title: Fibroblasts maintained in 3 dimensions show a better differentiation state and higher sensitivity to estrogens.
abstract: Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related to cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of β-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential.
authors.affiliation: Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Montani
authors.affiliation: Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia, Italy.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Steimberg
authors.affiliation: Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia, Italy.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Boniotti
authors.affiliation: Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia, Italy; Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Biasiotto
authors.affiliation: Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia, Italy; Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
authors.forename: Isabella
authors.initials: I
authors.lastname: Zanella
authors.affiliation: Integrated Systems Engineering (ISE), Milan, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Diafera
authors.affiliation: IRGB-CNR, Milan, Italy; IRCCS-Multimedica, Milan, Italy.
authors.forename: Ida
authors.initials: I
authors.lastname: Biunno
authors.affiliation: Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia, Italy; Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Caimi
authors.affiliation: Laboratory of Tissue Engineering, Anatomy and Physiopathology Unit, Department of Clinical and Experimental Sciences, School of Medicine, University of Brescia, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Mazzoleni
authors.affiliation: Laboratory of Biotechnology, Department of Laboratory Medicine, Civic Hospital of Brescia, Italy. Electronic address: diego.dilorenzo@yahoo.it.
authors.forename: Diego
authors.initials: D
authors.lastname: Di Lorenzo
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D004967:Estrogens / Q000502:physiology*; D005109:Extracellular Matrix / Q000502:physiology*; D005347:Fibroblasts / Q000502:physiology*; D020869:Gene Expression Profiling; D051379:Mice; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D004967:Estrogens; D012313:RNA
keywords: 3 dimension; Bioreactor; ECM; Estrogen receptor; Fibroblasts; Microgravity