Minute‐Scale Repeated Metabolic Probing of Living Cells Enabled by Rapid Parahydrogen‐Based Hyperpolarization

ABSTRACT Hyperpolarized (HP) 13 C nuclear magnetic resonance (NMR) spectroscopy enables real‐time observation of metabolic fluxes but is typically limited to single‐shot measurements due to complex preparation procedures and low experimental throughput. Here, we established a rapid and experimentally accessible workflow for temporally controlled HP measurements in living cells. Using SABRE‐SHEATH at 0.4 µT, we achieved 7.7% ± 0.2% 13 C polarization of 50 mM [1‐ 13 C]pyruvate within 60 s. Simple 1:50 dilution with phosphate‐buffered D 2 O yielded cell‐compatible solutions that retained 5.3% ± 0.4% polarization of 1.3 mM pyruvate without multi‐step purification. Combined with a simplified agarose bead immobilization approach, this enabled four injections of HP pyruvate into the same HeLa cell population within 7 min. Under rapid (≈2 min) reinjection intervals, the pyruvate‐to‐lactate conversion progressively declined, whereas stable metabolic conversion was maintained at 20 min intervals with intermittent cell medium perfusion. These findings demonstrate that rapid repeated substrate delivery can transiently exhaust cellular metabolic conversion capacity. This experimentally accessible operating mode enables the study of short‐term metabolic dynamics that remain obscured in conventional single‐shot HP‐NMR or long timescale thermally polarized NMR measurements.