673 - Replate: a multi-compression cartilage-on-chip model for medium-trhoughput mechano-response assessment

Purpose (the aim of the study): Arthritis is a degenerative joint disease affecting more than 10% of the world’s population. Within the joint, one of the primary tissues affected is the cartilage. Here, biomechanical stimulation, among other variables, plays a significant role in the development, progression and potential aggravation of the disease. It is still largely unknown how an inflammatory environment affects the mechano sensing of chondrocytes nor is it known how drug responsiveness is influenced by loading of the cartilage. Hence, we have engineered a user friendly, medium-throughput multi-compression cartilage-on-chip platform for addressing these questions.
Methods: The REPLATE multi-compression cartilage-on-chip comprises 3 cartridges (Figure 1). Each cartridge comprises 8 individual cartilage-on-chip units connected to a pressure system via a single inlet for simultaneous mechanical actuation. As the REPLATE hosts a maximum of 3 cartridges, a total of 24 cartilage-on-chip units per REPLATE can be tested simultaneously under three independent loading regimes. Each cartilage-on-chip unit presents a mechanical actuation unit, a cell-hydrogel chamber for loading human chondrocytes embedded in an appropriate hydrogel, and a perfusion channel. We first studied membrane deformation in the 8 units to check homogeneous mechanical loading in each of the cartridges. Here, pressures (0, 100, 200, 300, 400, 500, 600, 700 mbar) were applied using an external pressure pump. Different cell-hydrogel combinations including hyaluronic acid-tyramine / dextran-tyramine and agarose hydrogels were used. Deformation was assessed by measuring the displacement of 16 µm beads embedded in the hydrogel at day 0 (Figure not shown). Next, human chondrocytes and chondrosarcoma cells were mixed at increasing cell density with one of the 2 hydrogels after which the solutions were injected in the cell-hydrogel chamber of the cartilage on chip unit to assess cell survival and cartilage matrix production. Cell membrane disruption upon mechanical loading was assessed using a cell tracking dye. In addition, chondrocytes were cultured in the platform in static and dynamic conditions (0 mbar, 200 mbar, 400 mbar) until day 7 and phalloidin was used to stain the cytoskeleton. Lastly, qPCR was performed for the three conditions.
Results: The first multi-compression cartilage-on-chip model presenting up to 24 units fitting in the format of a 96 well plate was developed. Simultaneous mechanical stimulation of each of the cartilage on chip units in a cartridge was easily achieved via the common inlet. Limited variation was observed in membrane deformation between the individual units of a cartridge. This variation varied 10% for low pressures (0 to 300 mbar) and 15% for higher pressures (Figure not shown). Cell seeding was successful for different cell types (chondrocytes and chondrosarcoma), at different concentrations (5, 10, 25x106 cells/mL) and with different hydrogels (HA-TA/Dex-TA and agarose) (Figure 2). In each condition homogenous cell distribution was observed in the cell-hydrogel chamber of the chip. Cell tracker together with Phalloidin and DAPI staining showed cellular integrity as well as survival of the cells up to day 7 (Figure 3). Moreover, phalloidin/DAPI staining provided in-depth analysis of the morphological data at the single cell level. Lastly, ACAN, SOX9, COL2A1, COL1A1, COL10A1 were investigated at the qPCR level showing chondrocytes gene expression differences upon static (0 mbar) and dynamic (200 and 400 mbar).
Conclusions: We have successfully developed a medium throughput multi-compression model for the investigation of mechanobiology of chondrocytes and chondrosarcoma cells. The platform allows for real-time monitoring of the cells up to single cell level as well as to do on chip imaging at high resolution.