An in vitro expansion method and application of canine nk cells with high efficiency in killing tumor cells

Abstract

The application belongs to the technical field of bioengineering, and specifically discloses an in-vitro expansion method and application of dog NK cells with high tumor cell killing efficiency, which comprises the following steps: recovering K562 engineering cells stored by freezing in sequence; taking 3 mL of dog peripheral blood to obtain dog peripheral blood mononuclear cells (PBMC); activating and expanding culture of dog NK cells with high tumor cell killing efficiency; collecting the expanded dog NK cells with high tumor cell killing efficiency, and storing them in a freezing medium at a cell density of 1×10 8 The application can obtain dog NK cells with high expansion multiple from only 3 mL of dog peripheral blood; the number of dog NK cells of a healthy volunteer dog sample is 3.02×10 8 after 11 days of continuous expansion culture, and the expansion multiple is 1008; the total number of dog NK cells obtained from a tumor volunteer dog sample reaches 6.84×10 7 , and the expansion multiple is 340; and the dog NK cells obtained by the application have high tumor cell killing efficiency.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method and application for the in vitro expansion of canine NK cells that efficiently kill tumor cells. Background Technology

[0002] Natural killer (NK) cells, as important cells of innate immunity, are mainly distributed in peripheral blood, liver, and spleen. NK cells are not restricted by the major histocompatibility complex (MHC) and can recognize various tumor cells, senescent cells, and viruses without antibody dependence, and kill target cells. They play an important role in the body's anti-tumor, antiviral, immune regulation, and clearance of senescent cells.

[0003] In human medicine, NK cells are effective in treating hematologic malignancies and have also shown therapeutic potential in solid tumors. Some researchers have discovered that canine NK cells possess anti-tumor potential, such as in canine mammary cancer, osteosarcoma, melanoma, adenocarcinoma, and glioma.

[0004] Although adoptive immunotherapy using canine NK cells holds great promise, ethical and invasive concerns have limited veterinary research to date, with most studies using NK cells isolated from peripheral blood. Since NK cells comprise only 2.5%–15% of peripheral lymphocytes, and in tumor treatment applications, multiple infusions (7.5 × 10⁻⁶ cells per cell line) are necessary. 6 ~1×10 8 The NK cell count is low ( / kg), thus requiring a large amount of NK cells, which limits its clinical application.

[0005] Because research on canine NK cells is still in its early stages and the biological characteristics of canine NK cells are not fully understood, current methods for in vitro expansion of canine NK cells mainly draw on research experience in human medicine. Currently, common in vitro expansion methods for NK cells include the antibody-cytokine mixture method and the feeder cell-cytokine mixture method. The former generates activation signals by adding multiple antibodies and cytokines, thereby stimulating NK cell proliferation. However, this method produces NK cells with low purity, and in the case of challenging samples, the failure rate is high due to overstimulation and depletion of NK cells caused by excessive cytokines, resulting in higher production costs. The latter method generates activation signals by adding NK cells that recognize sensitive feeder cells and cytokines. This method is simpler, avoids exogenous contamination, has higher expansion efficiency, and lower cost. The memory generated by NK cells through contact with feeder cells can increase cytotoxicity, making it the best choice for in vitro expansion of canine NK cells with high tumor cytotoxicity.

[0006] Therefore, in order to meet the needs of research on anti-tumor therapy and future clinical applications, it is urgent to develop an in vitro expansion method for canine NK cells with high expansion efficiency, high cell purity, and strong killing effect on tumor cells. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method and application for the in vitro expansion of canine NK cells that efficiently kill tumor cells.

[0008] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0009] One of the technical solutions of this invention provides an in vitro expansion method for canine NK cells that efficiently kill tumor cells, comprising the following steps:

[0010] S1. Reviving cryopreserved K562 engineered cells;

[0011] S2. Take 3 mL of canine peripheral blood and separate canine peripheral blood mononuclear cells (PBMCs).

[0012] S3. Canine peripheral blood mononuclear cells (PBMCs) were resuspended in the activation composition and / or amplification composition and cultured continuously at 37°C and 5% CO2 for 11 days. After amplification culture on day 11, the cells were centrifuged at 700g for 5 minutes to obtain canine NK cells. The activation composition consisted of K562 engineered cells, Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The number of K562 engineered cells was 0.5–1 times that of canine peripheral blood mononuclear cells (PBMCs). The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine was 94:5:1. The concentrations of canine interleukin-2 were 1000–2500 IU / mL, vitamin B6 was 20.5–30.8 ng / mL, and vitamin C was 100–200 μg / mL. The amplification composition consisted of Advance RPMI... The medium consisted of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine was 94:5:1. The concentration of canine interleukin-2 was 1000–2500 IU / mL, the concentration of vitamin B6 was 20.5–30.8 ng / mL, and the concentration of vitamin C was 100–200 μg / mL.

[0013] S4. Collect the expanded, highly efficient tumor-killing canine NK cells, at a cell density of 1×10⁶. 8 Cells / mL are stored in cryopreservation solution.

[0014] Further, step S1 specifically includes:

[0015] After removing the frozen K562 engineered cells from the -80℃ freezer, place them in a 37℃ water bath and shake rapidly to thaw. Centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend in PBS, stain with trypan blue and count the cells. Divide the cells into tubes according to the required number, centrifuge at 1200 rpm for 5 min, discard the supernatant, and set aside for later use.

[0016] Further, step S2 specifically includes:

[0017] S21. Obtain 3 mL of fresh canine peripheral blood from volunteer dogs, dilute it with 3 mL of DPBS buffer, and slowly add the diluted canine peripheral blood to the upper layer of 6 mL of lymphocyte separation medium. Centrifuge at 700g for 30 min.

[0018] S22. After centrifugation, the middle white membrane layer was extracted into a centrifuge tube and washed twice with DPBS buffer to obtain washed canine peripheral blood mononuclear cells (PBMCs).

[0019] S23. Add red blood cell lysis buffer to the washed canine peripheral blood mononuclear cells (PBMCs), lyse at 4°C for 5 min, centrifuge at 300g for 10 min and discard the supernatant to obtain lysed canine peripheral blood mononuclear cells (PBMCs).

[0020] S24. Wash the lysed canine peripheral blood mononuclear cells (PBMCs) twice with DPBS buffer, centrifuge at 1200 rpm for 5 min, discard the supernatant, and obtain canine peripheral blood mononuclear cells (PBMCs).

[0021] Furthermore, step S3 specifically includes:

[0022] S31, Day 0: Canine peripheral blood mononuclear cells (PBMCs) were resuspended with the activation composition to achieve a cell density of 1 × 10⁻⁶. 6 Cells / mL, incubated at 37°C with 5% CO2, to a final volume of 6mL;

[0023] S32. On day 3, collect cells into 15mL centrifuge tubes, centrifuge at 700g for 5min, discard the supernatant, resuspend the cell clusters with the amplification composition, and incubate at 37℃ with 5% CO2.

[0024] S33, Day 5: Replenish with fresh amplification composition;

[0025] S34. On day 7, collect cells in 50mL centrifuge tubes, centrifuge at 700g for 5min, discard the supernatant and resuspend the cell clusters in DPBS buffer, count the cells to calculate the number of K562 engineered cells required for secondary stimulation; resuspend the cells using the activation composition and culture at 37℃ in 5% CO2.

[0026] S35. From day 8 to day 10, observe the cell status, change the medium and adjust the cell density. Replenish the amplification composition every 2 days to achieve a final cell density of 0.05 × 10⁻⁶ cells / day. 5 / mL~1×10 6 / mL;

[0027] S36. On day 11, the expanded cells were collected, centrifuged at 700g for 5 minutes, and the supernatant was discarded to obtain canine NK cells that effectively kill tumor cells.

[0028] The second technical solution of the present invention provides canine NK cells that are highly efficient at killing tumor cells, obtained by the above method.

[0029] The third technical solution of the present invention provides the application of highly efficient canine NK cells for killing tumor cells in the preparation of drugs for killing tumor cells.

[0030] Preferably, the tumor cells include, but are not limited to, canine breast cancer cells, canine osteosarcoma cells, canine melanoma cells, canine adenocarcinoma cells, and canine glioma cells.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) This invention requires only 3 mL of canine peripheral blood. The small amount of blood drawn can simultaneously meet the NK cell culture needs of small and large dogs. The expansion fold can also meet the autologous reinfusion needs of healthy and sick dogs, which is beneficial to the future clinical application of canine NK cell reinfusion.

[0033] (2) The canine NK cell culture composition of the present invention has simple components and does not require excessive cytokines and antibodies; the culture method provided by the present invention is simple and convenient.

[0034] (3) The canine NK cell amplification rate of the present invention is high. Using the above technical solution, after continuous amplification and culture for 11 days, the number of canine NK cells in healthy volunteer dog samples was 3.02 × 10⁻⁶. 8 The amplification factor was 1008-fold, and the total number of canine NK cells obtained from the tumor volunteer dog sample reached 6.84 × 10⁶. 7 One, with an amplification factor of 340 times.

[0035] (4) The canine NK cells obtained by this invention have a highly efficient tumor cell killing effect. The canine NK cells obtained by this invention have a 92% killing effect on K562 tumor cells at an effector-to-target ratio of 40:1, a 98% killing effect on the D17 solid tumor cell line (canine osteosarcoma), and a 63% killing effect on the CMT1211 cell line (canine mammary tumor), demonstrating the great potential of the canine NK cells obtained by this culture and expansion composition and method in the treatment of canine tumor diseases.

[0036] (5) No K562 engineered cells remain, ensuring high safety. The canine NK cells obtained by this invention can be used for adoptive immunotherapy after being expanded and cultured for 11 days, cryopreserved, and then thawed and cultured for 2 days, with no K562 engineered cells remaining. Attached Figure Description

[0037] Figure 1 Morphology of canine NK cells at different expansion and culture stages: A, Day 0; B, Day 7; C, Day 9; D, Day 11.

[0038] Figure 2 The growth curves of canine NK cells during different sample culture processes are shown.

[0039] Figure 3 The results of flow cytometry identification of canine NK cells after 11 days of amplification.

[0040] Figure 4 To investigate the killing effect of canine NK cells expanded for 7 days and revived for 2 days on K562, D17 and CMT1211 tumor cells at different effector-target ratios.

[0041] Figure 5 Morphological diagram of target cells after canine NK cells were co-cultured with K562, D17 and CMT 1211 tumor cell lines for 24 hours after 2 days of resuscitation culture.

[0042] Figure 6 Results of STR residue detection in canine NK cells 2 days after resuscitation and culture. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0044] Unless otherwise specified, the reagents designed in the following examples are all commercially available. For simplicity, some operations have not been described in detail, including the parameters, steps, and instruments used, all of which are well known and reproducible by those skilled in the art.

[0045] Explanation of the source of experimental materials:

[0046] K562 engineered cells: purchased from Sinocare Biotechnology (Heilongjiang) Co., Ltd.;

[0047] D17 cell line: purchased from ATCC;

[0048] CMT1211 cell line: a gift from China Agricultural University;

[0049] Advance RPMI 1640 culture medium is a commercially available product from GIBCO, catalog number 12633012;

[0050] Canine interleukin-2: purchased from Shandong Aibeirunkang Biotechnology Co., Ltd.;

[0051] LDH detection kit: purchased from Promega (Beijing) Biotechnology Co., Ltd.

[0052] Example 1

[0053] 1) Reviving cryopreserved K562 engineered cells

[0054] After removing the frozen K562 engineered cells from the -80℃ freezer, place them in a 37℃ water bath and shake them rapidly to thaw. Centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend in PBS, stain with trypan blue and count the cells. Divide the cells into tubes according to the required number, centrifuge at 1200 rpm for 5 min, discard the supernatant, and set aside.

[0055] 2) Canine peripheral blood mononuclear cells (PBMCs) were isolated;

[0056] ① Obtain 3 mL of fresh canine peripheral blood from volunteer dogs, dilute it with 3 mL of DPBS buffer, slowly add the diluted canine peripheral blood to the upper layer of 6 mL of lymphocyte separation medium, and centrifuge at 700g for 30 min.

[0057] ② After centrifugation, the middle white membrane layer was extracted into a centrifuge tube and washed twice with DPBS buffer to obtain washed canine peripheral blood mononuclear cells (PBMCs).

[0058] ③ Add red blood cell lysis buffer to the washed canine peripheral blood mononuclear cells (PBMCs), lyse at 4°C for 5 min, centrifuge at 300g for 10 min and discard the supernatant to obtain the lysed canine peripheral blood mononuclear cells (PBMCs).

[0059] ④ Wash the lysed canine peripheral blood mononuclear cells (PBMCs) twice with DPBS buffer, centrifuge at 1200 rpm for 5 min, discard the supernatant, and obtain canine peripheral blood mononuclear cells (PBMCs).

[0060] 3) Activation and expansion culture of canine NK cells that efficiently kill tumor cells

[0061] ① On day 0, canine peripheral blood mononuclear cells (PBMCs) were resuspended with the activation composition to achieve a cell density of 1 × 10⁻⁶. 6 Cells / mL, incubated at 37°C with 5% CO2, to a final volume of 6mL;

[0062] ② On day 3, cells were collected in 15mL centrifuge tubes, centrifuged at 700g for 5min, the supernatant was discarded, and the cell clusters were resuspended in the amplification composition and cultured at 37℃ with 5% CO2. In this example, the amplification composition consisted of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine was 94:5:1. The content of canine interleukin-2 was 1000 IU / mL, the content of vitamin B6 was 30.8 ng / mL, and the content of vitamin C was 200 μg / mL.

[0063] ③ On day 5, replenish with fresh amplification composition;

[0064] ④ On day 7, cells were collected in 50mL centrifuge tubes, centrifuged at 700g for 5min, the supernatant was discarded, and the cell clusters were resuspended in DPBS buffer. Cell counts were performed to calculate the number of K562 engineered cells required for secondary stimulation. Cells were resuspended using the activation composition and cultured at 37℃ in 5% CO2. In this example, the activation composition consisted of K562 engineered cells, Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The number of K562 engineered cells was 0.5 times that of canine peripheral blood mononuclear cells (PBMCs). The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine was 94:5:1. The content of canine interleukin-2 was 1000 IU / mL, the content of vitamin B6 was 30.8 ng / mL, and the content of vitamin C was 200 μg / mL.

[0065] ⑤ Observe the cell status from day 8 to day 10, change the medium and adjust the cell density, and replenish the amplification composition every 2 days to achieve a final cell density of 0.05 × 10⁻⁶. 5 / mL~1×10 6 / mL;

[0066] ⑥ On day 11, collect the expanded cells, centrifuge at 700g for 5 minutes, discard the supernatant, and obtain highly efficient tumor-killing canine NK cells. Propagate the cells at a rate of 1×10⁻⁶. 8 Cells / mL are stored in cryopreservation solution.

[0067] Example 2

[0068] The difference from Example 1 is that the activation composition used consists of K562 engineered cells, Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The number of K562 engineered cells is 0.5 times that of canine peripheral blood mononuclear cells (PBMCs). The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine is 94:5:1. The content of canine interleukin-2 is 2500 IU / mL, the content of vitamin B6 is 30.8 ng / mL, and the content of vitamin C is 100 μg / mL. The amplification composition consists of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The volume ratio of 1640 culture medium, fetal bovine serum, and glutamine was 94:5:1. The content of canine interleukin-2 was 2500 IU / mL, vitamin B6 was 30.8 ng / mL, and vitamin C was 100 μg / mL.

[0069] Example 3

[0070] The difference from Example 1 is that the activation composition used consists of K562 engineered cells, Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The number of K562 engineered cells is 0.5 times that of canine peripheral blood mononuclear cells (PBMCs). The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine is 94:5:1. The content of canine interleukin-2 is 2500 IU / mL, the content of vitamin B6 is 20.5 ng / mL, and the content of vitamin C is 100 μg / mL. The amplification composition consists of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The volume ratio of 1640 culture medium, fetal bovine serum, and glutamine was 94:5:1. The content of canine interleukin-2 was 2500 IU / mL, vitamin B6 was 20.5 ng / mL, and vitamin C was 100 μg / mL.

[0071] Comparative Example 1

[0072] The difference from Example 1 is that the number of K562 engineered cells in the activation composition used is twice that of canine peripheral blood mononuclear cells (PBMCs).

[0073] Comparative Example 2

[0074] The difference from Example 1 is that the activation composition used consists of K562 engineered cells, Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The number of K562 engineered cells is twice that of canine peripheral blood mononuclear cells (PBMCs). The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine is 94:5:1. The content of canine interleukin-2 is 2500 IU / mL, the content of vitamin B6 is 30.8 ng / mL, and the content of vitamin C is 200 μg / mL. The amplification composition consists of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The volume ratio of 1640 culture medium, fetal bovine serum, and glutamine was 94:5:1. The content of canine interleukin-2 was 2500 IU / mL, vitamin B6 was 30.8 ng / mL, and vitamin C was 100 μg / mL.

[0075] The cell counts of different activation and amplification compositions at different time points in Examples 1-3, Comparative Examples 1 and 2 were statistically analyzed, and the results are shown in Table 1. Morphological images of canine NK cells at different amplification stages were obtained as follows. Figure 1 As shown.

[0076] Table 1

[0077]

[0078] From Table 1 and Figure 1 It can be seen that as the amplification process proceeds, the activated canine NK cells increase in size, appearing irregular or comma-shaped, and the cell density increases, growing in clusters. The cell clusters become larger and larger with the amplification time, and no feeder cells were observed on the 7th and 11th days of amplification culture.

[0079] Furthermore, the expansion fold and cell phenotype of canine NK cells obtained in Examples 1-3, Comparative Example 1, and Comparative Example 2 were analyzed by flow cytometry:

[0080] 1) Folding of canine NK cell expansion

[0081] Cell counts were performed and growth curves were plotted using trypan blue staining on days 7, 9, and 11 of amplification culture, as shown in Table 1. Figure 2 As shown, the cell count increased rapidly on day 7, with the total cell count in healthy volunteer dogs reaching 7.03 × 10⁻⁶ on day 11. 8 The amplification factor was 234-fold, while the total number of cells in the tumor-bearing volunteer dogs reached 1.58 × 10⁻⁶ cells at 11 days. 8 One, with an amplification factor of 79 times.

[0082] 2) Flow cytometry detection of canine NK cell purity

[0083] Take 1×10⁻⁶ cells after 11 days of amplification culture. 6 Cells were washed once with DPBS and resuspended in 100 μl of DPBS containing 2% FBS. Flow cytometry antibodies against canine CD3, CD21, and CD94 were added to the cell suspension, and the cells were incubated in the dark for 30 min. Cells were then washed twice with 1 mL of DPBS and resuspended in 200 μl of DPBS. The proportion of CD3-CD21-CD94+ canine NK cells in the total cell count was determined using flow cytometry. The experimental results are shown in Table 1 and [Table data would be inserted here]. Figure 3 As shown, CD3-CD21- cells accounted for 98.85% of the cell population, and within the CD3-CD21- cell population, CD94+ NK cells, representing mature cells, accounted for 43.03%. Based on the CD3-CD21-CD94+ cell percentages, the number of NK cells in healthy volunteer dogs at day 11 was 3.02 × 10⁻⁶. 8 The number of cells increased 1008-fold, while the number of NK cells in the tumor-bearing volunteer dogs was 6.84 × 10⁶ at day 11. 7 One, amplified 340 times.

[0084] The above results demonstrate that the in vitro amplification and culture composition of the present invention can efficiently and significantly stimulate the proliferation of NK cells and amplify mature canine NK cells, even in poor samples from canines with tumors, achieving satisfactory amplification folds.

[0085] Application Example 1

[0086] To verify whether the drug obtained in this invention can be used to prepare a tumor-killing drug, this embodiment uses K562 cells, D17 cells, and CMT1211 cells as examples to test the killing effect of expanded canine NK cells on tumor cells, as detailed below:

[0087] Add 5 × 10⁻⁶ to a 96-well plate containing the amplification composition. 3 K562 cells and D17 cells were added to each well, and then canine NK cells that had been expanded for 7 days and revived for 2 days were added to the wells at E:T ratios of 40:1, 20:1, 10:1, 5:1, 2.5:1, and 1.25:1, respectively. The cells were then incubated for 4 hours. The NK cell control group, experimental group, background group, volume correction group, target cell self-release group, and target cell maximum release group were set up according to the LDH detection kit (CytoTox96 Non-Radioactive CytotoxicityAssay). Each group was set up with 3 replicates. After incubation, the value at 492nm was detected by a microplate reader, and the killing rate was calculated according to the formula.

[0088] Test results as follows Figure 4As shown, the killing rate of canine NK cells after 2 days of resuscitation and culture was higher than that after 7 days of expansion. At a ratio of 40:1, the killing rate of canine NK cells was the highest. The killing rate of canine NK cells after 2 days of resuscitation and culture was 92% against K562 cells, 98% against D17 canine osteosarcoma cells, and 63% against the CMT1211 canine mammary tumor cell line.

[0089] Two days after resuscitation, canine NK cells were co-cultured with K562, D17, and CMT1211 for 24 hours. The morphology of the target cells was as follows: Figure 5 As shown, after 24 hours of co-culture with canine NK cells, adherent D17 cells and CMT1211 cells showed incomplete cell membrane rupture, unclear boundaries, nucleus fragmentation, and sparse granular debris; K562 cells, surrounded by activated canine NK cells, lost their viable cell luster, showed incomplete cell membrane rupture, vacuolated cytoplasm, and eventually ruptured and died.

[0090] In addition, in this embodiment, after cryopreserving and thawing canine NK cells and continuing to culture them for 2 days, STR testing was used to detect the residual feeder cells, and the results were as follows. Figure 6 As shown. By Figure 6 It can be seen that after 11 days of expansion, STR testing using cryopreservation and resuscitation techniques revealed no residual K562 engineered cells used as feeder cells, indicating high safety.

[0091] The above results indicate that the canine NK cells obtained using this invention have strong cytotoxicity against tumor cells, showing great potential for future application in adjuvant therapy for tumor diseases and can be used to prepare drugs that kill tumor cells.

[0092] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for in vitro expansion of canine NK cells that efficiently kill tumor cells, characterized in that, Includes the following steps: S1. Thawing frozen K562 engineered cells: After removing the frozen K562 engineered cells from the -80℃ freezer, place them in a 37℃ water bath and shake rapidly to thaw. Centrifuge at 1200 rpm for 5 min, discard the supernatant, resuspend in PBS, stain with trypan blue, count the cells, divide into tubes according to the required number of cells, centrifuge at 1200 rpm for 5 min, discard the supernatant, and set aside. S2. Take 3 mL of canine peripheral blood and separate canine peripheral blood mononuclear cells (PBMCs). S21. Obtain 3 mL of fresh canine peripheral blood from volunteer dogs, dilute it with 3 mL of DPBS buffer, and slowly add the diluted canine peripheral blood to the upper layer of 6 mL of lymphocyte separation medium. Centrifuge at 700g for 30 min. S22. After centrifugation, the middle white membrane layer was extracted into a centrifuge tube and washed twice with DPBS buffer to obtain washed canine peripheral blood mononuclear cells (PBMCs). S23. Add red blood cell lysis buffer to the washed canine peripheral blood mononuclear cells (PBMCs), lyse at 4°C for 5 min, centrifuge at 300g for 10 min and discard the supernatant to obtain lysed canine peripheral blood mononuclear cells (PBMCs). S24. Wash the lysed canine peripheral blood mononuclear cells (PBMCs) twice with DPBS buffer, centrifuge at 1200 rpm for 5 min, discard the supernatant, and obtain canine peripheral blood mononuclear cells (PBMCs). S3. Canine peripheral blood mononuclear cells (PBMCs) were resuspended in the activation and amplification compositions and cultured continuously at 37°C and 5% CO2 for 11 days. After amplification culture on day 11, the cells were centrifuged at 700g for 5 minutes to obtain canine NK cells. The activation composition consisted of K562 engineered cells, Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The number of K562 engineered cells was 0.5–1 times that of canine PBMCs. The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine was 94:5:

1. The concentrations of canine interleukin-2 were 1000–2500 IU / mL, vitamin B6 was 20.5–30.8 ng / mL, and vitamin C was 100–200 μg / mL. The amplification composition consisted of Advance RPMI… The medium consisted of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin-2, vitamin B6, and vitamin C. The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine was 94:5:

1. The concentration of canine interleukin-2 was 1000–2500 IU / mL, the concentration of vitamin B6 was 20.5–30.8 ng / mL, and the concentration of vitamin C was 100–200 μg / mL. S31, Day 0: Canine peripheral blood mononuclear cells (PBMCs) were resuspended with the activation composition to achieve a cell density of 1 × 10⁻⁶. 6 Cells / mL were cultured at 37°C with 5% CO2 until a final volume of 6 mL. S32. On day 3, collect cells into 15 mL centrifuge tubes, centrifuge at 700g for 5 min, discard the supernatant, resuspend the cell clusters with the amplification composition, and incubate at 37℃ with 5% CO2. S33, Day 5: Replenish with fresh amplification composition; S34. On day 7, cells were collected in 50 mL centrifuge tubes, centrifuged at 700g for 5 min, the supernatant was discarded and the cell clusters were resuspended in DPBS buffer. Cell counts were performed to calculate the number of K562 engineered cells required for secondary stimulation. Cells were resuspended using the activation composition and cultured at 37°C in 5% CO2. S35. From day 8 to day 10, observe the cell status, change the medium and adjust the cell density. Replenish the amplification composition every 2 days to achieve a final cell density of 0.05 × 10⁻⁶ cells / day. 5 / mL~1×10 6 / mL; S36. On day 11, the expanded cells were collected, centrifuged at 700g for 5 minutes, and the supernatant was discarded to obtain canine NK cells that are highly effective at killing tumor cells. S4. Collect the expanded, highly efficient tumor-killing canine NK cells, at a cell density of 1×10⁶. 8 Cells / mL are stored in cryopreservation solution.

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