title: Concentration of Rat Bone Marrow Nucleated Cells Using Hypo-osmotic Hemolysis in Distilled Water.
abstract: BACKGROUND
  Bone marrow aspirates contain primarily red blood cells. To achieve efficient cell transplantation for regeneration, the red blood cells need to be removed from the aspirates. Cell isolation is typically performed using density gradient centrifugation. However, this method entails issues of clinical safety and convenience. This study describes an efficient method to concentrate bone marrow nucleated cells by hypo-osmotic hemolysis.
  
  
  METHODS
  The optimal hemolysis conditions were determined by diluting the bone marrow suspensions with distilled water in various dilution ratios. Then, the resulting cell fractions were transplanted in a rat cranial defect model to evaluate their effects on bone formation and their angiogenic effects.
  
  
  RESULTS
  The optimal hemolysis conditions were a 3.3-fold dilution in distilled water and a hypo-osmotic exposure time of 45 seconds. Nucleated cells obtained using this method included granulocytes and mononuclear cells. These cells contain cytoplasmic angiogenic factors, including vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor. In a rat cranial defect model, callus formation and angiogenesis were significantly increased following transplantation of concentrated marrow nucleated cells in this manner.
  
  
  CONCLUSIONS
  These results suggest angiogenic and osteogenetic effects of transplanting marrow nucleated cells using this hypo-osmotic method.
authors.affiliation: Department of Plastic and Reconstructive Surgery, Saitama Medical Center, Saitama Medical University, Kawagoe City, Saitama, Japan; Department of Plastic and Reconstructive Surgery, National Hospital Organization Tokyo Medical Center, Meguro-ward, Tokyo, Japan; and Department of Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ward, Tokyo, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Ohnishi
authors.affiliation: Department of Plastic and Reconstructive Surgery, Saitama Medical Center, Saitama Medical University, Kawagoe City, Saitama, Japan; Department of Plastic and Reconstructive Surgery, National Hospital Organization Tokyo Medical Center, Meguro-ward, Tokyo, Japan; and Department of Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ward, Tokyo, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Ochiai
authors.affiliation: Department of Plastic and Reconstructive Surgery, Saitama Medical Center, Saitama Medical University, Kawagoe City, Saitama, Japan; Department of Plastic and Reconstructive Surgery, National Hospital Organization Tokyo Medical Center, Meguro-ward, Tokyo, Japan; and Department of Plastic and Reconstructive Surgery, Keio University School of Medicine, Shinjuku-ward, Tokyo, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kishi