CN103160458A

CN103160458A - Low-serum medium suitable for growth of Vero cells

Info

Publication number: CN103160458A
Application number: CN2011104218558A
Authority: CN
Inventors: 汤承; 岳华; 苏晓蕊
Original assignee: Southwest Minzu University
Current assignee: Southwest Minzu University
Priority date: 2011-12-15
Filing date: 2011-12-15
Publication date: 2013-06-19

Abstract

The invention develops a low-serum medium suitable for growth of Vero cells. An amount of calf serum in conventional culturing is reduced from 10 % (V component) to 2 %, and conventionally added animal source components are replaced by plant source components and recombinant protein. The low-serum medium can reduce amount of serum, and thus not only reduces cost, but also minimizes possibilities that the serum in the medium carries exogenous pathogen contamination and reduces a lot of other proteins in the serum, thereby benefiting separation and purification of downstream products. By using a plant protein hydrolyzate and a recombinant epidermal growth factor to replace animal source components, side effects of animal source components can be reduced, and safety of biological products can be improved. The medium provided by the invention not only enables the Vero cells to grow well, but also reduces cost, minimizes exogenous pathogen contamination, and improves production stability of cell products, thereby providing a high-quality medium for producing of biological products with the Vero cells as virus host or expression carrier.

Description

A kind of low blood serum medium of suitable Vero Growth of Cells

1. technical field

The present invention relates to cell non-serum or low serum free culture system technical field, specifically about a kind of low blood serum medium of suitable Vero Growth of Cells, be used for the propagation of the low serum free culture system of Vero cell and virus.

2. background technology

The Vero cell is the clone of the production vaccine of the World Health Organization and China's biological products rules approval, and it has convenient sources, and biological safety is high,, virus multiplication titre advantages of higher responsive to multiple Viral infection.Use the calf serum of final concentration 10% (volume) in common Vero cell culture medium.But the contained chemical composition of serum is uncertain, also exists difference between each batch, and serum is expensive simultaneously, also easily causes bacterium, fungi, virus and mycoplasma contamination; In addition, in serum, the protein of a large amount of complicated components brings very large difficulty can for the separation and purification of the biological products such as vaccine, cytokine and monoclonal antibody.

Therefore, use no or little the trend that serum becomes the Vero cell cultures as far as possible.Prior art often by adding the animal source compositions such as bovine serum albumin, Transferrins,iron complexes, Regular Insulin, reduces the amount of serum.But the protein of animal source composition has a significant impact the security of biological products (as vaccine).The contriver develops the low blood serum medium of a kind of Vero cell, can either make Growth of Cells good, and form is normal, has reduced again pollution, improves the stability that cellular product is produced, and reduces the later stage purifying process, reduces production costs.

3. summary of the invention

The present invention is intended to overcome the shortcoming that must use the high-content calf serum could promote growth, poor stability in the cultivation of Vero cell routine, and a kind of new low blood serum medium is provided.

This substratum is under the condition of DMEM (available from HyClone company) as basic medium, on the basis of original 10% calf serum, serum-concentration is reduced to 2%, add again the soybean plants protolysate of 2-10g/L in substratum, the Urogastron of 20-100 μ g/L, the Sodium Selenite of 25-50nM, the glutamine of 2-8mM, the ironic citrate of 50-200 μ M, the vitamins C of 50-200 μ M, the thanomin of 150-300 μ M, beta-mercaptoethanol 0-50 μ M, Sodium.alpha.-ketopropionate 0.5-2mM.

Compare with the cultural method that reduces amount of serum by introducing the animal-origin component, the low blood serum medium of the present invention's development, introducing be the protein-soybean plants protolysate, recombinant human epidermal growth factor and some chemosynthesis materials of plant-sourced.Soybean plants protolysate is a kind of ultrafiltration, do not contain the animal ingredient medium additives, and it is to be come by the digestive process production of height optimization, has used aborning unique mixed enzyme, contains 15 seed amino acid compositions.The source of recombinant human epidermal growth factor and other added ingredientss is clear and definite, uniform component.Use these compositions to substitute the growth that zoogenous composition can promote the Vero cell.Therefore, the low blood serum medium of Vero cell of the present invention does not produce the side effect of following protein ingredient, and security is good.The present invention develops this, and the low blood serum medium of Vero cell of the present invention does not produce the side effect of following protein ingredient, and security is good.The low blood serum medium of the present invention's development is compared plesiomorphism with 10% calf serum of cellar culture, has reduced pollution, and the separation, the security that are conducive to the cell derived product are good; Compare with the serum free medium of commercialization, reduced cost, be suitable for use as the virus host of producing the biological products such as vaccine, the cultivation of expression vector.

4. description of drawings

Fig. 1 is the 48 hours pictures of Vero cell cultures that compare with 10% calf serum in embodiment two, Fig. 2 is low 48 hours pictures of blood serum medium Vero cell cultures of self-control in embodiment two, and Fig. 3 compares 48 hours pictures of Vero cell cultures with commercial serum-free in embodiment two.

5. embodiment

Below in conjunction with embodiment, the present invention is described in further detail:

Embodiment 1:

The present invention is by 5% calf serum, soybean plants protolysate (V component) 5g/L, and Urogastron 20 μ g/L, glutamine 2mM forms, beta-mercaptoethanol 30 μ M.

Present embodiment has reduced the amount of the calf serum of cellar culture use, adopt soybean plants protolysate, its cost is lower than the use conventional medium, and it is dead too early to have solved simultaneously the cell that causes because of the use that reduces calf serum, the undersaturated problem of form.

Embodiment 2:

The present invention is by 2% calf serum, soybean plants protolysate (V component) 5g/L, Urogastron 60 μ g/L, glutamine 4mM, Sodium Selenite 50nM, ironic citrate 100 μ M, vitamins C 100 μ M, thanomin 200 μ M form, beta-mercaptoethanol 30 μ M, Sodium.alpha.-ketopropionate 1mM.

Present embodiment further reduces the amount of serum, increases plant protein hydrolysate and Urogastron, has increased the density of cell, has improved specific growth rate, thereby has improved the vaccine output of field of medicaments.

Compound method:

1) being formulated in when using soybean plants protolysate to be used for basic grown cultures under laboratory scale of soybean plants protolysate mother liquor, need to use storage liquid concentrated and sterilization.In our laboratory with the concentration of 100g/L as storage liquid.Take 10g soybean plants protolysate powder, it is dissolved in the 100ml basic medium, fully carrying out disinfection without protein bound syringe type strainer with 0.22 μ m after the dissolving.If not soluble in basic medium, the incubator that can solution be placed in 37 ℃ before filtration kept about 30 minutes.Then labelled, be placed on 4 ℃ of Refrigerator stores, standby.

2) preparation of Urogastron is dissolved in Urogastron in the acetic acid of 10mM, and is labelled with the strainer filtration sterilization of 0.22mm, is placed on-20 ℃, can preserve at least 2 years.Be distributed into tubule in necessary situation, avoid multigelation during use as far as possible.

3) preparation of glutamine takes the 2.922g glutamine, is dissolved in the 100ml tri-distilled water, and is labelled with the 5ml centrifuge tube packing after autoclaving with the filtration sterilization of 0.22mm strainer ,-20 ℃ of preservations.

4) at first the preparation of substratum adds 2% calf serum in basic medium, add respectively again soybean plants protolysate, Urogastron, glutamine, vitamins C, ironic citrate, thanomin and Sodium Selenite, adjusting PH is 7.2-7.4, be settled to final volume with basic medium, in 2 ℃ of-8 ℃ of preservations.

Claims

1. the low blood serum medium of a suitable Vero Growth of Cells, it is characterized in that adding the calf serum of 2ml take DMEM as basic medium in basic medium, then add the soybean plants protolysate of 5g/L, the Urogastron of 60 μ g/L, the Sodium Selenite of 30nM, the glutamine of 4mM, the ironic citrate of 100 μ M, the vitamins C of 100 μ M, the thanomin of 200 μ M, beta-mercaptoethanol 30 μ M, Sodium.alpha.-ketopropionate 1mM.Request is carried out patent protection to the formula of this novel low blood serum medium.