title: Metabolomics guides rational development of a simplified cell culture medium for drug screening against Trypanosoma brucei.
abstract: In vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra- and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; α = 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
authors.forename: Darren J
authors.initials: DJ
authors.lastname: Creek
authors.forename: Brunda
authors.initials: B
authors.lastname: Nijagal
authors.forename: Dong-Hyun
authors.initials: DH
authors.lastname: Kim
authors.forename: Federico
authors.initials: F
authors.lastname: Rojas
authors.forename: Keith R
authors.initials: KR
authors.lastname: Matthews
authors.forename: Michael P
authors.initials: MP
authors.lastname: Barrett
mesh_terms: D000818:Animals; D003470:Culture Media / Q000737:chemistry*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D020128:Inhibitory Concentration 50; D055442:Metabolome / Q000187:drug effects*; D055432:Metabolomics / Q000379:methods*; D021261:Parasitic Sensitivity Tests / Q000379:methods; D010419:Pentamidine / Q000494:pharmacology; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism
chemical_list: D003470:Culture Media; D014344:Trypanocidal Agents; D010419:Pentamidine