In-vitro amplification method of dog peripheral blood source specific immune cells

Abstract

The invention discloses an in-vitro amplification method of dog peripheral blood source specific immune cells, and belongs to the technical field of immune cell culture. According to the method, dog peripheral blood serves as a cell source, and efficient amplification of specific immune cells is achieved within 14 days through the steps of lymphocyte separation, immune cell activation, staged density regulation and control culture, culture medium supplementing and the like. By optimizing the combination and concentration of cell factors and accurately regulating and controlling the cell density at different culture stages, the problems of low amplification efficiency, weak specific killing ability, short cell activity maintenance time and the like of the dog immune cells in the prior art are solved. The amplification multiple of the finally obtained specific immune cells can reach 125 times or more, the cell viability is kept 90% or more, the proportion of CD3-NKp46 + phenotype cells exceeds 70%, the application requirements of clinical anti-tumor immune cell preparations can be met, and an efficient and stable cell preparation scheme is provided for dog tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of immune cell culture technology, specifically to an in vitro expansion method for specific immune cells derived from canine peripheral blood, which is particularly suitable for the preparation of specific anti-tumor immune cell preparations required for canine tumor immunotherapy, and belongs to the interdisciplinary field of pet medical and bioengineering technology. Background Technology

[0002] With the rapid development of the pet industry, the health management of pet dogs has received increasing attention, and tumors have become one of the major diseases threatening the lives and health of dogs. In recent years, the incidence of tumors in pet dogs has shown a significant upward trend. Data shows that the incidence of tumors in dogs over 7 years old is as high as 30%-50%. Common types of tumors include lymphoma, mammary tumors, soft tissue sarcomas, melanomas, etc., of which malignant tumors account for more than half, seriously affecting the quality of life of dogs and even leading to death.

[0003] Currently, clinical treatments for canine tumors mainly include surgical resection, chemotherapy, radiotherapy, and preliminary immunotherapy. However, these traditional treatment methods all have significant limitations and drawbacks:

[0004] Surgical resection is only suitable for early-stage localized tumors. It cannot completely remove tumors that have metastasized or grown diffusely, and the recurrence rate after surgery is high.

[0005] Chemotherapy: Commonly used drugs include cyclophosphamide and doxorubicin. Although they can inhibit the proliferation of tumor cells to a certain extent, they have significant toxic side effects and are prone to causing a series of adverse reactions such as vomiting, diarrhea, and decreased immune function in dogs. This leads to poor tolerance and low treatment compliance in affected dogs, which seriously affects the treatment effect.

[0006] Radiotherapy: It has a certain control effect on local solid tumors, but the equipment required for this method is expensive, the clinical adoption rate is low, and the penetration of radiation into deep tumors is limited. It is easy to damage the surrounding normal tissues during the treatment process, causing secondary damage.

[0007] Immunotherapy, as an emerging treatment method, has advantages such as high specificity and few side effects. One major approach is the in vitro expansion and reinfusion of canine immune cells. However, existing methods for in vitro expansion of canine immune cells face numerous technical bottlenecks, primarily manifested in low expansion efficiency, making it difficult to obtain a sufficient number of therapeutic cells; weak specific tumor-killing ability, resulting in poor therapeutic effects; and short cell viability maintenance time after in vitro culture, making it difficult to sustain anti-tumor effects after reinfusion. These shortcomings prevent current immunotherapy methods from meeting the clinical demand for highly effective and safe anti-tumor immune cell preparations.

[0008] Therefore, in order to address the shortcomings of existing canine tumor treatment technologies, especially the core technical problem of in vitro expansion of immune cells in immunotherapy, developing an efficient, stable, and highly specific method for in vitro expansion of canine immune cells is of great practical significance and has broad application prospects for improving the treatment effect of canine tumors and improving the quality of life of sick dogs. Summary of the Invention

[0009] The core objective of this invention is to overcome the shortcomings of existing technologies for in vitro expansion of canine immune cells, such as low expansion efficiency, weak ability to specifically kill tumor cells, and short duration of cell viability. This invention provides a method for in vitro expansion of specific immune cells derived from canine peripheral blood. By optimizing the cell culture system and precisely controlling culture parameters, this method achieves efficient expansion of specific immune cells, obtaining highly active and specific anti-tumor immune cells. This provides high-quality cell preparations for canine tumor immunotherapy, addressing a challenge in clinical treatment.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A method for in vitro expansion of specific immune cells derived from canine peripheral blood, comprising the following steps:

[0012] Includes the following steps:

[0013] (1) Lymphocytes were isolated and purified from canine peripheral blood;

[0014] (2) The lymphocytes were activated in vitro using an activation solution;

[0015] (3) The activated immune cells were placed in the expansion medium for staged culture for 14 days. Fresh expansion medium was added regularly and the cell density was adjusted to obtain specific immune cells.

[0016] The activation solution comprises basal culture medium, canine interleukin-2 (Canine IL-2), canine interleukin-12 (Canine IL-12), canine interleukin-17A (Canine IL-17A), and activation magnetic beads (Gibco, 11163D); the amplification culture medium comprises basal culture medium, canine interleukin-2, canine interleukin-12, canine interleukin-17A, and fetal bovine serum.

[0017] Further, in step (1), canine peripheral blood lymphocyte separation solution is used to separate lymphocytes. The separation process includes sample dilution, density gradient centrifugation, cell washing and purification in sequence. The process parameters of the density gradient centrifugation are 800-1000g centrifugation for 20-30min.

[0018] Furthermore, the specific method of sample dilution is as follows: take a 50ml centrifuge tube, mix the sample diluent with the canine peripheral blood sample at a volume ratio of 1:2; the cell washing step is as follows: after density gradient centrifugation, collect the upper lymphocyte suspension, add PBS buffer, centrifuge at 250g for 8-10min, discard the supernatant to obtain purified lymphocytes.

[0019] Furthermore, the concentration ranges of each component in the activation solution are as follows: the concentration range of canine interleukin-2 is 100–1000 IU / ml, the concentration range of canine interleukin-12 is 5–10 ng / ml, and the concentration range of canine interleukin-17A is 5–20 IU / ml; the activation magnetic beads are Gibco brand 11163D model ACTIVATOR.

[0020] Further, the specific operations of the activation treatment in step (2) include: resuspending the purified lymphocytes with the activation solution and adjusting the cell density to 2-4. Place at 37℃ and 5% The activation incubation was carried out in the culture environment, and the activation incubation start diary was day D0.

[0021] Furthermore, the concentration ranges of each component in the amplification medium are as follows: the concentration range of canine interleukin-2 is 200-300 IU / ml, the concentration range of canine interleukin-12 is 50-100 ng / ml, the concentration range of canine interleukin-17A is 20-200 IU / ml, and the volume fraction of fetal bovine serum is 5%; the basal medium is X-VIVO 15 medium.

[0022] Furthermore, the specific scheme for phased regulation of cell density in step (3) is as follows:

[0023] Day 2: Add 1 to 2 times the volume of fresh amplification medium and mix gently;

[0024] Day 4: Adjust cell density to... ;

[0025] Day 6: Adjust cell density to... ;

[0026] Day 8: Adjust cell density to... ;

[0027] Days 10 and 12: Adjust cell density to [specific values]. .

[0028] Furthermore, the canine peripheral blood lymphocyte separation solution is a canine peripheral blood lymphocyte separation solution kit produced by Tianjin Haoyang Biotechnology Co., Ltd., with the product number LTS1079.

[0029] Furthermore, the specific immune cells are CD3-NKp46+ phenotype cells, and the proportion of these phenotype cells at day D14 is not less than 70%.

[0030] Furthermore, by day 14 of culture, the specific immune cells have expanded by at least 125 times and have a cell viability of at least 90%.

[0031] The in vitro expansion method for canine peripheral blood-derived specific immune cells of the present invention has the following significant advantages compared with the prior art:

[0032] The in vitro expansion method for canine peripheral blood-derived specific immune cells of this invention overcomes the core deficiencies of existing canine tumor treatment technologies and has significant industrial application prospects. This method can be scaled up for use in biopharmaceutical companies to produce cell preparations for canine tumor immunotherapy, providing veterinary hospitals with highly efficient and safe treatment products. Simultaneously, it can serve as a standardized technical platform for research institutions to conduct research on canine immune cells, promoting technological advancements in the field of canine tumor immunotherapy. With the increasing demand for precision treatment in the pet healthcare industry, the technological achievements of this invention will have broad market potential and social value, providing strong support for improving the health and quality of life of pet dogs. Attached Figure Description

[0033] Figure 1 is a comparison of the cell expansion folds of the method of the present invention (Group A) and the conventional method (Group B).

[0034] Figure 2 is a comparison of cell viability between the method of the present invention (Group A) and the conventional method (Group B).

[0035] Figure 3 is a comparison of the proportion of CD3-NKp46+ phenotype cells between the method of the present invention (Group A) and the conventional method (Group B). Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to specific embodiments, but this invention includes, but is not limited to, these embodiments.

[0037] Example 1

[0038] 1. Isolating immune cells from peripheral blood or apheresis blood using lymphocyte separation fluid.

[0039] (1) The lymphocyte separation medium used was the dog peripheral blood lymphocyte separation medium kit (catalog number: LTS1079) from Tianjin Haoyang Biotechnology.

[0040] (2) Sample dilution: Take a 50ml centrifuge tube and dilute the sample diluent with the blood sample at a volume ratio of 1:2 and mix well.

[0041] (3) Take 15ml of separation solution and add it to the bottom of a 50ml sample separation tube.

[0042] (4) Use a pipette to slowly add the blood sample onto the separation liquid, 800-1000g, and centrifuge for 20-30 minutes.

[0043] (5) After centrifugation, pour the supernatant into a new centrifuge tube, add 10 ml of PBS, and mix the cells.

[0044] (6) Centrifuge at 250g for 8-10 minutes and discard the supernatant.

[0045] 2. Activating immune cells using magnetic beads.

[0046] (1) Resuspend the cells in solution 1 and adjust the cell density to 2-4. .

[0047] (2) Components of Solution 1:

[0048] The immune cell culture medium was X-VIVO 15 medium.

[0049] Canine IL-2: 100~1000IU / ml

[0050] Canine IL-12: 5–10 ng / ml

[0051] Canine IL-17A: 5–20 IU / ml

[0052] Activation of magnetic beads (ACTIVATOR, Gibco, 11163D)

[0053] 3. After the immune cells are activated, the cell culture density is continuously adjusted.

[0054] Solution 1 activated the immune cells on day D0, and then cultured them until day D14. During this period, Solution 2 was added every 2 days to adjust the cell density to a fixed value.

[0055] (1) Add 1 to 2 times the volume of solution to D2.

[0056] (2) D4 adjusts the cell density to 8

[0057] (3) D6 adjusts the cell density to 1

[0058] (4) Adjust the cell density to 1.1 on day 8.

[0059] (5) Adjust cell density to 1.2 on days 10 and 12.

[0060] (6) Components of solution 2:

[0061] The immune cell culture medium was X-VIVO 15 medium.

[0062] Canine IL-2: 200-300 IU / ml

[0063] Canine IL-12: 50-100 ng / ml

[0064] Canine IL-17A: 20–200 IU / ml

[0065] Fetal bovine serum: 5%

[0066] 4. Results

[0067] The existing plan and the conventional plan were compared. The existing plan was group A, and the conventional plan was group B.

[0068] (1) Amplification factor and viability

[0069] Table 1. Amplification factor

[0070] Days D0 D2 D4 D6 D8 D10 D12 D14 A cell number 2e7 2.2e7 3e7 5e7 1e8 6e8 1e9 2.5e9 B cell number 2e7 2.2e7 2.8e7 3.6e7 6e7 2.3e8 5.5e8 1.1e9

[0071] Table 2. Cell viability

[0072] Days D0 D2 D4 D6 D8 D10 D12 D14 A viability 95% 90% 86.2% 87.6% 90.2% 92.1% 91.8% 92.5% B viability 95% 89.6% 82.7% 78.2% 80.5% 83% 82.1% 81.5%

[0073] See Figure 1 and Figure 2 Tables 1 and 2 are provided, and Figure 1 is a comparison of the cell expansion folds of the method of this invention (Group A) and the conventional method (Group B). The horizontal axis represents the number of culture days (D0 to D14), and the vertical axis represents the number of cells. As shown in the figure, the cell number in Group A increased from 2 on day D0... It continued to grow rapidly, reaching 2.5 by day 14. The number of cells in group B increased slowly, reaching only 1.1 on day 14; The cell expansion rate and final expansion fold of group A were significantly higher than those of group B.

[0074] Figure 2 shows a comparison of cell viability between the method of this invention (Group A) and the conventional method (Group B). The horizontal axis represents the number of culture days (D0 to D14), and the vertical axis represents cell viability (%). The cell viability of Group A remained stable throughout the culture period, fluctuating from 95% on day D0 to 92.5% on day D14, and generally maintained above 90%. The cell viability of Group B continuously decreased from 95% on day D0, dropping to 78.2% on day D6, and only 81.5% on day D14. The stability and final value of cell viability in Group A were both superior to those in Group B.

[0075] Based on the final D14 cell expansion fold, this protocol can expand to 125 times (compared to only about 50 times using conventional methods), which is sufficient to achieve the cell infusion volume for a single dog, meeting clinical needs.

[0076] Cell viability: Relatively stable, ultimately maintaining a viability of over 90%; compared with previous human viability data, this is also a relatively high value.

[0077] Table 3 Cell phenotypes

[0078] Days D0 D8 D14 A group CD3-NKp46+ phenotype 2.1% 67% 72.5% B group CD3-NKp46+ phenotype 2.1% 13.2% 20.8%

[0079] (2) Cell phenotype: see Figure 3 Table 3 and Figure 3 show a comparison of the proportion of CD3-NKp46+ phenotype cells using the method of this invention (Group A) and the conventional method (Group B). The horizontal axis represents the number of culture days (D0, D8, D14), and the vertical axis represents the proportion of CD3-NKp46+ phenotype cells (%). In Group A, the proportion of CD3-NKp46+ phenotype cells increased rapidly from 2.1% on day D0, reaching 67% on day D8, and further increasing to 72.5% on day D14. In Group B, the proportion of this phenotype cell increased slowly, reaching only 13.2% on day D8 and 20.8% on day D14. The specific cell induction efficiency of Group A was significantly higher than that of Group B. It can be seen that the cell phenotype was detected on days D8 and 14, and the final proportion could reach over 70%, indicating that the vast majority of cells had been transformed into target cells, which can effectively activate the body's immune system and kill tumors.

[0080] In summary, as a preferred embodiment, after separating canine peripheral blood with canine lymphocyte separation medium, it was first activated with solution 1, and then amplified and cultured with solution 2 until day 14. The final amplification fold, viability and phenotype all showed good performance.

[0081] Significantly improved amplification efficiency: This invention optimizes the combination and concentration ratio of cytokines, combined with a phased density control strategy, to enable specific immune cells to amplify by more than 125 times within a 14-day culture period, which is far higher than the approximately 50 times of conventional methods. This can meet the number of cells required for a single canine cell reinfusion therapy and solves the core problem of insufficient cell amplification in existing technologies.

[0082] Stable and prolonged cell viability: The culture system of this invention provides a suitable nutritional environment and growth conditions for immune cells, maintaining stable cell viability throughout the entire culture cycle, from approximately 95% on day D0 to over 90% on day D14, significantly higher than the 81.5% viability on day D14 achieved by conventional methods. Highly viable cells, after reinfusion, can rapidly adapt to the in vivo environment, continuously exert their anti-tumor effects, and prolong the duration of therapeutic efficacy.

[0083] High proportion of specific immune cells: By day 14 of culture, the proportion of CD3-NKp46+ phenotype cells exceeds 70%. These cells have a strong ability to specifically kill tumor cells, accurately identify and eliminate tumor cells in dogs, while avoiding damage to normal tissue cells, thus improving the specificity and safety of immunotherapy.

[0084] The culture system is standardized and has good reproducibility: This invention clarifies the process parameters for each step, including the type of separation solution, centrifugation conditions, types and concentration ranges of cytokines, and phased cell density control values, etc., and establishes a standardized culture process, avoiding the randomness of human operation, with good experimental reproducibility, which can meet the needs of large-scale production.

[0085] Simple to operate and cost-controllable: The reagents used in this invention are all commercially available products and do not require special customization; the culture process does not require complicated equipment and operations, and can be completed with only conventional cell culture equipment and techniques, which reduces the technical threshold and production costs, making it easy to promote and apply in clinical institutions and biotechnology companies;

[0086] Wide range of applications: This invention is applicable to peripheral blood samples from dogs of different breeds and ages. Whether it is peripheral blood from healthy dogs or dogs with tumors, highly active and specific immune cells can be amplified using this method, providing support for immunotherapy of various canine tumors, and has broad application prospects.

[0087] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for in vitro expansion of specific immune cells derived from canine peripheral blood, characterized in that, Includes the following steps: (1) Lymphocytes were isolated and purified from canine peripheral blood; (2) The lymphocytes were activated in vitro using an activation solution; (3) The activated immune cells were placed in the expansion medium for staged culture for 14 days. Fresh expansion medium was added regularly and the cell density was adjusted to obtain specific immune cells. The activation solution comprises basal culture medium, canine interleukin-2 (Canine IL-2), canine interleukin-12 (Canine IL-12), canine interleukin-17A (Canine IL-17A), and activation magnetic beads (Gibco, 11163D); the amplification culture medium comprises basal culture medium, canine interleukin-2, canine interleukin-12, canine interleukin-17A, and fetal bovine serum.

2. The in vitro amplification method according to claim 1, characterized in that, In step (1), canine peripheral blood lymphocyte separation solution is used to separate lymphocytes. The separation process includes sample dilution, density gradient centrifugation, cell washing and purification. The process parameters of density gradient centrifugation are 800-1000g centrifugation for 20-30min.

3. The in vitro amplification method according to claim 2, characterized in that, The specific method for sample dilution is as follows: Take a 50ml centrifuge tube and mix the sample diluent with the canine peripheral blood sample at a volume ratio of 1:

2. The cell washing step is as follows: After density gradient centrifugation, collect the upper lymphocyte suspension, add PBS buffer, centrifuge at 250g for 8-10 minutes, discard the supernatant to obtain purified lymphocytes.

4. The in vitro amplification method according to claim 1, characterized in that, The concentration ranges of each component in the activation solution are as follows: canine interleukin-2 concentration range is 100-1000 IU / ml, canine interleukin-12 concentration range is 5-10 ng / ml, and canine interleukin-17A concentration range is 5-20 IU / ml; the activation magnetic beads are Gibco brand 11163D ACTIVATOR.

5. The in vitro amplification method according to claim 1, characterized in that, The specific operations for activation treatment in step (2) include: resuspending the purified lymphocytes in the activation solution and adjusting the cell density to 2-4. Place at 37℃ and 5% The activation incubation was carried out in the culture environment, and the activation incubation start diary was day D0.

6. The in vitro amplification method according to claim 1, characterized in that, The concentration ranges of each component in the amplification medium are as follows: canine interleukin-2 concentration range is 200-300 IU / ml, canine interleukin-12 concentration range is 50-100 ng / ml, canine interleukin-17A concentration range is 20-200 IU / ml, and fetal bovine serum volume fraction is 5%; the basal medium is X-VIVO 15 medium.

7. The in vitro amplification method according to claim 1, characterized in that, The specific scheme for phased regulation of cell density in step (3) is as follows: Day 2: Add 1 to 2 times the volume of fresh amplification medium and mix gently; Day 4: Adjust cell density to... ; Day 6: Adjust cell density to... ; Day 8: Adjust cell density to... ; Days 10 and 12: Adjust cell density to [specific values]. .

8. The in vitro amplification method according to claim 2, characterized in that, The canine peripheral blood lymphocyte separation solution is a canine peripheral blood lymphocyte separation solution kit produced by Tianjin Haoyang Biotechnology Co., Ltd., with the product number LTS1079.

9. The in vitro amplification method according to claim 1, characterized in that, The specific immune cells are CD3-NKp46+ phenotype cells, and the proportion of these phenotype cells at day D14 is not less than 70%.

10. The in vitro amplification method according to claim 1, characterized in that, By day 14 of culture, the specific immune cells should have expanded by at least 125 times and have a cell viability of at least 90%.

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