Collagenase isolation method delays endothelial to mesenchymal transition

Abstract

Purpose : Cell therapy as a treatment for corneal endotheliopathies requires amplification of functional corneal endothelial cells (CEC). To avoid endothelial to mesenchymal transition (EMT), we hypothesized that using an isolation method that preserves cell junctions such as collagenase digestion of Descemet membrane would help to maintain the endothelial phenotype. Thus, we compared EDTA isolation and collagenase digestion methods.

Methods : Human corneas (N = 10) from our local eye bank were used to isolate CEC using either EDTA (0.02%, 45 min) or collagenase A, (1 mg/ml, 2-4h). CEC were counted before seeding to determine the percentage of isolated cells. Cell circularity used to analyse cell morphology was measured at every passage using phase contrast images (P0 to P3; 100 images/condition). At P3, cells were fixed and immunofluorescence directed against ZO-1 (tight junction marker) and α-SMA (EMT marker) was performed. Finally, in vitro functionality was assessed using transendothelial electric resistance (TEER) throughout the P3 culture. For the TEER measurements, controls included fibroblasts (a population that does not form a cell-barrier) and ARPE-19 (a population that consistently forms a barrier of 40 Ωcm²),

Results : A significantly higher percentage of isolated cells was obtained using the collagenase digestion method compared to EDTA (EDTA: 40±5 %; collagenase: 54±7 %; p=0.02). Cell morphology analysis showed that CEC isolated using collagenase were more circular (endothelial) than those isolated in EDTA at P0 (EDTA: 0.67±0.07; collagenase: 0.80±0.03; p=0.02) and P3 (EDTA: 0.58±0.03; collagenase: 0.70±0.05; p=0.01). At P3, ZO-1 staining revealed that it was not localized at cells junctions for EDTA and collagenase method. Positive α-SMA staining was observed and there was no difference in α-SMA expressing area percentage for both isolation methods (EDTA: 6±4 %; collagenase: 6±5 %). There was also no difference in TEER measure (EDTA: 40±20 Ωcm2; collagenase: 42±20 Ωcm2).

Conclusions : This study shows that isolation of CEC using collagenase helps to conserve the endothelial phenotype throughout cell expansion. The delayed TEM given by collagenase isolation is a step forward to clinical use of cultured CEC.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.