WO2020103439A1

WO2020103439A1 - Low-protein serum-free cell culture medium

Info

Publication number: WO2020103439A1
Application number: PCT/CN2019/091341
Authority: WO
Inventors: 苏恩本
Original assignee: 苏恩本
Priority date: 2018-11-21
Filing date: 2019-06-14
Publication date: 2020-05-28
Also published as: CN109234223B; CN109234223A

Abstract

Provided is a low-protein serum-free cell culture medium, the culture medium containing a basal physiological buffer mixture and protein components, wherein the protein components comprise bovine serum albumin, transferrin and insulin, and the total addition amount of the protein components does not exceed 30 mg/L. The only three protein components contained in the culture medium are relatively low in content, which is conducive to separating and purifying a product, to improving the quality of the product, to supporting hybridoma cells well, and to increasing the highest cell density and antibody production of hybridoma cells.

Description

Low protein serum-free cell culture medium

Technical field

The present application relates to the technical field of cell culture, in particular, to a low-protein serum-free cell culture medium.

Background technique

Cell culture is to simulate cell growth environment in vitro to achieve cell in vitro culture. Animal cell in vitro culture technology is widely used in the field of cell and tissue physiology, biochemistry and pharmacological activity research and the industrial production of biological products such as antibodies, hormones and vaccines. Among them, the preparation technology of cell culture medium is a vital part of cell culture technology in vitro. Although the traditional serum-containing medium contains various growth factors, plasma proteins, peptides and hormones, a large number of components necessary for cell growth and value-added, the serum has large batch-to-batch differences, potential contamination risks, unclear components, and is not conducive to the separation and purification of the target product And it is easy to be infected by viruses and mycoplasma infection. The serum-free medium has a relatively clear composition and a simple preparation process. It can be widely used in the field of modern biotechnology.

The composition of serum-free medium is relatively clear, the quality is consistent and the protein content is low, which is conducive to improving the stability of cell product production and making the cell product easy to purify. Optimized so that different cells can each continue to grow at a high density in the environment most conducive to their growth or expression of the product of interest.

There are also many related reports on serum component replacements that promote cell growth. Albumin is the main added factor in many serum-free media, which regulates osmotic pressure and protects cells from mechanical damage. Li Ping et al. (Li Ping, Jiang Lin, Huang Siyang, etc., Research on Serum-free Culture of Chinese Hamster Ovary Engineering Cells [J] Advances in Microbiology and Immunology 2004, 32 (4): 46 ～ 50) Experiments show that albumin has obvious Stabilize cell growth and increase cell line product expression. Generally, serum-free medium for suspension culture contains bovine serum albumin. Hu Xuemei et al. (Hu Xuemei, Zhang Yuanxing, Effect of Amino Acids on Chinese Hamster Ovary Cells in Serum-Free Medium [J] Journal of East China University of Science and Technology 1996, 22 (6): 283 ～ 285) Orthogonal test method was used to investigate serum-free CHO cell culture The effect of 15 kinds of amino acids and their concentration on cell culture, the results show that the added arginine, leucine, proline and methionine can obviously promote the growth of cells, while the high concentration of serine and tryptophan Acid has a significant inhibitory effect on cell growth. Insulin can promote the synthesis of RNA, protein and fatty acids, inhibit cell apoptosis, and is an important cell survival factor. Jan et al. (Jan L, Haggstrorm L. Specific growth rate as a parameter) for tracing growth-limiting substances in animal cells [J]. Journal of Biotechnology 1995, 42: 163-165) believes that insulin is rapidly depleted in batch culture It is the main reason why the specific growth rate of cells decreases. Selenium has an obvious role in the growth of mammalian cells. The trace element selenium participates in the process of glutathione peroxidase and peroxide dismutase, eliminating the damage of cells caused by oxidase and oxygen free radicals. Xue Qingshan (Edited by Xue Qingshan, Principles and Techniques of In Vitro Culture [M]. Beijing Science and Technology Press, 2001, 162 ～ 164) pointed out that the proper addition of selenite can promote cell growth, but if the concentration is too high, it will produce cells Toxic effects.

At present, the commercial serum-free medium is mainly imported, which is expensive, and the formula is confidential, which is not conducive to the optimization of the subsequent cultivation process. There are not many domestic serum-free medium manufacturers, and there is a problem that the market feedback cell is not adaptable. Some scientific research Institutions or R & D units are only for development and self-use. Therefore, independent research and development of serum-free medium that can support the rapid growth of hybridoma cells and high expression of monoclonal antibodies, and related process optimization and amplification to meet the needs of a large number of monoclonal antibodies expressed are critical to the target protein industrialization. In addition, in general, different cell lines have their unique nutritional needs, and no one culture medium will fully meet the needs of all cell lines or maximize the expression potential of the cells. Therefore, independent research and development and optimization of specific cell lines Personalized serum-free medium is also an important part of large-scale cell culture.

In view of this, this application is hereby submitted.

Application content

The serum medium has the problems of large batch-to-batch variation, potential contamination risk, unclear composition, is not conducive to the separation and purification of the target product and is easily infected. The protein-free serum-free medium with a completely clear chemical composition is highly targeted. In other words, it is not very versatile. Generally, one medium is only suitable for the cultivation of a certain type of cell. In addition, the cells are in a completely serum-free medium Easily affected by physical mechanical factors and chemical factors.

The purpose of the present application is to overcome the shortcomings of the above medium and prepare a low protein serum-free cell culture medium.

In order to achieve the above purpose of this application, the following technical solutions are specifically adopted:

The present application relates to a low-protein serum-free cell culture medium, which contains a basic physiological buffer mixture and protein components;

The protein component includes bovine serum albumin, transferrin, and insulin, and the total added amount of the protein component does not exceed 30 mg / L.

The medium is suitable for animal cell culture, especially mammalian cell culture.

Compared with the prior art, the beneficial effects of this application are:

(1) The content of only three protein components is very low, which is conducive to the separation and purification of the product and improves the quality of the product;

(2) Clear composition and convenient preparation, suitable for large-scale production and application;

(3) Strong versatility;

(4) It can support hybridoma cells well, and its highest cell density and antibody production can reach or exceed the effect of GIBCO brand commercial medium;

(5) Support long-term subculture of hybridoma cells, which can be directly cultured without adaptation.

BRIEF DESCRIPTION

In order to more clearly explain the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, so they are not It should be regarded as a limitation on the scope. For those of ordinary skill in the art, without paying any creative work, other related drawings can be obtained based on these drawings.

FIG. 1 is a growth curve of hybridoma cells 1C3 cultured in serum-free medium in an embodiment of the present application;

2 is a graph showing the change curve of glucose concentration, cell density, cell viability, and monoclonal antibody expression of hybridoma cells DF92 cultured in serum-free medium according to an example of the present application with culture time;

3 is a graph showing changes in glucose concentration, cell density, cell viability, and monoclonal antibody expression of hybridoma cells 1A4 cultured using Gibco serum-free medium in the prior art with culture time;

4 is a graph showing the change curve of glucose concentration, cell density, cell viability, and monoclonal antibody expression of hybridoma cells 1A4 fermented and cultured with serum-free medium in an example of the present application with the culture time.

detailed description

Before describing the cell cultures and methods of the present application, it should be understood that the present application is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

In some embodiments, the total added amount of the protein component is 18-30 mg / L, and 20 mg / L, 22 mg / L, 24 mg / L, 26 mg / L, and 28 mg / L can also be selected.

In some embodiments, the protein component includes bovine serum albumin 10.0-20.0 mg / L, transferrin 2.5-5.5 mg / L, and insulin 5.0-12.0 mg / L;

In some embodiments, the protein component includes bovine serum albumin 13.0-17.0 mg / L, transferrin 4.5-5.5 mg / L, and insulin 9.0-11.0 mg / L;

In some embodiments, the basic physiological buffer mixture is based on DMEM / F12 as a basic medium, and one or more of amino acids, vitamins, inorganic salts, trace elements, and lipids are added;

In some embodiments, the volume ratio of DMEM to F12 in DMEM / F12 is 0.8-1.2;

The volume ratio of DMEM to F12 in the DMEM / F12 is 1.

In some embodiments, the lipid is selected from the group consisting of:

Arachidonic acid 10-50μg / L, cholesterol 1500-3500μg / L, linoleic acid 50-250μg / L, linolenic acid 50-250μg / L, oleic acid 50-250μg / L, palmitoleic acid 50-250μg / L , Stearic acid 50-250μg / L, ethanolamine 1500-3500μg / L.

In some embodiments, the lipid is selected from the group consisting of:

Arachidonic acid 10-40μg / L, cholesterol 2500-3500μg / L, linoleic acid 130-230μg / L, linolenic acid 150-250μg / L, oleic acid 130-230μg / L, palmitoleic acid 75-175μg / L , Stearic acid 50-150μg / L, ethanolamine 2200-3200μg / L.

In some embodiments, the lipid is selected from the group consisting of:

Arachidonic acid 20-30μg / L, cholesterol 2800-3200μg / L, linoleic acid 160-200μg / L, linolenic acid 180-210μg / L, oleic acid 170-190μg / L, palmitoleic acid 115-135μg / L , Stearic acid 80-120μg / L, ethanolamine 2500-2900μg / L.

In some embodiments, the amino acid is selected from the group consisting of:

Arginine 36-58mg / L, cystine 8-12mg / L, histidine 9-15mg / L, isoleucine 13-23mg / L, leucine 13-23mg / L, lysine 14 -22mg / L, methionine 4-7mg / L, phenylalanine 9-15mg / L, threonine 18-22mg / L, tryptophan 3-5mg / L, tyrosine 9-16mg / L, Valine 18-22mg / L, Glycine 4-7mg / L, Alanine 5-9mg / L, Asparagine 8-12mg / L, Aspartic acid 8-12mg / L, Glutamate 8 -12mg / L, proline 5-9mg / L, serine 5-9mg / L.

In some embodiments, the amino acid is selected from the group consisting of:

Arginine 40-54mg / L, cystine 9-11mg / L, histidine 11-14mg / L, isoleucine 16-20mg / L, leucine 16-20mg / L, lysine 16 -20mg / L, methionine 5-6mg / L, phenylalanine 11-13mg / L, threonine 19-21mg / L, tryptophan 3-5mg / L, tyrosine 11-14mg / L, Valine 19-21mg / L, Glycine 5-6mg / L, Alanine 6-8mg / L, Asparagine 9-11mg / L, Aspartic acid 9-11mg / L, Glutamate 9 -11mg / L, proline 6-8mg / L, serine 6-8mg / L.

In some embodiments, the vitamin is selected from the group consisting of:

Choline chloride, calcium pantothenate, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine, inositol;

In some embodiments, the concentration of the above vitamins may be selected from 0.7-1.8 mg / L, and may also be selected from 0.8 mg / L, 0.9 mg / L, 1.0 mg / L, 1.1 mg / L, 1.2 mg / L, 1.3 mg / L, 1.4mg / L, 1.5mg / L, 1.6mg / L, 1.7mg / L; the concentration of each vitamin can be the same or different.

In some embodiments, the vitamin is selected from the group consisting of:

Choline chloride 0.7-1.8mg / L, calcium pantothenate 0.7-1.8mg / L, folic acid 0.7-1.8mg / L, nicotinamide 0.7-1.8mg / L, pyridoxal hydrochloride 0.7-1.8mg / L, nuclear yellow It is 0.7-1.8mg / L, thiamine 0.7-1.8mg / L and inositol 0.7-1.8mg / L.

In some embodiments, the vitamin is selected from the group consisting of:

Choline chloride 1.0-1.4mg / L, calcium pantothenate 0.8-1.4mg / L, folic acid 1.0-1.4mg / L, nicotinamide 1.0-1.4mg / L, pyridoxal hydrochloride 1.0-1.4mg / L, nuclear yellow 1.0-1.4mg / L, thiamine 1.0-1.4mg / L, inositol 1.0-1.4mg / L.

In some embodiments, the inorganic salt is selected from the group consisting of:

Ferric citrate 5-15mg / L, sodium pyruvate 40-60mg / L, calcium chloride 1-5mg / L, magnesium sulfate 1-5mg / L, potassium chloride 4-10mg / L, sodium chloride 80-100mg / L, sodium dihydrogen phosphate 100-200mg / L.

Ferric citrate 8-12mg / L, sodium pyruvate 45-55mg / L, calcium chloride 2-4mg / L, magnesium sulfate 2-4mg / L, potassium chloride 6-8mg / L, sodium chloride 85-100mg / L, sodium dihydrogen phosphate 100-140mg / L.

In some embodiments, the culture medium contains one or more trace elements selected from the group consisting of:

Selenium, molybdate, chromium, cobalt, nickel, zinc, copper, manganese, barium, gallium, lithium, tin, titanium, bromine, iodine, vanadium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, zirconium, Cadmium and aluminum;

In some embodiments, the trace element is selenium, added in the form of selenite, and the content is 3-8 μg / L.

In some embodiments, the basic physiological buffer mixture further includes a buffer and / or an anti-shear protection agent;

In some embodiments, the buffer is sodium bicarbonate 1.0-1.8g / L and Hepes3.2-4.5mg / L;

In some embodiments, the anti-shear protection agent is Pluronic F-68700-2000 mg / L.

According to an aspect of the present application, the present application also relates to a method of culturing cells, comprising: culturing the cells in the medium as described above;

In some embodiments, the cells are pluripotent stem cells, embryonic stem cells, bone marrow stromal cells, hematopoietic progenitor cells, lymphatic stem cells, bone marrow stem cells, T cells, B cells, macrophages, liver cells, pancreatic cells, cancer cells And cell lines;

In some embodiments, wherein the cell line is selected from the group consisting of:

CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, T cell line, B cell line, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, HT1080, L929, fusion tumor and cancer cell lines.

In a specific embodiment, the cell is a hybridoma cell. A hybridoma cell is an infinitely passaged cell capable of secreting a single unique antibody. The monoclonal antibody secreted by it is widely used in vaccine production, in vitro diagnosis, and biomedicine. The medium enables hybridoma cells to grow normally and express monoclonal antibodies under suspension conditions. On the one hand, it meets the needs of monoclonal antibodies in the in vitro diagnostic reagent industry, and on the other hand, it can reduce costs.

The embodiments of the present application will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific conditions are indicated in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be followed. The reagents or instruments used do not indicate the manufacturer, are all conventional products that are commercially available.

Example 1

Screening and optimization of serum-free medium components.

Using a 6-well plate and a 3 mL culture system, the hybridoma cell line 1D7 was cultured, and the viable cell density in the culture system was sampled at 72 hours. Using the L16 (45) orthogonal test method, referring to relevant literature materials, after screening, 5 types of additives that promote cell culture were determined. The 5 types of additives that promote cell culture were: A, 8 types of lipids; B , 8 vitamins; C, 3 proteins; D, 19 amino acids; E, 7 inorganic salts.

The test was based on DMEM / F12 (v / v, 1: 1), and pre-added 6μg / L selenite, 1.5g / L sodium bicarbonate and 3.8mg / L Hepes, and Pluronic F-681300mg / L. Orthogonal test method is used to optimize the five selected additives, each factor (A, B, C, D, E) is set to four different concentration levels (1,2,3,4, the concentration of additives contained from low To high).

In A1, arachidonic acid 10 μg / L, cholesterol 500 μg / L, linoleic acid 80 μg / L, linolenic acid 80 μg / L, oleic acid 80 μg / L, palmitoleic acid 50 μg / L, stearic acid 50 μg / L, ethanolamine 1000 μg / L.

In A2, arachidonic acid 15μg / L, cholesterol 1000μg / L, linoleic acid 100μg / L, linolenic acid 120μg / L, oleic acid 120μg / L, palmitoleic acid 80μg / L, stearic acid 60μg / L, ethanolamine 1500μg / L.

A3 arachidonic acid 20μg / L, cholesterol 2000μg / L, linoleic acid 150μg / L, linolenic acid 160μg / L, oleic acid 160μg / L, palmitoleic acid 100μg / L, stearic acid 80μg / L, ethanolamine 2000μg / L.

In A4, arachidonic acid 25μg / L, cholesterol 3000μg / L, linoleic acid 180μg / L, linolenic acid 200μg / L, oleic acid 180μg / L, palmitoyl acid 125μg / L, stearic acid 100μg / L, ethanolamine 2700μg / L.

B1 choline chloride 0.2mg / L, calcium pantothenate 0.2mg / L, folic acid 0.2mg / L, nicotinamide 0.2mg / L, pyridoxal hydrochloride 0.2mg / L, riboflavin 0.2mg / L, thiamine 0.2mg / L, inositol 0.2mg / L

B2 Choline Chloride 0.4mg / L, Calcium Pantothenate 0.4mg / L, Folic Acid 0.4mg / L, Nicotinamide 0.4mg / L, Pyridoxal Hydrochloride 0.4mg / L, Riboflavin 0.4mg / L, Thiamine 0.4mg / L, Inositol 0.4mg / L

B3 choline chloride 0.8mg / L, calcium pantothenate 0.8mg / L, folic acid 0.8mg / L, nicotinamide 0.8mg / L, pyridoxal hydrochloride 0.8mg / L, riboflavin 0.8mg / L, thiamine Element 0.8mg / L, inositol 0.8mg / L

B4 choline chloride 1.2mg / L, calcium pantothenate 1.2mg / L, folic acid 1.2mg / L, nicotinamide 1.2mg / L, pyridoxal hydrochloride 1.2mg / L, riboflavin 1.2mg / L, thiamine 1.2mg / L, Inositol 1.2mg / L

In C1, BSA is 5 mg / L, transferrin is 2 mg / L, and insulin is 3 mg / L;

In C2, BSA is 10 mg / L, transferrin is 2 mg / L, and insulin is 5 mg / L;

In C3, BSA is 15 mg / L, transferrin is 5 mg / L, and insulin is 8 mg / L;

In C4, BSA is 15 mg / L, transferrin is 5 mg / L, and insulin is 10 mg / L.

D1 arginine 24mg / L, cystine 5mg / L, histidine 6mg / L, isoleucine 9.5mg / L, leucine 9mg / L, lysine 9mg / L, methionine 3.5mg / L, Phenylalanine 5.5mg / L, Threonine 9.5mg / L, Tryptophan 2mg / L, Tyrosine 7mg / L, Valine 10mg / L, Glycine 2.5mg / L, Alanine 3.5mg / L of aspartic acid, 5mg / L of asparagine, 5mg / L of aspartic acid, 5mg / L of glutamic acid, 3mg / L of proline, 3mg / L of serine

D2 arginine 48mg / L, cystine 10mg / L, histidine 12mg / L, isoleucine 19mg / L, leucine 18mg / L, lysine 18mg / L, methionine 7mg / L, phenylalanine 11mg / L, threonine 19mg / L, tryptophan 4mg / L, tyrosine 14mg / L, valine 20mg / L, glycine 5mg / L, alanine 7mg / L , Asparagine 10mg / L, aspartic acid 10mg / L, glutamic acid 10mg / L, proline 6mg / L, serine 6mg / L

D3 arginine 96mg / L, cystine 20mg / L, histidine 24mg / L, isoleucine 38mg / L, leucine 36mg / L, lysine 36mg / L, methionine 14mg / L, phenylalanine 22mg / L, threonine 38mg / L, tryptophan 8mg / L, tyrosine 28mg / L, valine 40mg / L, glycine 10mg / L, alanine 14mg / L , Asparagine 20mg / L, aspartic acid 20mg / L, glutamic acid 20mg / L, proline 12mg / L, serine 12mg / L

D4 arginine 192mg / L, cystine 40mg / L, histidine 48mg / L, isoleucine 76mg / L, leucine 72mg / L, lysine 72mg / L, methionine 28mg / L, phenylalanine 44mg / L, threonine 76mg / L, tryptophan 16mg / L, tyrosine 56mg / L, valine 80mg / L, glycine 20mg / L, alanine 28mg / L , Asparagine 40mg / L, aspartic acid 40mg / L, glutamic acid 40mg / L, proline 24mg / L, serine 24mg / L

E1 iron citrate 12mg / L, sodium pyruvate 50mg / L, calcium chloride 4mg / L, magnesium sulfate 3mg / L, potassium chloride 7mg / L, sodium chloride 100mg / L, sodium dihydrogen phosphate 120mg / L

In E2, iron citrate 18mg / L, sodium pyruvate 75mg / L, calcium chloride 6mg / L, magnesium sulfate 4.5mg / L, potassium chloride 7mg / L, sodium chloride 150mg / L, sodium dihydrogen phosphate 150mg / L

E3 iron citrate 27mg / L, sodium pyruvate 112.5mg / L, calcium chloride 9mg / L, magnesium sulfate 6.75mg / L, potassium chloride 7mg / L, sodium chloride 200mg / L, sodium dihydrogen phosphate 200mg / L

In E4, iron citrate 40.5mg / L, sodium pyruvate 168.75mg / L, calcium chloride 13.5mg / L, magnesium sulfate 10mg / L, potassium chloride 7mg / L, sodium chloride 250mg / L, dihydrogen phosphate Sodium 250mg / L

The results are shown in Table 1.

Table 1 Orthogonal test optimization

Comparing the average value of the density of living cells in the culture system at 72 hours of culture, the results of orthogonal test showed that among the 16 groups of different combinations of added factors, the combination of A4B4C4D2E1 promoted the density of living cells the most. According to the size of the range R, the influence of each factor on the density of living cells was determined, and the order of the influence of each factor was C> A> D> B> E. On this basis, the optimal level of the five different additive factors is determined as: A4B4C4D2E1. The concentration of the additive combined at the optimal level was performed as the preferred concentration of the culture medium, followed by the cell culture of Example 2.

Example 2

Serum-free medium preparation and cell culture

1. Medium Preparation

Add the following substances to DMEM / F12 (v / v, 1: 1) as the basic medium:

Bovine serum albumin 15.0mg / L, transferrin 5mg / L, insulin 10mg / L, arginine 48mg / L, cystine 10mg / L, histidine 12mg / L, isoleucine 19mg / L, Leucine 18mg / L, Lysine 18mg / L, Methionine 7mg / L, Phenylalanine 11mg / L, Threonine 19mg / L, Tryptophan 4mg / L, Tyrosine 14mg / L , Valine 20mg / L, glycine 5mg / L, alanine 7mg / L, asparagine 10mg / L, aspartic acid 10mg / L, glutamic acid 10mg / L, proline 6mg / L, serine 6mg / L, choline chloride 1.2mg / L, calcium pantothenate 1.2mg / L, folic acid 1.2mg / L, nicotinamide 1.2mg / L, pyridoxal hydrochloride 1.2mg / L, riboflavin 1.2mg / L, Thiamin 1.2mg / L, inositol 1.2mg / L, iron citrate 12mg / L, sodium pyruvate 50mg / L, calcium chloride 4mg / L, magnesium sulfate 3mg / L, potassium chloride 7mg / L, chlorine Sodium chloride 100mg / L, sodium dihydrogen phosphate 120mg / L, arachidonic acid 25μg / L, cholesterol 3000μg / L, linoleic acid 180μg / L, linolenic acid 200μg / L, oleic acid 180μg / L, palmitic acid 125μg / L, stearic acid 100μg / L, ethanolamine 2700μg / L, selenite 6μg / L, sodium bicarbonate 1.5g / L and Hepes 3.8mg / L, Pluronic F-681300mg / L. After the medium is prepared according to the conventional method, it is sterilized by filtering with a 0.22 μm microporous filter.

2. Cell culture

Remove the frozen hybridoma cell line 1C3 cells from the liquid nitrogen irrigation, and quickly place them in a 37 ° C water bath. After complete thawing, centrifuge to remove the supernatant. Gently pipette the cells with a small amount of the above serum-free medium to mix the cells. Transfer to a square bottle, and then subculture in a CO2 incubator at 37 ° C and 5% CO2 humidity saturation.

3. Compared with GIBCO brand serum-free medium Hybridoma-SFM

The above 1C3 cells were expanded and introduced into 4 square flasks. When they were grown to the logarithmic phase, they were inoculated into 1.5L spinner flasks at an initial culture density of about 70 × 104cells / ml, and then placed at 37 ℃, 5% CO2 humidity saturated incubator suspension culture.

As shown in FIG. 1, the lower curve is the growth curve of the hybridoma cells cultured in the serum-free medium described in this application, and the upper curve is the growth curve of the hybridoma cells cultured in GIBCO Hybridoma-SFM medium. The whole cultivation process lasts 8 days. Serum-free medium and GIBCO Hybridoma-SFM medium live cell density of this application reached 537 × 104cells / ml and 551 × 104cells / ml respectively at 120h. The culture effect of the serum-free medium described in this application is almost equal to that produced by foreign manufacturers The cultivation effect of the same culture medium, meanwhile, the serum-free culture medium of the present application has relatively low cost, clear chemical composition, and extremely low protein content.

Example 3

Fed-batch culture of hybridoma cell lines to prepare monoclonal antibodies

The serum-free medium described in the present application is used for the hybridoma cell lines DF92 and 1A4 for fermentor suspension culture, and the medium formula is the same as in Example 2. DF92 uses a 14L bioreactor of Guangzhou Qizhi Biological Company, the initial culture volume is 2L, the control conditions are: Temp 37 ± 0.5 ℃, DO 50% ± 5%, pH 6.9 ± 0.1, Stir speed 80rpm, 1A4 uses Applikon 2L biological reaction The initial culture volume is 2L, and the control conditions are: Temp 37 ± 0.5 ℃, DO 50% ± 5%, pH 6.9 ± 0.1, Stir speed 80rpm. In the suspension culture process, the fed-batch culture method is adopted. Sampling and monitoring the relevant data at intervals of about 24h. These data include glucose concentration, cell density, cell viability and monoclonal antibody expression in the culture medium. The culture results of the two cells are shown in Figures 2 and 4 (Figure 3 shows the prior art Medium control), add feed medium according to the glucose consumption index in the medium, and the feed interval is about 24h from the start of feed. When the cell viability in the fermentor drops below 60%, the fed batch culture operation of the fermentor is stopped, the supernatant is collected by centrifugation and filtered, and the expression level is determined and the monoclonal antibody is purified. The results show that the serum-free medium described in this application can be normally used for hybridoma cell suspension culture and the preparation of monoclonal antibodies. Under the same culture conditions, the cell growth status and antibody production are comparable to or better than those of the purchased control medium After purchasing the culture medium, subsequent experiments also proved that the monoclonal antibody prepared using the serum-free medium can be normally used in the field of in vitro diagnostic reagents.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand: It can still modify the technical solutions described in the foregoing embodiments, or equivalently replace some or all of the technical features therein; and these modifications or replacements do not deviate from the essence of the corresponding technical solutions from the technical solutions of the embodiments of the present application Scope.

Claims

A low-protein serum-free cell culture medium, characterized in that the culture medium contains a basic physiological buffer mixture and protein components;

The protein component includes bovine serum albumin, transferrin, and insulin, and the total added amount of the protein component does not exceed 30 mg / L.
The low-protein serum-free cell culture medium according to claim 1, wherein the protein component comprises bovine serum albumin 10.0-20.0 mg / L, transferrin 2.5-5.5 mg / L and insulin 5.0-12.0 mg / L;

The protein components include bovine serum albumin 13.0-17.0 mg / L, transferrin 4.5-5.5 mg / L and insulin 9.0-11.0 mg / L.
The low-protein serum-free cell culture medium according to claim 1 or 2, wherein the basic physiological buffer mixture uses DMEM / F12 as the basic medium, and includes amino acids, vitamins, inorganic salts, trace elements, One or more of lipids;

The volume ratio of DMEM and F12 in the DMEM / F12 is 0.8-1.2; the volume ratio of DMEM and F12 in the DMEM / F12 is 1.
The low-protein serum-free cell culture medium according to claim 3, wherein the lipid is selected from the group consisting of:

Arachidonic acid 10-50μg / L, cholesterol 1500-3500μg / L, linoleic acid 50-250μg / L, linolenic acid 50-250μg / L, oleic acid 50-250μg / L, palmitoleic acid 50-250μg / L , Stearic acid 50-250μg / L, ethanolamine 1500-3500μg / L.
The low-protein serum-free cell culture medium according to claim 3, wherein the amino acid is selected from the group consisting of:

Arginine 36-58mg / L, cystine 8-12mg / L, histidine 9-15mg / L, isoleucine 13-23mg / L, leucine 13-23mg / L, lysine 14 -22mg / L, methionine 4-7mg / L, phenylalanine 9-15mg / L, threonine 18-22mg / L, tryptophan 3-5mg / L, tyrosine 9-16mg / L, Valine 18-22mg / L, Glycine 4-7mg / L, Alanine 5-9mg / L, Asparagine 8-12mg / L, Aspartic acid 8-12mg / L, Glutamate 8 -12mg / L, proline 5-9mg / L, serine 5-9mg / L.
The low-protein serum-free cell culture medium according to claim 3, wherein the vitamin is selected from the group consisting of:

Choline chloride 0.7-1.8mg / L, calcium pantothenate 0.7-1.8mg / L, folic acid 0.7-1.8mg / L, nicotinamide 0.7-1.8mg / L, pyridoxal hydrochloride 0.7-1.8mg / L, nuclear yellow It is 0.7-1.8mg / L, thiamine 0.7-1.8mg / L and inositol 0.7-1.8mg / L.
The low-protein serum-free cell culture medium according to claim 3, wherein the inorganic salt is selected from the group consisting of:

Ferric citrate 5-15mg / L, sodium pyruvate 40-60mg / L, calcium chloride 1-5mg / L, magnesium sulfate 1-5mg / L, potassium chloride 4-10mg / L, sodium chloride 80-100mg / L, sodium dihydrogen phosphate 100-200mg / L.
The low-protein serum-free cell culture medium according to claim 3, wherein the culture medium contains one or more trace elements selected from the group consisting of:

Selenium, molybdate, chromium, cobalt, nickel, zinc, copper, manganese, barium, gallium, lithium, tin, titanium, bromine, iodine, vanadium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, zirconium, Cadmium and aluminum;

The trace element is selenium, added in the form of selenite, and the content is 3-8 μg / L.
The low-protein serum-free cell culture medium according to claim 3, wherein the basic physiological buffer mixture further comprises a buffer and / or an anti-shear protection agent;

The buffer solution is sodium bicarbonate 1.0-1.8g / L and Hepes 3.2-4.5mg / L;

The anti-shear protection agent is Pluronic F-68700-2000mg / L.
A method for culturing cells, comprising: culturing cells in the low-protein serum-free cell culture medium according to any one of the preceding claims;

The cells are pluripotent stem cells, embryonic stem cells, bone marrow stromal cells, hematopoietic progenitor cells, lymphatic stem cells, bone marrow stem cells, T cells, B cells, macrophages, liver cells, pancreatic cells, cancer cells, and cell lines;

The cell line is selected from the group consisting of:

CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, T cell line, B cell line, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, HT1080, L929, fusion tumor and cancer cell lines.