title: High-Throughput Screening Assay for the Identification of Compounds Enhancing Collagenous Extracellular Matrix Production by ATDC5 Cells.
abstract: Extracellular matrix (ECM) provides not only structural support to the cells but also signals that regulate their fate. In many tissue engineering approaches, it remains challenging to achieve the right amount and type of ECM secreted by the cells to faithfully mimic the native tissue. In this article, we describe how to design and perform a high-throughput assay to screen for molecules capable of enhancing collagenous ECM (cECM) production. We chose ATDC5 cells to validate the assay since we want to use this chondrogenic cell line later for tissue engineering of hypertrophic cartilage. We used a fluorescently labeled collagen-binding probe to quantify total collagen content in ATDC5 cultures. The LOPAC(1280) library of pharmaceutically active compounds was screened using insulin (a known inducer of cECM in ATDC5 cells) as positive control. After screening and validation, the small-molecule tetradecylthioacetic acid (TTA) was shown to enhance cECM production by ATDC5 cells at both gene expression and protein level. Moreover, when combined with insulin, TTA showed a synergistic effect on cECM production. In contrast, exposure of human primary chondrocytes and human mesenchymal stromal cells to TTA did not induce cECM secretion. In conclusion, we have developed an HTS assay to screen for compounds capable of enhancing cECM production and discovered TTA as a potent enhancer of cECM secretion by ATDC5 cells.
authors.affiliation: 1 Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, The Netherlands .
authors.forename: Bach Q
authors.initials: BQ
authors.lastname: Le
authors.affiliation: 1 Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, The Netherlands .
authors.forename: Hugo
authors.initials: H
authors.lastname: Fernandes
authors.affiliation: 2 Laboratory for Cell and Tissue Engineering, Department of Biomedical Engineering, Eindhoven University of Technology , Eindhoven, The Netherlands .
authors.forename: Carlijn V C
authors.initials: CV
authors.lastname: Bouten
authors.affiliation: 3 Department of Developmental BioEngineering, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, The Netherlands .
authors.forename: Marcel
authors.initials: M
authors.lastname: Karperien
authors.affiliation: 1 Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, The Netherlands .
authors.forename: Clemens
authors.initials: C
authors.lastname: van Blitterswijk
authors.affiliation: 1 Department of Tissue Regeneration, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente , Enschede, The Netherlands .
authors.forename: Jan
authors.initials: J
authors.lastname: de Boer
mesh_terms: D002356:Cartilage / Q000166:cytology; D002454:Cell Differentiation; D002460:Cell Line; D003094:Collagen / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism*; D015870:Gene Expression; D057166:High-Throughput Screening Assays; D006801:Humans; D023822:Tissue Engineering
chemical_list: D003094:Collagen