Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium |
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Authors: | Tatiana A Egorova-Zachernyuk Giel J C G M Bosman Willem J DeGrip |
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Institution: | (1) Protein Labelling Innovation (PLI), BioScience Park, Archimedesweg 27, 2333 CM Leiden, The Netherlands;(2) Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences and Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands |
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Abstract: | Uniform stable-isotope labeling of mammalian cells is achieved via a novel formulation of a serum-free cell culture medium
that is based on stable-isotope-labeled autolysates and lipid extracts of various microbiological origin. Yeast autolysates
allow complete replacement of individual amino acids and organic acids in a chemically defined medium (DMEM/F12), enabling
a cost-effective formulation of a stable-isotope-labeled culture medium for mammalian cells. In addition, biomass-derived
hydrolysates, autolysates, and lipid extracts of various classes of algae were explored as cell culture components, both separately
and in combination with yeast autolysates. Optimal autolysate concentrations were established. Such novel medium formulations
were tested on mammalian cell lines, often used for recombinant protein production, i.e., Chinese hamster ovary (CHO) and
human embryonic kidney (HEK 293). Special attention was paid to the adaptation of these mammalian cell lines to serum-free
media. Formulation of the novel proprietary cell culture medium PLIm, based on yeastolates instead of individual amino acids
and organic acids, allows a four- to eightfold cost reduction for 15N and 13C,15N stable-isotope-labeling, respectively, in CHO cells and a three- to sixfold cost reduction in HEK 293 cells. A high level
of stable-isotope enrichment of mammalian cells (>90%) was achieved within four passages by complete replacement of carbon
and nitrogen sources in the medium with their stable-isotope-labeled analogs. These conditions can be used to more cost-effectively
produce labeled recombinant proteins in mammalian cells. |
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