Abstract
Purpose :
Increased intraocular pressure (IOP), arising from reduced functionality of outflow tissues, is one of the primary causes underlying the progression of glaucoma. Various current methods can help to alleviate elevated IOP, but improvements to efficacy are urgently needed. We aim to identify compounds that directly target a major site of resistance to aqueous humor outflow, Schlemm’s canal endothelial cells (SCEC).
Methods :
We propose a multi-layer microfluidic design for high-content screening of multiple compounds, to identify those that increase SCEC monolayer hydraulic conductivity. The device comprises of upper and lower channels laser cut from polymethyl methacrylate sheets, with a porous polyethylene terephthalate membrane sandwiched in between, allowing flow between the layers. A glass coverslip at the bottom provides optical access to the lower channel and membrane, upon which SCECs are cultured. The layers of the device are assembled using 3M adhesive tape. Hydraulic conductivity can be measured based on the difference in velocity of micron sized particles in perfusate in the lower channel upstream and downstream of the membrane using particle tracking velocimetry (PTV).
Results :
First, we identified the optimal method to fabricate the devices. Although polydimethylsiloxane is commonly used for microfluidic designs our alternative design/materials can be assembled in 5 minutes per device and is compatible with large scale manufacturing. Through live-dead AOPI assay we observed no difference in the viability of SCECs cultured in presence of tape as compared to tissue culture plastic.
Flow rates through EC monolayers in vitro are too small to measure using standard techniques. Our PTV approach was validated by applying known flow rates to the channels, and visualising particle location across multiple frames. The maximum particle velocities at each location were compared to an analytical solution for water through a rectangular channel to extract flow rates. Further validation is required for accuracy, but initial testing indicates feasibility of the approach.
Conclusions :
We have designed an innovative approach to measure hydraulic conductivity of multiple parallel SCEC monolayers using microfluidic methods. Once fully validated, we can perform high-content screening of various compounds simultaneously.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.