Биоинженерия / Биомеханика_микрофлюидные_устройства / статьи / 21_chin2011

Hydrogen peroxide is a poorly reactive oxidizing agent but it is

cell membrane-permeable. It is generated by several enzymatic

systems inside the cells, such as phagocytic oxidases in the plasma

membrane, sulfydryl oxidase in the endoplasmic reticulum, lipid

oxygenase in cytosol, etc. Under normal metabolic condition,

hydrogen peroxide is involved in the regulation of the signal

transduction events.4 For example, hydrogen peroxide inhibits

crucial phosphatases that attenuate signal propagation from

activated growth factor receptors. However, similar to super-

oxide anions, excess hydrogen peroxide will cause cellular

toxicity. Hydrogen peroxide can react with ferrous iron to form

hydroxyl radicals, which are short-lived ROS and can react with

DNA molecules, membrane lipids, and carbohydrates. This

means that the intracellular concentration of hydrogen peroxide

needs to be tightly regulated. In cells, excess hydrogen peroxide is

broken down by catalase and glutathione (GSH/Px) to yield

water molecules.

Overall, the antioxidant defence system of the cell ensures the

ROS level is kept at the basal and nontoxic level during normal

aerobic metabolism. This includes also non-enzymatic systems

such as glutathione, ascorbate, vitamin, etc. However, the

oxidative stress is generated when the intracellular level of ROS

exceeds the antioxidant ability of the defence system of the cell

and causes the loss of homeostasis. Oxidative stress can lead to

cell damage, apoptosis, and modifications to cellular proteins,

lipids, and DNA. The level of oxidative stress of the cell can be

determined by monitoring the morphological change of the

mitochondria since mitochondria are very important organelles

in the pathway of mitochondria-dependent cell apoptosis.5

Fig. 1 Schematic illustrations of (a) the design of the microfluidic chip,

Normally, mitochondria in endothelial cells form a filamentous

and (b) the hemodynamic Lab-on-a-chip system with configurable flow

reticular network and constantly undergo morphological

profile to mimic the blood flow in a blood vessel.

changes through fusion and fission.6,7 During apoptosis, mito-

chondria are fragmented via fission, and an individual mito-

different buffer media or dye solutions can be easily injected into

chondrion becomes swollen and loses its membrane potential.

the microchip under predefined flow rates.16 In this paper, the

There are

several reasons for the

excess levels of

ROS

designed Lab-on-a-chip system mimics the flow rate of blood in

production in

cells. First, short term

elevation of ROS

level

the artery at the resting condition or after an exhaustive exercise.

occurs after exhaustive exercise.8 Second, the presence of exog-

Different concentrations of glucose were added into the flow to

enous ROS sources can also elevate the ROS level, such as

mimic hyperglycemic conditions in diabetes patients. The shear-

ultraviolet light, ionizing radiation, inflammatory cytokines,

induced cellular responses under different extracellular condi-

environmental toxins, etc. Third, diseases can also increase the

tions were studied by investigating the ROS production level in

ROS level, such as hypertension,9 renal failure, diabetes,10,11

the endothelial cells and by monitoring the morphological

cardiac disease, etc. Therefore, researchers have put in efforts to

changes of mitochondria using fluorescence imaging technique.

investigate the relationship between the elevated ROS level and

the ROS-related diseases, and further search for potential drugs

with antioxidant ability.12 For example, high glucose level13 in

Materials and methods

diabetes patients and high sodium level14 in renal failure patients

Hemodynamic Lab-on-a-chip system

have been proven to increase the ROS production level in cells.

In addition, different antioxidants or ROS scavengers were

The design of the hemodynamic Lab-on-a-chip system is shown

studied and shown to reduce ROS production level. However,

in Fig. 1a. The chip consists of three-layer structures, namely

these studies were conducted under either a static condition in

bottom layer with a microfluidic network for cell loading and

culture dishes or under a constant shear stress,15 which were

liquid injection, middle layer with pneumatic valves and top layer

different from the physiological condition with pulsatile shear

with pneumatic connecting channels to control a series of

stress.

pneumatic valves simultaneously. The microfluidic chip was

In order to study the cellular responses of endothelial cells

fabricated in polydimethylsiloxane (PDMS) material using the

under a physiological pulsatile shear force-induced condition,

standard soft lithography process.17 The triple-layered PDMS

this paper introduces a hemodynamic Lab-on-a-chip system.

slab was then bonded to a coverslip by exposing to air plasma for

This system is biocompatible with highly controllable micro-

15 s using a handheld corona treater (BD-25, Electro-Technic

fluidic platform so that cells can be cultured in the microchip

Products). The coverslip was further bonded to a glass slide for

with a controlled perfusion of culture medium. Furthermore,

better robustness in operation. For the microfluidic networks, it

This journal is ª The Royal Society of Chemistry 2011

Lab Chip, 2011, 11, 1856–1863 | 1857

Initially, endothelial cells were loaded, cultured and stained

prior to the application of shear stress and chemical treatments.

When the cells were ready for the shear stress and chemical

Fig. 2 Phase images of endothelial cells (a) seeded in the microchannel

treatments, the two inlets were injected with normal culture

and (b) cultured in the microchannel for 48 hours.

medium and culture medium with 20 mM glucose solution,

respectively. With the concentration gradient network, three

plus 0.05% EDTA (Gibco, USA). Harvested cells were concen-

chemical conditions can be achieved, i.e. normal culture medium,

trated by centrifugation and then resuspended in the growth

culture medium with 10 mM glucose and culture medium with

medium at the concentration of about 1 107 cells ml 1. The cell

20 mM glucose. The time course experiments were conducted in

suspension was injected into the inlets of the microfluidic chip

the following manner. In the beginning, the first three rows of

using a glass syringe (Hamilton, USA). The chips were then

valves (valves 1 to 3) were closed and the last row of valves (valve 4)

incubated in the cell culture incubator with medium replenish-

was opened. Therefore, the liquid injected into the inlet can flow

ment every 24 hours until the cells in the microchannel reached

through the 4 culture sites at each branch of the micro-

almost full confluence for the experiments. Growth of the

channel. The first data point was measured at 15 min after the

endothelial cells in the microchannels was monitored under the

shear stress treatment. At this stage, the valves in the last row

phase-contrast microscope (IX81, Olympus). Fig. 2a and b show

(valve 4) were closed and the valves in the third rows (valve 3)

the endothelial cells after initial seeding and after 48 hours

were kept open as shown in Fig. 1a. The cell culture sites in

culture in the microchannel with a well formed monolayer of

the last row (R4) were disconnected from the microfluidic flow

cells. In monitoring the ROS level within the endothelial cells, it

in which the medium was flowing into the side branch at the

is critical to avoid the occurrence of hypoxia due to the lack of

downstream of the third row of culture sites to the outlets.

oxygen in the culture chamber because oxidative burst and NO

Subsequently, the second data point was measured at 30 min.

generation have proven to be the initial response to ischemia in

At this stage, the valves in the third rows (valve 3) were

endothelial cells.18 This hypoxia occurrence in endothelial cells

closed and the valves in the second rows (valve 2) were kept

can be avoided because the PDMS material is highly oxygen

open. The cell culture sites in the third row (R3) were

permeable, which ensures that a sufficient amount of oxygen is

disconnected. Similarly, each row of cell culture sites could be

supplied into the culture chamber.19

disconnected from the main microfluidic flow at each time

point by controlling the pneumatic valves. With this densely

Measurement of ROS by H2DCFDA

designed microfluidic network, one single chip can perform

a series of experiments with different chemical treatments and

The intracellular ROS level was measured using a cell-perme-

time course.

able fluorescent dye of 20,70-dichlorodihydrofluorescein diac-

A pulsation free precision pump (Nemesys, Cetoni) was used

etate (H2DCFDA) (D399, Invitrogen). The dye remains

for cell loading and media injection as shown in Fig. 1b. The flow

nonfluorescent until the acetate groups are removed by intra-

profile of the pump was configured to mimic the blood flow in

cellular esterases, and oxidation occurs due to the presence of

a blood vessel. A bubble trapper was connected between the

ROS (hydrogen peroxide and hydroxyl radicals) within the

precision pump and the microfluidic chip. The bubble trapper

cells. Prior to the chemical

and shear stress experiments, the

could significantly prevent bubbles from flowing into the chip,

cultured endothelial cells

in the microfluidic chips were

which can cause the endothelial cells in the microchannel to

detach from the coverslip and flow out of the chip. A heating

plate was used to maintain the temperature of the microfluidic

chip at 37 C for long-term observation under the microscope.

Protocol for culturing endothelial cells

Low glucose (1.0 mg ml 1) DMEM (Sigma, USA) was mixed

1 : 1 (v/v) with F-12 Ham nutrient mixture (Sigma, USA), the

mixture was supplemented with 10% (v/v) fetal bovine serum

(FBS, Gibco, USA) and 1% (v/v) penicillin/streptomycin (Gibco,

USA) to formulate the complete growth medium for culturing

human vascular endothelial cell line CRL1730 isolated from

umbilical vein. The endothelial cells were grown in the complete

Fig. 3 H2DCFDA is hydrolyzed inside the cell, retained in the cyto-

medium in a T-75 cell culture flask (NUNC, Denmark) at 37 C

plasm, and then oxidized by ROS, leading to the emission of green

with 5% CO2. Confluent cells were trypsinized with 0.25% trypsin

fluorescence.

1858 | Lab Chip, 2011, 11, 1856–1863

This journal is ª The Royal Society of Chemistry 2011

velocity profile for one heart beat cycle was then plotted and

compared with the theoretical profile as shown in Fig. S1‡. It

shows that the measured flow profile has the same trend as the

theoretical profile. The flow for one cycle under the condition of

faster heart beat (140 bpm) and higher shear stress (30 dyne cm 2)

is shown in Movie M1 (ESI‡) (frame rate: 300 fps). The temporal

responses of the endothelial cells in ROS production level under

different shear treatment conditions are shown in Fig. 6. The

static condition without a fluidic flow serves as a negative control

of the experiment and it can be seen that the fluorescence

intensity increased when the dye molecules stained the cells for

longer than 15 min. To exclude this intensity increment, the

normalized fluorescence intensities for the other three shear

treatment conditions were subtracted with that of the negative

control. Fig. 7a shows the intracellular ROS level when the

Fig. 6 Temporal responses of the ROS level in endothelial cells under

endothelial cells were being exposed to pulsatile shear stress of 30

different shear stress profiles.

dyne cm 2 for different time periods. The ROS level was

increased at the first 60 min by nearly 4-fold (Fig. 6). Prolonged

Experimental results and discussions

exposure of the endothelial cells to the shear flow up to 2 hours

Shear-induced cellular responses

resulted in a sustained elevation in ROS levels (Fig. 6), which is

similar to the one reported previously.15 The ROS levels of

Three different shear treatment conditions were selected to

endothelial cells under different shear treatment conditions for

investigate the shear-induced responses on cultured endothelial

up to 120 min are shown in Fig. 6 and 7b. By comparing the ROS

cells in the microchannel: (1) a constant shear stress of 30 dyne

levels in various shear treatment conditions at the 60 min time

cm 2 with a steady flow rate of 8.46 mL s 1, (2) a normal physi-

point, there is no significant increase of ROS level between the

ological pulsatile shear stress of 15 dyne cm 2 (mean) with

constant shear stress of 30 dyne cm 2 and the negative control,

a frequency of 70 bpm as shown in Fig. 5, and (3) a fast pulsatile

which has no shear stress as the culture medium was maintained

shear stress of 30 dyne cm 2 (mean) with a frequency of 140 bpm

in a static condition in the microchannel (0.36/0.34). In contrast,

to mimic the condition under exhaustive exercise. The pulsatile

the increment was more substantial when the endothelial cells

flow profile was verified by tracking the displacement of the

were being exposed to physiological pulsatile shear stress

suspended microbeads with a diameter of 5 mm in the micro-

conditions. The ROS level was increased by 1.7 fold (0.56/0.34)

channel. A high-speed camera (Photron, FASTCAM SA3) was

and 2.2 fold (0.76/0.34) at pulsatile shear stress of 15 and 30 dyne

employed to capture images of the microbeads in the micro-

cm 2, respectively (Fig. 6). This results show that it is necessary to

channel at 6000 fps. The velocity of the microbeads at different

investigate the effect of shear stress on the endothelial cells under

time points was calculated based on the displacement of the

a physiological condition, which can truly reflect the pulsatile

microbeads within a known time period. Based on the calculated

pattern of the blood flow in the artery. In addition, reports

velocity and the cross-sectional area of the microchannel, the

stating that pulsatile shear stress is critical to maintain the

flow rate of the flow in the microchannel was determined. The

functionalities of endothelial cells in the artery have been

View Article Online

intracellular ROS level was studied using real-time fluorescence

3

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