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Medium and method suitable for vero cell microcarrier suspension amplification culture

A technology of amplified culture and microcarriers, applied in the field of cell engineering, to achieve good application value, clear components, and improve product quality

Active Publication Date: 2019-02-12
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the serum-free microcarrier suspension culture of Vero cells has been achieved, only 5 liters of total volume bioreactor culture has been achieved, no scale-up culture has been involved, and the maximum cell density of 1.2×106 cells has only been achieved at 2 g / l cytodex1 microcarriers count / ml

Method used

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  • Medium and method suitable for vero cell microcarrier suspension amplification culture
  • Medium and method suitable for vero cell microcarrier suspension amplification culture
  • Medium and method suitable for vero cell microcarrier suspension amplification culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] A kind of low-cost culture medium suitable for Vero cell square bottle, roller bottle culture, microcarrier suspension culture and microcarrier suspension scale-up culture of the present embodiment, it comprises 21 kinds of amino acids, 11 kinds of inorganic salts, 12 kinds of vitamins, 1 1 protein hydrolyzate, 2 lipids, 2 buffer components, 7 additives; materials described below were purchased from Sigma.

[0100] A specific embodiment of the serum-free medium of this example, labeled V-SFM, was prepared using the ingredients and amounts listed in Table 1.

[0101] Table 1

[0102] Element

concentration

calcium chloride

116 mg / L

50 x major salts

20ml / L

10000×minor salts

0.1ml / L

200 x vitamins and other substances

5ml / L

glucose

4500mg / L

Glutamine

484 mg / L

10 x Amino Acids

100ml / L

10% Pluronic F-68

10ml / L

sodium bicarbonate

2600mg / L

soybean hydrolyzate ...

Embodiment 2

[0119] A kind of low-cost culture medium suitable for Vero cell square bottle, roller bottle culture, microcarrier suspension culture and microcarrier suspension scale-up culture of the present embodiment, it comprises 21 kinds of amino acids, 11 kinds of inorganic salts, 12 kinds of vitamins, 1 1 protein hydrolyzate, 2 lipids, 2 buffer components, 7 additives; materials described below were purchased from Sigma.

[0120] The ingredients are as follows, expressed in mg / l:

[0121]

[0122]

[0123]

[0124] The above components are dissolved in pyrogen-free ultrapure water for preparation, and a low-cost medium suitable for Vero cell square bottle, roller bottle culture, microcarrier suspension culture and microcarrier suspension scale-up culture can be obtained. Use the above-mentioned V-SFM medium to add trace serum to culture Vero cells in a domestic 25-liter stirred bioreactor, when the cell density reaches 3.5×10 6 After the number of cells / ml, the microcarriers w...

Embodiment 3

[0127] Use the medium in Example 1 to adhere to culture Vero cells in T25 tissue culture flasks

[0128] Vero cells cultured in MEM medium containing 10% (v / v) newborn bovine serum in a T25 tissue culture flask were digested with 0.25% (w / v) trypsin for 8 minutes. The low serum medium containing 1% (v / v) newborn bovine serum and the MEM medium containing 10% (v / v) newborn bovine serum listed in Table 1 were used to resuspend the cells, and the cell density was adjusted to 2.5×10 5 Cell count / ml. Inoculate in T25 tissue culture flask at 5 ml / bottle, culture statically at 37°C and 5% CO2, observe cell morphology with an inverted microscope and take pictures for record. After cultivating for 72 hours, Vero cells confluent into a monolayer in the low serum medium containing 1% (v / v) newborn bovine serum of the present invention and MEM medium containing 10% (v / v) newborn bovine serum, such as Figure 3A and Figure 3B shown.

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Abstract

The invention discloses a culture medium applicable to suspension and magnification cultivation of Vero cell microcarriers. The culture medium comprises amino acid, inorganic salt, vitamins, protein hydrolysate, lipid, buffer components and additives. The culture medium not only is applicable to static cultivation in a Vero square bottle and cultivation in a roller bottle, but also is supportive for suspension cultivation of the microcarriers and magnification cultivation of the microcarriers in a reactor, and can be used as a serum-free virus maintenance medium to realize serum-free virus production.

Description

technical field [0001] The invention relates to the technical field of cell engineering, in particular to a medium for supporting Vero cell adherent growth and virus proliferation and a method for using it, more specifically for Vero cell microcarrier suspension culture and microcarrier suspension scale A culture medium for amplified culture and a method for realizing large-scale culture by using the culture medium. Background technique [0002] Vero cells have a clear source, a clear history, and no tumorigenicity within a certain generation. It is a cell line approved by the World Health Organization (WHO) and my country's biological products regulations for the production of human and animal vaccines. It is the most important virus vaccine production at this stage. of the cell matrix. Vero cells are sensitive to a variety of viruses, and the virus proliferation titers are high. At present, various domestic biological products industries using Vero cells as the production...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/02
Inventor 张大鹤滕小锘龚迪
Owner 苏州沃美生物有限公司
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