A novel micropatterned substrate for maintaining chondrocyte phenotype

Author: Ram Saraswat, PhD Student in the Department of Nanoscience and Nanoengineering

Mentor: Dr. Scott Wood, Department of Nanoscience and Nanoengineering

One major limitation hindering the translation of in vitro osteoarthritis research into successful clinical disease-modifying therapies is that chondrocytes rapidly spread and lose their phenotype under standard monolayer conditions. Current strategies to maintain rounded morphologies of chondrocytes in culture either unnaturally restrict adhesion or are impractical for use in many assays to study the transient cell signaling events that regulate tissue health. To address the limitations of current techniques, we have developed a unique composite thin-film cell culture platform, the CellWell, to model articular cartilage that utilizes micropatterned hemispheroidal wells, precisely sized to fit individual cells (12–18 μm diameters), to promote physiologically spheroidal chondrocyte morphologies while maintaining compatibility with standard cell culture and analytical techniques. CellWells were constructed of 15-μm-thick 5% agarose films embedded with electrospun poly(vinyl alcohol) (PVA) nanofibers. Transmission electron microscope (TEM) images of PVA nanofibers revealed a mean diameter of 60.9 ± 24 nm, closely matching the observed 53.8 ± 29 nm mean diameter of human ankle collagen II fibers. Using AFM nanoindentation, CellWells were found to have compressive moduli of 158 ± 0.60 kPa at 15 μm/s indentation, closely matching published stiffness values of the native pericellular matrix. Primary human articular chondrocytes taken from ankle cartilage were seeded in CellWells and assessed at four weeks. Chondrocytes maintained their rounded morphology in CellWells (mean aspect ratio of 0.87 ± 0.1) more effectively than those seeded under standard conditions (0.65 ± 0.3). The CellWell’s design, with open, hemispheroidal wells in a thin film substrate of physiological stiffness, combines the practical advantages of two-dimensional (2D) culture systems with the physiological advantages of 3D systems. Future work will be focused on validating the maintenance of chondrocyte phenotypic markers in the CellWell.