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Methods for producing cell lines stable in serum-free medium suspension culture

A technology of suspension culture and culture medium, applied in biochemical equipment and methods, tissue culture, botany equipment and methods, etc., which can solve the problems of reduced virus productivity and long-term cultivation difficulties of cell inoculum

Inactive Publication Date: 2007-01-17
SCHERING AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One hurdle is the difficulty in maintaining cell inoculum in long-term culture
A second hurdle is that viral productivity tends to decrease significantly once producer cells are maintained in suspension

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1: Adaptation of adherent A549 cells to suspension culture in serum-free and animal-material-free medium

[0099] Following standard protocols for culturing adherent cells by trypsinization, A549 cells were thawed and passaged in Medium 1 (Table 1 ) in T-75 flasks. The acclimatization process takes 3-6 weeks to complete. To initiate the suspension adaptation process, adherent cells were gradually weaned by serial passage of cells through media containing decreasing levels of serum. This was done by diluting Medium 1 (see Table 1 ) with increasing volumes of serum-free and animal material-free suspension medium Medium 2 (see Table 1 ) at each cell culture passage. As a result, serum levels decreased stepwise, from an initial 10% fetal bovine serum (FBS) level by 50% at each passage, until a final FBS concentration of less than 0.3%. Each passage takes 3 to 5 days. Cells are passaged until some cells become non-adherent (eg, do not attach to the surface of the ...

Embodiment 2

[0106] Example 2: Comparison of the amount of cell aggregation of A549 cells from different cell lines in suspension culture

[0107] During the suspension adaptation of A549 cells in serum-free and animal material-free medium to produce the adapted A549 suspension cell line, cells that do not bind large cell masses are selectively retained. Subpopulations of cells or cell lines that do not attach to surfaces are selected and propagated in suspension culture in serum-free and animal material-free media. Desired cell populations are enriched by multiple rounds of selection by stopping agitation of the culture and allowing large cell clumps to settle to the bottom of the flask and subculturing the cells that remain in suspension. The resulting cells of the adapted A549 cell line were less aggregated than non-adapted A549 cells in the same suspension medium (see, eg, Table 3).

[0108] A549 adherent cells were digested with trypsin, washed once with medium 1 (see Table 1), and t...

Embodiment 3

[0110] Example 3: Production of CRAV by A549S cells cultured in suspension in serum-free and animal-material-free medium

[0111] Virus production from A549S cells was performed in Erlenmeyer flasks on an orbital shaker and in stirred tank bioreactors. In both cases, production was performed by infecting the cultures with viral inoculum.

[0112] For virus production in shake flasks, maintain temperature (37 °C), CO by placing the shaker in a tissue culture incubator. 2 level (5%) and humidity. Suspension A549 cells were grown in batch mode to approximately 1.8 × 10 6 to 2.4×10 6 density of cells / ml. Prior to virus inoculation, approximately 90% of the initial culture volume was exchanged with serum-free and animal material-free medium (Medium 2, see Table 1) by centrifugation. Take 1×10 8 Viruses were inoculated at a final concentration of virus particles / ml, equivalent to a virus particle to cell ratio of approximately (40 to 50):1. Two hours after virus inoculation, ...

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Abstract

The present invention provides methods for adapting cells, such as A549 cells, to growth in serum-free and animal material-free medium suspension culture. The present invention provides methods for preparing viruses, such as adenovirus, from the A549 cells adapted for growth in serum-free and animal material-free medium in suspension culture.

Description

field of invention [0001] The present invention relates to methods of growing cells in culture and using the cells to produce viruses. Background of the invention [0002] There are two major obstacles in the development of suspension methods for the production of viral vectors. One obstacle is the difficulty in maintaining cell inoculum in long-term culture. A second hurdle is that viral productivity tends to decrease significantly once producer cells are maintained in suspension. [0003] Methods of adapting A549 cells to serum-free media in static culture are known in the art. For example, in Siegfried et al., (Siegfried, et al., (1994) J. Biol. Chem. 269(11):8596-8603), the A549 cell line was adapted to serum-free medium in static culture. In this method, A549 cells are first adapted to minimal Eagle's medium containing 1% fetal bovine serum over a period of one month. When approaching confluence, these A549 cell monolayers were washed with saline and placed in serum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/06C12N7/01C12N7/02C12N15/861
CPCC12N2710/10052C12N7/00C12N2710/10051
Inventor Z·刘R·L·小龙利M·P·桑托洛M·沃洛奇
Owner SCHERING AG
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