Evaluation of clonogenic stromal progenitor cells (CFU-F) recovered from sequential enzymatic disaggregation of BM plugs. (A) Average mononucleated cell yields obtained from either standard flushing methods or from each successive digestion (n = 4). (B) Incidence of CFU-F obtained from either standard flushing technique (5 × 106 mononuclear cells/well) or from each fraction of digested marrow plugs (2.5 × 105 mononuclear cells/well) plated in triplicate. (C) Recovery of CFU-F from flushed BM and each fraction of DBM calculated per femur-tibia pair. (D) Incidence of CFU-F obtained from flushed BM versus the pool of DBM fractions (1-3; n = 8). (E) Recovery of CFU-F from flushed BM and the pool of DBM fractions (1-3) calculated per femur-tibia pair. Only colonies containing more than 50 stromal cells are scored. CFU-F data are presented both as incidence of clonogenic cells (CFU-F/1 × 106 mononuclear cells) and as the total number of CFU-F recovered from a given number of bones (CFU-F per total nucleated cells). (F) CFU-F incidence quantified by limit dilution analysis. Limit dilution assays were performed by plating BM mononuclear cells at various doses (500, 1000, 2500, 5000, and 10 000 cells/well in 24-well plates) with 24 replicates per dilution (n = 3). Plates were scored and negative wells enumerated. Data were analyzed with L-Calc 1.1.1 software (StemCell Technologies) and plotted as a negative linear relationship to identify the frequency of colony-forming cells. Data are mean ± SD. Statistical analysis of CFU-F incidence was performed with SigmaStat Version 3.5.
