title: Pseudotyping and culture conditions affect efficiency and cytotoxicity of retroviral gene transfer to human corneal endothelial cells.
abstract: OBJECTIVE
  To evaluate retroviral vectors as a tool to transduce normal human corneal endothelial cells (HCECs) and to optimize transduction to increase gene transfer efficiency.
  
  
  METHODS
  Enhanced green fluorescent protein (EGFP) encoding retroviral vectors based on HIV-1 or murine leukemia virus (MLV), pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or a modified foamy virus envelope protein (FV Env), and prototype foamy virus (PFV) were produced. Transduction was performed in four HCEC culture media that were previously described for specific cultivation of HCECs or organ culture of donor corneas, namely enriched HCEC growth medium F99(HCEC), its unsupplemented basal medium F99, MEM + 2% fetal calf serum (FCS) (MEM), and Human Endothelial-SFM (SFM). Transduction efficiency was evaluated by marker gene transfer assay, and cytotoxic effects of virus infection were evaluated by means of resazurin conversion assay.
  
  
  RESULTS
  PFV- and HIV-1-based vectors showed superior transduction efficiency compared with MLV-based vectors. Pseudotyping with a modified FV Env increased transduction efficiency compared with pseudotyping with VSV-G. In medium SFM, transduction efficiency of PFV, HIV-1-/FV Env, and MLV-based vectors was markedly reduced compared with the other culture media. When cells were cultured in F99-based media, cell viability was reduced by retroviral transduction compared with uninfected or mock infected controls, but remained unaffected when cells were cultured in SFM and was even increased when cells were cultured in MEM.
  
  
  CONCLUSIONS
  HIV-1-based vectors pseudotyped with FV Env can efficiently be used to transduce primary HCECs in vitro. However, transduction efficiency is dependent on culture conditions and impairs metabolic activity and viability of HCECs in vitro.
authors.affiliation: Institute of Anatomy, Medical Faculty Carl Gustav Carus, TU Dresden, Dresden, Germany. monika.valtink@tu-dresden.de
authors.forename: Monika
authors.initials: M
authors.lastname: Valtink
authors.forename: Nicole
authors.initials: N
authors.lastname: Stanke
authors.forename: Lilla
authors.initials: L
authors.lastname: Knels
authors.forename: Katrin
authors.initials: K
authors.lastname: Engelmann
authors.forename: Richard H W
authors.initials: RH
authors.lastname: Funk
authors.forename: Dirk
authors.initials: D
authors.lastname: Lindemann
mesh_terms: D000293:Adolescent; D000328:Adult; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008875:Middle Aged; D009924:Organ Culture Techniques; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D055815:Young Adult
chemical_list: D016895:Culture Media, Serum-Free