solution (containing IL-2500U/ml) is supplemented every other day, and target cells are harvested on 14-15 days of culture.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (4)
1. A method for efficiently amplifying NK cells in vitro by taking engineered erythrocytes as trophoblast cells is characterized by comprising the following steps: the protein such as erythrocyte surface modification IL-15,4-1BB, CD335 monoclonal antibody and CD2 monoclonal antibody is used for forming the engineered erythrocyte trophoblast cell, and the engineered erythrocyte trophoblast cell can be used for repeatedly stimulating PBMC to obtain a large amount of high-purity NK cells in vitro.
2. The engineered erythrotrophoblast cell for efficient in vitro expansion of NK cells according to claim 1, wherein: the protein combination of the engineered erythrocyte surface modification comprises IL-15,4-1BB, CD335 monoclonal antibody and CD2 monoclonal antibody.
3. The engineered erythrotrophoblast cell for efficient in vitro expansion of NK cells according to claim 1, wherein: the engineered erythrocytes are hydroformylated using low concentrations of glutaraldehyde, and the surface-modified proteins are combined with IL-15,4-1BB, CD335 mab and CD2 mab, which are prepared by the following method:
the method comprises the following steps: shaking fresh red blood cells with 0.025% glutaraldehyde at room temperature for 45 min;
step two: after being centrifugally washed by 300g of PBS solution for 8min, the mixture is respectively reacted with 10 mu g/ml of CD335 monoclonal antibody, 10 mu g/ml of IL-15, 5 mu g/ml of CD2 monoclonal antibody and 10 mu g/ml of 4-1BB for 2 hours;
step three: washing with PBS solution for 8min at 300g to obtain engineered erythrocyte, and storing the engineered erythrocyte trophoblast cell in 2-8 deg.C environment.
4. The engineered erythrotrophoblast cells for efficiently expanding NK cells in vitro according to claim 1, which can be used for repeatedly stimulating PBMCs to obtain a large amount of high-purity NK cells in vitro, and the specific use method is as follows:
the method comprises the following steps: mononuclear cells were obtained from cord and peripheral blood as engineered erythrotrophoblast cells/MNC 2: 1, bottle-laying and culturing in proportion;
step two: and (3) supplementing a fresh culture solution every other day, and culturing on the 7 th day according to the ratio of the engineered erythrocyte trophoblast cells/MNC 2: 1 to re-stimulate the cultured cells of interest;
step three: supplementing liquid every other day, and culturing for 14-15 days to obtain target cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2024087760A1 (en) * | 2022-10-26 | 2024-05-02 | 北京睿脉医药科技有限公司 | Method for coupling therapeutic molecules to surfaces of mature erythrocytes and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2024087760A1 (en) * | 2022-10-26 | 2024-05-02 | 北京睿脉医药科技有限公司 | Method for coupling therapeutic molecules to surfaces of mature erythrocytes and use |
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