title: Presence of xenogenic mouse RNA in RPE and IPE cells cultured on mitotically inhibited 3T3 fibroblasts.
abstract: OBJECTIVE
  Mitotically inhibited 3T3 fibroblasts are used as feeder layers to culture a variety of cells. However, transplantation of human cells cultured on mitotically arrested mouse cells poses potential risks, such as disease transfer and contamination with 3T3 cells. Bovine RPE and IPE cells were cultured on mitomycin-treated 3T3 fibroblasts, to examine cell characteristics and contamination by 3T3 products.
  
  
  METHODS
  IPE or RPE cells cultured on mitomycin-treated 3T3 fibroblasts were evaluated for adhesion, morphology, and tight junction formation by microscopy and immunohistochemistry. ROS phagocytosis was used to examine functional activity. Gene expression was evaluated by quantitative real-time PCR.
  
  
  RESULTS
  In the presence of 3T3 fibroblasts, primary IPE and RPE cells adhere, spread and acquire a hexagonal shape within 12 hours. When cultured on 3T3 fibroblasts, IPE and RPE cells exhibited stable expression of pigment epithelial genes, but expression of mouse collagen type I was also observed.
  
  
  CONCLUSIONS
  Culturing IPE and RPE cells on mitomycin-treated 3T3 fibroblasts resulted in rapid adhesion and growth of primary pigment cells. However, the presence of potentially hazardous xenogeneic mRNA of mouse origin in the cultures limits the use of these cells for transplantation.
authors.affiliation: IZKF (Interdisziplinäres Zentrum für Klinische Forschung) Aachen, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Johnen
authors.forename: Lucia
authors.initials: L
authors.lastname: Wickert
authors.forename: Marion
authors.initials: M
authors.lastname: Meier
authors.forename: Anna Katharina
authors.initials: AK
authors.lastname: Salz
authors.forename: Peter
authors.initials: P
authors.lastname: Walter
authors.forename: Gabriele
authors.initials: G
authors.lastname: Thumann
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival; D018920:Coculture Techniques; D003094:Collagen / Q000235:genetics; D024042:Collagen Type I; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D020869:Gene Expression Profiling; D007498:Iris / Q000166:cytology*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D016685:Mitomycin / Q000494:pharmacology; D008938:Mitosis / Q000187:drug effects*; D041681:NIH 3T3 Cells; D010587:Phagocytosis / Q000502:physiology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012374:Rod Cell Outer Segment / Q000378:metabolism; D013552:Swine; D019108:Tight Junctions; D062826:Zonula Occludens-1 Protein
chemical_list: D000477:Alkylating Agents; D024042:Collagen Type I; D008565:Membrane Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; D016685:Mitomycin; D003094:Collagen