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

The high variation often observed in the ex vivo fibroblastic-colony formin g unit (CFU-f) assay is likely to be due to both biological and experimenta l variation. To determine whether we could improve experimental methods we developed an alternative method of bone marrow cell (BMC) isolation employi ng a centrifugation step. The osteogenic capacity of centrifugally isolated BMC was compared to that of BMC that were isolated using the standard "flu shing" technique using the CFU-f assay. The centrifugation method was found to be both quick and simple to perform and allowed simultaneous preparatio n of all samples. Centrifugually isolated BMC gave rise to approximately 10 0% more cfu-ap and cfu-f in cultures from both tibiae and femurae. The prop ortion of alkaline phosphatase positive colonies remained the same and colo ny morphologies were similar for both isolation methods. Histological compa rison of the flushed and spun bones showed that after the flushing procedur e many cells remained in the marrow cavity especially in the trabecular are a. In contrast, centrifugation completely emptied the marrow space of all c ells 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 instea d of the frequency as is the norm. Using these methods to isolate BMC for e x vivo investigations should lead to a reduction in CFU-f number variation due to the isolation method.