Abstract
The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.
Publication types
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Comparative Study
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Evaluation Study
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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COS Cells / cytology
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COS Cells / drug effects
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COS Cells / metabolism*
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COS Cells / physiology
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Cell Adhesion / drug effects
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Cell Adhesion / physiology
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Cell Culture Techniques / instrumentation
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Cell Culture Techniques / methods*
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Cell Division / drug effects
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Cell Division / physiology
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Chlorocebus aethiops / genetics
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Coated Materials, Biocompatible / chemical synthesis
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Coated Materials, Biocompatible / pharmacology*
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E-Selectin / biosynthesis
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E-Selectin / genetics
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Gene Expression Regulation / drug effects
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Gene Expression Regulation / physiology
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Granulocyte-Macrophage Colony-Stimulating Factor / biosynthesis
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Granulocyte-Macrophage Colony-Stimulating Factor / genetics
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Green Fluorescent Proteins
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Humans
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Immunoglobulin M / biosynthesis
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Immunoglobulin M / genetics
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Luminescent Proteins / biosynthesis
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Luminescent Proteins / genetics
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Methylamines / pharmacology
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Mice / genetics
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Microspheres
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P-Selectin / biosynthesis
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P-Selectin / genetics
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Particle Size
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Polystyrenes / pharmacology
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Protein Engineering / methods
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Quality Control
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Recombinant Proteins / biosynthesis*
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Recombinant Proteins / genetics
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Transfection / methods*
Substances
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Coated Materials, Biocompatible
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E-Selectin
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Immunoglobulin M
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Luminescent Proteins
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Methylamines
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P-Selectin
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Polystyrenes
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Recombinant Proteins
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Green Fluorescent Proteins
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Granulocyte-Macrophage Colony-Stimulating Factor
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trimethylamine