Noninvasive and repetitive measurement of cellular metabolite from human osteosarcoma cells (MG-63) using 3.0 tesla proton (1 H) MR spectroscopy.

PURPOSE: This study suggests a noninvasive and repetitive measurement method using 1 H magnetic resonance spectroscopy to monitor changes in cellular metabolites within a single sample. METHODS: Longitudinal acquisition of cellular metabolites from three-dimensional cultured human osteosarcoma (MG-63) cells was conducted using 3.0 Tesla 1 H MRS for 2 weeks at three time points: days 1, 7, and 14. During the MR spectroscopy (MRS) scan, cell specimen temperatures were kept constant at 37°C by a lab-developed magnetic resonance compatible thermostatic device. A DNA assay and live/dead staining of the cell specimens were carried out at each time point to verify the MRS measurements. RESULTS: Cell viability in the proposed device did not significantly differ from that of cells in a conventional incubator (P = 0.946). Cell proliferation and choline concentration increased during the first week, but remained constant during the second week. Lactate did not change during the first week, but increased during the second week. Likewise, cell viability remained constant until day 7, then decreased. CONCLUSION: The proposed MRS technique results in a survivable environment for longitudinal studies of cells and provides a new way to measure metabolomic changes over time in single specimens of cells. Magn Reson Med 76:1912-1918, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Distinguishing tendon and ligament fibroblasts based on 1H nuclear magnetic resonance spectroscopy.

Tendon and ligament (T/L) have been known to be obviously different from each other in tissue level. However, due to the overlapping gene markers, distinction in cellular level has not been clearly verified yet. Recently, the use of nuclear magnetic resonance (NMR) spectroscopy has shown the potential to detect biological markers in cellular level. Therefore, in this study we applied a non-invasive technique based on NMR spectroscopy to establish biomarkers to distinguish between T/L fibroblasts. In addition the cellular morphologies and gene expression patterns were also investigated for comparison through optical microscopy and real-time polymerase chain reaction (PCR). No difference was observed from morphology and real-time PCR results, either as expected. However, we found clear differences in their metabolomic spectra using 1H NMR spectroscopy. The calculated integral values of fatty acids (with chemical shifts at ~0.9, 1.26, 1.59, 2.05, 2.25, and 2.81 ppm), lactate (~1.33 ppm), and leucine (~2.72 ppm) were significantly different between the two types of fibroblasts. To be specific tendon group exhibited higher level of the metabolite than ligament group. In conclusion, in-cell metabolomic evaluation by NMR technique used in this study is believed to provide a promising tool in distinguishing cell types, especially T/L cells, which cannot be classified by conventional biological assays.

Cytotoxicity of TSP in 3D Agarose Gel Cultured Cell.

PURPOSE: A reference reagent, 3-(trimethylsilyl) propionic-2, 2, 3, 3-d4 acid sodium (TSP), has been used frequently in nuclear magnetic resonance (NMR) and magnetic resonance spectroscopy (MRS) as an internal reference to identify cell and tissue metabolites, and determine chemical and protein structures. This reference material has been exploited for the quantitative and dynamic analyses of metabolite spectra acquired from cells. The aim of this study was to evaluate the cytotoxicity of TSP on three-dimensionally, agarose gel, cultured cells. MATERIALS AND METHODS: A human osteosarcoma cell line (MG-63) was selected, and cells were three dimensionally cultured for two weeks in an agarose gel. The culture system contained a mixture of conventional culture medium and various concentrations (0, 1, 3, 5, 7, 10, 20 30 mM) of TSP. A DNA quantification assay was conducted to assess cell proliferation using Quant-iT PicoGreen dsDNA reagent and kit, and cell viability was determined using a LIVE/DEAD Viability/Cytotoxicity kit. Both examinations were performed simultaneously at 1, 3, 7 and 14 days from cell seeding. RESULTS: In this study, the cytotoxicity of TSP in the 3D culture of MG-63 cells was evaluated by quantifying DNA (cell proliferation) and cell viability. High concentrations of TSP (from 10 to 30 mM) reduced both cell proliferation and viability (to 30% of the control after one week of exposure), but no such effects were found using low concentrations of TSP (0-10 mM). CONCLUSIONS: This study shows that low concentrations of TSP in 3D cell culture medium can be used for quantitative NMR or MRS examinations for up to two weeks post exposure.

Oxygen-induced frequency shifts in hyperoxia: a significant component of BOLD signal.

In comparison to the well-documented significance of intravascular deoxyhemoglobin (deoxyHgb), the effects of dissolved oxygen on the blood-oxygen-level-dependent (BOLD) signal have not been widely reported. Based on the fact that the prolonged inspiration of high oxygen fraction gas can result in up to a sixfold increase of the baseline tissue oxygenation, the current study focused on the influence of dissolved oxygen on the BOLD signal during hyperoxia. As results, our in vitro study revealed that the r1 and r2 (relaxivities) of the oxygen-treated serum were 0.22 mM(-1) · s(-1) and 0.19 mM(-1) · s(-1) , respectively. In an in vivo experiment, hyperoxic respiration induced negative BOLD contrast (i.e. signal decrease) in 18-42% of measured brain regions, voxels with accompanying significant decreases in both the T(*)2 (-12.1% to -19.4%) and T1 (-5.8% to -3.3%) relaxation times. In contrast, the T(*)2 relaxation time significantly increased (11.2% to 14.0%) for the voxels displaying positive BOLD contrast (in 41-50% of the measured brain), which reflected a hyperoxygenation-induced reduction in tissue deoxyHgb concentration. These data imply that hyperoxia-driven BOLD signal changes are primarily determined by the counteracting effects of extravascular oxygen and intravascular deoxyHgb. Oxygen-induced magnetic susceptibility was further demonstrated by the study of 1 min hypoxia, which induced BOLD signal changes opposite to those under hyperoxia. Vasoconstriction was more common in voxels with negative BOLD contrast than in voxels with positive contrast (% change of blood volume, -9.8% to -12.8% versus 2.0% to 2.2%), which further suggests that negative BOLD contrast is mainly evoked by an increase in extravascular oxygen concentration. Conclusively, frequency shifts, which are induced by the accumulation of oxygen molecules and associated magnetic field inhomogeneity, are a significant source of the negative BOLD contrast during hyperoxia.

Response of the primary auditory and non-auditory cortices to acoustic stimulation: a manganese-enhanced MRI study.

Structural and functional features of various cerebral cortices have been extensively explored in neuroscience research. We used manganese-enhanced MRI, a non-invasive method for examining stimulus-dependent activity in the whole brain, to investigate the activity in the layers of primary cortices and sensory, such as auditory and olfactory, pathways under acoustic stimulation. Male Sprague-Dawley rats, either with or without exposure to auditory stimulation, were scanned before and 24-29 hour after systemic MnCl2 injection. Cortex linearization and layer-dependent signal extraction were subsequently performed for detecting layer-specific cortical activity. We found stimulus-dependent activity in the deep layers of the primary auditory cortex and the auditory pathways. The primary sensory and visual cortices also showed the enhanced activity, whereas the olfactory pathways did not. Further, we performed correlation analysis of the signal intensity ratios among different layers of each cortex, and compared the strength of correlations between with and without the auditory stimulation. In the primary auditory cortex, the correlation strength between left and right hemisphere showed a slight but not significant increase with the acoustic simulation, whereas, in the primary sensory and visual cortex, the correlation coefficients were significantly smaller. These results suggest the possibility that even though the primary auditory, sensory, and visual cortices showed enhanced activity to the auditory stimulation, these cortices had different associations for auditory processing in the brain network.

Evaluation of metabolomic changes as a biomarker of chondrogenic differentiation in 3D-cultured human mesenchymal stem cells using proton (1H) nuclear magnetic resonance spectroscopy.

PURPOSE: The purpose of this study was to evaluate the metabolomic changes in 3D-cultured human mesenchymal stem cells (hMSCs) in alginate beads, so as to identify biomarkers during chondrogenesis using (1)H nuclear magnetic resonance (NMR) spectroscopy. MATERIALS AND METHODS: hMSCs (2×10(6) cells/mL) were seeded into alginate beads, and chondrogenesis was allowed to progress for 15 days. NMR spectra of the chondrogenic hMSCs were obtained at 4, 7, 11, and 15 days using a 14.1-T (600-MHz) NMR with the water suppression sequence, zgpr. Real-Time polymerase chain reaction (PCR) was performed to confirm that that the hMSCs differentiated into chondrocytes and to analyze the metabolomic changes indicated by the NMR spectra. RESULTS: During chondrogenesis, changes were detected in several metabolomes as hMSC chondrogenesis biomarkers, e.g., fatty acids, alanine, glutamate, and phosphocholine. The metabolomic changes were compared with the Real-Time PCR results, and significant differences were determined using statistical analysis. We found that changes in metabolomes were closely related to biological reactions that occurred during the chondrogenesis of hMSCs. CONCLUSIONS: In this study, we confirm that metabolomic changes detected by (1)H-NMR spectroscopy during chondrogenic differentiation of 3D-cultured hMSCs in alginate beads can be considered as biomarkers of stem cell differentiation.