While several murine osteocyte-like cell lines are available and techniques for isolating osteocytes from mouse bone have been described, few models exist for studying human osteocytes in vitro. We have developed a method for isolating osteocytes from bone taken from patients undergoing knee arthroplasty. Trabecular bone was dissected and washed to remove marrow and subjected to sequential digestions in collagenase/EDTA. Cells were harvested after each digest, plated on collagen coated wells and cultured over a 5 day time course. Osteocyte gene expression was analysed by RT-PCR and cell morphology examined by phalloidin staining. Cells harvested from digests 1 and 2 expressed low levels of the osteocyte markers SOST and DMP1, with increased levels observed in digests 3 and 4. The highest levels of these markers were observed in digests 5 and 6, with a 20-fold increase in DMP1 and a 9-fold increase in SOST mRNA compared to digest 1. FGF23 mRNA expression was absent in the early digests but was observed from digest 3 onwards, increasing up to 150-fold in expression in digest 6. The osteocyte markers PHEX and MEPE were also expressed in the isolated cells and were increased in the later digests. The cells isolated in digests 1 and 2 displayed a mixed morphology, with osteoblast-like cells and some dendritic osteocyte-like cells after 5 days of culture. Digests 3-6 contained many highly dendritic cells, which were initially observed after 2 days of culture and increased in number and dendricity after 5 days. Treatment of isolated cells from digests 3-6 with PTH or 1,25(OH)2vitaminD3 for 24 hours resulted in the downregulation of SOST and upregulation of FGF23 mRNA levels, respectively, similar to osteocytes in vivo. In conclusion, we have developed a reproducible method of isolating osteocytes from human bone. Such cells will be invaluable for furthering osteocyte research.
Disclosure: The authors declared no competing interests. This work was supported by the National Health and Medical Research Council of Australia (grant number APP1047796).