Method for in vitro expansion of natural killer cells

Abstract

The present application relates to a method for in vitro expansion of natural killer cells, comprising: providing a peripheral blood mononuclear cell sample; culturing the peripheral blood mononuclear cell sample in a first expansion culture solution, wherein the first expansion culture solution comprises a platelet-rich plasma activating solution (PRP+) and an NKCC-1 culture solution; culturing the peripheral blood mononuclear cell sample in a second expansion culture solution, wherein the second expansion culture solution comprises the platelet-rich plasma activating solution (PRP+) and an NKCC-2 culture solution; and culturing the peripheral blood mononuclear cell sample in an NKCC-2 culture solution.

Description

Technical Field

[0001] This invention relates to an in vitro expansion method for natural killer cells, and more particularly to an in vitro expansion method for increasing the number and purity of natural killer cells from peripheral blood mononuclear cell samples. Background Technology

[0002] Natural killer cells (NK cells) are an important cell type in the innate immune system. They are large granular lymphocytes that differentiate from hematopoietic stem cells and are mainly distributed in peripheral blood and spleen. They have the ability to quickly identify and destroy abnormal cells, including virus-infected cells and tumor cells, and play an important role in the first line of immune defense.

[0003] NK cells determine whether to attack target cells by balancing the signals of surface activating receptors and surface inhibitory receptors. They can exert cytotoxicity on target cells by releasing intracellular toxic granules (such as perforin and granzymes). NK cells can also secrete cytokines such as interferon-γ to enhance the activity of macrophages and adaptive immune cells. In addition, NK cells have the ability to attack tumor cells, which makes them play an important role in the field of cancer treatment.

[0004] In clinical practice, NK cells are typically obtained from peripheral blood mononuclear cells (PBMCs) and expanded in vitro to increase their number for applications in cancer immunotherapy, chronic viral infection control, and post-transplant immune regulation. However, current NK cell expansion and culture techniques suffer from problems such as poor expansion efficiency, insufficient cell purity, and difficulty in maintaining viability. Therefore, how to effectively and stably expand the number of NK cells and maintain their viability is an important topic of ongoing research in this field to achieve wider clinical applications. Summary of the Invention

[0005] In view of this, the present invention provides a method for in vitro expansion of natural killer cells, comprising:

[0006] Provide a peripheral blood mononuclear cell sample;

[0007] The peripheral blood mononuclear cell sample was cultured in a first amplification culture medium, wherein the first amplification culture medium included a platelet-rich plasma activation medium and an NKCC-1 culture medium.

[0008] The culture was carried out using a second amplification culture medium, the second amplification culture medium comprising the platelet-rich plasma activation medium and NKCC-2 culture medium; and

[0009] Cultured in NKCC-2 medium.

[0010] In some specific embodiments, the platelet-rich plasma activating solution includes platelet growth factor and platelet exosomes.

[0011] In some specific embodiments, the concentration of the platelet-rich plasma activating solution in the first amplification culture medium is 9 to 11% (v / v).

[0012] In some specific embodiments, the first amplification culture medium is cultured for 5 to 7 days.

[0013] In some specific embodiments, the concentration of the platelet-rich plasma activating solution in the second amplification culture medium is 9 to 11% (v / v).

[0014] In some specific embodiments, the second amplification culture medium is cultured for 3 to 5 days.

[0015] In some specific embodiments, the NKCC-2 culture medium is cultured for 3 to 5 days.

[0016] In some specific embodiments, platelet exosomes are further added to the first amplification culture medium and the second amplification culture medium.

[0017] In some specific embodiments, the preparation of the platelet-rich plasma activating solution includes:

[0018] Centrifuge a whole blood sample at 720×g for 5 minutes and collect the supernatant to obtain plasma.

[0019] The plasma was centrifuged at 1440×g for 10 minutes to precipitate platelets, and the supernatant was platelet-deficient plasma.

[0020] Excess platelet-deficient plasma was removed and retained to adjust the platelet concentration to 1 × 10⁻⁶ / mL. 9 One platelet is collected to obtain platelet-rich plasma;

[0021] Add CaCl2 solution to activate the platelet-rich plasma by shaking.

[0022] Centrifuge the activated platelet-rich plasma at 2300×g for 5 minutes, collect the supernatant and mix it with an equal volume of the ischemic platelet plasma; and

[0023] The mixed sample was further centrifuged at 2300×g for 5 minutes, and the supernatant was the platelet-rich plasma activation solution.

[0024] In some specific embodiments, glass beads are further added to the CaCl2 solution for oscillation and activation.

[0025] In one aspect, the present invention provides a natural killer cell, which is obtained by in vitro expansion as described above.

[0026] The beneficial effects of the in vitro expansion method for natural killer cells of the present invention are as follows:

[0027] The in vitro expansion method for natural killer cells provided by this invention improves the expansion efficiency of natural killer cells by adding platelet-rich plasma activation solution. At the same time, through cytotoxicity experiments, it is confirmed that after expanding NK cells by the method of this invention, the expanded cells can still maintain their cytotoxic activity against tumor cells. Moreover, compared with traditional expansion methods, the cells obtained by the in vitro expansion method of this invention have stronger cytotoxic activity.

[0028] In clinical practice, the amplification technology of this invention can obtain a sufficient quantity of natural killer cells for patients to use, which is beneficial for the relevant treatment course and has great potential for the future development of immune cell therapy. Attached Figure Description

[0029] Figure 1 This is a flowchart of the in vitro expansion method for natural killer cells according to the present invention;

[0030] Figure 2 This is a flowchart illustrating the preparation process of the platelet-rich plasma activating solution of the present invention;

[0031] Figure 3A This is a percentage graph of proteins in platelet-rich activated plasma obtained by size-exclusion chromatography (SEC).

[0032] Figure 3B This is a graph showing the particle size and concentration analysis of fractions 1 to 8 after collection;

[0033] Figure 4 This is a graph showing the change in the total number of cells after amplification culture in the PRP+ group (experimental group) and Ctrl group (control group) of Example 2;

[0034] Figure 5 This is a graph showing the percentage of immune cells after amplification and culture in the PRP+ group (experimental group) and Ctrl group (control group) of Example 2;

[0035] Figure 6 This is a graph showing the cytotoxic activity of the PRP+ group (experimental group) and the Ctrl group (control group) in Example 3. Detailed Implementation

[0036] Other technical contents, features and effects of the present invention will be clearly presented in the following detailed description of the preferred embodiments with reference to the accompanying drawings.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] In view of the difficulty of efficiently expanding natural killer cells in vitro and maintaining their cytotoxic activity using existing technologies, this invention provides a method for expanding natural killer cells in vitro. Please refer to [link to relevant documentation]. Figure 1 It includes:

[0039] Provide a peripheral blood mononuclear cell sample (step 11);

[0040] The peripheral blood mononuclear cell sample was cultured in a first amplification culture medium, wherein the first amplification culture medium included a platelet-rich plasma activation medium and NKCC-1 culture medium (step 12).

[0041] The culture was carried out using a second amplification culture medium, which included the platelet-rich plasma activation medium and NKCC-2 culture medium (step 13); and

[0042] Cultured in NKCC-2 medium (step 14).

[0043] In some specific embodiments, the peripheral blood mononuclear cell sample is derived from the peripheral blood of a subject.

[0044] In some specific embodiments, the platelet-rich plasma activating solution includes platelet growth factor and platelet exosomes.

[0045] In some specific embodiments, the platelet growth factors in the platelet-rich plasma activation solution include, but are not limited to, platelet-derived growth factor (PDGF), transforming growth factor-Beta (TGF-β), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), and fibroblast growth factor (FGF). In some specific embodiments, the platelet exosomes in the platelet-rich plasma activation solution may be prepared using a platelet exosome process and then added additionally.

[0046] In some specific embodiments, the concentration of the platelet-rich plasma activating solution in the first amplification culture medium is 9 to 11% (v / v), for example: 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, or 11% platelet-rich plasma activating solution. In some specific embodiments, the first amplification culture medium is cultured for 5 to 7 days, for example: 5 days, 6 days, or 7 days.

[0047] In some specific embodiments, the concentration of the platelet-rich plasma activating solution in the second amplification culture medium is 9 to 11% (v / v), for example: 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, or 11% platelet-rich plasma activating solution. In some specific embodiments, the second amplification culture medium is cultured for 3 to 5 days, for example: 3 days, 4 days, or 5 days.

[0048] In some specific embodiments, the NKCC-2 culture medium is cultured for 3 to 5 days, for example: 3 days, 4 days or 5 days.

[0049] In some specific embodiments, the total culture time for the in vitro expansion method of natural killer cells described in this invention is 11 to 17 days, for example: 11 days, 12 days, 13 days, 14 days, 15 days, 16 days or 17 days.

[0050] In some specific embodiments, the first and second amplification cultures are further supplemented with platelet exosomes. In some specific embodiments, the platelet exosomes are prepared by particle size chromatography from platelet-rich plasma activation solution.

[0051] In some specific embodiments, the preparation of the platelet-rich plasma activating solution includes:

[0052] Centrifuge a whole blood sample at 700 to 800 × g for 3 to 5 minutes, and collect the supernatant to obtain plasma;

[0053] Centrifuge the plasma at 1000 to 2000 × g for 8 to 10 minutes to precipitate platelets, wherein the supernatant is platelet-deficient plasma;

[0054] Excess platelet-deficient plasma was removed and retained to adjust the platelet concentration to approximately 1 × 10⁻⁶ per milliliter. 9 One platelet is collected to obtain platelet-rich plasma;

[0055] Add CaCl2 solution to activate the platelet-rich plasma by shaking.

[0056] Centrifuge the activated platelet-rich plasma at 2000 to 3000 × g for 3 to 5 minutes, collect the supernatant and mix it with an equal volume of the ischemic platelet plasma; and

[0057] The mixed sample is further centrifuged at 2000 to 3000 × g for 3 to 5 minutes, and the supernatant is the platelet-rich plasma activation solution.

[0058] In some specific embodiments, the volume ratio of the ischemic plasma to the supernatant is 4:6 to 6:4.

[0059] In some specific embodiments, the volume of the platelet-rich plasma is 3 to 5 times the volume of the CaCl2 solution. In some specific embodiments, glass beads are further added to the CaCl2 solution for agitation and activation.

[0060] In some specific embodiments, the preparation of platelet-rich plasma activation solution further includes vacuum freeze-drying the platelet-rich plasma activation solution to obtain the platelet-rich plasma activation solution in powder form.

[0061] Similarly, the present invention provides a natural killer cell, which is obtained by in vitro expansion method as described above.

[0062] It should be understood that the foregoing general description and the following detailed description are exemplary and illustrative, but not intended to limit the claims of this invention. Certain details of one or more embodiments of the invention are set forth in the following description. Other features or advantages of the invention will be apparent from the following non-exhaustive list of representative embodiments, and also from the appended claims.

[0063] Unless otherwise defined below, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. References to techniques used herein are intended to indicate techniques commonly understood in the art, including variations or equivalents of those techniques or subsequently developed alternatives, as will be apparent to those skilled in the art.

[0064] It should be noted that, as used herein, the singular terms “a,” “an,” and “the” include multiple indicators unless explicitly limited to one. Unless the context clearly indicates otherwise, the term “” is used interchangeably with the terms “and / or”.

[0065] As used herein, the terms “about,” “approximately,” or “nearly” essentially mean that the stated value or range is within 3%, preferably within 1%, and more preferably within 0.5%. The numerical values ​​provided herein are approximate and are intended to be inferred even if the terms “about,” “approximately,” or “nearly” were not used.

[0066] As used herein, the term "comprising" is open-ended, indicating that such embodiments may include additional elements. Conversely, the term "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except trace impurities). The term "substantially consisting of" is partially closed-ended, indicating that such embodiments may also include elements that do not substantially alter the essential characteristics of such embodiments.

[0067] Unless otherwise defined herein, scientific and technical terms used in connection with this document should have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms should include plural terms, and plural terms should include singular terms. Generally, the nomenclature used to connect the following techniques, as well as techniques in biochemistry, enzyme science, molecular and cell biology, microbiology, immunology, protein and nucleic acid chemistry, and flow cytometry, are known and frequently used in the art. Unless otherwise stated, the methods and techniques of this invention can generally be performed according to conventional methods known in the art and are described in the various general and more specific references cited and discussed in this specification.

[0068] As used herein, the terms "NKCC-1 culture medium" and "NKCC-2 culture medium" are derived from the KBM NK Cell Culture Kit (product number: KJN-16030210) from Kohjin Bio. The NKCC-1 culture medium contains sodium selenite, potassium nitrate, cyanocobalamin, monoethanolamine, linoleic acid, and oleic acid. The NKCC-2 culture medium contains iron(II) sulfate heptahydrate, cobalt(II) chloride hexahydrate, hexaammonium heptamolybdate tetrahydrate, sodium selenite, and cyanocobalamin.

[0069] As used herein, the term "amplification" refers to any process that increases the number or purity of cells. As used herein, the term "amplification medium" refers to any culture medium used to amplify specific cells, including basal culture medium and specific active ingredients, in this case referring to natural killer cells.

[0070] As used herein, the term "platelet-rich plasma (PRP)" refers to the fraction of plasma obtained after centrifugation with a high platelet concentration, including but not limited to those with a platelet concentration of 5 × 10⁻⁶. 8 cells / mL ~5×10 9 cells / mL, for example 6 × 10 8 cells / mL, 7×10 8 cells / mL, 8×10 8 cells / mL, 9×10 8 cells / mL, 1×10 9 cells / mL, 2×10 9 cells / mL, 3×10 9 cells / mL, 4×10 9 cells / mL, 5×10 9cells / mL. As used herein, the term "platelet-poor plasma (PPP)" refers to the fraction of plasma with a lower platelet concentration obtained after centrifugation. As used herein, the terms "platelet-rich plasma activated solution," "PRP+," "PRP Plus," and "advanced PRP" are used interchangeably to refer to platelet-rich plasma that has undergone specific treatment. This process involves activation treatment (e.g., calcium chloride treatment) to release growth factors and other active substances from platelets, followed by centrifugation to collect the supernatant, which is then mixed with platelet-poor plasma. For specific preparation methods, please refer to Example 1.

[0071] As used herein, the term "peripheral blood mononuclear cell" (PBMC) refers to any cell in the blood, excluding bone marrow, that has a round nucleus, including but not limited to lymphocytes (such as T cells, B cells, and NK cells), monocytes, and dendritic cells, but excluding erythrocytes and platelets.

[0072] The present invention is further illustrated by the following embodiments, which should not be construed as further limiting in any way. The entire contents of all referenced documents (including references, approved patents, published patent applications, and co-filed patent applications) cited in this application are expressly incorporated herein by reference.

[0073] Example 1: Platelet-rich plasma activating solution (PRP+)

[0074] In this embodiment, after collecting whole blood samples from the subjects, platelet-rich plasma activation solution was prepared using a platelet-rich plasma process, and further analysis and preparation of platelet exosomes were carried out.

[0075] Preparation of platelet-rich plasma activation solution

[0076] Please see Figure 2 This is a flowchart illustrating the preparation method of the platelet-rich plasma activating solution of the present invention. The preparation method includes:

[0077] Centrifuge a whole blood sample at 720×g for 5 minutes and collect the upper layer to obtain plasma (step 21);

[0078] Centrifuge the plasma at 1440×g for 10 minutes to precipitate platelets, wherein the supernatant is platelet-deficient plasma (step 22);

[0079] Excess platelet-deficient plasma was removed and retained to adjust the platelet concentration to 1 × 10⁻⁶ / mL. 9 Count the platelets to obtain platelet-rich plasma (step 23);

[0080] Add CaCl2 solution to activate the platelet-rich plasma by shaking (step 24);

[0081] Centrifuge the activated platelet-rich plasma at 2300×g for 5 minutes, collect the supernatant and mix it with an equal volume of the platelet-deficient plasma (step 25); and

[0082] The mixed sample was further centrifuged at 2300×g for 5 minutes, and the supernatant was the platelet-rich plasma activation solution (step 26).

[0083] The platelet-rich plasma activation solution can be further freeze-dried to produce platelet-rich plasma cryo-crystals for preservation and subsequent use.

[0084] Analysis and preparation of platelet exosomes

[0085] In this embodiment, the platelet-rich activated plasma solution obtained by the above preparation method was subjected to size-exclusion chromatography (SEC) using a chromatography column (qEV2 Gen 2 column 70nm, purchased from IZON, New Zealand), and fractions 1 to 8 were collected, with each fraction having a volume of 1 mL and a total collected volume of 8 mL.

[0086] In this embodiment, the OD280 of each fraction was measured to confirm the separation and purification effect through protein content. Please refer to the results for further information. Figure 3A The protein content of fractions 1 to 8 accounted for less than 0.03% of the total protein content of the sample, indicating that fractions 1 to 8 have a high-purity separation effect and can effectively separate protein impurities.

[0087] Exosomes are extracellular vesicles (EVs) with particle sizes typically ranging from 30 to 150 nm. In this embodiment, the collected distillate was further analyzed to confirm the particle size and content. The results can be found in [link to relevant documentation]. Figure 3B The collected fractions (fractions 1 to 8) showed that the main particle size was concentrated between 50 and 200 nm, with a significant peak at 73 nm, indicating that the main component of the collected fractions was platelet exosomes. Quantitative calculations revealed that the concentration of platelet exosomes in the collected fractions was approximately 1.61 × 10⁻⁶ per milliliter. 12 indivual.

[0088] This embodiment confirms that the platelet-rich plasma activation solution contains platelet exosomes, and that high-purity and high-concentration platelet exosomes can be collected by particle size sieving chromatography.

[0089] Example 2: Expansion of Natural Killer Cells

[0090] In this embodiment, the in vitro expansion method for natural killer cells described in this invention was used to expand and culture peripheral blood mononuclear cell samples for 14 days. The expansion effect of natural killer cells (labeled: CD3- and CD56+) was analyzed by flow cytometry, and the proportions of T cells (labeled: CD3+ and CD56-), NKT cells (labeled: CD3+ and CD56+), and other cells (labeled: CD3- and CD56-) were detected.

[0091] In this embodiment, peripheral blood mononuclear cells are isolated and purified from the blood sample of the subject, and then natural killer cells are expanded. The expansion method includes:

[0092] Provide a peripheral blood mononuclear cell sample;

[0093] The peripheral blood mononuclear cell sample was cultured for 6 days in the first amplification culture medium of the present invention, wherein the first amplification culture medium includes 10% platelet-rich plasma activation medium and NKCC-1 culture medium.

[0094] The culture was then carried out for 8 days using the second amplification culture medium of the present invention, which includes 10% platelet-rich plasma activation medium and NKCC-2 culture medium; and

[0095] The platelets were then cultured in NKCC-2 medium. The preparation method of the platelet-rich plasma activating solution used in this example is as described in Example 1.

[0096] In this embodiment, 6.5 × 10⁶ cells were taken from a peripheral blood mononuclear cell sample on day 0 of the amplification culture. 6 Cells were cultured in the first expansion medium for 6 days to activate natural killer cells, and cell counts and culture medium were added on days 3 and 6. On day 6 of expansion culture, the culture medium was replaced with the second expansion medium for 4 days to further amplify the natural killer cells. Subsequently, the cells were cultured in NKCC-2 medium for 4 days, and cell counts and culture medium were added on days 8, 10 and 14 of expansion culture.

[0097] In this embodiment, peripheral blood mononuclear cell samples were divided into two groups for cell expansion culture. The PRP+ group (experimental group) was cultured using the aforementioned in vitro expansion method, while the Ctrl group (control group) had its platelet-rich plasma-activating medium in the first expansion culture medium replaced with 10% autologous serum (Auto Serum, AS), and its platelet-rich plasma-activating medium in the second expansion culture medium replaced with 10% autologous plasma (Auto Plasma, AP). This was to compare the effect of platelet-rich plasma-activating medium on the expansion effect of natural killer cells. The expansion method used in the Ctrl group is a conventional method for expanding natural killer cells.

[0098] In some specific embodiments, platelet exosomes may be additionally added to the first and second amplification cultures. In some specific embodiments, the platelet exosomes are prepared as described in Example 1.

[0099] Please see Figure 4 On day 0 of amplification culture, the total number of cells in both the PRP+ group and the Ctrl group was 6.5 × 10⁻⁶. 6 However, after 14 days of amplification culture, the total number of cells in the PRP+ group increased to 2.52 × 10⁻⁶. 9 The number of cells increased by approximately 388 times, while the total number of cells in the Ctrl group increased by 1.75 × 10⁶. 9 The number of cells was only increased by approximately 269 times, demonstrating that the in vitro amplification method described in this invention can effectively amplify the number of cells.

[0100] Additionally, please see Figure 5 On day 0 of the expansion culture, the number of natural killer cells in both the PRP+ group and the Ctrl group was 1.43 × 10⁻⁶. 6 The PRP+ group, comprising 22% of the total cells, expanded to 1.31 × 10⁻⁶ natural killer cells after 14 days of amplification culture. 9 The number of natural killer cells in the Ctrl group was 52%, representing approximately 916-fold expansion, compared to 7.17 × 10⁶ natural killer cells in the Ctrl group. 8 The number of cells was 41%, representing approximately 501-fold increase, indicating that the in vitro amplification method described in this invention has a better effect on the amplification of natural killer cells and can obtain natural killer cells with higher purity. The experimental results also confirmed that adding platelet-rich plasma activating solution to the amplification culture medium can achieve a more effective amplification effect.

[0101] Example 3: Toxicity of Natural Killer Cells

[0102] In this embodiment, natural killer cells cultured by the in vitro expansion method described in this invention are subjected to a cell cytotoxicity experiment to detect their cytotoxic effect on antigens.

[0103] This embodiment is divided into a PRP+ group (experimental group) and a Ctrl group (control group). The amplification method for each group is as described in Example 2. The cytotoxicity efficiency of natural killer cells in each group is tested at an effector-to-target ratio (ET ratio) of 0.5:1.

[0104] This embodiment uses IMDM culture medium ( The company (product number: 10-016-CM) prepared immune cells (NK cells) to a cell density of 5 × 10⁴ cells / mL, while target cells (K562 cells) were stained and then prepared in IMDM medium to a cell density of 1 × 10⁴ cells / mL. 5 The cells were prepared at a ratio of immune cells to target cells of 0.5:1 per milliliter, and then co-cultured at 37°C for 4 hours. The cytotoxicity of the cells was then analyzed by flow cytometry. The experiment was performed in triplicate (n=3). The formula for calculating cytotoxicity is as follows:

[0105]

[0106] Please see Figure 6 The cytotoxic activity of the PRP+ group was 59.1%, while that of the Ctrl group was only 51.1%. The experimental results show that the in vitro expansion method of natural killer cells described in this invention can effectively maintain the activity of natural killer cells, and the natural killer cells expanded and cultured with platelet-rich plasma activation solution have better cytotoxic activity.

[0107] In summary, the in vitro expansion method for natural killer cells described in this invention can effectively expand natural killer cells in vitro and maintain their cytotoxic activity. In particular, the addition of the platelet-rich plasma activating solution described in this invention can more effectively expand natural killer cells and improve their purity, while achieving better cytotoxic activity.

[0108] The present invention has been disclosed above through the above embodiments, which are only some preferred embodiments of the present invention. However, they are not intended to limit the present invention. Any equivalent changes or modifications made by any person skilled in the art after understanding the foregoing technical features and embodiments of the present invention without departing from the spirit and scope of the present invention shall still fall within the scope of the present invention. The patent protection scope of the present invention shall be determined by the claims appended to this specification.

Claims

1. A method for in vitro expansion of natural killer cells, characterized in that, include: Provide a peripheral blood mononuclear cell sample; The peripheral blood mononuclear cell sample was cultured in a first amplification culture medium, which included a platelet-rich plasma activation medium and an NKCC-1 culture medium. The culture was carried out using a second amplification culture medium, the second amplification culture medium comprising the platelet-rich plasma activation medium and NKCC-2 culture medium; and Cultured in NKCC-2 medium.

2. The in vitro amplification method according to claim 1, characterized in that, The platelet-rich plasma activation solution includes platelet growth factor and platelet exosomes.

3. The in vitro amplification method according to claim 1, characterized in that, The concentration of the platelet-rich plasma activating solution in the first amplification culture medium is 9 to 11% v / v.

4. The in vitro amplification method according to claim 1, characterized in that, The first amplification culture medium is cultured for 5 to 7 days.

5. The in vitro amplification method according to claim 1, characterized in that, The concentration of the platelet-rich plasma activating solution in the second amplification culture medium is 9 to 11% v / v.

6. The in vitro amplification method according to claim 1, characterized in that, The second amplification culture medium is cultured for 3 to 5 days.

7. The in vitro amplification method according to claim 1, characterized in that, The NKCC-2 culture medium was cultured for 3 to 5 days.

8. The in vitro amplification method according to claim 1, characterized in that, Platelet exosomes were further added to the first and second amplification culture media.

9. The in vitro amplification method according to claim 1, characterized in that, The preparation of the platelet-rich plasma activation solution includes: Centrifuge a whole blood sample at 720×g for 5 minutes and collect the supernatant to obtain plasma. The plasma was centrifuged at 1440×g for 10 minutes to precipitate platelets, and the supernatant was platelet-deficient plasma. Excess platelet-deficient plasma was removed and retained to adjust the platelet concentration to 1 × 10⁻⁶ / mL. 9 1 platelet to obtain platelet-rich plasma; Add CaCl2 solution to activate the platelet-rich plasma by shaking. Centrifuge the activated platelet-rich plasma at 2300×g for 5 minutes, collect the supernatant and mix it with an equal volume of the ischemic platelet plasma; and The mixed sample was further centrifuged at 2300×g for 5 minutes, and the supernatant was the platelet-rich plasma activation solution.

10. A natural killer cell, characterized in that, It is obtained by in vitro amplification method as described in any one of claims 1 to 9.

Images