title: Multilayer spheroids to quantify drug uptake and diffusion in 3D.
abstract: There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil's inhibition of Pgp and GJIC were similar (IC50: 8 μM). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.
authors.affiliation: Department of Molecular Pharmacology, Physiology and Biotechnology, ‡Center for Biomedical Engineering, and §School of Engineering, Brown University , Providence, Rhode Island 02912, United States.
authors.forename: Toni-Marie
authors.initials: TM
authors.lastname: Achilli
authors.forename: Stephanie
authors.initials: S
authors.lastname: McCalla
authors.forename: Julia
authors.initials: J
authors.lastname: Meyer
authors.forename: Anubhav
authors.initials: A
authors.lastname: Tripathi
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Morgan
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism; D002229:Carbenoxolone / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D016572:Cyclosporine / Q000378:metabolism; D004058:Diffusion; D005452:Fluoresceins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008139:Loperamide / Q000378:metabolism; D061986:MCF-7 Cells; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D018874:Spheroids, Cellular / Q000378:metabolism*; D014700:Verapamil / Q000378:metabolism
chemical_list: D018435:ATP Binding Cassette Transporter, Subfamily B; D005452:Fluoresceins; D004364:Pharmaceutical Preparations; D008139:Loperamide; D016572:Cyclosporine; D014700:Verapamil; D002229:Carbenoxolone; C007740:fluorexon