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Published on: February 4, 2015
Presence of xenogenic mouse RNA in RPE and IPE cells cultured on mitotically inhibited 3T3 fibroblasts
Sandra Johnen1, Lucia Wickert, Marion Meier
1IZKF (Interdisziplinäres Zentrum für Klinische Forschung) Aachen, Germany.
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.
Insights
Culturing bovine retinal pigment epithelial (RPE) and iris pigment epithelial (IPE) cells on mouse 3T3 fibroblasts promotes rapid cell growth. However, contamination with mouse mRNA hinders their use in transplantation.
Area of Science:
- Cell Biology
- Tissue Engineering
- Xenotransplantation
Background:
- Mitotically inhibited 3T3 fibroblasts serve as feeder layers for cell culture.
- Transplanting human cells cultured on mouse feeder cells carries risks of disease transfer and contamination.
- Bovine RPE and IPE cells were investigated for culture on 3T3 fibroblasts.
Purpose of the Study:
- To evaluate the characteristics of bovine RPE and IPE cells cultured on mitomycin-treated 3T3 fibroblasts.
- To assess potential contamination by 3T3 fibroblast products.
Main Methods:
- Microscopy and immunohistochemistry were used to assess cell adhesion, morphology, and tight junction formation.
- Functional activity was evaluated using reactive oxygen species (ROS) phagocytosis.
- Gene expression was analyzed via quantitative real-time PCR.
Main Results:
- Bovine IPE and RPE cells rapidly adhered, spread, and adopted a hexagonal shape on 3T3 fibroblasts within 12 hours.
- Stable expression of pigment epithelial genes was observed in cultured cells.
- Expression of mouse collagen type I was detected, indicating xenogeneic contamination.
Conclusions:
- Culturing on 3T3 fibroblasts facilitates rapid adhesion and growth of primary bovine pigment cells.
- The presence of xenogeneic mouse mRNA in the cultures poses a significant risk.
- This contamination limits the clinical application of these cells for transplantation.
