Fast Filtration of Bacterial or Mammalian Suspension Cell Cultures for Optimal Metabolomics Results

Abstract

The metabolome offers real time detection of the adaptive, multi-parametric response of the organisms to environmental changes, pathophysiological stimuli or genetic modifications and thus rationalizes the optimization of cell cultures in bioprocessing. In bioprocessing the measurement of physiological intracellular metabolite levels is imperative for successful applications. However, a sampling method applicable to all cell types with little to no validation effort which simultaneously offers high recovery rates, high metabolite coverage and sufficient removal of extracellular contaminations is still missing. Here, quenching, centrifugation and fast filtration were compared and fast filtration in combination with a stabilizing washing solution was identified as the most promising sampling method. Different influencing factors such as filter type, vacuum pressure, washing solutions were comprehensively tested. The improved fast filtration method (MxP® FastQuench) followed by routine lipid/polar extraction delivers a broad metabolite coverage and recovery reflecting well physiological intracellular metabolite levels for different cell types, such as bacteria (Escherichia coli) as well as mammalian cells chinese hamster ovary (CHO) and mouse myeloma cells (NS0).The proposed MxP® FastQuench allows sampling, i.e. separation of cells from medium with washing and quenching, in less than 30 seconds and is robustly designed to be applicable to all cell types. The washing solution contains the carbon source respectively the 13C-labeled carbon source to avoid nutritional stress during sampling. This method is also compatible with automation which would further reduce sampling times and the variability of metabolite profiling data.

Antibiotic tolerance is associated with a broad and complex transcriptional response in E. coli

“…Samples were treated with Tween (0.2%) and chloramphenicol (5 µg/ml) then immediately filtered out of solution (using rapid filtration similar to REFs 100 , 101 ) and resuspended in lysis buffer containing 25 mM Tris pH 8.0, 25 mM NH 4 Cl, 10 mM MgOAc, 0.8% Triton X-100, 0.1 U/µL RNase-free DNase I, 1 U/μL Superase-In, ~ 60 U/µl ReadyLyse Lysozyme, 1.55 mM Chloramphenicol, and 17 μΜ 5′-guanylyl imidodiphosphate (GMPPNP); lysis buffer was modified from REF 102 . Rapid filtration allows for the harvesting of any whole-cell; this minimizes RNA degradation because samples are immediately frozen in liquid nitrogen have suspension in lysis buffer.…”

Metabolite Profiling Reveals the Glutathione Biosynthetic Pathway as a Therapeutic Target in Triple-Negative Breast Cancer

“…Cells (5–6 biological replicates) were harvested at a density of ~ 70–80% at either 24 or 48 hours after seeding to lumox plates (Sarstedt) as previously described (8). Cells were washed twice with 0.9% NaCl and extracted in dichloromethane:ethanol (0.82:1) as previously described (9). Extracts were flash frozen in liquid nitrogen according to (10).…”

Antibiotic tolerance is associated with a broad and complex transcriptional response inE. coli

“…Samples were treated with Tween (0.2%) and chloramphenicol (5 µg/ml) then immediately filtered out of solution (using rapid filtration similar to REFs 100–101 ) and resuspended in lysis buffer containing 25 mM Tris pH 8.0, 25 mM NH 4 Cl, 10 mM MgOAc, 0.8% Triton X-100, 0.1 U/µL RNase-free DNase I, 1 U/μL Superase-In, ∼60 U/µl ReadyLyse Lysozyme, 1.55 mM Chloramphenicol, and 17 μM 5′-guanylyl imidodiphosphate (GMPPNP); lysis buffer was modified from REF 102 . Rapid filtration allows for the harvesting of any whole-cell; this minimizes RNA degradation because samples are immediately frozen in liquid nitrogen have suspension in lysis buffer.…”