As chondrocytes fail to retain their chondrogenic potential in two-dimensional monolayer cultures, several three-dimensional culture systems have been employed for investigating the physiology and pathophysiology in articular cartilage tissues. In this study, we introduced a polyethylene glycol-coated microfabricated chip that enables spheroid formation from ATDC5 cell line, commonly used as a model for in vitro chondrocyte research. ATDC5 cells cultured in our devices aggregated immediately and generated a single spheroid per well within 24 h. Most cells in spheroids cultured in differentiation medium were viable and the circular shape and smooth surface of the spheroid were maintained up to 14 d in culture. We also detected potent hypoxia conditions, a key factor in chondrogenesis, in whole lesions of ATDC5 spheroids. Expression of chondrogenesis-related genes and type X collagen protein was significantly increased in ATDC5 spheroids grown in differentiation medium, compared with monolayer-cultured ATDC5 cells. We also demonstrated that the differentiation medium-induced Akt protein phosphorylation was upregulated in ATDC5 cells cultured in our spheroid device, suggesting that enhancement of chondrogenic potential in ATDC5 spheroids results from PI3/Akt signaling activation. These results indicated that our spheroid culture system could constitute a high-throughput strategy approach towards elucidating the molecular mechanisms that regulate chondrogenesis.

由于软骨细胞不能在二维单层培养物中保留其成软骨潜能，因此已经采用了几种三维培养系统来研究关节软骨组织的生理学和病理生理学。在这项研究中，我们介绍了一种涂有聚乙二醇的微制造芯片，该芯片能够从ATDC5细胞系中形成球状体，通常用作体外模型软骨细胞研究。在我们的设备中培养的ATDC5细胞会立即聚集并在24小时内每孔产生一个球状体。在分化培养基中培养的球状体中的大多数细胞是活的，并且球状体的圆形形状和光滑表面在培养中保持长达14 d。我们还在ATDC5椭球体的整个病变中检测到了强缺氧条件，这是软骨形成的关键因素。与单层培养的ATDC5细胞相比，在分化培养基中生长的ATDC5球体中与软骨形成相关基因和X型胶原蛋白的表达明显增加。我们还证明了分化培养基诱导的Akt蛋白磷酸化在我们的球形装置中培养的ATDC5细胞中被上调，提示ATDC5球体中的软骨形成电位增强是由PI3 / Akt信号激活引起的。这些结果表明我们的球体培养系统可以构成一种高通量的策略方法，以阐明调节软骨形成的分子机制。