Separation and concentration of murine hematopoietic stem cells (CFUS) using a combination of density gradient sedimentation and counterflow centrifugal elutriation

Abstract

To obtain concentrated suspensions of pluripotent hematopoietic stem cells *CFUS) from murine bone marrow, density gradient centrifugal sedimentation (DGCS) was combined with counterflow centrifugal elutriation (CE). This combination provided a 7.6 fold enrichment of the CFUS concentration. For DGCS, Percoll a suspension of silica particles coated with polyvinylpyrrolidone was used. For fractionation by the CE an elutriator rotor (JE-6, Beckman) was used for further concentration of the cells harvested from the DGCS. Bone marrow erythropoiesis was suppressed by transfusion plethora initiated 5-6 days before the bone marrow was harvested. These two physical separation procedures combined with transfusion plethora to suppress erythropoiesis are effective in producing an enriched fraction of CFUS without change in distribution of the histologic type of colonies.