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Robust Analytical Methods for the Accurate Quantification of the Total Biomass Composition of Mammalian Cells

  • Diana Széliová
  • Harald Schoeny
  • Špela Knez
  • Christina Troyer
  • Cristina Coman
  • Evelyn Rampler
  • Gunda Koellensperger
  • Robert Ahrends
  • Stephen Hann
  • Nicole Borth
  • Jürgen Zanghellini
  • David E. RuckerbauerEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2088)

Abstract

Biomass composition is an important input for genome-scale metabolic models and has a big impact on their predictive capabilities. However, researchers often rely on generic data for biomass composition, e.g. collected from similar organisms. This leads to inaccurate predictions, because biomass composition varies between different cell lines, conditions, and growth phases. In this chapter we present protocols for the determination of the biomass composition of Chinese Hamster Ovary (CHO) cells. These methods can easily be adapted to other types of mammalian cells. The protocols include the quantification of cell dry mass and of the main biomass components, namely protein, lipid, DNA, RNA, and carbohydrates. Cell dry mass is determined gravimetrically by weighing a defined number of cells. Amino acid composition and protein content are measured by gas chromatography mass spectrometry. Lipids are quantified by shotgun mass spectrometry, which provides quantities for the different lipid classes and also the distribution of fatty acids. RNA is purified and then quantified spectrophotometrically. The methods for DNA and carbohydrates are simple fluorometric and colorimetric assays adapted to a 96-well plate format. To ensure quantitative results, internal standards or spike-in controls are used in all methods, e.g. to account for possible matrix effects or loss of material. Finally, the last section provides a guide on how to convert the measured data into biomass equations, which can then be integrated into a metabolic model.

Key words

Biomass composition DNA RNA Amino acids Lipids Carbohydrates Chinese Hamster Ovary cells 

Notes

Acknowledgements

DS, SH, NB, JZ, and DER acknowledge support by the Federal Ministry for Digital and Economic Affairs (bmwd), the Federal Ministry for Transport, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, Government of Lower Austria and ZIT—Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG. DS received additional funding from the PhD program BioToP (Biomolecular Technology of Proteins) of the Austrian Science Fund (FWF Project W1224). The funding agencies had no influence on the conduct of this research.

Supplementary material

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465987_1_En_7_MOESM3_ESM.csv (12 kb)
Caption of the data object (CSV 12 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Diana Széliová
    • 1
    • 2
  • Harald Schoeny
    • 3
  • Špela Knez
    • 4
  • Christina Troyer
    • 5
  • Cristina Coman
    • 6
  • Evelyn Rampler
    • 3
  • Gunda Koellensperger
    • 3
  • Robert Ahrends
    • 6
  • Stephen Hann
    • 1
    • 2
  • Nicole Borth
    • 1
    • 2
  • Jürgen Zanghellini
    • 5
    • 7
    • 8
  • David E. Ruckerbauer
    • 1
    • 2
    Email author
  1. 1.Austrian Centre of Industrial BiotechnologyViennaAustria
  2. 2.University of Natural Resources and Life SciencesViennaAustria
  3. 3.University of ViennaViennaAustria
  4. 4.University of LjubljanaLjubljanaSlovenia
  5. 5.University of Natural Resources and Life SciencesViennaAustria
  6. 6.Leibniz Institut für Analytische Wissenschaften - e.V.DortmundGermany
  7. 7.Austrian Biotech University of Applied SciencesTullnAustria
  8. 8.Austrian Centre of Industrial BiotechnologyViennaAustria

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