Mouse skin digestion compound enzyme and mouse skin T cell separation method

Abstract

The invention belongs to the technical field of T cell separation, and particularly discloses a mouse skin digestion compound enzyme and a mouse skin T cell separation method, pancreatin is added when skin tissue is digested, Percoll density gradient centrifugation is performed after the skin tissue is digested, beta-ME is added into digestive juice, and vitamin C is added into the digestive juice. Compared with the prior art, T cells obtained through separation are remarkably increased.

Description

Technical Field

[0001] The invention relates to the technical field of T cell separation, and in particular to a mouse skin digestion complex enzyme and a method for separating mouse skin T cells. Background Art

[0002] T lymphocytes are an important part of the immune system. When they become diseased and produce clonal hyperplasia, and accumulate and proliferate mainly in skin tissue, it leads to cutaneous T-cell lymphoma.

[0003] Therefore, skin T cells play an important role in disease models such as dermatitis, skin cancer, and some autoimmune diseases and have been widely studied. However, the content of T cells in the skin is low, making it difficult to isolate and obtain sufficient numbers. At the same time, T cells are prone to death and their activity is easily damaged during the isolation process. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problems existing in the background technology. To this end, a method for separating mouse skin digestive complex enzyme and mouse skin T cells is provided.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A mouse skin digestion complex enzyme, the mouse skin digestion complex enzyme consisting of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, deoxyribonuclease at a final concentration of 0.1 mg / ml, type IV collagenase at a final concentration of 2 mg / ml, hyaluronidase at a final concentration of 0.5 mg / ml, and pancreatin at a final concentration of 0.25%; Alternatively, the mouse skin digestion complex enzyme is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1% , β-ME at a final concentration of 100 µM, vitamin C at a final concentration of 50 µM, deoxyribonuclease at a final concentration of 0.1 mg / ml, collagenase type IV at a final concentration of 2 mg / ml, and hyaluronidase at a final concentration of 0.5 mg / ml; Alternatively, the mouse skin digestion complex enzyme is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, deoxyribonuclease at a final concentration of 0.1 mg / ml, collagenase type IV at a final concentration of 2 mg / ml, hyaluronidase at a final concentration of 0.5 mg / ml, β-ME at a final concentration of 100 µM, and trypsin at a final concentration of 0.25%.

[0006] A method for isolating mouse skin T cells comprises the following steps: Step S1: prepare a fresh solution of the mouse skin digestion complex enzyme as described in claim 1 and place it on ice; When the prepared mouse skin digestion complex enzyme consists of the following components at final concentrations: RPMI 1640 medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, deoxyribonuclease at a final concentration of 0.1 mg / ml, type IV collagenase at a final concentration of 2 mg / ml, hyaluronidase at a final concentration of 0.5 mg / ml, and trypsin at a final concentration of 0.25%, then jump to steps S2-S7; Step S2: Euthanize the mice, remove the long hair and villi on the back, apply depilatory cream, leave it on for 5 minutes, then rinse it with double distilled water, and cut off 3 cm 2 The free fat layer of the mouse back skin was gently scraped off with forceps to obtain the mouse skin tissue; Step S3: Place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 Pour the mixture into a 50 ml centrifuge tube, add 5 ml mouse skin digestion complex enzyme solution, and place at 37 ° C, 225 rpm shaker for 45 minutes; Step S4: Prepare digestion stop solution: RPMI 1640 medium containing fetal bovine serum at a final concentration of 10%, pre-cool on ice, remove the skin tissue from the shaker, and add 10 ml of the stop solution to stop digestion; Step S5: the skin tissue solution was vortexed for 10 s and then placed on a 100 μm filter, and the filtrate was centrifuged at 300 g for 10 min at 4 °C; Step S6: Purify the mouse skin T cells in the precipitate by percoll density gradient centrifugation: resuspend the precipitated cells in 4 mL of 30% Percoll, transfer to a 15 ml centrifuge tube pre-added with 3 ml of 60% Percoll, centrifuge at 4°C, 800 g for 20 min, aspirate the upper layer solution, collect the middle layer T cells into a new 15 ml centrifuge tube, add 10 ml of digestion stop solution, shake gently to wash the cells, centrifuge at 4°C, 300 g for 10 min, and the precipitate is the separated T cells; Step S7: resuspend the precipitate with PBS solution, add flow cytometry antibody staining, and perform flow cytometry analysis.

[0007] The following is a technical solution further defined by the method of the present invention. When the prepared mouse skin digestion complex enzyme is composed of the following components at the final concentration: RPMI 1640 culture medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, β-ME at a final concentration of 100 µM, vitamin C at a final concentration of 50 µM, deoxyribonuclease at a final concentration of 0.1 mg / ml, collagenase type IV at a final concentration of 2 mg / ml, and hyaluronidase at a final concentration of 0.5 mg / ml, then jump to steps S8-S12; Step S8: Euthanize the mice, remove the long hair and villi on the back, apply depilatory cream, leave it for 5 minutes, then rinse it with double distilled water, and cut off 3 cm 2 The free fat layer of the mouse back skin was gently scraped off with forceps to obtain the mouse skin tissue; Step S9: Place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 Pour the mixture into a 50 ml centrifuge tube, add 5 ml mouse skin digestion complex enzyme solution, and place at 37 ° C, 225 rpm shaker for 45 minutes; Step S10: Prepare digestion stop solution: RPMI 1640 medium containing fetal bovine serum at a final concentration of 10% and β-ME at a final concentration of 100 µM, pre-cool on ice, remove the skin tissue from the shaker, and add 10 ml of the stop solution to stop digestion; Step S11: the skin tissue solution was vortexed for 10 seconds and then placed on a 100 μm filter screen for grinding and filtration. The filtrate was centrifuged at 4° C. and 300 g for 10 minutes to obtain the separated mouse skin T cells; Step S12: resuspend the precipitate with PBS solution, add flow cytometry antibody staining, and perform flow cytometry analysis.

[0008] The following is a technical solution further defined by the method of the present invention, when the prepared mouse skin digestion complex enzyme is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, deoxyribonuclease at a final concentration of 0.1 mg / ml, collagenase type IV at a final concentration of 2 mg / ml, hyaluronidase at a final concentration of 0.5 mg / ml, β-ME at a final concentration of 100 µM, and pancreatin at a final concentration of 0.25%, then jump to steps S13-S18; Step S13: Euthanize the mice, remove the long hair and villi on the back, apply depilatory cream, leave it for 5 minutes, then rinse it with double distilled water, and cut off 3 cm 2 The free fat layer of the mouse back skin was gently scraped off with forceps to obtain the mouse skin tissue; Step S14: Place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 Pour the mixture into a 50 ml centrifuge tube, add 5 ml mouse skin digestion complex enzyme solution, and place at 37 ° C, 225 rpm shaker for 45 minutes; Step S15: Prepare a digestion stop solution: containing fetal bovine serum with a final concentration of 10% and β-ME with a final concentration of 100 µM, pre-cool it on ice, remove the skin tissue from the shaker, and add 10 ml of the stop solution to stop the digestion; Step S16: the skin tissue solution was vortexed for 10 seconds and then placed on a 100 μm filter for grinding and filtration, and the filtrate was centrifuged at 300 g at 4°C for 10 minutes; Step S17: Purify the mouse skin T cells in the precipitate by percoll density gradient centrifugation: resuspend the precipitated cells in 4 mL of 30% Percoll, transfer to a 15 ml centrifuge tube pre-added with 3 ml of 60% Percoll, centrifuge at 4°C, 800 g for 20 min, aspirate the upper layer solution, collect the middle layer T cells into a new 15 ml centrifuge tube, add 10 ml of digestion stop solution, shake gently to wash the cells, centrifuge at 4°C, 300 g for 10 min, and precipitate the separated T cells; Step S18: resuspend the precipitate with PBS solution, add flow cytometry antibody staining, and perform flow cytometry analysis.

[0009] The following is a technical solution further defined in the method of the present invention, wherein the PBS solution contains BSA with a final concentration of 1%.

[0010] The following is a technical solution further defined by the method of the present invention, wherein the PBS solution contains BSA with a final concentration of 1% and β-ME with a final concentration of 100 µM.

[0011] Compared with the prior art, the present invention has the following technical effects: 1. The present invention adds pancreatic enzymes when digesting skin tissue: when digesting skin tissue, pancreatic enzymes help to decompose adhesion molecules on the cell surface and promote the release of immune cells; when used together with collagenase, the separation efficiency of T cells in the skin can be improved; 2. The present invention performs Percoll density gradient centrifugation after digesting the skin tissue: The main components of Percoll are polyvinyl alcohol and silica particles, which can form gradients with different densities to separate cells of different densities. By using Percoll density gradient centrifugation, the immune cells in the skin tissue digestion fluid can be further purified, and the proportion of T cell populations can be increased during flow cytometry analysis; 3. The present invention adds β-ME to the digestive fluid: improves the activity of T cells through anti-oxidation, improves the viability of T cells by maintaining the reduced state of cells, protects T cell surface receptors and functional proteins, and promotes T cell release by reducing the formation of disulfide bonds; 4. The present invention adds vitamin C to the digestive juice to reduce T cell oxidative damage, maintain the reducing environment in T cells, and improve T cell effector function through anti-oxidation effect.

[0012] The present invention is further described below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 It is a schematic diagram of a mouse hair removal experiment of the present invention; Figure 2 It is a schematic diagram of an experiment of applying a depilatory cream to mice of the present invention; Figure 3 It is a schematic diagram of an experiment of cutting the back skin of a mouse according to the present invention; Figure 4 It is a schematic diagram of an experiment in which mouse skin tissue of the present invention is placed in a clean culture dish; Figure 5 It is a schematic diagram of an experiment in which the shredded skin tissue of the present invention is poured into a centrifuge tube, a mouse skin digestion complex enzyme solution is added, and then the mixture is digested on a shaking table; Figure 6 It is a schematic diagram of an experiment of taking out skin tissue from a shaking table and adding a stop solution to stop digestion according to the present invention; Figure 7 It is a schematic diagram of an experiment in which a skin tissue solution of the present invention is placed on a filter screen for grinding and filtering; Figure 8 This is a schematic diagram of an experiment of centrifuging the filtrate, resuspending the precipitated cells, transferring them to a centrifuge tube, aspirating the upper layer solution after centrifugation, and collecting the middle layer T cells into a new centrifuge tube; Fig. 9 This is a schematic diagram of an experiment of adding a digestion stop solution, gently shaking and washing cells, and centrifuging the sediment; Fig.10 It is a schematic diagram of the cell population ratio of the improved method of the present invention and the control group of the prior art; Fig.11 It is a bar chart diagram of the cell percentage of the improved method of the present invention and the control group of the prior art. DETAILED DESCRIPTION

[0015] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0016] Example 1 The present embodiment provides a mouse skin digestion complex enzyme, which is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum with a final concentration of 10%, penicillin / streptomycin double antibody solution with a final concentration of 1%, HEPES buffer with a final concentration of 1%, GlutaMAX with a final concentration of 1%, NEAA with a final concentration of 1%, deoxyribonuclease with a final concentration of 0.1 mg / ml, type IV collagenase with a final concentration of 2 mg / ml, hyaluronidase with a final concentration of 0.5 mg / ml, and pancreatin with a final concentration of 0.25%.

[0017] A method for isolating mouse skin T cells, comprising the following steps: 1. Prepare a fresh solution of mouse skin digestion complex enzyme in Example 1 and place it on ice.

[0018] 2. If Figure 1 As shown, mice were euthanized and the long hair and villi on the back were removed; Figure 2 As shown, apply the depilatory cream, leave it on for 5 minutes and then rinse it off with double distilled water; Figure 3As shown, cut 3cm 2 The free fat layer was gently scraped off from the back skin of the mouse using forceps to obtain the mouse skin tissue.

[0019] 3. If Figure 4 As shown, place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 size, pour into a 50ml centrifuge tube, add 5ml mouse skin digestion complex enzyme solution, such as Figure 5 Digestion was performed at 37°C, 225 rpm in a shaker for 45 min as indicated.

[0020] 4. Prepare digestion stop solution: RPMI 1640 medium containing a final concentration of 10% fetal bovine serum, pre-cool on ice, remove the skin tissue from the shaker, and add 10 ml of the stop solution to stop digestion. Figure 6 shown.

[0021] 5. Vortex the skin tissue solution for 10 seconds and then grind and filter it on a 100 μm filter. Figure 7 The filtrate was centrifuged at 300 g for 10 min at 4 °C.

[0022] 6. Purify the mouse skin T cells in the precipitate by percoll density gradient centrifugation: resuspend the precipitated cells in 4 mL of 30% Percoll, transfer to a 15 ml centrifuge tube pre-added with 3 ml of 60% Percoll, centrifuge at 4°C, 800 g for 20 min, aspirate the upper layer solution, collect the middle layer T cells into a new 15 ml centrifuge tube, as shown in Figure 6. Figure 8 As shown, add 10 ml of digestion stop solution and shake gently to wash the cells, centrifuge at 300 g for 10 min at 4°C, and separate the T cells by precipitation. Fig. 9 shown.

[0023] 7. Resuspend the precipitate in PBS solution (containing 1% BSA at a final concentration), add flow cytometry antibody staining, and perform flow cytometry analysis.

[0024] In summary, pancreatic enzymes are added when digesting skin tissue: when digesting skin tissue, pancreatic enzymes help break down adhesion molecules on the cell surface and promote the release of immune cells; when used together with collagenase, it can improve the separation efficiency of T cells in the skin.

[0025] After digestion of skin tissue, perform Percoll density gradient centrifugation: The main components of Percoll are polyvinyl alcohol and silica particles, which can form gradients with different densities to separate cells of different densities. Through Percoll density gradient centrifugation, immune cells in skin tissue digestion fluid can be further purified, increasing the proportion of T cell populations during flow cytometry analysis.

[0026] Example 2 The present embodiment provides a mouse skin digestion complex enzyme, which is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum with a final concentration of 10%, penicillin / streptomycin double antibody solution with a final concentration of 1%, HEPES buffer with a final concentration of 1%, GlutaMAX with a final concentration of 1%, NEAA with a final concentration of 1%, β-ME with a final concentration of 100 µM, vitamin C with a final concentration of 50 µM, deoxyribonuclease with a final concentration of 0.1 mg / ml, type IV collagenase with a final concentration of 2 mg / ml, and hyaluronidase with a final concentration of 0.5 mg / ml.

[0027] A method for isolating mouse skin T cells, comprising the following steps: 1. Prepare a fresh solution of mouse skin digestion complex enzyme of Example 2 and place it on ice.

[0028] 2. If Figure 1 As shown, mice were euthanized and the long hair and villi on the back were removed; Figure 2 As shown, apply the depilatory cream, leave it on for 5 minutes and then rinse it off with double distilled water; Figure 3 As shown, cut 3cm 2 The free fat layer was gently scraped off from the back skin of the mouse using forceps to obtain the mouse skin tissue.

[0029] 3. If Figure 4 As shown, place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 size, pour into a 50ml centrifuge tube, add 5ml mouse skin digestion complex enzyme solution, such as Figure 5 Digestion was performed at 37°C, 225 rpm in a shaker for 45 min as indicated.

[0030] 4. Prepare digestion stop solution: RPMI 1640 medium, containing 10% fetal bovine serum and 100µM β-ME, pre-cool on ice, remove the skin tissue from the shaker, and add 10 ml of stop solution to stop digestion. Figure 6 shown.

[0031] 5. Vortex the skin tissue solution for 10 seconds and then grind and filter it on a 100 μm filter. Figure 7 As shown, the filtrate was centrifuged at 300 g for 10 min at 4°C, and the separated mouse skin T cells were obtained by precipitation.

[0032] 6. Resuspend the pellet in PBS solution (containing 1% BSA and 100 µM β-ME), add flow cytometry antibody staining, and perform flow cytometry analysis.

[0033] In summary, β-ME is added to the digestive fluid: 1) Improve T cell activity through antioxidant effects β-ME is a powerful antioxidant that can effectively reduce the oxidative stress of cells during culture. During cell culture, oxidative stress may cause cell damage, especially affecting the integrity of cell membranes, proteins and DNA. By adding β-ME, harmful substances such as hydrogen peroxide in the culture environment can be neutralized, protecting cells from oxidative damage, thereby improving cell vitality.

[0034] 2) Improve T cell viability by maintaining the reduced state of cells β-ME, as a reducing agent, can maintain the balance of the reducing state in cells. In cell culture, some cells may become dysfunctional or die due to redox imbalance. β-ME helps maintain the reducing environment in cells by reacting with disulfide bonds in cells (especially disulfide bonds in proteins), thereby enhancing the cell's ability to adapt to environmental changes. This is essential for the survival and functional maintenance of immune cells such as T cells.

[0035] 3) Protect T cell surface receptors and functional proteins During T cell culture, β-ME can help protect cell surface receptors and other functional proteins from oxidation, especially during immune response, surface molecules such as T cell receptors (TCR) need to maintain a certain reduction state in order to correctly bind to the MHC molecules of antigen presenting cells. By adding β-ME, these key proteins can be ensured not to be oxidized, thereby maintaining the function and proliferation of T cells.

[0036] 4) Promote T cell release by reducing disulfide bond formation β-ME can destroy disulfide bonds in the skin tissue matrix, reduce adhesion between tissue cells, and promote the release of T cells from the tissue.

[0037] Add vitamin C to digestive juice: 1) Reduce T cell oxidative damage through antioxidant effects Vitamin C is a powerful antioxidant that can remove free radicals and oxidants produced during cell culture, such as harmful substances such as hydrogen peroxide. Oxidative stress is a common source of cell damage during T cell culture, especially under high-density culture. Vitamin C helps maintain the health of T cells and increases their survival rate and function by reducing these oxidative damages.

[0038] 2) Maintaining a reducing environment within T cells Vitamin C is a powerful reducing agent that can maintain the reduced state within T cells, prevent the formation of disulfide bonds within the cells, maintain the reduced state of proteins including the T cell receptor (TCR) and other proteins involved in the immune response, and maintain T cell functions including TCR activation and signal transduction.

[0039] 3) Improve T cell effector function Vitamin C can promote T cells to produce cytokines and increase cytokine secretion, thereby enhancing the immune response of T cells, and improve the effector function of T cells by regulating intracellular metabolic pathways.

[0040] Example 3 The present embodiment provides a mouse skin digestion complex enzyme, which is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum with a final concentration of 10%, penicillin / streptomycin double antibody solution with a final concentration of 1%, HEPES buffer with a final concentration of 1%, GlutaMAX with a final concentration of 1%, NEAA with a final concentration of 1%, deoxyribonuclease with a final concentration of 0.1 mg / ml, type IV collagenase with a final concentration of 2 mg / ml, hyaluronidase with a final concentration of 0.5 mg / ml, β-ME with a final concentration of 100 µM, and pancreatin with a final concentration of 0.25%.

[0041] A method for isolating mouse skin T cells, comprising the following steps: 1. Prepare a fresh solution of mouse skin digestion complex enzyme in Example 3 and place it on ice.

[0042] 2. If Figure 1 As shown, mice were euthanized and the long hair and villi on the back were removed; Figure 2 As shown, apply the depilatory cream, leave it on for 5 minutes and then rinse it off with double distilled water; Figure 3 As shown, cut 3cm 2 The free fat layer was gently scraped off from the back skin of the mouse using forceps to obtain the mouse skin tissue.

[0043] 3. If Figure 4 As shown, place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 size, pour into a 50ml centrifuge tube, add 5ml mouse skin digestion complex enzyme solution, such as Figure 5 Digestion was performed at 37°C, 225 rpm in a shaker for 45 min as indicated.

[0044] 4. Prepare digestion stop solution: containing 10% fetal bovine serum and 100µM β-ME at a final concentration, pre-cool on ice, remove the skin tissue from the shaker, and add 10ml of stop solution to stop digestion. Figure 6 As shown; 5. Vortex the skin tissue solution for 10 seconds and then grind and filter it on a 100 μm filter. Figure 7 The filtrate was centrifuged at 300 g for 10 min at 4°C as indicated.

[0045] 6. Purify the mouse skin T cells in the precipitate by percoll density gradient centrifugation: resuspend the precipitated cells in 4 mL of 30% Percoll, transfer to a 15 ml centrifuge tube pre-added with 3 ml of 60% Percoll, centrifuge at 4°C, 800 g for 20 min, aspirate the upper layer solution, collect the middle layer T cells into a new 15 ml centrifuge tube, as shown in Figure 6. Figure 8 As shown, add 10 ml of digestion stop solution and gently shake to wash the cells, centrifuge at 300 g for 10 min at 4°C, and separate the T cells obtained by precipitation. Fig. 9 shown.

[0046] 7. Resuspend the pellet in PBS solution (containing 1% BSA and 100 µM β-ME), add flow cytometry antibody staining, and perform flow cytometry analysis.

[0047] In summary, pancreatic enzymes are added when digesting skin tissue: when digesting skin tissue, pancreatic enzymes help break down adhesion molecules on the cell surface and promote the release of immune cells. When used with collagenase, it can improve the efficiency of T cell isolation in the skin.

[0048] Add β-ME to digestion solution and stop solution: 1) Improve T cell activity through antioxidant effects β-ME is a powerful antioxidant that can effectively reduce the oxidative stress of cells during culture. During cell culture, oxidative stress may cause cell damage, especially affecting the integrity of cell membranes, proteins and DNA. By adding β-ME, harmful substances such as hydrogen peroxide in the culture environment can be neutralized, protecting cells from oxidative damage, thereby improving cell vitality.

[0049] 2) Improve the survival rate of T cells by maintaining the reduced state of cells β-ME, as a reducing agent, can maintain the balance of the reducing state in cells. In cell culture, some cells may become dysfunctional or die due to redox imbalance. β-ME helps maintain the reducing environment in cells by reacting with disulfide bonds in cells (especially disulfide bonds in proteins), thereby enhancing the cell's ability to adapt to environmental changes. This is essential for the survival and functional maintenance of immune cells such as T cells.

[0050] 3) Protect T cell surface receptors and functional proteins During T cell culture, β-ME can help protect cell surface receptors and other functional proteins from oxidation, especially during immune response, surface molecules such as T cell receptors (TCR) need to maintain a certain reduction state in order to correctly bind to the MHC molecules of antigen presenting cells. By adding β-ME, these key proteins can be ensured not to be oxidized, thereby maintaining the function and proliferation of T cells.

[0051] 4) Promote T cell release by reducing disulfide bond formation β-ME can destroy disulfide bonds in the skin tissue matrix, reduce adhesion between tissue cells, and promote the release of T cells from the tissue.

[0052] After digestion of skin tissue, perform Percoll density gradient centrifugation: The main components of Percoll are polyvinyl alcohol and silica particles, which can form gradients with different densities to separate cells of different densities. Through Percoll density gradient centrifugation, immune cells in skin tissue digestion fluid can be further purified, increasing the proportion of T cell populations during flow cytometry analysis.

[0053] like Fig.10 and 11 As shown, the analysis results of the methods of Example 1, Example 2 and Example 3 showed that compared with the control group (without the addition of trypsin, β-ME; without purification by Percoll density gradient centrifugation), the T cells isolated from the improved groups of Example 1, Example 2 and Example 3 increased significantly.

[0054] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, all equivalent changes made according to the shape, structure and principle of the present invention without departing from the content of the technical solution of the present invention should be included in the protection scope of the present invention.

Claims

1. A mouse skin digestion complex enzyme, characterized in that: The mouse skin digestion complex enzyme is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, deoxyribonuclease at a final concentration of 0.1 mg / ml, type IV collagenase at a final concentration of 2 mg / ml, hyaluronidase at a final concentration of 0.5 mg / ml, and trypsin at a final concentration of 0.25%; Alternatively, the mouse skin digestion complex enzyme is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1% , β-ME at a final concentration of 100 µM, vitamin C at a final concentration of 50 µM, deoxyribonuclease at a final concentration of 0.1 mg / ml, collagenase type IV at a final concentration of 2 mg / ml, and hyaluronidase at a final concentration of 0.5 mg / ml; Alternatively, the mouse skin digestion complex enzyme is composed of the following components at final concentrations: RPMI 1640 culture medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, deoxyribonuclease at a final concentration of 0.1 mg / ml, collagenase type IV at a final concentration of 2 mg / ml, hyaluronidase at a final concentration of 0.5 mg / ml, β-ME at a final concentration of 100 µM, and trypsin at a final concentration of 0.25%.

2. A method for isolating mouse skin T cells, characterized in that: The following steps are involved: Step S1: prepare a fresh solution of the mouse skin digestion complex enzyme as described in claim 1 and place it on ice; When the prepared mouse skin digestion complex enzyme consists of the following components at final concentrations: RPMI 1640 medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, deoxyribonuclease at a final concentration of 0.1 mg / ml, type IV collagenase at a final concentration of 2 mg / ml, hyaluronidase at a final concentration of 0.5 mg / ml, and trypsin at a final concentration of 0.25%, then jump to steps S2-S7; Step S2: Euthanize the mice, remove the long hair and villi on the back, apply depilatory cream, leave it on for 5 minutes, then rinse it with double distilled water, and cut off 3 cm 2 The free fat layer of the mouse back skin was gently scraped off with forceps to obtain the mouse skin tissue; Step S3: Place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 Pour the mixture into a 50 ml centrifuge tube, add 5 ml mouse skin digestion complex enzyme solution, and place at 37 ° C, 225 rpm shaker for 45 minutes; Step S4: Prepare digestion stop solution: RPMI 1640 medium containing fetal bovine serum at a final concentration of 10%, pre-cool on ice, remove the skin tissue from the shaker, and add 10 ml of the stop solution to stop digestion; Step S5: the skin tissue solution was vortexed for 10 s and then placed on a 100 μm filter, and the filtrate was centrifuged at 300 g for 10 min at 4 °C; Step S6: Purify the mouse skin T cells in the precipitate by percoll density gradient centrifugation: resuspend the precipitated cells in 4 mL of 30% Percoll, transfer to a 15 ml centrifuge tube pre-added with 3 ml of 60% Percoll, centrifuge at 4°C, 800 g for 20 min, aspirate the upper layer solution, collect the middle layer T cells into a new 15 ml centrifuge tube, add 10 ml of digestion stop solution, shake gently to wash the cells, centrifuge at 4°C, 300 g for 10 min, and the precipitate is the separated T cells; Step S7: resuspend the precipitate with PBS solution, add flow cytometry antibody staining, and perform flow cytometry analysis.

3. The method for isolating mouse skin T cells according to claim 2, characterized in that: When the prepared mouse skin digestion complex enzyme consists of the following components at the following final concentrations: RPMI 1640 medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 100 µM β-ME, vitamin C at a final concentration of 50 µM, deoxyribonuclease at a final concentration of 0.1 mg / ml, collagenase type IV at a final concentration of 2 mg / ml, and hyaluronidase at a final concentration of 0.5 mg / ml, then jump to steps S8-S12; Step S8: Euthanize the mice, remove the long hair and villi on the back, apply depilatory cream, leave it for 5 minutes, then rinse it with double distilled water, and cut off 3 cm 2 The free fat layer of the mouse back skin was gently scraped off with forceps to obtain the mouse skin tissue; Step S9: Place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 Pour the mixture into a 50 ml centrifuge tube, add 5 ml mouse skin digestion complex enzyme solution, and place at 37 ° C, 225 rpm shaker for 45 minutes; Step S10: Prepare digestion stop solution: RPMI 1640 medium containing fetal bovine serum at a final concentration of 10% and β-ME at a final concentration of 100 µM, pre-cool on ice, remove the skin tissue from the shaker, and add 10 ml of the stop solution to stop digestion; Step S11: the skin tissue solution was vortexed for 10 seconds and then placed on a 100 μm filter screen for grinding and filtration. The filtrate was centrifuged at 4° C. and 300 g for 10 minutes to obtain the separated mouse skin T cells; Step S12: resuspend the precipitate with PBS solution, add flow cytometry antibody staining, and perform flow cytometry analysis.

4. The method for isolating mouse skin T cells according to claim 2, characterized in that: When the prepared mouse skin digestion complex enzyme consists of the following components at the final concentrations: RPMI 1640 medium, fetal bovine serum at a final concentration of 10%, penicillin / streptomycin double antibody solution at a final concentration of 1%, HEPES buffer at a final concentration of 1%, GlutaMAX at a final concentration of 1%, NEAA at a final concentration of 1%, deoxyribonuclease at a final concentration of 0.1 mg / ml, collagenase type IV at a final concentration of 2 mg / ml, hyaluronidase at a final concentration of 0.5 mg / ml, β-ME at a final concentration of 100 µM, and trypsin at a final concentration of 0.25%, jump to steps S13-S18; Step S13: Euthanize the mice, remove the long hair and villi on the back, apply depilatory cream, leave it for 5 minutes, then rinse it with double distilled water, and cut off 3 cm 2 The free fat layer of the mouse back skin was gently scraped off with forceps to obtain the mouse skin tissue; Step S14: Place the mouse skin tissue in a clean culture dish, add 1 ml of mouse skin digestion complex enzyme solution to moisten it, and cut it into 1 mm pieces with surgical scissors. 2 Pour the mixture into a 50 ml centrifuge tube, add 5 ml mouse skin digestion complex enzyme solution, and place at 37 ° C, 225 rpm shaker for 45 minutes; Step S15: Prepare a digestion stop solution: containing fetal bovine serum with a final concentration of 10% and β-ME with a final concentration of 100 µM, pre-cool it on ice, remove the skin tissue from the shaker, and add 10 ml of the stop solution to stop the digestion; Step S16: the skin tissue solution was vortexed for 10 seconds and then placed on a 100 μm filter, and the filtrate was centrifuged at 300 g for 10 minutes at 4°C; Step S17: Purify the mouse skin T cells in the precipitate by percoll density gradient centrifugation: resuspend the precipitated cells in 4 mL of 30% Percoll, transfer to a 15 ml centrifuge tube pre-added with 3 ml of 60% Percoll, centrifuge at 4°C, 800 g for 20 min, aspirate the upper layer solution, collect the middle layer T cells into a new 15 ml centrifuge tube, add 10 ml of digestion stop solution, shake gently to wash the cells, centrifuge at 4°C, 300 g for 10 min, and precipitate the separated T cells; Step S18: resuspend the precipitate with PBS solution, add flow cytometry antibody staining, and perform flow cytometry analysis.

5. The method for isolating mouse skin T cells according to claim 2, characterized in that: The PBS solution contained BSA at a final concentration of 1%.

6. A method for isolating mouse skin T cells as claimed in claim 3 or 4, characterized in that: The PBS solution contained BSA at a final concentration of 1% and β-ME at a final concentration of 100 µM.

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