Method for in vitro expansion of cytokine-induced killer cells

Abstract

The present application relates to a method for in vitro expansion of cytokine-induced 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 interferon-gamma, a platelet-rich plasma activating solution (PRP+) and RPMI culture solution; culturing again in a second expansion culture solution, wherein the second expansion culture solution comprises interleukin-2, muromonab-CD3, the platelet-rich plasma activating solution (PRP+) and RPMI culture solution; and culturing again in a third expansion culture solution, wherein the third expansion culture solution comprises interleukin-2, the platelet-rich plasma activating solution (PRP+) and RPMI culture solution.

Description

Technical Field

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

[0002] Cytokine-induced killer cells (CIKs) are a group of immune cells that combine the characteristics of T cells and natural killer cells. They can effectively recognize and kill tumor cells. Because CIKs have anti-tumor activity, high cytotoxicity and relatively low autotoxicity, they are regarded as a potential immunotherapy tool in clinical practice.

[0003] CIK cells can be used in clinical trials for various tumors and have shown excellent efficacy. CIK cells are usually expanded and activated by peripheral blood mononuclear cells (PBMCs) or umbilical cord blood cells through specific cytokine induction. However, current in vitro expansion technology is difficult to efficiently expand CIK cells while maintaining their cytotoxic activity. Therefore, how to effectively expand the number of CIK cells and maintain their activity is an important issue that needs to be addressed in this field to achieve wider clinical application. Summary of the Invention

[0004] In view of this, the present invention provides a method for in vitro expansion of cytokine-activated killer cells, comprising:

[0005] Provide a peripheral blood mononuclear cell sample;

[0006] The peripheral blood mononuclear cell sample was cultured in a first amplification culture medium, wherein the first amplification culture medium included interferon-γ (IFNγ), a platelet-rich plasma activating medium (PRP+), and RPMI culture medium.

[0007] The culture was performed using a second amplification culture medium comprising interleukin 2 (IL-2), anti-CD3 monoclonal antibody (OKT3), platelet-rich plasma activation medium, and RPMI medium; and

[0008] The culture was carried out in a third amplification culture medium, which included interleukin-2 (IL-2), platelet-rich plasma activation medium, and RPMI culture medium.

[0009] In some specific embodiments, the platelet-rich plasma activating solution includes platelet growth factor and platelet exosomes.

[0010] In some specific embodiments, the interferon-γ concentration of the first amplification culture medium is 900 to 1100 U / mL and the concentration of the platelet-rich plasma activation medium is 9 to 11% (v / v).

[0011] In some specific embodiments, the first amplification culture medium is cultured for 1 to 2 days.

[0012] In some specific embodiments, the second amplification culture medium has an interleukin-2 concentration of 400 to 600 U / mL, a moromumab-CD3 concentration of 40 to 60 ng / mL, and the platelet-rich plasma activation medium has a concentration of 9 to 11% (v / v).

[0013] In some specific embodiments, the second amplification culture medium is cultured for 4 to 6 days.

[0014] In some specific embodiments, the concentration of interleukin-2 in the third amplification culture medium is 400 to 600 U / mL and the concentration of the platelet-rich plasma activation medium is 1 to 3% (v / v).

[0015] In some specific embodiments, the third amplification culture medium is cultured for 9 to 11 days.

[0016] In some specific embodiments, platelet exosomes are further added to the first amplification culture medium, the second amplification culture medium and the third 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 1 platelet 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] The beneficial effects of the in vitro expansion method for cytokine-induced killer cells according to the present invention are as follows:

[0025] The in vitro expansion method for cytokine-induced killer cells provided by this invention improves the expansion efficiency of cytokine-induced killer cells by adding platelet-rich plasma activating solution. At the same time, through toxicity test, it is confirmed that CIK cells expanded by the method described in this invention can still maintain their cytotoxic activity against tumor cells.

[0026] In clinical practice, the amplification technology of this invention can effectively obtain a sufficient quantity of cytokine-induced killer cells for patients to use, thereby facilitating the relevant treatment course. It has great potential for the future development of immune cell therapy. Attached Figure Description

[0027] Figure 1 This is a flowchart of the in vitro expansion method for cytokine-induced killer cells according to the present invention;

[0028] Figure 2 This is a flowchart illustrating the preparation process of the platelet-rich plasma activating solution of the present invention;

[0029] Figure 3A This is a percentage graph of proteins in platelet-rich activated plasma obtained by size-exclusion chromatography (SEC).

[0030] Figure 3B This is a graph showing the particle size and concentration analysis of fractions 1 to 8 after collection;

[0031] 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 FBS group (control group) of Example 2;

[0032] Figure 5 This is a graph showing the percentage of effector memory T cells (TEM) on day 16 of expansion culture in the PRP+ group (experimental group) and FBS group (control group) of Example 2.

[0033] Figure 6 This is a graph showing the cytotoxic activity of the PRP+ group (experimental group) and the FBS group (control group) in Example 3. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] In view of the shortcomings of current technology, this invention provides a method for in vitro expansion of killer cells induced by cytokines. Please refer to [link to relevant documentation]. Figure 1 It includes:

[0037] Provide a peripheral blood mononuclear cell sample (step 11);

[0038] The peripheral blood mononuclear cell sample was cultured in a first amplification culture medium, wherein the first amplification culture medium included interferon-γ, a platelet-rich plasma activating medium and RPMI culture medium (step 12).

[0039] The culture was carried out using a second amplification culture medium comprising interleukin-2, moromumab-CD3, the platelet-rich plasma activating medium, and RPMI culture medium (step 13); and

[0040] The culture was carried out in a third amplification culture medium, which included interleukin-2, platelet-rich plasma activation medium, and RPMI culture medium (step 14).

[0041] In some specific embodiments, the peripheral blood mononuclear cell sample is derived from the peripheral blood of a subject.

[0042] In some specific embodiments, the platelet-rich plasma activating solution includes platelet growth factor and platelet exosomes.

[0043] 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.

[0044] In some specific embodiments, the interferon-γ concentration of the first amplification culture medium is 900 to 1100 U / mL and the concentration of the platelet-rich plasma activation solution is 9 to 11% (v / v), for example: 900 U / mL, 920 U / mL, 940 U / mL, 960 U / mL, 980 U / mL, 1000 U / mL, 1020 U / mL, 1040 U / mL, 1060 U / mL. Interferon-γ at concentrations of 1080 U / mL or 1100 U / mL and platelet-rich plasma activation medium at concentrations of 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%. In some specific embodiments, the first amplification culture medium is cultured for 1 to 2 days.

[0045] In certain specific embodiments, the concentration of interleukin-2 in the second amplification culture medium is 400 to 600 U / mL, the concentration of moromumab-CD3 is 40 to 60 ng / mL, and the concentration of the platelet-rich plasma activating solution is 9 to 11% (v / v), for example: 400 U / mL, 420 U / mL, 440 U / mL, 460 U / mL, 480 U / mL, 500 U / mL, 520 U / mL, 540 U / mL, 560 U / mL, 580 U / mL, or 600 U / mL of interleukin-2, 40 ng / mL, 42 ng / mL, etc. Moromab-CD3 at concentrations of g / mL, 44 ng / mL, 46 ng / mL, 48 ng / mL, 50 ng / mL, 52 ng / mL, 54 ng / mL, 56 ng / mL, 58 ng / mL, or 60 ng / mL, and platelet-rich plasma activation medium at concentrations of 9%, 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%. In some specific embodiments, the second amplification culture medium is cultured for 4 to 6 days.

[0046] In certain specific embodiments, the concentration of interleukin-2 in the third amplification culture medium is 400 to 600 U / mL, and the concentration of the platelet-rich plasma activation solution is 1 to 3% (v / v), for example: 400 U / mL, 420 U / mL, 440 U / mL, 460 U / mL, 480 U / mL, 500 U / mL, 520 U / mL, 540 U / mL, 560 U / mL, 580 U / mL, or 600 U / mL of interleukin-2 and 1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3% of platelet-rich plasma activation solution. In some specific embodiments, the third amplification culture medium is cultured for 9 to 11 days.

[0047] In some specific embodiments, platelet exosomes are further added to the first, second, and third amplification culture media. In some specific embodiments, the platelet exosomes are prepared by particle size chromatography from platelet-rich plasma activation solution.

[0048] In some specific embodiments, the preparation of the platelet-rich plasma activating solution includes:

[0049] Centrifuge a whole blood sample at 700 to 800 × g for 3 to 5 minutes, and collect the supernatant to obtain plasma;

[0050] Centrifuge the plasma at 1000 to 2000 × g for 8 to 10 minutes to precipitate platelets, wherein the supernatant is platelet-deficient plasma;

[0051] Excess platelet-deficient plasma was removed and retained to adjust the platelet concentration to approximately 1 × 10⁻⁶ per milliliter. 9 1 platelet to obtain platelet-rich plasma;

[0052] Add CaCl2 solution to activate the platelet-rich plasma by shaking.

[0053] 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

[0054] 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.

[0055] In some specific embodiments, the volume ratio of the ischemic plasma to the supernatant is 4:6 to 6:4.

[0056] 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.

[0057] 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.

[0058] Similarly, the present invention provides a cytokine-induced killer cell, which is obtained by in vitro expansion method as described above.

[0059] 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.

[0060] 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.

[0061] 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 otherwise clearly indicates, the term “or” is used interchangeably with the terms “and / or.”

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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, the specific cells referred to are cytokine-induced killer cells.

[0066] 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 8cells / 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 9 cells / 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.

[0067] 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.

[0068] As used herein, the term "cytokine-induced killer cells" refers to cells produced by culturing peripheral blood lymphocytes (PBLs) with cytokines (such as IFN-γ, anti-CD3 antibodies, IL-2, and IL-1). These cells are heterogeneous and possess potent anti-tumor activity. The cells mentioned include, but are not limited to, natural killer T cells (labeled: CD3+ and CD56+), T cells (labeled: CD3+ and CD56-), and natural killer cells (labeled: CD3- and CD56+).

[0069] 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.

[0070] Example 1: Platelet-rich plasma activating solution (PRP+)

[0071] 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 platelet exosomes were further analyzed and prepared.

[0072] Preparation of platelet-rich plasma activation solution

[0073] 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:

[0074] Centrifuge a whole blood sample at 720×g for 5 minutes and collect the upper layer to obtain plasma (step 21);

[0075] Centrifuge the plasma at 1440×g for 10 minutes to precipitate platelets, wherein the supernatant is platelet-deficient plasma (step 22);

[0076] 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);

[0077] Add CaCl2 solution to activate the platelet-rich plasma by shaking (step 24);

[0078] 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

[0079] The mixed sample was further centrifuged at 2300×g for 5 minutes, and the supernatant was the platelet-rich plasma activation solution (step 26).

[0080] The platelet-rich plasma activation solution can be further freeze-dried to produce platelet-rich plasma cryo-crystals for preservation and subsequent use.

[0081] Analysis and preparation of platelet exosomes

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Example 2: Cytokine-induced expansion of killer cells

[0087] In this embodiment, the in vitro expansion method for cytokine-induced killer cells described in this invention was used to expand and culture peripheral blood mononuclear cell samples for 16 days. The expansion effect of cytokine-induced killer cells was analyzed by flow cytometry, and the proportion of effector memory T cells (labeled: CD45RO+ and CCR7-) in the total number of T cells was detected.

[0088] In this embodiment, peripheral blood mononuclear cells are isolated and purified from the blood sample of the subject, and then cytokine-induced killer cell amplification is performed. The amplification method includes:

[0089] Provide a peripheral blood mononuclear cell sample;

[0090] The peripheral blood mononuclear cell sample was cultured for 1 day in the first amplification culture medium of the present invention, wherein the first amplification culture medium includes 1000 U / mL of interferon-γ, 10% platelet-rich plasma activation medium and RPMI culture medium.

[0091] The culture was then carried out for 5 days using the second amplification culture medium of the present invention, which included 500 U / mL of interleukin-2, 50 ng / mL of moromumab-CD3, 10% platelet-rich plasma activation medium and RPMI culture medium.

[0092] The cells were then cultured for 10 days using the third amplification culture medium described in this invention. This third amplification culture medium includes 500 U / mL interleukin-2, 2% platelet-rich plasma activating solution, and RPMI culture medium. The preparation method of the platelet-rich plasma activating solution used in this embodiment is as described in Example 1.

[0093] In this embodiment, 2.4 × 10⁻⁶ cells were taken from a peripheral blood mononuclear cell sample on day 0 of the amplification culture. 7 Cells were cultured in the first expansion culture medium for one day to activate cytokines and induce killer cells; on day 1 of expansion culture, 1.6 × 10⁶ cells were taken from the activated sample. 7 Cells were cultured in a second expansion medium for 5 days to further activate cytokine-induced killer cells in the sample; on day 6 of expansion culture, they were cultured in a third expansion medium for 10 days to amplify the number of cytokine-induced killer cells, and cell counts and culture medium replenishment were performed on days 8, 10 and 13.

[0094] 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 FBS group (control group) had its platelet-rich plasma activating solution in the first, second, and third expansion culture media replaced with 10% fetal bovine serum (FBS) to compare the effect of platelet-rich plasma activating solution on the expansion of cytokines-induced killer cells.

[0095] In some specific embodiments, platelet exosomes may be additionally added to the first, second, and third amplification culture media. In some specific embodiments, the platelet exosomes are prepared as described in Example 1.

[0096] Please see Figure 4 On day 1 of expansion culture, the total number of cells in both the PRP+ group and the FBS group was 1.6 × 10⁻⁶. 7 However, after 16 days of expansion culture, the total number of cells in the PRP+ group increased to 3.8 × 10⁻⁶. 9 The number of cells increased by approximately 239 times, compared to 1.3 × 10⁶ cells in the FBS group. 9 The number of cells was only about 79-fold increased. The results show that the in vitro amplification method described in this invention can effectively amplify cytokine-induced killer cells, and adding platelet-rich plasma-activating medium to the amplification culture medium produces a more effective amplification effect compared to not adding it.

[0097] On the other hand, cytokine-induced killer cells mainly include natural killer T cells (labeled: CD3+ and CD56+) and T cells (labeled: CD3+ and CD56-). Therefore, in this embodiment, flow cytometry was used to perform more detailed sorting analysis on different labels. The results showed that after 16 days of expansion culture, the number of natural killer T cells in the PRP+ group increased from 1.92 × 10⁻⁶ before culture. 6 The amplification was 7.22 × 10⁻⁶. 8 The number of natural killer T cells in the FBS group was only 1.92 × 10⁻⁶ before culture. 6 The amplification was 4.68 × 10⁻⁶. 8 The number of T cells in the PRP+ group increased from 1.08 × 10⁶ before culture. 7 The amplification was 2.81 × 10⁻⁶. 9 The number of T cells in the FBS group was only 1.08 × 10⁶ compared to before culture. 7 The amplification was 8.19 × 10⁻⁶. 8 The results showed that the PRP+ group had better expansion of natural killer T cells and T cells than the FBS group, which also confirms that the in vitro expansion method described in this invention can effectively expand cytokine-induced killer cells.

[0098] In addition, after the cells were expanded and cultured, the proportion of effector memory T cells among the T cells was further detected by flow cytometry. Please refer to [link to relevant documentation]. Figure 5 In the PRP+ group, effector memory T cells accounted for 84% of all T cells, compared to only 66% in the FBS group. This demonstrates that adding platelet-rich plasma activating solution to induce cytokine-induced killer cell expansion can effectively increase the proportion of effector memory T cells. Effector memory T cells (TEM) can rapidly activate and initiate an immune response upon contact with the same antigen, significantly shortening the immune response time and exhibiting a longer lifespan. Therefore, a significant increase in effector memory T cells can effectively enhance the efficacy of cytokine-induced killer cells against antigens while maintaining a longer duration of effectiveness.

[0099] Example 3: Cytokines-induced cytotoxic activity of killer cells

[0100] In this embodiment, cytokine-induced killer cells cultured by the in vitro expansion method described in this invention are subjected to a cytotoxicity experiment to detect their cytotoxicity effect on antigens.

[0101] This embodiment is divided into a PRP+ group (experimental group) and an FBS group (control group). The amplification method for each group is as described in Example 2. The cytokine-induced killer cell cytotoxicity efficiency of each group is tested at an effector-to-target ratio (ETRatio) of 50:1, 25:1 and 12.5:1, respectively.

[0102] This embodiment uses IMDM culture medium ( The company (product number: 10-016-CM) formulated immune cells (CIK cells) to a cell density of 5×10⁻⁶. 6 Cells / mL, and after staining the target cells (K562 cells), they were cultured in IMDM medium to a cell density of 1×10⁻⁶ cells / mL. 5 Cells / mL were prepared according to the established effector-to-target ratio for each group, 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:

[0103]

[0104] Please see Figure 6 In the PRP+ group, the cytotoxic activity was 99.1%, 97.3%, and 85.6% in effector-to-target ratios of 50:1, 25:1, and 12.5:1, respectively, indicating that the in vitro expansion method described in this invention can effectively maintain the cytotoxic activity of cytokines-induced killer cells. On the other hand, compared with the FBS group, the cytotoxic activity was 87.7%, 61.5%, and 42.9% in effector-to-target ratios of 50:1, 25:1, and 12.5:1, respectively. The experimental results show that cytokines-induced killer cells expanded and cultured with platelet-rich plasma activation solution have better cytotoxic activity.

[0105] In summary, the in vitro expansion method for cytokine-induced killer cells described in this invention can effectively expand cytokine-induced killer cells in vitro and maintain their cytotoxic activity. In particular, after adding the platelet-rich plasma activating solution described in this invention, the cytokine-induced killer cells can be expanded more effectively, while increasing the proportion of effector memory T cells (TEM). This not only results in a longer cell half-life but also achieves better cytotoxic activity.

[0106] 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 killer cells induced by cytokines, 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 interferon-γ, a platelet-rich plasma activating medium, and RPMI culture medium. The culture was performed using a second amplification culture medium comprising interleukin-2, moromumab-CD3, the platelet-rich plasma activating medium, and RPMI medium; and The culture was carried out in a third amplification culture medium, which included interleukin-2, platelet-rich plasma activation medium, and RPMI culture 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 interferon-γ concentration in the first amplification culture medium is 900 to 1100 U / mL, and the concentration of the platelet-rich plasma activation solution 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 1 to 2 days.

5. The in vitro amplification method according to claim 1, characterized in that, The second amplification culture medium has an interleukin-2 concentration of 400 to 600 U / mL, a moromumab-CD3 concentration of 40 to 60 ng / mL, and a platelet-rich plasma activation solution concentration of 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 4 to 6 days.

7. The in vitro amplification method according to claim 1, characterized in that, The third amplification culture medium has an interleukin-2 concentration of 400 to 600 U / mL and the platelet-rich plasma activation solution has a concentration of 1 to 3% v / v.

8. The in vitro amplification method according to claim 1, characterized in that, The third amplification culture medium is cultured for 9 to 11 days.

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

10. 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 One platelet is collected 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.

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