A serum-free culture medium for SP2 / 0 cell culture and its preparation method and application

Abstract

The present application relates to the field of animal cell culture medium, and specifically discloses a serum-free culture medium for SP2 / 0 cell culture and a preparation method and application. The serum-free culture medium for SP2 / 0 cell culture is calculated per 1L, and the raw materials used for its preparation include 10.15g of MEM-α dry powder containing nucleosides, 50-1000μg heparin sodium, 200-4000mg recombinant human serum albumin and corresponding amounts of arachidonic acid, tocopherol acetate, Pluronic F-68, transferrin, recombinant human insulin, sodium selenite, ethanolamine, ascorbic acid, hydrocortisone, etc., and the remainder is water for cell culture. The serum-free culture medium for SP2 / 0 cell culture of the present application can meet the requirements that SP2 / 0 cells can be stably suspended and cultured under serum-free conditions without taming. During the SP2 / 0 cell culture process, when reaching high density, the cells do not aggregate, and the multiplication is stable.

Description

Technical Field

[0001] The present application relates to the field of animal cell culture medium, and in particular to a serum-free culture medium for SP2 / 0 cell culture and a preparation method and application thereof. Background Art

[0002] SP2 / 0 cells are mouse myeloma cells. SP2 / 0 cells are used to prepare mouse monoclonal antibodies and are also a common method in the preparation of monoclonal antibodies. However, SP2 / 0 cells themselves cannot secrete antibodies. Only hybridoma cells that have been successfully established after fusion with B lymphocytes that secrete positive antibodies can secrete antibodies.

[0003] In the related art, when SP2 / 0 cells are expanded and cultured in vitro, the culture medium used needs to be supplemented with a certain concentration of serum. Serum is a highly complex mixture composed of many components, including growth factors, hormones, trace elements, adhesion and extension factors, etc., which play a variety of biological activities. However, due to the presence of animal serum components in serum culture medium, it is easy to cause exogenous contamination, cell morphological variation and other problems, and there are potential safety hazards of carrying pathogens and large differences between different batches, which increases the difficulty of subsequent production processes.

[0004] In recent years, commercial SP2 / 0 cell serum-free culture medium has been prone to problems such as cell aggregation and unstable multiplication when SP2 / 0 cells are cultured at high density. Summary of the invention

[0005] The purpose of the present application is to provide a serum-free medium for SP2 / 0 cell culture and a preparation method and application thereof in order to solve the above technical problems. The serum-free medium for SP2 / 0 cell culture can satisfy the requirements of stable suspension culture of SP2 / 0 cells under serum-free conditions, and when the SP2 / 0 cells reach a high density during culture, the cells do not aggregate and multiply stably, while reducing the production cost of the large-scale preparation of the serum-free medium for SP2 / 0 cell culture.

[0006] The technical solution of this application

[0007] In a first aspect, the present application provides a serum-free culture medium for SP2 / 0 cell culture, using the following technical solution:

[0008] A serum-free medium for SP2 / 0 cell culture, wherein the composition and content of each raw material used for its preparation are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 0.2-3μg, linoleic acid 1-25μg, linolenic acid 1-25μg, myristic acid 1-25μg, oleic acid 1-25μg, palmitic acid 1-25μg, stearic acid 1-25μg, cholesterol 0.1-2mg, Tween 800 0.2-8mg, tocopherol acetate 1-25mg, Pluronic F-68 1-30mg, heparin sodium 50-1000μg, transferrin 1-15mg, recombinant human insulin 1-15mg, recombinant human serum albumin 200-4000mg, sodium selenite 0.1-1.5nmol, ethanolamine 10-150μmol, ascorbic acid 1-12mg, hydrocortisone 0.0005-0.01mg, sodium bicarbonate 2.2g, the balance is water for cell culture;

[0009] The Pluronic F-68 has a product number of P7061 and is produced by Sigma-Aldrich.

[0010] By adopting the above technical scheme, the above serum-free culture medium for SP2 / 0 cell culture is based on MEM-α culture medium, and arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, cholesterol, transferrin, recombinant human insulin and recombinant human serum albumin promote cell metabolism and provide basic components for SP2 / 0 cell growth; sodium selenite and ethanolamine can stimulate the growth and proliferation of SP2 / 0 cells and accelerate the growth and proliferation rate of cells; ascorbic acid and tocopherol acetate can provide antioxidant protection measures for SP2 / 0 cells; sodium heparin can reduce the aggregation of cells under high-density growth conditions; and combined with raw material components such as Tween 80, Pluronic F-68, and hydrocortisone, the serum-free culture medium for SP2 / 0 cell culture can achieve high-density culture, that is, the cell density reaches 1.3×10 6 cells / mL-3.6×10 6 cells / mL, and the cell viability was greater than 80%. It had the characteristics of good anti-aggregation effect and good multiplication stability. The cell agglomeration rate was less than 6%. The cell density of SP2 / 0 cells could still increase by more than 1 time after five passages.

[0011] Preferably, in the raw material components used in the above-mentioned serum-free culture medium for SP2 / 0 cell culture, the content of heparin sodium is 100-200 μg per 1 L of serum-free culture medium for SP2 / 0 cell culture.

[0012] By adopting the above technical solution, when SP2 / 0 cells are cultured in the serum-free medium for SP2 / 0 cell culture, high-density culture can be achieved, that is, the cell density reaches 2.0×10 6 cells / mL and above, and the cell viability was greater than 80%, and it had the characteristics of good anti-aggregation effect and good multiplication stability. The cell agglomeration rate was less than 3%, and the cell density of SP2 / 0 cells could still increase by more than 1 time after five passages.

[0013] Preferably, in the raw material components used in the above-mentioned serum-free culture medium for SP2 / 0 cell culture, the content of recombinant human serum albumin in each 1L of serum-free culture medium for SP2 / 0 cell culture is 1000-4000 mg.

[0014] By adopting the above technical solution, when SP2 / 0 cells are cultured in the serum-free medium for SP2 / 0 cell culture, high-density culture can be achieved, that is, the cell density reaches 2.8×10 6 cells / mL-3.6×10 6 cells / mL, and the cell viability can reach 92.1-93.0%. It has the characteristics of good anti-aggregation effect and good multiplication stability. The cell agglomeration rate is lower than 2.1%. When the cell density of SP2 / 0 cells is passaged for four times, it can still increase by more than 1.25 times.

[0015] Preferably, the above-mentioned serum-free medium for SP2 / 0 cell culture, calculated per 1L, the composition and content of each raw material used in its preparation are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 0.5-2μg, linoleic acid 5-10μg, linolenic acid 5-10μg, myristic acid 5-10μg, oleic acid 5-10μg, palmitic acid 5-10μg, stearic acid 5-10μg, cholesterol 0.15-0.22mg, Tween 80 0.5-2.2mg, tocopherol acetate 5-7mg, Pluronic F-68 5-10mg, sodium heparin 100-200μg, transferrin 3-5mg, recombinant human insulin 2-5mg, recombinant human serum albumin 1000-4000mg, sodium selenite 0.3-0.5nmol, ethanolamine 20-50μmol, ascorbic acid 5-10mg, hydrocortisone 0.0005-0.001mg, sodium bicarbonate 2.2g, and the remainder is water for cell culture.

[0016] By adopting the above technical solution, when SP2 / 0 cells are cultured in the serum-free medium for SP2 / 0 cell culture, high-density culture can be achieved, that is, the cell density reaches 2.9×10 6 cells / mL-3.6×10 6cells / mL, the cell density was relatively increased by 7.4%-33.3% and 11.5%-38.5% compared with the serum-containing medium for SP2 / 0 cells in comparative example 1 and the SFM serum-free medium (produced by Gibco) for SP2 / 0 cells in comparative example 2; the cell viability was greater than 90%, and the cell agglomeration rate was 1.6-2.1%.

[0017] Preferably, the above-mentioned serum-free medium for SP2 / 0 cell culture, calculated per 1L, the composition and content of each raw material used for its preparation are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 2μg, linoleic acid 10μg, linolenic acid 10μg, myristic acid 10μg, oleic acid 10μg, palmitic acid 10μg, stearic acid 10μg, cholesterol 0.22mg, Tween 802.2mg, tocopherol acetate 7mg, Pluronic F-68 10mg, heparin sodium 100μg, transferrin 5mg, recombinant human insulin 5mg, recombinant human serum albumin 1000mg, sodium selenite 0.5nmol, ethanolamine 50μmol, ascorbic acid 10mg, hydrocortisone 0.001mg, sodium bicarbonate 2.2g, and the balance is water for cell culture.

[0018] By adopting the above technical solution, when SP2 / 0 cells are cultured in the serum-free medium for SP2 / 0 cell culture, high-density culture can be achieved, that is, the cell density reaches 3.6×10 6 cells / mL, and the cell viability was greater than 92%. It had the characteristics of good anti-aggregation effect and good multiplication stability. The cell agglomeration rate was 1.6%. After five passages, the cell density of SP2 / 0 cells could still increase by more than 1.25 times.

[0019] In a second aspect, the present application provides a method for preparing a serum-free medium for SP2 / 0 cell culture, using the following technical solution:

[0020] The above-mentioned method for preparing a serum-free medium for SP2 / 0 cell culture specifically comprises the following preparation steps:

[0021] S1: Dissolving transferrin, recombinant human insulin, sodium selenite and ethanolamine in cell culture water respectively to obtain 100x transferrin concentrated mother solution, 100x recombinant human insulin concentrated mother solution, 100x sodium selenite concentrated mother solution and 100x ethanolamine concentrated mother solution;

[0022] S2: dissolving arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, cholesterol, Tween 80, tocopherol acetate and Pluronic F-68 in cell culture water in sequence to obtain mother solution I;

[0023] S3: dissolving recombinant human serum albumin, ascorbic acid and hydrocortisone in cell culture water to obtain a recombinant human serum albumin solution with a concentration of 100 mg / mL, an ascorbic acid solution with a concentration of 0.1 mg / L and a hydrocortisone solution with a concentration of 100 mg / mL;

[0024] S4: adding the MEM-α dry powder containing nucleosides to the cell culture water, stirring until the MEM-α dry powder containing nucleosides is completely dissolved, to obtain MEM-α culture medium;

[0025] The volume ratio of the cell culture water used in the preparation of the MEM-α medium to the total volume of the serum-free medium to be prepared for SP2 / 0 cell culture is 70-80:100;

[0026] S5: Into the MEM-α medium obtained in S4, add the 100x concentrated transferrin mother solution, 100x concentrated recombinant human insulin mother solution, 100x concentrated sodium selenite mother solution and 100x concentrated ethanolamine mother solution obtained in S1, and the 100 mg / mL recombinant human serum albumin solution, 0.1 mg / L ascorbic acid solution and 100 mg / mL hydrocortisone solution obtained in S3 in sequence, and stir until completely clear to obtain mixture I;

[0027] S6: Add the mother solution I obtained in S2 to the mixture I obtained in S5, and stir until completely clear to obtain a mixture II;

[0028] S7: adding sodium bicarbonate to the mixture II obtained in S6, and stirring until completely dissolved to obtain a mixture III;

[0029] S8: adding cell culture water to the mixture III obtained in S7, and accurately adjusting the volume to 100% of the volume of the serum-free medium for SP2 / 0 cell culture to be prepared, to obtain a crude serum-free medium for SP2 / 0 cell culture;

[0030] S9: filtering and sterilizing the crude serum-free medium for SP2 / 0 cell culture obtained in S8, controlling the bacterial endotoxin to ≤10EU / mL, and obtaining a serum-free medium for SP2 / 0 cell culture;

[0031] The filter membrane used in the above-mentioned filtration sterilization is a 0.22 μm syringe filter;

[0032] The 100x transferrin concentrated mother solution, 100x recombinant human insulin concentrated mother solution, 100x sodium selenite concentrated mother solution and 100x ethanolamine concentrated mother solution obtained in the above S1 are prepared and stored at -20°C;

[0033] The mother solution I obtained in the above S2 is stored at -20°C;

[0034] The recombinant human serum albumin solution with a concentration of 100 mg / mL obtained in S3 was stored at -20°C.

[0035] The following matters should be noted during the preparation of the serum-free medium for SP2 / 0 cell culture:

[0036] First, ensure that the MEM-α dry powder containing nucleosides is completely dissolved, that is, the stirring time cannot be too short. If the MEM-α dry powder containing nucleosides is not completely dissolved, it will affect the use of serum-free medium for SP2 / 0 cell culture;

[0037] Second, ensure the order of operations during the preparation process and strictly follow the order of the preparation steps, otherwise the serum-free medium used for SP2 / 0 cell culture may become turbid and insoluble.

[0038] By adopting the above-mentioned technical scheme, the preparation method of the serum-free culture medium for SP2 / 0 cell culture of the present application has a simple preparation process, convenient operation, high preparation efficiency, and is convenient for large-scale preparation. At the same time, it is carried out strictly in accordance with the operating sequence of the preparation process, and the consistency and stability of each batch of serum-free culture medium for SP2 / 0 cell culture can be ensured.

[0039] In a third aspect, the present application provides an application of a serum-free medium for SP2 / 0 cell culture in SP2 / 0 cell culture, using the following technical solution:

[0040] Application of a serum-free medium for SP2 / 0 cell culture in SP2 / 0 cell culture specifically comprises the following steps:

[0041] S1: Preparation of serum-free medium for SP2 / 0 cell culture

[0042] After the preparation of the serum-free medium for SP2 / 0 cell culture according to the above-mentioned preparation method is completed, the obtained serum-free medium for SP2 / 0 cell culture is tested to have a pH of 6.8-7.5 and an osmotic pressure of 280-350mOsm / L, which can meet the requirements of SP2 / 0 cell culture;

[0043] S2: Recovery of SP2 / 0 cells

[0044] The cryopreserved tube containing SP2 / 0 cells was quickly shaken and thawed in a 37°C water bath, and the thawed liquid containing SP2 / 0 cells was added to the serum-free medium for SP2 / 0 cell culture obtained in S1 and mixed evenly. The mixing volume ratio of the thawed liquid containing SP2 / 0 cells to the serum-free medium for SP2 / 0 cell culture was 1:9. The supernatant was discarded by centrifugation, and 1-2 mL of serum-free medium for SP2 / 0 cell culture was added and mixed by pipetting to obtain cell suspension I;

[0045] The obtained cell suspension I was added to a 10 cm culture dish, and then 8 mL of serum-free medium for SP2 / 0 cell culture was added, and the dish was placed in a cell culture incubator at 37° C. and 5% CO2 by volume for 16 h, and then the medium was changed to obtain cell suspension II;

[0046] S3: Expansion of SP2 / 0 cells

[0047] The cell suspension II obtained in S2 is added to the serum-free medium for SP2 / 0 cell culture obtained in S1, and then the suspension culture is carried out at a speed of 80-130 r / min under the conditions of a CO2 volume percentage concentration of 5% and a temperature of 37° C. until the culture ends at 5-7 days;

[0048] The amount of the cell suspension II is based on the number of SP2 / 0 cells added to each mL of serum-free medium for SP2 / 0 cell culture: 2×10 5 cells for calculation.

[0049] By adopting the above technical scheme, when SP2 / 0 cells are cultured in a serum-free medium for SP2 / 0 cell culture, the SP2 / 0 cells do not need to be acclimated and cultured and can be directly transferred from a serum medium to a serum-free medium for SP2 / 0 cell culture for culture, and high-density culture can be achieved, i.e., the cell density reaches 1.3×10 6 cells / mL-3.6×10 6 cells / mL, and the cell viability was greater than 80%. It had the characteristics of good anti-aggregation effect and good multiplication stability. The cell agglomeration rate was less than 6%. The cell density of SP2 / 0 cells could still increase by more than 1 time after five passages.

[0050] Beneficial technical effects of this application

[0051] A serum-free medium for SP2 / 0 cell culture of the present application can realize SP2 / 0 cells without acclimation culture, that is, directly transfer from serum culture medium to serum-free medium for SP2 / 0 cell culture of the present application for culture;

[0052] Furthermore, the serum-free medium for SP2 / 0 cell culture of the present application can achieve high-density culture of SP2 / 0 cells. When SP2 / 0 cells are cultured, the maximum cell density can reach 3.6×10 6 cells / mL, and the cell viability was greater than 80% when cultured at high density;

[0053] Furthermore, the serum-free medium for SP2 / 0 cell culture of the present application has a good anti-aggregation effect on cells during high-density culture of SP2 / 0 cells, and the cell agglomeration rate is less than 6%;

[0054] Furthermore, the serum-free medium for SP2 / 0 cell culture of the present application has good multiplication stability during the culture process of SP2 / 0 cells, and the cell density of SP2 / 0 cells can still increase by more than 1 times after five passages;

[0055] Furthermore, the serum-free culture medium for SP2 / 0 cell culture of the present application has a low production cost, and especially when the serum-free culture medium for SP2 / 0 cell culture is prepared on a large scale, the production cost will be further reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 It is the cell density diagram of Examples 1-8 and Comparative Examples 1-2;

[0057] Figure 2 It is a graph of cell viability of Examples 1-8 and Comparative Examples 1-2;

[0058] Figure 3 It is a graph of cell agglomeration rate of Examples 1-8 and Comparative Examples 1-2;

[0059] Figure 4 It is a test diagram of multiplication stability of Examples 1-8 and Comparative Examples 1-2;

[0060] Figure 5 It is the cell density diagram of Example 1, Examples 9-11, and Comparative Example 3;

[0061] Figure 6 It is a graph of cell viability of Example 1, Examples 9-11, and Comparative Example 3;

[0062] Figure 7 It is a graph of cell agglomeration rate of Example 1, Examples 9-11, and Comparative Example 3;

[0063] Figure 8a is a cell morphology diagram of Example 1;

[0064] Figure 8b is a cell morphology diagram of Example 9;

[0065] Figure 8cis a cell morphology diagram of Example 10;

[0066] Figure 8d is a cell morphology diagram of Example 11;

[0067] Figure 8e is a cell morphology diagram of Comparative Example 3;

[0068] Fig. 9 It is the cell density diagram of Example 1, Examples 12-14, and Comparative Example 4;

[0069] Fig.10 It is a graph of cell viability of Example 1, Examples 12-14, and Comparative Example 4;

[0070] Fig.11 It is a graph of cell agglomeration rate of Example 1, Examples 12-14, and Comparative Example 4;

[0071] Fig.12 It is a test chart of the multiplication stability of Example 1, Examples 12-14, and Comparative Example 4. DETAILED DESCRIPTION

[0072] The present application is further described in detail below in conjunction with embodiments, but the present application is not limited thereto.

[0073] Except for the special raw materials listed in the following table, all the raw materials used in the embodiments of the present application are commercially available and are required to be cell culture grade (bacterial endotoxin ≤ 10EU / mL);

[0074] Raw material name Part Number Manufacturer or supplier MEM-α dry powder containing nucleosides PM150421P Wuhan Prosai Life Science Co., Ltd. Pluronic F-68 P7061 Sigma-Aldrich Heparin Sodium H104201 Shanghai Aladdin Biochemical Technology Co., Ltd. Transferrin T8010 Beijing Solable Technology Co., Ltd. Recombinant human insulin P3378 BioSky Biotech Co., Ltd. Recombinant human serum albumin R283928 Shanghai Aladdin Biochemical Technology Co., Ltd. Australian serum CY103 Shanghai Yamei Biopharmaceutical Technology Co., Ltd.

[0075] Raw materials used in each example: MEM-α dry powder containing nucleosides, sodium selenite, ethanolamine, ascorbic acid, hydrocortisone, heparin sodium, arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, Tween 80, tocopherol acetate, stored at 0-8°C;

[0076] Transferrin, stored at 4°C;

[0077] Recombinant human insulin, recombinant human serum albumin, cholesterol, stored at -20℃.

[0078] After the serum-free medium for SP2 / 0 cell culture obtained in each embodiment of the present application is used for SP2 / 0 cell culture:

[0079] 1. Test methods for cell density, cell viability and cell aggregation rate of SP2 / 0 cells:

[0080] Take 10 μL of the culture medium during the expansion of SP2 / 0 cells and 10 μL of trypan blue staining solution, pipette and mix well to obtain a mixed solution, then transfer the mixed solution to a Countstar counting plate, put the Countstar counting plate into a fully automatic cell analyzer, and measure the cell density, cell viability and cell aggregation rate;

[0081] The above-mentioned fully automatic cell analyzer is Countstar Mira BF, produced by Shanghai Ruiyu Biotechnology Co., Ltd.;

[0082] The above-mentioned Countstar counting plate has a specification of 75.5 mm × 25.5 mm (L × W) and is produced by Shanghai Ruiyu Biotechnology Co., Ltd.

[0083] 2. SP2 / 0 cell multiplication stability test method

[0084] Starting from the expansion culture of SP2 / 0 cells, SP2 / 0 cells were cultured periodically, and the cell density after 2 days of each subculture was compared with the initial cell density of 2×10 5 Difference in cells / mL;

[0085] The SP2 / 0 cells were cultured periodically, i.e., the initial cell number of the SP2 / 0 cells was 2×10 5 cells / mL, the total culture volume was 20 mL, and after culturing for 2 days, a cell suspension I was obtained, and a sample was taken to determine the cell density of the cell suspension I;

[0086] For the first passage, the cell suspension I was replaced with fresh serum-free medium for SP2 / 0 cell culture until the cell density reached 2×10 5 cells / mL, the total culture volume was 20 mL, and after culturing for 2 days, a cell suspension II was obtained, and a sample was taken to determine the cell density of the cell suspension II;

[0087] For the second passage, the cell suspension II was replaced with fresh serum-free medium for SP2 / 0 cell culture until the cell density reached 2×10 5 cells / mL, the total culture volume was 20 mL, and after culturing for 2 days, a cell suspension III was obtained, and a sample was taken to determine the cell density of the cell suspension III;

[0088] For the third passage, the cell suspension III was replaced with fresh serum-free medium for SP2 / 0 cell culture until the cell density reached 2×10 5 cells / mL, the total culture volume was 20 mL, after culturing for 2 days, cell suspension IV was obtained, and the cell density of cell suspension IV was measured by sampling;

[0089] The above-mentioned subculturing process was repeated until the cell density of SP2 / 0 cells doubled to less than 1, and subculturing was terminated.

[0090] 3. pH test

[0091] The pH value is measured using a pH meter. The specific pH value measurement method is as follows:

[0092] (1) Turn on the pH meter and calibrate it with an acid-base indicator;

[0093] (2) Place the serum-free medium for SP2 / 0 cell culture to be tested on a magnetic stirrer to make the serum-free medium for SP2 / 0 cell culture uniform;

[0094] (3) Place the electrode in serum-free medium for SP2 / 0 cell culture and press the measurement button;

[0095] (4) Keep the electrode in the solution for 1-2 minutes to ensure accurate readings can be taken;

[0096] (5) Record the pH value, clean the pH meter electrode, and turn off the instrument;

[0097] The pH meter is a pH meter FE28-Standard, manufactured by Mettler-Toledo;

[0098] 4. Osmotic pressure test

[0099] Take 200 μL of serum-free medium for SP2 / 0 cell culture and use a freezing point osmometer to test the osmotic pressure of the serum-free medium for SP2 / 0 cell culture. The specific osmotic pressure test method is as follows:

[0100] (1) Place the freezing point osmometer on a stable table and ensure that the instrument is in a horizontal state;

[0101] (2) Turn on the power switch and wait for the instrument to preheat for a while before using it;

[0102] (3) Pour the serum-free medium for SP2 / 0 cell culture to be tested into the sample pool, being careful not to exceed the marked line;

[0103] (4) Adjust the temperature control device to gradually reduce the temperature;

[0104] (5) When the solution begins to freeze, the instrument will automatically detect the temperature at which the freezing point drops;

[0105] (6) Read the temperature value on the display screen and use the calculation function to obtain the osmotic pressure value of the serum-free medium used for SP2 / 0 cell culture;

[0106] The above-mentioned freezing point osmometer, model BS100, is produced by Shanghai Precision Instrument Co., Ltd.;

[0107] 5. Bacterial endotoxin test

[0108] Endotoxin detection kit (Limulus amebocyte lysate colorimetric method) (Biyuntian Biotechnology Co., Ltd., catalog number: C0276S) was used for detection;

[0109] Detection standard: When bacterial endotoxin is ≤10EU / mL, it meets the requirements for the preparation of serum-free medium for SP2 / 0 cell culture;

[0110] If the bacterial endotoxin test does not meet the standards, it is necessary to re-filter and then re-test for bacterial endotoxins until it meets the requirements for the preparation of serum-free medium for SP2 / 0 cell culture. Example

[0111] Examples 1-8

[0112] A serum-free medium for SP2 / 0 cell culture, calculated per 1L, the raw material components and contents used in its preparation are as follows:

[0113]

[0114]

[0115]

[0116] The method for preparing the serum-free medium for SP2 / 0 cell culture comprises the following preparation steps:

[0117] S1: Dissolving transferrin, recombinant human insulin, sodium selenite and ethanolamine in cell culture water respectively to obtain 100x transferrin concentrated mother solution, 100x recombinant human insulin concentrated mother solution, 100x sodium selenite concentrated mother solution and 100x ethanolamine concentrated mother solution;

[0118] S2: dissolving arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, cholesterol, Tween 80, tocopherol acetate and Pluronic F-68 in cell culture water in sequence to obtain mother solution I;

[0119] S3: dissolving recombinant human serum albumin, ascorbic acid and hydrocortisone in cell culture water to obtain a recombinant human serum albumin solution with a concentration of 100 mg / mL, an ascorbic acid solution with a concentration of 0.1 mg / L and a hydrocortisone solution with a concentration of 100 mg / mL;

[0120] S4: adding the MEM-α dry powder containing nucleosides to the cell culture water, stirring until the MEM-α dry powder containing nucleosides is completely dissolved, to obtain MEM-α culture medium;

[0121] S5: Into the MEM-α medium obtained in S4, add the 100x concentrated transferrin mother solution, 100x concentrated recombinant human insulin mother solution, 100x concentrated sodium selenite mother solution and 100x concentrated ethanolamine mother solution obtained in S1, and the 100 mg / mL recombinant human serum albumin solution, 0.1 mg / L ascorbic acid solution and 100 mg / mL hydrocortisone solution obtained in S3 in sequence, and stir until completely clear to obtain mixture I;

[0122] S6: Add the mother solution I obtained in S2 to the mixture I obtained in S5, and stir until completely clear to obtain a mixture II;

[0123] S7: adding sodium bicarbonate to the mixture II obtained in S6, and stirring until completely dissolved to obtain a mixture III;

[0124] S8: adding cell culture water to the mixture III obtained in S7, and accurately adjusting the volume to 100% of the volume of the serum-free medium for SP2 / 0 cell culture to be prepared, to obtain a crude serum-free medium for SP2 / 0 cell culture;

[0125] S9: filtering and sterilizing the crude serum-free medium for SP2 / 0 cell culture obtained in S8 to control the bacterial endotoxin to ≤10EU / mL, thereby obtaining a serum-free medium for SP2 / 0 cell culture;

[0126] The filter membrane used in the above-mentioned filtration sterilization is a 0.22 μm syringe filter;

[0127] The 100x transferrin concentrated mother solution, 100x recombinant human insulin concentrated mother solution, 100x sodium selenite concentrated mother solution and 100x ethanolamine concentrated mother solution obtained in the above S1 are prepared and stored at -20°C;

[0128] The mother solution I obtained in the above S2 is stored at -20°C;

[0129] The recombinant human serum albumin solution with a concentration of 100 mg / mL obtained in S3 was stored at -20°C.

[0130] The use of the above-mentioned serum-free medium for SP2 / 0 cell culture in SP2 / 0 cell culture specifically comprises the following steps:

[0131] S1: Preparation of serum-free medium for SP2 / 0 cell culture

[0132] The serum-free medium for SP2 / 0 cell culture obtained after preparation according to the above-mentioned preparation method of the serum-free medium for SP2 / 0 cell culture was tested to have a pH between 6.8-7.5 and an osmotic pressure between 280-350 mOsm / L, which met the culture requirements of SP2 / 0 cells;

[0133] S2: Recovery of SP2 / 0 cells

[0134] The cryopreservation tube containing SP2 / 0 cells was quickly shaken and thawed in a 37°C water bath, and the thawed liquid containing SP2 / 0 cells was added to the serum-free medium for SP2 / 0 cell culture and mixed evenly. The mixing ratio of the thawed liquid containing SP2 / 0 cells and the serum-free medium for SP2 / 0 cell culture was 1:9. The supernatant was discarded by centrifugation, and 1-2 mL of the serum-free medium for SP2 / 0 cell culture was added and mixed by pipetting to obtain a cell suspension I;

[0135] The obtained cell suspension I was added to a 10 cm culture dish, and then 8 mL of serum-free medium for SP2 / 0 cell culture was added, and the dish was placed in a cell culture incubator at 37° C. and 5% CO2 by volume for 16 h, and then the medium was changed and the cell density and cell viability were checked to obtain cell suspension II;

[0136] The above-mentioned cryopreserved tubes containing SP2 / 0 cells were from the Cell Bank of the Chinese Academy of Sciences;

[0137] S3: Expansion of SP2 / 0 cells

[0138] The cell suspension II obtained in S2 is added to the serum-free medium for SP2 / 0 cell culture obtained in S1, and then batch culture is performed at a CO2 volume percentage concentration of 5% and a temperature of 37° C. with a rotation speed of 80-130 r / min until 7 days;

[0139] The amount of the cell suspension III is based on the number of SP2 / 0 cells added to each mL of serum-free medium for SP2 / 0 cell culture: 2×10 5 cells for calculation.

[0140] Comparative Example 1

[0141] A serum-containing culture medium for SP2 / 0 cells, wherein the raw material component is MEM-α culture medium supplemented with 10% Australian serum by volume;

[0142] The MEM-α culture medium is a MEM-α dry powder containing nucleosides added to cell culture water and stirred until the MEM-α dry powder containing nucleosides is completely dissolved;

[0143] The ratio of the above-mentioned MEM-α dry powder containing nucleosides to the water used for cell culture is 10.15 g:1 L.

[0144] The use of the serum-containing medium for SP2 / 0 cells in SP2 / 0 cell culture specifically comprises the following steps:

[0145] S1: Preparation of serum-containing culture medium

[0146] The MEM-α dry powder containing nucleosides is added to the cell culture water, and stirred until the MEM-α dry powder containing nucleosides is completely dissolved to obtain the MEM-α culture medium;

[0147] The ratio of the above-mentioned MEM-α dry powder containing nucleosides to the water for cell culture is 10.15 g: 1 L;

[0148] The Australian serum was mixed with the MEM-α medium obtained above at a volume ratio of 10:90 to obtain a serum-containing medium for SP2 / 0 cells;

[0149] S2: Recovery of SP2 / 0 cells

[0150] The same as step S2 of the application in SP2 / 0 cell culture in Example 1;

[0151] S3: Expansion of SP2 / 0 cells

[0152] Same as step S3 of the application in SP2 / 0 cell culture in Example 1.

[0153] Comparative Example 2

[0154] An SFM serum-free medium for SP2 / 0 cell culture, wherein the SFM serum-free medium (manufactured by Gibco) is selected;

[0155] The use of the above-mentioned SFM serum-free medium for SP2 / 0 cells (produced by Gibco) in SP2 / 0 cell culture specifically includes the following steps:

[0156] S1: Recovery of SP2 / 0 cells

[0157] The same as step S2 of the application in SP2 / 0 cell culture in Example 1;

[0158] S2: Expansion culture of SP2 / 0 cells

[0159] Same as step S3 of the application in SP2 / 0 cell culture in Example 1.

[0160] SP2 / 0 cells were cultured using the serum-free medium for SP2 / 0 cell culture obtained in Examples 1-8, the serum-containing medium for SP2 / 0 cells in Comparative Example 1, and the SFM serum-free medium for SP2 / 0 cells (produced by Gibco) in Comparative Example 2. During the expansion of the SP2 / 0 cell culture, samples were taken every day to measure the cell density and cell viability. The cell density and cell viability graphs obtained are shown in Figures 1 and 2. Figure 1 , Figure 2 As shown, the horizontal axis is the culture time, and the vertical axis is the cell density and cell viability;

[0161] from Figure 1 As can be seen from the cell density graph, when the serum-free medium for SP2 / 0 cell culture obtained in Example 1-8 was used to culture SP2 / 0 cells, the cell density reached a peak of 2.2×10 6 cells / mL-3.6×10 6 cells / mL, and the cell density began to decrease after the 5th day; when SP2 / 0 cells were cultured in the serum-containing medium for SP2 / 0 cells in Comparative Example 1, the cell density was 2.7×10 6 cells / mL; when SP2 / 0 cells were cultured in SFM serum-free medium (manufactured by Gibco) for SP2 / 0 cells in Comparative Example 2, the cell density was 2.6×10 6 This indicates that the serum-free culture medium for SP2 / 0 cell culture of the present application can be used for high-density culture of SP2 / 0 cells.

[0162] In particular, when SP2 / 0 cells were cultured using the serum-free medium for SP2 / 0 cell culture obtained in Example 1, the cell density obtained after 5 days of culture was relatively increased by 33.3% and 38.5% compared with the cell density obtained when the serum-containing medium for SP2 / 0 cells of Comparative Example 1 and the SFM serum-free medium for SP2 / 0 cells (produced by Gibco) of Comparative Example 2 were used for culture.

[0163] from Figure 2 As can be seen from the cell viability graph, when SP2 / 0 cells were cultured using the serum-free culture medium for SP2 / 0 cells obtained in Examples 1-8, the serum-containing culture medium for SP2 / 0 cells in Comparative Example 1, and the SFM serum-free culture medium (produced by Gibco) for SP2 / 0 cells in Comparative Example 2, the SP2 / 0 cell culture process was very stable, and the SP2 / 0 cell viability was above 95% at the end of the first day of culture; the SP2 / 0 cell viability was greater than 80% on the 5th day of culture; and the SP2 / 0 cell viability dropped to below 80% on the 7th day of culture.

[0164] SP2 / 0 cells were cultured using the serum-free medium for SP2 / 0 cell culture obtained in Examples 1-8, the serum-containing medium for SP2 / 0 cells in Comparative Example 1, and the SFM serum-free medium for SP2 / 0 cells (produced by Gibco) in Comparative Example 2. Samples were taken on the 5th day of culture to determine the cell agglomeration rate. The test results are as follows: Figure 3 As shown;

[0165] from Figure 3 As can be seen from the cell agglomeration rate graph, when the serum-free medium for SP2 / 0 cell culture obtained in Examples 1-8 is used to culture SP2 / 0 cells, the SP2 / 0 cells have a good anti-agglomeration effect, and their cell agglomeration rates are all lower than 6%. When the SP2 / 0 cells are cultured using the serum-containing medium for SP2 / 0 cells of Comparative Example 1, the cell agglomeration rate of the SP2 / 0 cells is 8%; when the SP2 / 0 cells are cultured using the SFM serum-free medium for SP2 / 0 cells (produced by Gibco) of Comparative Example 2, the cell agglomeration rate of the SP2 / 0 cells is 3%. This shows that when the serum-free medium for SP2 / 0 cell culture of the present application is used to culture SP2 / 0 cells, it has a good cell anti-agglomeration effect.

[0166] SP2 / 0 cells were cultured using the serum-free medium for SP2 / 0 cell culture obtained in Examples 1-8, the serum-containing medium for SP2 / 0 cells in Comparative Example 1, and the SFM serum-free medium (manufactured by Gibco) for SP2 / 0 cells in Comparative Example 2. The SP2 / 0 cells were tested for multiplication stability during the expansion of the SP2 / 0 cells. The test results are shown in FIG. Figure 4 As shown, the horizontal axis is the culture time, and the vertical axis is the cell density;

[0167] from Figure 4 It can be seen from the doubling stability test graph that when SP2 / 0 cells are cultured using the serum-free medium for SP2 / 0 cell culture obtained in Examples 1-8, the serum-containing medium for SP2 / 0 cells in Comparative Example 1, and the SFM serum-free medium for SP2 / 0 cells (produced by Gibco) in Comparative Example 2, the SP2 / 0 cells have good doubling stability, and the cell density of the SP2 / 0 cells can still increase by more than 1 times after five passages. This shows that when SP2 / 0 cells are cultured using the serum-free medium for SP2 / 0 cell culture of the present application, the doubling stability is good.

[0168] Based on the test results of cell density, cell viability and cell agglomeration rate in Examples 1-8 above, it can be concluded that:

[0169] The raw material composition and content of the serum-free medium used for SP2 / 0 cell culture are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 0.5-2μg, linoleic acid 5-10μg, linolenic acid 5-10μg, myristic acid 5-10μg, oleic acid 5-10μg, palmitic acid 5-10μg, stearic acid 5-10μg, cholesterol 0.15-0.22mg, Tween 80 0.5-2.2mg, tocopherol acetate 5-7mg, Pluronic F-68 5-10mg, sodium heparin 100-200μg, transferrin 3-5mg, recombinant human insulin 2-5mg, recombinant human serum albumin 1000-4000mg, sodium selenite 0.3-0.5nmol, ethanolamine 20-50μmol, ascorbic acid 5-10mg, hydrocortisone 0.0005-0.001mg, sodium bicarbonate 2.2g, the remainder is water for cell culture:

[0170] On the fifth day of culture, the cell density reached 2.9×10 6 cells / mL-3.6×10 6 cells / mL, the cell density was relatively increased by 7.4%-33.3% and 11.5%-38.5% compared with the serum-containing medium for SP2 / 0 cells in comparative example 1 and the SFM serum-free medium (produced by Gibco) for SP2 / 0 cells in comparative example 2; the cell viability was above 90%, and the cell agglomeration rate was 1.6-2.1%.

[0171] Examples 9-11

[0172] A serum-free medium for SP2 / 0 cell culture, wherein the raw material components used for its preparation are the same as those in Example 1 except that the content of sodium heparin is different from that in Example 1. The content of sodium heparin is specifically shown in the following table:

[0173] Raw material components Example 1 Example 9 Example 10 Embodiment 11 Heparin sodium (μg) 100 1000 200 500

[0174] Comparative Example 3

[0175] A serum-free culture medium for SP2 / 0 cell culture, wherein the raw material components used for its preparation are the same as those in Example 1 except that the content of heparin sodium is 0 μg.

[0176] SP2 / 0 cells were cultured using the serum-free medium for SP2 / 0 cell culture obtained in Example 1, Examples 9-11, and Comparative Example 3. During the expansion of the SP2 / 0 cell culture, samples were taken every day to measure the cell density and cell viability. The cell density and cell viability graphs obtained are shown in Figures 1 and 2. Figure 5 , Figure 6 As shown, the horizontal axis is the culture time, and the vertical axis is the cell density and cell viability;

[0177] from Figure 5 and Figure 6 It can be seen that the cell density of SP2 / 0 cells cultured in serum-free medium with or without sodium heparin can reach 2.0×10 after 5 days of culture. 6 cells / mL, and the cell viability was higher than 80%.

[0178] SP2 / 0 cells were cultured using the serum-free medium for SP2 / 0 cell culture obtained in Example 1, Examples 9-11, and Comparative Example 3 of the present application. When the SP2 / 0 cells were expanded and cultured to the fifth day, samples were taken to determine the cell agglomeration rate. The test results are as follows: Figure 7 As shown;

[0179] from Figure 7 As can be seen from the cell agglomeration rate graph, adding sodium heparin to the serum-free medium for SP2 / 0 cell culture in Examples 1 and Examples 9-11 can significantly improve the cell agglomeration, with a cell agglomeration rate of less than 6%, while the cell agglomeration rate in Comparative Example 3 without adding sodium heparin is 8%. The reason for this may be that sodium heparin is a highly sulfated glycosaminoglycan with a negative charge, which can promote the suspension of single SP2 / 0 cells by changing the charge on the cell surface and reduce the cell agglomeration rate; and the anticoagulant property of sodium heparin also helps to maintain the cell dispersion state during cell culture and reduce the phenomenon of cell agglomeration.

[0180] The cell morphology of SP2 / 0 cells cultured to day 5 in Example 1, Example 9, Example 10, Example 11, and Comparative Example 3 was derived from the fully automatic cell analyzer. Figure 8a , 8b , 8c, 8d, 8e;

[0181] from Figure 8a , 8b , 8c, and 8d, it can be seen that when the amount of heparin sodium added was 100-1000 μg, the anti-aggregation effect of SP2 / 0 cells was better, which was Figure 7 The cell agglomeration rate data of the corresponding embodiments in the above table are consistent. In particular, when the amount of heparin sodium added is 100-200 μg, the cell agglomeration rate of SP2 / 0 cells is the lowest, which is less than 3%.

[0182] from Figure 8a , Figure 8e By comparison, it can be seen that the cell agglomeration rate was 8.0% without the addition of sodium heparin, that is, obvious cell agglomeration occurred, while the addition of sodium heparin could significantly improve the cell agglomeration situation, and the cell agglomeration rate was 1.6%, that is, the cell agglomeration rate decreased by 80%.

[0183] Examples 12-14

[0184] A serum-free medium for SP2 / 0 cell culture, wherein the raw material components used for its preparation are the same as those in Example 1 except that the content of recombinant human serum albumin is different from that in Example 1. The content of recombinant human serum albumin is specifically shown in the following table:

[0185] Raw material components Example 1 Example 12 Example 13 Embodiment 14 Recombinant human serum albumin (mg) 1000 4000 200 500

[0186] Comparative Example 4

[0187] A serum-free culture medium for SP2 / 0 cell culture, wherein the raw material components used for its preparation are the same as those in Example 1 except that the content of recombinant human serum albumin is 0 μg.

[0188] SP2 / 0 cells were cultured using the serum-free medium for SP2 / 0 cell culture obtained in Example 1, Examples 12-14, and Comparative Example 4. During the expansion of the SP2 / 0 cell culture, samples were taken every day to measure the cell density and cell viability. The cell density and cell viability graphs obtained are shown in Figures 1 and 2. Fig. 9 , Fig.10 As shown, the horizontal axis is the culture time, and the vertical axis is the cell density and cell viability;

[0189] from Fig. 9 As can be seen in the cell density graph, per 1L of serum-free medium for SP2 / 0 cell culture:

[0190] Without recombinant human serum albumin, the cell density of SP2 / 0 cells was 8×10 5 cells / mL;

[0191] The recombinant human serum albumin is 200-4000 mg. After 5 days of culture, the cell density of SP2 / 0 cells can reach 1.3×10 6 cells / mL-3.6×10 6 cells / mL, which is higher than the initial cell density (2.0×10 5 cells / mL) increased by 6.5-18 times;

[0192] Recombinant human serum albumin is 200-500 mg. After 5 days of culture, the cell density of SP2 / 0 cells can reach 1.3×10 6 cells / mL-1.7×10 6 cells / mL, which is higher than the initial cell density (2.0×10 5 cells / mL) increased by 6.5-8.5 times;

[0193] The recombinant human serum albumin is 1000-4000 mg. After 5 days of culture, the cell density of SP2 / 0 cells can reach 2.8×10 6 cells / mL-3.6×10 6 cells / mL, which is higher than the initial cell density (2.0×10 5 cells / mL) increased by 14-18 times;

[0194] from Fig.10 As can be seen from the cell viability chart, for every 1L of serum-free medium used for SP2 / 0 cell culture:

[0195] Without recombinant human serum albumin, the cell viability of SP2 / 0 cells was 65.0% at 5 days of culture;

[0196] The recombinant human serum albumin was 200-4000 mg, and the cell viability of SP2 / 0 cells was greater than 84.0% after 5 days of culture;

[0197] The recombinant human serum albumin is 1000-4000 mg, and the cell viability of SP2 / 0 cells can reach 92.1-93.0% after 5 days of culture.

[0198] SP2 / 0 cells were cultured using the serum-free medium for SP2 / 0 cell culture obtained in Example 1, Examples 12-14 and Comparative Example 4. When the SP2 / 0 cells were cultured to the fifth day, samples were taken to determine the cell agglomeration rate. The test results are as follows: Fig.11 As shown;

[0199] from Fig.11 It can be seen that when SP2 / 0 cells are cultured using the serum-free medium for SP2 / 0 cell culture obtained in Example 1, Examples 12-14 and Comparative Example 4, the SP2 / 0 cells have a good anti-aggregation effect, and their cell agglomeration rates are all lower than 2.1%. This shows that the content of recombinant human serum albumin has little effect on cell aggregation.

[0200] When the serum-free medium for SP2 / 0 cell culture obtained in Example 1, Examples 12-14, and Comparative Example 4 was used to culture SP2 / 0 cells, a multiplication stability test was performed on the SP2 / 0 cells during the expansion of the SP2 / 0 cells. The test results are as follows: Fig.12 As shown, the horizontal axis is the culture time, and the vertical axis is the cell density;

[0201] from Fig.12It can be seen that when SP2 / 0 cells are cultured using the serum-free medium for SP2 / 0 cell culture obtained in Example 1 and Examples 12-14, the cell density of SP2 / 0 cells can still increase by more than 1.25 times after four passages; while when SP2 / 0 cells are cultured using the serum-free medium for SP2 / 0 cell culture without recombinant human serum albumin obtained in Comparative Example 4, the SP2 / 0 cells multiply slowly, and the cell density of SP2 / 0 cells after one passage is only 1 times of the initial cell amount. This shows that the serum-free medium for SP2 / 0 cell culture of the present application increases the multiplication stability of SP2 / 0 cells during culture due to the addition of recombinant human serum albumin.

[0202] In summary, the serum-free culture medium for SP2 / 0 cell culture of the present application can realize the SP2 / 0 cell culture without the need for acclimation, that is, directly transfer from the serum culture medium to the serum-free culture medium for SP2 / 0 cell culture of the present application for high-density culture, and due to the synergistic effect of recombinant human serum albumin and sodium heparin, the SP2 / 0 cells have good anti-aggregation effect and good multiplication stability during the high-density culture process.

[0203] The above are all preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Therefore, all equivalent changes and improvements made within the scope of the present invention should be included in the protection scope of the present application.

Claims

1. A serum-free medium for SP2 / 0 cell culture, characterized in that: Calculated per 1L, the composition and content of each raw material used in its preparation are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 0.2-3μg, linoleic acid 1-25μg, linolenic acid 1-25μg, myristic acid 1-25μg, oleic acid 1-25μg, palmitic acid 1-25μg, stearic acid 1-25μg, cholesterol 0.1-2mg, Tween 80 0.2-8mg, tocopherol acetate 1-25mg, Pluronic F-68 1-30mg, heparin sodium 50-1000μg, transferrin 1-15mg, recombinant human insulin 1-15mg, recombinant human serum albumin 200-4000mg, sodium selenite 0.1-1.5nmol, ethanolamine 10-150μmol, ascorbic acid 1-12mg, hydrocortisone 0.0005-0.01mg, sodium bicarbonate 2.2g, the balance is water for cell culture; The Pluronic F-68 has a product number of P7061 and is produced by Sigma-Aldrich.

2. A serum-free medium for SP2 / 0 cell culture as claimed in claim 1, characterized in that: Calculated per 1L, the composition and content of each raw material used in its preparation are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 0.2-3μg, linoleic acid 1-25μg, linolenic acid 1-25μg, myristic acid 1-25μg, oleic acid 1-25μg, palmitic acid 1-25μg, stearic acid 1-25μg, cholesterol 0.1-2mg, Tween 80 0.2-8mg, tocopherol acetate 1-25mg, Pluronic F-68 1-30mg, sodium heparin 100-200μg, transferrin 1-15mg, recombinant human insulin 1-15mg, recombinant human serum albumin 200-4000mg, sodium selenite 0.1-1.5nmol, ethanolamine 10-150μmol, ascorbic acid 1-12mg, hydrocortisone 0.0005-0.01mg, sodium bicarbonate 2.2g, and the balance is water for cell culture.

3. A serum-free medium for SP2 / 0 cell culture as claimed in claim 1, characterized in that: Calculated per 1L, the composition and content of each raw material used in its preparation are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 0.2-3μg, linoleic acid 1-25μg, linolenic acid 1-25μg, myristic acid 1-25μg, oleic acid 1-25μg, palmitic acid 1-25μg, stearic acid 1-25μg, cholesterol 0.1-2mg, Tween 80 0.2-8mg, tocopherol acetate 1-25mg, Pluronic F-68 1-30mg, sodium heparin 50-1000μg, transferrin 1-15mg, recombinant human insulin 1-15mg, recombinant human serum albumin 1000-4000mg, sodium selenite 0.1-1.5nmol, ethanolamine 10-150μmol, ascorbic acid 1-12mg, hydrocortisone 0.0005-0.01mg, sodium bicarbonate 2.2g, and the balance is water for cell culture.

4. The serum-free medium for SP2 / 0 cell culture according to claim 1, characterized in that: Calculated per 1L, the composition and content of each raw material used in its preparation are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 0.5-2μg, linoleic acid 5-10μg, linolenic acid 5-10μg, myristic acid 5-10μg, oleic acid 5-10μg, palmitic acid 5-10μg, stearic acid 5-10μg, cholesterol 0.15-0.22mg, Tween 80 0.5-2.2mg, tocopherol acetate 5-7mg, Pluronic F-68 5-10mg, sodium heparin 100-200μg, transferrin 3-5mg, recombinant human insulin 2-5mg, recombinant human serum albumin 1000-4000mg, sodium selenite 0.3-0.5nmol, ethanolamine 20-50μmol, ascorbic acid 5-10mg, hydrocortisone 0.0005-0.001mg, sodium bicarbonate 2.2g, and the remainder is water for cell culture.

5. A serum-free medium for SP2 / 0 cell culture as claimed in claim 4, characterized in that: Calculated per 1L, the composition and content of each raw material used in its preparation are as follows: MEM-α dry powder containing nucleosides 10.15g, arachidonic acid 2μg, linoleic acid 10μg, linolenic acid 10μg, myristic acid 10μg, oleic acid 10μg, palmitic acid 10μg, stearic acid 10μg, cholesterol 0.22mg, Tween 80 2.2mg, tocopherol acetate 7mg, Pluronic F-68 10mg, heparin sodium 100μg, transferrin 5mg, recombinant human insulin 5mg, recombinant human serum albumin 1000mg, sodium selenite 0.5nmol, ethanolamine 50μmol, ascorbic acid 10mg, hydrocortisone 0.001mg, sodium bicarbonate 2.2g, and the remainder is water for cell culture.

6. A method for preparing a serum-free medium for SP2 / 0 cell culture according to any one of claims 1 to 5, characterized in that: The method comprises the following preparation steps: S1: Dissolving transferrin, recombinant human insulin, sodium selenite and ethanolamine in cell culture water respectively to obtain 100x transferrin concentrated mother solution, 100x recombinant human insulin concentrated mother solution, 100x sodium selenite concentrated mother solution and 100x ethanolamine concentrated mother solution; S2: dissolving arachidonic acid, linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, stearic acid, cholesterol, Tween 80, tocopherol acetate and Pluronic F-68 in cell culture water in sequence to obtain mother solution I; S3: dissolving recombinant human serum albumin, ascorbic acid and hydrocortisone in cell culture water to obtain a recombinant human serum albumin solution with a concentration of 100 mg / mL, an ascorbic acid solution with a concentration of 0.1 mg / L and a hydrocortisone solution with a concentration of 100 mg / mL; S4: adding the MEM-α dry powder containing nucleosides to the cell culture water, stirring until the MEM-α dry powder containing nucleosides is completely dissolved, to obtain MEM-α culture medium; The volume ratio of the cell culture water used in the preparation of the MEM-α medium to the total volume of the serum-free medium to be prepared for SP2 / 0 cell culture is 70-80:100; S5: Into the MEM-α medium obtained in S4, add the 100x concentrated transferrin mother solution, 100x concentrated recombinant human insulin mother solution, 100x concentrated sodium selenite mother solution and 100x concentrated ethanolamine mother solution obtained in S1, and the 100 mg / mL recombinant human serum albumin solution, 0.1 mg / L ascorbic acid solution and 100 mg / mL hydrocortisone solution obtained in S3 in sequence, and stir until completely clear to obtain mixture I; S6: Add the mother solution I obtained in S2 to the mixture I obtained in S5, and stir until completely clear to obtain a mixture II; S7: adding sodium bicarbonate to the mixture II obtained in S6, and stirring until completely dissolved to obtain a mixture III; S8: adding cell culture water to the mixture III obtained in S7, and accurately adjusting the volume to 100% of the volume of the serum-free medium for SP2 / 0 cell culture to be prepared, to obtain a crude serum-free medium for SP2 / 0 cell culture; S9: The crude serum-free culture medium for SP2 / 0 cell culture obtained in S8 is filtered and sterilized, and the bacterial endotoxin is controlled to be ≤10EU / mL to obtain a serum-free culture medium for SP2 / 0 cell culture.

7. Use of the serum-free medium for SP2 / 0 cell culture according to any one of claims 1 to 5 in SP2 / 0 cell culture, characterized in that: The specific steps are as follows: S1: preparing a serum-free medium for SP2 / 0 cell culture by the method for preparing a serum-free medium for SP2 / 0 cell culture as claimed in claim 6; S2: Recovery of SP2 / 0 cells The cryopreserved tube containing SP2 / 0 cells was quickly shaken and thawed in a 37°C water bath, and the thawed liquid containing SP2 / 0 cells was added to the serum-free medium for SP2 / 0 cell culture obtained in S1 and mixed evenly. The mixing volume ratio of the thawed liquid containing SP2 / 0 cells to the serum-free medium for SP2 / 0 cell culture was 1:

9. The supernatant was discarded by centrifugation, and 1-2 mL of serum-free medium for SP2 / 0 cell culture was added and mixed by pipetting to obtain cell suspension I; The obtained cell suspension I was added to a 10 cm culture dish, and then 8 mL of serum-free medium for SP2 / 0 cell culture was added, and the dish was placed in a cell culture incubator at 37° C. and 5% CO2 by volume for 16 h, and then the medium was changed to obtain cell suspension II; S3: Expansion of SP2 / 0 cells The cell suspension II obtained in S2 is added to the serum-free medium for SP2 / 0 cell culture obtained in S1, and then the suspension culture is carried out at a speed of 80-130 r / min under the conditions of a CO2 volume percentage concentration of 5% and a temperature of 37° C. until the culture ends at 5-7 days; The amount of the cell suspension II is 2×10 cells per mL of serum-free medium for SP2 / 0 cell culture. 5 cells for calculation.

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