Method of replicating viruses in suspension cultures of dog kidney cells

a technology of kidney cell suspension and dog kidney, which is applied in the field of animal cells, can solve the problems of inability to achieve economical influenza virus replication as a prerequisite for vaccine production, limited virus yield that can be harvested for vaccine formulation, and time-, labor- and cost-intensive use of embryonated hens' eggs for vaccine production, etc., and achieves the effects of reducing the risk of cell culture contamination, reducing the expenditure of effort, and reducing the number of infections

Inactive Publication Date: 2011-04-28
WARTHEN MONTY R +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]Moreover, by the use of the cells according to the invention, on the one hand a change of medium before infection to remove serum can be dispensed with and on the other hand the addition of protease (where needed) can be carried out simultaneously with the infection. On the whole, only a single opening of the culture vessel for infection with viruses is necessary, whereby the danger of the contamination of the cell cultures is drastically reduced. The expenditure of effort that would be associated with the change of medium, the infection and the subsequent protease addition is furthermore decreased.
[0021]The cells used according to the invention are derived from substrate-dependent Cutter Laboratory Dog Kidney (CLDK) cells. As used herein, “substrate-dependent CLDK cells” and like terms are used interchangeably with “adherent CLDK cells,”“aCLDK cells” or like terms. aCLDK cells are different from suspension CLDK cells. Whereas the former grow adherently, the latter grow in suspension, such as in a shaker flask or other large-scale production container including without limitation disposable or non-disposable bioreactors or biocontainers. aCLDK cells require a surface on which to grow, such as the surface of a roller bottle or microcarriers or beads added to the medium. aCLDK cells do not grow in suspension. When inoculated into medium in a shaker flask placed on an oscillating platform, aCLDK cells may maintain some viability, but do not grow. Rather, aCLDK cells tend to clump together.
[0022]Suspension CLDK cells are also termed “sCLDK” cells or “sCLDK-SF” cells to indicate that they grow in a serum-free media. sCLDK cells are derived from aCLDK cells by passaging through serum-free medium in suspension as described herein. On account of these properties and their ability to serve as host cells for replicating viruses, sCLDK cells are suitable for economical replication of viruses in cell culture by means of a simple and cost-effective process. In contrast to aCLDK cells, sCLDK cells do grow in a shaker flask placed on an oscillating platform or

Problems solved by technology

The use of embryonated hens' eggs for vaccine production is time-, labor- and cost-intensive.
The constraining requirement of growing these cells adherently places a limitation on the yield of cells that can be grown and also consequently places a limitation on the yield of virus that can be harvested for formulation in a vaccine.
Economical influenza virus replication as a prerequisite for vaccine production cannot be accomplished, however, using the methodology described in U.S. RE 33,164, as the change of media, the subsequent infec

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1a

Adaptation of CLDK Cells for Serum-Free or Serum Substitute-Free Suspended Growth

[0052]Cutter Laboratory Dog Kidney (CLDK) cells are anchorage dependent, and hence require a substrate or surface on which to grow. Suitable substrates include the interior surfaces of containers such as T-flasks or roller bottles, or upon the surface of beads or microcarriers that can be added to a culturing container. Typically, these cells form monolayers on the substrate, and can be grown in static culture and roller bottle. These cells require serum to grow and are epithelial in morphology.

[0053]Two ampoules of Cutter Laboratories aCLDK cells were thawed, and then transferred to two 4 mL cryotubes such that there was 1 mL of thawed material in each tube. 1 mL of supplemented EX-CELL™ MDCK serum-free medium was added to each cryotube. “Supplemented EX-CELL™ MDCK serum-free medium” as used herein refers to a mixture of EX-CELL™ MDCK serum-free medium (SAFC Biosciences, Lenexa, Kans.) supplemented wit...

example 1b

Adaptation of aCLDK Cells for Serum-Free or Serum Substitute-Free Suspended Growth

[0058]The above CLDK adaptation procedure was subsequently repeated and completed with fewer passages. The following table summarizes the culture media and observed growth for this subsequent procedure.

TABLE 2Passage History to Adapt aCLDK Cells to sCLDK-SF Cells%Pluronic ®% Lipumin ™Cell PlantCell7-DayF68ADCF SerumDensitycount / GrowthPassageSurfactantSubstitute(cells / mL)mLFactor100NA7.94 × 105NA2005.00 × 1055.95 × 1051.193115.00 × 1052.65 × 1050.534112.50 × 1051.08 × 1064.325115.00 × 1054.15 × 1050.836113.00 × 1054.45 × 1051.487114.00 × 1052.35 × 1050.598112.00 × 1058.34 × 1054.179115.00 × 1052.09 × 1064.1810115.00 × 1051.67 × 1063.3411115.00 × 1053.35 × 1066.7012115.00 × 1053.25 × 1066.5013115.00 × 1053.90 × 1067.8014115.00 × 1057.10 × 10614.2015115.00 × 1057.28 × 10614.5616115.00 × 1056.84 × 10613.6817115.00 × 1056.68 × 10613.36180.50.55.00 × 1058.10 × 10616.20190.250.255.00 × 1057.37 × 10614.74200.1...

example 1c

Procedure for Recovering sCLDK-SF MCS Cells from Liquid Nitrogen Storage and Optimum Growth Propagation

[0059]1L of supplemented EX-CELL™ MDCK serum-free medium was prepared using an aseptic technique. Thereafter, 2-3 mLs of cells from the frozen ampoules of passage 41 of Example 1A were retrieved from liquid nitrogen and allowed to thaw. The thawed ampoules were sprayed with 70% alcohol and allowed to dry. A sterile ampoule snapper was used to break open all vials. Thereafter, a sterile pipette was used to transfer an ampoule of cells to a small sterile tube with cap (sterile 4.5 mL cryovial or equivalent). This step was repeated for the second and third ampoules.

[0060]With a sterile pipette, 1.0 mL of supplemented EX-CELL™ MDCK serum free medium was added to each tube. After these tubes containing the cell-medium mixture sat for approximately 3-5 minutes, a sterile pipette was used to transfer cell-medium mixture from one small sterile tube to a 15 mL centrifuge tube (Falcon or equ...

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Abstract

Animal cells are described which can be infected by viruses and which are adapted to growth in suspension in medium free of animal-derived components, such as serum-free medium. Processes for the replication of viruses in cell culture using these cells are furthermore described, as well as vaccines which contain the viruses or antigenic portions thereof obtainable by the process.

Description

FIELD OF THE INVENTION[0001]The present invention relates to animal cells which can be infected by viruses and are adapted to growth in suspension in medium free of animal-derived components, such as serum-free medium, and to processes for the replication of viruses in cell culture using these cells. The present invention further relates to the viruses obtainable by the process described and to vaccines which contain viruses of this type or constituents thereof.BACKGROUND[0002]Many vaccines including influenza vaccine for the treatment of humans and animals consist of one or more virus strains which have been replicated in embryonated hens' eggs. These viruses are isolated from the allantoic fluid of infected hens' eggs and their antigens are used in vaccines as intact virus particles, as virus particles disintegrated by detergents and / or solvents, as chemically or physically inactivated viruses, or as isolated, defined virus proteins as in subunit vaccines. The viruses are often in...

Claims

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Application Information

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IPC IPC(8): C12N7/02C12N5/02
CPCC12N7/00C12N2760/16351C12N2760/16251C12N2760/16151C12N2760/16152A61P31/16A61P37/04
Inventor WARTHEN, MONTY R.SKOTTA, WILLIAM
Owner WARTHEN MONTY R
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