Microfluidic cell culture system
for testing the presence of toxic
substances in water
Irfan A Khan
Advisors: Dr. Michael L. Norton
Dr. Bin Wang
BBSC234
04/09/2012
Background

Arsenic contamination in
Bangladesh

31 million pounds of toxic
chemicals were dumped into
the Ohio River in 2007
(Source: Ohio River
Foundation)

Dioxin, PCB, Metals and
Pesticides (Source: EPA)
Diagram courtesy: http://cnx.org
Introduction

Common hazardous environmental toxins
◦ Heavy metals e.g., Pb, Cd, Hg, As
◦ Pesticides and herbicides
◦ Poly-chlorinated biphenyls (PCB)

May remain in environment in undetectable
concentrations for indefinite period

Can reach toxic level inside animal body through
biomagnification and bioaccumulation
Why endothelial cell culture?

Nitric oxide production can be
stimulated in healthy cells in vitro

Endothelial cells are very
sensitive to low levels of toxins

Decreases nitric oxide
production if exposed to toxic
substances
SEM Cross-section of an arteriole
Image courtesy: http://db2.photoresearchers.com/preview/SB9558.html
Endothelial cells
Objectives
Short term objective:
To analyze effect of environmental toxins on nitric
oxide production in endothelial cells
Long term objective:
To test the effect of environmental toxins on
endothelial nitric oxide production in cells grown
inside a microfluidic chip
Hypothesis

Endothelial cell culture can be used to
develop a very sensitive water toxicity
testing method
Materials and methods

Human umbilical vein endothelial cell (HUVEC)
culture

Bradykinin (100µM final concentration)

Cadmium chloride (40 nM final concentration)

Confocal Microscope

DAF-FM DA probe for nitric oxide detection
The nitric oxide probe DAF-FM DA
Cell membrane
Esterase
DAF-FM DA
(No fluorescence)
DAF-FM
(Weekly fluorescent)
NO•
Benzotriazole derivative
(Fluorescent)
Results

30 minutes time course for
1. Cells exposed to bradykinin stimulation only
2. Cells incubated with 40nM CdCl2 for 5 hours
prior to bradykinin stimulation
30 min time course for effect of BK on cells
without cadmium exposure
30 min time course BK stimulation in
cells not exposed to cadmium
mean intensity
50
40
30
20
10
0
0

5
10
15
20
Time (minutes)
25
30
The fluorescence intensity does not decrease with time
30 min time course for effect of BK on cells
with cadmium exposure
30 min time course BK stimulation in
cells exposed to cadmium
mean intensity
50
40
30
20
10
0
0

5
10
15
20
Time (minutes)
Fluorescence intensity decreases with time
25
30
Conclusion

Endothelial cell culture can be used for
testing the presence of 40nM cadmium
chloride in water by confocal microscopy
Future works

Testing other environmental toxins

Endothelial cells inside microfluidic
channels

Testing same toxins with microfluidic
endothelial cell culture

Electrochemical probe will be adopted if
possible
Acknowledgement
Dr. Bin Wang
 David Neff
 Dr. Elmer Price
 Colton Koontz
 Dr. Michael Norton
 Dr. Masudur Rahman

Thank you
Questions please
Confocal imaging

Single focal plane 30 minutes time course

488nm laser excitation

515-555nm PMT emission window

ImageJ for image data analysis

MSExcel for plots
Confocal scanning microscopy
LASER
Path of scan
(Red line)
Focal planes
Microfluidic endothelial cell culture

Endothelial cells experience
5dyne/cm2 shear stress due to
friction with blood flow

Increases nitric oxide production
with increasing blood flow

The shear stress can be mimicked
inside a microfluidic chip
Endothelial cells lining
the arterial wall
http://db2.photoresearchers.com/preview/SB9558.html
Fig: SEM Cross-section of an arteriole
Microfluidic chips

The chips are designed by AutoCAD

COMSOL Multiphysics is used to simulate
proposed designs

The chips that give uniform fluid flow in
simulation and are easy to operate, are
fabricated in PDMS (Poly-dimethyl
siloxane) on glass slide
PDMS chips on glass slide

PDMS is a Crystal clear rubber like
material with high tensile strength
5mm
For eNOS activity monitoring in microfluidic chips
by fluorescence microscopy
Contro
l
Test
Cell growth area
Directions of fluid flow through the chip
Culture maintenance
Cell seeding
Toxicity testing
For eNOS activity monitoring in microfluidic chips
by fluorescence microscopy
Control
Media
Test
Cell growth area
Directions of fluid flow through the chip
Culture maintenance
Cell seeding
Toxicity testing
Media
Cell growth area

For real-time monitoring of
effect of substances on
endothelial cells inside a
microfluidic chip
Testing the DAF-FM DA probe

Incubated cells for 30 minutes with
5µM DAF-FM DA in dark, at 37⁰C &
5% CO2

Excitation filter of 465-495nm

Emission wavelength 505nm – 555 nm
No cadmium with BK time
course raw data
With cadmium with BK time
course raw data
Arsenic in groundwater: A threat to sustainable agriculture in South and
South-east Asia
Hugh Brammera, Peter Ravenscroftb
a
Former FAO Agricultural Development Adviser, Bangladesh, 1974-87. 37 Kingsway
Court, Hove, East Sussex, BN3 2LP, United Kingdom
b
Principal Consultant, Entec UK Ltd, Trinity House, Cambridge Business Park, Cowley
Road, Cambridge, CB4 0WZ, United Kingdom
Received 20 June 2008. Accepted 15 October 2008. Available online 24 December
2008.
http://dx.doi.org/10.1016/j.bbr.2011.03.031