posted on 2020-03-23, 18:08authored byRam Saraswat, Ishara Ratnayake, E. Celeste Perez, William M. Schutz, Zhengtao Zhu, S. Phillip Ahrenkiel, Scott T. Wood
One
major limitation hindering the translation of in vitro osteoarthritis
research into clinical disease-modifying therapies is that chondrocytes
rapidly spread and dedifferentiate under standard monolayer conditions.
Current strategies to maintain rounded morphologies of chondrocytes
in culture either unnaturally restrict adhesion and place chondrocytes
in an excessively stiff mechanical environment or are impractical
for use in many applications. 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
24 h. Chondrocytes maintained their rounded morphology in CellWells
(mean aspect ratio of 0.87 ± 0.1 vs three-dimensional (3D) control
[0.86 ± 0.1]) more effectively than those seeded under standard
conditions (0.65 ± 0.3), with average viability of >85%. 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. Through its ease of use and ability to maintain
the physiological morphology of chondrocytes, we expect that the CellWell
will enhance the clinical translatability of future studies conducted
using this culture platform.