CN100348718C - Culture medium without animal originating component and serum for HEK293 cell adhesion culture - Google Patents

Abstract

The invention discloses a serum-free culture medium without animal-derived components and supporting the attachment culture of HEK293 cells. The serum-free medium consists of F12 medium and insulin, amino acids, monosaccharides and disaccharides, trace elements, CaCl ₂ , hyaluronic acid, aurintricarboxylic acid, ethanolamine, yeast hydrolyzate, hydroxypropyl-β-cyclic Composition of dextrin and other medium additives. The serum-free medium provided by the present invention has the following advantages: ① It can support the attached growth of HEK293 cells on the surface of tissue culture flasks and microcarriers. ②It does not contain animal-derived ingredients, and the chemical composition is basically clear. ③The cells grow well, and the culture density and viability of the cells are basically the same as those in the medium containing serum. ④ low cost.

Description

A serum-free medium that supports the attachment culture of HEK293 cells without animal-derived components

technical field

The invention relates to a serum-free medium supporting the culture of mammalian cells, more specifically a serum-free medium supporting the attachment culture of HEK293 cells without animal-derived components, belonging to the technical field of cell engineering.

Background technique

Animal cell culture is a key technical system for life science research and biotechnology applications. Medium is the most direct and important environmental factor that determines the growth and metabolism of cells cultured in vitro. According to whether animal serum needs to be supplemented in the use of the medium, animal cell culture medium can be roughly divided into synthetic medium and serum free medium.

Since the advent of the 199 medium developed by Morgan et al. in 1950, there have been at least 20 commercial synthetic mediums for different cell types, including EMEM, αMEM, DMEM, IMEM, F12 and RPMI 1640, which are widely used. synthetic media. The composition of synthetic medium is generally as many as 50 kinds, mainly including carbohydrates, amino acids, vitamins, nucleic acid derivatives, lipids and inorganic salts. The characteristic of the use of synthetic medium is that it can generally only maintain the survival of cells for a short period of time, and it needs to be supplemented with 5-20% (v/v) animal serum to support the growth and proliferation of in vitro cultured cells. Due to the complex composition of animal serum, there are more than 500 types of proteins alone. In life science research, the addition of animal serum often interferes with experimental results and affects cell function and phenotype. From the perspective of the production process of animal cell expression products, the quality difference between batches of animal serum can cause instability in the production of animal cell expression products, and the protein components in animal serum increase the difficulty of recombinant protein purification and quality control; from Considering the safety of animal cell expression products, the use of animal serum brings the possibility of animal-derived pathogenic microorganisms contaminating the product.

In 1976, Hayashi and Sato reported for the first time that adding hormones to synthetic medium could support the growth of cultured cells in vitro, which opened the prelude to the research and application of serum-free medium for animal cells. Since the late 1980s, as people have deepened their understanding of cell metabolism and physiology in vitro, and the types and sources of cytokines that regulate cell growth, metabolism, and differentiation have become more abundant, commercial serum-free media have been commercialized in terms of quantity and quality. The quality has been significantly improved, and serum-free medium has been widely used in many research fields of life sciences and large-scale production of biotechnology drugs.

HEK293 cells (human embryonic kidney 293 cells) are derived from the human embryonic kidney epithelial-like cell line transformed by adenovirus type 5, and are continuous passage cells with attachment-dependent growth. In life science research, HEK293 cells are widely used as cell models for the study of cell biology and functional genomics. In recent years, with the development of the industrialization of biotechnology drugs and the deepening of gene therapy research, the importance of HEK293 cells as the expression system for the production of recombinant protein drugs and the main production cells of recombinant virus vectors has become increasingly apparent.

The serum-free medium for HEK293 cells reported in the literature can only support the suspension culture of HEK293 cells, and animal-derived additives such as bovine serum albumin (bovine serum albumin, BSA) are added to the medium. The serum-free medium containing BSA largely retains the above-mentioned problems that may arise from the application of animal-derived serum in animal cell culture. The commercial protein-free medium (CD 293 medium) developed by Invitrogen in the United States is only suitable for the suspension culture of HEK293 cells. So far, there are no reports on animal-derived component-free serum-free medium that supports the adherent culture of HEK293 cells at home and abroad.

Suspension culture of HEK293 cells is not conducive to providing sufficient nutrients for cell growth and metabolism through continuous perfusion of the medium and effectively controlling the accumulation of harmful cell metabolites, thereby achieving animal cell expression that improves cell culture density and cell metabolism efficiency The purpose of product production process optimization. On the other hand, the suspension of HEK293 cells changed the original in vitro culture characteristics of HEK293 cell anchorage-dependent growth, which not only affected the cell phenotype of HEK293 cells cultured in vitro, but also affected the cell biology and functional genome of HEK293 cells as cell models. unavoidable disruptions to scientific research.

Contents of the invention

The purpose of the present invention is to provide a serum-free medium suitable for culturing HEK293 cells in vitro, which has the characteristics of not containing animal-derived components, and can support the attachment-dependent growth and high-density culture of HEK293 cells.

The idea adopted in the present invention is: 1). None of the formulations of the synthetic medium is designed for HEK293 cells. Although it can basically meet the nutritional requirements of HEK293 cells in vitro, the components such as carbohydrates, amino acids and inorganic salts therein There is still a large room for adjustment and optimization; 2). The synthetic medium lacks components that can support animal cell attachment culture, and cannot be directly used to support animal cell attachment culture. Based on the above considerations, the serum-free medium of the present invention has added the following components in the F12 medium;

(1) Insulin: promotes the uptake of glucose and amino acids by cells, and promotes cell growth.

(2) Amino acids: The concentration of amino acids in the medium usually limits the maximum density and cell viability that can be achieved by cell growth. There may be significant differences in the requirements of different cells for amino acids. These amino acids include essential amino acids that cannot be synthesized by specific cell types. Amino acids also include non-essential amino acids that cells can synthesize but consume at a rate greater than the cell's synthetic capacity.

(3) Monosaccharides and disaccharides: Fructose can partially replace glucose as one of the energy sources for cell growth and metabolism, and reduce the accumulation of lactic acid in the medium due to anaerobic glycolysis of glucose. Trehalose is a non-reducing disaccharide formed by combining two glucose molecules. It has non-specific protective effects on biological macromolecules such as hormones, growth factors and enzymes, and can also be used as an energy source for cell metabolism.

(4) Trace elements: trace elements such as Cu, Fe, Se and Ze are mainly used as prosthetic groups of enzymes to participate in the regulation of cell metabolism. in:

Cu: prosthetic group of superoxide dismutase, has anti-oxidation effect.

Fe: Cytochrome, catalase, peroxidase, etc. and the prosthetic group of heme, participating in the composition of the mitochondrial respiratory chain.

Se: A component of glutathione reductase, which has an antioxidant effect.

Ze: As the prosthetic group of enzymes, it is related to the activity or structure of more than 200 enzymes, and participates in the regulation of cell growth, protein synthesis and nucleic acid metabolism.

(5) Ca: An important component of the intercellular substance, which mediates cell adhesion and extension by acting on cadherin and integrin on the surface of the cell membrane.

(6) Hyaluronic acid: an extracellular matrix component that provides an environment and a scaffold for cell growth and adhesion.

(7) Aurintricarboxylic acid: a metal chelating agent, capable of binding, transporting and transferring Fe ²⁺ , partially replacing the function of iron transporter, and also promoting cell adhesion and extension by binding and transporting Ca ²⁺ .

(8) Ethanolamine: a precursor substance for the synthesis of phospholipids and cell membranes, which can promote the growth of cells to a certain extent.

(9) Yeast hydrolyzate: provide nutrients such as amino acids and vitamins for cell growth, and promote cell growth.

(10) Hydroxypropyl-β-cyclodextrin: store and transport biological macromolecules, improve the stability and bioavailability of biologically active substances.

According to above inventive concept, the present invention is on the basis of F12 medium, adds above-mentioned substance in the medium by following concentration (mg/L), forms a kind of new HEK293 cell serum-free medium (Table 1).

Table 1 Animal-derived component-free serum-free medium supporting HEK293 cell-attached culture

Composition Content (mg/L) F12 Medium Insulin Glutamine Proline Arginine Serine Asparagine  118002.5～10250～50050～100200～40040～60 (preferably 50) 40～60 (preferably 50)

Fructose Trehalose CuSO ₄ 5H ₂ OFeSO ₄ 7H ₂ ONa ₂ SeO ₃ ZnSO ₄ 7H ₂ OCaCl ₂ Hyaluronic Aurin Tricarboxylic Ethanolamine Yeast Hydrolyzate Hydroxypropyl-β-Cyclodextrin  1000～2000100～5000.05～0.50.2～20.005～0.020.2～2100～200 (preferably 150) 25～502.5～52.5～5500～1000250～500

The components of the above serum-free medium are commercial chemical/biochemical reagents, and their suppliers and models are as follows:

F12 medium is a powder preparation product of GIBCO BRL Company in the United States, and the product number is 21700-075.

Insulin is a recombinant product expressed by E. Coli from Sigma Company of the United States, and the product number is: I0259.

Glutamine is a powder preparation product of GIBCO BRL Company in the United States, and the commodity number is: 21051-040.

Proline is a powder preparation product of Sigma Company in the United States, and the product number is: P0380.

Arginine is a powder preparation product of Sigma Company in the United States, and the commodity number is: A4474.

Serine is a powder preparation product of Sigma Company in the United States, and the product number is: S4311.

Asparagine is a powder preparation product of Sigma Company in the United States, and the commodity number is: A4284.

Fructose is a powder preparation product of Sigma Company in the United States, and the product number is: F3510.

Trehalose is a medical grade powder preparation product of Nanning Zhongnuo Biological Engineering Co., Ltd.

CuSO ₄ ·5H ₂ O is a powder preparation product of Sigma Company in the United States, and the product number is: C8027.

FeSO ₄ ·7H ₂ O is a powder preparation product of Sigma Company in the United States, and the product number is F8633.

Na ₂ SeO ₃ is a powder preparation product of Sigma Company in the United States, and the product number is: S1382.

ZnSO ₄ ·7H ₂ O is a powder preparation product of Sigma Company in the United States, and the product number is: Z0251.

CaCl ₂ is a powder preparation product of Sigma Company in the United States, and the product number is: C7902.

Hyaluronic acid is a medical-grade powder preparation product of Shandong Zhengda Freda Pharmaceutical Co., Ltd.

Aurine tricarboxylic acid is a powder preparation product of Sigma Company in the United States, and the commodity number is: A1895.

Ethanolamine is a liquid preparation product of Sigma Company in the United States, and the product number is: E0135.

Yeast hydrolyzate is a powder preparation product of Sigma Company in the United States, and the product number is: Y0500.

Hydroxypropyl-β-cyclodextrin is a medical-grade powder preparation product of Hangzhou Zhengjia Chemical Co., Ltd.

Compared with the existing serum-free medium for HEK293 cells, the serum-free medium provided by the present invention has the following advantages: ① It can support the attached growth of HEK293 cells on the surface of tissue culture flasks and microcarriers. ②It does not contain animal-derived ingredients, and the chemical composition is basically clear. ③The cells grow well, and the culture density and viability of the cells are basically the same as those of the synthetic culture containing serum.

④ low cost.

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Description of drawings

Figure 1A shows HEK293 cells grown in the serum-free medium of the present invention observed under an inverted microscope; Figure 1B shows HEK293 cells grown in serum-containing medium observed under an inverted microscope.

Figure 2 shows the changes in cell density of HEK293 cells cultured in the serum-free medium and the serum-containing medium of the present invention respectively. Density of HEK293 cells cultured in serum-free medium (○); density of HEK293 cells cultured in F12 medium containing 5% (v/v) calf serum (▲).

Figure 3 shows the changes in cell density and viability of HEK293 cells cultured in a 5L stirred tank bioreactor with serum-free medium.

Figure 4 shows the cell mass formed by HEK293 cells cultured in a 5L stirred tank bioreactor with serum-free medium.

Detailed ways

Embodiment 1, adhere to culture HEK293 cell in 100ml tissue culture bottle

Dissolve the medium components listed in Table 2 in 1000ml deionized water, and stir at room temperature for 30min to fully dissolve. After sterilizing by filtration with a 0.2 μm filter membrane, store in a refrigerator at 4°C.

Table 2 Serum-free medium for culturing HEK293 cells in 100ml tissue culture flasks

Composition Content (mg/L) F12 Medium Insulin Glutamine Proline Arginine Serine Asparagine   118002.5250502005050

Fructose Trehalose CuSO ₄ 5H ₂ OFeSO ₄ 7H ₂ ONa ₂ SeO ₃ ZnSO ₄ 7H ₂ OCaCl ₂ Hyaluronic Aurin Tricarboxylic Ethanolamine Yeast Hydrolyzate Hydroxypropyl-β-Cyclodextrin  10001000.050.20.0050.2150252.52.5500250

HEK293 cells (purchased from Invitrogen, USA) cultured in a 100 ml tissue culture flask with serum-free medium were digested with 0.25% (w/v) trypsin at room temperature for 2 min. Use serum-free medium to disperse and adjust the concentration of cell suspension to 2×10 ⁵ cells/ml, inoculate 100 ml tissue culture flasks at 10 ml/bottle, inoculate 7 culture flasks each time, and culture statically at 37°C and 5% CO ₂ , Cell morphology was observed with an inverted microscope and photographed for record. One flask of cells was taken every day and digested with 0.25% (w/v) trypsin, and then the cell density was counted with a cell density and viability automatic analysis system (Cedex A20, Innovatis). From the second day of culture, the remaining cells were replaced with fresh medium according to the color change of the medium at 50-100% of the culture volume every day. After 4 days of culture, HEK293 cells formed a dense monolayer, and the cell morphology showed characteristics of polygonal epithelioid cells (Fig. 1A).

HEK293 cells cultured in a 100ml tissue culture flask with F12 medium containing 5% (v/v) calf serum were digested with 0.25% (w/v) trypsin at room temperature for 5 min. Disperse with serum-free F12 medium containing 5% (v/v) calf serum and adjust the concentration of the cell suspension to 2×10 ⁵ cells/ml, inoculate 100ml tissue culture bottles at 10ml/bottle, and inoculate 7 Culture flasks were cultured statically at 37°C, 5% CO ₂ . Except that the above-mentioned serum-free medium was replaced by the F12 medium containing 5% (v/v) calf serum, the culture and counting methods of HEK293 cells were the same as the above-mentioned method, which was used as the control of the embodiment. After 4 days of culture, the morphology of HEK293 cells in F12 medium containing 5% (v/v) calf serum was the same as that of HEK293 cells cultured in serum-free medium (Fig. 1B). The growth kinetics and cell density of HEK293 cells in the serum-free medium were comparable to their culture effects in the F12 medium containing 5% (v/v) calf serum as a control in the example ( FIG. 2 ).

Example 2, HEK293 cells were cultured by continuous perfusion in a 5L stirred tank bioreactor

HEK293 cells cultured in a 2000 ml spinner bottle with the serum-free medium listed in Table 2 were digested with 0.25% (w/v) trypsin at room temperature for 2 min. Use the serum-free medium listed in Table 3 to disperse the cells and inoculate them in a stirred tank bioreactor with a working volume of 5 L and equipped with a built-in rotary filter (B. Braun Biotecn International, Germany), with an inoculation density of 4.5×10 ⁵ cells/ml. The temperature was set at 37°C, the dissolved oxygen concentration was controlled at 30-50%, and the pH was controlled at 7.1-7.2. The stirring speed is set at 35-50r/min on the 1st-3rd day of culture to make the HEK293 cells aggregate each other and form cell clusters. From the 4th day, according to the change of the average particle size of the HEK293 cell Gradually increase the stirring speed within the range.

Table 3 Serum-free medium for perfusion culture of HEK293 cells in 5L bioreactor

Composition Content (mg/L) F12 Medium Insulin Glutamine Proline Arginine Serine Asparagine Fructose Trehalose CuSO ₄ 5H ₂ OFeSO ₄ 7H ₂ ONa ₂ SeO ₃ ZnSO ₄ 7H ₂ OCaCl ₂ Hyaluronic Aurintricarboxylic Ethanolamine Yeast Hydrolyzate Hydroxypropyl-β-Cyclodextrin   1180010500100400505020005000.520.02215050551000500

Samples were taken every day with YSI 7100 multi-parameter bioanalysis system (YSI 7100, Yellow Springs Instruments) to detect the concentration of glucose and lactic acid in the medium, and Cedex A20 was used to measure cell density and cell viability. After culturing for 1 day, start the medium perfusion system of the bioreactor, use the built-in rotary filter with a micropore diameter of 75 μm in the bioreactor to intercept the cell mass, and adjust the perfusion rate to 0.15-1.5 volume/day according to the cell density and glucose concentration , implement serum-free continuous perfusion culture operation of HEK293 cells.

The whole culture process lasted for 17 days, the density of HEK293 cells increased from 4.5×10 ⁵ cells/ml at the time of inoculation to 13.7×10 ⁶ cells/ml at the end of culture, and the viability of HEK293 cells remained at more than 90 during the whole culture process. % level (Figure 3). The shape of HEK293 cells forms spherical cell clusters with relatively regular shape and relatively uniform size under the action of hydrodynamic force (Figure 4).

Specific embodiments of the present invention have described the content of the present invention in detail. For those of ordinary skill in the art, any obvious changes made to it without departing from the spirit of the present invention, especially the equivalent replacement of several steps, constitute an infringement of the patent right of the present invention, and will bear the corresponding legal liability.

Claims (2)

1. support the HEK293 cell to attach the serum-free culture medium without animal origin components of cultivation for one kind, it is characterized in that: form by following concentration by following material:

F12 substratum 11800mg/L

Regular Insulin 2.5～10mg/L

Glutamine 250～500mg/L

Proline(Pro) 50～100mg/L

Arginase 12 00～400mg/L

Serine 40～60mg/L

L-asparagine 40～60mg/L

Fructose 1000～2000mg/L

Trehalose 100～500mg/L

CuSO ₄·5H ₂O 0.05～0.5mg/L

FeSO ₄·7H ₂O 0.2～2mg/L

Na ₂SeO ₃ 0.005～0.02mg/L

ZnSO ₄·7H ₂O 0.2～2mg/L

CaCl ₂ 100～200mg/L

Hyaluronic acid 25～50mg/L

Aurin tricarboxylic acid 2.5～5mg/L

Thanomin 2.5～5mg/L

Yeast hydrolyate 500～1000mg/L

Hydroxy propyl-Beta-cyclodextrine 250～500mg/L.

2. a kind of serum-free culture medium without animal origin components of supporting that the HEK293 cell attaches and cultivates according to claim 1 is characterized in that: be made up of by following concentration following material:

F12 substratum 11800mg/L

Regular Insulin 2.5～10mg/L

Glutamine 250～500mg/L

Proline(Pro) 50～100mg/L

Arginase 12 00～400mg/L

Serine 50mg/L

L-asparagine 50mg/L

Fructose 1000～2000mg/L

Trehalose 100～50mg/L

CuSO ₄·5H ₂O 0.05～0.5mg/L

FeSO ₄·7H ₂O 0.2～2mg/L

Na ₂SeO ₃ 0.005～0.02mg/L

ZnSO ₄·7H ₂O 0.2～2mg/L

CaCl ₂ 150mg/L

Hyaluronic acid 25～50mg/L

Aurin tricarboxylic acid 2.5～5mg/L

Thanomin 2.5～5mg/L

Yeast hydrolyate 500～1000mg/L

Hydroxy propyl-Beta-cyclodextrine 250～500mg/L.

Images