[Subculture, cryopreservation and recovery of osteoclasts]

To explore the feasibility of passage, cryopreservation, and recovery of osteoclasts in order to develop new techniques facilitating osteoclast research.

Passage of osteoclasts: adult male SD rat(SPF grade, weight of 250 g) was sacrificed and the abdominal aorta was exposed for blood draw. Monocytes isolated from peripheral circulation was treated with RANKL and M-CSF for 2 weeks. After formation of osteoclasts, they were trypsinized with pipetting, centrifuged, re-suspended with α-MEM containing RANKL and M-CSF, and cultured in 6 well-plates and 35 mm culture dishes. Freezing of osteoclasts: trypsinized osteoclasts were centrifuged and resuspended with DMSO, FBS, α-MEM (1:2:7), and were stored in liquid nitrogen(-196 °C). Recovery of osteoclasts: frozen osteoclasts were taken out of liquid nitrogen tank and thawed quickly at 37 °C in water bath. After wash with PBS, the cells were resuspended with α-MEM containing RANKL and M-CSF, and were cultured in 6 well dishes and 35 mm culture dishes. Meanwhile, cells were checked with inverted phase contrast microscope and observed in the live cell station for real time imaging. TRAP staining was performed 3 days after plating.

Trypsinization together with pipetting and shaking can detach the adherent osteoclasts, and the resuspended cells can be used for passage and storage in liquid nitrogen. The passaged cells became fully attached to the culture dishes in 2 hours, and the multinucleated feature could be clearly seen. The osteoclasts recovered from liquid nitrogen could completely spread out for 2 to 3 hours so that the multinucleated cells were clearly seen. These cells were still TRAP positive.

Although osteoclasts strongly adhere to the bottom of culture dishes, a large majority of the osteoclasts can be detached after appropriate digestion with trypsin, pipetting and shaking. These cells can be used for passage and cryopreservation. After recovering from liquid nitrogen, these cells still preserve the viability and the feature of osteoclasts. The results provide a new and powerful tool for future study of osteoclast biology.