Monolayer culture followed by three-dimensional culture of chondrocytes: a two-step method

Abstract

Objective To explore an ideal method for culturing autologous chondrocytes while maintaining their cartilaginous phenotype by combining monolayer culture with three-dimensional culture. Methods Chondrocytes were cultured by routine monolayer culture method,then the chondrocytes of fourth passage were seeded into alginate beads for three-dimensional culture following standard protocols. The cell morphology was observed by inverted microscope at predefined time points. The cell viability and proliferation were tested by trypan blue and LIVE/DEAD○R Kit,respectively. And extracellular matrix formation was examined by toluidine blue staining. Results The cartilaginous phenotype of chondrocytes could only be maintained by passage 2 and 3 after monolayer culture. Three-dimensional culture could gradually regain and maintain the cartilaginous phenotype of de -differentiated chondrocytes ( P4). Cells of P1 -3 by monolayer culture could maintain 90% activity,which decreased from P4. The cytoactivity of cells by three-dimensional culture method was always above 90%. The total doubling rate till P6 by monolayer culture ( 28 days) was 44 times that by three-dimensional culture. The staining intensity of cartilaginous extracellular matrix was significantly decreased after P4 by monolayer culture ( P 0. 05). For three-dimensional method,the staining intensities of cartilaginous extracellular matrix at day 21 and 28 were significantly higher than that at day 7( P 0. 05). Conclusion Monolayer culture allows proper proliferation of chondrocytes,and following three-dimensional culture in alginate beads can regain the cartilaginous phenotype of chondrocytes,thus achieving the aim of both amplification and maintenance of the cartilaginous phenotype.