1. Purpose
• Most glaucoma models irreversibly damage the TM
and are, thus, inappropriate for regenerative medicine.
• Recently, Abu-Hassan et al.4 showed saponin, a
detergent, can produce glaucoma-like TM cellularity
after treatment with 0.01% w/v saponin for 5 minutes,
• Reproducibility and technical difficulties arise due to
rapid saponin exposure.
• Here, we present a modified saponin-based glaucoma
model using low concentration for 24 hours to achieve
glaucoma-like, reproducible changes in TM cellularity
without permanent damage to existing cells.
4 Abu-Hassan, et al. Induced Pluripotent Stem Cells Restore Function in a Human Cell Loss
Model of Open-Angle Glaucoma. Stem Cells (Dayton, Ohio),
33(3), 751–761. http://doi.org/10.1002/stem.1885
Acknowledgements
• NSF Graduate Research Fellowship
• Georgia Research Alliance
• President’s Undergraduate Research Award
• Prof. Dan Stamer, Duke University – hTM Cells
• Irsham Zaman – Live-Dead Imaging
Replicating Trabecular Meshwork Cellularity Changes in
Glaucoma: A Modified in vitro Model
Richard T. Vannatta1 , Eric J. Snider1, C. Ross Ethier1
1 Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University
Saponin Concentration Optimization Ex Vivo Saponin Treatments
Conclusions
Introduction
• Glaucoma affects over 70 million people worldwide,
and is the 2nd leading cause of blindness.1
• In primary open-angle glaucoma, the most common
form of glaucoma, a well-established risk factor is
increased intraocular pressure (IOP) which is regulated
by the trabecular meshwork (TM).
• Glaucomatous eyes exhibit over 50% reduction in TM
cellularity compared to age-matched healthy eyes.2,3
Loss of these cells may lead to tissue dysfunction and
increased IOP.
• TM-targeted regenerative therapies are potential
treatments for glaucoma, but first a model of TM
changes in glaucoma is required.
1 Vitale S, et al. Prevalence of visional impairment in the United
States. JAMA 2006;295(18):2158–2163.
2 Alvarado, J. et al. Invest Ophthalmol Vis Sci 21, 714-727 (1981).
3 Liton, P.B. et al. Exp Gerontol 40, 745-748 (2005).
• Porcine TM cells (pTM) isolated from fresh porcine tissue.
• Human TM cells (hTM) obtained from Dr. Stamer (Duke University).
• 24-hour treatment with 0.0016% saponin achieves repeatable
glaucomatous-like TM cellularity changes.
• Loss of TM’s phagocytic function without loss in proliferation was
observed, suggesting lingering effects of saponin on TM cells.
• Saponin treatment produced glaucomatous cellularity changes in an
ex vivo setting, but IOP homeostasis remained functional.
• Next steps will further investigate residual damage to surviving TM
cells and ex vivo optimization to better mimic glaucomatous IOP.
• Saponin
concentrations
between 0.001%
and 0.002%
applied for 24
hours (n=3).
• Similar trends
for pTM and
hTM cells.
• 0.0016% closely
mimics degree
of TM cell loss.
R² = 0.8398
R² = 0.8832
0%
20%
40%
60%
80%
100%
0.0015% 0.0016% 0.0017% 0.0018% 0.0019% 0.0020%
%Living
% Saponin (w/v)
hTM % Living
pTM % Living
• Furthermore, six independent experiments
on pTM cells were run to examine
repeatability.
• Mean % Living = 42.5%
• Coefficient of variation = 0.143
• For its repeatability and accuracy to
glaucomatous cellularity loss, 0.0016%
was utilized for all subsequent work.
S
Effects of Saponin on Function in Surviving TM Cells
Phagocytic activity and proliferation were examined following saponin treatment
• Phagocytic activity is
characteristic of TM.
• Cells grown with
fluorescent heat-killed
E. Coli. Internalized
particles quantified as
median fluorescence
(n=3).
• 24hr after treatment
show reduced function,
though regain after
72hr
• Anterior segment of fresh porcine eye
secured in custom-built culture dishes.
• IOP monitored throughout perfusion
regime:
1. Media at physiological flow rates
(2.5μL/min) ~ 24hrs
2. 0.0016% saponin at 2.5μL/min ~ 24hrs
3. Media at 2.5μL/min to stabilize ~ 24hrs
4. Media at 5μL/min for IOP challenge
Source: Mao, et al. 2011
• IOP Challenge: Inability to manage IOP is a risk-factor in glaucoma. Doubling flow rate
increases pressure until TM cells accommodate increased flow and pressure
stabilizes.
• Saponin treated eyes show increased pressure vs. controls but recover from IOP
challenge
Initial
Stabilization
Saponin
Treatment
• alamarBlue®
fluorescence monitored
every 15 min for 5 hrs
with plate reader.
• Minimal proliferation
decrease occurred in
hTM and pTM cells,
even after 24 hours.
Control 0.0016% Saponin 0.01% Saponin
Phagocytic Activity
Proliferation
Source: BrightFocus Foundation
2X Flow Rate
Live Dead
C = Cornea
S = Sclera
C
S
Poster # 404
Live Dead Imaging: En face images of micrographs of porcine eyes are shown with
sclera and cornea orientations as indicated. 0.0016% saponin partially decreases
cellularity in TM as compared to high saponin and media only controls.
0%
20%
40%
60%
80%
100%
120%
24 hr hTM 24 hr pTM 72 hr pTM
PhagocyticActivetyNormalizedto
UntreatedControls
Post-Treatment Time and Cell Type
0%
20%
40%
60%
80%
100%
120%
140%
160%
24 hr hTM 24 hr pTM 72 hr pTM
ProliferativeActivityNormalizedd
toUntreaetedControls
Post-Treatment Time and Cell Type
n = 6
0
5
10
15
20
25
30
0 1 2 3 4 5 6 7
IOP(mmHg)
Time (days)
Control
0.0016% Saponin
SS
CC