CN111826350A - In-vitro culture method for improving activity of cord blood-derived NK cell - Google Patents
In-vitro culture method for improving activity of cord blood-derived NK cell Download PDFInfo
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Abstract
The invention discloses an in vitro culture method for improving the activity of cord blood-derived NK (natural killer) cells, which comprises the following raw materials: a biological safety cabinet, a flow cytometer, a horizontal temperature control centrifuge, a sterilization pot, a CO2 incubator, an inverted microscope, a three-classification blood analyzer, a cell counter, a pipettor, a-20 ℃ refrigerator, a 4 ℃ refrigerator, a 75cm2 culture bottle, a 225cm2 culture bottle, a 2L culture bag, a 10ml pipette, a 50ml high-efficiency centrifuge tube, 15ml and 50ml centrifuge tubes, 250ml centrifuge tubes, medical sterile gloves, disposable sterile caps and sterile gloves, a serum-free culture medium Lonzax-VO VI 15, recombinant interleukin 2, recombinant interleukin 15, 0.4% trypan blue solution, normal saline, human lymphocyte separation fluid, gamma interferon and recombinant interleukin 1 alpha. The method has the advantages that the cost for culturing the NK cells is low, the proliferation speed is high by 13-15 days, the multiplication factor is high by about 270 times, the cell activity is high, and the cell phenotype is 91% because the cord blood NK cells are cultured by adding the mushroom polysaccharide to the related cell factors.
Description
Technical Field
The invention relates to the technical field of natural killer cells, in particular to an in-vitro culture method for improving the activity of cord blood-derived NK (natural killer) cells.
Background
Natural killer cells are important immune cells of the body, are not only related to tumor resistance, virus infection resistance and immune regulation, but also participate in the occurrence of hypersensitivity reaction and autoimmune diseases under certain conditions, and can recognize target cells and kill mediators.
The exact source of NK cells is not well understood and is generally considered to be derived directly from the bone marrow, the developmental maturation of which is dependent on the microenvironment of the bone marrow. Mouse and human in vitro experiments show that the culture of thymocytes in the presence of cytokines such as IL-2 in vitro can also induce NK cells. The spleen of the mouse can promote the differentiation of NK cells under the induction of IL-3 in vivo. NK cells are mainly distributed in peripheral blood and account for 5-10% of PBMC, and have NK activity in lymph nodes and bone marrow, but the level of the NK cells is lower than that of the peripheral blood.
Because the NK cell source and culture time problems cause higher culture cost and slower propagation speed, and the cultured cell activity is insufficient, an in vitro culture method for improving the activity of the NK cell from cord blood is provided to solve the problems.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides an in vitro culture method for improving the activity of cord blood-derived NK cell cells, and solves the problems.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: an in vitro culture method for improving the activity of cord blood-derived NK cell cells comprises the following raw materials: a biological safety cabinet, a flow cytometer, a horizontal temperature control centrifuge, a sterilization pot, a CO2 incubator, an inverted microscope, a three-classification blood analyzer, a cell counter, a pipettor, a-20 ℃ refrigerator, a 4 ℃ refrigerator, a 75cm2 culture bottle, a 225cm2 culture bottle, a 2L culture bag, a 10ml pipette, a 50ml high-efficiency centrifuge tube, 15ml and 50ml centrifuge tubes, 250ml centrifuge tubes, medical sterile gloves, disposable sterile caps and sterile gloves, a serum-free culture medium Lonzax-VO VI 15, recombinant interleukin 2, recombinant interleukin 15, 0.4% trypan blue solution, physiological saline, human lymphocyte separation fluid, gamma interferon, recombinant interleukin 1 alpha, recombinant interleukin 18, lentinan and cord blood;
a culture method of NK cells cultured in vitro from human umbilical cord blood sources comprises the following steps:
(1) centrifuging to separate cord blood with density gradient, adding gamma interferon, recombinant interleukin 15, recombinant interleukin 1 alpha, recombinant interleukin 18, recombinant interleukin 2, and lentinan to perform induced amplification culture on cord blood CBMC.
(2) And (5) culturing for a certain time, collecting NK cells, and detecting the NK cells.
Preferably, in the step (1), the cord blood treatment step comprises the following steps of: subpackaging the cord blood into 50ml centrifuge tubes, centrifuging by a centrifuge at 800g for 10 minutes, sucking the plasma layer of the centrifuged cord blood into a new 50ml centrifuge tube, placing at 56 ℃, and inactivating for 30 min; inactivated plasma, 1200g, 10 min; diluting the cell layer after the cord blood centrifugation by one time with physiological saline; using Ficoll and a 50ml high-efficiency separation tube, carrying out density gradient centrifugation, 800g, and 30 minutes; sucking the leucocyte layer into a new 50ml centrifuge tube by a pipette, washing the cells twice by normal saline, wherein the first time is 800g and the first time is 6 min; the second time is 600g, 6 min; resuspending the cell suspension in 20ml serum-free medium with cell density of 0.5-3.0 x 106/ml, and simultaneously supplementing 5% autologous plasma, 100 and 3000U/ml recombinant interleukin 2, 1-100ng/ml recombinant interleukin 15, 50-2000U/ml gamma interferon, 0.01-10n/ml recombinant interleukin 1 alpha, 0.1-20ng/ml recombinant interleukin 18; the cells were cultured in a CO2 incubator.
Preferably, the operation of supplementing the blood is carried out on the cells of the blood, the steps are that the cells are cultured to the 5 th day, the growth state of the cells is observed under a microscope, the cells grow well, the culture solution turns yellow, NK culture medium is supplemented to 125ml, 5% plasma is supplemented at the same time, 100-3000U/ml recombinant interleukin 2, 1-100ng/ml recombinant interleukin 15, 0.1-20ng/ml recombinant interleukin 18 and 0.1-5ml lentinan are added, and if the growth is normal or poor, decrement amplification or delay amplification is considered.
Preferably, the cells after fluid infusion are bagged, and the specific steps are as follows, culturing to the 7 th day, observing larger colonies under a microscope, expanding the cell volume from 125mL to 500mL, transferring to a 2L culture bag, and supplementing 5% plasma (not adding after use), 100-3000U/mL recombinant interleukin 2, 1-100ng/mL recombinant interleukin 15, 0.1-20ng/mL recombinant interleukin 18, 0.1-10mL lentinan; on the 9 th day of culture, larger colonies can be observed under a microscope, the cell volume is expanded from 500mL to 1500mL, and 5% plasma (not added after use) is supplemented, 100-3000U/mL recombinant interleukin 2, 1-100ng/mL recombinant interleukin 15, 0.1-20ng/mL recombinant interleukin 18 and 0.1-20mL lentinan are added.
Preferably, the bagged cells are bagged, and the specific steps are as follows, the total volume is expanded to 2.5L on the 11 th day of culture, then 100-3000U/mL recombinant interleukin 2 is added, the total volume is expanded to 3L on the 13 th day of culture, then 100-3000U/mL recombinant interleukin 2 is added, the cell suspension is tested and endotoxin is detected on the 15 th day of cell culture, a small amount of cell suspension is respectively extracted from the culture bottle and the bag by a 5mL injector, the cell suspension is added into a positive-placed flat dish, the cell suspension is placed into a 35 ℃ constant-temperature biochemical culture box, the placing date and the culture article are recorded, and the cell suspension is inverted after the night and kept for 48 hours.
Preferably, under normal conditions, the cell suspension is harvested at 15 th and 16 th days, and data analysis is carried out, which can be correspondingly advanced or delayed according to actual conditions.
(III) advantageous effects
Compared with the prior art, the invention provides an in vitro culture method for improving the activity of cord blood-derived NK cell and a preparation process thereof, and the method has the following beneficial effects:
because the relevant cell factors are added with the mushroom polysaccharide to culture the cord blood NK cells, the method has low cost for culturing the NK cells, high proliferation speed of 13-15 days, high proliferation multiple of about 270 times, high cell activity and 91% of cell phenotype.
Drawings
FIG. 1 is a cell proliferation fold map of an in vitro culture method for increasing the activity of cord blood-derived NK cell cells according to the present invention;
FIG. 2 is a flow phenotype map of an in vitro culture method for enhancing cord blood-derived NK cell activity according to the present invention;
FIG. 3 is a flow-type phenotypic map parameter of an in vitro culture method for increasing the activity of cord blood-derived NK cell cells according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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.
An in vitro culture method for improving the activity of cord blood-derived NK cell cells comprises the following raw materials: a biological safety cabinet, a flow cytometer, a horizontal temperature control centrifuge, a sterilization pot, a CO2 incubator, an inverted microscope, a three-classification blood analyzer, a cell counter, a pipettor, a-20 ℃ refrigerator, a 4 ℃ refrigerator, a 75cm2 culture bottle, a 225cm2 culture bottle, a 2L culture bag, a 10ml pipette, a 50ml high-efficiency centrifuge tube, 15ml and 50ml centrifuge tubes, 250ml centrifuge tubes, medical sterile gloves, disposable sterile caps and sterile gloves, a serum-free culture medium Lonzax-VO VI 15, recombinant interleukin 2, recombinant interleukin 15, 0.4% trypan blue solution, normal saline, human lymphocyte separation fluid, gamma interferon, recombinant interleukin 1 alpha, recombinant interleukin 18, lentinan and cord blood.
A culture method of NK cells cultured in vitro from human umbilical cord blood sources comprises the following steps:
(1) centrifuging to separate cord blood with density gradient, adding gamma interferon, recombinant interleukin 15, recombinant interleukin 1 alpha, recombinant interleukin 18, recombinant interleukin 2, and lentinan to perform induced amplification culture on cord blood CBMC.
The cord blood treatment step in the step (1) is as follows: subpackaging the cord blood into 50ml centrifuge tubes, centrifuging by a centrifuge at 800g for 10 minutes, sucking the plasma layer of the centrifuged cord blood into a new 50ml centrifuge tube, placing at 56 ℃, and inactivating for 30 min; inactivated plasma, 1200g, 10 min; diluting the cell layer after the cord blood centrifugation by one time with physiological saline; using Ficoll and a 50ml high-efficiency separation tube, carrying out density gradient centrifugation, 800g, and 30 minutes; sucking the leucocyte layer into a new 50ml centrifuge tube by a pipette, washing the cells twice by normal saline, wherein the first time is 800g and the first time is 6 min; the second time is 600g, 6 min; resuspending the cell suspension in 20ml serum-free medium with cell density of 0.5-3.0 x 106/ml, and simultaneously supplementing 5% autologous plasma, 100 and 3000U/ml recombinant interleukin 2, 1-100ng/ml recombinant interleukin 15, 50-2000U/ml gamma interferon, 0.01-10n/ml recombinant interleukin 1 alpha, 0.1-20ng/ml recombinant interleukin 18; the cells were cultured in a CO2 incubator.
Culturing the cells to the 5 th day, observing the growth state of the cells under a microscope without abnormal conditions, ensuring that the cells grow well, enabling a culture solution to turn yellow, supplementing an NK culture medium to 125ml, simultaneously supplementing 5% of plasma, 100 + 3000U/ml of recombinant interleukin 2, 1-100ng/ml of recombinant interleukin 15, 0.1-20ng/ml of recombinant interleukin 18 and 0.1-10ml of lentinan, and considering decrement amplification or delay amplification if the growth is normal or poor.
Bagging the cells after fluid infusion, specifically comprising the following steps of culturing to the 7 th day, observing a larger colony under a microscope, expanding the cell volume from 125mL to 500mL, transferring to a 2L culture bag, supplementing 5% plasma (not added after use), 100-3000U/mL recombinant interleukin 2, 1-100ng/mL recombinant interleukin 15, 0.1-20ng/mL recombinant interleukin 18 and 0.1-10mL lentinan; on the 9 th day of culture, larger colonies can be observed under a microscope, the cell volume is expanded from 500mL to 1500mL, and 5% plasma (not added after use) is supplemented, 100-3000U/mL recombinant interleukin 2, 1-100ng/mL recombinant interleukin 15, 0.1-20ng/mL recombinant interleukin 18 and 0.1-20mL lentinan are added.
The bagged cells are bagged, and the specific steps are as follows, the total volume is expanded to 2.5L on the 11 th day of culture, then 100 plus 3000U/mL recombinant interleukin 2 is added, the total volume is expanded to 3L on the 13 th day of culture, then 100 plus 3000U/mL recombinant interleukin 2 is added, the cell suspension is tested and tested for endotoxin on the 15 th day of cell culture, a small amount of cell suspension is respectively extracted from the culture bottle and the bag by a 5mL injector, and then the cell suspension is added into a flat dish which is arranged upright, and then the cell suspension is placed into a constant temperature biochemical incubator with the temperature of 35 ℃, the placing date and the culture article are recorded, and the cell suspension is inverted after the night and kept for 48 hours.
(2) And (3) collecting NK cells after culturing for a certain time, detecting the NK cells, harvesting cell suspensions at 15 th and 16 th days under normal conditions, and analyzing data, wherein the data can be correspondingly advanced or delayed according to actual conditions.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (6)
1. An in vitro culture method for improving the activity of cord blood-derived NK (natural killer) cells is characterized by comprising the following raw materials: a biological safety cabinet, a flow cytometer, a horizontal temperature control centrifuge, a sterilization pot, a CO2 incubator, an inverted microscope, a three-classification blood analyzer, a cell counter, a pipettor, a-20 ℃ refrigerator, a 4 ℃ refrigerator, a 75cm2 culture bottle, a 225cm2 culture bottle, a 2L culture bag, a 10ml pipette, a 50ml high-efficiency centrifuge tube, 15ml and 50ml centrifuge tubes, 250ml centrifuge tubes, medical sterile gloves, disposable sterile caps and sterile gloves, a serum-free culture medium Lonzax-VO VI 15, recombinant interleukin 2, recombinant interleukin 15, 0.4% trypan blue solution, physiological saline, human lymphocyte separation fluid, gamma interferon, recombinant interleukin 1 alpha, recombinant interleukin 18, lentinan and cord blood;
a culture method of NK cells cultured in vitro from human umbilical cord blood sources comprises the following steps:
(1) centrifuging and separating cord blood by density gradient, and adding gamma interferon, recombinant interleukin 15, recombinant interleukin 1 alpha, recombinant interleukin 18, recombinant interleukin 2 and lentinan to perform induced amplification culture on cord blood CBMC;
(2) and (5) culturing for a certain time, collecting NK cells, and detecting the NK cells.
2. The method for culturing in vitro the cells of NK derived from cord blood according to claim 1, wherein the cord blood treatment step in step (1) is as follows, and the blood is divided: subpackaging the cord blood into 50ml centrifuge tubes, centrifuging by a centrifuge at 800g for 10 minutes, sucking the plasma layer of the centrifuged cord blood into a new 50ml centrifuge tube, placing at 56 ℃, and inactivating for 30 min; inactivated plasma, 1200g, 10 min; diluting the cell layer after the cord blood centrifugation by one time with physiological saline; using Ficoll and a 50ml high-efficiency separation tube, carrying out density gradient centrifugation, 800g, and 30 minutes; sucking the leucocyte layer into a new 50ml centrifuge tube by a pipette, washing the cells twice by normal saline, wherein the first time is 800g and the first time is 6 min; the second time is 600g, 6 min; resuspending the cell suspension in 20-40ml serum-free medium with cell density of 0.5-3.0 x 106/ml, and simultaneously supplementing 5% autologous plasma, 100-3000U/ml recombinant interleukin 2, 1-100ng/ml recombinant interleukin 15, 50-2000U/ml gamma interferon, 0.01-10n/ml recombinant interleukin 1 alpha, 0.1-20ng/ml recombinant interleukin 18; the cells were cultured in a CO2 incubator.
3. The in vitro culture method for improving the activity of NK cells derived from cord blood as claimed in claim 2, wherein the operations of supplementing blood to the cells of the cord blood are performed by culturing the cells to the 5 th day, observing the growth state of the cells under microscope without abnormal condition, the cells grow well, the culture solution turns yellow, supplementing NK medium to 125ml, and supplementing 5% plasma, 100 and 3000U/ml recombinant interleukin 2, 1-100ng/ml recombinant interleukin 15, 0.1-20ng/ml recombinant interleukin 18, 0.1-5ml lentinan, and considering down-amplification or postponing amplification if the growth is normal or poor.
4. The in vitro culture method for improving the activity of NK cells derived from cord blood as claimed in claim 3, wherein the cells after fluid infusion are bagged, and the specific steps are that the culture is carried out to day 7, a larger colony can be observed under a microscope, the cell volume is enlarged from 125mL to 500mL, the cells are transferred to a 2L culture bag, and 5% plasma (not added after use) is supplemented, 100 + 3000U/mL recombinant interleukin 2, 1-100ng/mL recombinant interleukin 15, 0.1-20ng/mL recombinant interleukin 18, 0.1-10mL lentinan is added; on the 9 th day of culture, larger colonies can be observed under a microscope, the cell volume is expanded from 500mL to 1500mL, and 5% plasma (not added after use) is supplemented, 100-3000U/mL recombinant interleukin 2, 1-100ng/mL recombinant interleukin 15, 0.1-20ng/mL recombinant interleukin 18 and 0.1-20mL lentinan are added.
5. The in vitro culture method for improving the activity of NK cells derived from cord blood as claimed in claim 4, wherein the in vitro culture method for improving the activity of NK cells derived from cord blood comprises the following steps of bagging the bagged cells, expanding the total volume to 2.5L on the 11 th day of culture, adding 100 plus 3000U/mL recombinant interleukin 2, expanding the total volume to 3L on the 13 th day of culture, adding 100 plus 3000U/mL recombinant interleukin 2, culturing the cells for 15 th day, performing bacteria detection and endotoxin detection on the cell suspension, extracting a small amount of cell suspension from the culture bottle and the bag by using a 5mL syringe, adding the cell suspension into a positive plate, placing the cell into a 35 ℃ constant temperature biochemical incubator, recording the placing date and culture articles, and inverting after overnight preservation for 48 hours.
6. The method of claim 1, wherein the cell suspension is harvested at 15 or 16 days under normal conditions, and the data analysis is performed, which can be advanced or delayed according to actual conditions.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114395527A (en) * | 2021-12-20 | 2022-04-26 | 中国人民解放军联勤保障部队第九二〇医院 | NK cell culture medium and method for in-vitro amplification of NK cells |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999045102A1 (en) * | 1998-03-03 | 1999-09-10 | Institut Gustave Roussy | Method for activating natural killer (nk) cells |
| CN105238752A (en) * | 2015-10-22 | 2016-01-13 | 苏州科贝生物技术有限公司 | Culture system and culture method for efficient amplification in vitro of autologous NK cells |
| CN107460167A (en) * | 2017-09-21 | 2017-12-12 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of amplification method of the NK cells of panoistic cell |
| CN107574148A (en) * | 2016-07-05 | 2018-01-12 | 拜西欧斯（北京）生物技术有限公司 | A kind of NK (NK cells) culture medium and preparation method thereof |
| CN108893443A (en) * | 2018-07-24 | 2018-11-27 | 陕西九州细胞基因工程有限公司 | A kind of Efficient amplification method of cytokine induction umbilical cord blood natural killer |
| CN109294984A (en) * | 2018-10-25 | 2019-02-01 | 湖北创力药业有限公司 | A kind of Lentinan and preparation method thereof of internal efficient amplification NK cell |
-
2019
- 2019-04-17 CN CN201910308308.5A patent/CN111826350A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999045102A1 (en) * | 1998-03-03 | 1999-09-10 | Institut Gustave Roussy | Method for activating natural killer (nk) cells |
| CN105238752A (en) * | 2015-10-22 | 2016-01-13 | 苏州科贝生物技术有限公司 | Culture system and culture method for efficient amplification in vitro of autologous NK cells |
| CN107574148A (en) * | 2016-07-05 | 2018-01-12 | 拜西欧斯（北京）生物技术有限公司 | A kind of NK (NK cells) culture medium and preparation method thereof |
| CN107460167A (en) * | 2017-09-21 | 2017-12-12 | 山东省齐鲁细胞治疗工程技术有限公司 | A kind of amplification method of the NK cells of panoistic cell |
| CN108893443A (en) * | 2018-07-24 | 2018-11-27 | 陕西九州细胞基因工程有限公司 | A kind of Efficient amplification method of cytokine induction umbilical cord blood natural killer |
| CN109294984A (en) * | 2018-10-25 | 2019-02-01 | 湖北创力药业有限公司 | A kind of Lentinan and preparation method thereof of internal efficient amplification NK cell |
Non-Patent Citations (2)
| Title |
|---|
| 夏大静等: "IL-12联合小剂量IL-2对人外周血单个核细胞增殖及杀瘤活性的影响" * |
| 张晓明等: "白细胞介素2和黄芪多糖对人NK细胞活性和增殖的影响" * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114395527A (en) * | 2021-12-20 | 2022-04-26 | 中国人民解放军联勤保障部队第九二〇医院 | NK cell culture medium and method for in-vitro amplification of NK cells |
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