Chapter 14 Quenching Methods for the Analysis of Intracellular Metabolites Judith Wahrheit and Elmar Heinzle Abstract Sampling and quenching methods for intracellular metabolite analysis in mammalian cells in adherent and suspension culture are described. Quenching of adherent cells is achieved by application of hot air after removal of the supernatant by suction. For suspension cultures, the addition of excess ice-cold saline results in a rapid inactivation of metabolism and significant dilution of extracellular metabolites. Medium carryover is prevented by rinsing the cells with washing solution. Separation of supernatant from suspension cells via centrifugation is incomplete due to required short centrifugation time. Thus, it is necessary to determine the reproducible cell recovery after quenching. Key words Metabolome analysis, Quenching, Extraction, Intracellular metabolites, Dynamic metabolic flux analysis, Compartmentation, CHO cells, Suspension cells
1
Introduction Metabolic studies are of fundamental importance in physiological, pathophysiological, and toxicological studies, as well as in metabolic engineering approaches. Due to significant progress in instrumental analytics, in particular using chromatography and mass spectrometry techniques, metabolite analyses have become increasingly popular [1, 2]. Since metabolite profiles represent a kind of final outcome of cellular processes, they provide an integrated snapshot of the cellular physiology including all upstream network events. Therefore, metabolomics has become an indispensable tool in systems biology [3, 4]. Exometabolome data, in particular in combination with steady-state metabolic flux analysis (MFA) methods, can provide a quantitative description of the cell physiology [5]. Knowledge of intracellular metabolites is, however, an essential requirement to obtain a comprehensive understanding of metabolism, e.g., by applying dynamic MFA techniques [6]. The analysis of intracellular
Ralf Pörtner (ed.), Animal Cell Biotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 1104, DOI 10.1007/978-1-62703-733-4_14, © Springer Science+Business Media, LLC 2014
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metabolites will also expand our knowledge about metabolic compartmentation [7]. While extracellular metabolites are usually highly abundant and easily accessible in the supernatant, the analysis of intracellular metabolites is far more challenging. It comprises three main steps: (a) sampling, (b) metabolite extraction, and (c) metabolite analysis. This book chapter will focus on the sampling protocol which is the most critical part of the whole procedure and essential to derive representative data [8]. Two major issues have to be addressed during sampling. First, the high turnover rates require fast and efficient quenching. Quenching means the stop of metabolic reactions maintaining actual in vivo concentrations to obtain a representative snapshot of a metabolic state at a given time-point. Hot quenching techniques efficiently stop the metabolism by denaturation of enzymes; cold quenching methods aim at slowing down the metabolic reactions. The lower the temperature, the more complete is the freezing of metabolism. Solvents, e.g., cold methanol, are commonly used as quenching solution in protocols for microorganisms [9, 10]. However, due to lack of a cell wall, mammalian cells are more fragile and prone to cellular damage. The use of solvents for mammalian cells has been shown to lead to tremendous membrane damage and metabolite leakage [11, 12]. Due to low intracellular and high extracellular amounts of certain metabolites, e.g., media components, the separation of cells from the supernatant represents another critical issue. Thus, the second requirement is the preservation of cellular integrity until the separation is completed. For adherently growing cells, extracellular media can be removed by suction [13]. Separation of suspension cells can be achieved by fast filtration [14] or centrifugation [11, 12]. Cell separation via centrifugation has also been successfully applied in microscale lab-on-a-chip approaches [15]. The separation of extra- and intracellular metabolites usually includes washing steps to prevent sample contamination with highly abundant media components. However, metabolite losses due to unspecific leakage or secretion can occur during the process. The aim of this book chapter is to characterize simple quenching methods for mammalian cells that do not require extraordinary laboratory equipment. In the following, we will describe separate protocols for quenching mammalian cells in adherent and suspension cultures. However, the focus will be on suspension cells which are more relevant for industrial applications. The necessity for washing steps should be carefully considered depending on the metabolites of interest. When the metabolites of interest occur only intracellularly, washing steps can potentially be omitted. Since mammalian cells usually are cultivated in rather rich media containing a wide range of amino and organic acids, the presented protocols include washing steps. Furthermore, we will suggest extraction methods that cover a wide range of metabolites in the central
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metabolism. However, since an appropriate extraction method largely depends on the metabolites of interest, there is no universal solution available. It might have to be adjusted when a particular class of metabolites, e.g., more hydrophobic compounds, is of interest. A huge selection of extraction methods can be found in the literature [10, 11, 16, 17]. The third step, i.e., the metabolite analysis, is commonly performed using gas chromatography or liquid chromatography coupled with mass spectrometry [1, 2]. The analytical part will not be further discussed here. The protocols for adherent and suspension cells are schematically depicted in Fig. 1.
2
Materials
2.1 Quenching of Adherent Cells
1. Washing solution: 37 °C PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and a pH of 7.4). 2. Hot-air blower with a nozzle able to provide hot air at a temperature of 150 °C. 3. Extraction solution: 80 % methanol. 4. Dry-ice/acetone bath. Prepare a dry-ice/acetone bath in an appropriate glass or plastic vessel to achieve a temperature of −80 °C. A thermal insulation can be included by placing the vessel in a larger vessel and putting insulating material, e.g., mineral wool, in between (see Note 1). 5. Plate shaker. 6. 1.5 or 2 ml sample tubes. 7. Centrifuge suitable for 1.5 or 2 ml sample tubes. 8. Centrifugal vacuum concentrator.
2.2 Quenching of Suspension Cells
1. Ice-water bath. Prepare an ice-water bath in a polystyrene box to achieve a temperature of 0 °C (see Note 2 and 3). 2. Ice. Prepare a styropor box with ice. 3. Quenching and washing solution (QS): 0.9 % (w/v) sodium chloride solution in MilliQ water. A volume of 95 ml QS is needed in total for each sample. 4. 50 ml tubes and a centrifuge suitable for 50 ml tubes. Prepare one tube with 45 ml of QS (for quenching) and one tube with 50 ml of QS (for one washing step) for each sample. 5. Liquid waste vessel to collect the supernatant quenching solution after centrifugation. 6. Vacuum pump connected to a tube and a clean Pasteur pipette to aspirate the residual liquid after decanting the quenching and washing supernatants.
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Fig. 1 Schematic representation of the protocols for quenching adherent cell cultures (left side, A1–A6) and suspension cell cultures (right side, S1–S13a for extraction of intracellular metabolites or S1–S7b for determination of cell recovery) (adapted from [8])
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7. Dry-ice/acetone bath. See Subheading 2.1, item 4 and Note 1. 8. Extraction solutions: 100 % methanol (ESM), 100 % distilled water (ESW) [17]. Volumes of 1 ml ESM and 250 μl of ESW are needed for each sample. 9. Vortex. 10. Centrifugal vacuum concentrator. 11. PBS.
3
Methods
3.1 Quenching of Adherent Cells
The presented protocol has been validated for hepatic cells grown in six-well plates at a cell density of 1 × 106 cells per well. However, the method can be transferred to other adherently growing cells. Volumes can be adjusted when different well formats are used. However, the cell density and the cell number used for extraction might be critical depending on the sensitivity of the analytical system used downstream (see Note 4). 1. Prewarm the washing solution (PBS) to 37 °C and precool the extraction solution (80 % methanol) to −80 °C. 2. Tilt the six-well plate by an angle of 45° and remove supernatant with a pipette by putting the tip at the lowest point. 3. Wash cell layer: Tilt the six-well plate by an angle of 45° and rinse the cell layer by pipetting 1.5 ml of 37 °C PBS on top of the cell layer. Immediately collect wash supernatant with a pipette by putting the tip at the lowest point and discard. Repeat washing step once (see Note 5). 4. Quench with hot air for 5 s using the hot-air blower. 5. Add extraction solution and put the culture plate on a plate shaker for 10 min. 6. Collect extract in a 1.5 or 2 ml sample tube. 7. Centrifuge for 10 min at 4 °C and 10,000 × g. 8. Transfer supernatant in a fresh 1.5 or 2 ml sample tube. 9. Dry the extracts using a centrifugal vacuum concentrator at room temperature. 10. Proceed with an appropriate sample preparation for analysis via LC-MS or GC-MS.
3.2 Quenching of Suspension Cells
The presented protocol has been validated for CHO cells grown in baffled shake flasks at a cell density of 2 × 106 cells per ml. However, the method is applicable to all suspension cultures of mammalian cells. The cell number used for extraction might be critical depending on the sensitivity of the analytical system used downstream (see Notes 4 and 11).
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1. Precool QS (one tube with 45 ml and one tube with 50 ml for each sample) and ESW in ice-water bath to 0 °C and precool centrifuge to 0 °C (see Note 3). 2. Set centrifuge to 1 min and 2,000 × g (see Note 6). 3. Prewarm PBS to 37 °C in a water bath. 4. Precool ESM in dry-ice/acetone bath to −80 °C. 5. Take one tube with 45 ml precooled QS per sample and add 5 ml cell suspension (see Note 7). 6. Mix by inverting the tube. 7. Centrifuge for 1 min at 0 °C and 2,000 × g (see Notes 6 and 11). 8. Carefully decant supernatant in liquid waste vessel. After removing the supernatant, keep falcon upside down (see Note 8). 9. While keeping the tube upside down, aspirate residues of liquid using a Pasteur pipette connected to a vacuum pump without touching the cell pellet (see Note 9). 10. Carefully pour 50 ml of precooled QS on top of the cell pellet. Do not mix, resuspend, or shake (see Note 10). 11. Repeat steps 7–9. Proceed with step 12 for determination of cell recovery or with step 13 for analysis of intracellular metabolites. 12. Resuspend cell pellet in warm (37 °C) PBS and determine the total viable cell number after quenching using the trypan blue exclusion method (see Note 11). 13. Resuspend cell pellet in 500 μl ESM (100 % methanol at −80 °C) and transfer the entire content (solution and cell debris) into a fresh 2 ml tube (see Note 12). 14. Freeze immediately by placing the tube in liquid nitrogen. 15. Thaw on ice. 16. Vortex 30 s. 17. Centrifuge 1 min at 800 × g. 18. Transfer supernatant into a fresh tube and keep on ice. 19. Repeat steps 12–16 and pool the supernatants. Keep on ice. 20. Resuspend cell pellet in 250 μl ESW (100 % distilled water at 0 °C). 21. Repeat steps 13–15. 22. Centrifuge 1 min at 16,000 × g and pool the supernatants. 23. Centrifuge the pooled supernatants 1 min at 16,000 × g and transfer into a fresh tube. 24. Dry the extracts using a centrifugal vacuum concentrator at room temperature. 25. Proceed with an appropriate sample preparation for analysis via LC-MS or GC-MS.
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Notes 1. Special care must be taken when preparing the dry-ice/acetone bath due to squirting of acetone. Start by addition of initially small amounts of dry ice. 2. The overall time of the sampling and quenching procedure is critical. In particular for the quenching of suspension cells where several centrifugation steps are involved, the time from taking the sample to freezing the cells in liquid nitrogen has to be kept to a minimum. Distances between sterile bench, centrifuge, ice boxes, vacuum pump, liquid nitrogen, etc. should be as short as possible. When more than one sample at a time need to be handled, helping hands from (properly instructed) colleagues are indispensable. The success of the quenching procedure can be verified by determining the energy charge (EC) (Fig. 2) [8]. The EC value should be between 0.75 and 0.95. 3. All materials, quenching solution, centrifuge, etc. have to be properly precooled in advance (minimum 1 h before taking the first sample) to ensure an efficient quenching. The temperature of the mixture (cell suspension in quenching solution) should be below 4 °C all the time before freezing the sample in liquid nitrogen. Check the temperature with a thermometer. The success of the quenching procedure can be verified by determining the energy charge (Fig. 2) [8]. 4. Depending on the sensitivity of the analytical system used downstream, the cell number used for extraction is critical.
Fig. 2 Quenching efficiency was assessed by determining the energy charge EC = ([ATP] + ½[ADP])/([ATP] + [ADP] + [AMP]) (n = 3). ATP, ADP, and AMP in quenched cells were analyzed in a luminometer using luciferase-based enzyme assays (Promega, Mannheim, Germany) (adapted from [8])
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Thus, the minimal cell number required to obtain a significant analytical signal has to be verified in advance. 5. The necessity of washing steps should be carefully considered. Washing steps might result in metabolite losses due to unspecific metabolite leakage or secretion. In particular, when the metabolites of interest occur only intracellularly, such as the phosphorylated glycolytic intermediates, washing steps can potentially be omitted. In the case of adherent cells, particular care has to be taken, when the cells are cultivated on a special coating substrate, e.g., collagen coating, or even in 3D-like structure like collagen sandwich cultivation. The coating substrate might be able to store significant amounts of extracellular medium compounds which might falsify the results. We propose including a “background control,” i.e., a coated well without cells that is treated exactly as the sample wells to correct for potential extracellular contamination. 6. Centrifugal forces higher than 2,000 × g should be avoided to prevent unnecessary stress to the cells. 7. We propose using a large (ninefold) excess of quenching solution to achieve a rapid temperature shift and at the same time a significant dilution of extracellular metabolites. 8. With the applied centrifugation conditions, the cell pellet is not very tightly sticking to the bottom. Turn the tube upside down to prevent residual liquid from flowing back to the cell pellet. All movements have to be performed carefully. Avoid vigorous shaking that could potentially result in detaching the cells! 9. Culture medium is stickier than water. In particular after the first centrifugation step, decanting the supernatant is not sufficient to remove all liquid. Make sure to collect all drops that are sticking to the inside of the tube wall using the vacuum pump to prevent contamination of the sample with extracellular compounds. Never touch the cell pellet with the tip of the Pasteur pipette! 10. A variety of intracellular metabolites of interest is also abundant in the extracellular medium requiring washing. Washing further affects cell recovery. Rinsing the cell pellet is sufficient to prevent carryover of extracellular medium components (Fig. 3a, “rinse”). Washing by resuspension (Fig. 3a, “wash”) of the cell pellet does not remove more metabolites than rinsing the cell pellet without resuspension, however, leads to a significantly decreased cell yield (Fig. 3b) [8]. Thus, washing by resuspending the cell pellet has to be avoided. Some metabolites might be easily removed after quenching with an excess of quenching solution (Fig. 3a, citrate, “no wash”) or after one additional washing step (Fig. 3a, glucose, “1× rinse”). Other metabolites, in particular monocarboxylates (lactate, pyruvate),
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Fig. 3 (a) Carryover of media components without washing and after rinsing the cell pellet once (1× rinse) or twice (2× rinse) with 50 ml quenching solution or resuspending the cell pellet in 50 ml quenching solution (1× wash). Fractions of initial amounts of glucose, lactate, pyruvate, and citrate found after treatment. (b) Cell recovery after washing (adapted from [8])
Fig. 4 (a) Cell recovery after harvesting at 1,000 or 2,000 × g as a fraction of the initial cell number (n = 2). (b) Centrifugal acceleration and cell recovery (n = 2) (adapted from [8])
might “bleed” from the cells by concentration-driven transport of proton-linked monocarboxylate transporters (MCT) [14]. It has to be kept in mind that metabolite losses can occur during washing. When adding the QS on the pellet, pour the solution on the tube wall and not directly on the cell pellet (cf. Note 8). 11. The applied conditions with a centrifugation time of 1 min and a total volume of 50 ml are critical for the cell recovery after quenching. Only a part of the cells will be sedimented during such short centrifugation times (Figs. 3b and 4). The rest will be discarded with the supernatant. The short centrifugation times are necessary to keep the overall time of the sampling and quenching procedure short. Increasing the centrifugation speed to more than 2,000 × g does not improve the cell yield (Fig. 4b). Thus, centrifugal forces higher than 2,000 × g should be avoided to prevent unnecessary stress to the cells (cf. Note 6).
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Fig. 5 Mean viable cell diameters before and after quenching (n = 7) (adapted from [8])
If absolute metabolite amounts shall be calculated in the end, the cell recovery has to be determined for each sample. Due to high reproducibility of the centrifugation process (Fig. 4a), the viable cell number in the cell pellet used for extraction can be deduced from a biological replicate treated exactly as the sample and resuspended in PBS for cell counting instead of freezing in liquid nitrogen (Fig. 1, S7B–S8B). Since separation of cells via centrifugation is incomplete at the applied settings, the centrifugation steps might result in a biased selection of cells which has to be checked for each cell type and cell line. It has to be verified that the cell population recovered after quenching reflects the cell population in the initial cell suspension, e.g., by comparing the cell diameters before and after centrifugation (Fig. 5) [8] or by flow cytometry. 12. Methanol is dripping out of the pipette tip. Keep the tube with the extraction solution very close to the tube with the pellet to make sure that the correct volume is used for extraction. The volume should best be gravimetrically verified. When transferring the pellet into the fresh tube, collect the entire content. Do not leave little crumbs or liquid drops behind! If necessary cut the tip of a 1 ml micropipette tip to collect the cell crumbs that are too big for the opening of the 1 ml tip. References 1. Wittmann C (2002) Metabolic flux analysis using mass spectrometry. Adv Biochem Eng Biotechnol 74:39–64 2. Wittmann C (2007) Fluxome analysis using GC-MS. Microb Cell Fact 6:6 3. Kell DB (2004) Metabolomics and systems biology: making sense of the soup. Curr Opin Microbiol 7:296–307
4. Zamboni N, Sauer U (2009) Novel biological insights through metabolomics and 13C-flux analysis. Curr Opin Microbiol 12:553–558 5. Niklas J, Heinzle E (2012) Metabolic Flux Analysis in Systems Biology of Mammalian Cells. Adv Biochem Eng Biotechnol 127:109–132 6. Nöh K, Wahl A, Wiechert W (2006) Computational tools for isotopically instationary 13C
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9.
10. 11.
12.
labeling experiments under metabolic steady state conditions. Metab Eng 8: 554–577 Wahrheit J, Nicolae A, Heinzle E (2011) Eukaryotic metabolism: measuring compartment fluxes. Biotechnol J 6:1071–1085 Wahrheit, J., Heinzle, E., Sampling and quenching of CHO suspension cells for the analysis of intracellular metabolites. BMC Proc 2013, 7(Suppl 6):P42, doi:10.1186/1753 -6561-7-S6-P42 Hans MA, Heinzle E, Wittmann C (2001) Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 56:776–779 Villas-Boas SG, Nielsen J, Smedsgaard J et al (2007) Metabolome analysis: an introduction. Wiley-VCH, Hoboken, NJ Dietmair S, Timmins NE, Gray PP et al (2010) Towards quantitative metabolomics of mammalian cells: development of a metabolite extraction protocol. Anal Biochem 404: 155–164 Kronthaler J, Gstraunthaler G, Heel C (2012) Optimizing high-throughput metabolomic biomarker screening: a study of quenching solutions to freeze intracellular metabolism in CHO cells. Omics 16:90–97
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13. Hofmann U, Maier K, Niebel A et al (2008) Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations. Biotechnol Bioeng 100:344–354 14. Volmer M, Northoff S, Scholz S et al (2011) Fast filtration for metabolome sampling of suspended animal cells. Biotechnol Lett 33: 495–502 15. Rajabi N, Bahnemann J, Wahrheit J et al. Inertia-based media exchange and quenching of cells for the continuous preparation of cells in a lab-on-a-chip. In: Proceedings of the 3rd European Conference on Microfluidics, Heidelberg, Germany, 4–5 December 2012 16. Ritter JB, Genzel Y, Reichl U (2008) Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: optimization using experimental design. Anal Biochem 373:349–369 17. Sellick C, Knight D, Croxford A et al (2010) Evaluation of extraction processes for intracellular metabolite profiling of mammalian cells: matching extraction approaches to cell type and metabolite targets. Metabolomics 6: 427–438
1
Introduction Metabolic studies are of fundamental importance in physiological, pathophysiological, and toxicological studies, as well as in metabolic engineering approaches. Due to significant progress in instrumental analytics, in particular using chromatography and mass spectrometry techniques, metabolite analyses have become increasingly popular [1, 2]. Since metabolite profiles represent a kind of final outcome of cellular processes, they provide an integrated snapshot of the cellular physiology including all upstream network events. Therefore, metabolomics has become an indispensable tool in systems biology [3, 4]. Exometabolome data, in particular in combination with steady-state metabolic flux analysis (MFA) methods, can provide a quantitative description of the cell physiology [5]. Knowledge of intracellular metabolites is, however, an essential requirement to obtain a comprehensive understanding of metabolism, e.g., by applying dynamic MFA techniques [6]. The analysis of intracellular
Ralf Pörtner (ed.), Animal Cell Biotechnology: Methods and Protocols, Methods in Molecular Biology, vol. 1104, DOI 10.1007/978-1-62703-733-4_14, © Springer Science+Business Media, LLC 2014
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metabolites will also expand our knowledge about metabolic compartmentation [7]. While extracellular metabolites are usually highly abundant and easily accessible in the supernatant, the analysis of intracellular metabolites is far more challenging. It comprises three main steps: (a) sampling, (b) metabolite extraction, and (c) metabolite analysis. This book chapter will focus on the sampling protocol which is the most critical part of the whole procedure and essential to derive representative data [8]. Two major issues have to be addressed during sampling. First, the high turnover rates require fast and efficient quenching. Quenching means the stop of metabolic reactions maintaining actual in vivo concentrations to obtain a representative snapshot of a metabolic state at a given time-point. Hot quenching techniques efficiently stop the metabolism by denaturation of enzymes; cold quenching methods aim at slowing down the metabolic reactions. The lower the temperature, the more complete is the freezing of metabolism. Solvents, e.g., cold methanol, are commonly used as quenching solution in protocols for microorganisms [9, 10]. However, due to lack of a cell wall, mammalian cells are more fragile and prone to cellular damage. The use of solvents for mammalian cells has been shown to lead to tremendous membrane damage and metabolite leakage [11, 12]. Due to low intracellular and high extracellular amounts of certain metabolites, e.g., media components, the separation of cells from the supernatant represents another critical issue. Thus, the second requirement is the preservation of cellular integrity until the separation is completed. For adherently growing cells, extracellular media can be removed by suction [13]. Separation of suspension cells can be achieved by fast filtration [14] or centrifugation [11, 12]. Cell separation via centrifugation has also been successfully applied in microscale lab-on-a-chip approaches [15]. The separation of extra- and intracellular metabolites usually includes washing steps to prevent sample contamination with highly abundant media components. However, metabolite losses due to unspecific leakage or secretion can occur during the process. The aim of this book chapter is to characterize simple quenching methods for mammalian cells that do not require extraordinary laboratory equipment. In the following, we will describe separate protocols for quenching mammalian cells in adherent and suspension cultures. However, the focus will be on suspension cells which are more relevant for industrial applications. The necessity for washing steps should be carefully considered depending on the metabolites of interest. When the metabolites of interest occur only intracellularly, washing steps can potentially be omitted. Since mammalian cells usually are cultivated in rather rich media containing a wide range of amino and organic acids, the presented protocols include washing steps. Furthermore, we will suggest extraction methods that cover a wide range of metabolites in the central
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metabolism. However, since an appropriate extraction method largely depends on the metabolites of interest, there is no universal solution available. It might have to be adjusted when a particular class of metabolites, e.g., more hydrophobic compounds, is of interest. A huge selection of extraction methods can be found in the literature [10, 11, 16, 17]. The third step, i.e., the metabolite analysis, is commonly performed using gas chromatography or liquid chromatography coupled with mass spectrometry [1, 2]. The analytical part will not be further discussed here. The protocols for adherent and suspension cells are schematically depicted in Fig. 1.
2
Materials
2.1 Quenching of Adherent Cells
1. Washing solution: 37 °C PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, and a pH of 7.4). 2. Hot-air blower with a nozzle able to provide hot air at a temperature of 150 °C. 3. Extraction solution: 80 % methanol. 4. Dry-ice/acetone bath. Prepare a dry-ice/acetone bath in an appropriate glass or plastic vessel to achieve a temperature of −80 °C. A thermal insulation can be included by placing the vessel in a larger vessel and putting insulating material, e.g., mineral wool, in between (see Note 1). 5. Plate shaker. 6. 1.5 or 2 ml sample tubes. 7. Centrifuge suitable for 1.5 or 2 ml sample tubes. 8. Centrifugal vacuum concentrator.
2.2 Quenching of Suspension Cells
1. Ice-water bath. Prepare an ice-water bath in a polystyrene box to achieve a temperature of 0 °C (see Note 2 and 3). 2. Ice. Prepare a styropor box with ice. 3. Quenching and washing solution (QS): 0.9 % (w/v) sodium chloride solution in MilliQ water. A volume of 95 ml QS is needed in total for each sample. 4. 50 ml tubes and a centrifuge suitable for 50 ml tubes. Prepare one tube with 45 ml of QS (for quenching) and one tube with 50 ml of QS (for one washing step) for each sample. 5. Liquid waste vessel to collect the supernatant quenching solution after centrifugation. 6. Vacuum pump connected to a tube and a clean Pasteur pipette to aspirate the residual liquid after decanting the quenching and washing supernatants.
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Fig. 1 Schematic representation of the protocols for quenching adherent cell cultures (left side, A1–A6) and suspension cell cultures (right side, S1–S13a for extraction of intracellular metabolites or S1–S7b for determination of cell recovery) (adapted from [8])
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7. Dry-ice/acetone bath. See Subheading 2.1, item 4 and Note 1. 8. Extraction solutions: 100 % methanol (ESM), 100 % distilled water (ESW) [17]. Volumes of 1 ml ESM and 250 μl of ESW are needed for each sample. 9. Vortex. 10. Centrifugal vacuum concentrator. 11. PBS.
3
Methods
3.1 Quenching of Adherent Cells
The presented protocol has been validated for hepatic cells grown in six-well plates at a cell density of 1 × 106 cells per well. However, the method can be transferred to other adherently growing cells. Volumes can be adjusted when different well formats are used. However, the cell density and the cell number used for extraction might be critical depending on the sensitivity of the analytical system used downstream (see Note 4). 1. Prewarm the washing solution (PBS) to 37 °C and precool the extraction solution (80 % methanol) to −80 °C. 2. Tilt the six-well plate by an angle of 45° and remove supernatant with a pipette by putting the tip at the lowest point. 3. Wash cell layer: Tilt the six-well plate by an angle of 45° and rinse the cell layer by pipetting 1.5 ml of 37 °C PBS on top of the cell layer. Immediately collect wash supernatant with a pipette by putting the tip at the lowest point and discard. Repeat washing step once (see Note 5). 4. Quench with hot air for 5 s using the hot-air blower. 5. Add extraction solution and put the culture plate on a plate shaker for 10 min. 6. Collect extract in a 1.5 or 2 ml sample tube. 7. Centrifuge for 10 min at 4 °C and 10,000 × g. 8. Transfer supernatant in a fresh 1.5 or 2 ml sample tube. 9. Dry the extracts using a centrifugal vacuum concentrator at room temperature. 10. Proceed with an appropriate sample preparation for analysis via LC-MS or GC-MS.
3.2 Quenching of Suspension Cells
The presented protocol has been validated for CHO cells grown in baffled shake flasks at a cell density of 2 × 106 cells per ml. However, the method is applicable to all suspension cultures of mammalian cells. The cell number used for extraction might be critical depending on the sensitivity of the analytical system used downstream (see Notes 4 and 11).
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1. Precool QS (one tube with 45 ml and one tube with 50 ml for each sample) and ESW in ice-water bath to 0 °C and precool centrifuge to 0 °C (see Note 3). 2. Set centrifuge to 1 min and 2,000 × g (see Note 6). 3. Prewarm PBS to 37 °C in a water bath. 4. Precool ESM in dry-ice/acetone bath to −80 °C. 5. Take one tube with 45 ml precooled QS per sample and add 5 ml cell suspension (see Note 7). 6. Mix by inverting the tube. 7. Centrifuge for 1 min at 0 °C and 2,000 × g (see Notes 6 and 11). 8. Carefully decant supernatant in liquid waste vessel. After removing the supernatant, keep falcon upside down (see Note 8). 9. While keeping the tube upside down, aspirate residues of liquid using a Pasteur pipette connected to a vacuum pump without touching the cell pellet (see Note 9). 10. Carefully pour 50 ml of precooled QS on top of the cell pellet. Do not mix, resuspend, or shake (see Note 10). 11. Repeat steps 7–9. Proceed with step 12 for determination of cell recovery or with step 13 for analysis of intracellular metabolites. 12. Resuspend cell pellet in warm (37 °C) PBS and determine the total viable cell number after quenching using the trypan blue exclusion method (see Note 11). 13. Resuspend cell pellet in 500 μl ESM (100 % methanol at −80 °C) and transfer the entire content (solution and cell debris) into a fresh 2 ml tube (see Note 12). 14. Freeze immediately by placing the tube in liquid nitrogen. 15. Thaw on ice. 16. Vortex 30 s. 17. Centrifuge 1 min at 800 × g. 18. Transfer supernatant into a fresh tube and keep on ice. 19. Repeat steps 12–16 and pool the supernatants. Keep on ice. 20. Resuspend cell pellet in 250 μl ESW (100 % distilled water at 0 °C). 21. Repeat steps 13–15. 22. Centrifuge 1 min at 16,000 × g and pool the supernatants. 23. Centrifuge the pooled supernatants 1 min at 16,000 × g and transfer into a fresh tube. 24. Dry the extracts using a centrifugal vacuum concentrator at room temperature. 25. Proceed with an appropriate sample preparation for analysis via LC-MS or GC-MS.
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Notes 1. Special care must be taken when preparing the dry-ice/acetone bath due to squirting of acetone. Start by addition of initially small amounts of dry ice. 2. The overall time of the sampling and quenching procedure is critical. In particular for the quenching of suspension cells where several centrifugation steps are involved, the time from taking the sample to freezing the cells in liquid nitrogen has to be kept to a minimum. Distances between sterile bench, centrifuge, ice boxes, vacuum pump, liquid nitrogen, etc. should be as short as possible. When more than one sample at a time need to be handled, helping hands from (properly instructed) colleagues are indispensable. The success of the quenching procedure can be verified by determining the energy charge (EC) (Fig. 2) [8]. The EC value should be between 0.75 and 0.95. 3. All materials, quenching solution, centrifuge, etc. have to be properly precooled in advance (minimum 1 h before taking the first sample) to ensure an efficient quenching. The temperature of the mixture (cell suspension in quenching solution) should be below 4 °C all the time before freezing the sample in liquid nitrogen. Check the temperature with a thermometer. The success of the quenching procedure can be verified by determining the energy charge (Fig. 2) [8]. 4. Depending on the sensitivity of the analytical system used downstream, the cell number used for extraction is critical.
Fig. 2 Quenching efficiency was assessed by determining the energy charge EC = ([ATP] + ½[ADP])/([ATP] + [ADP] + [AMP]) (n = 3). ATP, ADP, and AMP in quenched cells were analyzed in a luminometer using luciferase-based enzyme assays (Promega, Mannheim, Germany) (adapted from [8])
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Thus, the minimal cell number required to obtain a significant analytical signal has to be verified in advance. 5. The necessity of washing steps should be carefully considered. Washing steps might result in metabolite losses due to unspecific metabolite leakage or secretion. In particular, when the metabolites of interest occur only intracellularly, such as the phosphorylated glycolytic intermediates, washing steps can potentially be omitted. In the case of adherent cells, particular care has to be taken, when the cells are cultivated on a special coating substrate, e.g., collagen coating, or even in 3D-like structure like collagen sandwich cultivation. The coating substrate might be able to store significant amounts of extracellular medium compounds which might falsify the results. We propose including a “background control,” i.e., a coated well without cells that is treated exactly as the sample wells to correct for potential extracellular contamination. 6. Centrifugal forces higher than 2,000 × g should be avoided to prevent unnecessary stress to the cells. 7. We propose using a large (ninefold) excess of quenching solution to achieve a rapid temperature shift and at the same time a significant dilution of extracellular metabolites. 8. With the applied centrifugation conditions, the cell pellet is not very tightly sticking to the bottom. Turn the tube upside down to prevent residual liquid from flowing back to the cell pellet. All movements have to be performed carefully. Avoid vigorous shaking that could potentially result in detaching the cells! 9. Culture medium is stickier than water. In particular after the first centrifugation step, decanting the supernatant is not sufficient to remove all liquid. Make sure to collect all drops that are sticking to the inside of the tube wall using the vacuum pump to prevent contamination of the sample with extracellular compounds. Never touch the cell pellet with the tip of the Pasteur pipette! 10. A variety of intracellular metabolites of interest is also abundant in the extracellular medium requiring washing. Washing further affects cell recovery. Rinsing the cell pellet is sufficient to prevent carryover of extracellular medium components (Fig. 3a, “rinse”). Washing by resuspension (Fig. 3a, “wash”) of the cell pellet does not remove more metabolites than rinsing the cell pellet without resuspension, however, leads to a significantly decreased cell yield (Fig. 3b) [8]. Thus, washing by resuspending the cell pellet has to be avoided. Some metabolites might be easily removed after quenching with an excess of quenching solution (Fig. 3a, citrate, “no wash”) or after one additional washing step (Fig. 3a, glucose, “1× rinse”). Other metabolites, in particular monocarboxylates (lactate, pyruvate),
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Fig. 3 (a) Carryover of media components without washing and after rinsing the cell pellet once (1× rinse) or twice (2× rinse) with 50 ml quenching solution or resuspending the cell pellet in 50 ml quenching solution (1× wash). Fractions of initial amounts of glucose, lactate, pyruvate, and citrate found after treatment. (b) Cell recovery after washing (adapted from [8])
Fig. 4 (a) Cell recovery after harvesting at 1,000 or 2,000 × g as a fraction of the initial cell number (n = 2). (b) Centrifugal acceleration and cell recovery (n = 2) (adapted from [8])
might “bleed” from the cells by concentration-driven transport of proton-linked monocarboxylate transporters (MCT) [14]. It has to be kept in mind that metabolite losses can occur during washing. When adding the QS on the pellet, pour the solution on the tube wall and not directly on the cell pellet (cf. Note 8). 11. The applied conditions with a centrifugation time of 1 min and a total volume of 50 ml are critical for the cell recovery after quenching. Only a part of the cells will be sedimented during such short centrifugation times (Figs. 3b and 4). The rest will be discarded with the supernatant. The short centrifugation times are necessary to keep the overall time of the sampling and quenching procedure short. Increasing the centrifugation speed to more than 2,000 × g does not improve the cell yield (Fig. 4b). Thus, centrifugal forces higher than 2,000 × g should be avoided to prevent unnecessary stress to the cells (cf. Note 6).
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Fig. 5 Mean viable cell diameters before and after quenching (n = 7) (adapted from [8])
If absolute metabolite amounts shall be calculated in the end, the cell recovery has to be determined for each sample. Due to high reproducibility of the centrifugation process (Fig. 4a), the viable cell number in the cell pellet used for extraction can be deduced from a biological replicate treated exactly as the sample and resuspended in PBS for cell counting instead of freezing in liquid nitrogen (Fig. 1, S7B–S8B). Since separation of cells via centrifugation is incomplete at the applied settings, the centrifugation steps might result in a biased selection of cells which has to be checked for each cell type and cell line. It has to be verified that the cell population recovered after quenching reflects the cell population in the initial cell suspension, e.g., by comparing the cell diameters before and after centrifugation (Fig. 5) [8] or by flow cytometry. 12. Methanol is dripping out of the pipette tip. Keep the tube with the extraction solution very close to the tube with the pellet to make sure that the correct volume is used for extraction. The volume should best be gravimetrically verified. When transferring the pellet into the fresh tube, collect the entire content. Do not leave little crumbs or liquid drops behind! If necessary cut the tip of a 1 ml micropipette tip to collect the cell crumbs that are too big for the opening of the 1 ml tip. References 1. Wittmann C (2002) Metabolic flux analysis using mass spectrometry. Adv Biochem Eng Biotechnol 74:39–64 2. Wittmann C (2007) Fluxome analysis using GC-MS. Microb Cell Fact 6:6 3. Kell DB (2004) Metabolomics and systems biology: making sense of the soup. Curr Opin Microbiol 7:296–307
4. Zamboni N, Sauer U (2009) Novel biological insights through metabolomics and 13C-flux analysis. Curr Opin Microbiol 12:553–558 5. Niklas J, Heinzle E (2012) Metabolic Flux Analysis in Systems Biology of Mammalian Cells. Adv Biochem Eng Biotechnol 127:109–132 6. Nöh K, Wahl A, Wiechert W (2006) Computational tools for isotopically instationary 13C
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13. Hofmann U, Maier K, Niebel A et al (2008) Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part I. Experimental observations. Biotechnol Bioeng 100:344–354 14. Volmer M, Northoff S, Scholz S et al (2011) Fast filtration for metabolome sampling of suspended animal cells. Biotechnol Lett 33: 495–502 15. Rajabi N, Bahnemann J, Wahrheit J et al. Inertia-based media exchange and quenching of cells for the continuous preparation of cells in a lab-on-a-chip. In: Proceedings of the 3rd European Conference on Microfluidics, Heidelberg, Germany, 4–5 December 2012 16. Ritter JB, Genzel Y, Reichl U (2008) Simultaneous extraction of several metabolites of energy metabolism and related substances in mammalian cells: optimization using experimental design. Anal Biochem 373:349–369 17. Sellick C, Knight D, Croxford A et al (2010) Evaluation of extraction processes for intracellular metabolite profiling of mammalian cells: matching extraction approaches to cell type and metabolite targets. Metabolomics 6: 427–438
