Uniform stable-isotope labeling in mammalian cells: formulation of a cost-effective culture medium

• Published in: Applied microbiology and biotechnology 2011, Vol.89 (2), p.397-406
• Main Authors: Egorova-Zachernyuk, Tatiana A, Bosman, Giel J. C. G. M, DeGrip, Willem J
• Format: Article
• Language: English
• Subjects: Adaptation; Algae; Amino acids; Animals; Autolysates; Biodiversity; Biological and medical sciences; Biomass; Biomedical and Life Sciences; Biotechnology; Carbon Isotopes - analysis; Carbon Isotopes - metabolism; Cell culture; Cell Culture Techniques - economics; Cell Culture Techniques - instrumentation; Cell growth; Cell Proliferation; Cells - chemistry; Cells - cytology; Cells - metabolism; CHO Cells; Cricetinae; Cricetulus; Culture Media, Serum-Free - chemistry; Culture Media, Serum-Free - economics; Culture Media, Serum-Free - metabolism; Culture medium formulation; Dietary minerals; extracts; Fundamental and applied biological sciences. Psychology; HEK293 Cells; Humans; Isotope Labeling; Isotopes; Isotopic enrichment; Life Sciences; Lipids; Mammalian cells; Mammals; metabolomics; Methods and Protocols; Microbial Genetics and Genomics; Microbiology; Nitrogen Isotopes - analysis; Nitrogen Isotopes - metabolism; Nitrogen sources; NMR; Nuclear magnetic resonance; Organic acids; Ovaries; Penicillin; Proprietary; Proteins; Stable-isotope labeling; Studies; Yeast; Yeasts
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-010-2896-5

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.