Comparison of different methods to isolate mouse limbal epithelial cells

Area of Science:

• Ophthalmology
• Stem Cell Biology
• Regenerative Medicine

Background:

• Limbal stem cells (LSCs) are crucial for corneal epithelium maintenance and repair.
• Existing protocols for human LSC isolation are established, but mouse LSC isolation requires optimization for effective downstream analysis.
• Mouse models are vital for studying corneal diseases and LSC function.

Purpose of the Study:

• To compare four distinct methods for isolating mouse limbal epithelial cells, including limbal stem cells (LSCs), in a single-cell suspension.
• To identify the most effective protocol for obtaining a high yield of viable LSCs from mouse corneas.
• To evaluate cell yield, viability, single-cell percentage, and stem/progenitor cell populations across different isolation techniques.

Main Methods:

• Four protocols were evaluated: (1) trypsin digestion of dissected limbal rim, (2) dispase digestion of limbal epithelial sheet after corneal epithelium removal by rotating bur, (3) dispase and collagenase digestion at 37°C, and (4) trephine-assisted epithelial removal followed by dispase and collagenase digestion.
• Key metrics assessed included cell yield, percentage of single cells, cell viability, and the number of K14+ and p63α-bright stem/progenitor cells.
• Comparative analysis was performed to determine the efficiency and suitability of each method.

Main Results:

• Method 1 yielded the lowest cell count, single-cell percentage, and LSC population, making it unsuitable.
• Methods 2, 3, and 4 isolated comparable numbers of K14+ and p63α-bright stem/progenitor cells.
• Method 2 demonstrated higher cell viability than Method 4, while Method 4 achieved the highest single-cell percentage. Method 3 yielded the lowest cell number.

Conclusions:

• Methods 2, 3, and 4 are recommended for isolating heterogeneous limbal stem cells (LSCs) from mouse corneas.
• The choice between methods 2, 3, and 4 depends on specific research needs regarding cell viability, yield, and single-cell suspension quality.
• Optimized mouse LSC isolation protocols are essential for advancing corneal research and therapeutic development.