Development of a bioreactor to examine the response of corneal cells to fluid shear stress

Abstract

Aims/Purpose: Mechanical stimulation plays an important role in regulating cell behavior. While several studies have examined how corneal epithelial cells respond to mechanical stimuli such as fluidic shear stress, little is known about how keratocytes respond. The purpose of this study was to design a bioreactor system to determine the effect of shear stress on the behavior of corneal epithelial cells and keratocytes in an in-vitro 3D corneal hydrogel model.

Methods: A bioreactor chamber was made of PDMS containing 4 wells. 10 μl cell seeded hydrogels, consisting of 3.5 mg/ml collagen type I and 10 mM PEG (polyethylene-glycol) containing 5 x 10⁴ keratocytes, were added to each well to replicate the stroma. A 20 μm PCL (polycaprolactone) electrospun membrane was placed on top of each hydrogel and seeded with 1.6 x 10³ corneal epithelial cells. The chamber was connected to a pump system to allow fluid to pass over the hydrogels at specified flow rates. Several tests were performed to validate the bioreactor and examine the cell response to fluid shear stress.

Results: The bioreactor was successfully tested for hydraulic sealing and sterility maintenance. The shear stress was calculated using a simple mechanical model and validated using computational fluid dynamics software. Preliminary studies using collagen hydrogels showed a high degree of contraction so PEG was introduced to the hydrogels to assist in stabilization. The response of corneal epithelial cells to different rates of shear stress was examined by analyzing the expression of YAP (Yes-Associated-Protein) and ZO-1 (Zonula-occludens-1), both of which are known to be mechano-sensitive.

Conclusions: The bioreactor has been developed and validated to apply shear stress to corneal hydrogel models. Further studies will further evaluate the response of epithelial cells and keratocytes to fluid flow at different shear rates.