Real-Time Metabolic Detection in Living Cells Using Hyperpolarized ¹³C NMR

We developed non-invasive method to measure real-time serum metabolism using advanced hyperpolarized NMR techniques. Our goal is creating accessible tools for various biological research applications. Several hyperpolarized NMR systems for serum metabolism have been reported. However, standardized and reproducible protocols for broader research implementation have not been available until now.

Our protocol enhances accessibility using commercial available components. We also provide comprehensive validation, including in system mixing advantages and repeated measurement capabilities for broader adaption. Our results enable real-time tracking of dynamic metabolic changes in living cells without distraction. This opens new possibilities for long-term drug studies in diverse metabolic probe applications.

Our approach has limitations, including prob options and automation requirements. Solving the challenges will establish cellular metabolic measurement techniques and rich our laboratory's DNP research efforts in the future.

[Narrator] To begin, add two milliliters of 0.25% volume-to-volume trypsin EDTA solution to a 10 centimeter dish. Tilt the dish gently to ensure the entire surface is covered. Then aspirate the excess trypsin ethylenediaminetetracetic acid solution. Place the dish in an incubator set at 37 degrees Celsius and incubate for two minutes. Then add 10 milliliters of culture medium to the dish. Using a pipette, gently pipette up and down to detach the cells from the surface and collect the entire cell suspension into a 15 milliliter conical tube. Now take approximately 10 microliters of the cell suspension and load it into a cell counter to determine the number of cells. Ensure that the measured count confirms a yield of approximately 10 million cells or adjust as needed. Centrifuge the remaining cell suspension at 120 x g for five minutes. After centrifugation, aspirate and discard the supernatant. Resuspend the resulting cell pellet in four milliliters of 2% weight-to-volume alginate solution, and pipette slowly to minimize bubble formation. Now add 10 milliliters of 50 millimolar calcium chloride solution to a 50 milliliter centrifuge tube. Secure the syringe to the cap of the centrifuge tube containing calcium chloride solution. Draw 300 microliters of the cell alginate mixture into a 0.5 milliliter syringe fitted with a three gauge 10 millimeter needle. Then close the centrifuge tube tightly with the syringe fixed to the cap, and place the assembly into the centrifuge to spin gently at 200 x g for five minutes. Remove the supernatant after centrifugation and resuspend the resulting gel in culture medium. Next, using a Pasteur pipette, transfer the cell encapsulated gel into an NMR tube and insert the sponge fixture to ensure a tight seal. Prewarm the medium reservoir containing 5% volume-to-volume deuterium oxide in a 37 degrees Celsius water bath. Begin circulation at a rate of approximately 500 microliters per hour to stabilize the conditions. Inspect the setup to ensure there are no leaks before proceeding. Add 18 microliters of carbon 13-labeled pyruvic acid at a concentration of 14.2 molar doped with 25 millimolar OX063 to a sample vial. Connect the vial to the fluid path of the dynamic nuclear polarization polarizer system. Next, insert the connected vial into the bore unit of the dynamic nuclear polarizer. Apply microwave irradiation at approximately 188 gigahertz with a power of 22 milliwatts for a duration of 60 to 80 minutes. Use the solid state nuclear magnetic resonance spectrometer integrated into the dynamic nuclear polarization system to monitor the carbon 13 signal intensity. Record the signal data every five minutes using the systems control software. Now rapidly dissolve the polarized sample in 3.2 milliliters of dissolution buffer preheated to biological temperature. Load the prepared sample into an NMR tube and insert the tube into the spectrometer. On the NMR console, initiate the automated procedure to perform locking, tuning, matching, and shimming with the sample in place to ensure optimal spectral resolution and signal stability. Load a carbon 13 pulse-acquire sequence with proton decoupling-enabled using a setting like 13-CPD. Set the flip angle to 90 degrees with a one microsecond pulse labeled as P1. Next, define the acquisition time as 1.376 seconds, spectral width as 23,663 hertz, and acquisition points, or TD, as 65,536. Set the relaxation delay D1 to zero seconds. Assign 150 time increments to TD1 for acquiring 150 sequential spectra in pseudo-2 dimensional mode, and adjust the receiver gain to a low value, such as approximately one, to avoid signal saturation caused by the hyperpolarized samples. Now stop the peristaltic pump to temporarily halt the circulation of the medium before injecting the hyperpolarized solution through the inlet tubing. Begin the spectral acquisition approximately 10 seconds before dissolving the hyperpolarized substrate to ensure signal capture upon arrival at the detection site. When the solution exits the dynamic nuclear polarization polarizer, draw one milliliter of it into a syringe. Switch the three-way valve at the bioreactor inlet to position B to direct the hyperpolarized solution into the NMR tube. After the injection is complete, switch the valve back to position A to resume medium circulation and prevent any backflow. Finally, confirm that the injection was successful after noting an increase in the free induction decay signal on the acquisition software. Ensure the final solution reaches a temperature between 308 and 313 Kelvin and a pH of approximately seven before transferring for NMR measurement. Hyperpolarized carbon 13 NMR spectra of the same SCCVII cell encapsulated sample were acquired at 1.5 hour intervals to evaluate the feasibility of repeated non-destructive measurements. The first NMR measurement showed a markedly increased lactate signal, indicating active pyruvate to lactate metabolic conversion. Subsequent measurements at 1.5 hour intervals revealed a progressive reduction in lactate signal intensity, indicating decreased metabolic activity over time. In HeLa cells, initial measurements confirmed successful pyruvate to lactate conversion with clearly discernible signal peaks. Although the signal decreased gradually due to T2 relaxation, the signal-to-noise ratio remained adequate throughout the 60 second acquisition, suggesting that the current protocol is broadly applicable and can serve as a versatile tool for real-time metabolic profiling in various cell types.