Abstract
Purpose: :
To develop a novel 3D in vitro model to study the mechanism of giant vacuole (GV) formation of human Schlemm's canal endothelial cells (HSCEC) in response to basal-to-apical transendothelial flow with and without the presence of pharmacological agents.
Methods: :
A microfluidics-based 3D cell culture system was designed with two microfluidic channels for delivering perfusate and a central region for housing a 3D collagen gel; this geometry permits the establishment of pressure gradients across the collagen gel. Confluent monolayers of HSCEC were generated by perfusing a cell suspension into one channel, allowing the cells to settle along the collagen surface followed by 2-3 days of static culture. Microvascular endothelial cells (HMVEC) were used as a control. Small reservoirs were used to impose pressure differentials and produce basal-to-apical flow across the monolayer. Two different sets of experiments were conducted: (1) observe GV dynamics and tracer passage across the monolayer and (2) study the effects of pharmacological agents on GV formation. In the first experiment, confluent monolayers were labeled with live cell membrane or cytosolic dye. Multichannel confocal images were acquired and time-lapse imaging of GV dynamics and the hydrodynamic filtration pattern of fluorescent microspheres (200nm) were recorded and analyzed. In the second experiment, cells were incubated with 50 uM Y27632 for 1 hour prior to basal-apical perfusion with the same solution. Monolayers were then perfusion-fixed, counter-stained and examined with confocal microscopy. After confocal images were taken, cells were then processed for transmission electron microscopy (TEM).
Results: :
Confocal and TEM micrographs confirmed that a confluent monolayer of HSCECs and HMVECs was formed. GV formation was observed when both types of cells were exposed to a basal-to-apical flow. Time-lapse microscopy confirmed the dynamic nature of GV formation. Tracer was observed crossing SCEC monolayer near GVs. More GVs were seen after Y27632 treatment.
Conclusions: :
We have developed a physiologically relevant in vitro model to investigate GV dynamics with real-time imaging capabilities and endpoint structure analysis using optical and transmission electron microscopy. Our ability to obtain transverse optical sections and TEM images in the same set of HSCEC and HMVEC has given us a unique perspective for studying GV dynamics and the effects of pharmacological agents.
Keywords: outflow: trabecular meshwork • microscopy: light/fluorescence/immunohistochemistry • microscopy: electron microscopy