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New technique for culturing corneal epithelial cells of normal mice
Takeshi Kobayashi1, Ryuji Yoshioka, Atsushi Shiraishi
1Department of Ophthalmology and Regenerative Medicine, Ehime University School of Medicine, Shitsukawa, Toon, Ehime, Japan.
Purpose:
To describe a new method of culturing mouse corneal epithelial cells (MCECs).
Methods:
MCECs were isolated from C57/BL6 mouse corneas and cultured on type-I collagen-coated plastic dishes in low-calcium progenitor cell targeting medium (CnT-50). Expression of the mRNAs of N-terminal truncated isoform of p63 (DNp63), cytokeratin 12 (K12), and cytokeratin 14 (K14) were determined by reverse transcription-polymerase chain reaction (RT-PCR). To examine the differentiation capabilities, passage 3 (P3) MCECs at confluence were subcultured on amniotic membrane (AM) in a differentiation medium (CnT-30) until confluence. At confluence, 1 mM calcium was added and cultured for 4 more days. The expression of K12 in the stratified MCECs was analyzed by immunostaining.
Results:
The MCECs cultured in CnT-50 proliferated until at least P10. The number of cells at confluency at P3 was 61.8 (SD +/-9.4, n=5) times that at P0. MCECs cultured on AM in CnT-30 with addition of calcium were stratified up to two to three layers, and the stratified MCECs expressed K12. DNp63 mRNA was continuously expressed throughout the different passages, and K12 mRNA was detected in P0 cells and the stratified MCECs on AM.
Conclusions:
Cultured MCECs maintain their proliferation and differentiation capabilities as well as their corneal epithelial cell characteristics. These results suggest that MCECs produced by this culturing method provide a useful experimental model which can enable further development of research of the corneal epithelium.
Insights
A new method successfully cultures mouse corneal epithelial cells (MCECs), maintaining their key characteristics. This technique provides a valuable model for corneal epithelium research.
Area of Science:
- Ophthalmology
- Cell Biology
- Tissue Engineering
Background:
- Mouse corneal epithelial cells (MCECs) are crucial for studying corneal diseases and regeneration.
- Existing culture methods may not fully preserve MCEC characteristics, limiting their utility in research.
Purpose of the Study:
- To establish and validate a novel method for culturing MCECs.
- To assess the proliferation and differentiation potential of MCECs cultured using the new method.
Main Methods:
- MCECs isolated from C57/BL6 mouse corneas were cultured on collagen-coated dishes in low-calcium medium (CnT-50).
- Gene expression (DNp63, K12, K14) analyzed via RT-PCR.
- Differentiation potential assessed by subculturing on amniotic membrane (AM) in differentiation medium (CnT-30) with calcium supplementation.
Main Results:
- MCECs demonstrated sustained proliferation up to passage 10.
- Significant cell expansion observed by passage 3.
- Stratified MCECs cultured on AM expressed K12, indicating successful differentiation.
Conclusions:
- The described culture method effectively maintains MCEC proliferation and differentiation capabilities.
- Cultured MCECs retain essential corneal epithelial cell markers.
- This method offers a robust experimental model for advancing corneal epithelium research.
