Biological cell factor activity increasing preparation method

Abstract

The invention belongs to the technical field of biological cell factors, and particularly relates to a biological cell factor activity increasing preparation method which comprises the following steps: S1, separation and culture of mesenchymal stem cells: putting healthy human umbilical cord, placenta and adipose tissue into a hundred-grade super clean bench, washing and disinfecting, adding 0.1-0.5% mixed collagenase, vibrating and digesting at 37 DEG C and 120-180rpm for 6-50min, and culturing the mesenchymal stem cells; adding the same amount of normal saline, screening with a 150-mesh screen, collecting a primary cell suspension, carrying out gradient centrifugation on the primary cell suspension for three times, adding the precipitate obtained by centrifugation into a special culture medium for resuspension, inoculating the cell suspension into a culture bottle, and culturing in a 5% CO2 incubator at 37 DEG C; under the action of the constant flow pump, the ultrafilters of different specifications are connected in series for use, the whole pipeline is completely in a sterile closed state, the recovery rate of factors can be increased, impurities such as saccharides and macromolecular proteins can be cut off, human intervention is reduced, the automation degree is high, and the device is suitable for large-scale concentration.

Description

Technical Field

[0001] The present invention relates to the technical field of biological cytokines, and in particular to a method for preparing a biological cytokine with increased activity. Background Art

[0002] Biological cytokines are low-molecular-weight soluble proteins produced by a variety of cells induced by immunogens, mitogens or other stimulants. They have multiple functions such as regulating innate immunity and adaptive immunity, hematopoiesis, cell growth and damaged tissue repair. Cytokines can be divided into interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors, etc. Many cytokines exert their effects in the body through paracrine, autocrine or endocrine pathways, and have multiple physiological characteristics such as pleiotropy, overlap, antagonism and synergy, forming a very complex cytokine regulatory network that participates in many important physiological functions of the human body.

[0003] Mesenchymal stem cells are a type of pluripotent stem cell that possess all the common properties of stem cells, namely, self-renewal and multidirectional differentiation capabilities. They are also the most widely used in clinical applications. Their combined use with hematopoietic stem cells can improve the success rate of transplantation and accelerate hematopoietic reconstruction. After patients receive high-dose chemotherapy, the infusion of mesenchymal stem cells along with hematopoietic stem cells can significantly accelerate the recovery of blood cells, with safety and no adverse reactions. Stem cells also contain a large number of bioactive substances. Reported stem cell lysates contain vascular endothelial growth factor, basic fibroblast growth factor, epidermal growth factor, IL-6 and IL-7, megakaryocyte colony-stimulating factor, tumor necrosis factor, interferon, deoxyribonucleic acid and ribonucleic acid, active peptides, and immune proteins. These substances can promote the growth and proliferation of stem cells in the body and activate them.

[0004] Currently, biological cytokines and active substances are added to beauty cosmetics, which not only have moisturizing and whitening effects, but also can repair damaged skin, eliminate skin wrinkles, shrink pores, and improve complexion. There are also some reports on the purification of stem cell culture supernatant and stem cell lysis, but the results are not good. For example, there are residual sugars in the supernatant, the culture supernatant contains fetal bovine serum, and the activity of cytokines decreases during the concentration process, resulting in poor repair effects and low safety of use. Summary of the Invention

[0005] Based on the technical problems such as the decrease in cytokine activity during the concentration process, which leads to poor repair effect and low safety, the present invention proposes a preparation method for increasing the activity of biological cytokines.

[0006] The present invention provides a method for preparing biological cytokine activity growth, comprising the following steps:

[0007] S1: Isolation and culture of mesenchymal stem cells: Put the healthy human umbilical cord, placenta, adipose tissue into a class 100 clean bench, after washing and disinfecting, add 0.1-0.5% mixed collagenase, shake and digest at 37°C, 120-180rpm for 6-50min, supplement with equal amount of normal saline, sieve through a 150 mesh screen, collect the cell suspension, centrifuge the cell suspension three times, collect the precipitate, resuspend in special culture medium, inoculate in culture bottles, and incubate in a 37°C, 5% CO2 incubator, when the cells reach 85% confluence, subculture the cells;

[0008] S2: Cell subculture: Discard the culture medium in the sterile culture bottle, wash the cell surface twice with 0.9% normal saline, add 5ml of 0.075%-0.125% trypsin, observe the cells under a microscope, add equal amount of culture medium to stop digestion, transfer the detached cells and culture medium to a centrifuge tube for centrifugation, resuspend the cell precipitate obtained by centrifugation in culture medium, inoculate for P1 generation culture, when the cells reach more than 85% confluence, digest the P1 generation cells and add them to the cryoprotective solution at a density of 50-80 million / ml for cryopreservation;

[0009] S3: Identification of mesenchymal stem cells: Randomly select P1-P10 generation cells for flow cytometry identification, take 100μl of cell suspension, add 5μl of antibody, incubate at room temperature for 20min, then add 400μl of phosphate buffered saline, mix well, transfer to a 1.5ml EP tube, centrifuge at 1200rpm for 6min, discard the supernatant, then add 400μl of phosphate buffered saline, blow and transfer to the loading tube for machine loading;

[0010] S4: Collection of supernatant: Recover a vial of mesenchymal stem cells from liquid nitrogen, inoculate the cells into a T175 culture bottle, add 40ml of culture medium, and incubate in a 37°C, 5% CO2 incubator, subculture the cells at a ratio of 1:3 every three days, inoculate the cells into a four-layer cell factory when the cells reach P4, and continuously culture to P10 generation, collect all the supernatant, label it, and then digest the cells, resuspend them in culture medium, and store them at -80°C for future use;

[0011] S5: Concentration of supernatant: Filter the collected stem cell culture supernatant through a 0.2μm filter membrane, then concentrate the stem cell supernatant through a Vivaflow50 rotary flow / tangential flow ultrafilter with a molecular weight of 50KD, then pass the concentrated liquid through a Vivaflow50 rotary flow / tangential flow ultrafilter with a molecular weight of 3KD, collect the retentate, which is the concentrated cytokine solution;

[0012] S6: Preparation of lyophilized powder: add lyophilization protective agent to the cytokine concentrate, shake at constant temperature to mix, then divide into packages, 2-3 ml per bottle, put into -80℃ ultra-low temperature freezer for 16-24 hours, freeze-dry, use Shanghai Yibai's YB-FD-1 freeze dryer, vacuum degree 1-10Pa, cold trap temperature -60℃ to 70℃, vacuum time is 18-36 hours.

[0013] As a preferred embodiment of the present invention, in S1: the mixed collagenase is a mixture of collagenase P and collagenase I, and the mixing mass ratio of the collagenase P to the collagenase I is 3:2.

[0014] As a preferred embodiment of the present invention, in S6: the lyoprotectant components are mannitol, dextran, human albumin, vitamin C and propylene glycol, the mannitol accounts for 15-25% of the total volume, the dextran accounts for 3-5% of the total volume, the human albumin accounts for 1-2% of the total volume, the vitamin C accounts for 1% of the total volume, and the propylene glycol accounts for 10-20% of the total volume.

[0015] As a preferred embodiment of the present invention, in S6: the lyoprotectant components are mannitol, dextran, human albumin, vitamin C and propylene glycol, the mannitol accounts for 15% of the total volume, the dextran accounts for 5% of the total volume, the human albumin accounts for 1% of the total volume, the vitamin C accounts for 1% of the total volume, and the propylene glycol accounts for 20% of the total volume.

[0016] As a preferred embodiment of the present invention, in S6: the lyoprotectant components are mannitol, dextran, human albumin, vitamin C and propylene glycol, the mannitol accounts for 20% of the total volume, the dextran accounts for 4% of the total volume, the human albumin accounts for 1.5% of the total volume, the vitamin C accounts for 1% of the total volume, and the propylene glycol accounts for 15% of the total volume.

[0017] As a preferred embodiment of the present invention, in S6: the lyoprotectant components are mannitol, dextran, human albumin, vitamin C and propylene glycol, the mannitol accounts for 25% of the total volume, the dextran accounts for 3% of the total volume, the human albumin accounts for 2% of the total volume, the vitamin C accounts for 1% of the total volume, and the propylene glycol accounts for 10% of the total volume.

[0018] As a preferred embodiment of the present invention, in S6: the prepared lyophilized powder is stored at 4-8°C.

[0019] The beneficial effects of the present invention are:

[0020] 1. The present invention adopts Vivaflow50 vortex flow / tangential flow ultrafilter to prepare cytokine concentrate. Under the action of a constant flow pump, ultrafilters of different specifications are used in series. The entire pipeline is completely in a sterile and closed state. It can not only improve the recovery rate of factors and cut off impurities such as sugars and large molecular proteins, but also reduce human intervention. It has a high degree of automation and is suitable for large-scale concentration.

[0021] 2. The present invention can effectively maintain the activity of stem cell growth factors and active substances by storing the freeze-dried powder at 4-8°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of a method for preparing biological cytokine activity growth proposed by the present invention;

[0023] Figure 2 This is a diagram of the separation and culture of mesenchymal stem cells in a biological cytokine activity growth preparation method proposed by the present invention;

[0024] Figure 3 This is a cell passage diagram of a method for preparing biological cytokine activity growth proposed by the present invention;

[0025] Figure 4 This is an identification diagram of mesenchymal stem cells prepared using a biological cytokine activity growth method proposed by the present invention;

[0026] Figure 5 This is a diagram of collecting the supernatant of a method for preparing biological cytokine activity growth proposed by the present invention;

[0027] Figure 6 This is a concentrated diagram of the supernatant of a method for preparing biological cytokine activity growth proposed by the present invention;

[0028] Figure 7 This is a diagram of the preparation of freeze-dried powder for the biological cytokine activity increase preparation method proposed by the present invention. DETAILED DESCRIPTION

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0030] Example 1:

[0031] Reference Figures 1-7

[0032] A method for preparing biological cytokine activity growth comprises the following steps:

[0033] S1: Isolation and Culture of Mesenchymal Stem Cells: Healthy human umbilical cord, placenta, and adipose tissue were placed in a Class 100 clean bench. After washing and disinfection, 0.1-0.5% mixed collagenase was added and digested at 37°C, 120-180 rpm, with shaking for 6-50 min. An equal amount of physiological saline was added and the cells were sieved through a 150-mesh sieve to collect the primary cell suspension. After three gradient centrifugations of the primary cell suspension, the pellet was resuspended in a dedicated culture medium and the cell suspension was inoculated into a culture flask. The cells were cultured in a 37°C, 5% CO2 incubator. Cells were passaged when they reached 85% confluence.

[0034] S2: Cell passaging: Pour out the culture medium in the sterile culture flask, wash the cell surface twice with 0.9% saline, add 5 ml of 0.075%-0.125% trypsin for digestion, and when the cells begin to round up under a microscope, add an equal amount of culture medium to terminate the digestion. Transfer the detached cells and culture medium to a centrifuge tube and centrifuge. Resuspend the cell pellet obtained by centrifugation in culture medium and inoculate it for P1 culture. When the cells grow to a confluence of more than 85%, digest the P1 cells and add cryoprotectant at a density of 5-8 million / ml for storage at low temperature for later use;

[0035] S3: Identification of mesenchymal stem cells: randomly select P1-P10 cells for flow cytometry identification. Pipette 100 μl of cell suspension, add 5 μl of antibody, incubate at room temperature for 20 min, then add 400 μl of phosphate buffer, vortex to mix, transfer to a 1.5 ml EP tube, centrifuge at 1200 rpm for 6 min, discard the supernatant, and then add 400 μl of phosphate buffer, pipette and transfer to a sample tube for the instrument;

[0036] S4: Collection of supernatant: Resuscitate a mesenchymal stem cell from liquid nitrogen, seed the cells into a T175 culture flask, add 40 ml of culture medium, and culture in a 37°C, 5% CO2 incubator. Subculture the cells at a ratio of 1:3 every three days. When the cells reach P4, seed the cells into a four-layer cell factory and continue to culture until P10. Collect all the supernatant and label it. At the same time, digest the cells, resuspend them in culture medium, and store them at -80°C until use.

[0037] S5: Supernatant concentration: The collected stem cell culture supernatant is filtered through a 0.22 μm filter membrane and then concentrated through a Vivaflow50 vortex flow / tangential flow ultrafilter with a molecular weight of 50 kDa. The concentrate is then passed through a Vivaflow50 vortex flow / tangential flow ultrafilter with a molecular weight of 3 kDa, and the retentate is collected as the cytokine concentrate.

[0038] S6: Preparation of lyophilized powder: add lyophilization protective agent to the cytokine concentrate, shake at constant temperature to mix, then divide into packages, 2-3 ml per bottle, put into -80℃ ultra-low temperature freezer for 16-24 hours, freeze-dry, use Shanghai Yibai's YB-FD-1 freeze dryer, vacuum degree 1-10Pa, cold trap temperature -60℃ to 70℃, vacuum time is 18-36 hours.

[0039] In a specific embodiment, in S1: the mixed collagenase is a mixture of collagenase P and collagenase I, and the mixing mass ratio of collagenase P to collagenase I is 3:2.

[0040] Furthermore, in S6: the freeze-drying protective agent components are mannitol, dextran, human albumin, vitamin C and propylene glycol, mannitol accounts for 15% of the total volume, dextran accounts for 5% of the total volume, human albumin accounts for 1% of the total volume, vitamin C accounts for 1% of the total volume, and propylene glycol accounts for 20% of the total volume.

[0041] Furthermore, in S6: the prepared lyophilized powder is stored at 4-8°C.

[0042] Example 2:

[0043] Reference Figures 1-7

[0044] A method for preparing biological cytokine activity growth comprises the following steps:

[0045] S1: Isolation and Culture of Mesenchymal Stem Cells: Healthy human umbilical cord, placenta, and adipose tissue were placed in a Class 100 clean bench. After washing and disinfection, 0.1-0.5% mixed collagenase was added and digested at 37°C, 120-180 rpm, with shaking for 6-50 min. An equal amount of physiological saline was added and the cells were sieved through a 150-mesh sieve to collect the primary cell suspension. After three gradient centrifugations of the primary cell suspension, the pellet was resuspended in a dedicated culture medium and the cell suspension was inoculated into a culture flask. The cells were cultured in a 37°C, 5% CO2 incubator. Cells were passaged when they reached 85% confluence.

[0046] S2: Cell passaging: Pour out the culture medium in the sterile culture flask, wash the cell surface twice with 0.9% saline, add 5 ml of 0.075%-0.125% trypsin for digestion, and when the cells begin to round up under a microscope, add an equal amount of culture medium to terminate the digestion. Transfer the detached cells and culture medium to a centrifuge tube and centrifuge. Resuspend the cell pellet obtained by centrifugation in culture medium and inoculate it for P1 culture. When the cells grow to a confluence of more than 85%, digest the P1 cells and add cryoprotectant at a density of 5-8 million / ml for storage at low temperature for later use;

[0047] S3: Identification of mesenchymal stem cells: randomly select P1-P10 cells for flow cytometry identification. Pipette 100 μl of cell suspension, add 5 μl of antibody, incubate at room temperature for 20 min, then add 400 μl of phosphate buffer, vortex to mix, transfer to a 1.5 ml EP tube, centrifuge at 1200 rpm for 6 min, discard the supernatant, and then add 400 μl of phosphate buffer, pipette and transfer to a sample tube for the instrument;

[0048] S4: Collection of supernatant: Resuscitate a mesenchymal stem cell from liquid nitrogen, seed the cells into a T175 culture flask, add 40 ml of culture medium, and culture in a 37°C, 5% CO2 incubator. Subculture the cells at a ratio of 1:3 every three days. When the cells reach P4, seed the cells into a four-layer cell factory and continue to culture until P10. Collect all the supernatant and label it. At the same time, digest the cells, resuspend them in culture medium, and store them at -80°C until use.

[0049] S5: Supernatant concentration: The collected stem cell culture supernatant is filtered through a 0.22 μm filter membrane and then concentrated through a Vivaflow50 vortex flow / tangential flow ultrafilter with a molecular weight of 50 kDa. The concentrate is then passed through a Vivaflow50 vortex flow / tangential flow ultrafilter with a molecular weight of 3 kDa, and the retentate is collected as the cytokine concentrate.

[0050] S6: Preparation of lyophilized powder: add lyophilization protective agent to the cytokine concentrate, shake at constant temperature to mix, then divide into packages, 2-3 ml per bottle, put into -80℃ ultra-low temperature freezer for 16-24 hours, freeze-dry, use Shanghai Yibai's YB-FD-1 freeze dryer, vacuum degree 1-10Pa, cold trap temperature -60℃ to 70℃, vacuum time is 18-36 hours.

[0051] In a specific embodiment, in S1: the mixed collagenase is a mixture of collagenase P and collagenase I, and the mixing mass ratio of collagenase P to collagenase I is 3:2.

[0052] Furthermore, in S6: the freeze-drying protective agent components are mannitol, dextran, human albumin, vitamin C and propylene glycol, mannitol accounts for 20% of the total volume, dextran accounts for 4% of the total volume, human albumin accounts for 1.5% of the total volume, vitamin C accounts for 1% of the total volume, and propylene glycol accounts for 15% of the total volume.

[0053] Furthermore, in S6: the prepared lyophilized powder is stored at 4-8°C.

[0054] Example 3:

[0055] Reference Figures 1-7

[0056] A method for preparing biological cytokine activity growth comprises the following steps:

[0057] S1: Isolation and Culture of Mesenchymal Stem Cells: Healthy human umbilical cord, placenta, and adipose tissue were placed in a Class 100 clean bench. After washing and disinfection, 0.1-0.5% mixed collagenase was added and digested at 37°C, 120-180 rpm, with shaking for 6-50 min. An equal amount of physiological saline was added and the cells were sieved through a 150-mesh sieve to collect the primary cell suspension. After three gradient centrifugations of the primary cell suspension, the pellet was resuspended in a dedicated culture medium and the cell suspension was inoculated into a culture flask. The cells were cultured in a 37°C, 5% CO2 incubator. Cells were passaged when they reached 85% confluence.

[0058] S2: Cell passaging: Pour out the culture medium in the sterile culture flask, wash the cell surface twice with 0.9% saline, add 5 ml of 0.075%-0.125% trypsin for digestion, and when the cells begin to round up under a microscope, add an equal amount of culture medium to terminate the digestion. Transfer the detached cells and culture medium to a centrifuge tube and centrifuge. Resuspend the cell pellet obtained by centrifugation in culture medium and inoculate it for P1 culture. When the cells grow to a confluence of more than 85%, digest the P1 cells and add cryoprotectant at a density of 5-8 million / ml for storage at low temperature for later use;

[0059] S3: Identification of mesenchymal stem cells: randomly select P1-P10 cells for flow cytometry identification. Pipette 100 μl of cell suspension, add 5 μl of antibody, incubate at room temperature for 20 min, then add 400 μl of phosphate buffer, vortex to mix, transfer to a 1.5 ml EP tube, centrifuge at 1200 rpm for 6 min, discard the supernatant, and then add 400 μl of phosphate buffer, pipette and transfer to a sample tube for the instrument;

[0060] S4: Collection of supernatant: Resuscitate a mesenchymal stem cell from liquid nitrogen, seed the cells into a T175 culture flask, add 40 ml of culture medium, and culture in a 37°C, 5% CO2 incubator. Subculture the cells at a ratio of 1:3 every three days. When the cells reach P4, seed the cells into a four-layer cell factory and continue to culture until P10. Collect all the supernatant and label it. At the same time, digest the cells, resuspend them in culture medium, and store them at -80°C until use.

[0061] S5: Supernatant concentration: The collected stem cell culture supernatant is filtered through a 0.22 μm filter membrane and then concentrated through a Vivaflow50 vortex flow / tangential flow ultrafilter with a molecular weight of 50 kDa. The concentrate is then passed through a Vivaflow50 vortex flow / tangential flow ultrafilter with a molecular weight of 3 kDa, and the retentate is collected as the cytokine concentrate.

[0062] S6: Preparation of lyophilized powder: add lyophilization protective agent to the cytokine concentrate, shake at constant temperature to mix, then divide into packages, 2-3 ml per bottle, put into -80℃ ultra-low temperature freezer for 16-24 hours, freeze-dry, use Shanghai Yibai's YB-FD-1 freeze dryer, vacuum degree 1-10Pa, cold trap temperature -60℃ to 70℃, vacuum time is 18-36 hours.

[0063] In a specific embodiment, in S1: the mixed collagenase is a mixture of collagenase P and collagenase I, and the mixing mass ratio of collagenase P to collagenase I is 3:2.

[0064] Furthermore, in S6: the freeze-drying protective agent components are mannitol, dextran, human albumin, vitamin C and propylene glycol, mannitol accounts for 25% of the total volume, dextran accounts for 3% of the total volume, human albumin accounts for 2% of the total volume, vitamin C accounts for 1% of the total volume, and propylene glycol accounts for 10% of the total volume.

[0065] Furthermore, in S6: the prepared lyophilized powder is stored at 4-8°C.

[0066] In the present invention, a Vivaflow50 vortex flow / tangential flow ultrafilter is used to prepare a cytokine concentrate. Under the action of a constant flow pump, ultrafilters of different specifications are used in series. The entire pipeline is completely in a sterile and closed state. This not only improves the recovery rate of the factors and removes impurities such as sugars and large molecular proteins, but also reduces human intervention. The degree of automation is high and it is suitable for large-scale concentration. The freeze-dried powder is stored at 4-8°C, which can effectively maintain the activity of stem cell growth factors and active substances.

[0067] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for increasing the activity of biological cytokines, characterized in that: The following steps are involved: S1: Isolation and Culture of Mesenchymal Stem Cells: Healthy human umbilical cord, placenta, and adipose tissue were placed in a Class 100 clean bench. After washing and disinfection, 0.1-0.5% mixed collagenase was added and digested at 37°C, 120-180 rpm, with shaking for 6-50 min. An equal amount of physiological saline was added and the cells were sieved through a 150-mesh sieve to collect the primary cell suspension. After three gradient centrifugations of the primary cell suspension, the pellet was resuspended in a dedicated culture medium and the cell suspension was inoculated into a culture flask. The cells were cultured in a 37°C, 5% CO2 incubator. Cells were passaged when they reached 85% confluence. S2: Cell passaging: Pour out the culture medium in the sterile culture flask, wash the cell surface twice with 0.9% saline, add 5 ml of 0.075%-0.125% trypsin for digestion, and when the cells begin to round up under a microscope, add an equal amount of culture medium to terminate the digestion. Transfer the detached cells and culture medium to a centrifuge tube and centrifuge. Resuspend the cell pellet obtained by centrifugation in culture medium and inoculate it for P1 culture. When the cells grow to a confluence of more than 85%, digest the P1 cells and add cryoprotectant at a density of 5-8 million / ml for storage at low temperature for later use; S3: Identification of mesenchymal stem cells: randomly select P1-P10 cells for flow cytometry identification. Pipette 100 μl of cell suspension, add 5 μl of antibody, incubate at room temperature for 20 min, then add 400 μl of phosphate buffer, vortex to mix, transfer to a 1.5 ml EP tube, centrifuge at 1200 rpm for 6 min, discard the supernatant, and then add 400 μl of phosphate buffer, pipette and transfer to a sample tube for the instrument; S4: Collection of supernatant: Resuscitate a mesenchymal stem cell from liquid nitrogen, seed the cells into a T175 culture flask, add 40 ml of culture medium, and culture in a 37°C, 5% CO2 incubator. Subculture the cells at a ratio of 1:3 every three days. When the cells reach P4, seed the cells into a four-layer cell factory and continue to culture until P10. Collect all the supernatant and label it. At the same time, digest the cells, resuspend them in culture medium, and store them at -80°C until use. S5: Supernatant concentration: The collected stem cell culture supernatant is filtered through a 0.22 μm filter membrane and then concentrated through a Vivaflow50 vortex flow / tangential flow ultrafilter with a molecular weight of 50 kDa. The concentrate is then passed through a Vivaflow50 vortex flow / tangential flow ultrafilter with a molecular weight of 3 kDa, and the retentate is collected as the cytokine concentrate. S6: Preparation of lyophilized powder: add lyophilization protective agent to the cytokine concentrate, shake at constant temperature to mix, then divide into packages, 2-3 ml per bottle, put into -80℃ ultra-low temperature freezer for 16-24 hours, freeze-dry, use Shanghai Yibai's YB-FD-1 freeze dryer, vacuum degree 1-10Pa, cold trap temperature -60℃ to 70℃, vacuum time is 18-36 hours.

2. The method for increasing the activity of biological cytokines according to claim 1, characterized in that: In S1, the mixed collagenase is a mixture of collagenase P and collagenase I, and the mixing mass ratio of the collagenase P to the collagenase I is 3:

2.

3. The method for increasing the activity of biological cytokines according to claim 1, characterized in that: In S6: the freeze-dried protective agent components are mannitol, dextran, human albumin, vitamin C and propylene glycol, the mannitol accounts for 15-25% of the total volume, the dextran accounts for 3-5% of the total volume, the human albumin accounts for 1-2% of the total volume, the vitamin C accounts for 1% of the total volume, and the propylene glycol accounts for 10-20% of the total volume.

4. The method for increasing the activity of biological cytokines according to claim 1, characterized in that: In S6: the freeze-dried protective agent components are mannitol, dextran, human albumin, vitamin C and propylene glycol, the mannitol accounts for 15% of the total volume, the dextran accounts for 5% of the total volume, the human albumin accounts for 1% of the total volume, the vitamin C accounts for 1% of the total volume, and the propylene glycol accounts for 20% of the total volume.

5. The method for increasing the activity of biological cytokines according to claim 1, characterized in that: In S6: the freeze-dried protective agent components are mannitol, dextran, human albumin, vitamin C and propylene glycol, the mannitol accounts for 20% of the total volume, the dextran accounts for 4% of the total volume, the human albumin accounts for 1.5% of the total volume, the vitamin C accounts for 1% of the total volume, and the propylene glycol accounts for 15% of the total volume.

6. The method for increasing the activity of biological cytokines according to claim 1, characterized in that: In S6: the freeze-dried protective agent components are mannitol, dextran, human albumin, vitamin C and propylene glycol, the mannitol accounts for 25% of the total volume, the dextran accounts for 3% of the total volume, the human albumin accounts for 2% of the total volume, the vitamin C accounts for 1% of the total volume, and the propylene glycol accounts for 10% of the total volume.

7. The method for increasing the activity of biological cytokines according to claim 1, characterized in that: In S6: the prepared lyophilized powder is stored at 4-8°C.

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