w ¼ 200 mm to

study cells up to 24

m

m in height (e.g. oviductal

15

epithelial cells).

In addition, our device spans a shear stress

range of 0.7–130 dynes cm 2 (0.07–13 Pa), which also covers the

physiological shear stresses (1–70 dynes cm 2, 0.1–7 Pa)16,17 in

resting conditions. It exceeds this range to accommodate for

increased wall shear stress during exercise17 and high shear

pathologic states, e.g. thrombosis (70 to >100 dynes cm 2, 7 to

>10 Pa).16

This paper outlines the design and development of our novel

and simple multishear microdevice that can be used as a tool to

simultaneously investigate the effect of a wide range of shear

stresses on cell behaviour. The utility of this device was validated

by studying the effect of shear stress on the von Willebrand

factor (vWF, a large multimeric glycoprotein involved in platelet

Fig. 2 The microfluidic device with ten parallel channels.

adhesion and aggregation) production of HUVECs over a range

of 0.7–130 dynes cm 2 (0.07–13 Pa).

(where m is the viscosity, and L is the length of the channel). As

Experimental

all channels were designed to possess identical width and height,

the relationship between flow rate and resistance between two

Design and fabrication

channels was simplified to LAQA ¼ LBQB. Based on this equality,

The device has one inlet, one outlet and ten channels (Fig. 1). The

and the condition that the sum of flow rates in the individual

channels equal the main inlet flow rate into the device, the

flow rate within a microchannel is given by Q ¼ DP/R, where Q is

channel lengths were calculated to create a two order of magni-

the flow rate, DP is the pressure drop across the channel, and R is

tude difference in flow rates between channel 1 and channel 10,

the channel resistance. With a common reservoir outlet (Fig. 1

which in turn gave two orders of magnitude difference in shear

and Fig. 2), the pressure drop of each channel was designed to be

stress, according to tw ¼ 6Qm/(wh2), where tw is the shear stress,

equal (i.e. DPA ¼ DPB, where DPA and DPB are the pressure

and m is the viscosity of the media (0.001 N s m 2). The Reynolds

drops in channel A and channel B, respectively. Similarly, QARA

number (Re ¼ nDhr/m, where v is the velocity, Dh is the hydraulic

¼ QBRB, where QA and RA are the flow rate and resistance in

diameter (Dh

2wh/(w + h)), and r is the density of the media

channel A, and QB and RB are the flow rate and resistance in

3 ¼

channel B). The resistance of a rectangular microchannel with an

(998.2 kg m

)), and the entrance length (le ¼ (0.5 + 0.065Re)Dh)

aspect ratio of 2 (i.e. w z h, where w is the width and h is the

were also calculated

to ensure

laminar

flow and established

Poiseuille flow profiles in the channels.

height) can be found by:

To validate the device design, fluid flow profiles in the device

R

¼

(12 mL/wh3)[1

(h/w)((192/p5)PN

(1/n5)tanh(npw/

were obtained using micro particle image velocimetry (mPIV).

2h))] 1

n¼1,3,5

For these measurements, the fluid was seeded with red fluores-

cent particles of mean diameter 0.49 mm. A TSI PIVCAM 13 - 8

CCD (1280 1024 pixel resolution) camera synchronised with

a dual-head Nd:YAG pulse laser was used to obtain sequential

images. The time delay between two images was 40 ms. A total of

25 instantaneous velocity vector fields were extracted from the

particle images and ensemble-averaged to calculate the mean

local velocity. A 32 32 (pixel) interrogation window with

50–75% overlapping was used, corresponding to a spatial reso-

lution of 400 mm2. Velocity fields were then obtained using

Matlab, and wall and fluid shear stresses were derived from plots

of2 velocity versus

displacement (i.e. t

¼

dv

z/dx ¼ 2vmaxx/((w/

2

2

2) ) since vz ¼ vmax[1 (x /((w/2) ))]).

The device was fabricated using conventional microfabrication

techniques involving SU-8 photolithography and poly-

dimethylsiloxane (PDMS) soft lithography. Briefly, the designs

were generated using AutoCAD 2007 (Autodesk) and printed on

transparencies with 4000 dpi resolution (Graphic Skills, Bris-

bane, Australia). The transparencies were used as masks in

contact photolithography and a 365 nm light source (OAI, San

Fig. 1 Schematic of the multishear device containing ten channels with

Jose, CA, USA) was used to yield masters composed of a positive

relief of SU-8 photoresist on a silicon wafer.

varying lengths (H ¼ height; Z1 ¼ channel width; Z2 ¼ channel spacing;

The pattern created on the silicon wafers was replicated in

L1, 2, 3, 4, 5, 6, 7, 8, 9, 10

¼

length of channels). AA0

is the cross-sectional

view of this device.

poly-dimethylsiloxane (i.e. silicone elastomer). The silicone

1898

| Lab Chip, 2009, 9, 1897–1902

This journal is ª The Royal Society of Chemistry 2009

elastomer (Sylgard 184, Dow Corning, Midland, MI, USA) was

average of three separate experiments. Student t-test was

mixed 10 : 1 (w/w) with a silicone elastomer curing agent. The

peformed and the results are represented as mean SE.

mixture was poured over the patterned wafer to completely cover

the pattern and placed in the oven for 40 min at 60 C to cure.

Immunofluorescence labeling

The PDMS replica of the network design was cut out and finally

bonded to a clean glass slide using an air plasma to produce

After exposure to shear, the cells in the shear devices were rinsed

a sealed microfludic network. In addition, a special mould

with PBS. They were then fixed with 4% paraformaldehye in PBS

featuring an inverted cone, to allow easy removal of air bubbles

solution for 10 min and permeabilised with 1% Triton-X100 in

in the device, was made for the reservoir outlet. The PDMS

PBS for 10 min. The device was incubated with normal goat

replica of the reservoir was bonded to the rest of the device after

serum for 30 min to prevent nonspecific binding. The von Wil-

being treated with air plasma (Fig. 2).

lebrand factor of the cells was labelled with indirect immuno-

fluorescence staining. Firstly, polyclonal rabbit anti-human vWF

Cells

antibody (DakoCytomation, Denmark) at a 1 : 400 dilution was

used to incubate the cells for 45 min, at room temperature

HUVECs were harvested from human umbilical cord veins.

( 20 C). Alexa Fluor 633 (lem ¼ 515 nm) conjugated goat anti-

Briefly, the cells were isolated by perfusing the veins with a digest

rabbit IgG (Molecule Probes, USA) was used to label the cells as

solution (0.2% w/v Collagenase Type 2 (Worthington) in Hank’s

the second antibody at a dilution of 1 : 250 for 30 min, at room

balanced salts solution (Sigma)) for 10 min at 37 C. The

temperature. Cells were thereafter rinsed with PBS. Phalloidine-

harvested cells were grown in medium RPMI 1640 (Gibco) with

rhodamine (2 U mL 1, lem ¼ 565 nm) was used to label the

10% fetal bovine serum (Gibco), 100 mg mL 1 endothelial cell

F-actin microfilaments of the cytoskeleton of the HUVECs at

growth supplement (ECGS, BD BioSciences), 50 mg mL 1

a dilution of 1 : 50 for 30 min. DNA staining was carried out

heparin (Sigma), and 100 mg mL 1 penicillin/streptomycin. Only

using Hoescht (33342, Invitrogen) for 10 min.

cells from passages 4 to 8 were used in our experiments. Cell

Images were acquired with a Qimaging CCD camera (Retiga

culture was conducted in a humidified 5% CO2 and 95% air

Exi FAST 1394) connected to an Olympus BX61 inverted

incubator at 37 C.

microscope. The fluorescent images obtained from the Qimaging

CCD camera were analysed with Invivo and Image-Pro Plus 6.1

Shear experiment

software to obtain mean fluorescence intensity (MFI) values.

Image J software was used to obtain cell and nuclear sizes (the

After bonding, the devices were sterilised by autoclaving and

area covered per cell and nucleus), perimeters (the length of

allowed to cool down overnight in a laminar flow hood. The

outline around a cell and nucleus), and circularities (a measure of

channels of the device were then coated with fibronectin (human

elongation, where a circularity value of 1.0 indicates a perfect

plasma, Sigma) via injection of a solution of 100 mg mL 1

circle, and decreasing values indicate increasing elongation).

fibronectin in PBS into the device and incubated for 1 h at room

temperature. The channels were then washed with PBS to remove

Results and discussion

unattached fibronectin. A cell suspension of 1 106 cells mL 1

was injected into the fibronectin coated channels and incubated

Measured and calculated superficial velocities and shear stresses

at 37 C for 6 h.

are shown in Fig. 4. As designed, the device delivers shear stresses

Microfluidic devices have the advantage of minimising sample

spanning over at least two orders of magnitude (0.7–130 dynes

volume, and are thus especially ideal when samples are scarce

cm 2, 0.07–13 Pa). This range of shear stresses exceeds the range

and expensive. However, under the most stringent condition of

of shear stresses deemed to be physiological (1–70 dynes cm 2,

no perfusion where all nutrient transport into the channels is via

0.1–7 Pa) within in vivo vasculature. It also exceeds the range of

diffusion, small dimensions are likely to impede nutrient

shear stresses achieved by former systems, and has the advantage

delivery.18,19 This is particularly evident during the cell attach-

of confined shear stresses in individual channels for accurate data

ment period. The cell attachment period was selected after cells

acquisition.4,9,10,18

were observed at 4, 6, 8, 12, and 24 h without perfusion in the

The HUVECs were evenly distributed in all channels after 6 h

microchannels. The cells appeared attached by 4 h. They spread

of cell attachment. While cell attachment is still evident in all

and retained an appearance that was similar to those cultured in

channels of the device (Fig. 3) after 20 h of perfusion, substantial

macroscopic conditions for up to 8 h. However, at 12 h, 40% of

reductions in cell density are noted in channel 1 (130 dynes cm 2,

the cells appeared to round up. By 24 h, more than 90% of the

13 Pa). This shear stress obviously exceeds the limit of HUVEC

cells detached. From this observation, the cell attachment period

adhesion. This highlights the versatility of this multishear

of 6 h was selected.

microdevice in detachment studies. It can be used to investigate

The shear device was connected to a peristaltic pump (403U/

the ability of endothelial cells to withstand exposure to a range of

VM4 Watson Marlow Pump, Sweden). Silicone tubing (20 cm,

shear stresses, which is important to the viability of pre-seeded

1500 mm ID, 1000 mm thick) was connected to the system for

vascular grafts, as seeded cells can be washed away once blood

bubble free oxygen supply. Perfusion was conducted at an inlet

flow is restored to the graft.20

flow rate of 20 mL h 1 (steadily ramped up from 0 mL h 1 in 1

Von Willebrand factor is a large multimeric plasma protein

min) on sub-confluent layers of HUVECs. The cell seeding inlet

produced by endothelial cells and it is involved in promoting

(Fig. 1) was not used during perfusion. The cells were then fixed

platelet adhesion to damaged vessel walls. According to the

and immunostained (Fig. 3). The data presented represents the

results (Fig. 5), the vWF content of HUVECs increases with

This journal is ª The Royal Society of Chemistry 2009

Lab Chip, 2009, 9, 1897–1902 | 1899

On a final note, there is more cell elongation and actin filament

3

J. E. Moore, Jr., E. Burki, A. Suciu, S. Zhao, M. Burnier,

alignment in the direction of flow at higher shear levels (Fig. 3

H. R. Brunner and J. J. Meister, Ann. Biomed. Eng., 1994, 22, 416.

4

S. Usami, H. H. Chen, Y. Zhao, S. Chien and R. Skalak, Ann. Biomed.

and Fig. 6C). This agrees with findings in a number of studies.22,25

Eng., 1993, 21, 77.

In addition, in a small 2 h perfusion study, the cells were observed

5

C. Urbich, E. Dernbach, A. Reissner, M. Vasa, A. M. Zeiher and

to migrate downstream in the direction of flow. Future studies

S. Dimmeler, Arterioscler. Thromb. Vasc. Biol., 2002, 22, 69.

will incorporate

time lapse microscopy to investigate cell

6

J. Seebach, P. Dieterich, F. Luo, H. Schillers, D. Vestweber,

H. Oberleithner, H. J. Galla and H. J. Schnittler, Lab Invest., 2000,

behaviour with shear over time.

80, 1819.

7

M. Morigi, C. Zoja, M. Figliuzzi, M. Foppolo, G. Micheletti,

Conclusions

M. Bontempelli, M. Saronni, G. Remuzzi and A. Remuzzi, Blood,

1995, 85, 1696.

This multishear

microdevice delivers

physiologically

relevant

8

C. G. Galbraith, R. Skalak and S. Chien, Cell Motil. Cytoskeleton,

shear stresses, spanning over two orders of magnitude for any

1998, 40, 317.

9

J. Y. Ji, H. Jing and S. L. Diamond, Circ. Res., 2003, 92, 279.

one flow rate, making it a very convenient and time saving tool

10

E. Gutierrez, B. G. Petrich, S. J. Shattil, M. H. Ginsberg,

for mapping or screening cell behaviour. In addition, it requires

A. Groisman and A. Kasirer-Friede, Lab Chip, 2008, 8, 1486.

minimal

sample

volume,

making

it

attractive

for

studies

11

D. P. Gaver, 3rd and S. M. Kute, Biophys. J., 1998, 75, 721.

12

G. Kaplanski, C. Farnarier, A. M. Benoliel, C. Foa, S. Kaplanski and

involving

scarce

and expensive samples. To date,

this device

P. Bongrand, J. Cell Sci., 1994, 107(Pt 9), 2449.

covers the widest range of shear stresses in comparison to other

13

W. Van Driessche, P. De Smet and G. Raskin, Pfluegers Arch., 1993,

published shear devices. We used it to study HUVECs and our

425, 164.

results showed that there was a shear-dependent regulation of

14

E. B. Hunziker and R. K. Schenk, J. Physiol., 1989, 414, 55.

15

H. G. Verhage, M. L. Bareither, R. C. Jaffe and M. Akbar, Am.

vWF, and cell elongation and actin filament alignment in the

J. Anat., 1979, 156, 505.

direction of shear.

16

A. M. Malek, S. L. Alper and S. Izumo, Jama, 1999, 282, 2035.

17

A. Gnasso, C. Carallo, C. Irace, M. S. De Franceschi, P. L. Mattioli,

Acknowledgements

C. Motti and C. Cortese, Atherosclerosis, 2001, 156, 171.

18

H. Lu, L. Y. Koo, W. M. Wang, D. A. Lauffenburger, L. G. Griffith

The authors wish to acknowledge Dr Nicholas Timmins and

and K. F. Jensen, Anal. Chem., 2004, 76, 5257.

19

G. M. Walker, H. C. Zeringue and D. J. Beebe, Lab Chip, 2004, 4, 91.

Dr Ian Aird from the Australian Institute for Bioengineering and

20

P. Feugier, R. A. Black, J. A. Hunt and T. V. How, Biomaterials,

Nanotechnology (AIBN) for donating HUVECs, and funding

2005, 26, 1457.

from Australian

Research

Council

(ARC) Discovery

Grants

21

M. Galbusera, C. Zoja, R. Donadelli, S. Paris, M. Morigi, A. Benigni,

M. Figliuzzi, G. Remuzzi and A. Remuzzi, Blood, 1997, 90, 1558.

Scheme.

22

R. J. Sun, S. Muller, X. Wang, F. Y. Zhuang and J. F. Stoltz, Clin.

Hemorheol. Microcirc., 2000, 23, 1.

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