糖原合成酶激酶3β对炎症状态下牙周膜干细胞成骨分化能力的影响
Effects of GSK..
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糖原合成酶激酶3β对炎症状态下牙周膜干细胞成骨分化能力的影响
Effects of GSK3β on Osteogenic differentiation of human Periodontal Ligament Stem Cells in Inflammatory Microenvironment
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Nano Express
Biocompatible micro-sized cell culture chamber for the detection of nanoparticle-induced IL8 promoter activity on a small cell population
Yvonne Kohl, Gertie J Oostingh, Adam Sossalla, Albert Duschl, Hagen von Briesen* and Hagen Thielecke
Corresponding author:
von Briesen
Department of Cell Biology and Applied Virology, Fraunhofer Institute for Biomedical Engineering, 66386 St. Ingbert, Germany
Department of Molecular Biology, University of Salzburg, 5020 Salzburg, Austria
Department of Medical Engineering and Neuroprosthetics, Fraunhofer Institute for Biomedical Engineering, 66386 St. Ingbert, Germany
Vanguard AG, 12623 Berlin, Germany
For all author emails, please .
Nanoscale Research Letters 2011, 6:505&
doi:10.6X-6-505
The electronic version of this article is the complete one and can be found online at:
Received:20 April 2011
Accepted:23 August 2011
Published:23 August 2011
& 2011 K licensee Springer.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In most conventional in vitro toxicological assays, the response of a complete cell population is averaged, and
therefore, single-cell responses are not detectable. Such averaging might result in
misinterpretations when only individual cells within a population respond to a certain
stimulus. Therefore, there is a need for non-invasive in vitro systems to verify the toxicity of nanoscale materials. In the present study, a micro-sized
cell culture chamber with a silicon nitride membrane (0.16 mm2) was produced for cell cultivation and the detection of specific cell responses.
The biocompatibility of the microcavity chip (MCC) was verified by studying adipogenic
and neuronal differentiation. Thereafter, the suitability of the MCC to study the
effects of nanoparticles on a small cell population was determined by using a green
fluorescence protein-based reporter cell line. Interleukin-8 promoter (pIL8) induction,
a marker of an inflammatory response, was used to monitor immune activation. The validation
of the MCC-based method was performed using well-characterized gold and silver nanoparticles.
The sensitivity of the new method was verified comparing the quantified pIL8 activation
via MCC-based and standard techniques. The results proved the biocompatibility and
the sensitivity of the microculture chamber, as well as a high optical quality due
to the properties of Si3N4. The MCC-based method is suited for threshold- and time-dependent analysis of nanoparticle-induced
IL8 promoter activity. This novel system can give dynamic information at the level
of adherent single cells of a small cell population and presents a new non-invasive
in vitro test method to assess the toxicity of nanomaterials and other compounds.
PACS: 85.35.Be, 81.16.Nd, 87.18.Mp
Keywords: micro-sized
nanoparticlesBackground
There is a growing interest in improved test methods to assess biological effects
of nanoparticles. Studies of cellular processes and determination of toxic effects
of nanomaterials on cells are commonly based on examining the response of a cellular
population, such as a cell monolayer, tissue, or organ [-]. In many biological assays, such as colorimetric, fluorometric, or chemiluminescent
assays, the data are a result of the mean response of the complete cell population.
In those assays, the signal of a single cell is lost in the signal caused by the large
cell sample. A detectable signal, above the background noise, can be due to the response
of a specific subset of cells within the population or by a response of the complete
cell population. Especially when performing biological studies with nanoparticles,
there might be a large variation in the response of the individual cells based on
whether or not they came in contact with nanoparticles and, in addition, on the level
of exposure, which is known to play an important role. Since an altered response in
a low number of cells can be the trigger for certain diseases, such as autoimmunity,
cancer, and neuronal diseases, the analysis of nanoparticle-induced responses of individual
cells is of main importance [,]. Therefore, cell-based assays that can detect the response of a low number of individual
cells are required. In addition, in vitro studies demonstrated differences in the behavior of cells isolated or in a cell population
[-], showing that isolated single cells react in a different physiological manner compared
to cells within a monolayer or cell suspension. New methodologies have to be established
to bridge the gap between population and quantitative single-cell analysis. Technologies
for the characterization of single cells, such as capillary electrophoresis (2D, 3D),
polymerase chain reaction (PCR), single-cell gel electrophoresis, and elastography,
are already used, but these are invasive and often time-consuming techniques [-]. Invasive techniques destroy the cell and consequently do not permit the detection
of single living cells or to perform kinetics on one and the same cell. Flow cytometry
is used to investigate nanoparticle-induced effects at the single-cell level but is
not suitable for the characterization of adherent cells since the cells need to be
in suspension. Detachment of the cells from the surface of the cell culture dish might
alter their characteristics []. With regard to the application of single-cell analysis as pharmaceutical in vitro screening method, the goal of this study is the evaluation and validation of a non-invasive
technique to characterize cellular processes of adherent biological cells on an individual
level in a small defined cell population. Biological microelectromechanical systems
(Bio-MEMS) present a suitable approach for analyzing a small amount of cells on a
defined cell culture area. Recently, classical detection technologies like optical
and electrochemical analysis and mass spectroscopy have been combined with the chip
technology [-]. Dynamic single-cell culture arrays of isolated cells have enabled to determine the
level of produced or secreted proteins but do not simulate the physiological conditions
of a 2D cell culture [,]. Silicon nitride (Si3N4) has been used as matrix for cell-based assays due to its chemical, optical, and
mechanical properties []. Only few studies exist on the biocompatibility of Bio-MEM-materials [-]. Currently, no Bio-MEMS exist for long-term culturing, and long-term observation
of cell response features larger, more comparable cell culture area dimensions compared
to the micro-sized cell culture chamber presented in this paper [,-]. At current, no Bio-MEMS exist for long-term cultivation and non-invasive quantification
of specific cellular responses of adherent individual cells in a small defined cell
layer cultured on miniaturized Si3N4 membranes with cell culture areas smaller than 0.2 mm2. The use of a micro-sized chip-based cell culture system in combination with reporter
cells presents a powerful tool for the analysis of small cell populations and will
improve the evaluation of non-invasive in vitro test methods to observe sub-toxic effects on individual adherent cells in a small
cell population under physiological conditions. This article introduces a miniaturized
microcavity chip (MCC)-based method for the non-invasive analysis of nanoparticle-induced
effects of adherent single cells in a small defined cell layer.
Results and discussion
Fabrication of the miniaturized microcavity chip
An MCC was fabricated by semiconductor process technology (Figure ). The design focused on the improvement of the high-quality optical analysis of cellular
reactions of a small cell population compared to conventional cell culture chambers.
An 800-nm-thick transparent Si3N4 membrane forms the cell culture area with a surface of 0.16 mm2. Due to the positive optical and mechanical properties of Si3N4, the micro-sized culture chamber has optimal optical properties when using microscopic
analysis methods. The seven individual miniaturized cell culture chambers in each
cultivation segment guarantee a statistical analysis of the generated data. The MCC
represents an array of miniaturized cell culture chambers for permanent non-invasive
characterization of individual cells in a cell layer. The miniaturization of the cell
culture area guarantees the observation of the complete cell culture area (Figure
The miniaturized cell culture chamber. (a) Work flow of the fabrication. (b) Design of the MCC. The MCC contains 6 × 7 miniaturized cell culture chambers. (c) Photographic image of the microcavity chip. Scale bar 5mm.
Currently, the 800-nm-thick transparent Si3N4 membrane used in this study is the thinnest membrane layer available so far with good
optical properties, allowing easy analyzing of individual cells in a cell culture
layer with high optical quality. The six individual culture segments provide the opportunity
to analyze different materials or concentrations under identical physiological conditions
(Figure ).
Each of the six culture segments possesses seven individual microcavities which are
used as cell culture chambers (Figure ). The addition of a test substance in one of the six culture segments guarantees
a statistical analysis by the seven separate micro-sized cell culture chambers. The
size of the Si3N4 membranes of the cell culture area (400 × 400 μm) (Figure ) was chosen to observe the whole area with one microscopic image (888 × 666 μm) and
to guarantee a more physiologically realistic condition compared to single-cell analysis,
since about 200 to 250 cells are present in each cavity and thus a small monolayer
can be formed. To observe all cells of a cell layer in conventional cell culture chambers,
the whole area has to be scanned, which is a very time-consuming procedure. The advantage
of the miniaturized cell culture chamber is that the entire cell culture area can
be analyzed quickly with better optical quality and without any changes of the cell
Microscopic images of the miniaturized cell culture chamber with a Si3N4 membrane. (a) Photographic image. Scale bar 1,100 μm. (b) Phase contrast microscopic image. Scale bar 150 μm. (c) Scanning electron microscopic image. Scale bar 150 μm.
Another advantage of the MCC is that optimal focusing is possible, whereas polystyrene
membranes of conventional cell culture dishes only allow focusing in the center of
the cell culture area due to edge effects. The Si3N4-cell culture area of the miniaturized system possesses a square shape due to its
production process. Due to the etch process, the end walls are positioned in an angle
of 54.7° amplifying the optical properties of the cavity membrane due to the reduced
edge effects. Preliminary experiments showed that the round shape of conventional
cell culture chambers, like 96-well microplates or 384-well microplates, resulted
in edge effects, leading to unfocused microscopic images of the cells. Additionally,
the correlations between fluorescent and bright-field images did not conform to each
other when using conventional polystyrene cell culture chambers. In contrast, the
developed micro-sized cell culture chamber reduced the working distance during microscopy
due to the 800-nm-thin Si3N4 membrane. In addition, due to the square shape of the cell culture chambers, the edge
effects are minimized resulting in clear focused microscopic images with analogy bright-field
and fluorescent images with high optical quality. Furthermore, Si3N4 features minimal auto-fluorescence in comparison to polystyrene.
Currently, only few microsystems exist for non-invasive analysis of specific reactions
of individual cells in a small adherent cell population via optical methods [,]. Stangegaard et al. described a polymethylmethacrylate (PMMA) chip as micro cell
culture system with a cell culture area of 99 mm2 []. In comparison to the PMMA-micro cell culture system, the established MCC with its
800-nm-thin Si3N4 membranes offers a better optical quality and can also be used for scanning electron
microscopy (SEM). Compared to the conventional fluorescence-based analysis techniques,
the combination of a reporter cell line and the MCC presents a more sensitive and
cost-efficient in vitro method. Advantages of the quantitative analysis via MCC are the low sample volume,
the small amount of test materials, the capture of the complete cell culture area
with high optical quality, and thus the possibility to statistically analyze the variations
between the individual cell responses.
Analysis of the biocompatibility of the MCC
The biocompatibility of the evaluated microcavity chip was analyzed by culturing human
bronchial epithelial cells (A549 cells) in the miniaturized cell culture chamber for
48 h (Figure ). The cells adhered onto the Si3N4 membranes and showed characteristic morphologies. Scanning electron microscopic images
after 7 days of cultivation of A549 cells confirmed their adherence to the Si3N4 membrane (Figure ). Moreover, the cells did not only adhere to the Si3N4 membrane but also to the Si sides (Figure ). The viability of the A549 cells was verified after 7 days of proliferation via
fluorescein diacetate (FDA)/propidium iodide (PI) staining (Figure ). The viability after this prolonged incubation period was 96.2 ± 0.3%. Furthermore,
the suitability of the miniaturized cell culture chambers for cultivation and differentiation
of sensitive in vitro systems was determined.
Microscopic images of different cell types cultured in the miniaturized cell culture
chamber. (a) Scanning electron microscopic image of A549 cells on the Si-sidewalls. Scale bar
20 μm. (b) Scanning electron microscopic image of A549 cells after 7 days of culture on the
Si3N4 membrane. Scale bar 200 μm. (c) Scanning electron microscopic image of PC-12 cells 8 days after neuronal differentiation.
Scale bar 100 μm. Small box: bright-field image of neuronal differentiated PC-12 cells.
Scale bar 50 μm. (d) Fluorescence microscopic image of A549 cells after 7 days of cultivation after FDA/PI
staining. Scale bar 50 μm. (e) Bright-field microscopic image of proliferating hMSCs after 7 days. Scale bar 100
μm. (f) Scanning electron microscopic image of hMSCs after 18 days adipogenic differentiation.
Scale bar 100 μm. (g) Bright-field image of adipogenic differentiated hMSCs. Scale bar 20 μm.
As sensitive in vitro system, PC-12 cells (rat adrenal pheochromocytoma cells) were grown in the microcavity.
These cells are used as model cells in tissue engineering [,]. After adding the differentiation stimulus nerve growth factor to the cell culture
medium, the suspension cells started to adhere and form neuronal networks (Figure
). Mesenchymal stem cells (MSCs) were used as a model for a sensitive in vitro system []. The morphology of the human MSCs (hMSCs) during proliferation is comparable to the
morphology of the cells cultured on polystyrene membranes as it is common in conventional
cell culture chambers like 96-well microplates (Figure ). The adipogenesis was used to determine the effect of miniaturization on the differentiation
capacity of hMSCs. Human MSCs were cultured for 18 days in adipogenic differentiation
medium. Lipid droplets, which were formed as a result of adipocytes, are visible by
bright-field microscopy (Figure ). Scanning electron microscopy (SEM) images of the adipogenic differentiated hMSCs
show a clear increase of adipogenic differentiated hMSCs, also in the corner areas
of the microcavity (Figure ). The performed studies verify the biocompatibility of the Si3N4 membrane and the suitability of the microcavity for in vitro studies. A549 cells as well as hMSCs proliferate in the microcavity. Furthermore,
we are the first to demonstrate the possibility to induce adipogenic differentiation
of hMSCs as well as a neuronal differentiation of PC-12 cell in the microcavity with
a cell growth area of 0.16 mm2. Due to the high need for MSCs in the field of tissue engineering, the micro-sized
cell culture area opens new potential for culturing and differentiation of 3D MSC
cultures as well as studying stem cell niches using relatively low numbers of cells
which also allows the inclusion of more repetitions and treatments. Such studies could
provide insight in cancer stem cell research, since miniaturization allows a detailed
observation of the complete cell population in the cell culture chamber. The microchip
combined with neuronal cells provides a basis for new methods for research on neuronal
diseases like Alzheimer or Parkinson disease, for the development of new sensitive
drug screening methods and for the quantification of toxicodynamic and toxicokinetic
Application of the MCC for the analysis of nanoparticle-induced effects
After confirmation of the biocompatibility of the evaluated miniaturized cell culture
chambers, the system was validated for the non-invasive quantification of IL8 gene
promoter activations of individual cells of a small cell population. Currently, much
research is ongoing to determine potential effects of nanoparticles on health of workers
and consumers. The amounts of engineered nanoparticles with a range of different sizes
and shapes and made from different materials are steadily growing, and there is a
need to determine the biological response to these novel materials. In this respect,
the immune system is of special interest, since one of the main functions of the immune
system is to deal with foreign materials [].
In order to determine whether or not the MCC method could be suitable for the analysis
of nanoparticle-induced immunomodulatory effects, a stable transfected A549 reporter
cell line, containing the IL8 promoter sequence linked to the gene for green fluorescence
protein (pIL8-GFP), was established. The sequence of the IL8 promoter was placed before
the GFP sequence, whereby GFP was used as a reporter gene. IL8 promoter activation
resulted in the generation of GFP which was accumulated within the cell. Since the
original IL8 gene has not been replaced, the analysis of IL8 expression by conventional
methods is still feasible. Beyond that, the combination of the miniaturized cell culture
chamber and the transfected reporter cell line pIL8-GFP A549 allows the detection
of specific IL8 promoter activity of individual cells in a small adherent cell population.
First of all, the cells were stimulated by a pro-inflammatory stimulus to determine
whether the cells respond in an appropriate manner. Recombinant human tumor necrosis
factor alpha (rhTNF-alpha), a cytokine involved in local and systemic inflammations,
was added to the cell culture. The GFP expression of the transfected pIL8-GFP A549
cells verifies an IL8-coupled inflammatory response. The kinetics and stability of
GFP was determined by stimulating the A549 cells with the rhTNF-alpha. Stimulation
with rhTNF-alpha showed a dose-dependent increase in GFP production which peaked when
using 20 ng/ml TNF-alpha (unpublished observation). Moreover, the cell line could
be kept in culture for more than 1 month without a loss of responsiveness to general
cellular stimuli.
After 24 h exposure of the pIL8-GFP A549 cells with 20 ng/ml TNF-alpha, the GFP expression
was quantified via fluorescence spectrometry using a 96-well microplate and via fluorescence
microscopy using the micro-sized cell culture chamber. The comparison of the two different
methods results in a higher response when using the MCC-based technique (Figure ). By the miniaturized method, GFP expression was detectable in 59.2 ± 16.8% of the
cells in the microcavity compared to the untreated control (Figure ). Via a 96-well microplate, an increase of fluorescence intensity of 44.6 ± 9.7%
was proven (Figure ). Thereafter, the fluorescence intensity of 90 individual GFP-expressing pIL8-GFP
A549 cells was quantified after incubation with TNF-alpha (20 ng/ml) in the micro-sized
cell culture chamber. The fluorescence intensity of each individual cell was quantified
digitally as pixel number. The pixel number of the 90 analyzed cells varied between
0 and 2,700 pixels per cell. The histogram of the fluorescence intensity evidenced
that most stimulated cells have fluorescence intensities with values less than 270
pixels (Figure ). This result revealed that the MCC-based system is very sensitive and feasible for
quantifying GFP expression and distinguishing the fluorescence intensity of individual
cells in a small cell population.
GFP expression of TNF-alpha- and GC10-exposed pIL8-GFP A549 cells. pIL8-GFP A549 cells were cultured in the microcavities and exposed to 20 ng/ml TNF-alpha
or 30 μg/ml GC10 for 24 h under physiological conditions. In parallel, 10,000 pIL8-GFP
A549 cells were seeded in 96-well microplates and stimulated with 20 ng/ml TNF-alpha
and 30 μg/ml GC10 for 24 h. After the exposure time, the GFP expression of the pIL8-GFP
A549 cells in the microcavities was analyzed by fluorescence microscopy. The percentage
of GFP-expressing cells in the microcavity was calculated via the software analysis.
The GFP expression of the pIL8-GFP A549 cells in the 96-well micro plate was quantified
by fluorescence spectrometry. The percentage of GFP expression is pictured as alteration
to the untreated control (alteration to control/percent). (b) pIL8-GFP A549 cells were treated for 24 h with 20 ng/ml TNF-alpha in the microcavities.
The GFP expression of 90 individual cells was quantified. The classes of the fluorescence
intensities (x-axis: class of GFP intensity) and its frequency (y-axis: frequency) is presented.
Chemicals but especially particles can interact with single cells within a cell population
and only induce a response at a certain threshold concentration, which varies from
cell to cell, e. g., depending on the cell cycle stage or on previous exposures. Therefore,
the analysis and quantification of single-cell responses will provide important information
on the toxicity of the tested materials. The MCC-based method is therefore qualified
as new non-invasive in vitro method for analyzing single-cell responses of adherent cells under physiological conditions.
In order to detect the suitability to use the developed method for nanotoxicology
studies, two nano-sized materials (gold nanoparticles (GC10) and silver nanoparticles
(SC10)) have been used for validating the new non-invasive method. Before investigating
the effect of the nanoparticles on the IL8 promoter activation, they were characterized
physicochemically (Table ). The detected zeta-potential is a characteristic for uncoated nano-scaled gold and
correlates to the data described in the literature [-].
Physicochemical characterization of the used nanoparticles
To determine the inflammatory effect of GC10, pIL8-GFP A549 cells were cultured in
presence of 30 μg/ml nanoparticle suspension in the microcavities (0.16 mm2) and in the well of a 96-well plate (34 mm2) for 24 h. For every cavity, the total number of cells and the number of fluorescent
cells were determined by microscopy, and the ratio of fluorescent cells was calculated.
The quantification of the fluorescence and bright-field images resulted in an increased
amount of fluorescent cells (26.44 ± 4.09%) in comparison to the conventional method
(19.8 ± 18.5%) (Figure ). In addition, the fluorescence spectrometry resulted in a major standard deviation.
In contrast, the MCC-based method shows a small standard deviation, which indicates
that it is a very sensitive and reproducible system. For correlating the amount of
nanoparticles and the inflammatory status of a single cell, pIL8-GFP A549 cells were
incubated in 30 μg/ml GC10 or SC10 for 48 h. By fluorescence microscopy, it was observed
that the nanoparticles were not located homogeneously on the cells and on the membrane
(Figure ). However, no correlation was observed between the amount of nanoparticles on the
cells and the IL8 promoter activation. Nevertheless, the overlay of the bright-field
image (Figure ) and the fluorescence image (Figure ,
and ) of the GC10- and SC10-treated pIL8-GFP A549 cells in the microcavity allows quantification
of the fluorescence intensity and thus the inflammatory status of single cells.
Microscopic images of SC10- and GC10-treated pIL8-GFP A549 cells in the microcavity. pIL8-GFP A549 cells were treated for 48 h with (2) GC10 and (3) SC10 under physiological
conditions. (a) Bright-field image. Scale bar 50 μm. (b) Fluorescence image. Scale bar 50 μm. (c) Overlay of the bright-field and the fluorescence images. Scale bar 50 μm. (d) Overlay of the bright-field and the fluorescence images of individual GC10-exposed
pIL8-GFP A549 cells. Sections of this image are pictured in (d1 to d4). Beside individual
GFP-expressing pIL8-GFP A549 cells interacting with nanoparticle aggregates (d1, d2),
also GFP-expressing cells with few or less nanoparticle interaction were observed
The determination of the effect of miniaturization on nanoparticle-induced inflammatory
cell responses resulted in a basal amount of untreated pIL8-GFP A549 cells varying
between 8% and 11%, for all tested cell culture areas (Figure ). This is in agreement with previous experiences that A549 undergoes some degree
of activation by normal cell culture procedures and that IL8 induction is a particularly
sensitive signal. After MC100 exposure, the amount of GFP-expressing cells increased
slightly but was still at the level of the untreated control. After SC10 exposure,
the amount of fluorescent cells increased to 41.3 ± 5.1% (0.16 mm2), 36.0 ± 6.2% (11 mm2), and 43.3 ± 4.5% (34 mm2) (Figure ). The results obtained showed that the growth area had no influence on the cell response.
Effect of nanoparticles on pIL8-GFP A549 cells. (a) Effect of miniaturization on nanoparticle-induced inflammation in pIL8-GFP A549
cells. pIL8-GFP A549 cells were cultured on three different growth areas (0.16, 11,
and 34 mm2) and exposed to 20 μM SC10 and 20 μM MC100 for 24 h under physiological conditions.
The percentage of GFP-expressing cells per growth area was analyzed by fluorescence
microscopy. The amount of GFP-expressing pIL8-GFP A549 cells in relation to the cell
growth area is depicted. The results are presented as mean of three independent experiments
± SD. (b) Concentration-dependent effect of nanoparticles on mitochondrial dehydrogenase activity
of pIL8-GFP A549 cells. pIL8-GFP A549 cells were exposed to 0 to 50 μg/ml SC10 und
MC100 for 24 h under physiological conditions. Triton X-100 was used as positive control.
Via WST-1 assay the mitochondrial dehydrogenase activity was quantified. Untreated
cells were set as 100%. The results are presented as mean of three independent experiments
± SD compared to the untreated control.
The cytotoxic effect of SC10 and MC100 was evaluated using the WST-1 assay. MC100
induced a concentration-dependent cytotoxicity but a low decrease in mitochondrial
activity with a maximum reduction of 20% when cells were treated with 50 μg/ml MC100.
In contrast, SC10 had an IC50 value of 27 μg/ml in pIL8-GFP A549 cells, which correlated with the effect of SC10
on IL8 promoter activation (Figure
and ). The reduction in fluorescence intensity at higher concentrations could therefore
be caused by the cytotoxic effects of SC10. A maximal reduction of the mitochondrial
activity of 38% was found when cells were treated with 50 μg/ml SC10 (Figure ).
Concentration- and time-dependent effects of nanoparticles on the GFP expression of
pIL8-GFP A549 cells. (a) pIL8-GFP A549 cells were cultured in the microcavities and exposed to 30 μg/ml GC10
and SC10 for 48 h. The GFP expression of the pIL8-GFP A549 cells was analyzed via
fluorescence time-lapse microscopy. The percentage of GFP-expressing cells was quantified
using the software analysis. (b) pIL8-GFP A549 cells were treated with 0 to 50 μg/ml SC10 for 24 h in the microcavity
under physiological conditions. Parallel 10,000 pIL8-GFP A549 cells were cultured
and treated in a 96-well microplate with 0 to 50 μg/ml SC10. The GFP expression of
the pIL8-GFP A549 cells in the microcavities was analyzed by fluorescence microscopy
and the GFP expression of the cells in the microplate by fluorescence spectrometry.
Besides the threshold-dependent detection of inflammatory reactions, the usability
of the MCC-based system to determine time-dependent inflammatory processes was tested.
By time-lapse microscopy, SC10 induced a time-dependent increase of the amount of
GFP-expressing pIL8-GFP A549 cells in the microcavity. After 26 h, the percentage
of fluorescent cells decreased to the fluorescence level of untreated pIL8-GFP A549
cells (Figure ). The amount of GFP-expressing GC10-treated cells remained on the control level,
and after 30 h, the amount of fluorescent cells increased to 12.9 ± 4.1% and ranges
in the following 18 h between 9.8 ± 2.5% and 15.8 ± 0.5% (Figure ).
To test the use of the micro-sized cell culture for determination of threshold-dependent
effects, pIL8-GFP A549 cells were incubated with 0 to 50 μM SC10 for 24 h at 37°C.
Ten micrograms per milliliter of SC10 induced an increase in fluorescence intensity
of 25%, and 20 μg/ml induced a significant increase of 40% (Figure ), whereas concentrations higher than 40 μg/ml caused no significant increase in fluorescence
intensity compared to the untreated control. Inflammatory effects as well as cytotoxic
effects are threshold-dependent effects. Low concentrations leading to an inflammatory
process could cause cytotoxic effects, but normally this is not the case. If a cytotoxic
effect is induced, the concentration is often too high to activate the inflammation-specific
pathways in the cells. In our experiments, the exposure time of 24 h concentrations
up to 20 μg/ml resulted in a significant IL8 promoter activation quantified as GFP
expression and concentrations higher than 30 μg/ml resulted in a significant decrease
of cell viability as analyzed by WST-1 assay resulting in less GFP expression (Figure
The combination of the miniaturized cell culture chamber and the transfected reporter
cell line pIL8-GFP A549 realizes the establishment of a chip-based in vitro method as non-invasive technique for detecting inflammatory processes of adherent
cells in a small cell population. Compared to the 96-well microplates, the new miniaturized
cell culture chamber enables a fast and sensitive quantification of IL8 promoter activations
that is based on the analysis of individual cells within a population.
It has been described that the physical properties of nanoscale materials can interfere
with the analysis of toxicological parameters [,]. The MCC-based method is based on optical analysis followed by digital quantification
of the induced GFP expression of every individual cell in the microcavity. One advantage
of the miniaturized method is the recording of the complete cell culture area in one
image. Hence, every individual cell response is involved in the assessment of the
inflammatory status. By observing every individual cell, the interference of the physical
properties of the nanoparticles with the fluorescence spectrometric analysis was avoided.
Besides reproducibility and sensitivity, the use of the miniaturized system for the
detection of threshold-dependent effects was tested. The comparison of the data obtained
using a 96-well microplate and the developed micro-sized cell culture chamber verified
the suitability of the microcavities as biocompatible cell culture chamber with better
optical quality and the suitability of the MCC in combination with the transfected
reporter cell line pIL8-GFP A549 as new non-invasive in vitro method for the continuous observation of GFP expression and the quantification of
concentration- and time-dependent nanoparticle-induced IL8 promoter activation in
adherent cells of a small cell population.
Conclusions
The goal of this study was to establish a biocompatible micro-sized cell culture chamber
and to prove its applicability to determine nanoparticle-induced effects of an individual
cell of a small cell population.
The need to develop non-invasive in vitro methods to detect nanoparticle-induced effects of a small cell population is high
and can be illustrated in conjunction with the European chemical regulation REACH
[]. Previous methods for nanotoxicity studies are OECD sta most
of these ignore the individual differences within a cell population and can therefore
lead to misinterpretations. The development of the MCC, described in this manuscript,
allowed us to culture cells in a way in which their behavior is comparable to that
observed in conventional cell culture systems. Compared to macro-scale cell culture
chambers, the MCC offers the opportunity for culturing, long-term observation, and
manipulation of a small amount of cells on a defined cell culture area. Using this
non-invasive system, individual cells could easily be observed and specific cell reactions
could be quantified.
The bottom of the miniaturized cell culture chambers was made of Si3N4 membranes to ensure biocompatibility as well as excellent optical properties for cell
analysis. The small size of cavities enables a high number of cavities on each chip
and facilitates the performance of many independent assays on one plate. The funnel-shaped
cavities avoid the appearance of meniscuses, and therefore, the total cell growth
area can be used for analysis. Moreover, the miniaturization allows the microscopic
analysis of the entire cell population in the cavity in one microscopic field. The
detection of the complete cell layer guarantees reproducible results without any subjective
choice of representative areas of the cell monolayer.
Besides the advantage of convenient handling, the microscopic analysis of small amounts
of cells and nanomaterials increase the through-put rate of the experiments, resulting
in a time- and cost-effective method. The established cell culture chamber (0.16 mm2) is a biocompatible chamber with the thinnest (800 nm) transparent cell culture layer
existing, resulting in high optical quality. Therefore, the proposed in vitro method bridged the gap between population measurements and quantitative single-cell
analysis. Such a non-invasive system could be used to investigate the nano(immuno-)toxicity
on an individual cell level, followed by selective quantitative analysis of the induced
intensity.
Fabrication of the miniaturized cell culture chamber
A miniaturized microcavity chip (MCC) (length 3 cm, width 2.5 cm) was fabricated by
semiconductor process technology. Base material was a 500- μm-thick & 100 & orientated
silicon (Si) wafer, coated double-sided with an 800-nm-thick silicon nitride (Si3N4) layer. Design and fabrication are shown in Figure . The MCC consist of six cavities (length of the outline 4,000 × 4,000 μm, depth 400
μm), where each have seven separate funnel-shaped microcavities (length of the outline
400 × 400 μm, depth 100 μm). These represent the miniaturized cell culture chambers
with a Si3N4 membrane and a growth area of 0.16 mm2. Prior to the application of the MCC for biological analysis, they were autoclaved
at 121°C, 2 bar, for 15 min.
Cell lines and culture conditions
All cell culture reagents were obtained from Invitrogen (Karlsruhe, Germany), unless
stated otherwise. The human lung epithelial carcinoma cells A549 (ATCC no. 107) were
cultured in RPMI medium supplemented with L-glutamine (4 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% (v/v) fetal calf serum (FCS). PC-12 cells (rat adrenal pheochromocytoma cells, ATCC no.
159) were cultured in RPMI medium supplemented with L-glutamine (4 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), 10% (v/v) horse serum, and 5% (v/v) FCS. For neuronal differentiation, RPMI medium was supplemented with 0.5% horse
serum, 0.25% FCS, and 1% nerve growth factor. Human mesenchymal stem cells (hMSCs)
were isolated from the bone marrow of human thighbone of human donors as described
in literature []. The thighbones were kindly provided from the Protestant hospital in Zweibrücken
(Germany) from Dr. M. Maue and Dr. Hassinger. Dr. E. Gorjup (Fraunhofer IBMT, St.
Ingbert, Germany) isolated the hMSCs with a declaration of consent of each patient.
hMSCs were cultured in alpha-MEM supplemented with penicillin (50 U/ml), streptomycin
(50 μg/ml), and 15% (v/v) heat-inactivated FCS (proliferation medium). For adipogenic differentiation, the
proliferation medium was exchanged by differentiation medium (alpha-MEM supplemented
with penicillin (50 U/ml), streptomycin (50 μg/ml) and 10% (v/v) FCS, 100 ng/ml insulin, 100 mM dexamethasone, 200 μM indomethacin, and 500 μM
isobuthylmethylxanthine. Stable clones of pIL8 GFP-transfected A549 cells (A549 pIL8
GFP, see below) were cultured in the RPMI medium (supplemented with L-glutamine (4 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10% (v/v) FCS) in the presence of G418 (0.5 mg/ml final concentration).
Cells were maintained in a 5% CO2 humidified atmosphere at 37°C.
Experimental procedure
The experimental design is schematically depicted in Figure . To reduce the evaporation of the cell culture medium in the micro-sized cell culture
chambers, a biocompatible cell culture chamber was positioned on the top of the MCC.
Each chamber of the covered silicone FlexiPerm(R) chamber (Greiner Bio-One, Frickenhausen, Germany) includes seven individual miniaturized
microcavities for statistical analysis of the experimental data. For each experiment,
100 μl cell suspension (100,000 cells/ml) were placed in each of the six culture segments.
After 30 min, the cells adhered onto the Si3N4 membrane. The segments of the cell culture chamber were filled with 100 μl cell culture
medium. After 24 h of cell proliferation, the cells were exposed to the nanoparticles
by aspirating the medium, washing the cells with PBS and adding the nanoparticle-containing
medium. After the exposure time, the cells were analyzed microscopically. The total
number of cells and the number of fluorescent cells were counted, and the percentage
of GFP-expressing cells was calculated.
Schematic experimental design. To reduce the evaporation of the cell culture medium, a biocompatible cell culture
chamber was positioned on the top of the MCC. Each chamber of the covered silicone
FlexiPerm(R) chamber includes seven individual miniaturized microcavities for statistical analysis
of the experimental data. For each experiment, 100 μl cell suspension (100,000 cells/ml)
were placed in each of the six culture segments. After 30 min, the cells adhered on
the Si3N4 membrane. The segments of the cell culture chamber were filled with 100 μl cell culture
medium. After 24 h of cell proliferation, the cells were exposed to the nanoparticles
by aspirating the medium, washing the cells with PBS, and adding the nanoparticle-containing
medium. After the exposure time, the cells were analyzed microscopically.
Generation of the stably transfected reporter gene cell line
The host cells used for this study were A549 cells (ATCC no. 107). The human A549
cell line was transfected with an expression vector encoding green fluorescence protein
(GFP) and an insert that encodes for the IL8 promoter region. The pTurboGFP-PRL expression
vector was obtained from Evrogen (Moscow, Russia). This construct is a circular bacterial
DNA which contains genes coding for ampicillin and neomycin resistance, allowing selection
in respective bacteria and after transfection in human cells. Essential is that the
construct also contains the GFP gene, as a reporter gene. The IL8 promoter sequence
was amplified from human genomic DNA (Roche Diagnostics GmbH, Mannheim, Germany) by
reversed transcriptase polymerase chain reactions (RT-PCR) using the forward primer
5'-ata ctc gag ggg tac ctt cgt cat act ccg tat ttg ata agg aac a-3' and the reverse
primer 5'-aga att cgc ata gat ctt ccg gtg gtt tct tcc tgg ctc tt-3', containing the
restriction enzyme sequences for Xho I and Eco RI, respectively, to allow cloning
into the multiple cloning site. PCR with these primers resulted in an IL8 promoter
fragment of 250 bp (NCBI NM 000584). The promoter fragment was chosen to include the
main regulatory sites required for functional control of transcription. After cloning
and plasmid isolation using standard techniques, A549 cells were transfected using
Effectene (Qiagen, Hilden, Germany) following the distributors' instructions. After
transfection, cells were cultured in the presence of 0.5 g/l G418 (gentamicin). The
single-cell-derived clones of viable cells containing the insert, as verified by RT-PCR,
were expanded. Batches of the stably transfected cell lines were frozen in liquid
nitrogen, and individual aliquots of the stably transfected cells were not placed
in culture for more than 1 month.
Physicochemical characterization of the nanoparticles
To ensure a good comparability of our results with those obtained in other studies,
we have chosen nanoparticles which have been selected by the National Institute of
Standards and Technology as certified reference materials for preclinical biomedical
research. The commercially available gold nanoparticle solutions, synthesized by the
Frens method [], were purchased from BBInternational (Cardiff, UK). These particles were spherical
gold nanoparticles, 10 nm in size (type gold colloid GC10). The utilized silver nanoparticles
(SC10) with a diameter of 10 nm were purchased from PlasmaChem GmbH (Berlin, Germany).
The iron oxide nanoparticles (MC100) with a mean diameter of 100 nm possess a starch
shell and are also commercially available (Chemicell GmbH, Berlin, Germany). The colloidal
aqueous nanoparticle suspensions were sterile filtered to exclude any bacterial contamination
(pore size 0.22 μm). The nanoparticles were characterized by dynamic light scattering,
surface charge (zeta potential), and by absorption spectroscopy. For determining the
size distribution of the nanoparticles, dynamic light scattering measurements have
been performed on a Malvern Zeta Sizer Nano ZS (Malvern Instruments Ltd., Worcestershire,
UK) using disposable clear zeta cells (DTS 1060C). The average diameter and polydispersity
index (PDI) were provided by the instrument using general purpose analysis. The zeta
average diameter and PDI reported herein were obtained as the average of three independent
measurements (10 repetitions per measurement) performed on each sample. Zeta potential
measurements were performed using a Malvern Instruments Zetasizer Nano (Malvern Instruments
Ltd), operating with a variable-power (5 to 50 mW) He-Ne laser at 632 nm. Measurements
were taken in zeta cells (DTS 1060C) at 25°C and repeated three times (10 repetitions
per measurement) for each sample. UV-visible (UV/Vis) absorption spectra were taken
on a two-beam UV/Vis spectrometer (Lambda 950, Perkin Elmer, WalthamMassachusetts,
USA). The UV-visible absorption spectra of both gold nanoparticle suspensions were
recorded at room temperature. For the experiments, the wavelength ranging from 250
to 700 nm was used.
Determination of the mitochondrial activity
The mitochondrial function of the incubated cells was analyzed using the WST-1 assay
(Roche Diagnostics GmbH). This assay is based on the cleavage of stable tetrazolium
salt WST-1 by metabolically active cells to an orange formazan dye. The WST-1 assay
was performed according to the manufacturer's instructions, with appropriate controls.
After nanoparticle exposure, the cells were incubated with the ready-to-use WST-1
reagent for 4 h. After this incubation period, formazan formation was quantified by
absorbance measurements at 650 nm. The net absorbance change taken from the wells
of untreated cultured cells was scaled to 100% cell viability.
Determination of cell viability by FDA/PI staining
To differentiate between viable and dead cells, fluorescein diacetate (FDA)/propidium
iodide (PI) (Sigma-Aldrich, Deisenhofen, Germany) staining was performed. FDA, a membrane
permeable dye, is metabolized by viable cells to a green fluorescent dye. PI intercalates
in nucleic acids and is unable to penetrate the cell membrane and therefore stains
membrane-damaged cells only. Cell viability of A549 cells was determined by culturing
the cells for 48 h on the Si3N4 membrane at 37°C and 5% CO2. Afterwards, the cells were treated with the fluorescent dye mixture for 15 s. Thereafter,
the cells were washed once with PBS to remove excessive dye molecules. The cells were
observed under a Zeiss Observer Z1 fluorescence microscope (Zeiss, Jena, Germany)
using exFDA 470 nm/emFDA 525 nm and exFDA 555 nm/emFDA 602 nm. The percentage of the viable and dead cells was analyzed using the cell imaging
analysis software.
Time-lapse microscopy
Time-dependent nanoparticle-induced GFP expression was quantified using time-lapse
microscopy. The system Biostation IM-Q (Nikon, Düsseldorf, Germany) is composed of
a microscope, an incubator, and a high-resolution camera. The combination of an LED
and a fluorescence filter provides the opportunity for fluorescence quantification.
pIL8-GFP A549 cells were exposed to 30 μg/ml GC10 or SC10 in the micro culture chamber
at 37°C for 48 h. Every hour, a phase contrast and a fluorescent (ex 472 nm/em 520
nm) image of the entire cell culture area were taken of the same section of the sample.
The amount of fluorescent cells was quantified via the software analysis.
Scanning electron microscopy
For SEM of adipogenic differentiated hMSCs, neuronal differentiated PC-12 cells, and
A549 cells, the cells were fixed with cacodylate/glutaraldehyde buffer and contrasted
using 2% osmium tetroxide and 1% tannic acid. After dehydrating the cells with ethanol,
they were dried in a critical point dryer CPD-7501 (Quorum Technologies Ltd., East
Sussex, UK) and covered with gold. Samples were examined in a scanning electron microscope
EM 109T (Zeiss) using secondary electron mode.
Quantification of pIL8-GFP induction
For quantification of an induced inflammatory reaction, the reporter cells pIL8-GFP
A549 were used. The induction of the promoter is linked to the production of the inflammatory
cytokine, and consequently, the intensity of the IL8 promoter activation is proportional
to the GFP expression, which could be quantified microscopically and fluorometrically.
For fluorometric quantification of GFP expression 300 to 10,000 cells per well were
exposed to GC10 or SC10 (0 to 50 μg/ml) for 24 h at 37°C. After washing the cells
with PBS, the GFP intensity (ex 485 nm/em 535 nm) was quantified fluorometrically
using a Tecan plate reader (Tecan Deutschland GmbH, Crailsheim, Germany). RhTNF-alpha,
as inflammation stimulating agent, was used as positive control. The IL8 promoter
activation of pIL8-GFP A549 cells cultured in the micro cell culture chambers was
analyzed after SC10 or GC10 exposure (24 h) by fluorescence microscopy.
Statistical analysis
Independent experiments were performed three times in triplicates (n = 9), and the data are presented as mean ± SD. Statistical significance was established
as p & 0.01. Statistical tests were performed using the Mann-Whitney U test.
Abbreviations
Bio-MEM: biological microelect DNA: d FCS:
GC: gold col GFP: green
hMSCs: human me MC: magnetite col MCC: microcavity
OECD: Organisation for Economic Co-operation and D PBS: phosphate-buffered
PCR: polym pIL8: interleukin-8 PMMA: po
REACH: Registration: Evaluation: Authorisation and Restriction of C rhTNF-alpha:
recombinant tumor necrosis factor- SC: silver col Si3N4: TNF: tumor necrosis factor.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
YK carried out the cytotoxicity studies, performed the nanoparticle characterization,
designed the miniaturized cell culture chambers, participated in their fabrication,
and wrote the main parts of the manuscript. GO carried out the transfection of the
reporter cell line and was involved in the preparation of the manuscript. AS fabricated
the microcavity chip. AD and HT obtained funding for this project and helped to draft
the manuscript. HB helped to draft the manuscript. All authors read and approved the
final manuscript.
Acknowledgements
We thank Dipl.-Chem. Rainer Lilischkis (University of Life Science Kaiserslautern,
Institute for Information & Microsystems Technology, Germany) for his assistance with
SEM Dr. M. Maue and Dr. Hassinger (Protestant Hospital, Zweibrücken,
Germany) for kindly provi and Dr. Erwin Gorjup (Fraunhofer IBMT,
St. Ingbert, Germany) for isolating the hMSCs. This work was supported by the EU under
the Framework VI project DIPNA (Development of an Integrated Platform for Nanoparticle
Analysis to verify their possible toxicity and eco-toxicity) (STRP 032131 DIPNA).
References
Rajwade JM,
Paknikar KM:
Cellular responses induced by silver nanoparticles: in vitro studies. Toxicol Lett 2008,
179:93-100.
Toxicological effects of inorganic nanoparticles on human lung cancer A549 cells. J Inorg Biochem 2009,
103:463-471.
Rogerieux F,
Maillot-Marechal E,
Boczkowski J,
Lacroix G,
Comparative toxicity of 24 manufactured nanoparticles in human alveolar epithelial
and macrophage cell lines. Part Fibre Toxicol 2009,
Lewinski N,
Cytotoxicity of nanoparticles. Small 2008,
Bonacchi D,
Lorenzi G,
Hermanns MI,
Kirkpatrick CJ:
Gold nanoparticles induce cytotoxicity in the alveolar type-II cell lines A549 and
NCIH441. Part Fibre Toxicol 2009,
In vitro evaluation of the cytotoxicity of iron oxide nanoparticles with different
coatings and different sizes in A3 human T lymphocytes. Sci Total Environ 2010,
Anderson EC,
Hessman C,
Monroe MM,
The role of colorectal cancer stem cells in metastatic disease and therapeutic response. Cancers (Basel) 3:319-339.
Rajaraman R,
Guernsey DL,
Rajaraman MM,
Rajaraman SR:
Stem cells, senescence, neosis and self-renewal in cancer. Cancer Cell Int 2006,
Fitzpatrick LA,
Individual parathyroid cells are more sensitive to calcium than a parathyroid cell
population. Endocrinology 1990,
Petrasek D,
Samtaney R,
Glandular regulation of interstitial diffusion: a model and simulation of a novel
physiological mechanism. Am J Physiol Endocrinol Metab 2002,
283:E195-206.
Maercklein P,
Fitzpatrick LA:
Paracrine interactions among parathyroid cells: effect of cell density on cell secretion. J Bone Miner Res 1994,
9:971-976.
Arkhipov SN,
Berezovski M,
Jitkova J,
Krylov SN:
Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single
cells. Cytometry A 2005,
La Rocca R,
Lakshmikanth T,
Gentile F,
Tallerico R,
Zambetti LP,
Candeloro P,
De Angelis F,
Carbone E,
Di Fabrizio E:
Monitoring human leukocyte antigen class I molecules by micro-Raman spectroscopy at
single-cell level. J Biomed Opt 2010,
15:027007.
De Angelis F,
Candeloro P,
Patrini M,
Lazzarino M,
Maksymov I,
Liberale C,
Andreani LC,
Di Fabrizio E:
Nanoscale chemical mapping using three-dimensional adiabatic compression of surface
plasmon polaritons. Nature Nanotechnology 2010,
Tittmann BR,
Scheuchenzuber W:
Technique for rapid in vitro single-cell elastography. Ultrasound Med Biol 2006,
Dovichi NJ:
Cell cycle-dependent protein fingerprint from a single cancer cell: image cytometry
coupled with single-cell capillary sieving electrophoresis. Anal Chem 2003,
Aebersold R,
Dovichi NJ:
Identification of proteins in single-cell capillary electrophoresis fingerprints based
on comigration with standard proteins. Anal Chem 2003,
Krylov SN,
Arriaga E,
Palcic MM,
Dovichi NJ:
Single-cell analysis avoids sample processing bias. J Chromatogr B Biomed Sci Appl 2000,
741:31-35.
Armstrong DW:
Single-cell detection: test of microbial contamination using capillary electrophoresis. Anal Chem 2007,
Hansmann ML,
Rajewsky K,
Kuppers R:
Single-cell PCR analysis of T helper cells in human lymph node germinal centers. Am J Pathol 2000,
Micro/nanofluidic device for single-cell-based assay. Biomed Microdevices 2005,
Buchholz TA,
Hancock D,
Gamma-radiation-induced single cell DNA damage as a measure of susceptibility to lung
cancer: a preliminary report. Int J Oncol 2000,
17:399-404.
Hofstadler SA,
Severs JC,
Swanek FD,
Analysis of single cells with capillary electrophoresis electrospray ionization Fourier
transform ion cyclotron resonance mass spectrometry. Rapid Commun Mass Spectrom 1996,
10:919-922.
<a target="_blank" href="http://dx.doi.org/10.1002/(SICI):8Publisher&Full&Text
Recent developments in optical detection methods for microchip separations. Anal Bioanal Chem 2007,
387:183-192.
Davalos RV,
Carroll-Portillo A,
Martino A,
Apblett CA:
Impedimetric and optical interrogation of single cells in a microfluidic device for
real-time viability and chemical response assessment. Biosens Bioelectron 2008,
23:845-851.
Vandaveer WRt,
Pasas-Farmer SA,
Fischer DJ,
Frankenfeld CN,
Recent developments in electrochemical detection for microchip capillary electrophoresis. Electrophoresis 2004,
Di Carlo D,
Dynamic single cell culture array. Lab Chip 2006,
Porous silicon: a resourceful material for nanotechnology. Recent Pat Nanotechnol 2008,
2:128-136.
Davidsson R,
Bengtsson M,
Laurell T,
Microfluidic biosensing systems. Part I. Development and optimisation of enzymatic
chemiluminescent micro-biosensors based on silicon microchips. Lab Chip 2004,
4:481-487.
Davidsson R,
Johansson B,
Passoth V,
Bengtsson M,
Laurell T,
Microfluidic biosensing systems. Part II. Monitoring the dynamic production of glucose
and ethanol from microchip-immobilised yeast cells using enzymatic chemiluminescent
micro-biosensors. Lab Chip 2004,
4:488-494.
Fischer R,
Steinert S,
Stubenrauch M,
Hofmann GO,
Cell cultures in microsystems: biocompatibility aspects. Biotechnol Bioeng 2011,
108:687-693.
Stangegaard M,
Petronis S,
Jorgensen AM,
Christensen CB,
A biocompatible micro cell culture chamber (microCCC) for the culturing and on-line
monitoring of eukaryote cells. Lab Chip 2006,
Uhlemann J,
Biocompatibility of glass and silicon. Biomed Tech (Berl) 1998,
43(Suppl):438-440.
Current application of micro/nano-interfaces to stimulate and analyze cellular responses. Ann Biomed Eng 2010,
Gomez-Sj?berg R,
Leyrat AA,
Pirone DM,
Versatile, fully automated, microfluidic cell culture system. Anal Chem 2007,
Kawazoe N,
Wozniak MJ,
Imaizumi Y,
Tateishi T,
Adipogenic differentiation of mesenchymal stem cells on micropatterned polyelectrolyte
surfaces. J Nanosci Nanotechnol 2009,
9:230-239.
Nanoliter scale microbioreactor array for quantitative cell biology. Biotechnology and Bioengineering 2005,
Schaffer D,
Cliffel D,
Baudenbacher F:
NanoLiterBioReactor: long-term mammalian cell culture at nanofabricated scale. Biomed Microdevices 2004,
6:325-339.
Perozziello G:
Ca2+ mediates the adhesion of breast cancer cells in self-assembled multifunctional microfluidic
chip prepared with carbohydrate beads. Micro and Nanosystems 2010,
2:261-268.
Perozziello G,
Boccazzi P,
Sinskey AJ,
Geschke O,
Jensen KF:
Microbioreactors for bioprocess development. JALA 2007,
12:143-151.
Effect of carbon nanotube coating of aligned nanofibrous polymer scaffolds on the
neurite outgrowth of PC-12 cells. Cell Biol Int 2011,
35:741-745.
Waddell RL,
Collins KL,
Doctor JS:
Using PC12 cells to evaluate poly(caprolactone) and collagenous microcarriers for
applications in nerve guide fabrication. Biotechnol Prog 2003,
Inhibition of proliferation and differentiation of mesenchymal stem cells by carboxylated
carbon nanotubes. ACS Nano 2010,
Oostingh GJ,
Italiani P,
Colognato R,
Stritzinger R,
Pfaller T,
Favilli F,
Leppens H,
Lucchesi D,
Nelissen I,
Thielecke H,
Puntes VF,
Boraschi D:
Problems and challenges in the development and validation of human cell-based assays
to determine nanoparticle-induced immunomodulatory effects. Part Fibre Toxicol 2011,
Pfaller T,
Oostingh GJ,
Time evolution of the nanoparticle protein corona. ACS Nano 2010,
Diegoli S,
Manciulea AL,
Preece JA:
Interaction between manufactured gold nanoparticles and naturally occurring organic
macromolecules. Sci Total Environ 2008,
402:51-61.
Leifert A,
Fischler M,
Brandau W,
Jahnen-Dechent W:
Size-dependent cytotoxicity of gold nanoparticles. Small 2007,
Lockwood PE,
Messer RL,
Wataha JC:
In vitro biological effects of sodium titanate materials. J Biomed Mater Res B Appl Biomater 2007,
83:505-511.
Schulze C,
Schaefer UF,
Wohlleben W,
Interaction of metal oxide nanoparticles with lung surfactant protein A. Eur J Pharm Biopharm 2010,
77:376-383.
Alternative testing - the intelligent way to REACH compliance. Proc. 6th World Congress on Alternatives & Animal Use in the Life Sciences 2008,
AATEX 14:799-803.
Pittenger MF,
Mackay AM,
Jaiswal RK,
Douglas R,
Moorman MA,
Simonetti DW,
Marshak DR:
Multilineage potential of adult human mesenchymal stem cells. Science 1999,
284:143-147.
Controlled nucleation for the regulation of the particle size in monodisperse gold
suspensions. Nature Physical Science 1973,
241:20-22.
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