title: Slowing the Onset of Hypoxia Increases Colony Forming Efficiency of Connective Tissue Progenitor CellsIn Vitro.
abstract: BACKGROUND
  Survival and colony formation by transplanted tissue derived connective tissue progenitor cells (CTPs) are thought to be important factors in the success of clinical tissue engineering strategies for bone regeneration. Transplantation of cells into defects larger than a few millimeters expose cells to a profoundly hypoxic environment. This study tested the hypothesis that delaying the onset of hypoxia will improve the survival and performance of CTPsin vitro.
  
  
  METHODS
  To mimic declines seen in an avascularin vivobone defect, colony forming efficiency by marrow derived nucleated cells was assessed under osteogenic conditions. Variation in the rate of oxygen decline from an oxygen tension of 21% to 0.1% oxygen was explored using an incubator with programmable active control of gas concentrations. The effect of doping cultures with defined concentrations of RBCs was also used to evaluate the potential for RBCs to serve as a natural buffer in the setting of declining oxygen levels.
  
  
  RESULTS
  A delay in onset of hypoxia over 96 hours resulted in a 3-fold increase in the relative colony forming efficiency (rCFE) of CTPs as compared to an immediate onset of hypoxia. The presence of RBCsin vitroinhibited the rCFE of CTPs. Given the negative effects of RBCs, methods of RBC removal were evaluated and compared for their effectiveness of RBC removal and retention of colony forming efficiency.
  
  
  CONCLUSIONS
  These data suggest that conditions of hypoxia compromise colony forming efficiency in marrow derived CTPs. However, slowing the rate of decline of oxygen preserved colony forming efficiency at levels achieved in a stable normoxic (3% O2) environment. These data also suggest that RBCs are detrimental to the rCFE of CTPs and that buffy coat is an effective and preferred method for removing RBCs from marrow aspirates while preserving CTPs. These findings may inform clinical strategies for CTP transplantation.
authors.affiliation: Department of Biomedical Engineering, Cleveland Clinic, 9500 Euclid Ave ND20, Cleveland, OH 44195.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Heylman
authors.affiliation: Department of Biomedical Engineering, Cleveland Clinic, 9500 Euclid Ave ND20, Cleveland, OH 44195.
authors.forename: Tonya N
authors.initials: TN
authors.lastname: Caralla
authors.affiliation: Department of Biomedical Engineering, Cleveland Clinic 9500, Euclid Ave ND20, Cleveland, OH 44195.
authors.forename: Cynthia A
authors.initials: CA
authors.lastname: Boehm
authors.affiliation: Department of Biomedical Engineering, Cleveland Clinic, 9500 Euclid Ave ND20, Cleveland, OH 44195.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Patterson
authors.affiliation: Department of Biomedical Engineering, Cleveland Clinic, 9500 Euclid Ave ND20, Cleveland, OH 44195.
authors.forename: George F
authors.initials: GF
authors.lastname: Muschler
keywords: blood buffy coat; bone marrow; cell therapy; cellular hypoxia; colony forming unit; lymphoprep; progenitor cell; red blood cells