Comparison of different methods to isolate mouse limbal epithelial cells for single-cell analysis

Abstract

Purpose : Single-cell analysis provides an emerging technology to extract stem-cell specific signals. The preparation of single-cell suspension for the analysis on mouse limbal epithelial cells (LECs) is difficult and has not been established yet. Here we compared different methods to isolate mouse LECs into single-cell suspension.

Methods : Male and female mice at around 6 weeks old were used in this study. After euthanization, the central corneal epithelium was removed by a rotating burr and the eyeballs were collected followed by the removal of excess conjunctiva. The eyeballs were divided into 3 groups. In group 1 (G1), the intact eyeballs were incubated in DispaseII for overnight at 4 °C, followed by trypsin digestion with pipetting using 30 gauge needles. In group 2 (G2), the intact eyeballs were incubated in DispaseII for 1 hour and DispaseII plus collagenaseA for another hour at 37°C, followed by trypsin digestion with pipetting using 30 gauge needles. In group 3 (G3), the corneoscleral rims were dissected from eyeballs, cut into pieces, and incubated in trypsin for 10 cycles. The cells collected were analyzed for cell number, cell viability, percentage of single cells, percentage of small cells (cell diameter≤11µm), and percentage of epithelial progenitor cells expressing cytokeratin 14. The remaining tissue was examined by Haemotoxylin and Eosin (H&E) staining.

Results : The whole corneoscleral epithelial sheets could be peeled off in G1 and G2, which was confirmed by H&E staining. G2 harvested the highest number of cells than G1 and G3 (G1: 1.8x10⁵ cells, G2: 2.4x10⁵ cells, G3: 2.4x10⁴ cells, P<0.001). G1 and G2 had more live cells (G1: 88%, G2:86%, G3: 75%, P<0.0001), more small cells (G1: 49%, G2:58%, G3: 21%, P<0.05), and more K14⁺ cells (G1: 52%, G2: 46%, G3: 41%) than G3. G2 contained more single cells than G1 (G1: 59%, G2: 74%, G3: 61%, P<0.01). The histology on the remaining tissue showed that the basal limbal epithelial cells were completely removed in G1 and G2.

Conclusions : Dispase and collagenase digestion of the intact eyeball (G2) seems a better method to obtain mouse limbal epithelial cells in single-cell suspension.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.