title: Lipofection of rabbit chondrocytes and long lasting expression of a lacZ reporter system in alginate beads.
abstract: OBJECTIVE
  Our aim was to investigate the maintenance of the transfection status of non-viral transfected chondrocytes in an alginate culture system.
  
  
  METHODS
  Chondrocytes harvested from rabbit knees were isolated by sequential digestion and cultivated in monolayer culture. At 60-70% cell density, chondrocytes were transfected with different transfection systems (FuGENE6, CaCl2, Lipofectin). A lac Z expression vector (pcDNA 3.1/Myc-His+ lacZ) was used as a reporter system. In order to improve transfection rates, hyaluronidase (4 U/ml) was used prior and during the transfection procedure. Thereafter, transfected cells were either kept in monolayer culture or embedded in alginate beads and kept in culture for up to the next 30 weeks.
  
  
  RESULTS
  Transfection efficiency was maximal using FuGENE6TM/DNA at a ratio of 3:2 and hyaluronidase (4 U/ml). Transfection efficiency reached up to 40.8% (+/- 3.2%) after 36 h. In alginate beads lac Z positive cells declined to 8.5% +/- 3.3% after 4 weeks and to 4.6% +/- 3.2% after 12 weeks of culturing. After 30 weeks 3% of chondrocytes still expressed lac Z. In contrast, during culturing in monolayer, no lac Z expression was detectable after 4 weeks. Differentiation status of the chondrocytes was confirmed by histology and immunohistochemistry methods.
  
  
  CONCLUSIONS
  After successful gene transfer to rabbit chondrocytes the alginate system made it possible to culture lipofected chondrocytes phenotypically stable. Genetically engineered chondrocytes express the lac Z reporter gene over a period of at least 30 weeks. This transfection and culture system provides a promising tool to further investigate the over-expression of growth factors and enzyme inhibitors.
authors.affiliation: Dept. of Orthopaedic Surgery/RKU, University of Ulm, Oberer Eselsberg 45, 89081 Ulm, Germany. johannes.stoeve@medizin.uni-ulm.de
authors.forename: J
authors.initials: J
authors.lastname: Stöve
authors.forename: J
authors.initials: J
authors.lastname: Fiedler
authors.forename: K
authors.initials: K
authors.lastname: Huch
authors.forename: K-P
authors.initials: KP
authors.lastname: Günther
authors.forename: W
authors.initials: W
authors.lastname: Puhl
authors.forename: R
authors.initials: R
authors.lastname: Brenner
mesh_terms: D000464:Alginates; D000818:Animals; D002356:Cartilage / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards; D019902:Chondrocytes / Q000378:metabolism*; D018014:Gene Transfer Techniques / Q000592:standards*; D020723:Glucuronic Acid; D006603:Hexuronic Acids; D006614:Hindlimb; D007596:Joints; D007763:Lac Operon / Q000235:genetics*; D008863:Microspheres; D011817:Rabbits; D014162:Transfection / Q000379:methods / Q000592:standards
chemical_list: D000464:Alginates; D006603:Hexuronic Acids; D020723:Glucuronic Acid