CN117106718A - NK cell culture medium for targeting lung, culture method and application - Google Patents
NK cell culture medium for targeting lung, culture method and application Download PDFInfo
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- CN117106718A CN117106718A CN202311227614.9A CN202311227614A CN117106718A CN 117106718 A CN117106718 A CN 117106718A CN 202311227614 A CN202311227614 A CN 202311227614A CN 117106718 A CN117106718 A CN 117106718A
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Abstract
The invention discloses a lung-targeted NK cell culture medium, a culture method and application. The culture medium of the invention consists of basal culture medium and activating factors, wherein the activating factors comprise IL-7, SCF, IL-2, IL-15, IL-18, IL-21, L-ascorbic acid, hydrocortisone, betulinic acid, TLR-7 agonist, TLR-8 agonist, TLR-9 agonist and inactivated autologous plasma. The lung-targeting NK cell culture method comprises the following steps: firstly, coating a cell culture container by using a pre-coating liquid containing a stimulating factor; then inoculating peripheral blood mononuclear cells, adding the culture medium to adjust the cell concentration, and performing amplification culture twice to obtain the cell. The NK cell proportion obtained by the culture method is more than 90%, the proportion of targeted lung marker chemokine positive cells is more than 94%, and when the ratio of effector cells to target cells is 10:1, the in-vitro killing rate of A549 lung cancer cells is more than 90%.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a lung-targeted NK cell culture medium, a lung-targeted NK cell culture method and application of the lung-targeted NK cell culture medium.
Background
Natural killer cells (natural killer cell, NK) are important immune cells of the body, not only associated with anti-tumor, anti-viral infection and immunomodulation, but also in some cases involved in the development of hypersensitivity reactions and autoimmune diseases, capable of recognizing and killing target cells.
NK cells have anti-tumor, antiviral and anti-aging effects, and are hot spots for current research and application. Many improvements to NK cell culture techniques have been mainly put in increasing proliferation factors and killing activities of NK cells. In recent years, research discovers that NK cells have the functions of tissue residence and targeting different organs, and as the survival time of the NK cells in vivo is relatively short, the NK cells which can be cultured to target different organs can cause the cells to gather in specific organs, thereby improving the effect of treating tumors and virus infection of different organs. We screened a medium and culture method that targets NK cells in the lung by testing a number of different culture methods.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides an activation culture medium for improving the natural killer cell lung targeting, which can be helpful for improving the natural killer cell lung targeting.
The invention also provides a natural killer cell culture method for targeting the lung.
The invention also provides a natural killer cell for targeting the lung.
The invention also provides a natural killer cell culture method of the targeted lung or application of the natural killer cells in preparing medicaments for treating lung cancer.
The invention also provides application of the activation culture medium in NK cell culture or preparation of an NK cell culture reagent.
In a first aspect of the invention, an activation medium for increasing natural killer cell lung targeting is provided, the activation medium consisting of a basal medium and an activating factor, the activating factor comprising IL-7, SCF, IL-2, IL-15, IL-18, IL-21, L-ascorbic acid, hydrocortisone, betulinic acid, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist and inactivated autologous plasma.
The activation medium according to the embodiment of the invention has at least the following beneficial effects: the invention can obviously improve NK cells (CD 3) by reasonably matching IL-2, IL-15, IL-18, IL-21, L-ascorbic acid, hydrocortisone, betulinic acid, TLR-7 agonist, TLR-8 agonist, TLR-9 agonist and inactivated autologous plasma of IL-7 and SCF, and the prepared activation culture medium is used for peripheral blood mononuclear cell culture - CD56 + ) And the obtained NK cells (CD 3 - CD56 + ) Has excellent targeting to lung cancer cells, wherein the proportion of targeted lung marker chemokines (CCR 5 and CXCR 3) positive cells is more than 94 percent.
The terms "IL-2", "IL-7", "IL-15", "IL-18", "IL-21" as used herein refer to the cytokines interleukin-2, interleukin-7, interleukin-15, interleukin-18, interleukin-21 and derivatives thereof.
In some embodiments of the invention, the final concentration of IL-7 in the activation medium is 5000-10000U/mL, the final concentration of SCF is 5-50U/mL, the final concentration of IL-2 is 10-1000U/mL, the concentration of IL-15 is 5-50 ng/mL, the final concentration of IL-18 is 5-50 ng/mL, the final concentration of IL-21 is 5-50 ng/mL, the final concentration of L-ascorbic acid (LAA) is 5-50 μg/mL, the final concentration of hydrocortisone is 0.1-10 μM, the final concentration of betulinic acid is 0.1-10 μg/mL, the final concentration of TLR-7 agonist is 0.1-10 μM, the final concentration of TLR-8 agonist is 0.1-10 μM, the final concentration of TLR-9 agonist is 0.1-10 μM, and the final concentration of plasma is 5-10 μM (TLR-10 μM).
Preferably, the final concentration of IL-7 in the activation medium is 5000 to 8000U/mL, the final concentration of SCF is 10 to 50U/mL, the final concentration of IL-2 is 100 to 500U/mL, the concentration of IL-15 is 10 to 50ng/mL, the final concentration of IL-18 is 10 to 50ng/mL, the final concentration of IL-21 is 10 to 50ng/mL, the final concentration of L-ascorbic acid is 10 to 50 μg/mL, the final concentration of hydrocortisone is 2 to 10 μM, the final concentration of betulinic acid is 2 to 10 μM, the final concentration of TLR-7 agonist is 0.1 to 5 μM, the final concentration of the TLR-8 agonist is 0.1 to 5 μM, the final concentration of TLR-9 agonist is 0.1 to 5 μM, and the final concentration of the inactivated autologous plasma is 5 to 10% (v/v).
Preferably, the final concentration of IL-7 in the activation medium is 5000U/mL, the final concentration of SCF is 10U/mL, the final concentration of IL-2 is 200U/mL, the concentration of IL-15 is 20ng/mL, the final concentration of IL-18 is 20ng/mL, the final concentration of IL-21 is 20ng/mL, the final concentration of L-ascorbic acid is 20 μg/mL, the final concentration of hydrocortisone is 5 μM, the final concentration of betulinic acid is 5 μg/mL, the final concentration of TLR-7 agonist is 1 μM, the final concentration of TLR-8 agonist is 1 μM, the final concentration of TLR-9 agonist is 1 μM, and the final concentration of the inactivated autologous plasma is 5% (v/v).
In some embodiments of the invention, a final concentration of the inactivated autologous plasma of 5-10% (v/v) means that the volume fraction of the inactivated autologous plasma in the activation medium is 5-10%.
In some embodiments of the invention, the basal medium refers to a medium that provides basic nutrition and energy requirements to the cells being cultured, typically comprising nutrients such as inorganic salts, carbohydrates, amino acids, vitamins, and buffers. The basal medium of the present invention is a medium suitable for culturing NK cells.
In some preferred embodiments of the invention, the basal medium is a basal medium suitable for cell therapy applications, such as serum-free medium, more preferably serum-free, animal-derived material-free medium.
In some embodiments of the invention, the basal medium of the activation medium is SCGM medium. The SCGM culture medium is a serum-free culture medium without animal source substances, meets the GMP (Good Manufacturing Practice, pharmaceutical production quality control Specification) standard, and is suitable for cell culture of the invention.
In a second aspect of the present invention, there is provided a method of culturing natural killer cells targeted to the lung, comprising the steps of:
s1, coating a cell culture container by using a pre-coating liquid containing a stimulating factor;
s2, inoculating peripheral blood mononuclear cells into the cell culture container obtained in the step S1, adding the activation culture medium of the first aspect, and adjusting the final concentration of the peripheral blood mononuclear cells to be 3 multiplied by 10 6 ～5×10 6 Culturing for 15-17 days at a volume of one mL.
The culture method provided by the embodiment of the invention has at least the following beneficial effects:
(1) According to the culture method, human peripheral blood mononuclear cells are cultured in vitro for 16-18 days, so that NK cells can be amplified by 500-900 times, the proportion of NK cells reaches more than 90%, and the proportion of positive NK cells expressing targeted lung marker chemokines CCR5 and CXCR3 is more than 94%. In addition, the in vitro killing rate of NK cells to A549 lung cancer cells reaches more than 90 percent (the effect: target is 10:1)
(2) The natural killer cell culture method of the targeted lung is simple, does not need to design various fluid infusion culture mediums, and is convenient to operate and low in cost.
In some embodiments of the invention, in step S2, the activation medium is supplemented 2 to 3 times during the culturing period; in some preferred embodiments of the invention, the activation medium is supplemented twice.
In some embodiments of the invention, the first supplementation of the activation medium is a culture of days 3-4, and the final concentration of the peripheral blood mononuclear cells after supplementation of the activation medium is 1.5X10 6 ～3×10 6 And each mL.
In some embodiments of the invention, the second supplementation of the activation medium is from day 7 to day 8 of culture, after supplementation of the activation mediumThe final concentration of the peripheral blood mononuclear cells is 1.5X10 6 ～3×10 6 And each mL.
In some embodiments of the invention, the cell culture vessel may be replaced according to actual needs each time the activation medium is replenished, and in some preferred embodiments of the invention, the cell culture vessel may be replaced with a T175 flask when the activation medium is first replenished; the cell culture container may be replaced with a culture bag upon a second replenishment of the activation medium.
In some embodiments of the invention, the lung targeting natural killer cell culture method specifically comprises the following steps:
s1, coating a cell culture container by using a pre-coating liquid containing a stimulating factor;
s2, inoculating peripheral blood mononuclear cells into the cell culture container obtained in the step S1, adding the activation culture medium of the first aspect, and adjusting the final concentration of the peripheral blood mononuclear cells to be 3 multiplied by 10 6 ～5×10 6 Culturing the culture medium until the culture medium reaches 3 to 4 days;
s3, supplementing the activation culture medium and adjusting the final concentration of the peripheral blood mononuclear cells to be 1.5x10 6 ～3×10 6 Continuously culturing the culture medium in a T175 culture flask until the culture medium is 7-8 days;
s4, supplementing the activation culture medium and adjusting the final concentration of the peripheral blood mononuclear cells to be 1.5x10 6 ～3×10 6 And (3) culturing the natural killer cells in a culture bag until the culture bag is continued for 15-17 days, thus obtaining the natural killer cells targeting the lung.
In some embodiments of the invention, the stimulating factor comprises at least one of a CD16 antibody, fibronectin, NKp44 antibody, NKp46 antibody, CD137 antibody, and CD52 antibody.
The term "antibody" in the present invention includes glycosylated and non-glycosylated immunoglobulins relating to any isotype or subclass or antigen binding regions related to competition for specific binding with intact antibodies, including human, humanized, chimeric, multispecific, monoclonal, polyclonal antibodies and oligomers or antigen binding fragments thereof. Also included are proteins having antigen binding fragments or regions, such as Fab, fab ', F (ab') 2, fv, diabodies, fd, dAb, minibodies, single chain antibody molecules, complementarity Determining Region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides, which contain at least a portion of an immunoglobulin sufficient to confer binding to a specific antigen of a target polypeptide. The term "antibody" includes, but is not limited to, antibodies produced, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody.
In some embodiments of the invention, the final concentration of the CD16 antibody in the pre-coating solution is 1-50. Mu.g/mL, the final concentration of fibronectin is 1-50. Mu.g/mL, the final concentration of the NKp44 antibody is 1-50. Mu.g/mL, the final concentration of the NKp46 antibody is 1-50. Mu.g/mL, the final concentration of the CD137 antibody is 1-50. Mu.g/mL, and the final concentration of the CD52 antibody is 1-50. Mu.g/mL.
In some embodiments of the invention, the coating is for a period of 8 to 12 hours, preferably 8 hours.
In some embodiments of the invention, the coating temperature is 3 to 5 ℃, preferably 4 ℃.
In some embodiments of the invention, the cell culture vessel comprises at least one of a multi-well plate, a culture flask, a culture dish, and a culture bag.
In some embodiments of the invention, the multi-well plate comprises at least one of a 96-well plate, a 24-well plate, a 12-well plate, a 6-well plate; the culture bottle comprises at least one of a T25 culture bottle, a T75 culture bottle, a T175 culture bottle and a T225 culture bottle; the culture dish comprises a 100mm culture dish or a 150mm culture dish; the culture bag comprises at least one of a 100mL culture bag, a 200mL culture bag, a 500mL culture bag, a 1000mL culture bag and a 2L culture bag.
In the culture process, as the culture volume increases, the culture vessel can be replaced by a proper cell culture vessel according to the requirement so as to meet the cell growth condition.
In some embodiments of the invention, the peripheral blood mononuclear cells are derived from a human blood sample.
In some embodiments of the inventionIn an embodiment, the temperature of the culture is 36-38 ℃, and CO 2 The concentration is 4% -6% and the relative saturation humidity is 94% -98%.
Preferably, the temperature of the culture is 37 ℃, CO 2 The concentration was 5% and the relative saturation humidity was 95%.
The term "culturing" as used herein includes providing the chemical and physical conditions (e.g., temperature, gas), as well as growth factors, required for NK cell maintenance. Generally culturing NK cells involves providing NK cells with conditions for expansion (proliferation). Examples of chemical conditions that may support NK cell expansion include, but are not limited to, buffers, serum, nutrients, vitamins, antibiotics, cytokines, and other growth factors, which are periodically provided (or may be manually administered) in a cell culture medium suitable for NK cell expansion.
In a third aspect of the present invention, there is provided a lung-targeting NK cell obtained by the natural killer cell culturing method of the second aspect described above.
The NK cells according to the embodiment of the invention have at least the following beneficial effects: the proportion of NK cell targeted lung marker chemokines (CCR 5 and CXCR 3) positive cells obtained by the invention is more than 94%.
In a fourth aspect, the present invention provides an application of the natural killer cell targeted to the lung in preparing a medicament for treating lung cancer.
In a fifth aspect of the invention, there is provided the use of an activation medium according to the first aspect in NK cell culture or in the preparation of NK cell culture reagents.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a graph showing the growth of mononuclear cells according to examples and comparative examples of the present invention;
FIG. 2 is a cell morphology graph (scale 200 μm) of human Peripheral Blood Mononuclear Cells (PBMC) culture days 0, 3, 5, 7, 9, 11 and 16 according to example 1 of the present invention;
FIG. 3 is a graph showing the results of flow assay purity of NK cells at day 16 of culture in example 1 of the present invention;
FIG. 4 is a graph showing results of flow-through detection of NK cell expression targeting lung marker chemokines CCR5 and CXCR3 in example 1 and comparative example 1 of the present invention;
FIG. 5 is a graph showing the killing activity of NK cells of example 1 and comparative example 1 against A549 lung cancer cells of the present invention.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
The words "preferably," "more preferably," and the like in the present invention refer to embodiments of the invention that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
The embodiment provides a culture method of targeted lung NK cells, which comprises the following steps:
(1) Pretreatment of cell culture flasks
Phosphate buffer containing CD16 antibody, fibronectin (FN), NKp44 antibody, NKp46 antibody, CD137 antibody and CD52 antibody was added to T75 cell culture flask, and the liquid was allowed to spread well at the bottom of flask and kept at 4 ℃ overnight. Wherein the final concentration of CD16 antibody is 15. Mu.g/mL, the final concentration of fibronectin is 15. Mu.g/mL, the final concentration of NKp44 antibody is 15. Mu.g/mL, the final concentration of NKp46 antibody is 15. Mu.g/mL, the final concentration of CD137 antibody is 15. Mu.g/mL, and the final concentration of CD52 antibody is 15. Mu.g/mL.
(2) Isolation, activation and culture of peripheral blood mononuclear cells
And (3) carrying out differential centrifugation on 100ml of patient peripheral blood subjected to bacteria detection by a plate in a low-speed centrifugal machine at room temperature for 30 minutes to separate plasma and blood cells, uniformly mixing blood cell sediment with an equal volume of physiological saline, carrying out centrifugal separation by a Ficoll density gradient, washing by a buffer solution to obtain peripheral blood mononuclear cells (PBMC, wherein NK cells account for 7.41%), transferring upper plasma into a centrifuge tube, inactivating at 56 ℃ for 30 minutes, centrifuging at 4000rpm for 10 minutes, and taking the supernatant at 4 ℃ for later use (serving as inactivated autologous plasma for later use).
Inoculating Peripheral Blood Mononuclear Cells (PBMC) into T75 culture flask, adding appropriate amount of activating medium, standing at 37deg.C, and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
Wherein the PBMC inoculation concentration is 4.5X10 6 Each mL (total volume 10 mL) of the activation medium contained IL-7 at a concentration of 5000U/mL, SCF at a concentration of 10U/mL, IL-2 at a concentration of 200U/mL, IL-15 at a concentration of 20ng/mL, IL-18 at a concentration of 20ng/mL, IL-21 at a concentration of 20 μg/mL, LAA at a concentration of 20 μg/mL, hydrocortisone at a concentration of 5 μM, betulinic acid at a concentration of 5 μg/mL, TLR-7 agonist at a concentration of 1 μM, TLR-8 agonist at a concentration of 1 μM, TLR-9 agonist at a concentration of 1 μM, and inactivated autologous plasma at a concentration of 5%.
(3) Liquid supplementing culture on day 3
After 3 days of culture, the mononuclear cells are transferred into an uncoated T175 culture flask, and a proper amount of activating culture medium is supplemented for continuous culture, and the final concentration of PBMC is adjusted to be 3 multiplied by 10 6 And is kept at 37 ℃ and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
(4) Fluid infusion culture on day 7
After 7 days of culture, transferring the mononuclear cells into a culture bag, supplementing a proper amount of activating culture medium for continuous culture, and adjusting the final concentration of PBMC to 3×10 6 And is kept at 37 ℃ and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
(5) Cell collection
After 16 days of culture, the culture is finished, cells are collected, and NK cell purity, cell proliferation amount, tumor killing capacity and the like are sampled and detected.
Cell numbers were measured on days 0, 3, 5, 7, 9, 11 and 16 of culture using a full-automatic counter, respectively, and growth curves were plotted as shown in fig. 1. The results show that: on day 16 of culture, the concentration of cells was as high as 260.26 ×10 6 And (3) each mL (all cells collected were counted in 10mL physiological saline for resuspension).
Further, NK cells (CD 3) in cells collected after 16 days of culture were detected by a flow cytometry - CD56 + ) As shown in fig. 3, the results of the measurement showed that the NK cells in the collected cells were 91.2% in percentage, and the NK cells in the collected cells were amplified about 712 times compared to the NK cells in the initial peripheral blood PBMC at the time of inoculation (7.41%).
Example 2
The embodiment provides a culture method of targeted lung NK cells, which comprises the following steps:
(1) Pretreatment of cell culture flasks
Phosphate buffer containing CD16 antibody, FN, NKp44 antibody, NKp46 antibody, CD137 antibody and CD52 antibody was added to T75 cell culture flask, and the liquid was allowed to spread well at the bottom of flask and kept flat overnight at 4 ℃. Wherein the final concentration of CD16 antibody is 15. Mu.g/mL, the final concentration of fibronectin is 15. Mu.g/mL, the final concentration of NKp44 antibody is 15. Mu.g/mL, the final concentration of NKp46 antibody is 15. Mu.g/mL, the final concentration of CD137 antibody is 15. Mu.g/mL, and the final concentration of CD52 antibody is 15. Mu.g/mL.
(2) Isolation, activation and culture of peripheral blood mononuclear cells
And (3) carrying out differential centrifugation on 100ml of patient peripheral blood subjected to bacteria detection by a plate in a low-speed centrifugal machine at room temperature for 30 minutes to separate plasma and blood cells, uniformly mixing blood cell sediment with an equal volume of physiological saline, carrying out centrifugal separation by a Ficoll density gradient, washing by a buffer solution to obtain peripheral blood mononuclear cells (PBMC, wherein NK cells account for 7.41%), transferring upper plasma into a centrifuge tube, inactivating at 56 ℃ for 30 minutes, centrifuging at 4000rpm for 10 minutes, and taking the supernatant at 4 ℃ for later use (serving as inactivated autologous plasma for later use).
Inoculating Peripheral Blood Mononuclear Cells (PBMC) into T75 culture flask, adding appropriate amount of activating medium, standing at 37deg.C, and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
Wherein the PBMC inoculation concentration is 3.5X10 6 personal/mL (total)Volume 10 mL), the activation medium contained IL-7 at 5000U/mL, SCF at 10U/mL, IL-2 at 200U/mL, IL-15 at 20ng/mL, IL-18 at 20ng/mL, IL-21 at 20ng/mL, L-ascorbic acid (LAA) at 20 μg/mL, hydrocortisone at 5 μM, betulinic acid at 5 μg/mL, TLR-7 agonist at 1 μM, TLR-8 agonist at 1 μM, TLR-9 agonist at 1 μM, and inactivated autologous plasma at 5%.
(3) Liquid supplementing culture on day 3
After 3 days of culture, transferring the mononuclear cells into a T175 culture flask, supplementing a proper amount of activating culture medium for continuous culture, and adjusting the final concentration of PBMC to 2×10 6 And is kept at 37 ℃ and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
(4) Fluid infusion culture on day 7
After 7 days of culture, transferring the mononuclear cells into a culture bag, supplementing a proper amount of activating culture medium for continuous culture, and adjusting the final concentration of PBMC to 2×10 6 And is kept at 37 ℃ and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
(5) Cell collection
After 16 days of culture, the culture is finished, cells are collected, and NK cell purity, cell proliferation amount, tumor killing capacity and the like are sampled and detected.
Cell numbers were measured on days 0, 3, 5, 7, 9, 11 and 16 of culture using a full-automatic counter, respectively, and growth curves were plotted as shown in fig. 1. The results show that: on day 16 of culture, the concentration of cells reached 186.045 ×10 6 Each mL (all cells collected were counted in 10mL saline for resuspension) with NK cell ratio 83.35% amplified approximately 597.9-fold compared to the time of inoculation (NK cell ratio 7.41% in initial peripheral blood PBMCs).
Example 3
The embodiment provides a culture method of targeted lung NK cells, which comprises the following steps:
(1) Pretreatment of cell culture flasks
Phosphate buffer containing CD16 antibody, FN, NKp44 antibody, NKp46 antibody, CD137 antibody and CD52 antibody was added to T75 cell culture flask, and the liquid was allowed to spread well at the bottom of flask and kept flat overnight at 4 ℃. Wherein the final concentration of CD16 antibody is 15. Mu.g/mL, the final concentration of fibronectin is 15. Mu.g/mL, the final concentration of NKp44 antibody is 15. Mu.g/mL, the final concentration of NKp46 antibody is 15. Mu.g/mL, the final concentration of CD137 antibody is 15. Mu.g/mL, and the final concentration of CD52 antibody is 15. Mu.g/mL.
(2) Isolation, activation and culture of peripheral blood mononuclear cells
And (3) carrying out differential centrifugation on 100ml of patient peripheral blood subjected to bacteria detection by a plate in a low-speed centrifugal machine at room temperature for 30 minutes to separate plasma and blood cells, uniformly mixing blood cell sediment with an equal volume of physiological saline, carrying out centrifugal separation by a Ficoll density gradient, washing by a buffer solution to obtain peripheral blood mononuclear cells (PBMC, wherein NK cells account for 7.41%), transferring upper plasma into a centrifuge tube, inactivating at 56 ℃ for 30 minutes, centrifuging at 4000rpm for 10 minutes, and taking the supernatant at 4 ℃ for later use (serving as inactivated autologous plasma for later use).
Inoculating Peripheral Blood Mononuclear Cells (PBMC) into T75 culture flask, adding appropriate amount of activating medium, standing at 37deg.C, and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
Wherein the PBMC inoculation concentration is 4.5X10 6 Each mL (total volume 10 mL) of the activation medium contained IL-7 at a concentration of 5000U/mL, SCF at a concentration of 10U/mL, IL-2 at a concentration of 200U/mL, IL-15 at a concentration of 20ng/mL, IL-18 at a concentration of 40ng/mL, IL-21 at a concentration of 40 μg/mL, LAA at a concentration of 40 μg/mL, hydrocortisone at a concentration of 10 μM, betulinic acid at a concentration of 10 μg/mL, TLR-7 agonist at a concentration of 2 μM, TLR-8 agonist at a concentration of 2 μM, TLR-9 agonist at a concentration of 2 μM, and inactivated autologous plasma at a concentration of 5%.
(3) Liquid supplementing culture on day 3
After 3 days of culture, the mononuclear cells are transferred into a T175 culture flask to be supplemented with a proper amount of activated cultureCulture medium is continued, and final concentration of PBMC is adjusted to 3×10 6 And is kept at 37 ℃ and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
(4) Fluid infusion culture on day 7
After 7 days of culture, transferring the mononuclear cells into a culture bag, supplementing a proper amount of activating culture medium for continuous culture, and adjusting the final concentration of PBMC to 3×10 6 And is kept at 37 ℃ and CO 2 Culturing in an incubator with a concentration of 5% and a relative saturation humidity of 95%.
(5) Cell collection
After 16 days of culture, the culture is finished, cells are collected, and NK cell purity, cell proliferation amount, tumor killing capacity and the like are sampled and detected.
Cell numbers were measured on days 0, 3, 5, 7, 9, 11 and 16 of culture using a full-automatic counter, respectively, and growth curves were plotted as shown in fig. 1. The results show that: on day 16 of culture, the concentration of cells reached 199.43 ×10 6 The number of cells per mL (total cells collected were resuspended in 10mL of physiological saline) at an NK cell ratio of 85.68% was approximately 512.4-fold amplified compared to the time of inoculation (NK cells in initial peripheral blood PBMC: 7.41%), which was reduced relative to the cell expansion amount of example 1, presumably with the possibility of overstimulation of the active factors in the activation medium, a higher fraction of the activation medium resulted in proliferation of other immune cells such as T cells, and eventually the NK cell purity and expansion were lower.
Comparative example 1
This comparative example differs from example 1 in that the active factors of the activation medium only comprise IL-2, IL-15, SCF and autologous plasma, wherein the concentration of SCF is 10U/mL, the concentration of IL-2 is 200U/mL, the concentration of IL-15 is 20ng/mL, the autologous plasma is 5%, the other conditions being the same.
Detection example 1: positive ratio detection of CCR5 and CXCR3
Experimental methods
The flow cytometry was used to collect samples from example 1 and comparative example 1, respectivelyNK cells (CD 3) - CD56 + ) The positive proportion of CCR5 and CXCR3 was detected. Wherein the negative control is isotype control antibody to which only antibodies (CCR 5, CXCR 3) were added to the sample.
(II) results of experiments
The results of the assay are shown in FIG. 4, in which NK cells (CD 3) after 16 days of culture were cultured by the method of example 1 - CD56 + ) Positive ratios of CCR5 and CXCR3 were 98.9% and 94.5%, respectively, whereas NK cells (CD 3) after 16 days of culture using the method of comparative example 1 - CD56 + ) The positive ratios of CCR5 and CXCR3 were 82.1% and 72.3%, respectively, which were far lower than the positive ratios of NK cells CCR5 and CXCR3 cultured by the method of example 1, demonstrating that NK cells cultured by the method of example 1 have better targeting for lung cancer cells than NK cells cultured by the method of comparative example 1.
Detection example 2: detection of killing power of lung cancer cells
Experimental methods
The logarithmic growth phase a549 lung cancer cells (purchased from cell bank of academy of sciences of china) and NK cells obtained in example 1, comparative example 1 were collected and subjected to the following treatment:
(1) Taking an A549 lung cancer cell as a target cell and an NK cell as an effector cell, digesting the A549 lung cancer cell growing in the logarithmic phase with 0.25% of pancreatin to prepare single cell suspension, dyeing and counting trypan blue, and then adjusting the density of the cells to 1X 105 cells/mL;
(2) A549 cell suspensions were added to 96-well plates, 50uL per well, effector cells (NK) were added at different potency/target ratios (1:1, 5:1, 10:1), again 50uL per well:
(3) Simultaneously setting up natural release holes of effector cells and target cells, maximum release holes of target cells and natural release holes of culture medium, correcting volume of control holes, wherein the volume of each hole is 100 mu L, and 3 compound holes are respectively set up;
(4) The cells were exposed to 5% CO at 37 ℃ 2 Incubating in an incubator for 12 hours, and adding 10uL of lysate into each of the maximum release holes of the target cells 45min before the reaction is finished;
(5) After the reaction is finished, 50uL of LDH enzyme reaction solution and 50uL of supernatant are sucked from each hole, the mixture is added into another new 96-well plate, the mixture is subjected to light-proof reaction for 30min at room temperature, 50uL of reaction stopping solution is added, and an enzyme-labeled instrument is used for detecting the OD value of the mixture;
(6) The killing activity was calculated according to the following formula: killing activity (%) = (assay tube OD value-target cell natural release tube OD value-effector cell natural release tube OD value)/(target cell maximum release tube OD value-target cell natural release tube OD value) ×100%.
(II) results of experiments
The killing effect of the NK cells obtained by the methods of the embodiment 1 and the comparative example 1 on the A549 lung cancer cells is shown in fig. 5, and the result shows that the NK cells obtained by the method of the embodiment 1 have better killing effect on the A549 lung cancer cells compared with the comparative example 1, wherein when the proportion of effector cell nucleus target cells is 10:1, the killing activity of the NK cells obtained in example 1 was 92.67%, and since examples 2 and 3 were identical to the medium composition used in example 1, they had comparable killing activity against a549 lung cancer cells.
In summary, after the activation culture medium and the cell culture method are adopted to co-culture human peripheral blood mononuclear cells in vitro for 2 weeks, NK cells can be amplified by 500-900 times, the proportion of the NK cells is more than 90%, the killing activity of the NK cells can be enhanced, the killing rate of the NK cells on A549 lung cancer cells is more than 90% (the effect: the target is 10:1), the NK cells obtained by the culture method have the characteristic of targeting lung, and the proportion of positive cells expressing targeted lung marker chemokines CCR5 and CXCR3 is more than 94%.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. An activation medium for increasing natural killer cell lung targeting, the activation medium comprising a basal medium and an activating factor, the activating factor comprising IL-7, SCF, IL-2, IL-15, IL-18, IL-21, L-ascorbic acid, hydrocortisone, betulinic acid, a TLR-7 agonist, a TLR-8 agonist, a TLR-9 agonist, and inactivated autologous plasma.
2. The activation medium of claim 1, wherein the final concentration of IL-7 in the activation medium is 5000-10000U/mL, the final concentration of SCF is 5-50U/mL, the final concentration of IL-2 is 10-1000U/mL, the concentration of IL-15 is 5-50 ng/mL, the final concentration of IL-18 is 5-50 ng/mL, the final concentration of IL-21 is 5-50 ng/mL, the final concentration of L-ascorbic acid is 5-50 μg/mL, the final concentration of hydrocortisone is 0.1-10 μΜ, the final concentration of betulinic acid is 0.1-10 μg/mL, the final concentration of the IL-7 agonist is 0.1-10 μΜ, the final concentration of the IL-8 agonist is 0.1-10 μΜ, the final concentration of the TLR-9 agonist is 0.1-10 μΜ, and the final concentration of the autologous plasma is 5-10 v% (TLR v/v).
3. The activation medium of claim 1, wherein the basal medium of the activation medium is SCGM medium.
4. A method of culturing a lung-targeted natural killer cell, comprising the steps of:
s1, coating a cell culture container by using a pre-coating liquid containing a stimulating factor;
s2, inoculating peripheral blood mononuclear cells into the cell culture container obtained in the step S1, adding the activation medium according to any one of claims 1-3, and adjusting the final concentration of the peripheral blood mononuclear cells to 3X 10 6 ～5×10 6 Culturing for 15-17 days at a volume of one mL.
5. The method of claim 4, wherein the stimulating factor comprises at least one of a CD16 antibody, fibronectin, NKp44 antibody, NKp46 antibody, CD137 antibody, and CD52 antibody;
preferably, the final concentration of the CD16 antibody in the pre-coating solution is 1-50. Mu.g/mL, the final concentration of fibronectin is 1-50. Mu.g/mL, the final concentration of the NKp44 antibody is 1-50. Mu.g/mL, the final concentration of the NKp46 antibody is 1-50. Mu.g/mL, the final concentration of the CD137 antibody is 1-50. Mu.g/mL, and the final concentration of the CD52 antibody is 1-50. Mu.g/mL.
6. The method according to claim 4, wherein the time for coating is 8 to 12 hours;
preferably, the temperature of the coating is 3-5 ℃;
preferably, the cell culture vessel comprises at least one of a multi-well plate, a culture flask, a culture dish, and a culture bag.
7. The method according to claim 4, wherein the peripheral blood mononuclear cells are derived from a human blood sample; preferably, the temperature of the culture is 36-38 ℃, and CO 2 The concentration is 4% -6% and the relative saturation humidity is 94% -98%.
8. A lung-targeting NK cell obtained by the natural killer cell culture method of any one of claims 4 to 7.
9. Use of a culture method according to any one of claims 4 to 7 or a lung-targeting NK cell according to claim 8 for the preparation of a medicament for the treatment of lung cancer.
10. Use of an activation medium according to any one of claims 1 to 3 for NK cell culture or for the preparation of NK cell culture reagents.
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