Cytotoxicity of quantum dots assay on a microfluidic 3D-culture device basedon modeling diffusion process between blood vessels and tissues{

Jing Wu, Qiushui Chen, Wu Liu, Yandong Zhang and Jin-Ming Lin*

Received 3rd May 2012, Accepted 19th June 2012

DOI: 10.1039/c2lc40502d

In this work, a novel quantum dot (QD) cytotoxicity assay platform on a microfluidic three-

dimensional (3D) culture device via imitating the diffusion process between blood vessels and tissues

was developed. The device is composed of a main channel and two sets of cell culture chambers. The

cell culture chambers were located at different distances from the main channel and were divided into

‘‘close chambers’’ and ‘‘far chambers’’. HepG2 cells were cultured in an agarose matrix under 3D

conditions and kept at high viability for at least three days. Fluorescein sodium and fluorescein

isothiocyanate conjugated to bovine serum albumin (FITC-BSA) were used as models to demonstrate

the diffusion process between main channel and cell culture chambers. QD cytotoxicity was evaluated

by determining cell apoptosis, intracellular reactive oxygen species (ROS) and glutathione (GSH)

with specific fluorescence probes. Cell autophagy inhibitor 3-methyladenine (3-MA) could reduce cell

apoptosis at low concentrations of QDs, which proves that cell autophagy plays a key role in QD

cytotoxicity. The effect of a series of 3-MA solutions on cell apoptosis at QD concentration of

40 mg mL21 was investigated, which showed that the percentage of cell apoptosis decreased y15%

from 0 to 12 mM 3-MA. The device shows potential as a high-throughput, low-cost and time-saving

platform and constructs a more vivid biomimetic microenvironment for the QD cytotoxicity study.

Introduction

Engineered nanomaterials (NMs) have shown increasing poten-

tial in clinical aims such as diagnosis, imaging and drug

delivery.1–3 Quantum dots (QDs) are emerging as a new class

of NM and attracting great interest in biological imaging.4–6

However, cytotoxicity of QDs becomes a major impediment to

their universal application.7–9 Various mechanisms of QD

cytotoxicity have been studied, such as releasing of heavy metal

ions from CdSe or CdTe QDs,10,11 generation of reactive oxygen

species (ROS)12 and varying surface properties.13

The research on QD cytotoxicity commonly focused on

culturing cells in petri dishes or animal tests.14–16 The effects of

CdTe QDs capped with different chiral forms of the tripeptide

glutathione (GSH) on cytotoxicity and induction of autophagy

were examined by HepG2 cells cultured in petri dishes.17

Shuhendler et al. detected the cytotoxicity of PbSe QDs

encapsulated in stealth fatty ester on a breast tumor-bearing

animal model.18 However, the native cell microenvironments in

vivo consisting of complex cell–matrix and cell–soluble factor

interactions are greatly different from culturing cells in petri

dishes.19 Cytotoxicity studies on the animal model are now

challenging, as they are high-cost and time-consuming. Thus,

there is a great demand for developing a high-throughput, low-

cost, time-saving and more accurate in vitro platform to study

QD cytotoxicity.

Microfluidic technology provides prominent advantages, such

as rapid analysis, high integration, easy control and miniaturiza-

tion.20–22 It enables cells to live in a microenvironment with high

spatiotemporal precision and presents cells with a biochemical

and mechanical signal in a more physiologically relevant

context.19,20,23 Over past decades, the microfluidic device was

recognized as an excellent platform for cell-based bioassay that

had been widely expanded to cell metabolism,24,25 drug screen-

ing26–28 and stem cell tissue engineering.29,30 One of the most

important potential applications is implementing three-dimen-

sional (3D) cell culture and imitation of organs onto a

microfluidic device. 3D cell culture on a microfluidic device

could recreate the cell-culture microenvironments that help cells

retain their native tissue-specific functions and differentiated

state, and recapitulate tissue–tissue interfaces, spatiotemporal

chemical gradients and mechanical microenvironments of living

organs.31,32 For example, the critical functional alveolar–

capillary interface of the human lung was reconstituted on a

biomimetic microfluidic system by containing two closely

apposed microchannels.33 A multi-channel 3D microfluidic cell

culture system was also designed to represent the drug-

Beijing Key Laboratory of Microanalytical Method and Instrumentation,Department of Chemistry, Tsinghua University, Beijing, 100084, China.E-mail: jmlin@mail.tsinghua.edu.cn; Fax: +86-10-62792343;Tel: +86-10-62792343{ Electronic Supplementary Information (ESI) available:Characterization of cell culture chamber and CdTe-COOH QDs, cellculture, intracellular ROS and GSH detection. See DOI: 10.1039/c2lc40502d

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metabolizing and storage capabilities in the human body.34

Besides that, 3D cell culture was able to construct more complex

3D tissue structures to compare cell growth and migration under

different spatial composition.35 Thus, microfluidic technology

combined with 3D cell culture permits drug screening and toxin

testing in a more physiologically realistic microenvironment

under in vitro conditions and has the potential to replace animal

testing.31 However, to our knowledge, there is no report that

studies QD cytotoxicity on a microfluidic 3D-culture device.

The transport of QD to cells in the tissue from a feeding artery

is a two-step process. First, QDs flow near to their destination

through blood vessels. Then they cover the remaining distance

from the blood vessels to the cells in the tissues via diffusion and

convection.36 In this work, we have successfully developed a

microfluidic 3D-culture device for a QD cytotoxicity assay based

on reconstructing the diffusion process between blood vessels

and tissues. To provide proof of a QD cytotoxicity assay on a

biomimetic microsystem, a microfluidic 3D-culture device was

specifically designed that was composed of different heights of

main channel and cell culture chambers (Fig. 1). Cell culture

chambers were distributed with different gaps apart from the

main channel and were divided into ‘‘close chambers’’ and ‘‘far

chambers’’. The diffusion process was monitored in cell culture

chambers using fluorescein sodium and fluorescein isothiocya-

nate conjugated to bovine serum albumin (FITC-BSA) as model

molecules. HepG2 cells were cultured in both chambers under

3D conditions to demonstrate the effect of the diffusion process

of the QDs on cytotoxicity. Further study illustrated that the

cell autophagy inhibitor 3-methyladenine (3-MA) reduced cell

apoptosis at low concentrations of QDs. The designed micro-

device represents an innovative and low-cost detection platform

for QD cytotoxicity assay in a well defined 3D environment.

Experimental

Microfluidic 3D-culture device fabrication

Re-exposure technology was used in the fabrication. As shown in

Fig. 1, cell culture chambers were adjacent to the main channel.

The chambers were designed to be lower than the main channel.

Two steps of standard photolithography were used to make the

pattern on a silica wafer. Briefly, negative photoresist SU-8 2050

(Microchem, Newton, MA) was spun onto a silica wafer (Tianjin,

China) at a speed of 2000 rpm for 50 s using a spin-coater. After

baking at 65 uC for 10 min, UV light exposure and development

were done to generate the layer of cell culture chambers (38 mm

thick). Then another layer of SU-8 2050 photoresist was coated at

a speed of 1100 rpm for 50 s. The above process was repeated to

generate the main channel layer (71 mm thick). Silanization was

made to make the surface of the silica master to be hydrophobic.

A premixed 10 : 1 (by mass) poly-dimethylsiloxane (PDMS)

preploymer and curing agent (Dow Corning, Sylgard 184, Midland,

MI, USA) was poured onto the mold and degassed under vacuum for

30 min. After curing at 75 uC for 2 h in an oven, the PDMS was peeled

off carefully and cut into the designed shape. A flat-tipped syringe

needle was used to make the inlets and outlets of the channels. The

PDMS replica was sealed with a glass slide via oxygen plasma (PDC-

32 g, Harrick Plasma, Ithaca, NY, USA) treatment for 90 s. The

device was sterilized under UV light before use.

Microfluidic cell culture

When the HepG2 cells (Cancer Institute & Hospital Chinese

Academy of Medical Science, Beijing, China) reached conflu-

ence, two 60 cm2 dishes of cells were trypsinized. The cell

suspension was centrifuged, the supernatant was removed and

the remaining cells were resuspended at a concentration of 106

mL21. 100 mL 3% (w/v) low gelling temperature agarose (type

VII-A, Sigma, St. Louis, MO) solution in phosphate buffer saline

(PBS), 100 mL fetal bovine serum (FBS) and 100 mL PBS were

mixed to be used as the matrix of the 3D cell culture. Equal

volumes of cell suspension and matrix were mixed and then

infused into cell culture chambers. The cell infused device was

kept at 4 uC in the refrigerator for 10 min to accelerate the gelling

of the agarose. Finally, the cell culture medium was filled into the

main channel and a thin layer of the medium was coated on the

surface of the device to invade the evaporation of the medium in

the main channel. The device was put in a cell culture incubator

and the medium was refreshed each day. Cell viability was

characterized by a Live/Dead assay kit (Calcein-AM/EthD-1,

Invitrogen, CA, USA) every day and the data were analyzed by

program Image-Pro Plus 6.0.

Fluorescein sodium and FITC-BSA diffusion in agarose

The diffusion process in agarose was detected using fluorescein

sodium (Shanghai, China) and FITC-BSA (Beijing, China) as the

model. The final concentration of agarose in the matrix was 0.5%

Fig. 1 (a) Schematic representation of the diffusion process between

blood vessels and adjacent tissues. (b) Device design and a magnified

illustration of the cell culture chamber and the main channel. (c) Image of

an actual microfluidic 3D-culture device. (d) Confocal fluorescence

microscope images of HepG2 cells stained by DiI and calcein AM in a

microfluidic 3D-culture device.

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(w/v), so 0.5% (w/v) agarose in PBS solution was made and filled

into the cell culture chambers to conduct the experiments. After the

agarose solution gelled, 100 mM fluorescein sodium solution

(excitation, 490 nm; emission, .510 nm) and 2.75 mg mL21

FITC-BSA (excitation, 506 nm; emission, 529 nm) were injected

into the main channel with a pipette. The total injection volume was

10 mL. Fluorescence images were taken every 5 min for fluorescein

sodium and 10 min for FITC-BSA by a Leica DMI 4000 B

fluorescence microscope (Wetzlar, Germany) at the top of the cell

culture chambers. The fluorescein sodium experiment lasted for

120 min while the FITC-BSA experiment continued for 300 min.

Cytotoxicity of QD detection

Cell apoptosis, generation of ROS and reduction of GSH were

three indexes of QD cytotoxicity. They were detected by Hoechst

33342 (Invitrogen, CA, USA), dihydroethidium (DHE, Beijing,

China) and 2,3-naphthalenedicarboxaldehyde (NDA, Tokyo,

Japan), respectively. CdTe QDs coated by a carboxyl group

(noted as CdTe-COOH QDs) were used in this study. A stock

solution of CdTe-COOH QDs (5 mg mL21) was serially diluted

by a FBS-free medium into the desired concentrations (0, 10, 20,

30, 40, 50 mg mL21). After cells had been cultured in chambers

for 24 h, QD solutions were infused into the main channel to

replace the cell culture medium. Cells were treated by CdTe-

COOH QDs for 24 h under static conditions; 100 mM specific

fluorescence probe solutions in PBS were injected into the main

channel and incubated at 37 uC for 1 h. Fluorescence images

were acquired by a fluorescence microscope equipped with a

cooled CCD camera with software of Leica Application Suite,

LAS V2.7. Images of cell apoptosis were analyzed by program

Image-Pro Plus 6.0. Image analyses of ROS and GSH were

performed using commercially available image analysis software

(QCapture Pro, Version 5.1.1.14, Media Cybernetics, USA).

Characterization of cell autophagy

The stock solution of 3-MA (Sigma, St. Louis, MO) in PBS was

prepared beforehand. When used, it was diluted serially to the

desired concentrations. The cells in agarose had been treated by

3-MA (3 mM) for 5 h before the QD solutions were infused. Hoechst

33342 (100 mM) was used to characterize cell apoptosis after 24 h QD

treatment. Experiments of different concentrations of 3-MA (0, 3, 6,

9, 12 mM) treated on cells were done according to the above process.

Transmission electronic microscopy (TEM) was undertaken to

view the autophagic vesicles formed in cells. 20 mg mL21 QD

solution was selected to co-culture with cells for 24 h after 3-MA

(3 mM) treatment. Cells treated only with QD were prepared at

the same time. Cells without any treatment were used as a

control. All the samples were fixed overnight at 4 uC in 2.5%

glutaraldehyde and were prepared according to the standard

procedure for TEM viewing. A Hitachi H-7650B electronic

microscope was used to take the images.

Results and discussion

Design of microfluidic 3D-culture device

The microfluidic 3D-culture device was designed to perform QD

cytotoxicity based on a blood vessel and adjacent tissue model

(Fig. 1a). As proof-of-concept, the device mainly consisted of

two parts: the main channel represented blood vessels that could

deliver the cell culture medium and QD solutions, and the cell

culture chambers imitated adjacent tissues for QD targeting sites.

HepG2 cells were cultured in cell culture chambers in a 3D

matrix that had similar microenvironments to the extracellular

matrix (ECM) in tissues. Culture medium and QD solutions were

infused into the main channel and diffused into the cell culture

chambers. As shown in Fig. 1b, different heights of the main

channel and cell culture chambers were utilized to form a stop-

flow junction between them.37 The main channel is 71 mm high

and the cell culture chambers are 38 mm high (see ESI, Fig. S1{).

The surface tension at the junction ensured that the redundant

cell–agarose mixture remained in the cell culture chambers

instead of leaking into the main channel. To understand the

effect of the diffusion process on QD cytotoxicity, two different

lengths of stop-flow junctions were designed to study the

differences in QD cytotoxicity at the same QD concentration.

Close chambers are 0.8 mm away from the main channel and far

chambers are 2 mm away. The diffusion process of materials in

agarose was also examined and discussed in the next section.

Microfluidic 3D-culture has been widely used to create a more

vivid cell microenvironment in vitro. In this work, HepG2 cells

were cultured in agarose mixed with FBS, which could provide

sufficient nutrients to maintain high cell viability. In our

experiments, a Live/Dead assay kit (calcein AM/EthD-1) was

used to assess cell viability. As shown in Fig. 1d, HepG2 cells

were observed to be adherent to each other and spread into

Fig. 2 (a) Fluorescent images of HepG2 cells cultured in a microfluidic

3D-culture device for 1–3 days. Calcein-AM/EthD-1 was used to stain

cells (scale bar: 200 mm). (b) Viability of HepG2 cells cultured in different

chambers for 1–3 days. The standard error bars mean the variation of

three independent experiments.

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matrix. They maintain normal morphology and high viability in

the matrix for 3 days (Fig. 2a and 2b). After being cultured for

3 days, the viability of HepG2 cells was still above 85%. These

results indicate that a microfluidic 3D-culture is an efficient

strategy for a QD cytotoxicity study, owing to its biomimetic and

biocompatible cell microenvironment.

Imitation of diffusion process in tissues on the microfluidic 3D-

culture device

In the biological process, drug diffusion in tissues is a key factor

affecting drug efficiency. To imitate the diffusion process and

understand its effect on QD cytotoxicity, fluorescein sodium and

FITC-BSA were used to investigate the permeability of the

matrix. Fluorescein sodium and FITC-BSA were selected to

represent small molecule and biomacromolecule, respectively.

Agarose was used as the tissue matrix, due to its biocompat-

ibility,38 high porosity and ease of molecular diffusion.39 Close

chambers and far chambers were detected respectively to

understand how distance affected diffusion.

As shown in Fig. 3, the relative fluorescence intensities of both

fluorescein sodium and FITC-BSA in the close chambers are

stronger than those in the far chambers at the same time. It is

obvious that they diffuse more easily in the close chambers than

in the far chambers. Moreover, our results indicate that the

diffusion velocity of fluorescein sodium is much higher than that

of FITC-BSA. It is reasonable because biological macromole-

cules have a more intense interaction with agarose. Meanwhile,

this conclusion also indicates that QD diffusion in tissues might

be a key factor affecting its cytotoxicity, which will be further

discussed in the following experiments.

Study of QD induced cell apoptosis, intracellular ROS and GSH

variation using the cell 3D-culture based tissue model

Although QDs have been widely used in biological imaging and

targeted delivery systems, the potential cytotoxicity is still a

major problem, which hinders further application. QD cytotoxi-

city is reported to be concentration-dependent40 and is greatly

influenced by the QD diffusion process in tissue,18 according to

petri dish experiments and animal tests. In our work, a cell

3D-culture based tissue model on a microfluidic device was

constructed to investigate QD cytotoxicity visually. Some

characterization of CdTe-COOH QDs was illustrated in the

ESI (Fig. S2{). For the QD induced cell apoptosis assay,

Hoechst 33342 (lex = 340–380 nm, lem = 460 nm) was used as a

specific fluorescence probe. As shown in Fig. 4, cell apoptosis, in

both the close and far chambers, was obviously concentration-

dependent. Higher concentrations induced a larger number of

apoptotic cells. Moreover, our results also showed that HepG2

cells in the close chambers suffered more severe QD cytotoxicity

than those in far chambers (Fig. 4b). The cell apoptosis rate in

close chambers was always higher than that in the far chambers

under the same experimental conditions. These results demon-

strated that the QD diffusion process in tissues was a key factor

in QD cytotoxicity.

From a mechanistic perspective, ROS generation and GSH

level are two typical indexes to explain the toxic effect of

nanoparticles,41–43 and in vitro assays for them are predictive

for toxicity screening. In our experiments, two specific

fluorescence probes, DHE (lex = 535 nm, lem = 610 nm) and

NDA (lex = 460 nm, lem = 530 nm), were used to detect

intracellular ROS and GSH, respectively. As shown in Fig. 5,

with the increase of CdTe-COOH QDs, the quantity of ROS

augments while the amount of GSH decreases. More details on

the fluorescence images of ROS and GSH are given in the ESI

(Fig. S3{). In a normal intracellular microenvironment, ROS

generation is kept at low frequency and easily neutralized by in

vivo antioxidant defenses such as GSH and antioxidant

enzymes, while this balance could be easily broken down in

the presence of QDs. Besides, comparison between close

chambers and far chambers indicates again that the diffusion

process is a very important factor for the cytotoxicity of QDs.

Fig. 3 Fluorescence images and curves of fluorescein sodium (a) and FITC-BSA (b) diffuse in 0.5% (w/v) agarose. Experiments were repeated three

times in parallel.

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Consequently, the designed cell 3D-culture based tissue model

is an excellent platform for studying QD induced cytotoxicity

and clearly understanding the effect of the QD diffusion

process on cytotoxicity.

Effect of cell autophagy on QD cytotoxicity

Cell autophagy is an important cellular pathway that degrades

bulk cytoplasm, long-lived proteins, and entire organelles by

forming lysosomes. It is a cytoprotective mechanism that plays a

vital role in the cell programmed death process.44 Recent

research has pointed out that cell autophagy has always been

crucial in QD cytotoxicity.45,46 To prove this mechanism, 3-MA

was used as a cell autophagy inhibitor in HepG2 cells before QD

treatment. As shown in Fig. 6a, 3-MA obviously reduced cell

apoptosis although it did not block cell death. Cell apoptosis was

significantly decreased by 3-MA under relatively low QD

concentrations. However, the effect was not obvious when the

concentration of QDs was more than 40 mg mL21 (Fig. 6b and

6c). These results indicated that there might be other mechanisms

explaining the cytotoxicity of QDs in addition to cell autophagy.

The marked feature of cell autophagy is formation of

autophagsomes, a double-membrane-bound vesicle.47 Thereby,

the TEM images in Fig. 6d exactly define the cellular vacuole

formation and the morphological changes that occurred in

HepG2 cells treated by QDs with/without 3-MA pretreatment.

Cells without any treatment were used as a control. Cellular

vacuoles formed in the cytoplasm of QD treated cells, while

nothing emerged in the control (large cellular vacuoles are

marked by black arrows, Fig. 6d). In comparison, the number of

cellular vacuoles was strikingly smaller in cells treated with

3-MA before QD treatment. Cytoplasmic debris was internalized

in some autophagic vacuoles. During the whole process of

autophagy, it is obvious that the mitochondria and the

endoplasmic reticulum are injured. Some swollen mitochondria

could be seen in the cytoplasms of cells treated by QDs (marked

by white arrows, Fig. 6d) and the number was larger in samples

without 3-MA pretreatment.

To further study the effect of 3-MA on cytotoxicity at high

QD concentrations, cell apoptosis in 40 mg mL21 QD solution

was investigated by increasing the concentrations of 3-MA.

Fig. 7 showed that cell apoptosis had no noticeable decrease by

treating HepG2 cells with 3-MA in the range of 3–9 mM. In the

close chambers, when the HepG2 cells were pretreated with

Fig. 4 (a) Fluorescence images of cell apoptosis after being treated with

different concentrations of CdTe-COOH QDs for 24 h (scale bar:

200 mm). (b) Curves of cell apoptosis analyzed from the above

fluorescence images. The standard error bars mean the variation of

three independent experiments.

Fig. 5 Intracellular ROS generation (a) and GSH reduction (b) responding to different concentrations of CdTe-COOH QD treatment. The insets

show fluorescence images of ROS and GSH. The standard error bars mean the variation of three independent experiments (scale bar: 200 mm).

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12 mM 3-MA, the cell apoptosis rate decreased from 68% to

50%, compared with no 3-MA pretreatment. In the far

chambers, the cell apoptosis rate also decreased from 62% to

47% as the concentration of 3-MA increased from 0 to 12 mM.

Thus, cell autophagy is demonstrated to be an important

pathway leading to QD cytotoxicity on the microfluidic 3D-

culture device.

Conclusions

In summary, a microfluidic 3D-culture device was successfully

developed as a microengineering approach to studying QD

cytotoxicity in vitro. The diffusion process between the blood

vessels and the adjacent tissues was further modeled on it and

might offer new opportunities to investigate QD cytotoxicity

under a more accurate microenvironment. Differences in QD

cytotoxicity were observed in various chambers because of

differing diffusion distances. Cell autophagy was proven to be

one pathway leading to QD cytotoxicity by cell apoptosis data

and TEM images. In conclusion, this microfluidic 3D-culture

device is miniaturized and easily handled. It could potentially be

attached to high-throughput analysis systems and used for

screening of cellular responses to drugs or environmental toxins.

Acknowledgements

This work was supported by National Natural Science

Foundation of China (No. 20935002) and the Research Fund

for the Doctoral Program of Higher Education (No.

20110002110052).

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Fig. 6 (a) Fluorescence images of cells in close chambers stained by

Hoechst 33342 following QD and QD + 3-MA treatment (scale bar:

200 mm). (b) Histogram of cell apoptosis in close chambers following QD

and QD + 3-MA treatment. (c) Histogram of cell apoptosis in far

chambers following QD and QD + 3-MA treatment. The standard error

bars mean the variation of three independent experiments. (d) TEM

images of HepG2 cells treated with QDs (20 mg mL21) and QDs + 3-MA.

HepG2 cells without any treatment are used as a control. QD-triggered

vacuoles in the cytoplasm are indicated by the black arrows and

mitochondria are marked by the white arrows.

Fig. 7 Apoptosis of HepG2 cells treated with different concentrations

of 3-MA before being incubated with QDs (40 mg mL21). The standard

error bars mean the variation of three independent experiments.

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3480 | Lab Chip, 2012, 12, 3474–3480 This journal is � The Royal Society of Chemistry 2012

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