A cell leakproof porous poly(DL-lactic-co-glycolic acid) (PLGA)-collagen hybrid scaffold was prepared by wrapping the surfaces of a collagen sponge except the top surface for cell seeding with a bi-layered PLGA mesh. The PLGA -collagen hybrid scaffold had a structure consisting of a central collagen sponge formed inside a hi-layered PLGA mesh cup. The hybrid scaffold showed high mechanical strength. The cell seeding efficiency was 90.0% when human mesenchymal stem cells (MSCs) were seeded in the hybrid scaffold. The central collagen sponge provided enough space for cell loading and supported cell adhesion, while the bi-layered PLGA mesh cup protected against cell leakage and provided high mechanical strength for the collagen sponge to maintain its shape during cell culture. The MSCs in the hybrid scaffolds showed round cell morphology after 4 weeks culture in chondrogenic induction medium. Immu-nostaining demonstrated that type 11 collagen and cartilaginous proteoglycan were detected in the extracellular matrices. Gene expression analyses by real-time PCR showed that the genes encoding type II collagen, aggrecan, and SOX9 were upregulated. These results indicated that the MSCs differentiated and formed cartilage-like tissue when being cultured in the cell leakproof PLGA-collagen hybrid scaffold. The cell leakproof PLGA-collagen hybrid scaffolds should be useful for applications in cartilage tissue engineering. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 819-826, 2010