CFSE fluorescence staining to detect daily cell movement in normal human corneas

Abstract

Purpose: We used our human limbal stem cell labelling method previously described to investigate epithelial cell movements and record cell migration daily for one week.

Methods: Fresh normal human corneas from donors not suitable for transplantation were provided by the Melvin Jones Lions Eye Bank (Genoa, Italy) and used for the study. Carboxyfluorescein diacetate succinimidyl esters (CFSE) (Molecular Probes) was used to target corneal epithelial cells; fluorescence was digitally recorded for seven consecutive days (T1-T7), and the rate of cell movement was determined by tracing the different position of the CFSE-labelled limbal stem cells. A daily time-lapse sequence was digitally recorded.<br /> Cell movement was analyzed in frozen cross- sections by fluorescence microscopy.

Results: Daily average CFSE-labelled limbal stem cell movement was 0.073 cm ± 0.01 (± SD) / day.

Conclusions: CFSE staining allowed us to track corneal epithelial cells, from the limbal basal layer centripetally to the superficial epithelium and showed to be a reliable method able to observe and quantify the migration of epithelial cells on a daily basis. CFSE-labelled cells moved towards the corneal surface in one week.