10. For the first inhibition experiments, a wide concentration range of the substance should be used (e.g., 0.1/1/10/100 μ M). After the initial experiment, additional measuring points can be included for the determination of a valid IC50 value.
Emir Taghikhani et al.
11. The correct cell number for seeding MDCKII cells on ThinCerts® has to be elucidated for each cell line. It is impor- tant that the cells will form a tight monolayer after being cultured and right before the vectorial transport experiments.
If the monolayer is leaky, this will result in radioactivity in the apical compartment due to diffusion and not due to transport processes. Leakiness needs to be regularly checked using non- permeable markers (e.g., inulin) or by measuring tran- sepithelial resistance.
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Daniel F. Gilbert and Oliver Friedrich (eds.), Cell Viability Assays: Methods and Protocols, Methods in Molecular Biology, vol. 1601, DOI 10.1007/978-1-4939-6960-9_12, © Springer Science+Business Media LLC 2017
Chapter 12
Metabolite Profiling of Mammalian Cell Culture Processes to Evaluate Cellular Viability
Isobelle M. Evie, Alan J. Dickson, and Mark Elvin
Abstract
Metabolite profiling allows for the identification of metabolites that become limiting during cell culture and/or for finding bottlenecks in metabolic pathways that limit culture growth and proliferation. Here we describe one protocol with two different sampling methodologies for GC–MS-based metabolite profiling.
We also highlight an example of the types of datasets that are attainable and how such datasets can be evaluated to identify factors related to cell viability. We also demonstrate, via the same methodology, the accurate quantification of a number of metabolites of interest.
Key words Gas chromatography–mass spectrometry (GC–MS), Metabolites, Extracellular profile, Intracellular profile, Metabolomics, Metabolite profiling, Mammalian cells
1 Introduction
In mammalian cell culture maintaining optimum viability is essential in extending culture duration. Therefore assessment of viability throughout culture is an essential process. In order to maintain cellular proliferation and cell viability throughout culture, mam- malian cells require a supply of specific nutrients to meet their met- abolic demands [1], with metabolic activity seen as an indicator of cell health [2]. While successful attempts have been made to extend and maintain cell viability through optimization of commercial cell culture medium [3], metabolic bottlenecks remain. The depletion and exhaustion of metabolites limits cellular viability, particularly throughout long-term culture [4]. Such issues highlight the need for further understanding of the effects of culture conditions on cell viability. While assays can inform of cell death (e.g., vital dye exclusion [5]), metabolite profiling enables for an earlier indication of progression towards a poor viability.
Most commonly, mammalian cell culture is characterized by a highly glycolytic state paralleled by high rates of waste produc- tion [6]. When nutrients become limiting throughout culture,
cells cannot generate energy required for critical cellular processes.
Ultimately, nutrient limitations lead to a restricted ATP supply to the cells [4], limiting cellular growth and viability. Having predic- tive early metabolic indicators would allow timely intervention to prevent loss of viability.
With the development of Omics-based technologies [7], strin- gent methodologies are now available to assess the link between metabolic function and cell viability, with metabolic profiles found to favorably shift under optimized culture conditions [8]. Cells grown in optimized conditions are known to lead to increased ATP production [9], demonstrating the importance of metabolic status for cellular viability.
Nutrient starvation and by-product accumulation can trigger cell death through apoptosis, limiting cell viability [10]. To understand and address such metabolic consequences, metabolite profiling can aid in understanding the pattern of metabolism that occurs in differ- ent cell culture regimes [11]. Metabolite profiling strategies have been used to improve cell viability through understanding the meta- bolic demands of specific cell lines [1], identify apoptosis-inducing metabolites [12], and to identify cell- engineering targets [13].
Analytical mass spectrometry platforms such as gas chroma- tography–mass spectrometry (GC–MS) and liquid chromatogra- phy–mass spectrometry (LC–MS) are widely utilized for metabolite profiling [14, 15]. Such profiles allow for identification of metabo- lites, through comparison of individual peaks of the fragmentation pattern, to metabolomic databases and mass spectral libraries [16, 17].
In this protocol, we describe and demonstrate through exemplar data, a validated adapted method [18], enabling for parallel extra- cellular (footprint) and intracellular (fingerprint) metabolite profil- ing of metabolites from suspension-cultured mammalian cells.
This method involves the recovery of metabolites by direct sampling from spent medium (extracellular) and through quench- ing of cells (intracellular) to stop cellular metabolism before separation of the cells from the medium [18, 19]. Sampling is fol- lowed by methanol and water extractions of the metabolites from the quenched cells [18, 20]. The metabolite samples generated through this method are amenable to analysis by mass spectrome- try. Initially samples are derivatized after extraction, allowing for compounds of poor volatility, polarity, and stability to gain proper- ties more amendable to mass spectrometry analysis, enabling the detection of a wider range of metabolites [21].
Here we also demonstrate and describe through the use of GC–
MS, an example of the types of datasets that are attainable. Datasets received through the application of mass-spectrometry provide a broad semiquantitative profile of a range of metabolites. To allow for determination of the concentration of metabolites of interest, we also demonstrate the use of standards in tandem with the metabolic sam- ples analyzed, allowing for accurate quantification of metabolites.
139 The generated metabolic profiles enable the identification of metabolites that relate to the transition in changes to cell viability throughout different phases of cell culture. Such data can be used to identify factors related to the status of cell viability.
2 Materials
Prepare all solutions using ultrapure water (prepared by purifying deionized water, to attain a sensitivity of 18 M Ω -cm at room tem- perature) and use analytical grade reagents where possible. Prepare and store all reagents at room temperature (unless indicated otherwise).