Presence of xenogenic mouse RNA in RPE and IPE cells cultured on mitotically inhibited 3T3 fibroblasts

Invest Ophthalmol Vis Sci. 2011 Apr 25;52(5):2817-24. doi: 10.1167/iovs.10-6429.


Purpose: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylating Agents / pharmacology
  • Animals
  • Cattle
  • Cell Proliferation / drug effects
  • Cell Survival
  • Coculture Techniques
  • Collagen / genetics
  • Collagen Type I
  • Fibroblasts / cytology*
  • Fibroblasts / metabolism
  • Fluorescent Antibody Technique, Indirect
  • Gene Expression Profiling
  • Iris / cytology*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mice
  • Mitomycin / pharmacology
  • Mitosis / drug effects*
  • NIH 3T3 Cells
  • Phagocytosis / physiology
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Pigment Epithelium of Eye / cytology*
  • Pigment Epithelium of Eye / physiology
  • RNA, Messenger / metabolism*
  • Retinal Pigment Epithelium / cytology*
  • Retinal Pigment Epithelium / physiology
  • Reverse Transcriptase Polymerase Chain Reaction
  • Rod Cell Outer Segment / metabolism
  • Swine
  • Tight Junctions
  • Zonula Occludens-1 Protein


  • Alkylating Agents
  • Collagen Type I
  • Membrane Proteins
  • Phosphoproteins
  • RNA, Messenger
  • TJP1 protein, human
  • Tjp1 protein, mouse
  • Zonula Occludens-1 Protein
  • Mitomycin
  • Collagen