Isolating homogenous cells from mandibular condylar cartilage by LCM technique

Abstract

Aim: The tissue-engineered mandibular condylar cartilage (MCC) should ideally mimic the native zonal structure to achieve a functional condylar replacement. This study attempted to optimize a working protocol for isolating homogenous cell populations from MCC by using laser capture micro-dissection (LCM) technique. Methods: Frozen sections were prepared after harvesting MCC tissues from three SD rats. Cells from four identified zones were separately isolated (10 samples/zone) using LCM technique. tRNA was extracted for qPCR, and 3'/M ratio of GAPDH was calculated to evaluate RNA quality. Results: The morphology of tissue sections was preserved allowing accurate identification of all four zones of MCC, namely fibrous, proliferative, mature and hypertrophic chondrocytes zones. This improved morphology extended the range of cell types isolated via LCM technique. The average of the 3'/M GAPDH ratios, a better guide to RNA quality for small-quantity LCM-samples, was 1.46±0.41, 1.49±0.27, 1.75±0.50, and 1.60±0.43 for fibrous, proliferative, mature, and hypertrophic samples, respectively. Conclusion: The present study shows that the optimized LCM protocol could allow isolation of homogenous cells from various zones of MCC separately, thereby generating accurate molecular and genetic data for further study on tissue-engineered mandibular condylar cartilage.