Establishment of novel three-dimensional culture system for human chondrocytes using micro-space plate

Abstract

Purpose: It has been reported that monolayer culture of human articular chondrocytes results in dedifferentiation and loss of chondrogenic property. Therefore, the dedifferentiation is a big problem when the influence of cytokines and drugs were evaluated. In order to solve this problem, we performed a novel three-dimensional culture system for human chondrocytes using micro-space plates compared with monolayer culture. Methods: The micro-space plate has regularly arranged 200 mm×100 mm×50 mm square compartments on its surface. Human articular chondrocytes were cultured in flat plates or micro-space plates for 1 week. After culture, the level of collagen 1A1 (COL1A1) and aggrecan mRNA expressions were measured by real-time PCR, and cartilage matrix production was assessed by alcian blue staining. Furthermore, chondrocytes were cultured with IL-1β, IL-6+soluble IL-6 receptor (sIL-6R) in the presence or absence of high molecular hyaluronic acid (HA) for additional 24 h and then the production of MMP-3 in cell supernatant was measured by ELISA. Results: At the same passage of cells, human articular chondrocytes grown on micro-space plates in clusters after 5-7 days’ culture, whereas cells grown on flat plates in fibroblast-like morphology. In three-dimensional culture, the mRNA expression levels of COL1A1 and aggrecan were higher than those in monolayer culture. And the cartilage matrix production in three-dimensional culture was higher than that in monolayer culture. Moreover, the production of MMP-3 induced by IL-1β and IL-6+sIL-6R was higher in three-dimensional culture than in monolayer culture. Inhibitory activity of HA on MMP-3 production was similar between three-dimensional culture and monolayer culture. Over the subsequent passage, the expression levels of COL1A1 and aggrecan mRNA and MMP-3 production by IL-1β and IL-6+sIL-6R gradually decreased in monolayer culture, but they remained at higher levels in three-dimensional culture. Conclusions: In conclusion, the present study demonstrated that the novel three-dimensional culture using micro-space plates maintains chondrogenic properties. Three-dimensional culture using micro-space plates is a useful tool for chondrocyte culture.