Optimal magnitude of shear stress for human corneal epithelial cell proliferation in microfluidic culture system

Abstract

Purpose : Human corneal epithelial cells (HCEC), the outermost layer of cornea, are exposed to shear stress caused by eyelids during blinking and eye rubbing. They play an important role in corneal wound healing process against injury by external trauma or eye surgery. Previous in vitro studies with CECs and other cells suggest that shear stress may affect cell morphology and growth. However, the shear stress magnitudes were highly variable. Therefore, here, we investigated the effect of shear stress on HCEC proliferation by using in-vitro microfluidic culture system to find optimal magnitude of shear stress for in vitro study.

Methods : We used cell line of HCEC, telomerase-immortalized human corneal epithelial cells (hTCEpi) and microfluidic chamber system (Ibidi GmbH, Martinsried, Germany). After seeding 1 × 10⁵ cells/per μ-slide (surface area: 2.5 cm², volume: 200 μl), four different magnitudes of shear stress (0, 0.22, 0.4 and 0.8 dyn/cm²) were applied for 24 hours. After 24 hours shear stress, bromodeoxyuridine (BrdU) was applied to HCEC to measure cell proliferation. Then fixation with 3.7% paraformaldehyde, permeabilization with 0.1% triton X-100, treatment of anti-BrdU antibody and treatment of Cy3-conjugated secondary antibody were performed for BrdU staining. And then Cy3 signal was detected with confocal microscopy.

Results : The increment of HCEC cell density in μ-slides after shear stress was observed across all conditions; 0, 0.22, 0.4 and 0.8 dyn/cm² group, 161.60 ± 27.40 cells/mm², 448.05 ± 32.48 cells/mm², 270.80 ± 32.48 cells/mm² and 247.14 ± 36.77 cells/mm², respectively (Mean ± SEM, p<0.001). As magnitude of shear stress increased, Cy3 intensity observed in nucleus of HCEC increased up to 0.4 dyn/cm², but the difference between 0.22 and 0.4 dyn/cm² was not significant (p>0.05). In 0.8 dyn/cm² group, Cy3 intensity decreased compared with 0.4 dyn/cm² group (p<0.001).

Conclusions : We investigated the effect of fluid shear stress on HCEC proliferation with microfluidic culture system. We suggest that the optimal shear stress for HCEC culture system is in between 0.22 to 0.4 dyn/cm². In future study, we would like to investigate the molecular mechanism of shear stress-dependent cell proliferation.

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