A clinical canine natural killer cell cryopreservation solution and a preparation method and application thereof

Abstract

This invention belongs to the technical field of cell preservation, specifically relating to a clinical cryopreservation solution for canine natural killer cells, its preparation method, and its application. The invention uses physiological saline as a solvent and dextran 40, a complex amino acid, canine serum albumin, canine interleukin-2, and sodium bisulfite as active ingredients. The complex amino acid includes three or more of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine. The cell cryopreservation solution provided by this invention is free of serum and DMSO, has a stable and safe composition, and the components work synergistically to provide a stable survival environment for canine natural killer cells. After preservation at 4℃~8℃ for 72 hours, the viability of canine natural killer cells can be maintained at over 80%, achieving in vitro preservation of canine natural killer cells while retaining good killing activity against canine mammary tumor cells.

Description

Technical Field

[0001] This invention belongs to the technical field of cell preservation, specifically relating to a clinical canine natural killer cell cryopreservation solution, its preparation method, and its application. Background Technology

[0002] In recent years, cancer has been a persistent threat to canine health, a common cause of death in clinical practice, and severely impacts the survival and quality of life of pet dogs. While traditional cancer treatments such as surgery, radiotherapy, and chemotherapy can improve survival and prognosis to some extent, the quality of life for affected dogs remains difficult to guarantee. Adoptive immunotherapy is currently a hot research topic in cancer treatment. This involves adoptively transferring autologous or allogeneic expanded immune cells, such as T cells, dendritic cells, and natural killer cells, into the patient to restore the reduced number of immune cells caused by cancer and enhance the body's immunity. Natural killer cells can kill tumor cells without prior antigen sensitization, and allogeneic adoptive therapy does not cause graft-versus-host disease, thus showing great promise in the field of cancer immunotherapy.

[0003] As the phenotype of canine natural killer (NSC) cells has become clearer and in vitro expansion systems have matured, NSC cells are increasingly being used in canine tumor treatment. Studies have shown that canine NSC cells have good efficacy against osteosarcoma and mammary tumors, indicating a promising future for their application. However, the production and clinical use of canine NSC cells are often difficult to coordinate, requiring in vitro expanded cells to be transported to veterinary hospitals. Most treatment facilities only conduct quality control on delivered cell products before cell reinfusion / treatment, easily neglecting cell quality control and management throughout the entire transportation process. From cell collection to final treatment, under normal temperature or 37°C conditions, intracellular biochemical reactions are active, metabolism is vigorous, oxygen consumption is high, and energy consumption is significant. These physiological processes generate a large number of oxygen free radicals and lipid peroxides, which must be promptly removed; otherwise, changes in environmental pH and osmotic pressure will occur, leading to cell swelling and death. Therefore, a convenient and cost-effective method for preserving canine NSC cells is needed.

[0004] Currently, cell preservation methods mainly include cryopreservation and refrigeration. Cryopreservation involves storing cells at liquid nitrogen temperature (-196℃), typically requiring serum and dimethyl sulfoxide (DMSO). However, DMSO has cytotoxic, vascular, and hepatotoxic effects. If frozen cells are used directly after thawing, excessive DMSO can harm the dog's body. Furthermore, this cryopreservation method requires cryogenic equipment for freezing and thawing, and a thawing process is necessary before transplantation. The freeze-thaw and thawing processes cause some damage to all cells and tissues, posing a challenge to the equipment requirements of veterinary hospitals. Refrigeration uses low-temperature conditions (2-8℃), which reduces oxygen free radicals and lipid peroxides produced during normal cellular physiological processes. The delivered cells require no special treatment and can be used directly, making it less costly than cryopreservation. However, the refrigeration of canine natural killer cells requires a special preservation solution that is not only safe and easy to handle, but also ensures that the viability and killing activity are not significantly affected after preservation. Therefore, in order to ensure the cold storage and transportation of canine natural killer cells, it is particularly important to develop a cell cold storage solution that can efficiently preserve the activity of canine natural killer cells. Summary of the Invention

[0005] The purpose of this invention is to provide a clinical canine natural killer cell cryopreservation solution, its preparation method, and its application, which efficiently preserves the activity of canine natural killer cells and achieves short-term preservation of canine natural killer cells.

[0006] To achieve the above objectives, the present invention provides a cell cryopreservation solution that does not contain serum and DMSO, and includes active ingredients and solvents;

[0007] The active ingredients include dextran 40, complex amino acids, canine serum albumin, canine interleukin-2, and sodium bisulfite.

[0008] The complex amino acid includes three or more of the following: leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine.

[0009] The solvent includes physiological saline.

[0010] Preferably, the concentration of dextran 40 in the refrigerated preservation solution is 45–60 mg / mL;

[0011] The concentration of canine serum albumin in the refrigerated preservation solution is 10–25 mg / mL;

[0012] The concentration of canine interleukin-2 in the refrigerated preservation solution is 100–600 IU / mL;

[0013] The concentration of sodium bisulfite in the refrigerated preservation solution is 0.38–0.5 mg / mL;

[0014] The concentration of the complex amino acids in the refrigerated preservation solution is 9.61–27.2 mg / mL.

[0015] Preferably, the concentration of dextran 40 in the refrigerated preservation solution is 50 mg / mL;

[0016] The concentration of canine serum albumin in the refrigerated preservation solution was 20 mg / mL;

[0017] The concentration of canine interleukin-2 in the refrigerated preservation solution was 400 IU / mL;

[0018] The concentration of sodium bisulfite in the refrigerated preservation solution is 0.38 mg / mL.

[0019] Preferably, the composite amino acid is composed of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine; the mass ratio of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine in the composite amino acid is 3.08:1.35:2.18:1.35:0.45:1.8:2.55:4.65:1.65:0.75:1.5.

[0020] Alternatively, the complex amino acid is composed of leucine, isoleucine, phenylalanine, tryptophan, and glycine; the mass ratio of leucine, isoleucine, phenylalanine, tryptophan, and glycine in the complex amino acid is 3.08:1.35:2.18:0.45:2.55.

[0021] Alternatively, the complex amino acid is composed of threonine, tryptophan, methionine, lysine hydrochloride, arginine, histidine, and valine; the mass ratio of threonine, tryptophan, methionine, lysine hydrochloride, arginine, histidine, and valine in the complex amino acid is 1.35:0.45:1.8:4.65:1.65:0.75:1.5.

[0022] The present invention also provides a method for preparing the refrigerated preservation solution described in the above technical solution, wherein the active ingredient is dissolved using the solvent.

[0023] The present invention also provides the application of the cryopreservation solution described in the above technical solution or the cryopreservation solution obtained by the preparation method described in the above technical solution in the preservation of canine natural killer cells.

[0024] This invention also provides a method for preserving canine natural killer cells for clinical use, comprising the following steps:

[0025] The canine natural killer cells are mixed with the cryopreservation solution described in the above technical solution or the cryopreservation solution prepared by the above technical solution to obtain a cell suspension, which is then stored at 4–8°C.

[0026] Preferably, the density of canine natural killer cells in the cell suspension is 1×10⁻⁶. 6 ~1×10 7 per mL.

[0027] Preferably, the density of canine natural killer cells in the cell suspension is 5 × 10⁻⁶. 6 per mL.

[0028] The present invention also provides the application of the refrigeration preservation solution described in the above technical solution or the refrigeration preservation solution obtained by the preparation method described in the above technical solution in the preparation of canine mammary tumor cell killing products.

[0029] Beneficial effects:

[0030] This invention uses physiological saline as a solvent and dextran 40, complex amino acids, canine serum albumin, canine interleukin-2, and sodium bisulfite as active ingredients. The complex amino acids include three or more of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine. The cryopreservation solution provided by this invention is free of serum and DMSO, and its composition is stable and safe. The components in the resulting cell cryopreservation solution work synergistically to provide canine natural killer cells with a stable survival environment, the energy required for basal metabolism, and mechanical protection, thus stably maintaining cellular metabolic homeostasis and enabling in vitro preservation of canine natural killer cells, increasing their preservation time. Example results show that after preservation at 4℃~8℃ for 72 hours, the viability of canine natural killer cells can remain above 80%, retaining good proliferative activity. They can be directly infused into dogs, retaining good killing activity against canine mammary tumor cells, providing convenience for short-distance transportation and clinical application of canine natural killer cells. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0032] Figure 1 Microscopic image (100×) of canine natural killer cells after being preserved in the cell cryopreservation solution of Example 1 for 72 hours and then re-inoculated into culture flasks for 24 hours.

[0033] Figure 2This is a comparison of the killing activity of canine natural killer cells (NKCs) against canine mammary tumor cells after being preserved for 72 hours using the cell cryopreservation solution from Example 1, compared with that of canine NKCs that were not cryopreserved. Detailed Implementation

[0034] This invention provides a cell cryopreservation solution that does not contain serum or DMSO, and includes active ingredients and solvents;

[0035] The active ingredients include dextran 40, complex amino acids, canine serum albumin, canine interleukin-2, and sodium bisulfite.

[0036] The complex amino acid includes three or more of the following: leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine.

[0037] The solvent includes physiological saline.

[0038] In this invention, the concentration of dextran 40 in the cell cryopreservation solution is preferably 45-60 mg / mL, more preferably 50-55 mg / mL. This invention uses dextran 40 as the active ingredient, which can reduce the damage of low temperature to canine natural killer cells, maintain the osmotic pressure of the preservation solution, and prevent excessive cell aggregation during preservation.

[0039] In this invention, the concentration of canine serum albumin in the cell cryopreservation solution is preferably 10–25 mg / mL, more preferably 150–550 IU / mL, even more preferably 200–500 IU / mL, and most preferably 300–400 IU / mL. This invention uses canine serum albumin as the active ingredient to provide cells with mechanical protection and the energy required for basal metabolism.

[0040] In this invention, the concentration of canine interleukin-2 in the cell cryopreservation solution is preferably 100–600 IU / mL, more preferably 200–550 IU / mL, even more preferably 300–500 IU / mL, and most preferably 400 IU / mL. This invention uses canine interleukin-2 as the active ingredient, which can promote the activation and expansion of natural killer cells and affect the preservation status of canine natural killer cells.

[0041] In this invention, the concentration of sodium bisulfite in the cell cryopreservation solution is preferably 0.38–0.5 mg / mL, more preferably 0.4–0.45 IU / mL. This invention uses sodium bisulfite as the active ingredient to adjust the pH and osmotic pressure of the in vitro cryopreservation solution for canine natural killer cells.

[0042] In this invention, the concentration of the complex amino acids in the cell cryopreservation solution is preferably 9.61–27.2 mg / mL, more preferably 12.15–27 mg / mL, even more preferably 21.31–26 mg / mL, and most preferably 22–25 mg / mL. This invention uses complex amino acids as the active ingredient to provide canine natural killer cells with nutrients and the energy required for basal metabolism to maintain their normal life activities.

[0043] In this invention, the complex amino acid comprises three or more of the following: leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine. Preferably, it is composed of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine; or it is composed of leucine, isoleucine, phenylalanine, tryptophan, and glycine; or it is composed of threonine, tryptophan, methionine, lysine hydrochloride, arginine, histidine, and valine. When the composite amino acid is composed of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine, the preferred mass ratio of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine in the composite amino acid is 3.08:1.35:2.18:1.35:0.45:1.8:2.55:4.65:1.65:0.75:1.5; when the composite amino acid is composed of leucine, isoleucine, phenylalanine, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine, the preferred mass ratio of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine is 3.08:1.35:2.18:1.35:0.45:1.8:2.55:4.65:1.65:0.75:1.5. When the compound amino acid is composed of alanine, tryptophan, and glycine, the preferred mass ratio of leucine, isoleucine, phenylalanine, tryptophan, and glycine is 3.08:1.35:2.18:0.45:2.55. When the compound amino acid is composed of threonine, tryptophan, methionine, lysine hydrochloride, arginine, histidine, and valine, the preferred mass ratio of threonine, tryptophan, methionine, lysine hydrochloride, arginine, histidine, and valine is 1.35:0.45:1.8:4.65:1.65:0.75:1.5.

[0044] In this invention, when the complex amino acids include leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine, the concentration of leucine in the refrigerated preservation solution is preferably 3.08 mg / mL; the concentration of leucine in the refrigerated preservation solution is preferably 3.08 mg / mL; the concentration of isoleucine in the refrigerated preservation solution is preferably 1.35 mg / mL; the concentration of phenylalanine in the refrigerated preservation solution is preferably 2.18 mg / mL; and the concentration of threonine in the refrigerated preservation solution is preferably 1.35 mg / mL. The concentrations of valine, tryptophan, methionine, glycine, lysine hydrochloride, and valine in the refrigerated preservation solution are preferably 1.5 mg / mL, 0.45 mg / mL, 1.8 mg / mL, 2.55 mg / mL, 4.65 mg / mL, 1.65 mg / mL, and 0.75 mg / mL, respectively. This invention combines leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine to provide the essential amino acids for the life activities of canine natural killer cells, enabling them to obtain more sufficient nutrients and a more favorable environment for growth, thus better maintaining their normal biological activity.

[0045] This invention does not have strict requirements on the source of the components in the cell cryopreservation solution; conventionally obtained sources are acceptable. The combination of the above components in this invention results in a cell cryopreservation solution with safe and stable components, providing canine natural killer cells with sufficient nutrition, necessary activating substances, and a stable osmotic pressure and pH environment. This enables the in vitro preservation of canine natural killer cells while retaining good killing activity against canine mammary tumor cells.

[0046] The present invention also provides a method for preparing the refrigerated preservation solution described in the above technical solution, wherein the active ingredient is dissolved using the solvent.

[0047] In view of the function of the refrigeration preservation solution provided by the present invention, the application of the refrigeration preservation solution in the preservation of canine natural killer cells and / or in the preparation of canine mammary tumor cell killing products also fall within the protection scope of the present invention.

[0048] This invention also provides a method for preserving canine natural killer cells for clinical use, comprising the following steps:

[0049] The canine natural killer cells are mixed with the cryopreservation solution described in the above technical solution or the cryopreservation solution prepared by the above technical solution to obtain a cell suspension, which is then stored at 4–8°C.

[0050] In this invention, the density of canine natural killer cells in the cell suspension is preferably 1 × 10⁻⁶. 6 ~1×10 7 Cells / mL, more preferably 5 × 10⁻⁶ 6 per mL.

[0051] Using the cell cryopreservation solution provided by this invention, canine natural killer cells can be preserved for 72 hours at 4℃~8℃. The viability of the canine natural killer cells can be maintained at over 80%, and they retain good proliferative activity. They can be directly infused into dogs and retain good killing activity against canine mammary tumor cells, providing convenience for short-distance transportation and clinical application of canine natural killer cells.

[0052] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a clinical canine natural killer cell cryopreservation solution, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0053] Example 1

[0054] A clinical cryopreservation solution for canine natural killer cells, using physiological saline as a solvent, comprises the following components per 1 mL: dextran 40, a complex amino acid, canine serum albumin, canine interleukin-2, and sodium bisulfite. The complex amino acid consists of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine, with the following concentrations:

[0055] Dextran 4050 mg / mL, canine serum albumin 20 mg / mL, canine interleukin 2400 IU / mL, sodium sulfite 0.38 mL / mL, and the concentration of complex amino acids was 21.31 mg / mL;

[0056] The mass ratio of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine in the compound amino acid is 3.08:1.35:2.18:1.35:0.45:1.8:2.55:4.65:1.65:0.75:1.5, which corresponds to the following concentrations of each amino acid in the cell cryopreservation solution:

[0057] Leucine 3.08 mg / mL, Isoleucine 1.35 mg / mL, Phenylalanine 2.18 mg / mL, Threonine 1.35 mg / mL, Tryptophan 0.45 mg / mL, Methionine 1.8 mg / mL, Glycine 2.55 mg / mL, Lysine Hydrochloride 4.65 mg / mL, Arginine 1.65 mg / mL, Histidine 0.75 mg / mL, and Valine 1.5 mg / mL.

[0058] Example 2

[0059] A clinical canine natural killer cell cryopreservation solution, the only difference from Example 1, is that the content of canine interleukin-2 is 300 IU / mL.

[0060] Example 3

[0061] A clinical canine natural killer cell cryopreservation solution, the only difference from Example 1, is that the content of canine interleukin-2 is 150 IU / mL.

[0062] Example 4

[0063] A clinical canine natural killer cell cryopreservation solution, the only difference from Example 1, is that the content of canine interleukin-2 is 100 IU / mL.

[0064] Example 5

[0065] A clinical canine natural killer cell cryopreservation solution differs from Example 1 only in that the concentration of the complex amino acid is 9.61 mg / mL. The complex amino acid consists of leucine, isoleucine, phenylalanine, tryptophan, and glycine, with the following concentrations: leucine 3.08 mg / mL, isoleucine 1.35 mg / mL, phenylalanine 2.18 mg / mL, tryptophan 0.45 mg / mL, and glycine 2.55 mg / mL.

[0066] Example 6

[0067] A clinical canine natural killer cell cryopreservation solution differs from Example 1 only in that the concentration of the complex amino acid is 12.15 mg / mL. The complex amino acid consists of threonine, tryptophan, methionine, lysine hydrochloride, arginine, histidine, and valine, with the following concentrations: threonine 1.35 mg / mL, tryptophan 0.45 mg / mL, methionine 1.8 mg / mL, lysine hydrochloride 4.65 mg / mL, arginine 1.65 mg / mL, histidine 0.75 mg / mL, and valine 1.5 mg / mL.

[0068] Comparative Example 1

[0069] A clinical canine natural killer cell cryopreservation solution, the only difference from Example 1 is that canine interleukin-2 is omitted, while the other components and contents are the same as in Example 1.

[0070] Comparative Example 2

[0071] A clinical canine natural killer cell cryopreservation solution, the only difference from Example 1 is that dextran 40 is omitted, while the other components and contents are the same as in Example 1.

[0072] Comparative Example 3

[0073] A clinical canine natural killer cell cryopreservation solution, the only difference from Example 1 is that sodium sulfite is omitted, while the other components and contents are the same as in Example 1.

[0074] Comparative Example 4

[0075] A clinical canine natural killer cell cryopreservation solution, the only difference from Example 1 is that the complex amino acids are omitted, while the other components and contents are the same as in Example 1.

[0076] Comparative Example 5

[0077] A clinical canine natural killer cell cryopreservation solution, the only difference from Example 1 is that canine serum albumin is omitted, while the other components and contents are the same as in Example 1.

[0078] Comparative Example 6

[0079] A clinically applicable cryopreservation solution for canine natural killer cells, with the following composition and concentration compared to Example 1:

[0080] Dextran 4053 mg / mL, canine serum albumin 15 mg / mL, canine interleukin 2400 IU / mL, sodium sulfite 0.4 mg / mL, and the concentration of complex amino acids was 22.72 mg / mL;

[0081] The mass ratio of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine in the complex amino acid is 3.28:1.44:2.32:1.44:0.48:1.92:2.72:4.96:1.76:0.8:1.6, which means that the cell cryopreservation solution contains 3.28 mg / mL of leucine, 1.44 mg / mL of isoleucine, 2.32 mg / mL of phenylalanine, 1.44 mg / mL of threonine, 0.48 mg / mL of tryptophan, 1.92 mg / mL of methionine, 2.72 mg / mL of glycine, 4.96 mg / mL of lysine hydrochloride, 1.76 mg / mL of arginine, 0.8 mg / mL of histidine, and 1.6 mg / mL of valine.

[0082] Comparative Example 7

[0083] A clinically applicable cryopreservation solution for canine natural killer cells, with the following composition and concentration compared to Example 1:

[0084] Dextran 4056 mg / mL, canine serum albumin 10 mg / mL, canine interleukin 2400 IU / mL, sodium sulfite 0.43 mg / mL, and the concentration of complex amino acids was 24.15 mg / mL;

[0085] The mass ratio of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine in the complex amino acid is 3.49:1.53:2.47:1.53:0.51:2.04:2.89:5.27:1.87:0.85:1.7, which means that the cell cryopreservation solution contains 3.49 mg / mL of leucine, 1.53 mg / mL of isoleucine, 2.47 mg / mL of phenylalanine, 1.53 mg / mL of threonine, 0.51 mg / mL of tryptophan, 2.04 mg / mL of methionine, 2.89 mg / mL of glycine, 5.27 mg / mL of lysine hydrochloride, 1.87 mg / mL of arginine, 0.85 mg / mL of histidine, and 1.7 mg / mL of valine.

[0086] Example 7

[0087] Canine natural killer cells were preserved using the cell cryopreservation solutions from Examples 1-3 and Comparative Examples 1-3, respectively. The preservation methods are as follows:

[0088] The canine natural killer cells were mixed with cell cryopreservation solution and resuspended to achieve a cell density of 5 × 10⁻⁶. 6 The sample was divided into 2 mL cryovials, 1 mL per tube, and stored at 4°C.

[0089] Test Example 1

[0090] Canine natural killer cells stored for 24h, 48h, 72h, and 96h in Example 4 were stained with trypan blue, and the viability of the canine natural killer cells was determined by a cell counter. The results are shown in Table 1.

[0091] Table 1. Viability (%) of canine natural killer cells preserved in different cell cryopreservation solutions

[0092]

[0093] As shown in Table 1, the cell cryopreservation solutions of Examples 1-3 showed better preservation effects on canine natural killer cells than those of Comparative Examples 1-3. After 72 hours of preservation at 4°C, the viability of canine natural killer cells remained above 78%. In Examples 2-3, the concentration of canine interleukin-2 differed from that of Example 1, resulting in poorer preservation effects. The cell cryopreservation solution of Example 1 showed the best preservation effect, with a viability of 83.82% after 72 hours of preservation at 4°C, indicating that canine interleukin-2 promotes the activation and expansion of natural killer cells. Comparative Examples 1-3 altered the content of canine serum albumin and various nutrients, significantly reducing the viability of canine natural killer cells after 72 hours of preservation at 4°C. The synergistic effect of the components in the preservation solution provided by this invention provides a stable survival environment, the energy required for basal metabolism, and mechanical protection for canine natural killer cells, stably maintaining cellular metabolic homeostasis and extending the preservation time of canine natural killer cells.

[0094] Example 6

[0095] (1) The canine natural killer cells preserved for 72 hours using the cell cryopreservation solution from Example 1 in Example 4 were re-seeded into culture flasks and cultured for 24 hours. The growth of the canine natural killer cells after 72 hours of preservation at 4°C was observed. The results showed that the canine natural killer cells, after being preserved at 4°C for 72 hours in the preservation solution from Example 1, still retained good proliferative activity. Re-seeding into the culture medium allowed them to form canine natural killer cell colonies, demonstrating good proliferative capacity. Figure 1 ).

[0096] (2) Using canine mammary tumor cells as target cells and canine natural killer cells as effector cells, the killing activity of canine natural killer cells was investigated:

[0097] 1×10 4 Canine mammary tumor cells were seeded into 96-well plates and cultured overnight. After complete adherence, canine natural killer (NK) cells (without refrigeration) and canine NK cells (preserved at 4°C for 72 hours using the cell refrigeration solution from Example 1) were added at a target-to-efficacy ratio of 1:16. After co-culturing for 24 hours, the culture medium was discarded, and the cells were washed twice with PBS. Then, 100 μL of canine mammary tumor cell culture medium and 10 μL of CCK8 were added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance (OD) value was measured at 450 nm using a microplate reader. Three auxiliary wells were set up for each experiment. The cell killing rate was calculated according to the following formula. The results are shown below. Figure 2 As shown.

[0098] Cell killing rate (%) = 1 - (OD value of canine natural killer cell group - OD value of pure culture medium) / (OD value of pure tumor cell group - OD value of pure culture medium) × 100%.

[0099] according to Figure 2 As can be seen, after being stored at 4°C for 72 hours using the cell cryopreservation solution of Example 1, the killing rate of canine natural killer cells against canine mammary tumor cells was 58.09%, which was not significantly different from that of canine natural killer cells before cryopreservation, and the cells still maintained good killing activity.

[0100] As can be seen from the above, the cell cryopreservation solution provided by the present invention can provide a stable survival environment for canine natural killer cells, realize the in vitro preservation of canine natural killer cells, extend the preservation time of canine natural killer cells, and retain good killing activity of canine mammary tumor cells.

[0101] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A cell cryopreservation solution, characterized in that, No serum or DMSO added, including active ingredients and solvents; The active ingredients include dextran 40, complex amino acids, canine serum albumin, canine interleukin-2, and sodium bisulfite. The complex amino acid includes three or more of the following: leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine. The solvent includes physiological saline; The concentration of dextran 40 in the refrigerated preservation solution is 45-60 mg / mL; The concentration of canine serum albumin in the refrigerated preservation solution is 10-25 mg / mL; The concentration of canine interleukin-2 in the refrigerated preservation solution is 100~600 IU / mL; The concentration of sodium bisulfite in the refrigerated preservation solution is 0.38~0.5 mg / mL; The concentration of the complex amino acids in the refrigerated preservation solution is 9.61~27.2 mg / mL; The complex amino acid is composed of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine; the mass ratio of leucine, isoleucine, phenylalanine, threonine, tryptophan, methionine, glycine, lysine hydrochloride, arginine, histidine, and valine in the complex amino acid is 3.08:1.35:2.18:1.35:0.45:1.8:2.55:4.65:1.65:0.75:1.

5. Alternatively, the complex amino acid is composed of leucine, isoleucine, phenylalanine, tryptophan, and glycine; the mass ratio of leucine, isoleucine, phenylalanine, tryptophan, and glycine in the complex amino acid is 3.08:1.35:2.18:0.45:2.

55. Alternatively, the complex amino acid is composed of threonine, tryptophan, methionine, lysine hydrochloride, arginine, histidine, and valine; the mass ratio of threonine, tryptophan, methionine, lysine hydrochloride, arginine, histidine, and valine in the complex amino acid is 1.35:0.45:1.8:4.65:1.65:0.75:1.

5.

2. The refrigeration preservation solution according to claim 1, characterized in that, The concentration of dextran 40 in the refrigerated preservation solution is 50 mg / mL; The concentration of canine serum albumin in the refrigerated preservation solution was 20 mg / mL; The concentration of canine interleukin-2 in the refrigerated preservation solution was 400 IU / mL; The concentration of sodium bisulfite in the refrigerated preservation solution is 0.38 mg / mL.

3. The method for preparing the refrigeration preservation solution according to claim 1 or 2, characterized in that, The active ingredient is dissolved using the solvent.

4. The application of the cryopreservation solution according to claim 1 or 2 or the cryopreservation solution obtained by the preparation method according to claim 3 in the preservation of canine natural killer cells.

5. A method for preserving canine natural killer cells for clinical use, characterized in that, Includes the following steps: The canine natural killer cells are mixed with the cryopreservation solution described in claim 1 or 2 or the cryopreservation solution prepared by the method described in claim 3 to obtain a cell suspension, which is then stored at 4-8°C.

6. The preservation method according to claim 5, characterized in that, The density of canine natural killer cells in the cell suspension was 1×10⁻⁶. 6 ~1×10 7 per mL.

7. The preservation method according to claim 6, characterized in that, The density of canine natural killer cells in the cell suspension was 5 × 10⁻⁶. 6 per mL.

8. The use of the cryopreservation solution according to claim 1 or 2 or the cryopreservation solution obtained by the preparation method according to claim 3 in the preparation of a canine mammary tumor cell killing product.

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