Cell Sample Collection, Quenching and Metabolite Extraction in Metabolomics

The qualitative and quantitative analysis of small molecule metabolites inside and outside the cell using metabolomics analysis techniques can analyze the concentration changes of metabolites inside and outside the cell, and systematically observe the changes of metabolites at the cellular level from a holistic perspective. It has a wide range of applications in many fields such as environmental stress, drug efficacy, and disease pathogenesis. Cells are different from other samples in that they are rich in metabolites but low in content and not easy or incomplete to collect. Therefore, an effective and reliable processing method is needed to obtain more comprehensive metabolite information.

Cell Collection

Metabolites are abundant but less abundant in cells than in serum and tissue samples. A minimum of 10^-6 cells are typically cultured and harvested for metabolomics experiments. To ensure that metabolites are detected as fully and as possible by the instrument, it is recommended that 10^-7 cells be collected for metabolomic experiments.

There are 2 main methods commonly used for cell collection:

• Trypsin enzymatic digestion of cells
• Sterile spatula scraping of cells

It was shown that the extraction rate of most metabolites during trypsin treatment was lower than that of scraper scraping. The disruption of the cell membrane during proteolysis resulted in enhanced permeability of the cell membrane and leakage of metabolites, resulting in lower extraction efficiency. Therefore, trypsin treatment is not recommended during the study of cellular metabolomics. It is recommended to obtain cells by scraping with a spatula after removing the medium to minimize the loss of metabolites and to obtain more metabolite information.

Cell Quenching

Quenching is designed to inactivate enzymes in cells, terminate cellular metabolism, and reduce experimental error. The ideal quenching solvent should be able to immediately inhibit cellular metabolic activity without damaging the cell membrane and preventing leakage of intracellular metabolites.

Commonly used quenching methods:

• Adding low-temperature organic solvent
• Adding low-temperature isotonic solution
• Adding liquid nitrogen

A rapid rinse with saline prior to cell quenching improves sensitivity and does not affect metabolic changes. Immediately after the physiological saline rinse, a low-temperature quenching agent should be added to quench the cells to reduce metabolite changes.

Using methanol alone as a quencher can cause leakage of some metabolites, so it is not recommended to use 100% methanol alone as a quenching solution. It is usually recommended to use a solution that is isotonic with intracellular (e.g., 0.9% sterile saline), which can maintain the ionic strength inside and outside the cells and maintain cellular integrity while preventing leakage of intracellular metabolites as much as possible.

Protocol for quenching of suspension cultured mammalian cells (Sellick et al., 2011)

Cellular Metabolites Extraction

Cells are rich in metabolites and extraction methods are crucial. No single method has so far been able to be applied to the extraction of all metabolites. Selecting a suitable extraction method for metabolites extracts the intracellular metabolites as much as possible to minimize the loss of metabolites.

Selection of organic solvents

Different organic solvents extract different classes of metabolites and are generally selected based on the principle of similar solubility. The most widely used extraction method in the field of cellular metabolomics is liquid-liquid extraction. Commonly used extractants are methanol/water, acetonitrile/water, methanol/chloroform, etc. The extraction solvents can be selected according to the experimental purpose and optimized

Cell lysis

In order to obtain as much intracellular metabolites as possible, the cells should be made to undergo the maximum possible fragmentation. Commonly used fragmentation methods are:

• Repeated freeze-thawing
• Ultrasonic fragmentation
• Mechanical homogenization

Repeated freeze-thawing has an effect on metabolites due to large temperature changes. After cell lysis, cells are processed by centrifugation and the supernatant is taken for analysis or lyophilized. Cell samples can be repeatedly homogenized to ensure maximum extraction of metabolites. To increase the detection of the instrument, the extracts can also be concentrated to allow the maximum amounts of metabolites to be analyzed in a small volume. This can be achieved by vacuum concentration or low-temperature freeze-drying.

Overview of protocol for metabolite extraction from suspension cultured mammalian cells (Sellick et al., 2011).

Reference

1. Sellick, C. A., Hansen, R., Stephens, G. M., Goodacre, R., & Dickson, A. J. (2011). Metabolite extraction from suspension-cultured mammalian cells for global metabolite profiling. Nature protocols, 6(8), 1241-1249.