Purification of Primary Chondrocyte Cells extracted from knee joint of Sprague-Dawley rats

Harvesting of pure cells from primary tissue is difficult as isolated tissue contains various cell types. Articular cartilage is highly specialized tissue that functions by forming a smooth surface enabling joint movement. Cartilage is mostly made up of chonrocytes. However, when harvesting cartilage other tissues arising from the synovial membranes, tendons and even nerves. This makes acquisition of a pure chondrocyte culture challenging. Harvesting chondrocytes from neonatal rat joints is even more difficult because the larger joints such as the knee joint is roughly about 2 mm in diameter. Thus despite careful isolation from neonatal rat joints cartilage will be accompanied by a mixture of bone cells and non cartilaginous connective tissue. Cells derived from this tissue will therefore need to be purified. In this study the different adhesion affinity of dissimilar cells on solid surface was used to separate chondrocytes from other cell types. This was achieved by plating primary cell suspensions in tissue culture grade (TC) cell culture flasks and removing those unattached cells after 20 minutes. The unattached cells were then re-plated in a new TC grade cell culture flask. This process was repeated a further 6 times, after which the final cell suspension was allowed to attach to the surface for 100 minutes. This resulted in the generation of 8 cell cultures, plus one control in which the serial attachment process was not used. All 9 cultures were incubated at 370C for 4 days and then immunostained with monoclonal Anti S-100 ( -subunit – SIGMA) antibody and monoclonal anti collagen type-I antibody (SIGMA). Immunocytochemical staining showed that control cultures consisted of a mixture of chondrocytes and other cell types. However, of those cultures derived from the serial attachment process, chondrocytes only became evident after 100 minutes of cell attachment. By the 8th plating the cultures contained almost 100% chondrocytes. These results suggest that chondrocytes can be readily isolated and purified by a prolonged differential adhesion technique with a primary incubation extending for 140 minutes.