Basics of isolation and cultivation of chondrocytes according to good laboratory practice

Abstract

OBJECTIVES: The objective of the present study was to determine if chondrocytes isolated from human cartilage of five elderly patients (mean age 63) with osteoarthritis (stage 3) maintain their proliferation and chondrogenic potential. Isolation and cultivation of chondrocytes was performed according to good laboratory practice (GLP) standards. Methods: Chondrocytes were isolated from cartilage biopsy by enzymatic digestion. Cultivation of cells was performed in a controlled environment (cleanroom). Phenotype characterization of chondrocytes was achieved by flow cytometry analysis. Results: Three weeks after cultivation, polygonal structures typical of chondrocytes were observed, but spindle/fibroblast like morphology was also detected in cultured cells. Flow cytometric analysis showed that chondrocytes were positive for CD44 (98.35% ± 0.50), CD90 (97.15% ± 0.13) after first passage (P1) and the cells were negative for hematopoietic marker CD45 (0.21% ± 0.11). ConclusionS: Human articular chondrocytes obtained from five elderly patients with osteoarthritis maintained a chondrocyte phenotype and could be potentially used for autologous implantation. We have standardized the conditions for cultivation according to GLP standards to minimize the risk of in vitro cell contamination.