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 CyBi™-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. (Journal of Biomolecular Screening 2007:956-965)

Biomek Cell Workstation: A Flexible System for Automated 3D Cell Cultivation

“…However, published papers support manual cell detachment followed by automated bead formation with different alginate concentrations using encapsulators or droplet generators. 17,[19][20][21] Concerning this matter, Coward et al produced HeLa cell-alginate beads by dropping a cell-alginate solution in a stirred solution (0.204 M CaCl 2 in 0.15 M NaCl buffer) with a syringe pump of an Inotech IER-20 encapsulator (Inotech, Dottikon, Switzerland) at a flow rate of 5 mL of cell suspension/min using a 200 mm nozzle vibrating at 1295 Hz. 17 Kim et al manufactured the beads in a petri dish with human embryonic stem cells (hESCs) by dropping a cellalginate (1.1%) suspension into a 100 mM CaCl 2 /10 mM HEPES solution by an air-driven droplet generator (Nisco Engineering, Zurich, Switzerland) at a flow rate of 8 L/mL by a pressure of 100 kPa.…”

“…Nevertheless, the literature supports pellet formation consisting of bone marrowderived mesenchymal stem cells, 19 human nasal cartilage cells, 20 and cartilage cells from porcine femur bones. 21 The 3D constructs were generated with and without carrier material for different applications and cell lines. Regarding this, spheroid cultures [15][16][17][18] and pellet cultures [19][20][21] are 3D cell formats without carrier material.…”

“…21 The 3D constructs were generated with and without carrier material for different applications and cell lines. Regarding this, spheroid cultures [15][16][17][18] and pellet cultures [19][20][21] are 3D cell formats without carrier material. This is realizable by the domination of cell-cell contacts over cell-matrix bonds.…”

“…• Automated cell seeding by CyBi-Disk workstation 21 consequently followed the manual or automated cell seeding of cell suspension. The pellets were formed over time.…”

“…15,16 The alginate bead manufacturing is realized manually in six-well plates 13 or mainly in petri dishes by semiautomated formation processes. 17,[19][20][21] Regarding this, the cell detachment processes were manually performed followed by the automated droplet formation. Spheroids are self-aggregated clusters by domination of cell-cell interactions.…”