Performance Trade-offs in In Situ Chemostat NMR

Abstract

Investigating cell cultures with NMR requires high cell densities to provide adequate signal-to-noise, or scans must be summed over long time periods and short-term events are lost. The mixing within a chemostat can be used to shorten the time required to acquire informative in situ NMR spectra from cell cultures. However, performance trade-offs can occur between net signal, spectral resolution, and oxygenation due to sampling volume, conductivity, gas bubble, and fluid flow effects. These trade-offs and the effect of different mixing regimes were theoretically analyzed to quantify how device design decisions impact performance. The results were found to concur with data from cell-free NMR experiments performed in 18 mS/cm conductivity medium. The results also guided the redesign of an NMR bioreactor in terms of relative radio frequency (rf) coil and sample dimensions and other factors. The design, which entails using chemostat mixing to shunt sample through a rf coil in ca. 0.4 s, provides adequate oxygenation for the 4-16% (v/v) cell suspensions examined. Gains realized include lower conductive losses, better magnetic field homogeneity, and the exclusion of gas bubbles from the sampling zone. These gains enable the acquistion of spectra from dilute (3-4% v/v) Saccharomyces cerevisiae chemostat cultures in 6.9 min with high resolution in both the orthophosphate and the beta-NTP regions. Samples with 16% (v/v) cells also yield useful spectra within 0.5-1.0 min.

Development of a small-scale bioreactor: Application to in vivo NMR measurement

“…In addition, the smallscale bioreactor has to contain enough cells to be capable of performing noninvasive in vivo as well as ex vivo assays. Current noninvasive in vivo assays or those under development are based on highly sophisticated analytical equipment, such as optical sensors for UV, IR, Raman, and fluorescence spectroscopy, in situ microscopy (Ulber et al, 2003), dielectric spectroscopy (Ducommun et al, 2002), and in vivo nuclear magnetic resonance (NMR) (Castro et al, 1999). These different analytical techniques are highly complementary to obtain an adequate description of cell population physiological, nutritional, and metabolic states and dynamic behaviors.…”

In SituNMR Systems

“…(6) is defined in Eq. (7) as [7] where the parentheses denote activities. In solution, the two ionic species of inorganic phosphate interconvert rapidly (about 10 9 -10 10 s -1 ), and consequently are in fast exchange on the NMR timescale, so that a single resonance will be observed for inorganic phosphate according to Eq.…”

“…The resonances for fructose near 82 ppm indicate 5-ring tautomers of fructose and the resonance at 99 ppm indicates the 6-ring tautomers of fructose. Specific consumption rates of the α and β anomers of glucose can be determined easily from [1][2][3][4][5][6][7][8][9][10][11][12][13] C] labeled glucose, for example as was done in NMR experiments of yeast (26).…”

“…Many in situ reactors are based on simple modification to the sample tube configuration. The probe configuration can be more radically altered by placing the radiofrequency coil inside a micro-reactor (4) and although the sensitivity is improved, the resolution suffers (7).…”

“…This phenomena contributes to the feasibility of continuous-flow reactors. Impressively, a chemostat reactor for the NMR have been constructed so that cells in a given growth state can be studied (4,7). Simpler batch reactor designs are also available.…”