title: Robust Generation of Quiescent Porcine Valvular Interstitial Cell Cultures.
abstract: BACKGROUND
  Valvular interstitial cells (VICs) in the healthy aortic valve leaflet exhibit a quiescent phenotype, with <5% of VICs exhibiting an activated phenotype. Yet, in vitro culture of VICs on tissue culture polystyrene surfaces in standard growth medium results in rapid transformation to an activated phenotype in >90% of cells. The inability to preserve a healthy VIC phenotype during in vitro studies has hampered the elucidation of mechanisms involved in calcific aortic valve disease. This study describes the generation of quiescent populations of porcine VICs in 2-dimensional in vitro culture and their utility in studying valve pathobiology.
  
  
  RESULTS
  Within 4 days of isolation from fresh porcine hearts, VICs cultured in standard growth conditions were predominantly myofibroblastic (activated VICs). This myofibroblastic phenotype was partially reversed within 4 days, and fully reversed within 9 days, following application of a combination of a fibroblast media formulation with culture on collagen coatings. Specifically, culture in this combination significantly reduced several markers of VIC activation, including proliferation, apoptosis, α-smooth muscle actin expression, and matrix production, relative to standard growth conditions. Moreover, VICs raised in a fibroblast media formulation with culture on collagen coatings exhibited dramatically increased sensitivity to treatment with transforming growth factor β1, a known pathological stimulus, compared with VICs raised in either standard culture or medium with a fibroblast media formulation.
  
  
  CONCLUSIONS
  The approach using a fibroblast media formulation with culture on collagen coatings generates quiescent VICs that more accurately mimic a healthy VIC population and thus has the potential to transform the study of the mechanisms of VIC activation and dysfunction involved in the early stages of calcific aortic valve disease.
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Porras
authors.affiliation: Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors.forename: Nicole C A
authors.initials: NC
authors.lastname: van Engeland
authors.affiliation: Department of Chemical and Biological Engineering, Colorado School of Mines, Golden, CO.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Marchbanks
authors.affiliation: Tissue Engineering, Magdi Yacoub Institute, Imperial College, London, United Kingdom.
authors.forename: Ann
authors.initials: A
authors.lastname: McCormack
authors.affiliation: Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands.
authors.forename: Carlijn V C
authors.initials: CV
authors.lastname: Bouten
authors.affiliation: Tissue Engineering, Magdi Yacoub Institute, Imperial College, London, United Kingdom.
authors.forename: Magdi H
authors.initials: MH
authors.lastname: Yacoub
authors.affiliation: Tissue Engineering, Magdi Yacoub Institute, Imperial College, London, United Kingdom n.latif@imperial.ac.uk kmasters@wisc.edu.
authors.forename: Najma
authors.initials: N
authors.lastname: Latif
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin-Madison, Madison, WI n.latif@imperial.ac.uk kmasters@wisc.edu.
authors.forename: Kristyn S
authors.initials: KS
authors.lastname: Masters
mesh_terms: D000818:Animals; D001021:Aortic Valve / Q000378:metabolism* / Q000473:pathology*; D001024:Aortic Valve Stenosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D002114:Calcinosis / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D049109:Cell Proliferation; D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D005353:Fibronectins / Q000378:metabolism*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D007150:Immunohistochemistry; D008297:Male; D009124:Muscle Proteins / Q000096:biosynthesis / Q000235:genetics*; D058628:Myofibroblasts / Q000378:metabolism* / Q000473:pathology; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine
chemical_list: D015415:Biomarkers; D016207:Cytokines; D005353:Fibronectins; D009124:Muscle Proteins; D012313:RNA
keywords: aortic valve; calcific aortic valve disease; differentiation; myofibroblasts; quiescence; valvular interstitial cell