title: Development of a high-throughput screening assay based on the 3-dimensional pannus model for rheumatoid arthritis.
abstract: The 3-dimensional (3-D) pannus model for rheumatoid arthritis (RA) is based on the interactive co-culture of cartilage and synovial fibroblasts (SFs). Besides the investigation of the pathogenesis of RA, it can be used to analyze the active profiles of antirheumatic pharmaceuticals and other bioactive substances under in vitro conditions. For a potential application in the industrial drug-screening process as a transitional step between 2-dimensional (2-D) cell-based assays and in vivo animal studies, the pannus model was developed into an in vitro high-throughput screening (HTS) assay. Using the CyBitrade mark-Disk workstation for parallel liquid handling, the main cell culture steps of cell seeding and cultivation were automated. Chondrocytes were isolated from articular cartilage and seeded directly into 96-well microplates in high-density pellets to ensure formation of cartilage-specific extracellular matrix (ECM). Cell seeding was performed automatically and manually to compare both processes regarding accuracy, reproducibility, consistency, and handling time. For automated cultivation of the chondrocyte pellet cultures, a sequential program was developed using the CyBio Control software to minimize shear forces and handling time. After 14 days of cultivation, the pannus model was completed by coating the cartilage pellets with a layer of human SFs. The effects due to automation in comparison to manual handling were analyzed by optical analysis of the pellets, histological and immunohistochemical staining, and real-time PCR. Automation of this in vitro model was successfully achieved and resulted in an improved quality of the generated pannus cultures by enhancing the formation of cartilage-specific ECM. In addition, automated cell seeding and media exchange increased the efficiency due to a reduction of labor intensity and handling time.
authors.affiliation: Department of Bioprocess Engineering, Institute of Biotechnology, Technical University of Berlin, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Ibold
authors.forename: Simone
authors.initials: S
authors.lastname: Frauenschuh
authors.forename: Christian
authors.initials: C
authors.lastname: Kaps
authors.forename: Michael
authors.initials: M
authors.lastname: Sittinger
authors.forename: Jochen
authors.initials: J
authors.lastname: Ringe
authors.forename: Peter M
authors.initials: PM
authors.lastname: Goetz
mesh_terms: D000818:Animals; D001172:Arthritis, Rheumatoid / Q000473:pathology*; D001483:Base Sequence; D018920:Coculture Techniques; D017931:DNA Primers; D007150:Immunohistochemistry; D008954:Models, Biological; D016133:Polymerase Chain Reaction; D013552:Swine
chemical_list: D017931:DNA Primers