Abstract:

BACKGROUND: There are no detailed reports about how to rapidly isolate muscle tissues from the femur and tibia which directly influences the ischemic period of bone marrow cells in mice.
OBJECTIVE: To investigate the rapidest and feasible method for harvesting murine bone marrow cells (BMCs) in order to decrease the ischemia injury of BMCs.
METHODS: Muscle and connective tissues surrounding the femur and tibia were entirely removed by using an initiated retrograde method by which dissection of bones were started at ankle joints and ended at hip joints. The key point, in the whole isolating procedure, was “crossing → slipping → replicating” with appropriate force with ophthalmic scissors worked in coordination with fine tissue forceps. Time of entirely isolating the femur and tibia as well as time of harvesting BMCs were recorded. The obtained BMCs were cultured in 25 cm2 corning flasks using RPMI1640 with 10% fetal bovine serum, and after 72 hours, the cells were stained with trypan blue and the survival rate was detected with cell counter.
RESULTS AND CONCLUSION: Time of isolating the femurs and tibia and time of harvesting BMCs were (1.9±0.4) minutes and (14.7±1.8) minutes, respectively. No bone or marrow cavity was injured or contaminated. Compared with before culture, the number of BMCs after 72 hours increased significantly [(8.1±2.0)×10⁷ vs. (4.7±0.7)×10⁷, P < 0.01], while the cell survival rate did not change remarkably [(97.5±1.1)% vs. (95.4±1.0)%, P > 0.05]. No visible microbe was found in the culture media. To harvest mouse BMCs using the retrograde isolation of the femur and tibia is a practical, feasible and rapid method.