Cellular samples are important samples for metabolomics analysis. As the first site of biological metabolism, cells are of great research importance in metabolomic analysis. Metabolomics applied to cell samples plays an important role in numerous medical, animal and plant physiology and cell biology studies, such as the identification and detection of markers of diseases.
The process of untargeted metabolomics analysis based on ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) generally includes: sample collection and pretreatment, metabolite extraction, LC-MS full scan detection, data pre-processing, statistical analysis and differential structure identification. Sample collection and replicate setup are particularly important and directly related to experimental results as well as data analysis.
Scheme of a typical metabolomics workflow applicable to cell analysis (Bispo et al., 2021).
Metabolomics methods have been widely used for animal and plant tissues, yeast and bacteria, and different sample collection techniques have been developed for different tissues and cell types.
Unlike other samples, cell samples need to undergo a quenching process immediately after collection to rapidly inactivate the intracellular enzymes and thus terminate the changes of intracellular metabolites. The cell sample collection process can be performed with liquid nitrogen for rapid quenching. However, liquid nitrogen should not be poured directly into the cells. This will result in direct fragmentation of the cell membrane, allowing its contents to flow directly out and interfere with the later analysis of intracellular metabolites. The following will describe the collection of optimized cell samples and the referenceable way of cell culture fluid supernatant collection.
Cell Collection
For suspension cells, walled cells and other cells grown from culture medium, the separation of culture medium and cell body needs to be considered. And it is important to consider whether the metabolic activities within the cells continue after the collection is completed. Based on such factors, the step of quenching needs to be added to the collection process for general cell samples.
Cell samples were counted using a cell counter or cell counting system prior to collection. An amount of approximately 106 to 108 cells is consistent with metabolomic analysis.
Adherent cell collection procedure:
- Quickly pour off the culture medium and place the culture dish upside down on absorbent paper to absorb the culture fluid.
- Add pre-cooled PBS at 4°C and rinse 2-3 times repeatedly (if adding with a pipette, add against the wall of the culture dish to avoid washing up the cells) and pour off the PBS.
- Aspirate the remaining PBS with a pipette and quench the cells by exposing the bottom (outer wall) of the culture dish to liquid nitrogen (1*107 cells is appropriate).
- Add 500 µL of pre-cooled methanol-water (4:1, V/V), scrape off the cells with a cell spatula, and transfer to a 1.5 mL centrifuge tube with a pipette.
- Add another 500 µL of pre-cooled methanol-water (4:1, V/V) to the culture dish. Transfer as many of the remaining cells as possible to a 1.5 mL centrifuge tube. Seal the centrifuge tubes using sealing film (or you can quench the cells and transfer them directly to the centrifuge tubes by scraping them off without adding the extraction solution) and freeze at -80 degrees.
Notes:
1) If adding the extraction solution must be sealed tightly with sealing film.
2) If doing lipid, lipid mimetic targeting, targeted metabolism, etc., do not operate according to the methanol-water (4:1) method. Cells should be quenched and transferred directly to the centrifuge tube by scraping down without adding the extract.
3) When quenching cells with liquid nitrogen, please be careful with the quality of the centrifuge tube to prevent damage to the sample due to broken tubes.
4) Take cells of logarithmic growth period and culture them to the same period. It is recommended to prepare more samples for subsequent use.
5) If the culture dish is too large for 1 mL of reagent to be completely transferred when collecting the walled cells, it is recommended that the maximum volume of reagent added should not exceed 3 mL.
6) Adherent cells can also be collected using trypsin digestion followed by low-speed centrifugation to remove the culture supernatant. Then use PBS solution to wash 1-2 times, discard the PBS supernatant solution, and quench the collected cell precipitate with liquid nitrogen for subsequent experiments.
Suspension cell collection procedure:
- Transfer to an imported 15 mL centrifuge tube together with the culture medium and centrifuge at low speed for 5 min (below 3000 rpm) to allow the cells to settle at the bottom of the tube (1*107 cells is appropriate).
- Pour off the culture medium (as clean as possible) and rinse 2~3 times repeatedly with PBS.
- Mark the centrifuge tube. Insert the tip of the centrifuge tube into liquid nitrogen. Quench the cell precipitate for 1 min, then freeze at -80 degrees.
Notes:
1) Operate as quickly as possible. Pour the culture medium as cleanly as possible. If filter paper is available, it is recommended to use filter paper to aspirate the residual medium.
2) Take cells of logarithmic growth period and culture them until the same period. It is recommended to prepare more samples for subsequent use.
Preservation medium
Preservation media are used in metabolomics to preserve a sample's native metabolite profile shortly after it is collected from a cell. Preservation media are designed to inhibit or prevent further metabolism or enzymatic reaction of a metabolic sample. Additionally, preservation media are used to maintain a sample's metabolic stability by preventing the volatility of potential target metabolites. Various preservation media for metabolomics samples have been developed that employ various antioxidants and stabilizers.
The liquid preservation media option typically involves the addition of a stabilizing/anti-oxidant agents such as N-acetylcysteine or a mixture of Mg2+, Ca2+, and EDTA. This option is attractive because it requires minimal handling of the sample and can be used in studies involving high turnover workflows.
The freeze-dried option (e.g. freezing in liquid nitrogen) is more labour-intensive but also induces greater stabilization of the samples against metabolic degradation. It also has the advantage of preserving biologically relevant metabolites that may be depleted in the liquid form.
Freeze-thawing of cells is inevitable during storage and transportation, which may have a significant impact on the experimental results. If not necessary, we recommend to discard the supernatant and PBS from the sample and send the remaining cell precipitate directly to the sample for analysis. This will minimize the possibility of bias in the results due to the medium.
Cell Culture Supernatant Collection
Cell culture supernatant collection procedure:
1. Take about 2mL of cell culture fluid, centrifuge at low speed, and separate the cell precipitate.
2. Take 1mL (recommended amount, minimum 500μL) or more of supernatant, store the sample at -80°C, and send the sample on sufficient dry ice.
Notes:
Culture supernatant in general, the sample metabolite content is low and contains high salt and sugar content, which makes the analysis difficult and requires high requirements for both experimental instruments and experimental methods. The following points need to be noted.
1. After determining the need for metabolomic analysis, please contact your technical advisor to submit the contents of the culture medium to determine whether the metabolomic analysis operation can be performed.
2. When collecting samples, strictly avoid high speed centrifugation, violent shaking, too high or too low temperature resulting in cell fragmentation in the culture medium.
3. Avoid repeated thawing after collection. Store and transport at low temperature throughout, and use dry ice for long distance transportation until analysis.
Reference
- Bispo, Daniela SC, et al. "Metabolomic applications in stem cell research: A review." Stem Cell Reviews and Reports 17.6 (2021): 2003-2024.
For Research Use Only. Not for use in diagnostic procedures.
Contact Us
