Fabrication Of Cellular Aggregates In Spinner Flasks Towards Cartilage Regeneration

Cell-based therapy strategies hold great promise for cartilage tissue regeneration.However,to obtain a large quantity of chondrogenic cells has been challenging.Cellular aggregates not only provide an in vitro culture condition mimicking in vivo microenvironment,but also are readily applicable as cell-based therapy.By exploiting spinner flask,the aggregation kinetics and mechanism of both articular chondrocytes and mesenchymal stem cells(MSCs)were investigated.Three culture systems in spinner flask for bovine articular chondrocytes(bACs),rabbit mesenchymal stem cells(rMSCs)and mixture of bACs and rMSCs.Specifically,the effects of agitation rate and cell inoculation density on the aggregation kinetics,aggregates size,cell proliferation and extracellular matrix(ECM)production were studied.In addition,the influences of cell aggregation on chondrocyte phenotype and stemness of MSCs,as well as the molecular mechanism of aggregation were also explored.The results showed that suspension culture in spinner flask induced aggregation of bACs and rMSCs,Both bACs and rMSCs exhibited increased aggregation rate and aggregates size with decreasing agitation rate and increasing cell inoculum density.In addition,aggregates size also increased with extended culture time.Gene expression of both adhesion molecules based on real-time PCR analysis suggested that ?1-Integrin and Cadherin were involved in the formation of bACs and rMSCs aggregates,respectively.In aggregates,bACs exhibited improved chondrocyte differentiation based on upregulated gene expression of Collagen II and SOX-9,and rMSCs exhibited increased gene expression of the stemness marker,including of SOX-2,NANOG,and OCT-4.In spinner flask,hybrid aggregates composed of bACs and rMSCs were also fabricated,showing an rMSCs core and a bACs periphery.Taken together,cellular aggregates with chondrogenic potential were prepared in dynamic suspension culture using spinner flask,the key culture parameters to the aggregation process were identified,and the molecular mechanism of cell aggregation was revealed.This would lay solid foundation for large-scale production of cellular aggregates for cartilage regeneration.