Amplification cryopreservation method of natural killer cells
By using activation and expansion medium combined with monoclonal antibodies and cytokines, combined with frozen CS10 and human albumin, the problem of poor recovery of natural killer cells after frozen survival was solved, and efficient cell freezing and resuscitation was achieved.
Patent Information
- Application Number
- CN202510566567.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-05
AI Technical Summary
In the prior art, it is difficult to recover the viability and anti-tumor activity of fresh cells after cryopreservation after low temperature, and existing frozen liquids such as CS10 are not effective in the frozen NK cells.
Natural killer cells are activated and expanded cultured by using activation culture medium containing suitable monoclonal antibodies and cytokines. After the cell amplification culture culture, the frozen solution of CS10 and human albumin is frozen, including pre-coated culture containers with CD52 monoclonal antibody, and the addition of cytokines such as IL-2, IL-12, IL-15, and IL-18 for activation and expansion culture, and then frozen with CS10 and human albumin.
It realizes that natural killer cells can still restore high viability and high killer activity after freezing at low temperature, and improve the cell quality after freezing.
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Figure CN120424871A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of cell expansion technology, and in particular to a method for expanding and freezing natural killer cells. Background Art
[0002] Natural killer (NK) cells are the body's first line of defense against infection and tumors. They can directly kill tumor cells, virus-infected cells, and damaged cells without being restricted by the major histocompatibility complex (MHC). They also secrete multiple cytokines and participate in the regulation of adaptive immunity. Clinical trials have demonstrated that natural NK cells are highly effective in treating solid tumors.
[0003] Rapid in vitro expansion to obtain large numbers of activated NK cells is key to the clinical application of NK cell therapy for malignant tumors. However, NK cells, as a rare cell subset among lymphocytes, only account for 5%-15% of human peripheral blood lymphocytes. Although NK cell expansion is high when genetically modified K562 cells are used as trophoblast cells for NK cell culture, K562, as a cancer cell line, needs to be irradiated before NK cell culture, and its safety is always not guaranteed. Therefore, the exploration of safer and more efficient NK cell expansion methods in vitro is the basis for its clinical application. In addition to trophoblast cells, antibody-coupled cytokine methods are also widely used for in vitro NK cell expansion. However, this method currently has low expansion efficiency, with expansion multiples ranging from 200-500 times, and poor stability. In summary, how to achieve large-scale expansion and culture of NK cells with high purity and high anti-tumor activity in vitro is currently a key issue in NK cell therapy.
[0004] In addition to the aforementioned challenges in NK cell expansion and culture, cryopreservation of NK cells is a key challenge in the clinical treatment of malignant tumors. CS10 is a serum-free, animal component-free cryopreservation solution containing 10% DMSO. This cryopreservation solution has been widely used for cryopreservation of immune cells and provides excellent cryopreservation protection.
[0005] While CS10-based cryopreservation solutions can be used to cryopreserve immune cells like CAR-T cells, with high viability and activity restored upon thawing, cryopreservation of NK cells faces significant challenges. Even with the addition of protective agents like albumin to CS10, NK cells rarely regain the viability and anti-tumor activity of fresh cells upon thawing after cryopreservation. Summary of the Invention
[0006] In view of the shortcomings of the existing technology, the purpose of the embodiments of the present application is to provide a method for expanding and freezing natural killer cells to improve the current problem that natural killer cells are difficult to recover to the viability and anti-tumor activity of fresh natural killer cells after being frozen at low temperatures.
[0007] In a first aspect, embodiments of the present application provide a method for expanding and cryopreserving natural killer cells, comprising:
[0008] (1) pre-coating a culture container with a first monoclonal antibody and a second monoclonal antibody, wherein the first monoclonal antibody includes CD52 monoclonal antibody;
[0009] (2) inoculating umbilical cord blood mononuclear cells into a pre-coated culture container, adding an activation medium for activation culture, wherein the activation medium includes a basal medium, a first cytokine, a second cytokine, a phytohemagglutinin, and nutrients, wherein the first cytokine includes IL-2;
[0010] (3) transferring the cells activated in step (2) into an expansion medium for expansion culture, wherein the expansion medium includes a basal medium and IL-2;
[0011] (4) adding cytokines to the cells expanded and cultured in step (3) and culturing for 1-2 days, and collecting the cells to obtain natural killer cells; wherein the cytokines include at least two of IL-12, IL-15, and IL-18;
[0012] (5) The natural killer cells collected in step (4) are rinsed with physiological saline, resuspended in a freezing solution to adjust the cell density, and then frozen; wherein the freezing solution includes CS10 and human serum albumin, the volume of CS10 is 35%-65%, and the concentration of human serum albumin is 0.05-0.12 g / mL.
[0013] The present application activates and expands natural killer cells using an activation medium and an expansion medium containing appropriately matched monoclonal antibodies and cytokines, thereby efficiently obtaining high-purity and high-activity natural killer cells. After cell expansion and culture, the cells are cultured for 1-2 days with appropriate cytokines. At the same time, the natural killer cells are cryopreserved using a freezing solution containing an appropriate concentration of CS10 and human serum albumin. After low-temperature cryopreservation and subsequent thawing, the natural killer cells can still recover high viability and high killing activity.
[0014] In some embodiments of the present application, the concentration of IL-12 added in step (4) is 20-150 ng / mL, the concentration of IL-15 is 20-150 ng / mL, and the concentration of IL-18 is 50-500 ng / mL.
[0015] The present application uses cytokines at appropriate concentrations to culture the expanded cells for 1-2 days, which facilitates the subsequent recovery of the cells after low-temperature freezing and still allows them to recover high viability and high killing activity.
[0016] In some embodiments of the present application, the freezing solution also includes physiological saline.
[0017] In some examples of this application, the cell density was adjusted to 5×10 7 -15×10 7 pieces / mL.
[0018] In some embodiments of the present application, the second monoclonal antibody includes at least one of CD16 monoclonal antibody, CD56 monoclonal antibody, CD137 monoclonal antibody, and NKG2D monoclonal antibody.
[0019] The present application uses at least one monoclonal antibody selected from CD16 monoclonal antibody, CD56 monoclonal antibody, CD137 monoclonal antibody, and NKG2D monoclonal antibody and CD52 monoclonal antibody in combination with a pre-coated culture container, thereby facilitating subsequent activation culture of umbilical cord blood mononuclear cells.
[0020] In some embodiments of the present application, the concentration of each of the first monoclonal antibody and the second monoclonal antibody is 0.5-5 μg / mL.
[0021] The present application uses a first monoclonal antibody and a second monoclonal antibody of appropriate concentrations to pre-coat a culture container, thereby facilitating subsequent activation and culture of umbilical cord blood mononuclear cells.
[0022] In some embodiments of the present application, the second cytokine in the activation medium includes at least two of IL-12, IL-15, and IL-18.
[0023] The present application can efficiently activate and culture umbilical cord blood mononuclear cells by using at least two cytokines among IL-12, IL-15, and IL-18 in combination with IL-2, and in combination with phytohemagglutinin and nutrients.
[0024] In some embodiments of the present application, the concentration of IL-12 in the activation medium is 10-50 ng / mL, the concentration of IL-15 is 10-50 ng / mL, and the concentration of IL-18 is 50-200 ng / mL.
[0025] In some embodiments of the present application, the concentration of IL-2 in the activation culture medium is 200-1500 IU / mL, the concentration of phytohemagglutinin is 0.05-0.2 μg / mL, and the concentration of nutrients is 0.5%-10%; wherein the nutrients include platelet lysate or autologous plasma.
[0026] The present application can efficiently activate and culture umbilical cord blood mononuclear cells by using appropriate concentrations of cytokines, phytohemagglutinins and nutrients.
[0027] In some embodiments of the present application, the expansion culture medium further comprises human recombinant albumin and / or platelet lysate.
[0028] The present application can ensure efficient expansion and culture of umbilical cord blood mononuclear cells by adding human recombinant albumin and / or platelet lysate to the expansion culture medium.
[0029] In some embodiments of the present application, the concentration of IL-2 in the expansion medium is 200-1500 IU / mL, the concentration of human recombinant albumin is 0.02-0.1 g / mL, and the concentration of platelet lysate is 2%-12%.
[0030] The present application can efficiently expand and culture umbilical cord blood mononuclear cells by using appropriate concentrations of IL-2 and human recombinant albumin and / or platelet lysate.
[0031] In some embodiments of the present application, the expansion culture includes a first-stage expansion culture and a second-stage expansion culture; the first-stage expansion culture includes transferring the activated cells to an expansion culture medium containing 200-1500 IU / mL of IL-2 and 0.02-0.1 g / mL of human recombinant albumin and / or 2%-12% platelet lysate for expansion culture; the second-stage expansion culture includes transferring the cells that have undergone the first-stage expansion culture to an expansion culture medium containing 200-1500 IU / mL of IL-2 for expansion culture.
[0032] The present application adopts appropriate expansion culture medium to respectively carry out first stage expansion culture and second stage expansion culture, so as to efficiently expand and culture umbilical cord blood mononuclear cells.
[0033] In some embodiments of the present application, the amount of umbilical cord blood mononuclear cells seeded in the pre-coated culture container is 1.0×10 6 -1.5×10 6 indivual.
[0034] The present application facilitates subsequent efficient activation culture and expansion culture by inoculating an appropriate amount of umbilical cord blood mononuclear cells in a pre-coated culture container. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without creative work.
[0036] Figure 1This is a graph showing the cell viability results of NK cells provided in Experimental Example 2 of this application at 0 h and 24 h of refrigeration at 4°C after 3 months of cryopreservation and recovery.
[0037] Figure 2 This is a graph showing the cell yield results of NK cells provided in Experimental Example 2 of this application at 0 h and 24 h of refrigeration at 4°C after 3 months of cryopreservation.
[0038] Figure 3 This is a graph showing the killing activity of NK cells against HCT-8 tumor cells provided in Experimental Example 3 of this application at 0 h and 24 h after thawing after being frozen for 3 months and refrigerated at 4°C.
[0039] Figure 4 This is a graph showing the in vivo anti-tumor activity results of NK cells after 3-month cryopreservation and recovery provided in Experimental Example 4 of the present application; wherein A is tumor volume, and B is tumor weight.
[0040] Figure 5 This is a graph showing the in vivo anti-tumor activity results of NK cells after 3-month cryopreservation and recovery provided in Experimental Example 5 of the present application; wherein A is tumor volume, and B is tumor weight. DETAILED DESCRIPTION
[0041] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.
[0042] To improve the current problem that natural killer cells are difficult to recover to the viability and anti-tumor activity of fresh natural killer cells after cryopreservation, the present invention provides a method for amplifying and cryopreserving natural killer cells, including:
[0043] (1) pre-coating a culture container with a first monoclonal antibody and a second monoclonal antibody, wherein the first monoclonal antibody includes CD52 monoclonal antibody;
[0044] (2) inoculating umbilical cord blood mononuclear cells into a pre-coated culture container, adding an activation medium for activation culture, wherein the activation medium includes a basal medium, a first cytokine, a second cytokine, a phytohemagglutinin, and nutrients, wherein the first cytokine includes IL-2;
[0045] (3) transferring the cells activated in step (2) to an expansion medium containing 200-1500 IU / mL of IL-2 and 0.02-0.1 g / mL of human recombinant albumin and / or 2%-12% of platelet lysate for a first-stage expansion culture; then transferring the cells that have undergone the first-stage expansion culture to an expansion medium containing 200-1500 IU / mL of IL-2 for a second-stage expansion culture;
[0046] (4) adding cytokines to the cells expanded and cultured in step (3) and culturing for 1-2 days, and collecting the cells to obtain natural killer cells; wherein the cytokines include at least two of IL-12, IL-15, and IL-18;
[0047] (5) The natural killer cells collected in step (4) are rinsed with physiological saline, resuspended in a freezing solution to adjust the cell density, and then frozen; wherein the freezing solution includes CS10 and human serum albumin, the volume of CS10 is 35%-65%, and the concentration of human serum albumin is 0.05-0.12 g / mL.
[0048] By activating and expanding natural killer cells using activation medium and expansion medium containing appropriately matched monoclonal antibodies and cytokines, high-purity, highly active natural killer cells can be efficiently obtained. After cell expansion and culture, the cells are cultured for 1-2 days with appropriate cytokines. At the same time, the natural killer cells are cryopreserved using a freezing solution containing an appropriate concentration of CS10 and human serum albumin. After low-temperature cryopreservation and subsequent thawing, the natural killer cells can still recover high viability and high killing activity.
[0049] In some embodiments of the present application, the concentration of IL-12 added in step (4) is 20-150 ng / mL, the concentration of IL-15 is 20-150 ng / mL, and the concentration of IL-18 is 50-500 ng / mL. Using appropriate concentrations of cytokines to culture the expanded cells for 1-2 days facilitates subsequent recovery of the cells after cryopreservation, and they can still recover high viability and high cytotoxic activity.
[0050] In some embodiments of the present application, the freezing solution also includes physiological saline.
[0051] In some examples of this application, the cell density was adjusted to 5×10 7 -15×10 7 pieces / mL.
[0052] In some embodiments of the present application, the second monoclonal antibody comprises at least one of anti-CD16, anti-CD56, anti-CD137, and anti-NKG2D. Pre-coating a culture vessel with at least one of anti-CD16, anti-CD56, anti-CD137, and anti-NKG2D and anti-CD52 facilitates subsequent activation and culture of umbilical cord blood mononuclear cells.
[0053] In some embodiments of the present application, the concentration of each of the first monoclonal antibody and the second monoclonal antibody is 0.5-5 μg / mL. As an example, the concentration of each of the first monoclonal antibody and the second monoclonal antibody can be, but is not limited to, 0.5 μg / mL, 0.6 μg / mL, 0.8 μg / mL, 1 μg / mL, 1.2 μg / mL, 1.5 μg / mL, 1.8 μg / mL, 2 μg / mL, 2.2 μg / mL, 2.5 μg / mL, 2.8 μg / mL, 3 μg / mL, 3.2 μg / mL, 3.5 μg / mL, 3.8 μg / mL, 4 μg / mL, 4.2 μg / mL, 4.5 μg / mL, 4.8 μg / mL, and 5 μg / mL. Pre-coating the culture container with the first monoclonal antibody and the second monoclonal antibody at appropriate concentrations facilitates the subsequent activation and culture of umbilical cord blood mononuclear cells.
[0054] In some embodiments of the present application, the second cytokine in the activation culture medium includes at least two of IL-12, IL-15, and IL-18. The combination of at least two of these cytokines, IL-12, IL-15, and IL-18, with IL-2, along with lectins and nutrients, allows for efficient activation and culture of umbilical cord blood mononuclear cells.
[0055] In some embodiments of the present application, the concentration of IL-12 in the activation medium is 10-50 ng / mL, the concentration of IL-15 is 10-50 ng / mL, and the concentration of IL-18 is 50-200 ng / mL. As an example, the concentration of the second cytokine IL-12 can be, but is not limited to, 10 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 25 ng / mL, 28 ng / mL, 30 ng / mL, 35 ng / mL, 38 ng / mL, 40 ng / mL, 45 ng / mL, 48 ng / mL, and 50 ng / mL. The concentration of IL-15 can be, but is not limited to, 10 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, 25 ng / mL, 28 ng / mL, 30 ng / mL, 35 ng / mL, 38 ng / mL, 40 ng / mL, 45 ng / mL, 48 ng / mL, and 50 ng / mL. The concentration of IL-18 can be, but is not limited to, 50 ng / mL, 55 ng / mL, 58 ng / mL, 60 ng / mL, 65 ng / mL, 68 ng / mL, 70 ng / mL, 75 ng / mL, 78 ng / mL, 80 ng / mL, 85 ng / mL, 88 ng / mL, 90 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL, 180 ng / mL, 185 ng / mL, 190 ng / mL, 195 ng / mL, and 200 ng / mL.
[0056] In some embodiments of the present application, the concentration of IL-2 in the activation culture medium is 200-1500 IU / mL, the concentration of phytohemagglutinin is 0.05-0.2 μg / mL, and the concentration of nutrients is 0.5%-10%; wherein the nutrients include platelet lysate or autologous plasma. As an example, the concentration of IL-2 in the activation medium can be, but is not limited to, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL, 1000 IU / mL, 1050 IU / mL, 1100 IU / mL, 1150 IU / mL, 1200 IU / mL, 1250 IU / mL, 1300 IU / mL, 1350 IU / mL, 1400 IU / mL, 1450 IU / mL, or 1500 IU / mL. The concentration of lectin can be, but is not limited to, 0.05 μg / mL, 0.06 μg / mL, 0.08 μg / mL, 0.1 μg / mL, 0.12 μg / mL, 0.15 μg / mL, 0.16 μg / mL, 0.18 μg / mL, and 0.2 μg / mL. The concentration of nutrients can be, but is not limited to, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%. Using appropriate concentrations of cytokines, lectin, and nutrients can efficiently activate and culture umbilical cord blood mononuclear cells.
[0057] In some embodiments of the present application, the activation culture medium may further include 1-10 μg / mL transferrins and 5-25 μg / mL human recombinant insulin. As an example, the concentration of the transferrins in the activation culture medium may be, but is not limited to, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL. The concentration of human recombinant insulin may be, but is not limited to, 5 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 15 μg / mL, 18 μg / mL, 20 μg / mL, 22 μg / mL, 25 μg / mL.
[0058] In some embodiments of the present application, the expansion medium further comprises human recombinant albumin and / or platelet lysate. Adding human recombinant albumin and / or platelet lysate to the expansion medium can ensure efficient expansion and culture of umbilical cord blood mononuclear cells.
[0059] In some embodiments of the present application, the concentration of IL-2 in the expansion medium is 200-1500 IU / mL, the concentration of human recombinant albumin is 0.02-0.1 g / mL, and the concentration of platelet lysate is 2%-12%. As an example, the concentration of IL-2 in the expansion medium can be, but is not limited to, 200 IU / mL, 250 IU / mL, 300 IU / mL, 350 IU / mL, 400 IU / mL, 450 IU / mL, 500 IU / mL, 550 IU / mL, 600 IU / mL, 650 IU / mL, 700 IU / mL, 750 IU / mL, 800 IU / mL, 850 IU / mL, 900 IU / mL, 950 IU / mL, 1000 IU / mL, 1050 IU / mL, 1100 IU / mL, 1150 IU / mL, 1200 IU / mL, 1250 IU / mL, 1300 IU / mL, 1350 IU / mL, 1400 IU / mL, 1450 IU / mL, or 1500 IU / mL. The concentration of human recombinant albumin can be, but is not limited to, 0.02 g / mL, 0.03 g / mL, 0.04 g / mL, 0.05 g / mL, 0.06 g / mL, 0.07 g / mL, 0.08 g / mL, 0.09 g / mL, and 0.1 g / mL. The concentration of platelet lysate can be, but is not limited to, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, and 12%. Using appropriate concentrations of IL-2, human recombinant albumin, and / or platelet lysate, umbilical cord blood mononuclear cells can be efficiently expanded and cultured.
[0060] In some embodiments of the present application, the amount of umbilical cord blood mononuclear cells seeded in the pre-coated culture container is 1.0×10 6 -1.5×10 6 Inoculating an appropriate amount of umbilical cord blood mononuclear cells in a pre-coated culture container facilitates subsequent efficient activation and expansion culture.
[0061] In some embodiments of the present application, the basal culture medium is ImmunoCult TM -XF T Cell Expansion Medium.
[0062] The features and performance of the present application are further described in detail below with reference to the embodiments.
[0063] 1. The reagents and materials used in this application are as follows:
[0064] Reagents: Ficoll (Tianjin Haoyang), CD16 monoclonal antibody (Tongli Haiyuan), NKG2D monoclonal antibody (Jianwan protein), CD52 monoclonal antibody (Jianwan protein), ImmunoCult TM -XF T cell expansion medium (Stemcell), 1640 basal medium (Shanghai Darthill Biotechnology), IL-2 (Jiangsu Kingsley Pharmaceuticals), IL-12 (Jiangsu Jinsili Pharmaceuticals), IL-12 (Jiangwan Protein), IL-15 (Beijing Kexin Biotechnology), IL-18 (Beijing Kexin Biotechnology), human recombinant insulin (Beijing Kexin Biotechnology), transferrin (Wuhan Heyuan Biotechnology), platelet lysate (MillCreek), human recombinant albumin (Wuhan Heyuan Biotechnology), and human serum albumin (Rongsheng).
[0065] Cell lines: HCT-8 (purchased from ATCC), SKOV-3 (purchased from Procell).
[0066] 2. The detection methods in the test examples of this application include:
[0067] (1) Cell counting: Gently resuspend the expanded cultured cells in physiological saline and transfer them to a 50 mL tube. Take 10.5 μL of the cell suspension and add 10.5 μL of AO / PI dye. Detect the cell viability and cell concentration on a Countstar instrument.
[0068] (2) Detection of cell surface markers:
[0069] 1. Take a sample of cells and count them. Centrifuge the cell suspension at 1000 rpm for 5 minutes and discard the supernatant.
[0070] 2. Resuspend with appropriate PBS (containing 1% BSA), wash the cells once, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant;
[0071] 3. Resuspend the cells with appropriate PBS (containing 1% BSA) and adjust the cell concentration to about 2.0×10 7 / mL;
[0072] 4. Dispense the cell suspension from the previous step into 1.5mL EP tubes, 50μL each (10 cells per tube). 6 );
[0073] 5. Add 2 μL each of PerCP-CD45, FITC-CD3, APC-CD16, and PE-CD56 antibodies at the same time, place the mixed tube in a 4°C refrigerator, and incubate in the dark for 30 minutes;
[0074] 6. Add 1 mL of PBS to each tube to resuspend and wash the cells, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant;
[0075] 7. Add 200 μL of PBS to each tube to resuspend the cells and detect them on the instrument.
[0076] (3) NK cells’ ability to kill tumor cells:
[0077] 1. Tumor cell treatment: Remove target cells, discard the culture medium, wash once with 6 mL of PBS, and discard the solution; then add 0.5 mL of 0.25% trypsin to digest, observe under a microscope to digest into single cells, and then add 6 mL of complete culture medium to neutralize; use a pipette to mix the target cells and collect them into a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the solution; then add 6 mL of complete culture medium for killing to resuspend, centrifuge at 1000 rpm for 5 minutes, and discard the solution; wash the cells once, resuspend them in 1640 complete culture medium, count, and adjust the cell concentration to 1×10 5 / mL, collect into 15mL or 50mL centrifuge tube for later use; take 2mL or 4mL complete medium for killing, resuspend and count, and adjust the concentration to 1×10 5 Transfer the 50 mL of 4% HCl to a 15 mL or 50 mL centrifuge tube for later use. Refer to the Countstar Cell Counter SOP for counting methods. The target cells are HCT-8 cells.
[0078] 2. Effector (NK) cell treatment: Collect NK cells expanded to day 18 into a 15 mL centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the liquid, add 6 mL of complete killing medium, centrifuge at 1500 rpm for 5 min, discard the liquid; add 2 mL or 4 mL of complete killing medium to resuspend and count, and adjust the concentration to 2 × 10 5 / mL (efficacy-target ratio of 2:1) into a 1.5mL centrifuge tube or a 5mL EP tube for later use. The counting method refers to the "Countstar Cell Counter SOP".
[0079] 3. Cell plating: Prepare a 96-well U-shaped plate. The order of adding cell suspension and complete culture medium for killing is as follows: culture medium control well: 200 μL complete culture medium for killing, 5 replicates; culture medium correction well: 200 μL complete culture medium for killing, 5 replicates; effector-target ratio experimental well: 100 μL effector cell suspension + 100 μL target cell suspension, 5 replicates; effector cell spontaneous release well: 100 μL complete culture medium for killing + 100 μL effector cell suspension, 5 replicates; target cell Spontaneous cell release wells: 100 μL of 1640 complete medium + 100 μL of target cell suspension, 5 replicates; Maximum target cell release wells: 100 μL of 1640 complete medium + 100 μL of target cell suspension, 5 replicates; Add 200 μL of PBS to the outermost wells of the 96-well U-shaped plate to prevent evaporation of the medium in the experimental wells; Note: The cell suspension needs to be fully mixed before adding; If the number of target cells or effector cells is sufficient, use an 8-channel pipette to add them to reduce the gap between wells.
[0080] 4. Cell incubation: After gently shaking the culture plate, place it in a 37°C, 5% CO2 incubator and incubate for 3 hours and 15 minutes.
[0081] 5. Killing detection: incubate for 3h15min, take out the 96-well U-shaped plate, add 20μL / well of cell lysate to the "culture medium correction well" and "target cell maximum release well" respectively, and mix the target cell maximum release well by blowing after adding the lysate; then place it in a 37℃, 5% CO2 incubator and continue incubating for 45min; take out the substrate mixture from -20℃ in advance and restore it to room temperature, away from light; take out the 96-well U-shaped plate after incubation for 4h, centrifuge at 1500rpm for 4min; after centrifugation, use an 8-channel micropipette to aspirate 50μL / well of the detection supernatant into a 96-well ELISA plate; add 50μL of substrate mixture to each well of the ELISA plate (add substrate in the dark), incubate at room temperature in the dark, and time for 30-40 minutes; add 50μL / well stop solution; puncture large bubbles with a syringe needle, and measure the absorbance at a wavelength of 490nm within 1h after adding the stop solution.
[0082] 6. Result evaluation: First, remove outliers from the wells. Subtract the mean absorbance value of the "medium control well" from the mean absorbance value of the "target cell spontaneous release well" and the "effector cell spontaneous release well"; subtract the mean absorbance value of the "medium control well" from the mean absorbance value of each well in the effector-target ratio experiment; subtract the mean absorbance value of the "medium correction well" from the mean absorbance value of the "maximum target cell release well"; substitute the corrected values into the following formula to calculate the percentage of cytotoxicity produced by each well in the effector-target ratio experiment;
[0083] Cell killing rate (%) = (effector-target ratio experimental well - effector cell spontaneous release well - target cell spontaneous release well) / (target cell maximum release well - target cell spontaneous release well) × 100.
[0084] Example 1
[0085] 1. Obtaining CBMC (mononuclear cells) from umbilical cord blood
[0086] (1) Umbilical cord blood was divided equally and transferred into 50 mL centrifuge tubes. After equalization, the tubes were centrifuged at 3000 rpm for 15 min.
[0087] (2) After centrifugation, discard the red blood cells at the bottom and transfer the white blood cell layer and upper plasma layer as completely as possible to a 50 mL centrifuge tube. Add physiological saline to 35 mL and resuspend thoroughly. Prepare four 15 mL centrifuge tubes and add 4 mL of Ficoll separation solution to each tube. Slowly add to the Ficoll separation solution surface, taking care not to disrupt the surface layer. Centrifuge at 2000 rpm for 20 min.
[0088] (3) After centrifugation, discard the supernatant, aspirate the buffy coat layer into a new 50 mL centrifuge tube, add physiological saline to 50 mL, resuspend, and centrifuge at 1700 rpm for 5 min; repeat the above steps, take a small amount for counting, centrifuge at 1500 rpm for 5 min, and discard the remaining supernatant;
[0089] (4) Resuspend the CBMCs washed for the last time in culture medium and adjust the cell density to 1×10 6 / mL to obtain umbilical cord blood mononuclear cells.
[0090] 2. Activation and Expansion of NK Cells
[0091] (1) On day -1, 2 μg / mL of CD16 monoclonal antibody, NKG2D monoclonal antibody, and CD52 monoclonal antibody were added to 24-well plates for antibody pre-coating at 4°C overnight;
[0092] (2) On day 0, 0.4 mL of the extracted CBMCs were inoculated into a 24-well plate pre-coated with an antibody, and 500 IU / mL of IL-2, 20 ng / mL of IL-12, 20 ng / mL of IL-15, 100 ng / mL of IL-18, 2 μg / mL of transferrin, 10 μg / mL of human recombinant insulin, 2% platelet lysate, and 0.1 μg / mL of phytohemagglutinin were added for activation culture in an incubator at 37°C and 5% CO2.
[0093] (3) On the 4th and 6th days, ImmunoCult® containing 500 IU / mL IL-2, 5 μg / μL human recombinant albumin, and 2% platelet lysate were added to the 24-well plates, respectively. TM -XF T cell expansion complete medium, in a 37 ° C, 5% CO2 incubator for the first stage of expansion culture;
[0094] (4) On day 8, gently resuspend the cells and transfer them to a T25 flask. Add ImmunoCult® containing 500 IU / mL IL-2, 5 μg / μL human recombinant albumin, and 2% platelet lysate to the T25 flask. TM -XF T cell expansion complete medium, continue the first stage expansion culture in a 37°C, 5% CO2 incubator; and add the above medium on day 10;
[0095] (5) On day 12, gently resuspend the cells, transfer them to a T75 flask, and add ImmunoCult® containing 500 IU / mL IL-2 to the T75 flask. TM -XF T cell expansion complete medium, incubate in a 37°C, 5% CO2 incubator for the second stage of expansion; and add the above medium on day 14;
[0096] (6) On day 15, 20 ng / mL IL-12, 20 ng / mL IL-15, and 50 ng / mL IL-18 were added to the T75 flask and cultured in a 37°C, 5% CO2 incubator for 1 day;
[0097] (7) On the 16th day, the cells were counted, the cell status was observed under a microscope, and the cell proliferation was calculated. The cell density reached 2×10 6 -6×10 6 Cells were collected, rinsed with physiological saline, and then resuspended in freezing medium to adjust the cell density to 5×10 7 -15×10 7 / mL; wherein, the freezing solution includes CS10 and human serum albumin. Based on 1mL of freezing solution, the volume of CS10 is 0.5mL, and the volume of human serum albumin is 0.5mL (concentration is 0.1g / mL); and then frozen at -80°C.
[0098] Example 2
[0099] This embodiment provides a method for expanding NK cells, comprising the following steps:
[0100] (1) On day -1, 2 μg / mL of CD16 monoclonal antibody, NKG2D monoclonal antibody, and CD52 monoclonal antibody were added to 24-well plates for antibody pre-coating at 4°C overnight;
[0101] (2) On day 0, 0.4 mL of the extracted CBMCs were inoculated into a 24-well plate pre-coated with an antibody, and 500 IU / mL of IL-2, 20 ng / mL of IL-12, 20 ng / mL of IL-15, 100 ng / mL of IL-18, 2 μg / mL of transferrin, 10 μg / mL of human recombinant insulin, 2% platelet lysate, and 0.1 μg / mL of phytohemagglutinin were added for activation culture in an incubator at 37°C and 5% CO2.
[0102] (3) On the 4th and 6th days, ImmunoCult® containing 500 IU / mL IL-2, 5 μg / μL human recombinant albumin, and 2% platelet lysate were added to the 24-well plates, respectively. TM -XF T cell expansion complete medium, in a 37 ° C, 5% CO2 incubator for the first stage of expansion culture;
[0103] (4) On day 8, gently resuspend the cells and transfer them to a T25 flask. Add ImmunoCult® containing 500 IU / mL IL-2, 5 μg / μL human recombinant albumin, and 2% platelet lysate to the T25 flask. TM-XF T cell expansion complete medium, continue the first stage expansion culture in a 37°C, 5% CO2 incubator; and add the above medium on day 10;
[0104] (5) On day 12, gently resuspend the cells, transfer them to a T75 flask, and add ImmunoCult® containing 500 IU / mL IL-2 to the T75 flask. TM -XF T cell expansion complete medium, incubate in a 37°C, 5% CO2 incubator for the second stage of expansion; and add the above medium on day 14;
[0105] (6) On day 15, 50 ng / mL of IL-12, 50 ng / mL of IL-15, and 200 ng / mL of IL-18 were added to the T75 flask and cultured in a 37°C, 5% CO2 incubator for 1 day;
[0106] (7) On the 16th day, the cells were counted, the cell status was observed under a microscope, and the cell proliferation was calculated. The cell density reached 2×10 6 -6×10 6 Cells were collected, rinsed with physiological saline, and then resuspended in freezing medium to adjust the cell density to 5×10 7 -15×10 7 / mL; wherein, the freezing solution includes CS10 and human serum albumin. Based on 1mL of freezing solution, the volume of CS10 is 0.5mL, and the volume of human serum albumin is 0.5mL (concentration is 0.1g / mL); and then frozen at -80°C.
[0107] Example 3
[0108] This embodiment provides a method for expanding NK cells, comprising the following steps:
[0109] (1) On day -1, 2 μg / mL of CD16 monoclonal antibody, NKG2D monoclonal antibody, and CD52 monoclonal antibody were added to 24-well plates for antibody pre-coating at 4°C overnight;
[0110] (2) On day 0, 0.4 mL of the extracted CBMCs were inoculated into a 24-well plate pre-coated with an antibody, and 500 IU / mL of IL-2, 20 ng / mL of IL-12, 20 ng / mL of IL-15, 100 ng / mL of IL-18, 2 μg / mL of transferrin, 10 μg / mL of human recombinant insulin, 2% platelet lysate, and 0.1 μg / mL of phytohemagglutinin were added for activation culture in an incubator at 37°C and 5% CO2.
[0111] (3) On the 4th and 6th days, ImmunoCult® containing 500 IU / mL IL-2, 5 μg / μL human recombinant albumin, and 2% platelet lysate were added to the 24-well plates, respectively. TM -XF T cell expansion complete medium, in a 37 ° C, 5% CO2 incubator for the first stage of expansion culture;
[0112] (4) On day 8, gently resuspend the cells and transfer them to a T25 flask. Add ImmunoCult® containing 500 IU / mL IL-2, 5 μg / μL human recombinant albumin, and 2% platelet lysate to the T25 flask. TM -XF T cell expansion complete medium, continue the first stage expansion culture in a 37°C, 5% CO2 incubator; and add the above medium on day 10;
[0113] (5) On day 12, gently resuspend the cells, transfer them to a T75 flask, and add ImmunoCult® containing 500 IU / mL IL-2 to the T75 flask. TM -XF T cell expansion complete medium, incubate in a 37°C, 5% CO2 incubator for the second stage of expansion; and add the above medium on day 14;
[0114] (6) On day 15, 50 ng / mL of IL-12, 50 ng / mL of IL-15, and 500 ng / mL of IL-18 were added to the T75 flask and cultured in a 37°C, 5% CO2 incubator for 1 day;
[0115] (7) On the 16th day, the cells were counted, the cell status was observed under a microscope, and the cell proliferation was calculated. The cell density reached 2×10 6 -6×10 6 Cells were collected, rinsed with physiological saline, and then resuspended in freezing medium to adjust the cell density to 5×10 7 -15×10 7 / mL; wherein, the freezing solution includes CS10 and human serum albumin. Based on 1mL of freezing solution, the volume of CS10 is 0.5mL, and the volume of human serum albumin is 0.5mL (concentration is 0.1g / mL); and then frozen at -80°C.
[0116] Comparative Example 1
[0117] This comparative example provides a method for expanding natural killer cells, which differs from Example 1 in that: on the 15th day, 200 IU / mL of IL-2 is added to the T75 flask.
[0118] Comparative Example 2
[0119] This comparative example provides a method for expanding natural killer cells, which differs from Example 1 in that the cells expanded and cultured to day 16 in Example 1 are directly used, and the freezing medium only includes CS10.
[0120] Comparative Example 3
[0121] This comparative example provides a method for expanding natural killer cells. The method differs from Example 1 in that the cells expanded and cultured to day 16 in Example 1 are directly used. The freezing solution includes CS10, recombinant albumin, and normal saline. Based on 1 mL of freezing solution, the volume of CS10 is 0.5 mL, the volume of normal saline is 0.5 mL, and the concentration of recombinant albumin is 0.1 g / mL.
[0122] Comparative Example 4
[0123] This comparative example provides a method for expanding natural killer cells. The method differs from Example 1 in that the cells expanded and cultured to day 16 in Example 1 are directly used. The freezing solution includes CS10 and human serum albumin. Based on 1 mL of freezing solution, the volume of CS10 is 0.8 mL, and the volume of human serum albumin is 0.2 mL (concentration is 0.04 g / mL).
[0124] Comparative Example 5
[0125] This comparative example provides a method for expanding natural killer cells. The method differs from Example 1 in that the cells expanded and cultured to day 16 in Example 1 are directly used. The freezing solution includes CS10, human serum albumin, and normal saline. Based on 1 mL of freezing solution, the volume of CS10 is 0.2 mL, the volume of normal saline is 0.1 mL, and the volume of human serum albumin is 0.7 mL (concentration is 0.14 g / mL).
[0126] Test Example 1
[0127] In this experiment, cells expanded and cultured to day 16 in Examples 1-3 and Comparative Example 1 were assayed using a Countstar instrument to measure cell viability and viable cell concentration. Cell mass and viable cell count were calculated using the formula (cell mass = total cell concentration × volume, viable cell count = viable cell concentration × volume). Cell surface markers were assayed to determine the proportion of NK cells, and the NK cell expansion factor was calculated using the formula (expansion factor = number of NK cells after expansion / number of NK cells before expansion). The results are shown in Table 1.
[0128] Table 1 The proportion, cell viability and expansion times of NK cells before freezing
[0129] Experimental group NK cell ratio (%) Cell viability (%) Amplification factor Example 1 94.37±4.92 95.35±3.17 5329±356 Example 2 93.45±3.15 94.21±4.39 5215±475 Example 3 95.28±3.36 94.67±3.64 5503±587 Comparative Example 1 96.15±3.52 95.16±2.93 5238±762
[0130] As shown in Table 1, the natural killer cell expansion methods provided in Examples 1-3 and Comparative Example 1 all efficiently expanded and obtained natural killer cells, with no significant differences in the proportion, cell viability, or expansion factor of the natural killer cells obtained after 16 days of expansion and culture. Since Comparative Examples 2-5 directly used cells expanded and cultured to day 16 in Example 1, the expansion results in Comparative Examples 2-5 were identical to those in Example 1.
[0131] Test Example 2
[0132] After 3 months of cryopreservation, the cells in Examples 1-3 and Comparative Examples 1-5 were revived in a 37°C water bath. The revived cells were refrigerated at 0 h and 4°C for 24 h, and the cell mass and viable cell mass were detected. The cell viability and cell yield were calculated as follows: cell viability (%) = (viable cell mass / total cell mass) × 100%; cell yield (%) = (viable cell mass after resuscitation / viable cell mass after cryopreservation) × 100%. The results are shown in Table 1. Figure 1-Figure 2 shown.
[0133] From Table 1, Figure 1 and Figure 2 The results show that while there were no significant differences in the cell ratio, cell viability, and expansion factor of the natural killer cells expanded and cultured to day 16 between Examples 1-3 and Comparative Examples 1-5, the cells frozen in Examples 1-3, after thawing after three months of cryopreservation, had cell viability and cell yield exceeding 95% at 0 h; and after 24 hours of refrigeration at 4°C, the cell viability exceeded 85% and the cell yield exceeded 90%. In contrast, the cells frozen in Comparative Examples 1-5, after three months of cryopreservation and thawing, had significantly lower cell viability and cell yield at 0 h and after 24 hours of refrigeration at 4°C. The present application describes that the activation and expansion culture of natural killer cells is performed using an activation culture medium and an expansion culture medium containing appropriately matched monoclonal antibodies and cytokines, which can efficiently obtain high-purity and highly active natural killer cells. After cell expansion culture, the cells are cultured for 1-2 days with appropriate cytokines. At the same time, the natural killer cells are cryopreserved using a freezing solution containing an appropriate concentration of CS10 and human serum albumin. After low-temperature cryopreservation and subsequent revival, the natural killer cells can still be restored to a high viability.
[0134] Test Example 3
[0135] In this test example, the cells frozen in Example 1-3 and Comparative Example 1-5 were frozen for 3 months and then revived in a 37°C water bath. The revived cells were used to measure the killing activity of NK cells against HCT-8 cells using the LDH release method. The results are shown in Figure 2. Figure 3 shown.
[0136] The above test was also performed on the cells expanded and cultured to day 16 in Examples 1-3 and Comparative Examples 1-5. The results are shown in Table 2.
[0137] Table 2 Cytotoxicity of NK cells before cryopreservation
[0138]
[0139]
[0140] As shown in Table 2, the natural killer cells obtained by expansion and culture in Examples 1-3 and Comparative Examples 1-5 all had high tumor cell killing activities, with no significant difference.
[0141] Depend on Figure 3 The results show that while there was no significant difference in the cytotoxicity of natural killer cells against tumor cells after expansion and culture to day 16 in Examples 1-3 and Comparative Examples 1-5, the cells frozen in Example 1-3 and thawed after three months of cryopreservation exhibited a cytotoxicity of over 60% against tumor cells at 0 h, close to the cytotoxicity of cells before cryopreservation. After 24 h of refrigeration at 4°C, the cytotoxicity still exceeded 45%. In contrast, the cytotoxicity of cells frozen in Comparative Examples 1-5 and thawed after three months of cryopreservation showed significantly reduced activity against tumor cells at 0 h and after 24 h of refrigeration at 4°C. The present application describes that the activation and expansion culture of natural killer cells is performed using an activation medium and an expansion medium containing appropriately matched monoclonal antibodies and cytokines, which can efficiently obtain high-purity and highly active natural killer cells. After cell expansion culture, the cells are cultured for 1-2 days with appropriate cytokines. At the same time, the natural killer cells are cryopreserved using a freezing solution containing an appropriate concentration of CS10 and human serum albumin. After low-temperature cryopreservation and subsequent thawing, the natural killer cells can still recover high killing activity.
[0142] Test Example 4
[0143] In this test example, the cells frozen in Example 1 were frozen for 3 months and then revived in a 37°C water bath. The revived cells were used to treat a subcutaneous intestinal cancer transplanted mouse model to evaluate the in vivo anti-tumor activity of the frozen NK cells. The specific steps are as follows: HCT-8 intestinal cancer cells were used to form tumors in NCG mice. When the tumor volume reached 50 mm 3 Around 30 dpi, the patients were divided into two groups, including solvent group and cryopreserved NK cell treatment group. The patients were injected via tail vein once every 3 days. Each time, 2.0×10 7 Cells / cell. The results are as follows Figure 4 shown.
[0144] Depend on Figure 4The results show that the cells frozen in Example 1, after thawing for 3 months, can effectively reduce the size and weight of tumors in tumor-bearing mice when used to treat them. This indicates that the present application uses activation medium and expansion medium containing appropriately matched monoclonal antibodies and cytokines to activate and expand natural killer cells, efficiently obtaining high-purity, highly active natural killer cells. After cell expansion culture, the cells are cultured for 1-2 days with appropriate cytokines. At the same time, the natural killer cells are cryopreserved in a freezing solution containing an appropriate concentration of CS10 and human serum albumin. After low-temperature cryopreservation and subsequent thawing, the natural killer cells can still regain high cytotoxic activity.
[0145] Test Example 5
[0146] In this experimental example, the cells frozen in Example 1 were frozen for 3 months and then revived in a 37°C water bath. The revived cells were used to treat a mouse model of ovarian cancer peritoneal metastasis to evaluate the in vivo anti-tumor activity of frozen NK cells. The specific steps were as follows: ovarian cancer SKOV-3 cells were used to form tumors in the peritoneal cavity of NCG mice. Four days after the tumor cells were inoculated, the mice were divided into two groups according to the tumor fluorescence intensity evaluation, including a solvent group and a frozen and revived NK cell treatment group. The cells were injected intraperitoneally once every 3 days, and the frozen and revived NK cell treatment group was injected with 2.0×10 7 Cells / cell. The results are as follows Figure 5 shown.
[0147] Depend on Figure 5 The results show that the cells frozen in Example 1, after thawing after 3 months of cryopreservation, can effectively reduce tumor progression in tumor-bearing mice. This shows that the present application uses an activation medium and expansion medium containing appropriately matched monoclonal antibodies and cytokines to activate and expand natural killer cells, which can efficiently obtain high-purity and high-activity natural killer cells. After cell expansion culture, the cells are cultured for 1-2 days with appropriate cytokines. At the same time, the natural killer cells are cryopreserved in a freezing solution containing an appropriate concentration of CS10 and human serum albumin. After low-temperature cryopreservation and subsequent thawing, the natural killer cells can still recover high killing activity.
[0148] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.
Claims
1. A method for expanding and freezing natural killer cells, characterized in that: include: (1) pre-coating a culture container with a first monoclonal antibody and a second monoclonal antibody, wherein the first monoclonal antibody includes CD52 monoclonal antibody; (2) inoculating umbilical cord blood mononuclear cells into the pre-coated culture container, adding activation medium for activation culture, wherein the activation medium includes a basal medium, a first cytokine, a second cytokine, a phytohemagglutinin, and nutrients, wherein the first cytokine includes IL-2; (3) transferring the cells activated in step (2) into an expansion medium for expansion culture, wherein the expansion medium includes a basal medium and IL-2; (4) adding cytokines to the cells expanded and cultured in step (3) and culturing for 1-2 days, and collecting the cells to obtain natural killer cells; wherein the cytokines include at least two of IL-12, IL-15, and IL-18; (5) The natural killer cells collected in step (4) are rinsed with physiological saline, and then resuspended in a freezing solution to adjust the cell density and then frozen; wherein the freezing solution includes CS10 and human serum albumin, the volume of the CS10 is 35%-65%, and the concentration of the human serum albumin is 0.05-0.12 g / mL.
2. The amplification and freezing method according to claim 1, characterized in that: The concentration of the IL-12 added in the step (4) is 20-150 ng / mL, the concentration of the IL-15 is 20-150 ng / mL, and the concentration of the IL-18 is 50-500 ng / mL.
3. The amplification and freezing method according to claim 1, characterized in that: The freezing solution also includes physiological saline.
4. The amplification and freezing method according to claim 1, characterized in that: The cell density was adjusted to 5×10 7 -15×10 7 pieces / mL.
5. The amplification and freezing method according to claim 1, characterized in that: The second monoclonal antibody comprises at least one of CD16 monoclonal antibody, CD56 monoclonal antibody, CD137 monoclonal antibody, and NKG2D monoclonal antibody; Preferably, the concentration of each of the first monoclonal antibody and the second monoclonal antibody is 0.5-5 μg / mL.
6. The amplification and freezing method according to claim 1, characterized in that: The second cytokine in the activation medium includes at least two of IL-12, IL-15, and IL-18; Preferably, the concentration of IL-12 in the activation medium is 10-50 ng / mL, the concentration of IL-15 is 10-50 ng / mL, and the concentration of IL-18 is 50-200 ng / mL.
7. The amplification and freezing method according to claim 1, characterized in that: The concentration of the IL-2 in the activation medium is 200-1500 IU / mL, the concentration of the phytohemagglutinin is 0.05-0.2 μg / mL, and the concentration of the nutrient is 0.5%-10%; Wherein, the nutrients include platelet lysate or autologous plasma.
8. The amplification and freezing method according to claim 1, characterized in that: The expansion culture medium further comprises human recombinant albumin and / or platelet lysate; Preferably, the concentration of IL-2 in the expansion medium is 200-1500 IU / mL, the concentration of human recombinant albumin is 0.02-0.1 g / mL, and the concentration of the platelet lysate is 2%-12%.
9. The amplification and freezing method according to claim 8, characterized in that: The amplification culture includes a first-stage amplification culture and a second-stage amplification culture; The first stage of expansion culture comprises transferring the activated cells to an expansion culture medium containing 200-1500 IU / mL of IL-2 and 0.02-0.1 g / mL of human recombinant albumin and / or 2%-12% of platelet lysate for expansion culture; The second stage expansion culture comprises transferring the cells that have undergone the first stage expansion culture to an expansion culture medium containing 200-1500 IU / mL of IL-2 for expansion culture.
10. The amplification and freezing method according to claim 1, characterized in that: The amount of umbilical cord blood mononuclear cells inoculated in the pre-coated culture container is 1.0×10 6 -1.5×10 6 indivual.