title: High-throughput fluorescence imaging approaches for drug discovery using in vitro and in vivo three-dimensional models.
abstract: BACKGROUND
  High-resolution microscopy using fluorescent probes is a powerful tool to investigate individual cell structure and function, cell subpopulations and mechanisms underlying cellular responses to drugs. Additionally, responses to drugs more closely resemble those seen in vivo when cells are physically connected in three-dimensional (3D) systems (either 3D cell cultures or whole organisms), as opposed to traditional monolayer cultures. Combined, the use of imaging-based 3D models in the early stages of drug development has the potential to generate biologically relevant data that will increase the likelihood of success for drug candidates in human studies.
  
  
  METHODS
  The authors discuss current methods for the culturing of cells in 3D as well as approaches for the imaging of whole-animal models and 3D cultures that are amenable to high-throughput settings and could be implemented to support drug discovery campaigns. Furthermore, they provide critical considerations when discussing imaging these 3D systems for high-throughput chemical screenings.
  
  
  CONCLUSIONS
  Despite widespread understanding of the limitations imposed by the two-dimensional versus the 3D cellular paradigm, imaging-based drug screening of 3D cellular models is still limited, with only a few screens found in the literature. Image acquisition in high throughput, accurate interpretation of fluorescent signal, and uptake of staining reagents can be challenging, as the samples are in essence large aggregates of cells. The authors recognize these shortcomings that need to be overcome before the field can accelerate the utilization of these technologies in large-scale chemical screens.
authors.affiliation: a National Institutes of Health, National Center for Advancing Translational Sciences , Rockville, MD 20850, USA asimeono@mail.nih.gov.
authors.forename: Natalia J
authors.initials: NJ
authors.lastname: Martinez
authors.affiliation: a National Institutes of Health, National Center for Advancing Translational Sciences , Rockville, MD 20850, USA asimeono@mail.nih.gov.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Titus
authors.affiliation: a National Institutes of Health, National Center for Advancing Translational Sciences , Rockville, MD 20850, USA asimeono@mail.nih.gov.
authors.forename: Amanda K
authors.initials: AK
authors.lastname: Wagner
authors.affiliation: a National Institutes of Health, National Center for Advancing Translational Sciences , Rockville, MD 20850, USA asimeono@mail.nih.gov.
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D015195:Drug Design; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*
keywords: drug discovery; fluorescent imaging; high content; high-throughput screen; three-dimensional cellular models; whole-animal imaging