Table 2.

Summary of recent optimisation efforts of metabolite extraction procedures for cell culture metabolomics

References	Cell line/s	Samples	Cell washing	Quenching	Extraction	Analytical platform	Comments
2017
Kapoore et al. [65]	MDA-MB-231	Cells	Comparison of no wash, to one wash or two washes with ice-cold PBS or distilled water	Comparison of no quenching, to 100% methanol, 60% methanol both unbuffered and buffered with 0.85% AMBIC or 70 mM HEPES, and direct quenching with LN2	Culture media discarded → cells washed for 60 s → cells quenched (−50 °C) and scraped into quenching solution → addition of 100% methanol and freeze–thaw cycles for metabolite extraction → samples lyophilized prior to derivatisation for analysis	GC–MS Selected 11 metabolite classes	Compared five washing protocols and five quenching protocols for minimum metabolite leakage from a single breast cancer adherent mammalian cell line Optimised protocol was a single PBS wash and quenching with 60% methanol supplemented with 70 mM HEPES
2016
Muschet et al. [71]	THLE-2, HK-2, HepG2, SGBS	Cells	Washed twice with warmed PBS	88% methanol precooled on dry ice	Trypsinisation and cell scraping were compared. Extraction solution was the quenching methanol plus additional 88% methanol → homogenisation with 80 mg glass beads → centrifugation → stored at −80 °C	FIA-MS/MS, LC–MS/MS Targeted assay of 188 preselected metabolites	Cell harvesting methods of trypsinisation or cell scraping are tested with the same extraction procedure across four different human cell lines The effect of harvesting technique on metabolite extraction was dependant on the cell lines used, and the specific metabolites. Cell scraping better correlated to metabolite concentrations and cell numbers, than trypsinisation
Peterson et al. [73]	MDA-MB-231	Cells	Washed three times with ice-cold 0.9% saline	Ice-cold saline (quenching considered to occur during washing steps)	Chloroform:methanol:water (1:3:1) extraction solution, either added directly to cells, or following collection by scraping into saline → extraction by vortex mixer for 30 min → centrifugation → evaporated to dryness → stored at −80 °C	HPLC-QE Orbitrap MS, normal phase LC, positive and negative ESI, 32 min acquisition at 0.3 mL/min, full scan range 85–1275 m/z Untargeted analysis	Recommends the direct extraction of metabolites from adherent cells in the culture dish, as it was found to have a higher recovery of both polar and non-polar metabolites than removing cells from the dish before metabolite extraction
Garcia-Canaveras et al. [72]	HepG2	Cells	Single wash with cold PBS	Direct addition of LN2	Comparison of five extraction solutions: HEPES-EDTA; 100% methanol; and water:methanol:chloroform in a monophasic, biphasic and sequential phase extraction → cold extraction solution added directly to cells or to collected trypsinised cells and subject to three freeze–thaw cycles → centrifugation → evaporated to dryness → reconstitution of sample for appropriate analytical technique	LC–QTOF MS, reverse and normal phase LC, positive and negative ESI, full scan range 50–1200 m/z Untargeted analysis	Optimisation study for several steps of adherent mammalian cell metabolite extraction and analysis by LC–MS Optimal cell harvest and metabolite extraction procedure was found to be direct addition of extraction solution and scraping of cells over trypsinisation
2015
Kapoore et al. [66]	MDA-MS-436, MCF-7, HMEC-1	Cells	Washed twice with ice-cold PBS	60% methanol at −50 °C	Trypsinisation and cell scraping were compared. Extraction of metabolites from cell mass was via addition of 100% methanol followed by three freeze–thaw cycles: snap freezing in liquid nitrogen → thawing on dry ice → vortexing → centrifugation. Extraction procedure was repeated and the two extracts combined and freeze-dried (lyophilised)	GC–MS	Metabolite leakage is influenced by the cell line used. An extra protein precipitation step in the extraction procedure did not show any significant improvement in metabolite recoveries. Recommends the use of rapid, combined quenching and extraction approaches to minimise metabolite leakage occurring from cells
Madji Hounoum et al. [74]	NSC-34	Cells	Washed twice with either PBS at 37 °C or 0.9% (w/v) NaCl	100% methanol at −40 °C	Cells were scraped into quenching methanol → four different extraction solvents were compared: methanol:water (1:1), acetonitrile:water (1:1), dichloromethane:methanol:water (3:3:2), and dichloromethane:acetonitrile:water (3:3:2) → vortexing and sonication → centrifugation → dried in vacuum concentrator	NMR, GC–MS, LC–HRMS GC–MS: ZB-5 column, full scan range 50–500 m/z, untargeted analysis with compound matching to in-house standards library LC–HRMS: reverse phase LC, positive and negative ESI, full scan range 60–900 m/z	Optimisation study that focuses on simultaneous, combined-analysis-based metabolomics studies, and suitable extraction and sample preparation methods that best fit all different platforms A dual-phase extraction (methanol:dichloromethane:water) was selected, as the different phases could be analysed differently by multiple analytical platforms
Ser et al. [75]	HCT116	Cells and culture medium	Comparison of PBS, Millipore water or no wash	Comparison of cells placed on ice (4 °C) or dry ice (−80 °C)	Comparison of 80% methanol:20% water and 80%methanol:20%water + 5% formic acid → cells scraped → centrifugation → dried in vacuum concentrator → stored at −80 °C	LC–QEMS Reverse-phase LC, positive and negative ESI, full scan range 50–900 m/z	Different metabolite extraction procedure for medium samples: methanol:water (20:3) → vigorous vortexing → centrifugation → dried in vacuum centrifuge Extraction temperature (either −80 °C or 4 °C) does not change metabolite quantification. Recommends use of formic acid in extraction solvent, and the exclusion of a cell washing step
2014
Matheus et al. [70]	ECC1, HeLa	Cells	Cold PBS	Kept on ice	Cells were either trypsinised or scraped into PBS → centrifugation → resuspension in D2O → cells disrupted either by probe sonication or bath sonication → centrifugation → stored at −80 °C	NMR	Sonication of cells suspended in water was the only metabolite extraction procedure tested in this study, and was deemed suitable for NMR analysis
Rahman et al. [76]	HeLa, Hek293, MEF	Cells	Warmed PBS	Snap-frozen in liquid nitrogen	40% acetonitrile:40% methanol:20% water → cells scraped → agitation → centrifugation → dried in vacuum concentrator → stored at −80 °C	LC–MS/MS Reverse-phase LC, positive and negative ESI, 20 min acquisition at 0.5 mL/min flow rate	Does not compare several attempted extraction techniques, rather reports on one technique and the optimisation of MS parameters for the chosen procedure
2013
Bi et al. [77]	Panc-1	Cells	Isotonic saline at 37 °C	Liquid nitrogen	Trypsin detachment of cells or cell scraping into water → four different extraction solvents: acetonitrile, methanol, methanol: chloroform: water, and methanol: chloroform: acetonitrile → vortexing → centrifugation → dried under nitrogen	LC–QTOF-MS	Trypsinisation found to be inadequate due to substantial metabolite leakage. Flash quenching with liquid nitrogen, followed by methanol/chloroform/water extraction was optimal for analysis by LC–MS
2012
Hutschenreuther et al. [78]	MCF-7	Cells	PBS	Placed in −80 °C	Trypsin detachment of cells or cell scraping → centrifugation → comparison of different extraction solvents: ethanolic potassium hydroxide; water:methanol:chloroform; and 100% methanol → extract purification using 3 kDa membrane filters → dried in vacuum concentrator	GC–MS 5-MS column, full scan range 78–600 m/z	Recommends methanol as an extraction solvent, with direct quenching and cell scraping
2011
Dettmer et al. [63]	SW480	Cells	PBS or no wash	PBS or direct methanol	Seven different extraction solvents added to cell pellets or direct to cells; methanol:chloroform: water (1:1:0.1); 100% methanol; 80% methanol:20% water; 100% acetone; 80% acetone:20% water; methanol:isopropanol: water (1:1:0.1); and acid–base methanol. Three different cell disruption techniques were compared using methanol:water (80:20) as extraction solvent; freeze/thaw; ultrasonication; and homogenisation	GC–MS 5-MS column, full scan range 50–600 m/z, untargeted analysis with compound matching to in-house standards library	Extensive comparison of several extraction procedures; trypsinisation vs. cell scraping in buffer or directly into extraction solvent; seven different extraction solvents; freeze/thaw cycles vs. ultrasonication or homogenisation. Direct scraping of cells into methanol/water extraction solvent was chosen as the method of choice
Lorenz et al. [79]	INS-1	Cells	Deionised water at 37 °C or Krebs–Ringer-HEPES buffer at 37 °C	Direct addition of liquid nitrogen to culture dish	Different extraction solvents added to plate; 90% methanol:10% chloroform; 100% ethanol; 100% acetonitrile; 100% methanol → cells scraped → centrifugation	HPLC–MS	Extraction solvent 90% methanol:10% chloroform found to be superior for metabolite recovery and stability
Martineau et al. [67]	MCF-7, MDA-MB-468, SKBr3, ZR75-1	Cells	PBS	100% methanol	Cells scraped into methanol used for quenching → comparison of five different extraction solvents; acetonitrile:water (4:1); methanol:water (4:1); 100% methanol; perchloric acid (2% v/v in water); or methanol:dichloromethane:water (2:2:1.8) → vortexed → centrifuged → dried by nitrogen gas evaporation	NMR	Concludes that methanol:dichloromethane:water extraction solvent is the most suitable for NMR analysis of four different breast cancer cell lines
Sheikh et al. [80]	MCF-7	Cells	Ice-cold PBS	Kept on ice	Cell pellets suspended in water and lysed by freeze–thaw cycles → ultrasonication → methanol:chloroform:acetonitrile → dried in vacuum concentrator	UPLC–QTOF-MS Positive and negative ESI	Study states that a variety of extraction procedures were tested, but reports on only the one deemed most successful, which is then verified using seven other cancer cell lines. Conclusion is that this method “can be successfully used for cell metabolomics”
2010
Danielsson et al. [68]	INS-1 832/13	Cells	Ice-cold PBS	Methanol at −80 °C	Compared extraction solvents of 100% methanol or 82% methanol:18% water → Comparison of three cell disruption methods: stainless steel ball mill; snap-freezing in liquid nitrogen; and vortexing → centrifugation → dried in vacuum concentrator	GC–MS 5-MS column, full scan range 50–800 m/z	Optimised cell disruption, metabolite extraction and GC–MS settings with the aim of an unbiased, high-throughput method. Concluded that extraction with 82% methanol:18% water with cell disruption using the ball-mill was the most effective extraction procedure
Dietmair et al. [64]	CHO	Cells	PBS or no wash	Comparison of cold, isotonic saline (0.9% NaCl), cold 60% methanol, and cold 60% methanol buffered with 0.9% ammonium bicarbonate	Several different extraction solvents added to cell suspensions, a mixture of hot (>70 °C) or cold (<4 °C) solvents consisting: 50% acetonitrile:50% water; 100% methanol; 50% methanol:50% water; 50% methanol:50% chloroform; 80% methanol:20% water; 75% ethanol; 75% ethanol buffered with HEPES; 100% water; potassium hydroxide; and perchloric acid → dried in vacuum concentrator → resuspended in water and stored at −80 °C	HPLC	Extensive study that compares several quenching and extraction procedures that were chosen from literature. Concluded that quenching with cold saline effectively halted metabolism, and 50% acetonitrile:50% water was the superior extraction solvent
2009
Teng et al. [69]	MCF-7, MDA-MB-231	Cells	Ice-cold PBS	100% methanol	Cells scraped into quenching methanol → added extraction solvent methanol:chloroform:water (4:4:2.85) → only aqueous phase collected → dried in vacuum concentrator	NMR	Compares a direct quenching and collection method with trypsinisation before quenching and extraction. Concludes that direct quenching and collection of samples exhibits much greater recovery of metabolites than the conventional trypsinisation method (approx. 50-fold higher)