Microfluidic Modeling of the Human Schlemm’s Canal Endothelium - PDF Document

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  1. Microfluidic Modeling of the Human Schlemm’s Canal Endothelium Atur Patel, Chen-Yuan Yang, Haiyan Gong Department of Ophthalmology, Boston University School of Medicine AIMS BACKGROUND RESULTS • Primary open angle glaucoma (POAG) is a leading cause of blindness and the primary risk factor is chronic elevation of intraocular pressure, which results from increased aqueous outflow resistance. • T o utilize a 3D microfluidic cell culture device to construct an in vitro model of a monolayer of Schlemm’s canal endothelium. • T o determine if the Schlemm’s canal endothelium, on its own, poses a significant barrier to aqueous outflow and therefore contributes to increased outflow resistance in POAG. • Amonolayer of endothelial cells covering Schlemm’s canal is an important barrier in the aqueous outflow pathway. Characterizing how fluid movement across this endothelial layer is reduced in POAG is paramount to understanding the pathogenesis of the disease. HUVEC Monolayer SCEC Monolayer • T o determine how aqueous humor moves across the Schlemm’s canal endothelium using fluorescent tracers • The physiologic response of Schlemm’s Canal Endothelial Cells (SCECs) to diffusion or flow has not been well characterized. However, a similar cell line, Human Umbilical Vein Endothelial Cells (HUVECs), has been well characterized and thus serves as an ideal control cell line. Apical Side Basal Side Apical Side Basal Side Collagen Matrix Collagen Matrix (a) (b) METHODS Fluorescent Intensity Plot (70kDA Diffusive Permeability (70kDa Dextran) Dextran Particles Permeability SCEC (n=1) 3D Collagen Hydrogel Fluorescent 1.2 Cell Barrier 1.40E-05 Intensity 1.20E-05 Cell Monolayer 0.8 HUVEC (n=4) (cm/s) 1.00E-05 Apical Side Basal Side Normalized SCEC HUVEC 0.6 8.00E-06 Diffusive Basal Side 6.00E-06 250 px 0 px HUVEC 0.2 4.00E-06 Cell Monolayer 0 50 100 150 200 250 2.00E-06 2012) Distance (pixels) 0.00E+00 PDMS Media Figure 2. (a) HUVECs: Phalloidin and DAPI stain after 2 hours of media flow and 1 hour of fixative flow at 157 Pa. (b) SCECs: Phalloidin and DAPI stain after 2 hours of media flow and 1 hour of fixative flow at 157 pa. (c) 70 kDa fluorescent dextran passive diffusion (basal-apical) across a monolayer of HUVECs. Note the sharp border of green fluorescence on the left, across the monolayer, indicating a good cell barrier. (d) Normalized fluorescent intensity plotted against the pixel distance across the cell barrier (as shown on the red line in figure 2c) comparing SCECs and HUVECs. (e)Acomparison of the diffusive permeability between HUVECs used in this study, HUVECs from a previous study (Zervantonakis, 2012), and SCECs. (b) Apical Side (a) Channels Figure 1. (a) Schematic of a microfluidic system for establishing basal to apical flow across a monolayer of cells (Vickerman , 2012). (b) Picture of a microfluidic device after fabrication. The 3D microfluidic cell culture devices were constructed from polydimethylsiloxane (PDMS) and consisted of two parallel channels connected by a 3D collagen matrix embedded in a micro-pillar array. A monolayer of HUVEC or SCECs were seeded on one side of the gel matrix. CONCLUSION Diffusive permeability was characterized by passively allowing 70kDa fluorescently labeled dextran particles to diffuse across the collagen matrix and monolayer (basal to apically as in situ), taking fluorescent images every 10 mins. Diffusive permeability was then calculated by plotting the change in fluorescent intensity across the monolayer. A proof of concept for a microfluidic model of Schlemm’s canal was demonstrated in these experiments. HUVECs seeded on microfluidic devices were shown to form a monolayer barrier with a similar diffusive permeability as past studies indicating that our model has the potential to simulate the in-situ monolayer barrier of Schlemm’s Canal endothelium. Flow was characterized by setting up columns of media on the basal channels and letting it flow across the collagen matrix and monolayer, marking fluid levels every 30 mins. SCECs were shown to have a significantly higher diffusive permeability than HUVECs which indicates either a problem inherent to the cell line including collection methods and donor variability or SCECs may, in fact, not pose significant resistance to diffusion on their own. RESULTS Future studies should characterize flow (in addition to diffusion) across the monolayer barrier. Additionally, more imaging studies with confocal and electron microscopy should be acquired. Finally, more studies with SCECs are necessary with different donor sources such as monkey eyes to better characterize these cells. • HUVECs showed adequate cell-cell adhesion [demonstrated by VE-cadherin stain] on glass bottom dishes (not shown). • HUVECs and SCECs formed a monolayer of cells when seeded on the microfluidic device as expected (Figure 2a,b). • Fluorescently labeled dextran particles set to passively diffuse across a monolayer of HUVECs (basal-apically) showed a sharp drop in fluorescent intensity across the monolayer indicating a good cell barrier. (Figure 2c,d). The diffusive permeability of HUVECs was consistent with previous data (Figure 2e). ACKNOWLEDGEMENTS This work was supported in part by the MSSRP, NIH EY022634 grant, and The Massachusetts Lions Eye Research Fund. • The diffusive permeability of SCECs (1.4*10-5 cm/s) was significantly higher than that of HUVECs (6.6*10-6 cm/s) (Figure 2e). References: 1.Vickerman et al. Mechanism of a flow-gated angiogenesis switch: early signaling events at cell-matrix and cell-cell junctions. 2012 2. Zervantonakis et al. Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function. 2012