Abstract
Purpose :
Shear stress is known to regulate the behavior of many cell types. The anterior surface of the eye is subjected to shear stress through tear film movement and blinking; however, little is known about how this stress affects ocular cells. The aim of this study was to examine the effect of fluidic shear stress on limbal-derived epithelial stem cells.
Methods :
Human limbal stem cells were isolated from donated corneal scleral rims with donor consent for research purposes. Cells were obtained from three separate donors of varying ages. To examine the cellular response to fluidic shear stress, we employed a commercially available fluid flow system (IBIDI). A total of 500,000 cells were seeded onto µslides (IBIDI) and exposed to either high shear (2.43 dyn/cm2) or low shear (1.1 dyn/cm2) for 1 or 3 days. Cells not subjected to shear were utilized as a control. Subsequently, we analyzed the cells using RT-PCR and immunocytochemical staining.
Results :
After subjecting cells to shear stress for 1 to 3 days, gene expression associated with limbal stem cells significantly increased in cells derived from two out of the three donors. Notably, the 1-day low shear group exhibited the most significant upregulation of stem cell markers among the donors. The expression of mature corneal epithelial markers (cytokeratin 3 and 12) in response to shear stress was also found to be donor-dependent. In addition to variations in gene expression, cells stratified after 3 days of high shear stress. ZO-1 staining was stronger in cells subjected to low shear stress after 3 days although again this varied considerably between donors. TRPV4 was consistently and significantly upregulated across all shear groups and donors compared to static culture.
Conclusions :
This study demonstrates that both low shear and high shear stress significantly influence the expression of limbal stem cell and epithelial markers, cell stratification and barrier function. While there may be advantages to culturing cells under shear flow conditions, variations in the response to shear stress among cells from different donors highlight the importance of considering factors such as donor age, sex, and health in future studies involving human-derived cells.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.