title: Culture of human cells and synthesis of extracellular matrix on materials compatible with direct analysis by mass spectrometry.
abstract: The extracellular matrix (ECM) is a complex three-dimensional network of macromolecules synthesized by cells and is essential for the structure and the function of a tissue. The aim of our approach was to propose a surface allowing cell culture and subsequent analysis of ECM produced by cells directly on materials compatible with Surface Enhanced Laser Desorption Ionization-Time Of Flight (SELDI-TOF) mass spectrometry on a 96-well format. Surfaces were made of aluminium and spots of 2 mm in diameter were covered with specific chemical groups (silica, C(6) and C(12) alkyl groups, carboxyl, quaternary amine, or nitrilotriacetic acid groups). We found that among the chemically modified aluminium spots, only silica groups allowed the culture of human vascular cells. The wettability was an essential parameter for cell culture on the surfaces. Indeed, cells could only be cultured on surfaces presenting a moderate wettability with water contact angles of ca. 60 degrees. Then, by treatment of confluent cells with detergents (Triton X100 and deoxycholate), we were able to obtain ECM on the surfaces that were subsequently analyzed using a mass spectrometer, which is currently impossible with any type of cell culture system. As an example, the analysis of ECM from human vascular smooth muscle cells (hVSMCs) and human umbilical vein endothelial cells (HUVECs) appeared to be reproducible and evidenced different ECM patterns from the two cell types. Applications based on these materials can be proposed for biomarker discovery or characterization of cells for biomedical/diagnostic purposes.
authors.affiliation: Inserm U698, Cardiovascular Bio-engineering, X. Bichat Hospital, University Paris 7, Paris, F-75018, France.
authors.forename: Damien
authors.initials: D
authors.lastname: Lavigne
authors.forename: Luc
authors.initials: L
authors.lastname: Guerrier
authors.forename: Virginie
authors.initials: V
authors.lastname: Gueguen
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Michel
authors.forename: Egisto
authors.initials: E
authors.lastname: Boschetti
authors.forename: Olivier
authors.initials: O
authors.lastname: Meilhac
authors.forename: Didier
authors.initials: D
authors.lastname: Letourneur
mesh_terms: D000535:Aluminum / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D002478:Cells, Cultured; D003902:Detergents / Q000737:chemistry; D005109:Extracellular Matrix / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D010455:Peptides / Q000032:analysis; D040901:Proteomics; D012822:Silicon Dioxide / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013499:Surface Properties; D014357:Trypsin / Q000378:metabolism; D017155:Wettability
chemical_list: D000588:Amines; D003902:Detergents; D010455:Peptides; D012822:Silicon Dioxide; D000535:Aluminum; D014357:Trypsin; D009571:Nitrilotriacetic Acid