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Evaluation of cytotoxic effect of 5-fluorouracil on human carcinoma cells in microfluidic system
Sensors and Actuators B 160 (2011) 1544–1551
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Evaluation of cytotoxic effect of 5-fluorouracil on human carcinoma cells in microfluidic system E. Jedrych a,∗ , S. Flis b , K. Sofinska a , Z. Jastrzebski b , M. Chudy a , A. Dybko a , Z. Brzozka a a b
Department of Microbioanalytics, Institute of Biotechnology, Warsaw University of Technology, Poland Department of Pharmacology, National Medicines Institute, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 6 April 2011 Received in revised form 29 June 2011 Accepted 27 August 2011 Available online 6 September 2011 Keywords: Cytotoxic tests Microfluidic system Lab-on-a-chip Cancer treatment Cell culture 5-Fluorouracil
a b s t r a c t In this paper, we present cytotoxicity tests performed in a microanalytical hybrid system. The microchambers of cell culture were integrated with a concentration gradient generator (CGG), which created five different concentrations of evaluated agent in a single step. 5-Fluorouracil (5-FU) is one of the most important chemotherapeutic agents for cancer treatment, therefore it was tested in the microsystem. Independent tests of the cytotoxic effect of 5-FU were performed in this microsystem on different cell lines (human lung carcinoma cells – A549 and human colon carcinoma cells – HT-29). We also present the dependence of cell viability at different times of incubation with 5-FU. We proved that 5-FU has a stronger inhibition effect on A549 than on HT-29 cells. Moreover, the results obtained in the microfluidic system were compared with that of a macroscale. It can be concluded that the conditions of cultivation can affect the level of cytotoxicity of drugs, and the microsystem is a good example that could be used in this field of research. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Non-small cell lung carcinoma (NSCLC) and colorectal cancer (CRC) are one of the most common worldwide types of cancers which develop slowly, sometimes without any symptoms for many years. Therefore, in diagnosis both diseases are often detected at an advanced stage while tumours are inoperable and/or patients have metastasis [1,2]. In such cases, chemotherapy is the first-line treatment but unfortunately it is still unsatisfactory. Differences in a positive response to chemotherapy may depend on many factors such as tumour size, the number and the size of metastases or the gene profile (changes in the gene expression as a result of their mutation or methylation) [3]. Several approaches have been suggested to optimize chemotherapeutic treatment of NSCLC or CRC patients. One approach is to test the chemosensitivity of tumour cells in vitro to predict better the clinical response to drugs [4]. A limitation of such a high-throughput screening is availability and the volume of biological samples. In vitro tests (using 96-well plates) are unsatisfactory, as this method differs from the in vivo in several aspects, for example, there are more complicated interactions between cell–cell and cell–intercellular matrix in in vivo systems. This problem may be solved by using microfluidic cell culture system (MCCS; microsystem) as a promising alternative to the conventional cell culture methods (macrosystem) [5].
∗ Corresponding author. E-mail address: [email protected] (E. Jedrych). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.08.074
Microsystems (called lab-on-a-chip) have started to play an important role in cell biology and drug development [6–11]. The ability of microdevice geometry modification gives the possibility of growth factors, reagents and oxygen control inside the system, as well as hydrodynamic shear stress upon the cultured cells [12,13]. The usage of microfluidic systems allows to reduce the consumption of solvents and reagents. This is important for drug research where the tested drug and the volume of biological samples are limited [14,15]. Besides, the scale of microchannels corresponds well with the native cellular microenvironment, so microfluidic devices are suitable especially for biological applications particularly at the cellular level [16]. Moreover, a flow of medium and tested substances through microfluidic systems can simulate in vivo conditions. The integration of microsystem with control elements allows the automation of investigations such as cell culture, cell sorting, gene expression, biochemical separation and cytotoxicity tests [17–20]. Cytotoxicity tests are performed in the microsystems using the concentration generator gradient (CGG) [21]. Besides of this technique, pipetting in nanotiterplates or micro segmented flow techniques are also often used [22–25]. All these methods enable to handle small fluid volumes, reduced instrumental materials and keep low unit cost. Microfluidic systems enable an integration of functional components and the utilization of fluid dynamics to control molecules in both time and space. The general differences between CGG and microdroplets concern the profile of fluid flow [23]. A fluid flow through the microchannels creates parabolic velocity profile over the cross-section of the channel. Whereas, localization of reagents in the droplets is an effective
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way of eliminating this phenomenon and allow to specify reaction and incubation times. Application of microdroplet enables to reduce evaporation. Moreover it allows good mixing by segmentinternal convection [26]. However, the mixing by using microdroplets is more complicated than the use of two streams of liquid flow. The CGG creates a few different concentrations in a single step and allows to skip the step of preparing of many solutions used in the investigation. Pipetting procedures are replaced by drugs’ flow, which is controllable and simulate to human organism conditions. The microdevices with different geometry of CGG were used for the testing: bupivacaine and lidocaine on the mouse myoblast cells [27], cellular responses to hydrogen peroxide on the HeLA cells [28]. Toh et al. used a microfluidic system for hepatotoxicity of 5 model drugs (acetaminophen, diclofenac, quinidine, rifampin and ketoconazole) [29]. Creating a program for automatic administration of the substance will allow the simple and user-friendly usage of a microchip to cytotoxicity tests. Due to the fact that the miniaturization dedicated to cell analysis research offers many advantages, so the microfluidic system can be a better method than research in the macroscale. The aim of our study was to perform a series of cytotoxicity assays in a PDMS/glass microfluidic system using two human cancer cell lines. 5-Fluorouracil (5-FU) was used as one of the most popular and best known chemotherapeutic agents [30,31]. Moreover, 5-FU can also be used for tests of the synergistic effect with various drugs, which are significant for antitumour treatment. In the literature, we did not find tests performed in a microscale, where the 5-FU cytotoxic effect was investigated on the A549 (human lung carcinoma cell line) and HT-29 (human colon adenocarcinoma cell line). 5-FU like other pyrimidine antagonists is similar in structure to uracil, one of the four naturally occurring RNA bases. The mechanism of 5-FU action is associated with the inhibition of the thymidylate synthase (TS) and incorporation of 5-FU into RNA and DNA, resulting in DNA strand breakage and decreased protein synthesis which is responsible for its cytotoxic properties [32]. The obtained results (in micro- and macrosystem) have been correlated and the correlation coefficients were ≥0.9. However we observed differences between these two methods. Our investigation shows that conditions, in which cells are cultured and analyzed, have an influence on the cytotoxicity effects of 5-FU.
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(a width) × 50 m (a depth). The microsystem was composed of PDMS – poly(dimethylsiloxane) (Dow Corning Sylgrad 184) and the sodium glass (75 mm × 55 mm × 1 mm) (Helmand). The network of microchannels, which was fabricated in PDMS, enabled the introduction of cell suspension, culture medium and tested substances. The geometry of CGG included two inlets and five outlets, which permitted testing the influence of five different 5-FU concentrations in a single assay. The photolithography and the replica molding technique were used for microchannels’ network fabrication. The photosensitive material: the capillary film (Pro/Cap 50, Chromaline) was deposited on a sodium glass plate and exposed to UV light for 2.5 min through a photomask. Then, the exposed capillary film was developed by using water and dried. The obtained stamp with microchannels’ network was utilized for the replication in PDMS. PDMS prepolymer was mixed with the curing reagent in the weight ratio 10:1. Then, the degassed liquid mixture was poured onto the prepared stamp which was cured for 1 h at 60 ◦ C. After that, the PDMS replica was peeled off from the master and 1.3 mm diameter holes for tubings were drilled. The microchambers were fabricated in a hydrophilic glass plate assuring the place for good adhesion and proliferation of adherent cells. The most important advantage of the microchambers is the assurance of minimized hydrodynamic stress caused by the medium flow over the cell culture. The microchambers were fabricated using photolithography and wet etching method in the sodium glass plate. In the first stages, the sodium glass plate was washed with solvents (according to the following sequence: distilled water, acetone and 2-propanol) and heated at 200 ◦ C for 10 min. Next, the photoresist (S1818, Micro Chemicals) was spincoated onto a clean surface of the glass and the glass plate was pre-baked at 100 ◦ C for 3 min and exposed to UV light through a photomask for 180 s. Following this procedure, the pattern was developed using the A1518 developer (Micro Chemicals) and the plate was heated at 130 ◦ C for 5 min. Next, the microchambers were etched with the mixture of the NH4 F:HF (in 6:1 ratio) for 40 min. Finally, the photoresist was removed by flushing the structure with acetone. The PDMS plate along with the microchannel network and access holes, was bonded with the glass plate using surface plasma activation (Plasma Preen System Inc. II 973). 2.3. Microfluidic cell culture
2. Materials and methods 2.1. Biological material The A549 (human lung carcinoma cell line) and HT-29 (human colon adenocarcinoma cell line) were used as the model cells for both micro- and macroscale experiments. The cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in the RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco), 2 mM glutamax (Gibco), 100 U/ml penicillin, 100 g/ml streptomycin and 250 ng/ml amphoterycin (Gibco). The cells were cultured at 37 ◦ C in a humidified atmosphere including 5% CO2 . 2.2. Microfluidic device design and fabrication Design of the hybrid (PDMS/glass) microfluidic cell culture system for the cell testing of drugs was presented in details in our previous work [5], so only a brief description will be given here. The geometry of the microsystem consists of a matrix (5 × 5) of the culture microchambers (a diameter of 1 mm, a depth of 30 m) coupled with microchannels creating the concentration gradient generator (CGG). The dimensions of microchannels were 100 m
The microfluidic system was sterilized by exposure to UV light (Black Ray) for 20 min. To sterilize tubings and minimize contamination of the microdevice the microsystem was flushed with 70 vol.% ethyl alcohol at a flow rate of 50 l/min for 20 min. Then, the culture medium was introduced into the microdevice at a flow rate of 50 l/min for 20 min to assure optimal cell growth conditions. In order to introduce all fluids and cells, syringe pumps (NE 1000 New Era Pump Systems Inc.) were used. After the culture medium introduction, the microsystem was placed in a CO2 incubator for 2 h. Next, the cell suspensions of 1 × 106 A549 cells/ml and of 3 × 106 HT-29 cells/ml were prepared and they were introduced into the microchambers at a flow rate of 15 l/min. The sealed microfluidic device was placed in the incubator at 37 ◦ C and 5% CO2 . The medium in the microchambers was replaced every day (with a rate of 1.2 l/min for 50 min) to maintain suitable conditions for the cell culture. The temperature of the microsystem was controlled by the use of the heated microscope table. 2.4. Cytotoxicity assay 2.4.1. Microdevice test The A549 and HT-29 cells were cultured in independent microsystems in order to obtain suitable density (40,000 cell/cm2 ) for the cytotoxicity tests. After that, two different solutions of the
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Fig. 1. (A) Microfluidic array for cytotoxic assays. In the microsystem two different cell lines A549 (B) and HT-29 (C) were cultured and analyzed. Magnification of B and C – 5×. CGG – concentration gradient generator; PDMS – poly(dimethylsiloxane).
5-FU (0 and 300 M) were introduced into the microchambers through the CGG inlets with a flow rate of 1.2 l/min for 50 min. Consecutive series of culture chambers achieved 5-FU concentration of 0, 75, 150, 225 and 300 M. The microdevice with introduced 5-FU was placed in the cell culture incubator for the next 24 h and in the independent experiment for 48 h. After that time, the solution of propidium iodide (PI, Sigma–Aldrich) and calcein AM (CAM, Sigma–Aldrich) was introduced for 30 min and cell viability was determined using an inverted microscope (Olympus IX-71). The calcein AM is a non-fluorescent, hydrophobic compound that easily
permeates in live cells. The hydrolysis of calcein AM by intracellular esterases produces calcein, a hydrophilic, strongly fluorescent compound that is well-retained in the cell cytoplasm. PI intercalates in nucleic acids of necrotic cells and exhibits red fluorescence. The solution of 1 l of 2 mM CAM and 50 l of 1 mg/ml PI in 0.5 ml of culture medium was used to determine viability of the cells. Images of each microchamber were prepared using a fluorescent microscope at 5× and 10× magnification. Cell viability was determined by counting the number of green objects (live cells) and red objects (dead cells) with image processing software (cellFˆ , OLYMPUS).
Fig. 2. Viability of (A) A549 and (B) HT-29 cells in the microsystem after 24 h incubation with different concentration of 5-FU. Cells were stained by the double-staining method with calcein AM and propidium iodide (PI). Green fluorescence – live cells, red fluorescence – dead cells. Magnification at 10× (Olympus IX-71). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. (A) Comparison of cell survival of A549 and HT-29 cells in the presence of 5-fluorouracil (5-FU) in two cultivation systems. In both systems the cells were incubated with drug for 24 and 48 h, and then assessed by staining with calcein AM (CAM). Each point represents the mean ± SD (n ≥ 4), asterisks indicate P < 0.05, for comparison with control. The data were normalized to the control condition (cells incubated without 5-FU) at each time point and for each cell line. The level of viability of the cells was calculated by equation: number cells or OD (with drug)/number cells or OD (without drug–control) × 100%. In macroscale methods blank controls, for each drug concentrations, were also determined and subtract from appropriate OD values. (B) The control condition (cells incubated without 5-FU) at each time point and for each cell line are shown.
2.4.2. Calcein AM assay in macrosystem The cells were incubated with 5-fluorouracil (Sigma, St. Louis, MI, USA) for 24 and 48 h. Drug concentrations ranged from 75 to 300 M. 5-FU was dissolved in 100% dimethylsulfoxide (DMSO; Sigma) and then diluted with the medium for further experiments. In all experiments, control cells were incubated in the medium supplemented with DMSO alone. The final concentration of DMSO was maintained at 0.2% which had no effect on cellular growth and survival. The cells were seeded in black-walled 96-well plates at a density of 1 × 104 /well. After 24 h, growing cells attached to the bottom, were treated with different concentrations of freshly prepared test compounds in a complete medium (three wells per concentration) for 24 and 48 h. After incubation with the reagent, the medium was removed and the cells were treated with 50 l of CAM (2 M) for 30 min at 37 ◦ C. Following two washes in phosphate buffered
saline (PBS) the culture medium without phenol red was added. Next, the green fluorescence was measured with the fluorescence plate reader Ascent (Thermo Scientific, USA) at 485 nm (excitation) and 538 nm (emission). 2.5. Gas chromatography The same microsystem was used for cytotoxicity tests several times for the same cell line and the same time incubation. This was possible, as highly effective recovery and the reuse protocol for the microdevice was developed. The absorption of 5-FU in PDMS block (after several tests performed in the same microsystem) was measured, according to previously elaborated procedure [5]. Gas chromatography (GC) with a flame ionization detector (FID) was used to evaluate the level of absorption and the concentration of the absorbed 5-FU in PDMS block.
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in the microchambers. Fig. 1A shows the designed microsystem, which was used for two different cell line cultures and analysis. Fig. 1B and C shows that the cells A549 and HT-29 have adhered very well to the growth surface of the microchamber. The two cell lines selected for the investigation were good models because they represented two different types of cell growth and morphology. In order to remove the old medium and provide new nutrient substances for the cells, fresh culture medium (described in the experiment section) was introduced. All parameters concerning the preparation of the microsystem for the cell culture were previously optimized. 3.2. Cytotoxicity assay
Fig. 4. The data demonstrate correlation between macro- and microsystem, two different methods of cell cultivation, for measuring cell viability. A549 and HT-29 cells were incubated with 5-FU for 24 and 48 h. Each point represents the mean ± SD (n ≥ 4). The grey lines represent the linear regression R2 > 0.9.
2.6. Statistical analysis Experimental data are expressed as mean ± standard deviation (SD) from at least four independent experiments. Statistical significance was evaluated using one-way analysis of variance (ANOVA) followed by post hoc Tucky’s test. The regression analysis was performed to compare the results (SigmaPlot, Systat Software Inc, USA). P < 0.05 was considered as a statistically significant. 3. Results and discussion 3.1. Cell culture in the microsystem In this paper, we present A549 and HT-29 cell lines cultured in the microfluidic system. HT-29 cell line was derived from a different organ and its cells represent a dissimilar character of growth in comparison with A549 cells. HT-29 cells create characteristic clusters, whereas A549 cells steadily colonize the growth surface. The successful culture with both cell lines was performed in the microsystem. The conditions created in the microsystem allowed the adhesion and proliferation of both cultured cell lines. The suspension of both cell lines and the flow rate used for cell docking in the microchambers allows the introduction of the cells to each culture microchamber in a sufficient amount for future cultures. Moreover, seeded cell density allows the maintenance of cellular interactions so as to ensure proper cell growth. A549 and HT-29 cells adhesion and proliferation were observed within 24 h after seeding. The geometry of the microsystem and the applied procedures did not cause hydrodynamic stress to the cells cultured
Microfluidic cell culture is a promising technology for applications in the drug screening industry. Microfluidic systems can be used in the future as alternative tools for cell analysis and the analysis of new compounds. The key benefits include biological function improvement, higher quality cell-based data, reduced reagent consumption and lower costs. This investigation demonstrated how microfluidic cell culture design may be adopted as a method compatible with the standard 96-well plate format (macrosystem) [33]. Fig. 2 shows representative results of the cell viability test performed in the various microsystems. The toxic effect of 5-FU on the A549 and HT-29 cells was determined by CAM and PI. For each row of the microchambers matrix, a specific concentration of 5-FU was obtained. Viability of the cells, cultured in the microchambers, exposed to a gradient perfusion of 5-FU decreased with a higher concentration of the drug. The number of dead cells (red objects) after 24 h incubation with 5-FU increase with the drug concentration. 5-FU tested at concentrations ranging from 75 to 300 M, inhibited the survival of both cell types. However, HT-29 cells were less sensitive to the cytotoxic agent than A549 cells in both incubation times (i.e. 24 h and 48 h). The specific morphology of two different cell lines may be one of the reasons for these differences. HT-29 cells form specific clusters (Fig. 1C), therefore the compounds cannot be evenly distributed to all cells. However, A549 cells which adhere to the glass one by one were in direct contact with the tested compound. Moreover, the cells used in experiments have a different genetic status of genes. HT-29 cells acquire resistance to the new conditions (in this case to the cytostatic drug) more rapidly than A549 cells. This is the most likely cause of various cytostatic actions of 5-FU for these two cell lines. Generally, the inhibitory effect of 5-FU was dose- and timedependent. The strongest inhibition, approaching 80% after 48 h incubation, was observed for A549 cells exposed to 300 M 5-FU. The results of the cells viability were compared with the tests performed in static conditions using 96-well plates. Using this method, HT-29 cells were also less sensitive to 5-FU than A549 cells. In Fig. 3A the data obtained from at least four independent experiments are shown. In one experiment, the same concentration was analyzed five times in the microsystem – in one row of microchambers and three times in 96-well plate – in three wells. In Fig. 3B the control conditions (cells incubated without 5-FU) at each time point and for each cell line are shown. The result obtained in both systems (i.e. micro- and macrosystem) of cell cultivation (Fig. 3A) has a similar dependence of concentration of 5-FU and time incubation with the drug. But in each test, differences between both systems were observed. For example, the cell survival rate for the HT-29 line after 48 h incubation with 300 M 5-FU decreased to 65% and 50% in the micro- and macrosystem, respectively. The cytotoxic effects on cells in the macroscale were shown to be lower by about 10–20% than in the microscale, with the exception of A549 cells incubated at 75 M for 48 h period. For each cell line in both times of incubation and both methods (micro and macro)
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Table 1 The viability of A549 an HT-29 cells (percent ratio green to all cells) for four experiments after incubation with different time performed in the microsystems. A549 24h Micro1 Micro2 Micro3 Micro4
Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) A549 48h
Micro1 Micro2 Micro3 Micro4
Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility)
0 µM 94.39 3.55 91.28 3.64 98.19 5.64 95.70 5.33
75 µM 78.60 4.76 81.50 5.14 80.30 2.68 75.38 3.42
150 µM 54.75 13.70 73.82 3.12 76.34 6.56 74.35 7.43
225 µM 39.11 5.88 56.06 8.02 33.84 7.86 61.63 5.16
300 µM 21.02 3.23 49.35 18.04 20.31 1.34 53.55 9.43
0 µM 90.45 3.48 94.00 8.49 98.48 3.48 93.55 4.02
75 µM 66.42 12.78 80.00 8.60 75.56 12.78 56.56 5.63
150 µM 57.96 1.72 43.87 12.26 50.46 1.72 43.00 7.40
225 µM 46.06 3.61 47.28 6.19 36.94 3.61 22.29 4.27
300 µM 22.41 9.94 33.3 17.32 30.44 9.94 18.56 7.13
the IC50 were calculated. For A549 cells IC50 were determined as follows 251 M (24 h), 181 M of 5-FU (48 h) in the microsystem and 210 M (24 h) and 38.4 M of 5-FU (48 h) in the macrosystem. For HT-29 cells it equals: 810 M (24 h), 400 M of 5-FU (48 h) in the microsystem and 437.5 M (24 h) and 318 M of 5-FU (48 h) in the macrosystem. The obtained results indicate that, from a cellular perspective, the microenvironment in microfluidic cultures can be significantly different from those in traditional macroscale cultures. However, the results of regression analysis (performed between the data of cell viability) indicate significant correlation of 5-FU cytotoxicity between two evaluated systems because Pearson’s correlation coefficients (R2 ) were ≥0.9 (Fig. 4). Despite the observed differences between micro and macroscale, the developed system could be an alternative tool for the cell analysis. Here we investigated a few concentrations of 5-FU on two cancer cell lines. Both cell lines have shown dose-dependent effect. The drug concentrations used in our experiments allowed to compare macro- and microsystem as well as showed differences between the culture conditions. In the future experiments, higher number of drug concentrations are to be tested. It will allow for better referencing and better resolved dose/response functions. The geometry of the microsystem enables to obtain more concentrations by the addition of CGG single module. Moreover, using the developed microsystem it is possible to test more drug concentrations without the change of CGG geometry, but only by using of other range of concentration introduced to the microdevice. For each new-tested cell line or drug validation step is important for the evaluation of the results in comparison to conventional test procedure. We focused our investigation on the reason for these differences. Most likely, the discrepancy between the two systems could result from the differences in the cell culture conditions and different access of the anticancer drug to cells cultivated in the macrosystem. Tests performed on the plates were carried out in static conditions, while in the microsystem the microcultures were rinsed with medium, drugs and solution of CAM and PI. We observed that the flow exposure of a toxicant could affect the cells in a different manner relative to static exposure conditions, depending on the cell types and physicochemical properties of the toxicant. The flow conditions simulate the provision of drugs and substances into a living body, therefore the microfluidic device may be assumed as a quite simple model more resembling of in vivo conditions, which are not properly simulated by 96-well plates. The differences between micro and macroscale were observed by various groups. For example Beebe et al. tested three culture devices: 96 well plates, microwells and microchannels, which represent a potential culture platform that produces different
HT29 24h Micro1 Micro2 Micro3 Micro4
Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) HT29 48h
Micro1 Micro2 Micro3 Micro4
Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility)
0 µM 97.11 2.54 98.01 1.12 97.92 2.39 97.04 3.28
75 µM 86.46 5.56 90.23 4.49 90.50 4.37 88.22 3.61
150 85.20 0.73 90.98 5.45 90.39 6.03 84.95 7.47
225 µM 81.43 5.48 88.68 3.44 91.62 2.46 79.99 0.40
300 µM 72.63 8.56 90.99 1.63 90.94 0.25 71.19 0.45
0 µM 98.83 0.65 97.50 6.43 98.18 2.48 94.81 3.36
75 µM 84.66 5.13 89.60 2.42 77.42 18.64 61.42 5.18
150 µM 76.87 4.52 84.80 5.95 73.50 18.83 53.96 5.90
225 µM 75.86 3.85 77.30 4.95 67.34 16.11 54.29 8.63
300 µM 69.92 4.04 76.80 0.93 57.01 11.63 58.28 5.46
microenvironments. They observed various proliferations, glucose metabolism, signaling pathway activation and protein expression levels between cells cultured in traditional macroscale cultures and in microfluidic cultures [34]. Cooksey et al. described the study of the performance and variability of a cell-based toxicity assay in microfluidic devices. The authors quantified reduction in cellular green fluorescent protein (GFP) due to inhibition of ribosome activity by cycloheximide (CHX) [35]. The results were compared with macroscale and significant changes in GFP in the microfluidic chamber than in the dishes were faster detectable. The results indicate that from a cellular perspective, the microenvironment in microfluidic cultures can be significantly different from those in traditional macroscale cultures. Microfluidic devices for cell based assays have provided new types of microenvironments and new methods for controlling and observing the cellular responses. The results obtained in the microsystem allow to study and observe influence of the change of culture microenvironment on the cells. However, the results are dependent on the tested cell lines, density of culture and the time of incubation with drug. 3.3. Replication and reproducibility of microfluidic system The same microsystem was used for cytotoxicity tests several times for the same cell line and the same time incubation. We analyzed replication and reproducibility of the microsystem with parallel chambers for replicate measurements. The values shown in Figs. 3 and 4 (concerned microsystems) were obtained for each concentration from five microchambers in one device and at least four independent experiments. In Table 1, the viability of A549 an HT-29 cells (percent ratio green to all cells) for four experiments after incubation with different time is presented. SD shows the reproducibility of viability in the column of microchambers with the same concentration of the drug in one microsystem. The low differences between microchambers in the same concentration were observed. Moreover gas chromatography was used to evaluation of cytotoxic drug absorption in the block of polymer of microdevice. 5-FU used to test cytotoxicity could be absorbed in a PDMS block, which was utilized to the microsystem fabrication. Although procedures of recovery and reuse were performed, the absorbed 5-FU could have influence on research results. The level of 5-FU absorption in PDMS block was investigated using a gas chromatography. In the chromatogram of standard solution (Fig. 5A), there are peaks corresponding to 5-FU (time 1.90 min) and DMSO (time 1.19 min). In the tested sample, DMSO used for the extraction of absorbed substances from PDMS parts of the microsystem contained only DMSO
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Fig. 5. Gas chromatogram of 5-FU extracted by DMSO from PDMS. Standard solution (A) and the sample (B).
(time 1.19 min) (Fig. 5B). The PDMS block did not absorb the 5-FU, so usage of the same microchip for several cytotoxicity tests did not have cytotoxic influence on the subsequent cultures performed in the same microfluidic system. 4. Conclusions In this paper, we described the use of the microfluidic device for testing the toxicity of 5-fluorouracil, commonly used as an anticancer agent, and discuss its results in contrast to the traditional cytotoxicity assays performed in 96-well plates (macrosystem). The results show that the cells in macroscale are much more sensitive to the tested 5-fluorouracil than the cells tested in microscale. Although differences between the cytotoxicity effect of 5-FU on the cells in both culture conditions were observed, the regression analysis indicated a significant correlation of 5-FU cytotoxicity. Therefore, it can be concluded that the conditions of cultivation may affect the level of cytotoxicity of drugs and the microfluidic system can be an alternative tool for performing the cytotoxicity assays. Additionally, it was shown that the cells with different characteristics of growth can be cultivated in such a microdevice. The viability of two different cell lines (i.e. A549 and HT-29) was tested. We proved that the inhibitory effect of 5-FU as well as type of the cell line were both time and drug concentration dependable. The designed geometry of the microsystem enabled us to test different concentrations of 5-FU in a single step. This microfluidic system enables the performance of various toxicological examinations such as cell viability, cell death and real time imaging. Our results may be useful for biological and medical research. Creation of automatic administration of tested substances will allow the application of the microsystem in a laboratory as a userfriendly tool. In the future, the designed microsystem may be used for rapid, preliminary evaluation of toxic influence of tested compounds with a potential cytotoxic activity. This system can also be used in the first stages of drugs screening. Additionally, it can also be used to test the synergistic effect of chemotherapeutic agents, which will allow the reduction of time and costs of analyses. Acknowledgements The authors would like to thank Anna Jerzak from the Laboratory of Technological Processes (WUT) for GC measurement. This work was financially supported by Warsaw University of Technology.
References [1] G.A. Silvestri, M.P. Rivera, Chest 128 (2005) 3975–3984. [2] R. Labianca, S. Mosconi, M.C. Garassino, Ann. Oncol. 17 (2006) 51–54. [3] R. Napieralski, K. Ott, M. Kremer, K. Becker, A.L. Boulesteix, F. Lordick, J.R. Siewert, H. Höfler, G. Keller, Clin. Cancer Res. 13 (2007) 5095–5102. [4] Y. Kodera, S. Ito, M. Fujiwara, Y. Mochizuki, N. Ohashi, Y. Ito, G. Nakayama, M. Koike, Y. Yamamura, A. Nakao, Int. J. Clin. Oncol. 11 (2006) 449–453. [5] K. Ziolkowska, E. Jedrych, R. Kwapiszewski, J. Lopacinska, M. Skolimowski, M. Chudy, Sens. Actuators B 145 (2010) 533–542. [6] M.H. Wu, S.B. Huang, G.B. Lee, Lab Chip 10 (2010) 939–956. [7] B.H. Weig, R.L. Bardell, C.R. Cabrera, Adv. Drug Deliv. Rev 55 (2003) 349–377. [8] J. Pihl, M. Karlsson, D.T. Chiu, Drug Discov. Today 10 (2005) 1377–1383. [9] J. El-Ali, P.K. Sorger, K.F. Jensen, Nature 442 (2006) 403–441. [10] R. Baudoin, A. Corlu, L. Griscom, C. Legallais, E. Leclerc, Toxicol. In Vitro 21 (2007) 535–544. [11] L. Kang, B.G. Chung, R. Langer, A. Khademhosseini, Drug Discov. Today 13 (2008) 1–13. [12] G.M. Walker, H.C. Zeringue, D.J. Beebe, Lab Chip 4 (2004) 91–97. [13] J.H. Sung, M.L. Shuler, Bioprocess. Biosyst. Eng. 33 (2010) 5–19. [14] J.H. Qin, N.N. Ye, X. Liu, B.C. Lin, Electrophoresis 26 (2005) 3780–3788. [15] M.J. Powers, K. Domansky, M.R. Kaazempur-Mofrad, A. Kalezi, A. Capitano, A. Upadhyaya, P. Kurzawski, K.E. Wack, D.B. Stolz, R. Kamm, L.G. Geiffith, Biotechnol. Bioeng. 78 (2002) 257–269. [16] Ch. Yi, Ch.W. Li, S. Ji, M. Yang, Anal. Chim. Acta 560 (2006) 1–23. [17] A.Y. Fu, H.P. Chou, C. Spence, F.H. Arnold, S.R. Quake, Anal. Chem. 74 (2002) 2451–2457. [18] D.M. Thompson, K.R. King, K.J. Wieder, M. Toner, M.L. Yarmush, A. Jayaramen, Anal. Chem. 76 (2004) 4098–4103. [19] Zhang Ch, Z. Zhao, N.A. Abdul Rahim, D. Noort, H. Yu, Lab Chip 9 (2009) 3185–3192. [20] J. Komen, F. Wolbers, H.R. Franke, H. Andersson, I. Vermes, A. Berg van den, Biomed. Microdevices 10 (2008) 727–737. [21] N.L. Jeon, S. Dertinger, D.T. Chiu, I. Choi, A. Stroock, G. Whiteside, Langmuir 16 (2000) 8311–8316. [22] A. Funfak, J. Cao, O.S. Wolfbeis, K. Martin, J.M. Kohler, Mikrochim. Acta 164 (2009) 279–286. [23] A. Huebner, S. Sharma, M. Srisa-Art, F. Hollfelder, J.B. Edel, A.J. deMello, Lab Chip 8 (2008) 1244–1254. [24] W. Liu, H.J. Kim, E.M. Lucchetta, W. Du, R.F. Ismagilov, Lab Chip 9 (2009) 2153–2162. [25] A. Funfak, R. Hartung, J. Cao, K. Martin, K.H. Wiesmüller, O.S. Wolfbeis, J.M. Kohler, Sens. Actuators B 142 (2009) 66–72. [26] A. Funfak, J. Cao, A. Knauer, K. Martin, J.M. Kohler, J. Environ. Monit. 13 (2011) 410–415. [27] A. Tirella, M. Marano, F. Vozzi, A. Ahluwalia, Toxicol. In Vitro 22 (2008) 1957–1964. [28] H. Bang, W.G. Lee, H. Yun, Ch. Chung, J.K. Chang, D.Ch. Han, Microsyst. Technol. 14 (2008) 719–724. [29] Y.Ch. Toh, T.Ch. Lim, D. Tai, G. Xiao, D. Noort, H. Yu, Lab Chip 9 (2009) 2026–2035. [30] D.B. Longley, D.P. Harkin, P.G. Johnston, Nat. Rev. Cancer 3 (2003) 330–338. [31] N. Zhang, Y. Yin, S.J. Xu, W.S. Chen 13, Molecules (2008) 1551–1569. [32] P. Noordhuis, U. Holwerda, C.L. Van der Wilt, C.J. Van Groeningen, K. Smid, S. Meijer, H.M. Pinedo, G.J. Peters, Ann. Oncol. 15 (2004) 1025–1032. [33] P.J. Lee, N. Ghorashian, T.A. Gaige, P.J. Hung, JALA 12 (2007) 363–367.
E. Jedrych et al. / Sensors and Actuators B 160 (2011) 1544–1551 [34] A.L. x, D.J. Beebe, Int. Biol. 1 (2009) 182–195. [35] G.A. Cooksey, J.T. Elliott, A.L. Plant, Anal. Chem. 83 (2011) 3890–3896.
Biographies Elzbieta Jedrych was born in Poland in 1984. She received her MSc in chemical technology from the Department of Chemistry, Warsaw University of Technology (WUT), Poland in 2008. Currently she is PhD student in chemistry at the Department of Microbioanalytics, where she is a member of Chemical Sensors Research Group. Her current research interests are: designing and manufacturing of microfluidic Lab-on-a-Chip systems, developing of microsystems for cell culture and analysis. Sylwia Flis was born in Poland. She received her MSc in biology from the Warsaw University, Poland in 1998 and PhD in science of medicine at Institute of Pharmacology PAS in 2005. Currently she is PhD in science of medicine at the National Medicines Institute, Warsaw, Department of Pharmacology, Poland. Her current research interests are: biochemistry, genetics and molecular biology, medicine pharmacology, toxicology and pharmaceutics. ´ Kamila Sofinska was born in Poland in 1986. She is master student in chemical biotechnology at the Faculty of Chemistry, Warsaw University of Technology (WUT). Her current interest is “Lab-on-a-Chip” systems applications, such as cell engineering and medical diagnosis. Zenon Jastrzebski was born in Poland in 1944. He received his the veterinary doctor in Warsaw University of Life Sciences (SGGW), Poland in 1968, and doctor of agricultural sciences from the Institute of Genetics and Animal Breeding Polish Academy of Sciences in 1997. Currently he has been a head at the National Medicines Institute, Warsaw, Department of Pharmacology, Poland. His current research interests are: biochemistry, genetics and molecular biology agricultural and biological sciences, medicine, chemistry.
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Michal Chudy was born in Poland in 1973. He received his MSc in chemical technology from the Department of Chemistry, Warsaw University of Technology (WUT), Poland in 1997, and PhD in chemistry from the same faculty in 2001. Currently he has been an assistant professor at the Department of Microbioanalytics, where he is a member of Chemical Sensors Research Group. His current research interests are: various chemical sensors (ISE, ISFETs, CHEMFETs, SSE), designing and manufacturing of microfluidic Lab-on-a-Chip systems, developing new analytical methods for medical and bioanalytical application. Artur Dybko received his PhD in optoelectronics from the Faculty of Electronics, Warsaw University of Technology (WUT), Poland in 1996, and DSc in metrology from the same faculty in 2002. In 1990 he joined an optoelectronic company Sensomed, Poland as research and development manager. Since 1992 he started his PhD studies at the Faculty of Electronics (WUT). In 1996 he was employed as an assistant professors at the Faculty of Chemistry, Department of Analytical Chemistry (WUT). Currently he has been a university professor at the Department of Microbioanalytics, where he is a member of Chemical Sensors Research Group. His current research interests are: fibre optic chemical sensors, electrochemical sensors (ionselective electrodes, ion-sensitive field effect transistors), microfluidic structures for application in mTAS, low noise electronic circuits, application of computers in measurements. Zbigniew Brzozka obtained his PhD (1982) and his DSc (1991) from the Warsaw University of Technology (WUT). In 1998 he was appointed full professor of analytical chemistry in the Department of Analytical Chemistry WUT. He is head of the Chemical Sensor Research Group (CSRG). His current fields of interest are chemical membrane sensors, ion recognition, ion monitoring in clinical and environmental protection, and miniaturized analytical devices (-TAS). Among other areas, emphasis is now also being laid on the applications of polymer microfabrication technologies to microchemical analysis, such as Lab-on-a-Chip and (bio)electrochemical systems based on chemical reactions and/or various type of detection principle.
Contents lists available at SciVerse ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Evaluation of cytotoxic effect of 5-fluorouracil on human carcinoma cells in microfluidic system E. Jedrych a,∗ , S. Flis b , K. Sofinska a , Z. Jastrzebski b , M. Chudy a , A. Dybko a , Z. Brzozka a a b
Department of Microbioanalytics, Institute of Biotechnology, Warsaw University of Technology, Poland Department of Pharmacology, National Medicines Institute, Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 6 April 2011 Received in revised form 29 June 2011 Accepted 27 August 2011 Available online 6 September 2011 Keywords: Cytotoxic tests Microfluidic system Lab-on-a-chip Cancer treatment Cell culture 5-Fluorouracil
a b s t r a c t In this paper, we present cytotoxicity tests performed in a microanalytical hybrid system. The microchambers of cell culture were integrated with a concentration gradient generator (CGG), which created five different concentrations of evaluated agent in a single step. 5-Fluorouracil (5-FU) is one of the most important chemotherapeutic agents for cancer treatment, therefore it was tested in the microsystem. Independent tests of the cytotoxic effect of 5-FU were performed in this microsystem on different cell lines (human lung carcinoma cells – A549 and human colon carcinoma cells – HT-29). We also present the dependence of cell viability at different times of incubation with 5-FU. We proved that 5-FU has a stronger inhibition effect on A549 than on HT-29 cells. Moreover, the results obtained in the microfluidic system were compared with that of a macroscale. It can be concluded that the conditions of cultivation can affect the level of cytotoxicity of drugs, and the microsystem is a good example that could be used in this field of research. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Non-small cell lung carcinoma (NSCLC) and colorectal cancer (CRC) are one of the most common worldwide types of cancers which develop slowly, sometimes without any symptoms for many years. Therefore, in diagnosis both diseases are often detected at an advanced stage while tumours are inoperable and/or patients have metastasis [1,2]. In such cases, chemotherapy is the first-line treatment but unfortunately it is still unsatisfactory. Differences in a positive response to chemotherapy may depend on many factors such as tumour size, the number and the size of metastases or the gene profile (changes in the gene expression as a result of their mutation or methylation) [3]. Several approaches have been suggested to optimize chemotherapeutic treatment of NSCLC or CRC patients. One approach is to test the chemosensitivity of tumour cells in vitro to predict better the clinical response to drugs [4]. A limitation of such a high-throughput screening is availability and the volume of biological samples. In vitro tests (using 96-well plates) are unsatisfactory, as this method differs from the in vivo in several aspects, for example, there are more complicated interactions between cell–cell and cell–intercellular matrix in in vivo systems. This problem may be solved by using microfluidic cell culture system (MCCS; microsystem) as a promising alternative to the conventional cell culture methods (macrosystem) [5].
∗ Corresponding author. E-mail address: [email protected] (E. Jedrych). 0925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2011.08.074
Microsystems (called lab-on-a-chip) have started to play an important role in cell biology and drug development [6–11]. The ability of microdevice geometry modification gives the possibility of growth factors, reagents and oxygen control inside the system, as well as hydrodynamic shear stress upon the cultured cells [12,13]. The usage of microfluidic systems allows to reduce the consumption of solvents and reagents. This is important for drug research where the tested drug and the volume of biological samples are limited [14,15]. Besides, the scale of microchannels corresponds well with the native cellular microenvironment, so microfluidic devices are suitable especially for biological applications particularly at the cellular level [16]. Moreover, a flow of medium and tested substances through microfluidic systems can simulate in vivo conditions. The integration of microsystem with control elements allows the automation of investigations such as cell culture, cell sorting, gene expression, biochemical separation and cytotoxicity tests [17–20]. Cytotoxicity tests are performed in the microsystems using the concentration generator gradient (CGG) [21]. Besides of this technique, pipetting in nanotiterplates or micro segmented flow techniques are also often used [22–25]. All these methods enable to handle small fluid volumes, reduced instrumental materials and keep low unit cost. Microfluidic systems enable an integration of functional components and the utilization of fluid dynamics to control molecules in both time and space. The general differences between CGG and microdroplets concern the profile of fluid flow [23]. A fluid flow through the microchannels creates parabolic velocity profile over the cross-section of the channel. Whereas, localization of reagents in the droplets is an effective
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way of eliminating this phenomenon and allow to specify reaction and incubation times. Application of microdroplet enables to reduce evaporation. Moreover it allows good mixing by segmentinternal convection [26]. However, the mixing by using microdroplets is more complicated than the use of two streams of liquid flow. The CGG creates a few different concentrations in a single step and allows to skip the step of preparing of many solutions used in the investigation. Pipetting procedures are replaced by drugs’ flow, which is controllable and simulate to human organism conditions. The microdevices with different geometry of CGG were used for the testing: bupivacaine and lidocaine on the mouse myoblast cells [27], cellular responses to hydrogen peroxide on the HeLA cells [28]. Toh et al. used a microfluidic system for hepatotoxicity of 5 model drugs (acetaminophen, diclofenac, quinidine, rifampin and ketoconazole) [29]. Creating a program for automatic administration of the substance will allow the simple and user-friendly usage of a microchip to cytotoxicity tests. Due to the fact that the miniaturization dedicated to cell analysis research offers many advantages, so the microfluidic system can be a better method than research in the macroscale. The aim of our study was to perform a series of cytotoxicity assays in a PDMS/glass microfluidic system using two human cancer cell lines. 5-Fluorouracil (5-FU) was used as one of the most popular and best known chemotherapeutic agents [30,31]. Moreover, 5-FU can also be used for tests of the synergistic effect with various drugs, which are significant for antitumour treatment. In the literature, we did not find tests performed in a microscale, where the 5-FU cytotoxic effect was investigated on the A549 (human lung carcinoma cell line) and HT-29 (human colon adenocarcinoma cell line). 5-FU like other pyrimidine antagonists is similar in structure to uracil, one of the four naturally occurring RNA bases. The mechanism of 5-FU action is associated with the inhibition of the thymidylate synthase (TS) and incorporation of 5-FU into RNA and DNA, resulting in DNA strand breakage and decreased protein synthesis which is responsible for its cytotoxic properties [32]. The obtained results (in micro- and macrosystem) have been correlated and the correlation coefficients were ≥0.9. However we observed differences between these two methods. Our investigation shows that conditions, in which cells are cultured and analyzed, have an influence on the cytotoxicity effects of 5-FU.
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(a width) × 50 m (a depth). The microsystem was composed of PDMS – poly(dimethylsiloxane) (Dow Corning Sylgrad 184) and the sodium glass (75 mm × 55 mm × 1 mm) (Helmand). The network of microchannels, which was fabricated in PDMS, enabled the introduction of cell suspension, culture medium and tested substances. The geometry of CGG included two inlets and five outlets, which permitted testing the influence of five different 5-FU concentrations in a single assay. The photolithography and the replica molding technique were used for microchannels’ network fabrication. The photosensitive material: the capillary film (Pro/Cap 50, Chromaline) was deposited on a sodium glass plate and exposed to UV light for 2.5 min through a photomask. Then, the exposed capillary film was developed by using water and dried. The obtained stamp with microchannels’ network was utilized for the replication in PDMS. PDMS prepolymer was mixed with the curing reagent in the weight ratio 10:1. Then, the degassed liquid mixture was poured onto the prepared stamp which was cured for 1 h at 60 ◦ C. After that, the PDMS replica was peeled off from the master and 1.3 mm diameter holes for tubings were drilled. The microchambers were fabricated in a hydrophilic glass plate assuring the place for good adhesion and proliferation of adherent cells. The most important advantage of the microchambers is the assurance of minimized hydrodynamic stress caused by the medium flow over the cell culture. The microchambers were fabricated using photolithography and wet etching method in the sodium glass plate. In the first stages, the sodium glass plate was washed with solvents (according to the following sequence: distilled water, acetone and 2-propanol) and heated at 200 ◦ C for 10 min. Next, the photoresist (S1818, Micro Chemicals) was spincoated onto a clean surface of the glass and the glass plate was pre-baked at 100 ◦ C for 3 min and exposed to UV light through a photomask for 180 s. Following this procedure, the pattern was developed using the A1518 developer (Micro Chemicals) and the plate was heated at 130 ◦ C for 5 min. Next, the microchambers were etched with the mixture of the NH4 F:HF (in 6:1 ratio) for 40 min. Finally, the photoresist was removed by flushing the structure with acetone. The PDMS plate along with the microchannel network and access holes, was bonded with the glass plate using surface plasma activation (Plasma Preen System Inc. II 973). 2.3. Microfluidic cell culture
2. Materials and methods 2.1. Biological material The A549 (human lung carcinoma cell line) and HT-29 (human colon adenocarcinoma cell line) were used as the model cells for both micro- and macroscale experiments. The cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and were grown in the RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco), 2 mM glutamax (Gibco), 100 U/ml penicillin, 100 g/ml streptomycin and 250 ng/ml amphoterycin (Gibco). The cells were cultured at 37 ◦ C in a humidified atmosphere including 5% CO2 . 2.2. Microfluidic device design and fabrication Design of the hybrid (PDMS/glass) microfluidic cell culture system for the cell testing of drugs was presented in details in our previous work [5], so only a brief description will be given here. The geometry of the microsystem consists of a matrix (5 × 5) of the culture microchambers (a diameter of 1 mm, a depth of 30 m) coupled with microchannels creating the concentration gradient generator (CGG). The dimensions of microchannels were 100 m
The microfluidic system was sterilized by exposure to UV light (Black Ray) for 20 min. To sterilize tubings and minimize contamination of the microdevice the microsystem was flushed with 70 vol.% ethyl alcohol at a flow rate of 50 l/min for 20 min. Then, the culture medium was introduced into the microdevice at a flow rate of 50 l/min for 20 min to assure optimal cell growth conditions. In order to introduce all fluids and cells, syringe pumps (NE 1000 New Era Pump Systems Inc.) were used. After the culture medium introduction, the microsystem was placed in a CO2 incubator for 2 h. Next, the cell suspensions of 1 × 106 A549 cells/ml and of 3 × 106 HT-29 cells/ml were prepared and they were introduced into the microchambers at a flow rate of 15 l/min. The sealed microfluidic device was placed in the incubator at 37 ◦ C and 5% CO2 . The medium in the microchambers was replaced every day (with a rate of 1.2 l/min for 50 min) to maintain suitable conditions for the cell culture. The temperature of the microsystem was controlled by the use of the heated microscope table. 2.4. Cytotoxicity assay 2.4.1. Microdevice test The A549 and HT-29 cells were cultured in independent microsystems in order to obtain suitable density (40,000 cell/cm2 ) for the cytotoxicity tests. After that, two different solutions of the
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Fig. 1. (A) Microfluidic array for cytotoxic assays. In the microsystem two different cell lines A549 (B) and HT-29 (C) were cultured and analyzed. Magnification of B and C – 5×. CGG – concentration gradient generator; PDMS – poly(dimethylsiloxane).
5-FU (0 and 300 M) were introduced into the microchambers through the CGG inlets with a flow rate of 1.2 l/min for 50 min. Consecutive series of culture chambers achieved 5-FU concentration of 0, 75, 150, 225 and 300 M. The microdevice with introduced 5-FU was placed in the cell culture incubator for the next 24 h and in the independent experiment for 48 h. After that time, the solution of propidium iodide (PI, Sigma–Aldrich) and calcein AM (CAM, Sigma–Aldrich) was introduced for 30 min and cell viability was determined using an inverted microscope (Olympus IX-71). The calcein AM is a non-fluorescent, hydrophobic compound that easily
permeates in live cells. The hydrolysis of calcein AM by intracellular esterases produces calcein, a hydrophilic, strongly fluorescent compound that is well-retained in the cell cytoplasm. PI intercalates in nucleic acids of necrotic cells and exhibits red fluorescence. The solution of 1 l of 2 mM CAM and 50 l of 1 mg/ml PI in 0.5 ml of culture medium was used to determine viability of the cells. Images of each microchamber were prepared using a fluorescent microscope at 5× and 10× magnification. Cell viability was determined by counting the number of green objects (live cells) and red objects (dead cells) with image processing software (cellFˆ , OLYMPUS).
Fig. 2. Viability of (A) A549 and (B) HT-29 cells in the microsystem after 24 h incubation with different concentration of 5-FU. Cells were stained by the double-staining method with calcein AM and propidium iodide (PI). Green fluorescence – live cells, red fluorescence – dead cells. Magnification at 10× (Olympus IX-71). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 3. (A) Comparison of cell survival of A549 and HT-29 cells in the presence of 5-fluorouracil (5-FU) in two cultivation systems. In both systems the cells were incubated with drug for 24 and 48 h, and then assessed by staining with calcein AM (CAM). Each point represents the mean ± SD (n ≥ 4), asterisks indicate P < 0.05, for comparison with control. The data were normalized to the control condition (cells incubated without 5-FU) at each time point and for each cell line. The level of viability of the cells was calculated by equation: number cells or OD (with drug)/number cells or OD (without drug–control) × 100%. In macroscale methods blank controls, for each drug concentrations, were also determined and subtract from appropriate OD values. (B) The control condition (cells incubated without 5-FU) at each time point and for each cell line are shown.
2.4.2. Calcein AM assay in macrosystem The cells were incubated with 5-fluorouracil (Sigma, St. Louis, MI, USA) for 24 and 48 h. Drug concentrations ranged from 75 to 300 M. 5-FU was dissolved in 100% dimethylsulfoxide (DMSO; Sigma) and then diluted with the medium for further experiments. In all experiments, control cells were incubated in the medium supplemented with DMSO alone. The final concentration of DMSO was maintained at 0.2% which had no effect on cellular growth and survival. The cells were seeded in black-walled 96-well plates at a density of 1 × 104 /well. After 24 h, growing cells attached to the bottom, were treated with different concentrations of freshly prepared test compounds in a complete medium (three wells per concentration) for 24 and 48 h. After incubation with the reagent, the medium was removed and the cells were treated with 50 l of CAM (2 M) for 30 min at 37 ◦ C. Following two washes in phosphate buffered
saline (PBS) the culture medium without phenol red was added. Next, the green fluorescence was measured with the fluorescence plate reader Ascent (Thermo Scientific, USA) at 485 nm (excitation) and 538 nm (emission). 2.5. Gas chromatography The same microsystem was used for cytotoxicity tests several times for the same cell line and the same time incubation. This was possible, as highly effective recovery and the reuse protocol for the microdevice was developed. The absorption of 5-FU in PDMS block (after several tests performed in the same microsystem) was measured, according to previously elaborated procedure [5]. Gas chromatography (GC) with a flame ionization detector (FID) was used to evaluate the level of absorption and the concentration of the absorbed 5-FU in PDMS block.
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in the microchambers. Fig. 1A shows the designed microsystem, which was used for two different cell line cultures and analysis. Fig. 1B and C shows that the cells A549 and HT-29 have adhered very well to the growth surface of the microchamber. The two cell lines selected for the investigation were good models because they represented two different types of cell growth and morphology. In order to remove the old medium and provide new nutrient substances for the cells, fresh culture medium (described in the experiment section) was introduced. All parameters concerning the preparation of the microsystem for the cell culture were previously optimized. 3.2. Cytotoxicity assay
Fig. 4. The data demonstrate correlation between macro- and microsystem, two different methods of cell cultivation, for measuring cell viability. A549 and HT-29 cells were incubated with 5-FU for 24 and 48 h. Each point represents the mean ± SD (n ≥ 4). The grey lines represent the linear regression R2 > 0.9.
2.6. Statistical analysis Experimental data are expressed as mean ± standard deviation (SD) from at least four independent experiments. Statistical significance was evaluated using one-way analysis of variance (ANOVA) followed by post hoc Tucky’s test. The regression analysis was performed to compare the results (SigmaPlot, Systat Software Inc, USA). P < 0.05 was considered as a statistically significant. 3. Results and discussion 3.1. Cell culture in the microsystem In this paper, we present A549 and HT-29 cell lines cultured in the microfluidic system. HT-29 cell line was derived from a different organ and its cells represent a dissimilar character of growth in comparison with A549 cells. HT-29 cells create characteristic clusters, whereas A549 cells steadily colonize the growth surface. The successful culture with both cell lines was performed in the microsystem. The conditions created in the microsystem allowed the adhesion and proliferation of both cultured cell lines. The suspension of both cell lines and the flow rate used for cell docking in the microchambers allows the introduction of the cells to each culture microchamber in a sufficient amount for future cultures. Moreover, seeded cell density allows the maintenance of cellular interactions so as to ensure proper cell growth. A549 and HT-29 cells adhesion and proliferation were observed within 24 h after seeding. The geometry of the microsystem and the applied procedures did not cause hydrodynamic stress to the cells cultured
Microfluidic cell culture is a promising technology for applications in the drug screening industry. Microfluidic systems can be used in the future as alternative tools for cell analysis and the analysis of new compounds. The key benefits include biological function improvement, higher quality cell-based data, reduced reagent consumption and lower costs. This investigation demonstrated how microfluidic cell culture design may be adopted as a method compatible with the standard 96-well plate format (macrosystem) [33]. Fig. 2 shows representative results of the cell viability test performed in the various microsystems. The toxic effect of 5-FU on the A549 and HT-29 cells was determined by CAM and PI. For each row of the microchambers matrix, a specific concentration of 5-FU was obtained. Viability of the cells, cultured in the microchambers, exposed to a gradient perfusion of 5-FU decreased with a higher concentration of the drug. The number of dead cells (red objects) after 24 h incubation with 5-FU increase with the drug concentration. 5-FU tested at concentrations ranging from 75 to 300 M, inhibited the survival of both cell types. However, HT-29 cells were less sensitive to the cytotoxic agent than A549 cells in both incubation times (i.e. 24 h and 48 h). The specific morphology of two different cell lines may be one of the reasons for these differences. HT-29 cells form specific clusters (Fig. 1C), therefore the compounds cannot be evenly distributed to all cells. However, A549 cells which adhere to the glass one by one were in direct contact with the tested compound. Moreover, the cells used in experiments have a different genetic status of genes. HT-29 cells acquire resistance to the new conditions (in this case to the cytostatic drug) more rapidly than A549 cells. This is the most likely cause of various cytostatic actions of 5-FU for these two cell lines. Generally, the inhibitory effect of 5-FU was dose- and timedependent. The strongest inhibition, approaching 80% after 48 h incubation, was observed for A549 cells exposed to 300 M 5-FU. The results of the cells viability were compared with the tests performed in static conditions using 96-well plates. Using this method, HT-29 cells were also less sensitive to 5-FU than A549 cells. In Fig. 3A the data obtained from at least four independent experiments are shown. In one experiment, the same concentration was analyzed five times in the microsystem – in one row of microchambers and three times in 96-well plate – in three wells. In Fig. 3B the control conditions (cells incubated without 5-FU) at each time point and for each cell line are shown. The result obtained in both systems (i.e. micro- and macrosystem) of cell cultivation (Fig. 3A) has a similar dependence of concentration of 5-FU and time incubation with the drug. But in each test, differences between both systems were observed. For example, the cell survival rate for the HT-29 line after 48 h incubation with 300 M 5-FU decreased to 65% and 50% in the micro- and macrosystem, respectively. The cytotoxic effects on cells in the macroscale were shown to be lower by about 10–20% than in the microscale, with the exception of A549 cells incubated at 75 M for 48 h period. For each cell line in both times of incubation and both methods (micro and macro)
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Table 1 The viability of A549 an HT-29 cells (percent ratio green to all cells) for four experiments after incubation with different time performed in the microsystems. A549 24h Micro1 Micro2 Micro3 Micro4
Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) A549 48h
Micro1 Micro2 Micro3 Micro4
Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility)
0 µM 94.39 3.55 91.28 3.64 98.19 5.64 95.70 5.33
75 µM 78.60 4.76 81.50 5.14 80.30 2.68 75.38 3.42
150 µM 54.75 13.70 73.82 3.12 76.34 6.56 74.35 7.43
225 µM 39.11 5.88 56.06 8.02 33.84 7.86 61.63 5.16
300 µM 21.02 3.23 49.35 18.04 20.31 1.34 53.55 9.43
0 µM 90.45 3.48 94.00 8.49 98.48 3.48 93.55 4.02
75 µM 66.42 12.78 80.00 8.60 75.56 12.78 56.56 5.63
150 µM 57.96 1.72 43.87 12.26 50.46 1.72 43.00 7.40
225 µM 46.06 3.61 47.28 6.19 36.94 3.61 22.29 4.27
300 µM 22.41 9.94 33.3 17.32 30.44 9.94 18.56 7.13
the IC50 were calculated. For A549 cells IC50 were determined as follows 251 M (24 h), 181 M of 5-FU (48 h) in the microsystem and 210 M (24 h) and 38.4 M of 5-FU (48 h) in the macrosystem. For HT-29 cells it equals: 810 M (24 h), 400 M of 5-FU (48 h) in the microsystem and 437.5 M (24 h) and 318 M of 5-FU (48 h) in the macrosystem. The obtained results indicate that, from a cellular perspective, the microenvironment in microfluidic cultures can be significantly different from those in traditional macroscale cultures. However, the results of regression analysis (performed between the data of cell viability) indicate significant correlation of 5-FU cytotoxicity between two evaluated systems because Pearson’s correlation coefficients (R2 ) were ≥0.9 (Fig. 4). Despite the observed differences between micro and macroscale, the developed system could be an alternative tool for the cell analysis. Here we investigated a few concentrations of 5-FU on two cancer cell lines. Both cell lines have shown dose-dependent effect. The drug concentrations used in our experiments allowed to compare macro- and microsystem as well as showed differences between the culture conditions. In the future experiments, higher number of drug concentrations are to be tested. It will allow for better referencing and better resolved dose/response functions. The geometry of the microsystem enables to obtain more concentrations by the addition of CGG single module. Moreover, using the developed microsystem it is possible to test more drug concentrations without the change of CGG geometry, but only by using of other range of concentration introduced to the microdevice. For each new-tested cell line or drug validation step is important for the evaluation of the results in comparison to conventional test procedure. We focused our investigation on the reason for these differences. Most likely, the discrepancy between the two systems could result from the differences in the cell culture conditions and different access of the anticancer drug to cells cultivated in the macrosystem. Tests performed on the plates were carried out in static conditions, while in the microsystem the microcultures were rinsed with medium, drugs and solution of CAM and PI. We observed that the flow exposure of a toxicant could affect the cells in a different manner relative to static exposure conditions, depending on the cell types and physicochemical properties of the toxicant. The flow conditions simulate the provision of drugs and substances into a living body, therefore the microfluidic device may be assumed as a quite simple model more resembling of in vivo conditions, which are not properly simulated by 96-well plates. The differences between micro and macroscale were observed by various groups. For example Beebe et al. tested three culture devices: 96 well plates, microwells and microchannels, which represent a potential culture platform that produces different
HT29 24h Micro1 Micro2 Micro3 Micro4
Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) HT29 48h
Micro1 Micro2 Micro3 Micro4
Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility) Viability of cells [%] SD (reproducibility)
0 µM 97.11 2.54 98.01 1.12 97.92 2.39 97.04 3.28
75 µM 86.46 5.56 90.23 4.49 90.50 4.37 88.22 3.61
150 85.20 0.73 90.98 5.45 90.39 6.03 84.95 7.47
225 µM 81.43 5.48 88.68 3.44 91.62 2.46 79.99 0.40
300 µM 72.63 8.56 90.99 1.63 90.94 0.25 71.19 0.45
0 µM 98.83 0.65 97.50 6.43 98.18 2.48 94.81 3.36
75 µM 84.66 5.13 89.60 2.42 77.42 18.64 61.42 5.18
150 µM 76.87 4.52 84.80 5.95 73.50 18.83 53.96 5.90
225 µM 75.86 3.85 77.30 4.95 67.34 16.11 54.29 8.63
300 µM 69.92 4.04 76.80 0.93 57.01 11.63 58.28 5.46
microenvironments. They observed various proliferations, glucose metabolism, signaling pathway activation and protein expression levels between cells cultured in traditional macroscale cultures and in microfluidic cultures [34]. Cooksey et al. described the study of the performance and variability of a cell-based toxicity assay in microfluidic devices. The authors quantified reduction in cellular green fluorescent protein (GFP) due to inhibition of ribosome activity by cycloheximide (CHX) [35]. The results were compared with macroscale and significant changes in GFP in the microfluidic chamber than in the dishes were faster detectable. The results indicate that from a cellular perspective, the microenvironment in microfluidic cultures can be significantly different from those in traditional macroscale cultures. Microfluidic devices for cell based assays have provided new types of microenvironments and new methods for controlling and observing the cellular responses. The results obtained in the microsystem allow to study and observe influence of the change of culture microenvironment on the cells. However, the results are dependent on the tested cell lines, density of culture and the time of incubation with drug. 3.3. Replication and reproducibility of microfluidic system The same microsystem was used for cytotoxicity tests several times for the same cell line and the same time incubation. We analyzed replication and reproducibility of the microsystem with parallel chambers for replicate measurements. The values shown in Figs. 3 and 4 (concerned microsystems) were obtained for each concentration from five microchambers in one device and at least four independent experiments. In Table 1, the viability of A549 an HT-29 cells (percent ratio green to all cells) for four experiments after incubation with different time is presented. SD shows the reproducibility of viability in the column of microchambers with the same concentration of the drug in one microsystem. The low differences between microchambers in the same concentration were observed. Moreover gas chromatography was used to evaluation of cytotoxic drug absorption in the block of polymer of microdevice. 5-FU used to test cytotoxicity could be absorbed in a PDMS block, which was utilized to the microsystem fabrication. Although procedures of recovery and reuse were performed, the absorbed 5-FU could have influence on research results. The level of 5-FU absorption in PDMS block was investigated using a gas chromatography. In the chromatogram of standard solution (Fig. 5A), there are peaks corresponding to 5-FU (time 1.90 min) and DMSO (time 1.19 min). In the tested sample, DMSO used for the extraction of absorbed substances from PDMS parts of the microsystem contained only DMSO
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Fig. 5. Gas chromatogram of 5-FU extracted by DMSO from PDMS. Standard solution (A) and the sample (B).
(time 1.19 min) (Fig. 5B). The PDMS block did not absorb the 5-FU, so usage of the same microchip for several cytotoxicity tests did not have cytotoxic influence on the subsequent cultures performed in the same microfluidic system. 4. Conclusions In this paper, we described the use of the microfluidic device for testing the toxicity of 5-fluorouracil, commonly used as an anticancer agent, and discuss its results in contrast to the traditional cytotoxicity assays performed in 96-well plates (macrosystem). The results show that the cells in macroscale are much more sensitive to the tested 5-fluorouracil than the cells tested in microscale. Although differences between the cytotoxicity effect of 5-FU on the cells in both culture conditions were observed, the regression analysis indicated a significant correlation of 5-FU cytotoxicity. Therefore, it can be concluded that the conditions of cultivation may affect the level of cytotoxicity of drugs and the microfluidic system can be an alternative tool for performing the cytotoxicity assays. Additionally, it was shown that the cells with different characteristics of growth can be cultivated in such a microdevice. The viability of two different cell lines (i.e. A549 and HT-29) was tested. We proved that the inhibitory effect of 5-FU as well as type of the cell line were both time and drug concentration dependable. The designed geometry of the microsystem enabled us to test different concentrations of 5-FU in a single step. This microfluidic system enables the performance of various toxicological examinations such as cell viability, cell death and real time imaging. Our results may be useful for biological and medical research. Creation of automatic administration of tested substances will allow the application of the microsystem in a laboratory as a userfriendly tool. In the future, the designed microsystem may be used for rapid, preliminary evaluation of toxic influence of tested compounds with a potential cytotoxic activity. This system can also be used in the first stages of drugs screening. Additionally, it can also be used to test the synergistic effect of chemotherapeutic agents, which will allow the reduction of time and costs of analyses. Acknowledgements The authors would like to thank Anna Jerzak from the Laboratory of Technological Processes (WUT) for GC measurement. This work was financially supported by Warsaw University of Technology.
References [1] G.A. Silvestri, M.P. Rivera, Chest 128 (2005) 3975–3984. [2] R. Labianca, S. Mosconi, M.C. Garassino, Ann. Oncol. 17 (2006) 51–54. [3] R. Napieralski, K. Ott, M. Kremer, K. Becker, A.L. Boulesteix, F. Lordick, J.R. Siewert, H. Höfler, G. Keller, Clin. Cancer Res. 13 (2007) 5095–5102. [4] Y. Kodera, S. Ito, M. Fujiwara, Y. Mochizuki, N. Ohashi, Y. Ito, G. Nakayama, M. Koike, Y. Yamamura, A. Nakao, Int. J. Clin. Oncol. 11 (2006) 449–453. [5] K. Ziolkowska, E. Jedrych, R. Kwapiszewski, J. Lopacinska, M. Skolimowski, M. Chudy, Sens. Actuators B 145 (2010) 533–542. [6] M.H. Wu, S.B. Huang, G.B. Lee, Lab Chip 10 (2010) 939–956. [7] B.H. Weig, R.L. Bardell, C.R. Cabrera, Adv. Drug Deliv. Rev 55 (2003) 349–377. [8] J. Pihl, M. Karlsson, D.T. Chiu, Drug Discov. Today 10 (2005) 1377–1383. [9] J. El-Ali, P.K. Sorger, K.F. Jensen, Nature 442 (2006) 403–441. [10] R. Baudoin, A. Corlu, L. Griscom, C. Legallais, E. Leclerc, Toxicol. In Vitro 21 (2007) 535–544. [11] L. Kang, B.G. Chung, R. Langer, A. Khademhosseini, Drug Discov. Today 13 (2008) 1–13. [12] G.M. Walker, H.C. Zeringue, D.J. Beebe, Lab Chip 4 (2004) 91–97. [13] J.H. Sung, M.L. Shuler, Bioprocess. Biosyst. Eng. 33 (2010) 5–19. [14] J.H. Qin, N.N. Ye, X. Liu, B.C. Lin, Electrophoresis 26 (2005) 3780–3788. [15] M.J. Powers, K. Domansky, M.R. Kaazempur-Mofrad, A. Kalezi, A. Capitano, A. Upadhyaya, P. Kurzawski, K.E. Wack, D.B. Stolz, R. Kamm, L.G. Geiffith, Biotechnol. Bioeng. 78 (2002) 257–269. [16] Ch. Yi, Ch.W. Li, S. Ji, M. Yang, Anal. Chim. Acta 560 (2006) 1–23. [17] A.Y. Fu, H.P. Chou, C. Spence, F.H. Arnold, S.R. Quake, Anal. Chem. 74 (2002) 2451–2457. [18] D.M. Thompson, K.R. King, K.J. Wieder, M. Toner, M.L. Yarmush, A. Jayaramen, Anal. Chem. 76 (2004) 4098–4103. [19] Zhang Ch, Z. Zhao, N.A. Abdul Rahim, D. Noort, H. Yu, Lab Chip 9 (2009) 3185–3192. [20] J. Komen, F. Wolbers, H.R. Franke, H. Andersson, I. Vermes, A. Berg van den, Biomed. Microdevices 10 (2008) 727–737. [21] N.L. Jeon, S. Dertinger, D.T. Chiu, I. Choi, A. Stroock, G. Whiteside, Langmuir 16 (2000) 8311–8316. [22] A. Funfak, J. Cao, O.S. Wolfbeis, K. Martin, J.M. Kohler, Mikrochim. Acta 164 (2009) 279–286. [23] A. Huebner, S. Sharma, M. Srisa-Art, F. Hollfelder, J.B. Edel, A.J. deMello, Lab Chip 8 (2008) 1244–1254. [24] W. Liu, H.J. Kim, E.M. Lucchetta, W. Du, R.F. Ismagilov, Lab Chip 9 (2009) 2153–2162. [25] A. Funfak, R. Hartung, J. Cao, K. Martin, K.H. Wiesmüller, O.S. Wolfbeis, J.M. Kohler, Sens. Actuators B 142 (2009) 66–72. [26] A. Funfak, J. Cao, A. Knauer, K. Martin, J.M. Kohler, J. Environ. Monit. 13 (2011) 410–415. [27] A. Tirella, M. Marano, F. Vozzi, A. Ahluwalia, Toxicol. In Vitro 22 (2008) 1957–1964. [28] H. Bang, W.G. Lee, H. Yun, Ch. Chung, J.K. Chang, D.Ch. Han, Microsyst. Technol. 14 (2008) 719–724. [29] Y.Ch. Toh, T.Ch. Lim, D. Tai, G. Xiao, D. Noort, H. Yu, Lab Chip 9 (2009) 2026–2035. [30] D.B. Longley, D.P. Harkin, P.G. Johnston, Nat. Rev. Cancer 3 (2003) 330–338. [31] N. Zhang, Y. Yin, S.J. Xu, W.S. Chen 13, Molecules (2008) 1551–1569. [32] P. Noordhuis, U. Holwerda, C.L. Van der Wilt, C.J. Van Groeningen, K. Smid, S. Meijer, H.M. Pinedo, G.J. Peters, Ann. Oncol. 15 (2004) 1025–1032. [33] P.J. Lee, N. Ghorashian, T.A. Gaige, P.J. Hung, JALA 12 (2007) 363–367.
E. Jedrych et al. / Sensors and Actuators B 160 (2011) 1544–1551 [34] A.L. x, D.J. Beebe, Int. Biol. 1 (2009) 182–195. [35] G.A. Cooksey, J.T. Elliott, A.L. Plant, Anal. Chem. 83 (2011) 3890–3896.
Biographies Elzbieta Jedrych was born in Poland in 1984. She received her MSc in chemical technology from the Department of Chemistry, Warsaw University of Technology (WUT), Poland in 2008. Currently she is PhD student in chemistry at the Department of Microbioanalytics, where she is a member of Chemical Sensors Research Group. Her current research interests are: designing and manufacturing of microfluidic Lab-on-a-Chip systems, developing of microsystems for cell culture and analysis. Sylwia Flis was born in Poland. She received her MSc in biology from the Warsaw University, Poland in 1998 and PhD in science of medicine at Institute of Pharmacology PAS in 2005. Currently she is PhD in science of medicine at the National Medicines Institute, Warsaw, Department of Pharmacology, Poland. Her current research interests are: biochemistry, genetics and molecular biology, medicine pharmacology, toxicology and pharmaceutics. ´ Kamila Sofinska was born in Poland in 1986. She is master student in chemical biotechnology at the Faculty of Chemistry, Warsaw University of Technology (WUT). Her current interest is “Lab-on-a-Chip” systems applications, such as cell engineering and medical diagnosis. Zenon Jastrzebski was born in Poland in 1944. He received his the veterinary doctor in Warsaw University of Life Sciences (SGGW), Poland in 1968, and doctor of agricultural sciences from the Institute of Genetics and Animal Breeding Polish Academy of Sciences in 1997. Currently he has been a head at the National Medicines Institute, Warsaw, Department of Pharmacology, Poland. His current research interests are: biochemistry, genetics and molecular biology agricultural and biological sciences, medicine, chemistry.
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Michal Chudy was born in Poland in 1973. He received his MSc in chemical technology from the Department of Chemistry, Warsaw University of Technology (WUT), Poland in 1997, and PhD in chemistry from the same faculty in 2001. Currently he has been an assistant professor at the Department of Microbioanalytics, where he is a member of Chemical Sensors Research Group. His current research interests are: various chemical sensors (ISE, ISFETs, CHEMFETs, SSE), designing and manufacturing of microfluidic Lab-on-a-Chip systems, developing new analytical methods for medical and bioanalytical application. Artur Dybko received his PhD in optoelectronics from the Faculty of Electronics, Warsaw University of Technology (WUT), Poland in 1996, and DSc in metrology from the same faculty in 2002. In 1990 he joined an optoelectronic company Sensomed, Poland as research and development manager. Since 1992 he started his PhD studies at the Faculty of Electronics (WUT). In 1996 he was employed as an assistant professors at the Faculty of Chemistry, Department of Analytical Chemistry (WUT). Currently he has been a university professor at the Department of Microbioanalytics, where he is a member of Chemical Sensors Research Group. His current research interests are: fibre optic chemical sensors, electrochemical sensors (ionselective electrodes, ion-sensitive field effect transistors), microfluidic structures for application in mTAS, low noise electronic circuits, application of computers in measurements. Zbigniew Brzozka obtained his PhD (1982) and his DSc (1991) from the Warsaw University of Technology (WUT). In 1998 he was appointed full professor of analytical chemistry in the Department of Analytical Chemistry WUT. He is head of the Chemical Sensor Research Group (CSRG). His current fields of interest are chemical membrane sensors, ion recognition, ion monitoring in clinical and environmental protection, and miniaturized analytical devices (-TAS). Among other areas, emphasis is now also being laid on the applications of polymer microfabrication technologies to microchemical analysis, such as Lab-on-a-Chip and (bio)electrochemical systems based on chemical reactions and/or various type of detection principle.