Improved zonal chondrocyte production protocol integrating size-based inertial spiral microchannel separation and dynamic microcarrier culture for clinical application.

The zonal property of articular cartilage endows the tissue with biphasic mechanical properties to withstand shearing force and compressional loading. Current treatments for articular cartilage damage are not able to efficiently restore the zonal organisation and functionality. Size-based sorting of freshly isolated chondrocytes from full thickness (FT) cartilage using a spiral microfluidic device was shown to efficiently separate and enrich zonal chondrocytes. The translational application of this sorting protocol is challenging in the clinical setting due to the limited number of autologous chondrocytes from a patient. It is thus essential to explore the practicability of this sorting protocol on expanded chondrocytes. In this study, we first show that size-sorted zonal chondrocytes expanded on microcarriers in dynamic condition (dMC) were able to support comparable proliferation, while maintaining cell morphology, and the zonal cell size-phenotype relation, in contrast to expansion on a tissue culture plate. We further show that post-expansion size-based sorting can be applied on dMC-expanded FT chondrocytes, generating enriched zonal subpopulations that form phenotypically distinct cartilage constructs in the 3D hydrogel. This study demonstrates a novel scale-up zonal chondrocyte production protocol, incorporating size-based zonal chondrocyte separation and dMC platform, to maintain zonal chondrocytes' phenotypes better to support zonal repair of articular cartilage.