Centrifugal isolation of bone marrow from bone: An improved method for the recovery and quantitation of bone marrow osteoprogenitor cells from rat tibiae and femurae

The high variation often observed in the ex vivo fibroblastic-colony forming unit (CFU-f) assay is likely to be due to both biological and experimental variation. To determine whether we could improve experimental methods we developed an alternative method of bone marrow cell (BMC) isolation employing a centrifugation step. The osteogenic capacity of centrifugally isolated BMC was compared to that of BMC that were isolated using the standard "flushing" technique using the CFU-f assay. The centrifugation method was found to be both quick and simple to perform and allowed simultaneous preparation of all samples. Centrifugually isolated BMC gave rise to approximately 100% more cfu-ap and cfu-f in cultures from both tibiae and femurae. The proportion of alkaline phosphatase positive colonies remained the same and colony morphologies were similar for both isolation methods. Histological comparison of the flushed and spun bones showed that after the flushing procedure many cells remained in the marrow cavity especially in the trabecular area. In contrast, centrifugation completely emptied the marrow space of all cells except bone lining cells and osteoblasts. Thus the osteogenic capacity of the bone marrow can be expressed as the number of CFU-f per bone instead of the frequency as is the norm. Using these methods to isolate BMC for ex vivo investigations should lead to a reduction in CFU-f number variation due to the isolation method.