Optimization for Culturing Primary Articular Chondrocytes
Culturing primary chondrocytes extracted from articular cartilage tissue presents many challenges to researchers. Several techniques are widely used, including monolayer cultures or 3D substrates such as alginate beads. Choosing the correct substrate is important because primary cells may lose their phenotype or ability to maintain their cellular identity, and can dedifferentiate into fibroblast depending on the technique used. One of the most popular techniques widely used in cartilage research is the use of spherical beads made of 1.2% alginate. Alginate beads allow the chondrocytes to live in an environment similar to actual cartilage tissue, by allowing the cells to keep their phenotypical cortical morphology and produce their own extracellular matrix and interact with other cells via cell-matrix interactions. However, it is difficult to obtain a clean RNA extraction from cells cultured in alginate beads because the alginate interferes with analysis of RNA expression. Using another substrate that could maintain the desired phenotype and allows easy RNA extraction would be beneficial. Cytopore-2 beads are a microcarrier that is made of cellulose and allows for the cells to grow throughout the multiple pores of the bead. The main benefit of this substrate is that it allows easy and clean RNA extractions that require no washes, and the cells can live inside the beads for several weeks without passaging. The main purpose of this study is to optimize culturing conditions for primary chondrocytes. Bovine articular chondrocytes (BAC) were extracted from the metacarpophalangeal joint of 18-24 month old steers. The cells were grown on two 3D substrates: Alginate and Cytopore-2, and also on monolayer in a standard T-75 flask. In addition, some cells were passaged once or twice using trypsin-EDTA. Gene expression analysis and morphological characterization was carried out utilizing reverse transcription-PCR and Laser Scanning Confocal Microscopy, respectively. Following these experiments, Cytopore-2 beads were determined to be the optimal culturing technique based on RNA extraction methods and immunohistochemistry, because the chondrocytes were able to maintain the true phenotype of chondrocytes and not dedifferentiate into fibroblasts. These results provide a novel and alternative culturing technique for primary chondrocytes that can have an impact on future studies into the mechanism of osteoarthritis progression and treatments for cartilage damage and repair.