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Serum-free culture medium without animal origin components for culturing Vero cell micro-carrier

A serum-free culture medium and cell microcarrier technology, which is applied in the field of cell engineering, can solve the problems of secret medium composition formula and high price, and achieve the effects of low cost, basic clarity and clear chemical composition.

Inactive Publication Date: 2010-10-20
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the commercialized Vero cell serum-free medium exists to varying degrees, such as the medium is mostly provided in the form of liquid, the price is expensive, the composition of the medium is kept secret, and there is an adaptation process for cells to be transferred from serum-containing medium to serum-free medium, etc. question

Method used

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  • Serum-free culture medium without animal origin components for culturing Vero cell micro-carrier
  • Serum-free culture medium without animal origin components for culturing Vero cell micro-carrier
  • Serum-free culture medium without animal origin components for culturing Vero cell micro-carrier

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Experimental program
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Effect test

Embodiment 1

[0064] Embodiment 1 attaches and cultivates Vero cells in a 100ml tissue culture flask

[0065] 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.

[0066] Table 2 Serum-free medium for cultivating Vero cells in 100ml tissue culture flasks

[0067]

[0068] Component Content (mg / L)

[0069] 

[0070] DMEM / F12 (1:1) medium 15600

[0071] Epidermal growth factor 0.025

[0072] Insulin 2.5

[0073] Aurintricarboxylic acid 0.005

[0074] Serum amine 0.005

[0075] Glutamine 125

[0076] Cystine 25

[0077] Arginine 100

[0078] Methionine 50

[0079] Serine 25

[0080] Asparagine 50

[0081] Tyrosine 250

[0082] Fructose 1000

[0083] Trehalose 100

[0084] Vitamin C 25

[0085] Biotin 0.01

[0086] C...

Embodiment 2

[0095] Embodiment 2 Microcarrier immobilization culture Vero cells in 250ml stirred culture flask

[0096] Vero cells cultured in DMEM / F12 medium containing 5% (v / v) calf serum in a 100 ml tissue culture flask were digested with 0.25% (w / v) trypsin at room temperature for 2 min. Use serum-free medium listed in Table 3 and DMEM / F12 disperse cells containing 5% (v / v) calf serum respectively, along with pretreated Cytodex 1 microcarrier (used concentration is 2mg / ml) to inoculate 250ml stirring Culture flasks, inoculated at a density of 3×10 5 cells / ml, the culture volume is 100ml / bottle. 37°C, 5% CO2 , Suspension culture at 30r / min, according to the color change of the culture medium, replace the fresh medium with 50-100% of the culture volume every day, and the whole culture process lasts for 7 days. Samples were taken every 24 hours and the cell morphology was observed with an inverted microscope. The cell density was counted with a cell density and viability automatic analy...

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Abstract

The invention discloses a serum-free culture medium without animal origin components for culturing a Vero cell micro-carrier. The serum-free culture medium consists of a DMEM / F12 (1:1) culture medium and culture medium additive components such as epidermal growth factors, insulin, serotonin, aurin tricar-boxylic acid, biotin, vitamin C, amino acid, fructose, trehalose, trace elements, hydroxypropyl-beta-cyclodextrin and the like. The serum-free culture medium has the following advantages that: (1) the attachment growth of Vero cells in a tissue culture vessel and on the surface of the micro-carrier can be supported, and the cells can be transferred from a serum culture medium to the serum-free culture medium without a course of adaptation; (2) the serum-free culture medium contains no animal origin components, and has basically definite chemical components, and a low cost; and (3) the cells grow well, and the cellular morphology, the density and the vitality are basically the same as those of the serum culture medium.

Description

technical field [0001] The invention relates to a serum-free medium for Vero cell microcarrier culture, more specifically to a serum-free medium for Vero cell microcarrier immobilized culture without animal-derived components, belonging to the technical field of cell engineering. Background technique [0002] Vero cell (African green kidney cell, Vero cell) is a vaccine production cell line approved by the World Health Organization and my country's biological product regulations, and is the most important cell matrix for virus vaccine production at this stage. Compared with primary cells, diploid cells and other subcultured cell substrates used for vaccine production, Vero cells have the following characteristics: ①The source is convenient, can be continuously passaged, and the growth rate is fast; ②It is sensitive to infection by various viruses, High virus proliferation titer; ③Stable genetic traits, low degree of malignant transformation, high biological safety; ④Cultivat...

Claims

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Application Information

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IPC IPC(8): C12N5/071
Inventor 陈昭烈刘红刘兴茂李世崇叶玲玲吴本传王启伟
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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