Abstract
Purpose: :
The endothelial cells of Schlemm’s canal (SC) are likely important for IOP regulation, but these cells normally experience large strains (e.g. giant vacuole formation) that may affect their barrier function. We investigate whether biomechanical strain can be effectively imposed on and tolerated by cultured SC cells using an established cell-stretching device (Lee et al., Am J Physiol, 1996).
Methods: :
SC cells were isolated and cultured from human donor eyes using established methods (Stamer et al., IOVS, 1998). Two days prior to experiments, cells were seeded at low density on elastomeric membranes mounted within the stretcher and cultured in medium containing 10% serum. Membranes were pre-coated with collagen I either by passive adsorption or covalent chemistry, following Wipff et al. (Biomat., 2009). Phase contrast images were acquired immediately before and ≤2 min after 10% equibiaxial stretch was applied and maintained to the membrane. Viability was assessed using a fluorescent Live/Dead assay. In separate studies, we calibrated membrane strain by tracking fiducial markers on the membrane.
Results: :
SC cells attached and spread on elastomeric membranes similarly to cells seeded on TC plastic when collagen was covalently bound, but spreading was poor when collagen was passively adsorbed. Application of 10% equibiaxial strain (corresponding to 21% areal increase) lead to a significant increase in cell area (12.9±6.9%, mean±SD; n=31 cells in 3 independent experiments, p<10-10) compared to unstretched controls (-0.18±2.6%, n=25), but the area increase was significantly less than the expected 21% value (p<10-6). In contrast, strain estimates based on tracking the displacement of individual nuclei agreed with the expected 21% value (21.8±2.4%). While stretch was well tolerated by most cells, a small fraction died within 40 min after stretch (3 of 57 cells in 3 independent experiments) compared to unstretched controls (0 of 78 cells).
Conclusions: :
Systems to study SC cells in response to stretch must incorporate covalent binding of matrix proteins to the membrane and calibration of the strain field. Based on cell area changes, however, SC cells do not appear to experience the total membrane strain, possibly because of insufficient cell-substrate linkages or rapid retraction of the cell within 2 minutes of stretch. These limitations must be considered in any future studies that investigate the response of SC cells to lateral stretch.Acknownledments: We thank Prof. Dan Stamer and Kristin Perkumas (U. of Arizona) for expert provision of SC cells.
Keywords: outflow: trabecular meshwork • intraocular pressure • aqueous