title: Variability of permeability estimation from different protocols of subculture and transport experiments in cell monolayers.
abstract: BACKGROUND
  In vitro models with high predictive ability have been revealed as strong tools for pharmaceutical industry. However, the variability in permeability estimations complicates the comparison and combination of data from different laboratories and it makes necessary the careful validation of the model and the continuous suitability demonstration. The adequate standardization of pre-experimental, experimental and post-experimental factors might help to reduce the inter- and intra-laboratory variability in permeability values.
  
  
  METHODS
  The objective of this paper is the evaluation of the effect of passage number, experimental protocol, time after seeding and calculation method on the permeability values and their variability in transport experiments in Caco-2, MDCK and MDCK-MDR1 cells. Metoprolol, Lucifer yellow and Rhodamine-123 were used to check the performance of the cell lines. Protocols used differ mainly in the differentiation time and the filter support coating with collagen. Data was analyzed with sink and non-sink approaches. The final purpose was to explore pre-experimental, experimental and post-experimental conditions in order to select the best experimental scenarios for permeability assays.
  
  
  RESULTS
  Results indicated that for passive diffusion studies, coating helps cell differentiation in a more stable manner in all cell lines compared to protocol without coating which showed permeability changes with passages and more variable values. In both protocols the paracellular route became more restricted with higher passage numbers. Functionality of P-gp assessed with Rhodamine permeability did not change with passage number in Caco-2 cells with any of the protocols but increased in both protocols in MDCK and MDCK-MDR1 cells. Protocol without coating showed the less variable results in these cell lines. Rhodamine permeabilities increased with higher maturation times due to a higher expression of the transporter. Nevertheless for compounds absorbed by passive diffusion there was not a clear trend neither in permeability values nor in variability.
  
  
  CONCLUSIONS
  As a conclusion, we have confirmed the influence of maturation conditions and passage number in permeability values and in their variability. Based on our results protocol with coating would be more adequate for studies of compounds absorbed by passive diffusion but the protocol without coating gave us better results for studies about P-gp interactions.
authors.affiliation: Pharmaceutics and Pharmaceutical Technology Department, University of Valencia, Spain.
authors.forename: Davinia
authors.initials: D
authors.lastname: Oltra-Noguera
authors.affiliation: Pharmaceutics and Pharmaceutical Technology Department, University of Valencia, Spain; Department of Engineering, Pharmaceutics and Pharmaceutical Technology Area, Universidad Miguel Hernández de Elche, Elche, Spain.
authors.forename: Victor
authors.initials: V
authors.lastname: Mangas-Sanjuan
authors.affiliation: Pharmaceutics and Pharmaceutical Technology Department, University of Valencia, Spain.
authors.forename: Amparo
authors.initials: A
authors.lastname: Centelles-Sangüesa
authors.affiliation: Pharmaceutics and Pharmaceutical Technology Department, University of Valencia, Spain; Department of Engineering, Pharmaceutics and Pharmaceutical Technology Area, Universidad Miguel Hernández de Elche, Elche, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Gonzalez-Garcia
authors.affiliation: Pharmaceutics and Pharmaceutical Technology Department, University of Valencia, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Sanchez-Castaño
authors.affiliation: Department of Engineering, Pharmaceutics and Pharmaceutical Technology Area, Universidad Miguel Hernández de Elche, Elche, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Gonzalez-Alvarez
authors.affiliation: Pharmaceutics and Pharmaceutical Technology Department, University of Valencia, Spain.
authors.forename: Vicente-German
authors.initials: VG
authors.lastname: Casabo
authors.affiliation: Pharmaceutics and Pharmaceutical Technology Department, University of Valencia, Spain.
authors.forename: Virginia
authors.initials: V
authors.lastname: Merino
authors.affiliation: Department of Engineering, Pharmaceutics and Pharmaceutical Technology Area, Universidad Miguel Hernández de Elche, Elche, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Gonzalez-Alvarez
authors.affiliation: Department of Engineering, Pharmaceutics and Pharmaceutical Technology Area, Universidad Miguel Hernández de Elche, Elche, Spain. Electronic address: mbermejo@goumh.umh.es.
authors.forename: Marival
authors.initials: M
authors.lastname: Bermejo
mesh_terms: D000818:Animals; D001692:Biological Transport; D018938:Caco-2 Cells; D018929:Cell Culture Techniques; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004285:Dogs; D006801:Humans; D061985:Madin Darby Canine Kidney Cells
keywords: Caco-2; Cell monolayer; Culture standardization; MDCK; Permeability; Variability