Metabolomics is a downstream complement to genomics, transcriptomics, and proteomics, providing an overall assessment of the physiological state of biological systems. It represents the end point of genetic regulation and its effect on changes in enzymatic activity and endogenous biochemical responses in cells. Combining feature selection, pattern recognition, and multivariate data analysis methods, metabolomic analysis aims to provide a comprehensive assessment of changes in metabolite levels in blood, urine, tissue, or cells.
Cell samples are important samples for metabolomic analysis. As the first place for biological metabolism, cells have important research significance in metabolomic analysis. In recent years, in many medical, animal and plant physiology and cell biology research, such as the identification and detection of disease markers, metabolomics applied to cell samples has played an important role as a tool.
Mass Spectrometry-Based Study of Cell Samples
The non-targeted metabolomics analysis process based on ultra-high performance liquid chromatography-mass spectrometry generally includes: sample collection and pretreatment, metabolite extraction, LC-MS full scan detection, data preprocessing, statistical analysis and differences Structure Identification. The first step, sample collection and replicate settings, is particularly important, which is directly related to experimental results and data analysis.
Metabolomics approaches have been widely used in animal and plant tissues, yeast and bacteria, and different sample collection techniques have been developed for different tissues and cell types.
Different from other samples, the quenching process needs to be carried out immediately after the cell sample is collected, that is, the enzymes in the cells are rapidly inactivated, thereby stopping the changes of intracellular metabolites. During the collection of cell samples, liquid nitrogen can be used for rapid quenching, but liquid nitrogen cannot be directly poured into the cells, which will cause the cell membrane to be directly broken and the contents to flow out directly, which will affect the later analysis of intracellular metabolites.
The extraction process of metabolites from cells was optimized and verified, and the extraction effect of quenching process on metabolites, the leakage of metabolites during the process, the effect of removing contaminant impurities during pretreatment, and the results of sample mass spectrometry were evaluated. The following is a scientific approach to the collection of cell samples and the collection of cell culture supernatants.
I. Cell collection
For cells grown in medium such as suspension cells and adherent cells, it is necessary to consider the separation of the medium and the cell body, and whether the metabolic activity inside the cells continues after the collection is completed. Based on this factor, the general cell samples are During the collection process, a quenching step needs to be added.
The cell samples were counted by a cell counter or a cell counting system before collection, and the amount of about 5106~1108 cells was in line with the requirements of metabolomics analysis.
Adherent cell collection procedure:
1. Quickly pour off the culture medium, and place the culture dish upside down on absorbent paper to dry the culture medium.
2. Add pre-cooled PBS at 4°C and rinse repeatedly 2~3 times (if using a pipette, add it against the wall of the culture dish to avoid washing up the cells) and pour out the PBS.
3. Use a pipette to suck up the remaining PBS, touch the bottom (outer wall) of the culture dish with liquid nitrogen to quench the cells (1*107 cells is suitable),
4. Add 500 microliters of pre-cooled methanol-water (4:1, V/V), scrape the cells with a cell scraper, transfer them to a 1.5 mL centrifuge tube with a pipette,
5. Add 500 microliters of pre-cooled methanol-water (4:1, V/V) to the petri dish, transfer the remaining cells as much as possible to a 1.5 mL centrifuge tube, and seal the centrifuge tube with parafilm ( After quenching the cells, directly scrape the cells without adding the extract and transfer them to a centrifuge tube) -80 degrees cryopreservation.
(1) If the extract is added, it must be sealed with parafilm.
(2) For lipids, lipid targeted targeting, targeted metabolism, etc., the methanol-water (4:1) method should not be used. After quenching the cells, the cells should be directly scraped and transferred to a centrifuge tube without adding the extract.
(3) When quenching cells with liquid nitrogen, please be careful with the quality of the centrifuge tube to prevent the sample from being damaged due to breakage of the centrifuge tube
(4) Take the cells in the logarithmic growth phase 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 when collecting adherent cells, 1 mL of reagent cannot be completely transferred, and it is recommended to add the maximum reagent volume of no more than 3 mL.
(6) The adherent cells can also be collected by trypsin digestion, centrifuged at low speed, removed the culture medium supernatant, washed 1-2 times with PBS solution, discarded the PBS supernatant solution, and settled the collected cells in liquid nitrogen Follow-up experiments after quenching
Suspension cell collection procedure:
1. Transfer the culture medium to an imported 15 mL centrifuge tube, and centrifuge at low speed for 5 minutes (below 3000 rpm) to allow the cells to settle at the bottom of the centrifuge tube (1*107 cells is suitable).
2. Pour off the medium (try to be as clean as possible) and rinse with PBS for 2 to 3 times.
3. Label the centrifuge tube, insert the tip of the centrifuge tube into liquid nitrogen, quench the cell pellet for 1 min, and then freeze at -80°C.
(1) The operation should be as fast as possible, and the medium should be poured out as cleanly as possible. If there is filter paper, it is recommended to use filter paper to absorb the remaining medium.
(2) Take the cells in the logarithmic growth phase and culture them to the same period. It is recommended to prepare more samples for subsequent use.
The storage medium is closely related to the subsequent extraction results, and cells will inevitably freeze and thaw during storage and transportation, which may have a greater impact on the experimental results. If it is not necessary, we recommend discarding the supernatant and PBS, and directly sending the remaining cell pellets for analysis, which can try to avoid the deviation of the experimental results due to the medium. If the cells must be stored in a medium, it is recommended to use a mixture of methanol and water for storage.
In the process of cell isolation, due to the fragility of cells, some improper operations will lead to cell fragmentation, which will affect the process of metabolomics analysis. Therefore, the following precautions should be taken.
1. The whole process of sample collection should not be excessively violent. Do not vortex, high-speed centrifuge or ultrasound, and avoid repeated freezing and thawing to ensure that the cell membrane will not be damaged and reduce the impact of metabolomics analysis.
2. When washing with PBS, do not wash away the cells. After washing sufficiently, try to pour out the PBS completely.
3. Pay attention to control conditions and time during quenching. Some mammalian cell lines cannot accept quenching conditions below -45°C, which may lead to cell membrane fragmentation.
4. Do not pour liquid nitrogen directly into the petri dish to quench, it will cause cell breakage and metabolite outflow.
II. Collection of cell culture supernatant
1. Take about 2 mL of cell culture medium and centrifuge at low speed to separate the cell pellet.
2. Take 1 mL (recommended amount, at least 500 μL) of the supernatant, store the sample at -80°C, and send the sample with enough dry ice.
Due to its sample properties, the supernatant of the culture medium generally has a low content of metabolites and a high salt and sugar content, which makes the analysis more difficult, and requires high experimental instruments and experimental methods. The following points should be paid attention to. point:
1. After confirming that metabolomics analysis is required, please contact the pre-sales and submit the contents of the medium to determine whether metabolomics analysis can be performed.
2. When collecting samples, strictly avoid high-speed centrifugation, vigorous shaking, and high or low temperature to cause cell breakage in the medium.
3. Avoid repeated thawing after collection, store and transport at low temperature throughout the process, and use dry ice for long-distance transport until analysis.