Chapter 7: Isolation of Mutants of Cultured Mammalian Cells

Thompson, Larry H.; Baker, Raymond M.

doi:10.1016/s0091-679x(08)60052-7

Cited by 181 publications

(89 citation statements)

References 130 publications

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“…The density of cells inoculated at each concentration of MTX was critical in obtaining reproducible survival curves. Sparing effects at highcell densities under selective conditions have been described previously and are attributed to cross-feeding as well as normal contact inhibition (44 6 and 7, these maximum cell densities were reduced 10-fold. MTX-supplemented medium was changed every 2 to 3 days for a period of 10 days to remove cell debris which could reverse MTX toxicity by providing thymidine and preformed purines.…”

Section: Methodssupporting

confidence: 68%

Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea.

Brown¹,

Tlsty²,

Schimke³

1983

Mol. Cell. Biol.

170

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Section: Methodssupporting

confidence: 68%

Enhancement of methotrexate resistance and dihydrofolate reductase gene amplification by treatment of mouse 3T6 cells with hydroxyurea.

Brown¹,

Tlsty²,

Schimke³

1983

Mol. Cell. Biol.

170

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“…Such changes could be identified by simply looking at Giemsa stained metaphase spreads of chromosomes of as few of 20-30 cells [5][6][7][8]. These studies included the finding that mutations occur in CHO cells at a rate of 100 in 10 6 cells over a 30-day period of culture [9]. Very recent studies on cells of non-diploid origins, including genome sequence analysis both with immortalized cells and with patient derived cancer cells emphasized their enormous fluidity and mutational impacts [10][11][12][13].…”

Section: Chromosomal and Genomic Heterogeneity Of Cho Cells Other Cementioning

confidence: 99%

Cloning of CHO Cells, Productivity and Genetic Stability—A Discussion

Wurm

Wurm²

2017

Processes

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While many perceive mammalian cell culture-based manufacturing for biopharmaceuticals an established technology, numerous open questions remain to be solved. Genetic diversity and mutation rates in CHO cells have been underestimated since progeny of a clonal CHO cell become genetically diverse with each cell division. This is an important issue since products are made in bioreactors containing up to 10 14 cells that have divided for weeks. Regulatory interest in "Proof of Clonality" is a misguided and misunderstood concern in this context. We revisit decades of research on scope and rate of genetic changes in CHO cells and suggest approaches to minimize trends for genomic instability when establishing reliable manufacturing processes. A concept is proposed for transfection-derived cell populations containing relatively stable (The term "stable" will always be used in a context of relative stability-considering time frames of weeks to months at best.) "CHO master sequence" genomes (containing the desired DNAs of interest). Stable cell populations are to be selected for and maintained for the various phases of manufacturing under specific culture conditions reducing trends for the selection of diverse subpopulations. Such conditions are based on insights gained from population genetics, evolutionary landscape fitness principles, and a 40-year old model for evolution of error prone replicating systems-the Quasi-Species concept.

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“…The isolation and characterization of these cells has been published (7,21,22,25). Mutant cell lines were retested for resistance to diphtheria toxin by incubating cells with diphtheria toxin (100 ng/ml) for 24 h. The majority of mutant cells survived toxin treatment, while we estimate that >95 % of WTB cells were killed.…”

Section: Cellsmentioning

confidence: 99%