Assessment of cellular responses in three-dimensional cell cultures through chemical exchange saturation transfer and 1 H MRS.

The article from this special issue was previously published in NMR In Biomedicine , Volume 35, Issue 9, 2022. For completeness we are including the title page of the article below. The full text of the article can be read in Issue 35:9 on Wiley Online Library: https://doi.org/10.1002/nbm.4757.

Assessment of cellular responses in three-dimensional cell cultures through chemical exchange saturation transfer and 1 H MRS.

Metabolic responses to physiological changes have been detected using chemical exchange saturation transfer (CEST) imaging in clinical settings. Similarly to other MRI techniques, the CEST technique was based originally on phantoms from buffer solutions and was then further developed through animal experiments. However, CEST imaging can capture certain dynamics of metabolism that solution phantoms cannot model. Cell culture phantoms can fill the gap between buffer phantoms and animal models. In this study, we used 1 H NMR and CEST in a B0 field of 9.4 T to investigate HEK293T cells from two-dimensional (2D) cultures, three-dimensional (3D) cultures, and 3D cultures seeded with cell spheroids. Two CEST dips were observed: the magnitude of the amine dip at 2.8 ppm increased during the incubation period, whereas the hydroxyl dip at 1.2 ppm remained approximately the same or modestly increased. We also observed a CEST dip at 2.8 ppm from the 2D culture responding dramatically to doxorubicin treatment. By cross-validating with pH values and the concentrations of amine and hydroxyl protons extracted through 1 H NMR, we observed that they did not correspond to an increase in the amine pool. We believe that the denaturation or degradation of proteins from the fetal bovine serum increased the size of the amine pool. Although 3D culture conditions can be further improved, our study suggests that 3D cultures have the potential to bridge studies of solution phantoms and those on animals.

Chemical Exchange Saturation Transfer (CEST) Signal at -1.6 ppm and Its Application for Imaging a C6 Glioma Model.

The chemical exchange saturation transfer (CEST) signal at -1.6 ppm is attributed to the choline methyl on phosphatidylcholines and results from the relayed nuclear Overhauser effect (rNOE), that is, rNOE(-1.6). The formation of rNOE(-1.6) involving the cholesterol hydroxyl is shown in liposome models. We aimed to confirm the correlation between cholesterol content and rNOE(-1.6) in cell cultures, tissues, and animals. C57BL/6 mice (N = 9) bearing the C6 glioma tumor were imaged in a 7 T MRI scanner, and their rNOE(-1.6) images were cross-validated through cholesterol staining with filipin. Cholesterol quantification was obtained using an 18.8-T NMR spectrometer from the lipid extracts of the brain tissues from another group of mice (N = 3). The cholesterol content in the cultured cells was manipulated using methyl-ß-cyclodextrin and a complex of cholesterol and methyl-ß-cyclodextrin. The rNOE(-1.6) of the cell homogenates and their cholesterol levels were measured using a 9.4-T NMR spectrometer. The rNOE(-1.6) signal is hypointense in the C6 tumors of mice, which matches the filipin staining results, suggesting that their tumor region is cholesterol deficient. The tissue extracts also indicate less cholesterol and phosphatidylcholine contents in tumors than in normal brain tissues. The amplitude of rNOE(-1.6) is positively correlated with the cholesterol concentration in the cholesterol-manipulated cell cultures. Our results indicate that the cholesterol dependence of rNOE(-1.6) occurs in cell cultures and solid tumors of C6 glioma. Furthermore, when the concentration of phosphatidylcholine is carefully considered, rNOE(-1.6) can be developed as a cholesterol-weighted imaging technique.

Testing the predictive ability of the "spinal cord injury equation" in estimating vancomycin clearance.

PURPOSE: A new method of estimating drug clearance in patients with spinal cord injury (SCI) was tested against other methods through a retrospective analysis of its predictive ability in estimating vancomycin clearance. METHODS: A retrospective chart-based investigation was conducted at a hospital with a large SCI population. Using data collected from electronic patient records, 179 cases were reviewed, and 87 cases met the inclusion criteria: a diagnosis of longstanding SCI, administration of vancomycin via i.v. infusion during the 18-month study period, and at least one documented steady-state vancomycin concentration. Using the "spinal cord injury equation" (i.e., CL(SCI) equation), pharmacokinetic analysis was performed to determine patient-specific clearance values, which were compared to values resulting from five widely used methods of estimating the glomerular filtration rate in spinal cord-injured patients. The primary outcome measures were bias and precision, as indicated by statistical analyses to determine the mean prediction error (ME) and the square root of the mean squared prediction error (RMSE) of each method. RESULTS: Compared with the other evaluated methods, the CL(SCI) equation was found to be less biased and more precise, with the smallest calculated ME and RMSE values (p < 0.05). The five alternative methods significantly overestimated vancomycin clearance, by 45-92% (p < 0.05). The CL(SCI) method underestimated vancomycin clearance (by 6%) but not to a significant degree (p = 0.06). CONCLUSION: The study results suggest that the CL(SCI) equation for predicting vancomycin clearance was unbiased and may be more precise relative to other frequently used methods in the study population of patients with long-term SCI.