Profiling Convoluted Single-Dimension Proton NMR Spectra: A Plackett–Burman Approach for Assessing Quantification Error of Metabolites in Complex Mixtures with Application to Cell Culture

Single-dimension hydrogen, or proton, nuclear magnetic resonance spectroscopy (1D-¹H NMR) has become an attractive option for characterizing the full range of components in complex mixtures of small molecular weight compounds due to its relative simplicity, speed, spectral reproducibility, and noninvasive sample preparation protocols compared to alternative methods. One challenge associated with this method is the overlap of NMR resonances leading to “convoluted” spectra. While this can be mitigated through “targeted profiling”, there is still the possibility of increased quantification error. This work presents the application of a Plackett–Burman experimental design for the robust estimation of precision and accuracy of 1D-¹H NMR compound quantification in synthetic mixtures, with application to mammalian cell culture supernatant. A single, 20 sample experiment was able to provide a sufficient estimate of bias and variability at different metabolite concentrations. Two major sources of bias were identified: incorrect interpretation of singlet resonances and the quantification of resonances from protons in close proximity to labile protons. Furthermore, decreases in measurement accuracy and precision could be observed with decreasing concentration for a small fraction of the components as a result of their particular convolution patterns. Finally, the importance of a priori concentration estimates is demonstrated through the example of interpreting acetate metabolite trends from a bioreactor cultivation of Chinese hamster ovary cells expressing a recombinant antibody

Manipulating mammalian cell morphologies using chemical-mechanical polished integrated circuit chips

<p>Tungsten chemical-mechanical polished integrated circuits were used to study the alignment and immobilization of mammalian (Vero) cells. These devices consist of blanket silicon oxide thin films embedded with micro- and nano-meter scale tungsten metal line structures on the surface. The final surfaces are extremely flat and smooth across the entire substrate, with a roughness in the order of nanometers. Vero cells were deposited on the surface and allowed to adhere. Microscopy examinations revealed that cells have a strong preference to adhere to tungsten over silicon oxide surfaces with up to 99% of cells adhering to the tungsten portion of the surface. Cells self-aligned and elongated into long threads to maximize contact with isolated tungsten lines as thin as 180 nm. The orientation of the Vero cells showed sensitivity to the tungsten line geometric parameters, such as line width and spacing. Up to 93% of cells on 10 μm wide comb structures were aligned within ± 20° of the metal line axis. In contrast, only ~22% of cells incubated on 0.18 μm comb patterned tungsten lines were oriented within the same angular interval. This phenomenon is explained using a simple model describing cellular geometry as a function of pattern width and spacing, which showed that cells will rearrange their morphology to maximize their contact to the embedded tungsten. Finally, it was discovered that the materials could be reused after cleaning the surfaces, while maintaining cell alignment capability.</p

Concentration change of pyridoxamine with respect to UV fluence for oxygen-saturated (O2) and nitrogen-saturated (N2) media.

<p>Concentration change of pyridoxamine with respect to UV fluence for oxygen-saturated (O2) and nitrogen-saturated (N2) media.</p

Comparison of the cell culture performance.

<p>A) Time-course of viable cell density for cells grown in different fluence UV-treated nitrogen-saturated (N2) or oxygen-saturated media (O2) media. B) Total cell densities reached on Day 5 (end of exponential phase for most cultures). C) Viability of cells on Day 5. D) Total viable cell density. E) Growth rate during exponential growth phase (Days 1 through 5). The error bars represent the standard deviation for N = 6. *—Significant difference when compared to control for the same gas treatment group. #—Significant difference between N₂- and O₂-saturated groups at an individual fluence level.</p

Concentration change with respect to UV fluence for oxygen-saturated (light gray triangles) and nitrogen-saturated (dark gray circles) media for compounds with statistically significant regression trends.

<p>The error bars represent the standard deviation where N = 10.</p

Experimental apparatus.

<p>Experimental apparatus.</p

Reduction equivalent fluence (REF) based on MS2 bioassay of irradiated CD-CHO media with one UV reactor at two flow rates (50 mL/min and 100 mL/min) for both oxygen- and nitrogen-saturated media.

<p>Reduction equivalent fluence (REF) based on MS2 bioassay of irradiated CD-CHO media with one UV reactor at two flow rates (50 mL/min and 100 mL/min) for both oxygen- and nitrogen-saturated media.</p

Fluence rates estimated based on the single reactor pass clearance of MS2 at 50 mL/min.

<p>Fluence rates estimated based on the single reactor pass clearance of MS2 at 50 mL/min.</p

Concentration change of tryptophan, kynurenine, riboflavin, and lumichrome with respect to UV fluence for oxygen-saturated (O2) and nitrogen-saturated (N2) media.

<p>*—Significant difference between N₂ and O₂ saturated groups at an individual fluence level.</p