[Subculture, cryopreservation and recovery of osteoclasts].

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

[Imaging observation of live cells originating from osteoclasts of the blood system].

OBJECTIVE: To study the application of the live cell imaging method to observe the whole process of osteoclast formation induced by monocyte macrophages in the blood system in order to clarify the origin of osteoclasts and their cytodynamics. METHODS: Blood samples (8 ml) were collected from the abdominal aorta of male SD rats weighing 280 g. Mononuclear cells were obtained by density gradient centrifugation and induced by RANKL and M-CSF. The cells were cultured and divided into four groups: inverted phase contrast microscope (IPCM) group, TRAP group, SEM group and live cell imaging (LCI) group. Images of the IPCM group were captured by a digital microscopic imaging system and recorded daily. The TRAP group was identified by enzyme activity staining after a 21-day cultivation period. The SEM group was SEM-observed after a 21-day cultivation period. The LCI group was consecutively and dynamically observed for 35 days. RESULTS: After 2-week cultivation, IPCM observations showed the formation of numerous apocytes. These cells displayed round, fusiform, fan-shaped, elliptic or irregular gibbous profiles. TRAP staining showed that most apocytes and monocytes had positive(+)reaction. SEM observations showed many bone absorption lacunae, hollows and channels, in which many osteoclasts with absorption activity were observed. Live cell imaging observations found that multinuclear osteoclasts originating from peripheral blood were generated by fusion of monocytes and apocytes and intercross fusion of monocytes and apocytes,which occurred at the adherent stage of the cells. Cytodynamic observations showed that the cell form of osteoclasts was complex and changeable. CONCLUSION: RANKL and M-CSF can induce differentiation and formation from monocytes in rat peripheral blood into multinuclear osteoclasts with bone absorption activity. The osteoclasts were formed by various cell fusion processes at the adherent stage. The adherent property of osteoclasts is important for their survival and function. Osteoclasts have phagocytosis and their morphological structure is dynamically changeable, involving not only apocytes but monocytes. The osteoclast property of multinuclear giant cells formed by cell fusion may be a special biological behavior for their adaptation of functional needs and bone absorption efficiency. This experiment has further evidenced the theory of osteoclast origination in the blood system and provided new experimental clues for clarifying the cytodynamic and cytobiological properties of osteoclasts.

Association of apolipoprotein E 4 polymorphism with cerebral infarction in Chinese Han population.

AIM: To study the association between APOE polymorphisms and cerebral infarction through a case-control study among the Chinese Han population. METHODS: First-ever cerebral infarction patients (n=226) whose ages ranged from 40 to 60 years old were recruited from Department of Neurology, Zhongshan Hospital, Shanghai, and Zhejiang Chinese Traditional Medicine Hospital, Zhejiang, China. Unrelated healthy controls (n=201) were selected from the general population in the same area with similar age and sex distribution. APOE was amplified by one-stage PCR using the forward primer: 5'-GGC ACG GCT GTC CAA GGA GCT-3' and reverse primer: 5'-GAT GGC GCT GAG GCC GCG CT-3'. The PCR product was digested directly with 5 U of CfoI and separated by a 20 % polyacrylamide (acrylamide: bis-acrylamide=29:1) nondenaturing gel. RESULTS: Both cerebral infarction patient and control groups were in Hardy-Weinberg equilibrium. The allele frequency of APOE*2, APOE*3, and APOE*4 was 4.6 %, 81.9 %, and 13.5 % respectively in the patients with cerebral infarction; 5.7 %, 87.3 %, and 7.0 % respectively in the healthy control group. Compared with APOE3/3 subjects, APOE4/4 carriers had a 2.1-fold risk of cerebral infarction (odds ratio 2.1, 95 % confidence limits 1.3 to 3.4). The allele frequency of APOE*4 in the cerebral infarction patient group was significantly higher than that in the control group (13.5 % vs 7.0 %; P=0.002). CONCLUSION: APOE 4 is a risk factor for cerebral infarction among the Chinese Han population.