Determination of Intra- and Extracellular Metabolic Adaptations of 3D Cell Cultures upon Challenges in Real-Time by NMR

Abstract

NMR flow devices provide longitudinal real-time quantitative metabolome characterisation of living cells. However, discrimination of intra- and extracellular contributions to the spectra represents a major challenge in metabolomic NMR studies. The present NMR study demonstrates the possibility to quantitatively measure both metabolic intracellular fingerprints and extracellular footprints on human control fibroblasts by using a commercially available flow tube system with a standard 5 mm NMR probe. We performed a comprehensive 3D cell culture system characterisation. Diffusion NMR was employed for intra- and extracellular metabolites separation. In addition, complementary extracellular footprints were determined. The implemented perfused NMR bioreactor system allowed the determination of 35 metabolites and intra- and extracellular separation of 19 metabolites based on diffusion rate differences. We show the reliability and sensitivity of NMR diffusion measurements to detect metabolite concentration changes in both intra- and extracellular compartments during perfusion with different selective culture media, and upon complex I inhibition with rotenone. We also demonstrate the sensitivity of extracellular footprints to determine metabolic variations at different flow rates. The current method is of potential use for the metabolomic characterisation of defect fibroblasts and for improving physiological comprehension.

Methods for Oxygen Determination in an NMR Bioreactor as a Surrogate Marker for Metabolomic Studies in Living Cell Cultures

“…In our previous study we found that only 1–2% of total water in the sample volume within the sensitive NMR region was compartmentalized within cell membranes of the investigated fibroblasts, which is in agreement with previous studies (Pilatus et al, 1997, Van Zijl et al, 1991). Therefore, the oxygen content determined with the global T 1 relaxation time technique mainly reflects the oxygen level in the extracellular space, potentially obscuring the interpretation of possible oxygen changes in the vicinity of the cells.…”

“…We and others have previously demonstrated the capability to quantify oxygen content and oxygen consumption within 3D cell cultures, using relaxation time oxygen sensitivity of embedded fluorine atoms. 5 However, the deterioration of NMR signal quality caused by PFTBA beads in the 3D cell culture scaffold and a frequent insensitivity to oxygen changes rendered the prepared sample unusable, 18 prompting us to look for alternative solutions. In this study, we describe oxygen quantification approaches in living human 3D cell cultures in a perfused NMR bioreactor system: (I) T 1 estimation of water is implemented to determine the global concentration and consumption of oxygen throughout the NMR tube; solutions to suppress RD of very strong water resonance are discussed; (II) in situ intracellular oxygen content estimation using T 1 of slowly diffusing water molecules is implemented to determine local oxygen tension and consumption within 3D cell culture samples; and (III) spatially resolved oxygen levels along the tube using T 1 estimations of water profiles are determined to detect oxygen potential changes along the scaffold length.…”

“…Human fibroblasts, derived from skin biopsy of healthy controls in routine diagnostics, were supplied to the routine cell culture laboratory of the Center of Laboratory Medicine of the University Hospital Bern. The cells were cultivated in 2D as performed previously. , Briefly, the control fibroblast cell line was cultured in MEM supplemented with 10% fetal calf serum, 1× nonessential amino acids (100 μM glycine, l -alanine, l -asparagine, l -aspartic acid, l -glutamic acid, l -proline, l -serine) (GIBCO/Invitrogen, Carlsbad, CA, USA), 2 mM l -glutamine, 200 μM uridine, 1 mM sodium pyruvate, 100 U mL –1 penicillin, and 100 μg mL –1 streptomycin and 10 μg mL –1 chlortetracycline at 310 K and 5% CO 2 in a cell culture incubator. The medium was exchanged between two and three times a week, and fibroblasts were passaged using 0.05% trypsin–EDTA.…”

“…Similar to what also other groups have shown, , a perfused NMR bioreactor system was established based on the InsightMR flow unit and real-time metabolic changes of small molecules by 1 H NMR and concurrent oxygenation levels were determined during periodically flow-rate interruption experiments (“stop and go”), and upon applying a substrate inhibition protocol during constant perfusion. However, a deterioration of NMR signal quality caused by these microcarrier beads was observed, likely due to susceptibility differences, and a frequent insensitivity to oxygen changes was encountered, probably due to Ostwald ripening leading to the formation of large PFTBA droplets. , Owing to their both highly hydrophobic and lipophobic nature, PFCs require the formation of aqueous emulsions for biological studies. However, these emulsions can become unstable and lead to coalescence, resulting in larger droplets and lower PFCs, corresponding to a decreased sensitivity to oxygen changes.…”

“…In both cases, after polymerization, the 3D scaffold was gently released into a Petri dish containing 35 mL medium and incubated for 24 h. 5,18 Preparation for the NMR Measurement. The 3D scaffold was taken up from the Petri dish using an infusion tube and gently inserted into the InsightCell glass tube, clamped on the upper surface of a 1 cm-high bottom gel plug, as described elsewhere.…”