title: Variations of secretome profiles according to conditioned medium preparation: The example of human mesenchymal stem cell-derived adipocytes.
abstract: One challenging point in analyzing cellular secretome collected as conditioned medium is cross-contamination by cell culture media components, especially bovine serum proteins. A common approach for serum removal is to wash the cells, an alternative is to grow cells using serum-free conditions. Given that the sample processing may influence the phenotype of cells and thus the secretome, it is important to establish the optimal protocol for each cell type. In this study, we compared two methods for preparing conditioned medium from human adipocytes derived from mesenchymal stem cells. Cells were either washed twice with PBS or cultured the last four days of differentiation in serum-free adipogenic medium. Gene expression of the cells was evaluated by using real-time PCR and 1D LC-MS/MS was used to compare secreted proteins present in the culture supernatants. Surprisingly, results showed significant differences in gene expression patterns of the cells and in protein content of the conditioned media and suggested that PBS washes induced severe modifications of the phenotype of cells and thus changes in protein secretion profiles. These data emphasize the significant variations in protein species related to cell manipulations and underline the importance of procedure optimization prior to any proteomic investigation.
authors.affiliation: ULCO, PMOI, Boulogne-sur-mer, France.|University of Lille, PMOI Lille, France.
authors.forename: Aline
authors.initials: A
authors.lastname: Clabaut
authors.affiliation: ULCO, PMOI, Boulogne-sur-mer, France.|University of Lille, PMOI Lille, France.
authors.forename: Céline
authors.initials: C
authors.lastname: Grare
authors.affiliation: Mass spectrometry Laboratory, Institut Jacques Monod, UMR 7592, Univ Paris Diderot, CNRS, Paris, France.
authors.forename: Thibaut
authors.initials: T
authors.lastname: Léger
authors.affiliation: ULCO, PMOI, Boulogne-sur-mer, France.|University of Lille, PMOI Lille, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Hardouin
authors.affiliation: ULCO, PMOI, Boulogne-sur-mer, France.|University of Lille, PMOI Lille, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Broux
mesh_terms: D017667:Adipocytes / Q000187:drug effects* / Q000378:metabolism; D000328:Adult; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D005260:Female; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D020543:Proteome / Q000032:analysis* / Q000378:metabolism*; D040901:Proteomics; D055815:Young Adult
chemical_list: D017077:Culture Media, Conditioned; D020543:Proteome
keywords: Adipocyte; Conditioned medium; Mesenchymal stem cell; Secretome