Chondrocytes are highly specialized cells of mesenchymal origin that are responsible for producing,sustaining and degrading the cartilage extracelluar matrix (ECM)[1].The two main macromolecules in articular cartilage ECM are the large proteoglycan aggrecan and type Ⅱ collagen with specific function of tensile strength and resistance to compressive loads[2].Conventional 2D monolayer cultures are regarded as unsuitable,as monolayer expansion of articular chondrocytes does not support the chondrogenic phenotype during passaging.Instead,it is well accepted that high cell density three-dimensional (3D) culture favor the maintenance of the chondrocytes phenotype and support redifferentiation of dedifferentiated articular chondrocytes.Spheroids formation is one of the most well characterized cell models for 3D culture and drug screening due to its simplicity,reproducibility,and similarity to physiological tissue.Concave microwells have been paid more attention because of forming 3D cell spheroids easily without complicated manipulation.Here,we developed a novel,rapid,method to fabricate concave device and firstly form chondrocytes spheroids and maintain chondrogenic phenotype in simple manner.The design of the concave microwell device was illustrated in Fig1.The device including 20×20 concave microwells for forming 3D chondrocytes spheroids was fabricated by a simple and fast photo-etching fabrication technique.The concave PDMS-based microwell system was an attractive method,because it could be used to form the uniform size multi-cellular chondrocytes spheroids,which were an important factor to investigate the special function of chondrocytes or cartilage.To prove the system can maintain phenotype of the chondrocytes in 3D multi-cellular spheroids,we set the monolayer chondrocytes culture for 3 days in plastic as control group,and 3D chondrocytes multi-cellular spheroids harvesting from the concave microwells device for 3 days as experimental group to detect the expression of the type Ⅱ collagen and Aggrecan by immunofluorescence.The immunofluorescence results were demonstrated in Fig.3.Conventional monolayer culture chondrocytes almost had not expressed the type Ⅱ collagen and aggrecan.Nevertheless,chondrocytes spheroids showed higher expression of the type Ⅱ collagen and aggrecan.This concave device could form 3D chondrocyte spheroids and maintain phenotype commendably,indicating a useful platform for cartilages tissue engineering.