A method for preparing freeze-dried powder of bovine whole blood extract

Abstract

The invention provides a method for preparing a lyophilized powder of a bovine whole blood extract, comprising the following steps: (1) collecting healthy bovine fetal blood, culturing it with a blood analyzer, and counting CIK cells of the cultured bovine fetal blood; separating mononuclear cells with a lymphocyte separation solution; suspending the obtained mononuclear cells in a CIK cell serum-free culture solution to form a mixed solution; (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing it in a CO2 incubator to obtain a final culture solution; (3) subjecting the final culture solution to centrifugation and ultrafiltration to obtain a leukocyte extract; (4) mixing 100 parts by weight of the leukocyte extract, 3-8 parts by weight of mannitol, 2-6 parts by weight of trehalose, 1-4 parts by weight of nicotinamide, 0.2-1.9 parts by weight of tranexamic acid and 1-2 parts by weight of vitamin C to obtain a product; and (5) freeze-drying the product to obtain a lyophilized powder.

Description

Technical Field

[0001] The invention belongs to the technical field of freeze-dried powder, and in particular relates to a method for preparing freeze-dried powder of bovine whole blood extract. Background Art

[0002] Blood freeze-dried powder usually refers to a blood product or blood component that has been freeze-dried, such as thrombin freeze-dried powder, which is a local hemostatic drug made from prothrombin extracted from animal (such as cow or pig) blood. The following are some of the main uses of thrombin freeze-dried powder: Hemostatic effect: The main function of thrombin freeze-dried powder is to promote the rapid conversion of fibrinogen in the blood into fibrin, accelerate the blood coagulation process, and thus achieve a hemostatic effect. It is often used in small blood vessels that are not easy to ligate during surgery, gastrointestinal bleeding, traumatic bleeding, etc. Local application: It is suitable for a variety of bleeding conditions, including but not limited to nose bleeding, gum bleeding, skin cuts, surgical wound bleeding, etc., and it can be directly applied to the bleeding site to exert its rapid hemostatic effect. Treatment of gastrointestinal bleeding: In the treatment of gastrointestinal bleeding, in addition to direct application, it is also recommended to first give antacids to neutralize gastric acid and use acid inhibitors at the same time to optimize the treatment effect. Anti-inflammatory effect: Although the main effect is hemostasis, thrombin freeze-dried powder has also been pointed out to have a certain anti-inflammatory effect.

[0003] In the prior art, bovine serum is a light yellow transparent liquid obtained by centrifuging and filtering blood collected from cattle. It is rich in various nutrients and growth factors and is an indispensable raw material in the fields of cell culture, biopharmaceuticals, vaccine production, etc. Bovine serum has the characteristics of rich nutrition, significant growth promotion effect, and good batch stability, and is widely used in life science research and biotechnology industry. The existing bovine serum storage conditions are high and need to be stored in a cold storage before it can be transported. The preparation of blood freeze-dried powder is a complex and delicate process, which usually includes the following key steps: Collection and anticoagulation: For blood from animals, fresh blood needs to be collected first, and an anticoagulant (such as heparin) is added immediately to prevent blood coagulation. For example, in the preparation of Dachengqi Decoction animal plasma freeze-dried powder, heparin is used for anticoagulation immediately after blood collection. In the production of deer blood freeze-dried powder, a blood anticoagulant solution of a specific concentration is prepared, and then fresh deer blood is mixed with it, and other ingredients such as highly purified grain liquor may be added for homogenization and stirring. Pretreatment: The collected blood is subjected to necessary tests and tests to ensure that it meets the requirements of subsequent treatment, such as microbial indicators, active ingredient content, etc. Separation and purification: For example, in the case of Dachengqi Decoction animal plasma freeze-dried powder, plasma separation is required. The blood is separated into blood cells and plasma by means of centrifugation or filtration, and only the required part is retained for the next step. Solution preparation: The required active ingredients (such as plasma, thrombin, etc.) are mixed with suitable excipients (such as mannitol) and other necessary ingredients to prepare a solution to be freeze-dried. Freezing: The prepared solution is quickly frozen to an extremely low temperature, generally at -20°C or lower, to form solid ice crystals, which helps to maintain the stability of the ingredients. Vacuum freeze drying: The frozen solution is placed in a vacuum environment to remove water by sublimation. At this stage, the temperature is still maintained at a low level, and the water is pumped out by a vacuum pump to prevent the water from directly converting from liquid to gas, ensuring the dryness and stability of the final product. Crushing and packaging: After drying, the freeze-dried product is crushed into the required powder, and then packaged and sealed under sterile conditions to ensure the long-term stability and sterility of the product.

[0004] Culture medium refers to a nutrient matrix composed of different nutrients for the growth and reproduction of microorganisms, plants or animals (or tissues). It generally contains several major types of substances such as carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins and water. According to its composition and use, culture medium can be divided into basic culture medium, enrichment culture medium, selective culture medium, identification culture medium and anaerobic culture medium. The uses of culture medium mainly include: growth and reproduction of living organisms such as microorganisms, plant or animal cells; identification, classification and counting of microorganisms; storage of strains or maintenance of strain vitality; scientific research, such as exploring the physiological and biochemical reaction mechanisms in the life process. Liquid culture medium is a culture medium in liquid form, which is a solution composed of water and various nutrients. Compared with solid culture medium, liquid culture medium is more convenient to use, can provide a larger culture capacity, and can better control the culture conditions. The existing liquid culture medium has high storage conditions, cannot separate the solid and liquid in the culture medium, needs to be stored in a cold storage before it can be transported, and has a short shelf life, which is not convenient for storage. At the same time, it will also produce sediments that affect the use of subsequent staff.

[0005] Bovine serum is a light yellow transparent liquid obtained by centrifuging and filtering blood collected from cattle. It is rich in various nutrients and growth factors and is an indispensable raw material in the fields of cell culture, biopharmaceuticals, and vaccine production. Bovine serum is rich in nutrients, has a significant growth-promoting effect, and has good batch stability. It is widely used in life science research and biotechnology industries. The existing bovine serum storage conditions are high and it needs to be stored in a cold storage before it can be transported.

[0006] In the freeze-drying process of bovine serum, the key process parameters include pre-freezing temperature, vacuum degree, heating rate, etc. The reasonable control of these parameters is crucial to ensure the quality of freeze-dried products. If the pre-freezing temperature is too low, the ice crystals may be too large, affecting the drying effect; if the vacuum degree is too low, the sublimation rate may be reduced, prolonging the freeze-drying time; if the heating rate is too fast, the product temperature may be too high, affecting the product quality. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing a lyophilized powder of bovine whole blood extract. The present invention aims at the problem that the existing bovine serum storage conditions have high requirements. The lyophilized powder of bovine serum can be used as a nutrient component of a culture medium after adding purified water, can be stored at room temperature, does not need to be stored in a cold storage, is convenient and quick to use, and can be taken and used at any time.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0010] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0011] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0012] (3) The final culture medium is centrifuged at 3200-3500 r / min for 25-30 min, and then ultrafiltered at 10-20 KD to obtain a leukocyte extract;

[0013] (4) mixing 100 parts by weight of leukocyte extract, 3-8 parts by weight of mannitol, 2-6 parts by weight of trehalose, 1-4 parts by weight of nicotinamide, 0.2-1.9 parts by weight of tranexamic acid and 1-2 parts by weight of vitamin C to obtain a product;

[0014] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0015] Furthermore, the bovine fetal blood CIK cells cultured in step (1) are ≥5×10 per liter. 10 indivual.

[0016] Furthermore, the amount of IL 2 added is 3-5% of the mass of the mixed solution.

[0017] Furthermore, the amount of interferon added is 1-3% of the mass of the mixed solution.

[0018] Furthermore, the amount of CD3 monoclonal antibody added accounts for 0.1-0.9% of the mass of the mixed solution.

[0019] Furthermore, the pre-freeze drying is specifically as follows: pre-freeze in an ultra-low temperature refrigerator at -80--60°C for 10-12 hours, and then place in a vacuum freeze dryer at -30--25°C for 1-2 hours, and then place in a vacuum freeze dryer at a vacuum degree of 1.2×10 -2 -1.6×10 -2 mbar, freeze-dry for 10-12 h at -10--5 °C, maintain vacuum, and continue freeze-drying at 20 °C for 3-4 h.

[0020] Furthermore, in step (2), the culture is carried out at a temperature of 35-36° C. for 15-17 days.

[0021] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: the present invention solves the problem that the existing bovine serum storage conditions require high conditions. The bovine serum freeze-dried powder of the present invention can be used as a nutrient component of a liquid culture medium after adding purified water, and can be stored at room temperature without the need for cold storage. It is convenient and quick to use, and can be used whenever you want. In view of the problem that the existing bovine serum storage conditions require high conditions, the provided bovine serum freeze-dried powder can be stored at room temperature without the need for cold storage. It is convenient and quick to use, and can be used whenever you want. Moreover, the effect is better than the quality of serum before freeze-drying, has no toxic side effects, and the quality of the bovine serum freeze-dried powder does not change significantly when stored at room temperature for a short period of time.

[0022] The bovine whole blood extract freeze-dried powder of the present invention can be further used as an active ingredient in the preparation of products in multiple fields such as medicines and biological preparations. It can be prepared in any form, such as oral dosage forms: powders, tablets, capsules, soft capsules, aqueous medicines, syrups, tinctures, pills, powders, packets, or granules; topical preparations for external use: creams, ointments, emulsions, gels, semisolid pastes, patches, pastes, sprays or aerosols; injections: solutions, suspensions or emulsions, with good storage stability and high biological activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the growth status of the lyophilized HEK-293 cells in Example 1;

[0024] Figure 2 This is a schematic diagram of the growth status of the freeze-dried powder SP2 / 0-Ag / 14 cells in Example 1;

[0025] Figure 3 This is a schematic diagram of the growth status of the lyophilized Hela cells of Example 1;

[0026] Figure 4 This is a schematic diagram of the growth state of the freeze-dried powder Vero cells in Example 1;

[0027] Figure 5 Schematic diagram of HEK-293 cell growth status in traditional serum;

[0028] Figure 6 This is a schematic diagram of the growth status of SP2 / 0-Ag / 14 cells in traditional serum;

[0029] Figure 7 This is a schematic diagram of the growth status of Hela cells in traditional serum;

[0030] Figure 8 Schematic diagram of the growth status of Vero cells in traditional serum. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] This embodiment provides a method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0034] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0035] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0036] (3) The final culture medium was centrifuged at 3200 r / min for 25 min and then ultrafiltered at 10 KD to obtain a leukocyte extract;

[0037] (4) mixing 100 parts by weight of leukocyte extract, 3 parts by weight of mannitol, 2 parts by weight of trehalose, 1 part by weight of nicotinamide, 0.2 parts by weight of tranexamic acid and 1 part by weight of vitamin C to obtain a product;

[0038] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0039] The bovine fetal blood CIK cells cultured in step (1) are 5×10 per liter. 10 indivual.

[0040] The amount of IL 2 added was 3% of the mixed solution mass.

[0041] The amount of interferon added is 1% of the mass of the mixed solution.

[0042] The amount of CD3 monoclonal antibody added was 0.1% of the mass of the mixed solution.

[0043] The pre-freeze drying process is as follows: pre-freeze in a -80°C ultra-low temperature refrigerator for 10 h, then place in a vacuum freeze dryer at -30°C for 1 h, and keep in a vacuum of 1.2×10 -2 mbar, freeze-dry for 10 h at -10 °C, maintain vacuum, and continue freeze-drying at 20 °C for 3 h.

[0044] In the step (2), the culture is carried out at a temperature of 35° C. for 15 days.

[0045] Example 2

[0046] This embodiment provides a method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0047] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0048] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0049] (3) The final culture medium was centrifuged at 3500 r / min for 30 min, and then ultrafiltered at 20 KD to obtain a leukocyte extract;

[0050] (4) mixing 100 parts by weight of leukocyte extract, 8 parts by weight of mannitol, 6 parts by weight of trehalose, 4 parts by weight of nicotinamide, 1.9 parts by weight of tranexamic acid and 2 parts by weight of vitamin C to obtain a product;

[0051] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0052] The bovine fetal blood CIK cells cultured in step (1) are 6×10 per liter. 10 indivual.

[0053] The amount of IL 2 added was 5% of the mixed solution mass.

[0054] The amount of interferon added is 3% of the mixed solution mass.

[0055] The added amount of CD3 monoclonal antibody accounted for 0.9% of the mass of the mixed solution.

[0056] The specific pre-freeze drying process is as follows: pre-freeze in a -60°C ultra-low temperature refrigerator for 12 hours, then place in a vacuum freeze dryer, keep at -25°C for 2 hours, and keep the temperature at a vacuum degree of 1.6×10 -2 mbar, freeze-dry at -5°C for 12 h, maintain vacuum, and continue freeze-drying at 20°C for 4 h.

[0057] In the step (2), the culture is carried out at a temperature of 36° C. for 17 days.

[0058] Comparative Example 1

[0059] The difference between this comparative example and Example 1 is that the addition amount of IL 2 accounts for 7% of the mass of the mixed solution.

[0060] A method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0061] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0062] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0063] (3) The final culture medium was centrifuged at 3200 r / min for 25 min and then ultrafiltered at 10 KD to obtain a leukocyte extract;

[0064] (4) mixing 100 parts by weight of leukocyte extract, 3 parts by weight of mannitol, 2 parts by weight of trehalose, 1 part by weight of nicotinamide, 0.2 parts by weight of tranexamic acid and 1 part by weight of vitamin C to obtain a product;

[0065] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0066] The bovine fetal blood CIK cells cultured in step (1) are 5×10 per liter. 10 indivual.

[0067] The amount of IL 2 added was 7% of the mixed solution mass.

[0068] The amount of interferon added is 1% of the mass of the mixed solution.

[0069] The amount of CD3 monoclonal antibody added was 0.1% of the mass of the mixed solution.

[0070] The pre-freeze drying process is as follows: pre-freeze in a -80°C ultra-low temperature refrigerator for 10 h, then place in a vacuum freeze dryer at -30°C for 1 h, and keep in a vacuum of 1.2×10 -2 mbar, freeze-dry for 10 h at -10 °C, maintain vacuum, and continue freeze-drying at 20 °C for 3 h.

[0071] In the step (2), the culture is carried out at a temperature of 35° C. for 15 days.

[0072] Comparative Example 2

[0073] The difference between this comparative example and Example 1 is that the amount of interferon added accounts for 6% of the mass of the mixed solution.

[0074] A method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0075] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0076] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0077] (3) The final culture medium was centrifuged at 3200 r / min for 25 min and then ultrafiltered at 10 KD to obtain a leukocyte extract;

[0078] (4) mixing 100 parts by weight of leukocyte extract, 3 parts by weight of mannitol, 2 parts by weight of trehalose, 1 part by weight of nicotinamide, 0.2 parts by weight of tranexamic acid and 1 part by weight of vitamin C to obtain a product;

[0079] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0080] The bovine fetal blood CIK cells cultured in step (1) are 5×10 per liter. 10 indivual.

[0081] The amount of IL 2 added was 3% of the mixed solution mass.

[0082] The amount of interferon added is 6% of the mixed solution mass.

[0083] The amount of CD3 monoclonal antibody added was 0.1% of the mass of the mixed solution.

[0084] The pre-freeze drying process is as follows: pre-freeze in a -80°C ultra-low temperature refrigerator for 10 h, then place in a vacuum freeze dryer at -30°C for 1 h, and keep in a vacuum of 1.2×10 -2 mbar, freeze-dry for 10 h at -10 °C, maintain vacuum, and continue freeze-drying at 20 °C for 3 h.

[0085] In the step (2), the culture is carried out at a temperature of 35° C. for 15 days.

[0086] Comparative Example 3

[0087] The difference between this comparative example and Example 1 is that the added amount of CD3 monoclonal antibody accounts for 1.4% of the mass of the mixed solution.

[0088] A method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0089] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0090] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0091] (3) The final culture medium was centrifuged at 3200 r / min for 25 min and then ultrafiltered at 10 KD to obtain a leukocyte extract;

[0092] (4) mixing 100 parts by weight of leukocyte extract, 3 parts by weight of mannitol, 2 parts by weight of trehalose, 1 part by weight of nicotinamide, 0.2 parts by weight of tranexamic acid and 1 part by weight of vitamin C to obtain a product;

[0093] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0094] The bovine fetal blood CIK cells cultured in step (1) are 5×10 per liter. 10 indivual.

[0095] The amount of IL 2 added was 3% of the mixed solution mass.

[0096] The amount of interferon added is 1% of the mass of the mixed solution.

[0097] The amount of CD3 monoclonal antibody added accounted for 1.4% of the mixed solution mass.

[0098] The pre-freeze drying process is as follows: pre-freeze in a -80°C ultra-low temperature refrigerator for 10 h, then place in a vacuum freeze dryer at -30°C for 1 h, and keep in a vacuum of 1.2×10 -2 mbar, freeze-dry for 10 h at -10 °C, maintain vacuum, and continue freeze-drying at 20 °C for 3 h.

[0099] In the step (2), the culture is carried out at a temperature of 35° C. for 15 days.

[0100] Comparative Example 4

[0101] The difference between this comparative example and Example 1 is that the weight portion of mannitol is different.

[0102] A method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0103] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0104] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0105] (3) The final culture medium was centrifuged at 3200 r / min for 25 min and then ultrafiltered at 10 KD to obtain a leukocyte extract;

[0106] (4) mixing 100 parts by weight of leukocyte extract, 1 part by weight of mannitol, 2 parts by weight of trehalose, 1 part by weight of nicotinamide, 0.2 parts by weight of tranexamic acid and 1 part by weight of vitamin C to obtain a product;

[0107] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0108] The bovine fetal blood CIK cells cultured in step (1) are 5×10 per liter. 10 indivual.

[0109] The amount of IL 2 added was 3% of the mixed solution mass.

[0110] The amount of interferon added is 1% of the mass of the mixed solution.

[0111] The amount of CD3 monoclonal antibody added was 0.1% of the mass of the mixed solution.

[0112] The pre-freeze drying process is as follows: pre-freeze in a -80°C ultra-low temperature refrigerator for 10 h, then place in a vacuum freeze dryer at -30°C for 1 h, and keep in a vacuum of 1.2×10 -2 mbar, freeze-dry for 10 h at -10 °C, maintain vacuum, and continue freeze-drying at 20 °C for 3 h.

[0113] In the step (2), the culture is carried out at a temperature of 35° C. for 15 days.

[0114] Comparative Example 5

[0115] The difference between this comparative example and Example 1 is that the weight portion of niacinamide is different.

[0116] A method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0117] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0118] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0119] (3) The final culture medium was centrifuged at 3200 r / min for 25 min and then ultrafiltered at 10 KD to obtain a leukocyte extract;

[0120] (4) mixing 100 parts by weight of leukocyte extract, 3 parts by weight of mannitol, 2 parts by weight of trehalose, 7 parts by weight of nicotinamide, 0.2 parts by weight of tranexamic acid and 1 part by weight of vitamin C to obtain a product;

[0121] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0122] The bovine fetal blood CIK cells cultured in step (1) are 5×10 per liter. 10 indivual.

[0123] The amount of IL 2 added was 3% of the mixed solution mass.

[0124] The amount of interferon added is 1% of the mass of the mixed solution.

[0125] The amount of CD3 monoclonal antibody added was 0.1% of the mass of the mixed solution.

[0126] The pre-freeze drying process is as follows: pre-freeze in a -80°C ultra-low temperature refrigerator for 10 h, then place in a vacuum freeze dryer at -30°C for 1 h, and keep in a vacuum of 1.2×10 -2 mbar, freeze-dry for 10 h at -10 °C, maintain vacuum, and continue freeze-drying at 20 °C for 3 h.

[0127] In the step (2), the culture is carried out at a temperature of 35° C. for 15 days.

[0128] Comparative Example 6

[0129] The difference between this comparative example and Example 1 is that the weight portion of tranexamic acid is different.

[0130] A method for preparing a lyophilized powder of bovine whole blood extract, the preparation method comprising the following steps:

[0131] (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution;

[0132] (2) adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain the final culture solution;

[0133] (3) The final culture medium was centrifuged at 3200 r / min for 25 min and then ultrafiltered at 10 KD to obtain a leukocyte extract;

[0134] (4) mixing 100 parts by weight of leukocyte extract, 3 parts by weight of mannitol, 2 parts by weight of trehalose, 1 part by weight of nicotinamide, 2.5 parts by weight of tranexamic acid and 1 part by weight of vitamin C to obtain a product;

[0135] (5) The product is pre-freeze-dried under sterile conditions at -25°C. After drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain freeze-dried powder.

[0136] The bovine fetal blood CIK cells cultured in step (1) are 5×10 per liter. 10 indivual.

[0137] The amount of IL 2 added was 3% of the mixed solution mass.

[0138] The amount of interferon added is 1% of the mass of the mixed solution.

[0139] The amount of CD3 monoclonal antibody added was 0.1% of the mass of the mixed solution.

[0140] The pre-freeze drying process is as follows: pre-freeze in a -80°C ultra-low temperature refrigerator for 10 h, then place in a vacuum freeze dryer at -30°C for 1 h, and keep in a vacuum of 1.2×10 -2 mbar, freeze-dry for 10 h at -10 °C, maintain vacuum, and continue freeze-drying at 20 °C for 3 h.

[0141] In the step (2), the culture is carried out at a temperature of 35° C. for 15 days.

[0142] Performance Testing

[0143] Stability test

[0144] The freeze-dried powder prepared in the embodiment and the comparative example was subjected to accelerated testing and placed in a constant temperature and humidity incubator at a temperature of 25°C ± 2°C and a relative humidity of 65% ± 5% for 6 months to examine various indicators of the product. The accelerated test results of the product are shown in Table 1.

[0145] Table 1 Performance test results

[0146]

[0147] 2. Comparison of the effects of the lyophilized powder of Example 1 of the present invention as a culture medium and traditional serum in culturing different cells. See Table 2. Figure 1-8 .

[0148] Table 2 Test results

[0149]

[0150] The results show that, when the freeze-dried powder of Example 1 of the present invention and traditional serum were used to culture different types of cells, HEK-293, SP2 / 0-Ag / 14, Hela, and Vero cells grew well, and the monolayer cell outlines were clear; the serum freeze-dried powder was superior to the traditional serum.

[0151] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a freeze-dried powder of bovine whole blood extract, characterized in that: The preparation method comprises the following steps: (1) Collect healthy bovine fetal blood, culture it using a blood analyzer, and count CIK cells in the cultured bovine fetal blood; separate mononuclear cells using lymphocyte separation fluid; suspend the obtained mononuclear cells in CIK cell serum-free culture fluid to form a mixed solution; (2) Adding IL-2, interferon and CD3 monoclonal antibody to the mixed solution, and culturing in a CO2 incubator to obtain a final culture solution; the amount of IL2 added accounts for 3-5% of the mass of the mixed solution, the amount of interferon added accounts for 1-3% of the mass of the mixed solution, and the amount of CD3 monoclonal antibody added accounts for 0.1-0.9% of the mass of the mixed solution; (3) The final culture medium is centrifuged at 3200-3500 r / min for 25-30 min, and then ultrafiltered at 10-20 KD to obtain a leukocyte extract; (4) mixing 100 parts by weight of leukocyte extract, 3-8 parts by weight of mannitol, 2-6 parts by weight of trehalose, 1-4 parts by weight of nicotinamide, 0.2-1.9 parts by weight of tranexamic acid and 1-2 parts by weight of vitamin C to obtain a product; (5) The product is pre-freeze-dried under sterile conditions, and after drying, ice crystals are removed and the product is crushed, sealed, and stored at 4°C to obtain a freeze-dried powder; The number of bovine fetal blood CIK cells cultured in step (1) is ≥ 5×10 per liter. 10 indivual; Pre-freeze drying is specifically as follows: pre-freeze in an ultra-low temperature refrigerator at -80--60°C for 10-12 hours, then place in a vacuum freeze dryer, keep at -30--25°C for 1-2 hours, freeze-dry under vacuum at -10--5°C for 10-12 hours, maintain the vacuum degree, and continue freeze-drying.

2. The method for preparing the lyophilized powder of bovine whole blood extract according to claim 1, characterized in that: In the step (2), the culture is carried out at a temperature of 35-36°C.

3. The method for preparing the lyophilized powder of bovine whole blood extract according to claim 1, characterized in that: The number of bovine fetal blood CIK cells cultured in step (1) is ≥7×10 per liter. 10 indivual.

4. The method for preparing the lyophilized powder of bovine whole blood extract according to claim 1, characterized in that: The vacuum degree is 1.2×10 -2 -1.6×10 -2 mbar.

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