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Published on: October 28, 2012
A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures
Zhong Chen1, Xiaowen Lu1, Namratha Mylarapu1
1Department of Cellular Biology & Anatomy, Medical College of Georgia, Augusta University, Augusta, GA, USA.
Purpose:
This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures.
Methods:
Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method. Methods evaluated included 0.25% trypsin-EDTA, low temperature, mitomycin-C, and dispase. Degree of fibroblast contamination was examined using light microscopy evaluation of cell phenotype, immunofluorescence and western blotting using cell type-specific markers. Anti-pancytokeratin (PanCK) was used as the epithelial immunofluorescence label, and anti-α smooth muscle actin (αSMA) as the fibroblast immunofluorescence label. Epithelial western blot antibodies included PanCK, keratin 12, and E-cadherin, while αSMA, collagen 1A1 and collagen 3A1 were used to identify fibroblasts.
Results:
Fibroblast contamination of human and mouse primary cornea epithelial cell cultures was best controlled using the 0.25% trypsin-EDTA method. The other methods examined were not effective at eliminating cornea fibroblast contamination.
Conclusions:
Trypsin-EDTA digestion is a simple and effective method for controlling fibroblast contamination of cultured primary human and mouse corneal epithelial cells.
Insights
A simple trypsin-EDTA method effectively eliminates fibroblast contamination in human and mouse primary corneal epithelial cell cultures, enabling easier cell culture. This approach ensures pure cell cultures for research.
Area of Science:
- Cell Biology
- Tissue Engineering
- Ophthalmology Research
Background:
- Fibroblast contamination is a common challenge in primary cell cultures.
- Maintaining pure epithelial cell cultures is crucial for accurate research outcomes.
- Previous methods for fibroblast elimination have varied success across different cell types.
Purpose of the Study:
- To evaluate existing methods for eliminating fibroblast contamination in human and mouse primary corneal epithelial cells.
- To identify a simple, effective, and easy-to-implement method for culturing fibroblast-free corneal epithelial cells.
- To establish a reliable protocol for obtaining pure primary corneal epithelial cell cultures.
Main Methods:
- Isolation and culture of primary human and mouse corneal stromal and epithelial cells.
- Evaluation of purification methods including trypsin-EDTA, low temperature, mitomycin-C, and dispase.
- Assessment of fibroblast contamination using light microscopy, immunofluorescence (PanCK, αSMA), and western blotting (PanCK, K12, E-cadherin, αSMA, Col1A1, Col3A1).
Main Results:
- The 0.25% trypsin-EDTA method demonstrated superior efficacy in controlling fibroblast contamination.
- Other evaluated methods, including low temperature, mitomycin-C, and dispase, were ineffective.
- Successful elimination of fibroblast contamination was confirmed through cell-specific marker analysis.
Conclusions:
- Trypsin-EDTA digestion is a simple and effective strategy for eliminating fibroblast contamination.
- This method facilitates the culture of pure primary human and mouse corneal epithelial cells.
- The findings provide a straightforward protocol for researchers working with corneal epithelial cells.
