title: Scaling up the ex vivo expansion of human circulating CD34(+)progenitor cells with upregulation of angiogenic and anti-inflammatory potential.
abstract: OBJECTIVE
  The therapeutic application of CD34+ circulating progenitor cells (which includes endothelial progenitor cells) has been hampered by the quantity and quality of isolated circulating CD34(+) cells from the patient's peripheral blood. Our group had previously established a suspension culture system for human CD34(+) cells, with increased quantity and quality (QQ) of the angiogenic cell product. We successfully scaled up the expansion process with the use of culture bags because there is the need to move toward a dynamic and fully controlled bioreactor system to meet Good Manufacturing Practice (GMP) standards and attain clinically meaningful cell doses in a time- and cost-effective way.
  
  
  METHODS
  CD34(+) cells isolated from mobilized peripheral blood of healthy donors were expanded ex vivo for 7 days in QQ medium (serum-free) in cell culture bags (30 mL) and pre- and post-expansion cells were characterized by means of flow cytometry and quantitative polymerase chain reaction; angiogenic potential was assessed by use of the in vitro tube formation assay.
  
  
  RESULTS
  Our data show effective expansion of the cultured population (7-fold) while maintaining the stem/progenitor content and increasing the endothelial population. Moreover, post-expanded cells showed higher tube formation capacity compared with pre-expanded cells. In addition, an upregulation of the anti-inflammatory gene expression and a downregulation of pro-inflammatory genes were observed, which suggests that the increase in angiogenic potential is not paired with an increase in the inflammatory profile.
  
  
  CONCLUSIONS
  The QQ expansion method was successfully scaled up to cell culture bags and was able to meet GMP standards, with a higher in vitro angiogenic profile.
authors.affiliation: Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Márcia F
authors.initials: MF
authors.lastname: Mata
authors.affiliation: Department of Medicine, Case Cardiovascular Research Institute, Harrington-McLaughlin Heart & Vascular Institute, University Hospitals Case Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. Electronic address: cardiojp@gmail.com.
authors.forename: João Pedro
authors.initials: JP
authors.lastname: Lopes
authors.affiliation: Department of Medicine, Case Cardiovascular Research Institute, Harrington-McLaughlin Heart & Vascular Institute, University Hospitals Case Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Ishikawa
authors.affiliation: Department of Medicine, Case Cardiovascular Research Institute, Harrington-McLaughlin Heart & Vascular Institute, University Hospitals Case Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Mohamad A
authors.initials: MA
authors.lastname: Alaiti
authors.affiliation: Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Joaquim M S
authors.initials: JM
authors.lastname: Cabral
authors.affiliation: Department of Bioengineering and iBB-Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
authors.forename: Cláudia L
authors.initials: CL
authors.lastname: da Silva
authors.affiliation: Department of Medicine, Case Cardiovascular Research Institute, Harrington-McLaughlin Heart & Vascular Institute, University Hospitals Case Medical Center, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Costa
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism*; D019149:Bioreactors; D018929:Cell Culture Techniques; D002453:Cell Cycle; D049109:Cell Proliferation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D066026:Endothelial Progenitor Cells / Q000378:metabolism*; D005434:Flow Cytometry; D064368:Healthy Volunteers; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000276:immunology*; D018919:Neovascularization, Physiologic / Q000502:physiology*; D015854:Up-Regulation
chemical_list: D018952:Antigens, CD34; D016895:Culture Media, Serum-Free
keywords: CD34(+) progenitor cells; Good Manufacturing Practice; angiogenesis; ex vivo expansion; scale-up; serum-free