Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

The purpose of this article is to provide a detailed, stepwise visual protocol for extracting aqueous metabolites from cultured cancer cells for subsequent metabolome analysis. The main advantage of this technique is its simplicity and reliability for extracting metabolites from adherent cells. But above all, it's well optimized for metabolome analysis using capillary electrophoresis-mass spectrometry or CEMS.

Metabolomics is a powerful technique to identify biomarkers or detecting early-stage cancer and predicting chemotherapy response to cancer patients. We use cancer cells in this demonstration. However, this technique can also be applied to harvest metabolites from other adherent cells such as fibroblasts in iPS cells.

Metabolite concentrations are normalized based on the number of viable cells. So careful preparation of culture, this is the key for obtaining good reproducible data. Demonstrating the procedure will be Ami Maruyama, a technician from my laboratory.

To begin, plate HCC827 and PC-9 cells in 100-millimeter dishes containing 10 milliliters of RPMI-1640 medium supplemented with 10%fetal bovine serum. Place the dishes in an incubator at 5%carbon dioxide and 37 degrees Celsius for 24 hours to culture. After incubation, gently tilt the 100-millimeter culture dishes to aspirate the cell culture media.

Wash cells on each dish using two milliliters of phosphate-buffered saline solution without calcium and magnesium. Gently rock each dish so that the PBS solution completely covers the surface of the dish, then tilt the dishes to aspirate the wash buffer. Warm 0.25%trypsin-EDTA solution to 37 degrees Celsius.

With a five-milliliter serological pipette, add two milliliters of the warmed trypsin-EDTA solution to each dish. Gently rock each dish so that the trypsin completely covers the surface of the dish. Incubate the culture dishes at 37 degrees Celsius for approximately five minutes.

Then, add four milliliters of the pre-warmed complete growth medium to each dish. And gently pipette several times to re-suspend the cells in medium. Transfer each cell suspension to a separate 15-milliliter conical tube.

Centrifuge at 800 times g for five minutes. Discard the supernatant and re-suspend each cell pellet in two milliliters of the pre-warmed complete growth medium. Mix 10 microliters of the cell suspension with 10 microliters of 0.4%trypan blue solution, in a 1.5-milliliter microtube.

Load 10 microliters of the mixture into a cell counting chamber slide through capillary action. Insert the chamber slide into the automated cell counter and press the capture button to capture the image and display the results of the total number and percent viability of the cells. If the total cell number is above 0.5 million cells per milliliter, add further growth medium to the 15-milliliter conical tube to obtain the desired cell concentration.

Now, add two to five milliliters of the culture to each 100-millimeter cell culture dish to seed approximately one to 2 1/2 million cells per dish. Incubate the culture dishes in 5%carbon dioxide at 37 degrees Celsius for 18 hours. First, in a 15-milliliter volumetric flask, dilute a commercial internal standard solution containing L-Methionine sulfone and D-Camphor-10-sulfonic acid 1, 000-fold in ultrapure water.

Dissolve mannitol in ultrapure water to prepare a 0.05 grams per milliliter mannitol solution in ultrapure water as wash buffer. Then, into the filter cup of each centrifugal filter unit, pipette 250 microliters of ultrapure water. Cap the filter units tightly, and centrifuge at 9, 100 times g at four degrees Celsius for five minutes.

Check the volume of each filtrate. If significant filtrate has accumulated during the first short spin, the filter unit may be defective. Discard the filter unit and use a new filter unit instead.

Close the lids of the filter units tightly and centrifuge again at 9, 100 times g at four degrees Celsius for 30 minutes. Ensure that no ultrapure water remains in any of the filter cups, and remove the filtered ultrapure water in each collection tube with a pipette. Place the filter cups back into their collection tubes, and use the centrifugal filter units within an hour to avoid damage upon drying.

Take out the 100-millimeter culture dishes from the incubator and aspirate the cell culture medium from each dish. Add 10 milliliters of PBS culture medium with or without 250 micromolar diamide to each dish, taking care not to disturb the cell layer. Incubate the culture dishes at 37 degrees Celsius for 30 minutes.

After incubation, aspirate the cell culture medium from each 100-millimeter culture dish. Slightly tilt the dish, and gently add two milliliters of 5%mannitol wash buffer to the edge of each dish to wash the cells, taking care not to disturb the cell layer. Aspirate the wash buffer from each culture dish, and wash the cells again by slightly tilting the dish and gently adding 10 milliliters of wash buffer per dish.

Then, completely aspirate the wash buffer from the edge of each culture dish. Now, add 800 microliters of 99.7%methanol per culture dish, and gently rock each culture dish back and forth, to cover its entire surface. Leave the dishes at room temperature for 30 seconds.

Slowly add 550 microliters of the diluted internal standard solution per dish by immersing the tip of the pipette into the methanol and gently pipetting up and down, several times. Gently rock each culture dish back and forth to cover its entire surface, and leave the dishes at room temperature for 30 seconds. Then, transfer the extracted solution from each culture dish to a separate 1.5-milliliter microcentrifuge tube.

Centrifuge the tubes at 2, 300 times g at four degrees Celsius for five minutes. Transfer 700 microliters of each supernatant into two centrifugal filter units, with 350 microliters per unit. Centrifuge the filter tubes at 9, 100 times g at four degrees Celsius for approximately two hours, until no liquid remains in the filter cups.

Remove the filter cups, and tightly close the lids of the collection tubes. In this study, HCC827 and PC-9 cells grew equally for three hours. CEMS analysis indicates difference between diamide-treated cells and PBS-treated cells for both cell lines.

Among these, several intermediates in the pentose phosphate pathway and in upper glycolysis were significantly higher in the diamide-treated conditions, whereas a few tricarboxylic cycle intermediates were lower in the treated conditions. Metabolome profiles of intracellular metabolites revealed 175 and 150 differential metabolites in HCC827 and PC-9 cells, respectively. Following diamide treatment, the level of gluconic acid and oxidized glucose increased 12-fold in HCC827 cells and 10-fold in PC-9 cells.

Similarly, the level of glucose 6-phosphate, a phosphorylated glucose in the first hexokinase-catalyzed glycolysis product, also increased 6.3-and 3.5-fold in HCC827 and PC-9 cells, respectively. In addition, the levels of 6-phosphogluconate, the first intermediate in the pentose phosphate pathway, dramatically increased 89-fold in HCC827 cells and 231-fold in PC-9 cells. In contrast, levels of other glycolytic intermediates such as fructose 6-phosphate did not change in the diamide experimental condition.

Total nicotinamide adenine dinucleotide phosphate levels were nearly equivalent between diamide treatment and PBS control conditions. Using 5%mannitol solution, other than PBS, as wash buffer for washing cells is very important for CEMS analysis, because salt-based buffers interfere with the analysis and adversely affect measurement. This protocol is unsuitable for extracting hydrophobic metabolites like lipids, so we need to develop a protocol for conveniently extracting both hydrophilic and hydrophobic metabolites for more comprehensive metabolome analysis.

This technique realizes an easy extraction of metabolites from almost any adherent cells, and thus, is applicable for a variety of research such as cancer, stem cells, pharmacology, nutrition, and cosmetic science.