title: Evaluation of methods for cell harvesting and the biological properties at successive passages of canine bone marrow stromal cells.
abstract: OBJECTIVE
  To compare methods for harvesting canine bone marrow stromal cells (BMSCs) and determine the biological properties of canine BMSCs at successive passages in vitro.
  
  
  METHODS
  BMSCs collected from the femurs of 9 Beagles.
  
  
  METHODS
  A fibroblast assay was performed to compare 2 methods for harvesting BMSCs: the aspiration and perfusion method. Flow cytometric analysis was performed to evaluate the cell surface markers. Changes in proliferative activity were analyzed by examining radioactivity of hydrogen 3-thymidine. Cell senescence was studied via senescence-associated β-galactosidase staining, and differentiation properties (osteogenesis and adipogenesis) were estimated in association with passage.
  
  
  RESULTS
  The aspiration method yielded significantly more fibroblasts than the perfusion method. The cells harvested by both methods gave positive results for CD44 and CD90 and negative results for CD34 and CD45. After induction, the cells had osteogenic and adipogenic phenotypes. The biological properties of BMSCs harvested by the aspiration method were estimated in association with passage. With increasing number of passages, the proliferative activity was reduced and the proportion of cells with senescence-associated β-galactosidase staining was increased. The capacity of differentiation was reduced at passage 3.
  
  
  CONCLUSIONS
  The aspiration method was superior for collection of BMSCs. In early passages, canine BMSCs had the proliferative activity and potential of osteogenic and adipogenic differentiation, but this decreased with increased number of passages. Consideration of passage will be important to the success of any strategy that seeks to regenerate tissue though the use of BMSCs.
authors.affiliation: Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-58 Rinku Ourai Kita, Izumisano, Osaka, 598-8531, Japan.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Nishida
authors.forename: Yuki
authors.initials: Y
authors.lastname: Shoji
authors.forename: Michi
authors.initials: M
authors.lastname: Nakamura
authors.forename: Shingo
authors.initials: S
authors.lastname: Hatoya
authors.forename: Kikuya
authors.initials: K
authors.lastname: Sugiura
authors.forename: Jyoji
authors.initials: J
authors.lastname: Yamate
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Kuwamura
authors.forename: Takao
authors.initials: T
authors.lastname: Kotani
authors.forename: Masanari
authors.initials: M
authors.lastname: Nakayama
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Chizuka
authors.initials: C
authors.lastname: Ide
authors.forename: Toshio
authors.initials: T
authors.lastname: Inaba
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology* / Q000502:physiology*; D018929:Cell Culture Techniques / Q000662:veterinary*; D004285:Dogs / Q000502:physiology*; D013048:Specimen Handling / Q000379:methods / Q000662:veterinary*; D017154:Stromal Cells