A simple and low-cost method for culturing canine NK cells in vitro

Abstract

The application discloses a simple and low-cost method for culturing canine NK cells in vitro and belongs to the technical field of cell culture. The method comprises the following steps: separating NK cells from peripheral blood of a dog, culturing the NK cells with a canine NK cell activation culture medium for 24-48 hours, then continuing to culture the NK cells with a canine NK cell expansion culture medium, adding the canine NK cell expansion culture medium after 48 hours, expanding the cells in a bottle when the cells are obviously expanded, otherwise maintaining the cells in the original bottle after centrifugation, adding liquid and expanding the cells in a bottle or culturing the cells in the original bottle every other day, and harvesting the canine NK cells after 12-18 days. The method provided by the application does not need to activate the canine NK cells by using engineered trophoblast cells and multiple cell factors, and does not need to activate the cells by using an antibody, so that the method is simple and low in cost, and the cultured canine NK cells normally express NKp46, do not express CD3 and CD5, and have normal inhibitory and killing tumor abilities.

Description

Technical Field

[0001] This invention relates to a method for in vitro cell culture, specifically a simple and low-cost method for in vitro culture of canine NK cells, belonging to the field of cell culture technology. Background Technology

[0002] As the first line of defense in the immune system and a core component of the innate immune system, natural killer cells (NK cells) play a crucial role in tumor immunotherapy, antiviral infection, and immune regulation. Unlike T lymphocytes, NK cells can directly recognize and rapidly kill tumor cells and virus-infected cells without prior antigen sensitization, and are not restricted by the major histocompatibility complex (MHC). Coupled with their wide availability and good safety profile, NK cells are attracting increasing attention in tumor treatment and anti-infection fields. Human NK cells are continuously developing towards clinical applications, with several drugs targeting hematologic malignancies and solid tumors, including autologous NK cells, genetically modified NK cells, and iPSC-derived NK cells, currently in phase I or II clinical trials. Clinical data on canine peripheral blood-derived NK cells have been published; for example, Professor Robert Canter's team at the University of California, Davis, has published several clinical research results on canine NK cell therapy for pet melanoma and osteosarcoma, showing promising clinical therapeutic effects.

[0003] Compared to human NK cells, which have already entered widespread clinical trials and even been applied clinically, canine NK cells lag behind in both scientific research and clinical application. Research on in vitro culture of canine NK cells is relatively lacking. Furthermore, due to the significant interspecies differences between dogs and humans, the in vitro culture protocol for canine NK cells cannot be completely replicated for human NK cells, posing a greater challenge. Culture media typically suitable for human NK cells cannot support the normal expansion of canine NK cells. Commercially available canine species-specific activating antibodies and species-specific IL factors (IL2, IL15, IL12, etc.) are severely lacking. In canine NK cell culture, only antibodies against human antigens (such as anti-human CD16 or NKp46 antibodies) and human-derived IL molecules (such as recombinant human IL-2, IL-21, IL-15, and IL12) can be used to activate and culture canine NK cells. However, due to interspecies sequence differences, the activation and proliferation-promoting effects of anti-human antigen antibodies and human IL molecules on canine NK cells are relatively limited, severely affecting the expansion of canine NK cells and also impacting their function. Even if some commercially available canine IL protein molecules exist, the low demand and small production scale result in high selling prices, which is not conducive to cost control in canine NK cell preparation.

[0004] A novel canine NK cell culture method has been developed that eliminates the need for activation treatment with engineered feeder cells (such as IL15-41BB-K562), activation treatment with multiple factors such as IL-12 (10 ng / mL), IL-18 (50 ng / mL), IL-15 (50 ng / mL), and IL-2 (1000 U / mL), and antibody coating activation treatment. This method will effectively reduce the cost of in vitro culture of canine NK cells and is of great significance for promoting the clinical translation of canine NK cells. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a culture medium suitable for canine NK cell culture and a simple and low-cost method for in vitro culture of canine NK cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A simple and low-cost method for in vitro culture of canine NK cells includes the following steps:

[0008] (1) Isolate NK cells from peripheral blood of dogs, resuspend them in freshly prepared canine NK cell activation medium, then inoculate them into culture flasks, add freshly prepared canine NK cell activation medium, shake back and forth and left and right, and then incubate in a 37℃, 5% CO2 incubator for 24h-48h.

[0009] (2) After activation culture, freshly prepared canine NK cell expansion culture medium is slowly added along the side wall of the culture flask, and then placed in a 37℃, 5% CO2 incubator for 48h. Samples are taken and counted, and freshly prepared canine NK cell expansion culture medium is added. If the cells expand significantly, the flask is expanded or the cells are transferred to a cell culture bag for culture. If the cells do not expand significantly, the cells are centrifuged and the original flask is maintained for culture. Samples are taken and counted every 2 days and the medium is added. If the cells expand significantly, the flask is expanded or the cells are transferred to a cell culture bag for culture. If the cells do not expand significantly, the cells are centrifuged and the original flask is maintained for culture. Canine NK cells are harvested after 12-18 days of expansion culture.

[0010] The preparation method of the canine NK cell activation medium is as follows: PRMI 1640 is used as the basal medium, and recombinant human IL2, recombinant human IL-15, decitabine and fetal bovine serum are added at concentrations of 100 IU / mL, 1000 U / mL, 10 nM-20 nM and 10%, respectively.

[0011] The preparation method of canine NK cell expansion medium is as follows: PRMI 1640 is used as the basal medium, and recombinant human IL-15 and fetal bovine serum are added at concentrations of 2000 U / mL and 10%, respectively.

[0012] Preferably, in step (1), the culture flask is a T75 cell culture flask.

[0013] Preferably, in step (1), before culturing, the density of canine NK cells is adjusted to 2 × 10⁻⁶. 6 per mL.

[0014] Preferably, in step (1), the culture time is 48 hours.

[0015] Preferably, in step (2), when the canine NK cell expansion culture medium is added for the second time, the cell density is adjusted to 1.0 × 10⁻⁶. 6 cells / mL -1.5×10 6 per mL.

[0016] Preferably, in step (2), when replenishing fluid every 2 days thereafter, the cell density is adjusted to 0.5 × 10⁻⁶. 6 cells / mL -1.0×10 6 per mL.

[0017] Preferably, in step (2), canine NK cells are harvested after 18 days of amplification and culture.

[0018] The advantages of this invention are as follows: The method for culturing canine NK cells in vitro provided by this invention first pre-treats canine NK cells with decitabine to activate them, and then expands the canine NK cells with a culture medium containing only recombinant human IL15. The entire process no longer requires engineered feeder cells (such as IL15-41BB-K562) and multiple cytokines (such as IL-12 (10 ng / mL), IL-18 (50 ng / mL), IL-15 (50 ng / mL), IL-2 (1000 U / mL)) to activate the canine NK cells, nor does it require antibody coating activation. The operation is simple and the cost is low. Furthermore, the cultured canine NK cells normally express the NK cell characteristic marker NKp46 and do not express the T cell characteristic markers CD3 and CD5, thus having normal tumor inhibition and killing capabilities. Attached Figure Description

[0019] Figure 1 The images show the flow cytometry results of CD3, CD5, and NKp46 in NK cells isolated from canine peripheral blood. In the images, A is the flow cytometry result of CD3, B is the flow cytometry result of CD5, and C is the flow cytometry result of NKp46.

[0020] Figure 2 The images show the proliferation of canine NK cells harvested after 18 days of amplification and culture. In the images, A shows the proliferation of canine NK cells in the 10 nM DAC-activated group, B shows the proliferation of canine NK cells in the 20 nM DAC-activated group, and C shows the proliferation of canine NK cells in the control group without DAC activation.

[0021] Figure 3 This is a graph showing the fold expansion of canine NK cells harvested after 18 days of amplification culture in each group.

[0022] Figure 4 This is a graph showing the Vero cell count results for the control group and at each effector-target ratio;

[0023] Figure 5 This is a graph showing the statistical results of the killing rate of canine NK cells against Vero cells at different effect-to-target ratios;

[0024] Figure 6 These are in vivo imaging results of mice in the non-treatment control group and the NK cell-treated group 29 days after modeling. Detailed Implementation

[0025] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0026] I. Culture medium

[0027] 1. Canine NK cell activation culture medium

[0028] Using PRMI 1640 as the basal medium, recombinant human IL-2 and recombinant human IL-15 were first added at concentrations of 100 IU / mL and 1000 U / mL, respectively. Then, decitabine (DAC, NSC: 127716, CAS: 2353-33-5) was added at a concentration of 10 nM-20 nM. Finally, fetal bovine serum (FBS) was added at a concentration of 10% (v / v) to obtain canine NK cell activation medium.

[0029] 2. Canine NK cell expansion culture medium

[0030] Using PRMI 1640 as the basal medium, recombinant human IL-15 was first added at a concentration of 2000 U / mL, followed by fetal bovine serum (FBS) at a concentration of 10% (v / v) to obtain canine NK cell expansion medium.

[0031] II. Cultivation Methods

[0032] 1. Activation of canine NK cells

[0033] NK cells (canine NK cells) were isolated from canine peripheral blood, resuspended in freshly prepared canine NK cell activation medium, and then seeded into T75 cell culture flasks. Freshly prepared canine NK cell activation medium was then added to adjust the canine NK cell density to 2 × 10⁻⁶ cells / mL. 6 The sample is placed in a 37℃, 5% CO2 incubator and shaken back and forth and left and right for 24-48 hours.

[0034] 2. Expansion of canine NK cells

[0035] After activation culture, freshly prepared canine NK cell expansion medium was slowly added along the side wall of the T75 cell culture flask. The flask was then incubated at 37°C with 5% CO2 for 48 hours. Cells were then counted, and freshly prepared canine NK cell expansion medium was added to adjust the cell density to 1.0 × 10⁻⁶ cells / year. 6 cells / mL -1.5×10 6 If cell proliferation is significant, expand the culture flask or transfer the cells to a cell culture bag. If cell proliferation is not significant, centrifuge the cells and maintain the culture in the original flask. Subsequently, sample and count the cells every two days, replenish the culture medium (canine NK cell expansion medium), and adjust the cell density to 0.5 × 10⁶ cells / mL. 6 cells / mL -1.0×10 6 If the cell count is significantly increased (e.g., cells / mL), expand the culture flask or transfer the cells to a cell culture bag. If there is no significant cell expansion, centrifuge the cells and maintain the original culture flask. Harvest canine NK cells after 12-18 days of expansion culture.

[0036] Example

[0037] 1. Isolation of NK cells from canine peripheral blood mononuclear cells (PBMCs). Specifically:

[0038] (1) Collect 100cc of peripheral blood from a dog using an anticoagulant blood collection tube, add an equal volume of 2% BSA-PBS solution (preparation method below) to the fresh blood, dilute and mix well;

[0039] (2) Add 15 mL of Ficoll lymphocyte separation solution (hereinafter referred to as Ficoll) to a 50 mL test tube, and then slowly add 35 mL of diluted blood along the wall of the test tube, being careful not to disturb the surface layer of the Ficoll solution;

[0040] (3) Centrifuge at 750g speed for 20min, discard the first layer of supernatant, carefully transfer the white film layer solution into a new 50mL test tube, add 2% BSA-PBS solution to 50mL, and centrifuge at 350g speed and 4℃ temperature for 10min;

[0041] (4) Discard the supernatant, add 1 mL of 2% BSA-PBS solution to resuspend the cells, add washing buffer (preparation method below) to 50 mL, and centrifuge at 160 g speed and 4 °C for 15 min;

[0042] (5) Discard the supernatant, add 50 mL of 2% BSA-PBS solution to resuspend the cells, centrifuge at 300 g speed and 4℃ temperature for 10 min, discard the supernatant, and resuspend the cells with an appropriate amount of washing solution;

[0043] (6) Label NK cells with antibodies: cell count, per 1×10 7 Add 160 μL of washing buffer, 40 μL of CD5 monoclonal antibody (clone number: YKIX322.3, brand: eBioscience™) and 20 μL of FcR blocking agent to each cell. Incubate at 4°C in the dark for 20 min, gently pipetting the cells every 5 min. After incubation, add 1 × 10⁻⁶ cells per cell. 7 Add 2 mL of washing buffer to each cell, centrifuge at 300 g speed and 4 °C for 10 min, repeat washing twice to obtain antibody-labeled NK cells;

[0044] (7) Co-incubate magnetic beads with antibody-labeled NK cells: count cells, per 1×10 7 Antibody-labeled NK cells were resuspended in 160 μL of washing buffer, then 40 μL of magnetic beads (Anti-PE MicroBeads UltraPure, Miltenyi, catalog number: 130-105-639) were added, mixed well, and incubated at 4°C in the dark for 15 min, with the beads being pipetted every 5 min. After incubation, 1 × 10⁶ cells were added. 7 Add 2 mL of washing buffer to each cell, centrifuge at 300 g speed and 4 °C for 10 min, repeat washing twice to obtain cells co-incubated with magnetic beads, and resuspend the cells in 500 μL of washing buffer for co-incubation with magnetic beads.

[0045] (8) Isolation of NK cells via LD separation column: Install the cell sorting device, add 2 mL of washing buffer to the LD separation column and pass it through the column once. Add 500 μL of resuspended magnetic beads to the LD separation column and observe the liquid level. When the cells have completely entered the LD separation column, add 1 mL of washing buffer to pass through the column. When the liquid level shows that the cells have entered the separation part of the LD separation column and there is no further dripping, add 1 mL of washing buffer, collect the cells, and centrifuge at 300 g speed and 4 °C for 10 min to obtain canine NK cells. Resuspend the canine NK cells in PRMI 1640 medium and count them for later use.

[0046] Preparation method of 2% BSA-PBS solution: Add 2g bovine serum albumin (BSA) to every 100mL PBS buffer. After the BSA dissolves, filter the solution through a 0.45μm filter in a clean bench and store it aseptically for later use.

[0047] Preparation of washing solution: Add bovine serum albumin (BSA) and 0.05M ethylenediaminetetraacetic acid (EDTA) stock solution to PBS buffer to prepare PBS buffer containing 0.5% (w / v) BSA and 2mM EDTA. Filter the solution through a 0.45μm filter in a clean bench and store aseptically for later use.

[0048] The isolated canine NK cells were analyzed by flow cytometry for CD5, CD3, and NKp46 levels. Specifically:

[0049] (1) The isolated canine NK cells were centrifuged at 500g for 5 min, resuspended in PBS buffer, and divided into 0.5×10⁻⁶ cells per tube. 6 The cells were aliquoted into EP tubes, centrifuged at 500g for 5 min, and the cells were collected. The cells were resuspended in 400 μL of PBS buffer and centrifuged at 500g for 5 min. This process was repeated twice. The supernatant was discarded, and 200 μL of PBS buffer was added to each tube to resuspend the cells, thus obtaining a cell suspension.

[0050] (2) Detection of NKp46 positivity rate: Add 5 μL of APC fluorescent group-goat anti-mouse IgG (H+L) secondary antibody to 200 μL of cell suspension obtained above, incubate at 4℃ for 20 min, then centrifuge at 500g for 5 min, discard the supernatant, and resuspend the cells in 200 μL of PBS buffer for flow cytometry gating; Add 5 μL of APC fluorescent group-goat anti-mouse IgG (H+L) secondary antibody to 200 μL of cell suspension, incubate at 4℃ for 20 min, then centrifuge at 500g for 5 min, discard the supernatant, resuspend the cells in 200 μL of PBS buffer, then add 5 μL of anti-canine NKp46 (CD335) antibody, incubate at 4℃ for 20 min, then centrifuge at 500g for 5 min, discard the supernatant, resuspend the cells in 200 μL of PBS buffer, and then detect the NKp46 positivity rate using flow cytometry.

[0051] (3) CD5 positivity rate detection: Add 5 μL of PE fluorescent group-rat IgG2a kappa isotype control antibody to 200 μL of cell suspension obtained above, incubate at 4℃ for 20 min, then centrifuge at 500g for 5 min, discard the supernatant, resuspend the cells in 200 μL of PBS buffer for flow cytometry gating; add 5 μL of PE fluorescent group-anti-CD5 monoclonal antibody to 200 μL of cell suspension, incubate at 4℃ for 20 min, then centrifuge at 500g for 5 min, discard the supernatant, resuspend the cells in 200 μL of PBS buffer, and detect the CD5 positivity rate using flow cytometry.

[0052] (4) CD3 positivity rate detection: Add 5 μL of mouse IgG1 kappa isotype control antibody to 200 μL of cell suspension obtained above, incubate at 4℃ for 20 min, then centrifuge at 500g for 5 min, discard the supernatant, resuspend the cells in 200 μL of PBS buffer for flow cytometry gating; add 5 μL of FITC fluorescent group-anti-CD3e monoclonal antibody to 200 μL of cell suspension, incubate at 4℃ for 20 min, then centrifuge at 500g for 5 min, discard the supernatant, resuspend the cells in 200 μL of PBS buffer, and detect the CD3 positivity rate using flow cytometry.

[0053] The flow cytometry results of NK cells isolated from canine peripheral blood are shown below. Figure 1 .

[0054] Depend on Figure 1 The results showed that the positive rates of CD3, CD5, and NKp46 in the isolated canine NK cells were 0.72%, 0.83%, and 98.39%, respectively. In other words, CD5-negative canine NK cells were isolated.

[0055] 2. Activation of isolated CD5-negative canine NK cells with decitabine.

[0056] (1) Prepare canine NK cell activation culture medium and canine NK cell expansion culture medium with different components.

[0057] (i) 10 nM DAC activation medium: PRMI 1640 is used as the basic medium. First, recombinant human IL2 and recombinant human IL-15 are added at concentrations of 100 IU / mL and 1000 U / mL, respectively. Then, decitabine is added at a concentration of 10 nM. Finally, fetal bovine serum (FBS) is added at a concentration of 10% (v / v). Mix well to obtain the medium.

[0058] (ii) 20 nM DAC activation medium: PRMI 1640 is used as the basal medium. First, recombinant human IL2 and recombinant human IL-15 are added at concentrations of 100 IU / mL and 1000 U / mL, respectively. Then, decitabine is added at a concentration of 20 nM. Finally, fetal bovine serum (FBS) is added at a concentration of 10% (v / v). Mix well to obtain the medium.

[0059] (iii) Control activation medium without DAC addition: PRMI 1640 was used as the basal medium. Recombinant human IL2 and recombinant human IL-15 were added first at concentrations of 100 IU / mL and 1000 U / mL, respectively. Then fetal bovine serum (FBS) was added at a concentration of 10% (v / v). The mixture was then stirred to obtain the final product.

[0060] (iv) Canine NK cell expansion medium: PRMI 1640 was used as the basic medium. First, recombinant human IL-15 was added at a concentration of 2000 U / mL, and then fetal bovine serum (FBS) was added at a concentration of 10% (v / v). The mixture was then stirred.

[0061] (2) Activation of fresh canine NK cells

[0062] The isolated CD5-negative canine NK cells were divided into 9 portions, each containing 3 × 10⁶ cells. 6 CD5-negative canine NK cells were activated using freshly prepared 10 nM DAC activation medium (designated as the 10 nM DAC activation group), 20 nM DAC activation medium (designated as the 20 nM DAC activation group), and control activation medium without DAC (designated as the control group without DAC). The CD5-negative canine NK cells from each group were seeded into 6-well plates, and the corresponding activation medium was added to 1.5 mL to adjust the density of CD5-negative canine NK cells to 2 × 10⁶ cells / well. 6 The sample was prepared in 3 wells per group, with each well containing 1 sample per mL. The wells were shaken back and forth and left and right, and then incubated in a 37°C, 5% CO2 incubator for 48 hours.

[0063] (3) Expansion of fresh canine NK cells

[0064] After activation culture of each group of cells, 1.5 mL of freshly prepared canine NK cell expansion medium was slowly added along the well wall. The cells were then incubated at 37°C in a 5% CO2 incubator for 48 h. After centrifugation, samples were collected for counting, and freshly prepared canine NK cell expansion medium was added to adjust the cell density to 1.0 × 10⁶ cells / well. 6 cells / mL -1.5×10 6If cell proliferation is significant, cells are cultured in multiple wells. If cell proliferation is not significant, cells are centrifuged at 500g for 5 min, then cultured in the original wells. Samples are taken every 2 days for cell counting and replenishment of medium (canine NK cell expansion medium) to adjust the cell density to 0.5 × 10⁶ cells / mL. 6 cells / mL -1.0×10 6 If the cells are significantly expanded, they are cultured in multiple wells. If the cells are not significantly expanded, they are centrifuged at 500g for 5 minutes and then cultured in the original wells. After 18 days of expansion culture, canine NK cells are harvested.

[0065] The harvested canine NK cells were counted, and the total amount prepared was calculated.

[0066] The proliferation of canine NK cells harvested after 18 days of amplification culture is shown in the figure. Figure 2 The statistical results of the fold expansion of canine NK cells in each group are shown in the figure. Figure 3 .

[0067] Depend on Figure 2 and Figure 3 It can be seen that the addition of decitabine to the canine NK cell activation culture medium can significantly improve the proliferation capacity of canine NK cells, with the cell expansion fold increasing by 8-11 times compared with the absence of decitabine.

[0068] Canine NK cells treated with decitabine and IL2 only require IL15 to proliferate effectively. We hypothesize that the mechanism is that decitabine treatment demethylates key genes, alters the expression of key genes related to canine NK cell activation, and lowers the signal threshold required for activation and expansion.

[0069] III. In vitro antitumor evaluation of canine NK cells

[0070] 1. Cell preparation

[0071] (1) Vero cell resuscitation and passage

[0072] Vero cell resuscitation: Take one vial of Vero cells from a liquid nitrogen tank and place it into a cell transfer flask. Thaw the cells by rapid shaking in a 37°C water bath. Then transfer the cells to a 50mL centrifuge tube and add 9mL of DMEM high-glucose complete medium containing 10% (v / v) FBS to the centrifuge tube. Centrifuge at 200g speed and 20°C for 5min. Discard the supernatant and resuspend the cells in 10mL of DMEM high-glucose complete medium containing 10% (v / v) FBS. Transfer the cells to a 10cm Petri dish and incubate at 37°C in a 5% CO2 incubator for 3 days.

[0073] Vero cell digestion: Remove Vero cells from the incubator and observe the cells. When the confluence of Vero cells reaches 90%-100%, they can be passaged. Discard the cell solution in the 10cm petri dish, rinse the cells with 5mL PBS buffer, discard the rinse solution, add 2mL of EDTA solution containing 0.25% (w / v) trypsin, digest for 45s, discard the trypsin, place the petri dish in a 37℃, 5% CO2 incubator for 5min, and then resuspend the cells in 3mL of DMEM high-glucose complete medium containing 10% (v / v) FBS.

[0074] Vero cell counting: Take 20 μL of cell suspension and 20 μL of 0.4% (w / v) trypan blue solution, mix them thoroughly, and then count the cells on a cell counting device.

[0075] Vero cell passage: Resuspend the digested Vero cells in DMEM high-glucose complete medium containing 10% (v / v) FBS, and seed them into new T175 cell culture flasks at a volume ratio of 1:3-6. Add DMEM high-glucose complete medium containing 10% (v / v) FBS to the appropriate volume, and continue to culture in a 37℃, 5% CO2 incubator for 3 days.

[0076] (2) Canine NK cell culture

[0077] The isolated CD5-negative canine NK cells were added to freshly prepared 10 nM DAC activation medium, and the cell density was adjusted to 2 × 10⁶ cells / day. 6 Cells were cultured at a density of 1.0 × 10⁶ cells / mL, gently shaken back and forth and side to side, and then incubated at 37°C in a 5% CO₂ incubator for 48 hours. After cell activation culture, freshly prepared canine NK cell expansion medium was slowly added along the culture flask wall. The cells were then incubated at 37°C in a 5% CO₂ incubator for 48 hours, followed by centrifugation, cell counting, and further addition of freshly prepared canine NK cell expansion medium to adjust the cell density to 1.0 × 10⁶ cells / mL. 6 cells / mL -1.5×10 6 If the cell density is significantly increased (cells / mL), then expand the culture. If there is no significant cell expansion, centrifuge the cells at 500g for 5 minutes, then maintain the original culture in the flask. Take samples every 2 days to count and replenish the medium (canine NK cell expansion medium) to adjust the cell density to 0.5 × 10⁶ cells / mL. 6 cells / mL -1.0×10 6 If the cells are significantly expanded at a density of 10 cells / mL, then the cells are expanded for further culture. If the cells are not significantly expanded, then the cells are centrifuged at 500g for 5 minutes and then cultured in the original bottle. After 18 days of expansion culture, canine NK cells are harvested.

[0078] 2. Co-culture of canine NK cells and Vero cells

[0079] (1) Vero cell seeding and culture

[0080] 10,000 Vero cells were seeded into 24-well flat-bottomed culture plates, and an appropriate amount of DMEM high-glucose complete medium containing 10% (v / v) FBS was added to each well (the final volume of the medium was 400 μL). A total of 15 wells were set up and labeled as follows: control group (Vero cells alone), effector-to-target ratio 2.5:1 group, effector-to-target ratio 5:1 group, effector-to-target ratio 10:1 group, and effector-to-target ratio 20:1 group. Each group had 3 replicates. The plates were incubated at 37°C in a 5% CO2 incubator for 15 ± 1 h.

[0081] (2) Co-culture of canine NK cells (effect cells) and Vero cells (target cells)

[0082] Canine NK cells were resuspended in canine NK cell expansion medium and divided into four tubes, with the density of canine NK cells adjusted to 2.5 × 10⁶ cells / tube. 6 cells / mL (referred to as tube A), 5.0 × 10 6 cells / mL (referred to as tube B), 10.0 × 10 6 cells / mL (denoted as tube C) and 20.0 × 10 6 The suspension volume was 800 μL per tube (cells / mL). The DMEM high-glucose complete medium in the original 24-well flat-bottom culture plate was removed. 100 μL of canine NK cells from tubes A, B, C, and D were added to the wells of the effector-to-target ratio groups of 2.5:1, 5:1, 10:1, and 20:1, respectively. 300 μL of canine NK cell expansion medium was then added to each well. The wells of the control group were incubated with only 400 μL of canine NK cell expansion medium. The cells were incubated at 37°C in a 5% CO2 incubator for 15 ± 1 h.

[0083] (3) Digestion and collection of Vero cells

[0084] Remove the culture plate from the incubator after incubation, aspirate the culture medium, digest the remaining Vero cells in the wells, add 100 μL of trypsin to each well, digest into single cells, resuspend in serum-containing DMEM high-glucose complete medium, transfer to EP tubes for mixing, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cells in 100 μL of serum-containing DMEM high-glucose complete medium, and count using trypan blue staining.

[0085] 3. Experimental Results

[0086] Vero cell count results for each group are shown below. Figure 4 The statistical results of the killing rate of canine NK cells against Vero cells at various effector-to-target ratios are shown in the figure. Figure 5 .

[0087]

[0088] Depend on Figure 4 and Figure 5 It can be seen that the antitumor characteristics of canine NK cells harvested after 18 days of amplification and culture using the method provided by this invention remained unchanged, ensuring the normal quality of NK cell characteristics; as the effector-target ratio increased, the killing rate of canine NK cells against Vero cells steadily increased.

[0089] IV. In vivo antitumor evaluation of canine NK cells

[0090] 1. Laboratory animals and their housing conditions

[0091] NCG mice, SPF grade, 7 weeks old, female.

[0092] They are kept at 20-26℃ and 40-70% humidity, with a 12-hour light-dark cycle (12L:12D). They have free access to food and water and can adapt to being kept for 7 days.

[0093] 2. Tumor cell inoculation

[0094] Experimental cells: K562-Luc cells were revived, and the revival passage was Pn+14.

[0095] On the day of inoculation, K562-Luc cells in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS buffer. Then, they were inoculated into the tail vein of mice at a dose of 1 × 10⁻⁶ cells / mL. 6 A mouse model of K562-Luc leukemia was established by tail vein tumor bearing 200 μL / mouse. The survival rates of K562-Luc cells before and after tumor bearing were 97.8% and 95.9%, respectively.

[0096] 3. Canine NK cell culture

[0097] The isolated canine CD5-negative NK cells were added to freshly prepared 10 nM DAC activation medium, and the cell density was adjusted to 2 × 10⁶ cells / day. 6 Cells were cultured at a density of 1.0-1.5 × 10⁶ cells / mL, gently shaken back and forth and side to side, and then incubated at 37°C in a 5% CO₂ incubator for 48 hours. After cell activation, freshly prepared canine NK cell expansion medium was slowly added along the culture flask wall. The cells were then incubated at 37°C in a 5% CO₂ incubator for 48 hours, followed by centrifugation, cell counting, and further addition of freshly prepared canine NK cell expansion medium to adjust the cell density to 1.0-1.5 × 10⁶ cells / mL. 6 Cells were cultured at a density of [number] cells / mL, and expanded according to the volume of the cell suspension. If there was no significant cell expansion, the cells were centrifuged at 500g for 5 minutes, and then cultured in the original flask. Samples were taken every 2 days for counting and replenishment of medium (canine NK cell expansion medium) to adjust the cell density to 0.5-1.0 × 10⁶ cells / mL.6 Cells / mL were cultured to expand the cell suspension. If there was no significant cell expansion, the cells were centrifuged at 500g for 5 minutes and then cultured in the original bottle. After 18 days of expansion culture, canine NK cells were harvested.

[0098] 4. Group administration

[0099] On the 5th day after inoculation (modeling), in vivo fluorescence imaging was used to detect the tumor burden and perform data analysis. The average photon count was 5.79 × 10⁻⁶. 4 (p / sec / cm) 2 Eight mice were randomly divided into two groups: a non-treatment control group and an NK cell administration group, with four mice in each group. The NK cell administration group received canine NK cells harvested after 18 days of expansion culture on the same day (day 5 post-inoculation). The dosage of canine NK cells was 1×10⁻⁶. 4 Animals per kg were treated. The non-treatment control group received no treatment. After treatment, 0.1 mL / animal of IL2 was injected subcutaneously at 30 min, 48 h, and 96 h. The concentration of IL2 was 100 IU / mL.

[0100] 5. Experimental Results

[0101] Twenty-nine days after modeling, in vivo imaging results of mice in the non-treatment control group and the NK cell-treated group are shown in the figure. Figure 6 .

[0102] Depend on Figure 6 It is known that canine NK cells harvested after 18 days of amplification and culture using the method provided in this invention have anti-tumor effects.

[0103] The above in vitro and in vivo killing data show that the antitumor characteristics of canine NK cells harvested after 18 days of expansion and culture using the method provided in this invention remain unchanged, ensuring normal NK cell quality.

[0104] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A method for simply and inexpensively culturing canine NK cells in vitro, characterized by, The method comprises the following steps: (1) isolating NK cells from peripheral blood of a dog, resuspending the NK cells with freshly prepared dog NK cell activation culture medium, inoculating the NK cells into a culture bottle, adding freshly prepared dog NK cell activation culture medium, shaking the culture bottle, and then placing the culture bottle into a 37 DEG C, 5% CO2 incubator for culture for 24-48 hours; (2) after the activation culture is completed, slowly supplementing freshly prepared dog NK cell expansion culture medium along the side wall of the culture bottle, and then placing the culture bottle into a 37 DEG C, 5% CO2 incubator for culture for 48 hours; sampling and counting, supplementing freshly prepared dog NK cell expansion culture medium, and if the cells are obviously expanded, expanding the cells in a bottle or transferring the cells into a cell culture bag for culture; if the cells are not obviously expanded, centrifuging the cells and maintaining the cells in the original bottle for culture; subsequently, sampling and counting every 2 days to supplement liquid; if the cells are obviously expanded, expanding the cells in a bottle or transferring the cells into a cell culture bag for culture; if the cells are not obviously expanded, centrifuging the cells and maintaining the cells in the original bottle for culture; and harvesting dog NK cells after expansion culture for 12-18 days; wherein the dog NK cell activation culture medium is prepared by adding recombinant human IL2, recombinant human IL-15, decitabine and fetal bovine serum into PRMI 1640 as a basic culture medium, and the concentrations are 100 IU / mL, 1000 U / mL, 10-20 nM and 10%, respectively; the dog NK cell expansion culture medium is prepared by adding recombinant human IL-15 and fetal bovine serum into PRMI 1640 as a basic culture medium, and the concentrations are 2000 U / mL and 10%, respectively.

2. The method for simply and inexpensively culturing canine NK cells in vitro according to claim 1, characterized by, In step (1), the culture bottle is a T75 cell culture bottle.

3. The method for simply and inexpensively culturing canine NK cells in vitro according to claim 1, wherein In step (1), the density of the canine NK cells was adjusted to 2 x 10 6 cells / mL before culture.

4. The method for simply and inexpensively culturing canine NK cells in vitro according to claim 1, wherein In step (1), the culture time is 48 hours.

5. The method for simply and inexpensively culturing canine NK cells in vitro according to claim 1, wherein In step (2), the second time the canine NK cell expansion medium is added, the cell density is adjusted to 1.0 x 10 6 cells / mL - 1.5 x 10 6 cells / mL.

6. The method for simply and inexpensively culturing canine NK cells in vitro according to claim 1, wherein In step (2), the cell density was adjusted to 0.5 x 10 6 6 / mL - 1.0 x 10 6 6 / mL every 2 days when the medium was replenished.

7. The method for simply and inexpensively culturing canine NK cells in vitro according to claim 1, wherein the canine peripheral blood mononuclear cells are separated from the blood of a dog by using a Ficoll-Paque density gradient method. In step (2), the dog NK cells are harvested after expansion culture for 18 days. In step (1), the culture bottle is a T75 cell culture bottle. In step (1), the culture time is 48 hours. In step (2), the dog NK cells are harvested after expansion culture for 18 days.

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