Analysis of the efficiency of photodynamic therapy using a microsystem for mono-, co- and mixed cultures

Sensors and Actuators B 221 (2015) 1356–1365

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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Analysis of the efficiency of photodynamic therapy using a microsystem for mono-, co- and mixed cultures Elzbieta Jastrzebska ∗ , Magdalena Bulka, Natalia Rybicka, Kamil Zukowski Institute of Biotechnology, Department of Microbioanalytics, Faculty Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

a r t i c l e

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Article history: Received 27 March 2015 Received in revised form 4 July 2015 Accepted 7 August 2015 Available online 9 August 2015 Keywords: Photodynamic therapy (PDT) Coculture Monoculture Non-malignant-carcinoma models Reactive oxygen species

a b s t r a c t Analysis of phototoxicity in monoculture can be performed both in a macroscale and a microsystem. To improve this kind of research, we propose fully integrated microsystem for evaluation of the efficiency of PDT procedures on three kinds of cell cultures (monoculture, coculture and mixed culture). Thanks to this the influence of PDT procedures on non-malignant cells, cultured in different distances with carcinoma cells was examined. 5-aminolevulinic acid (ALA) as a precursor of photosensitizer was used. In vitro nonmalignant—carcinoma models with both A549-Balb/c 3T3 and A549-MRC5 cells were investigated in the designed microsystem. An influence of the carcinoma (A549) cells presence on the non-malignant cell viability was noticed for: A549-MRC5 coculture and A549-Balb/c 3T3 and A549-MRC5 mixed cultures. Our results confirm that the microsystem is useful for PDT procedures analysis not only in monoculture (like in standard laboratories) but also in coculture and mixed culture. The proposed microsystem is a new discovery, allowing us to establish, how different distances between two cell types influence on the efficiency of PDT procedure. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Development of biologically relevant models of human tissues and organs is an important step for drug discovery. The usage of microsystems (called lab-on-a-chip) enables to mimic in vivo conditions, therefore these systems could be a good way to develop alternative methods to animal testing [1–4]. Nowadays, the labon-a-chip systems are good tools for fundamental biological studies [5,6]. The main advantage of these tools is easy cell manipulation and precise control of a cellular environment. The cells in live organisms are exposed to various microenvironmental signals such as: cell–cell or cell–extracellular matrix interactions. An analysis of these factors is limited in the conventional methods (macroscale). Therefore, the microsystems could be a good solution for accurate, easy and fast investigation of cellular functions. Moreover, a flow of reagents, oxygen level and value of a hydrodynamic shear stress can be monitored in the microscale [7,8]. The microsystems for cell engineering must meet several important requirements [9,10]. First of all, they should be non-toxic for the cells. It means, that the construction materials have to be biocompatible and chemically inert. Polymers

∗ Corresponding author. Tel.: +48 222345757. E-mail address: [email protected] (E. Jastrzebska). http://dx.doi.org/10.1016/j.snb.2015.08.022 0925-4005/© 2015 Elsevier B.V. All rights reserved.

(i.e. poly(dimethylsiloxane)—PDMS, poly(methyl methacrylate)— PMMA, polycarbonate—PC), glass or silicone are used for fabrication of the microsystems. Transparency is the next feature of the microsystems, which should be provided. The usage of the transparent materials allows a real-time monitoring of cell proliferation and their growth. Therefore, PDMS is the most often used of the materials, which were the above mentioned. It results from the properties of PDMS such as: gas permeability, biocompatibility, transparency and stability in a wide range of temperature (from −50 ◦ C to 200 ◦ C). Moreover, PDMS can be integrated with other materials, for example with hydrophilic glass [11,12]. Lab-on-a-chip systems can be used as functional tools for cell culture, an analysis of their proliferation and cell based toxicity assays [13,14]. In the literature, we can find other applications of the microsystem, such as: cell lysis, single-cell analysis or organ culture [9,13,15]. Moreover, the microfluidic devices can also be useful for mimic human circulatory system [16]. In addition, cell–cell interactions and their migrations can be analysed in such microtools [17]. The investigation of a cell migration and cell–cell communications are important to understand the cell behaviour in live organisms. Therefore, the scientists have started to investigate this kind of cell functions in microscale. Cell movement can be driven by chemical gradients (i.e. chemotaxis) or physical parameters, i.e. mechanical stimulation, magnetic fields and electric fields (electrotaxis) [18,19]. Kaji et al. have presented a method for

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controlling in microscale cell–cell interactions. It was investigated how the regulation of these interactions influence on a tissue biological function [20]. In turn, Goers et al. noticed that cell–cell interactions in cocultures are strongly dependent on the extracellular environment and the distance between the cells [21]. Similarly, Yeh et al. have studied the fluid shear stress influence on cell coculture performed in different distance [22]. As was mentioned before, the microsystems are often used for cytotoxicity analysis of compounds (especially used for anticancer therapy). More and more frequently they are also used for examining of newly synthetized nanoparticles. However, other anticancer therapies, for example photodynamic therapy (PDT), are not often investigated in the microscale. PDT is one of a few methods applicable in oncological (i.e. head, neck, lung, esophagus, pancreas or bladder cancer) and non-oncological (i.e. lichen sclerosus, Staphylococcus aureus) treatments. To perform PDT three factors are required: a photosensitizer, a light that is absorbed by this photosensitizer and an intracellular oxygen [23,24]. This method is based on an introduction of the photosensitizer in an organism, where it is accumulated in the carcinoma cells. Next, after a light irradiation, the induced photosensitizer reacts with an intracellular oxygen. This leads generation of reactive oxygen species (ROS), which should be toxic for the carcinoma cells. Nowadays, PDT is already used for treatment of a few kinds of cancer, but this method ought to be still studied. In order to ensure the efficiency of PDT on various cell types, many photosensitizers are still tested in vitro and in vivo conditions. 5-aminolevulinic acid (ALA) is the most popular precursor of photosensitizer–protoporphyrin IX (PPIX) [25,26]. Besides this, verteporfin, temoporfin, phthalocyanine or bacteriochlorophyll are drugs used in PDT [27]. Moreover, newly synthesized compounds are also investigated (i.e. profens(six tetraphenyl derivatives of arylpropionic acids) [28], AcrDIMs (3,6-bis((1-alkyl-5-oxoimidazolidin-2-yliden)imino)acridine hydrochlorides) [29], new derivatives of tetrakis(hydroxyphenyl)porphyrin]) [30]. In addition, nanoparticles, loaded with photosensitizers, could improve the efficiency of PDT procedures [31]. For example, Bazylinska et al. have presented research about photocytotoxicity analysis of nanocarriers, which were loaded with cyanine photosensitizer [32]. Research proved that, accumulation of this compound in MCF7/WT cells and efficiency of PDT procedures is dependent on the construction of nanoparticles. In turn, Mohammadia et al. tested photosensitizing properties of ALA, conjugated with goldnanoparticles [33]. Despite the fact that many photosensitizers are discovered, PDT procedures are investigated in the microsystems only by a few scientific groups [34–36]. For example, scientists have presented a microchip for high-throughput PDT screening on cell monoculture [35]. The other group investigated PDT procedures on 3D breast cancer tissue model, containing human breast cancer cells (MCF-7) and primary adipose-derived stromal cells (ASCs) [36]. Although these existing microsystems could be used for PDT procedures analysis, they still need improvement in many aspects. First, the proposed microsystems limited the analysis of interactions and migrations between various kinds of cells. The investigation of the mutual influence of carcinoma and nonmalignant cells is limited. Therefore, we present the fully integrated microsystem for evaluation of the efficiency of PDT procedures on these two kinds of cells. It was analyzed how intercellular signals, which can be released by either cell types, influence on the cell viability. Moreover, the presented microsystem consists of a geometry of microchannels, which allows to perform three kinds of cell cultures (mono-, coculture and mixed culture) in a single chip. Thanks to this, the investigation of communications between non-malignant and carcinoma cells could be examined. The proposed microsystem is a new discovery, allowing us to establish,

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how different distances between two cell types influence on the efficiency of PDT procedure. 2. Material and methods 2.1. A microsystem design and fabrication Fig. 1(a) and (b) shows a geometry of the designed PDT microsystem. Its design and fabrication method were based on the protocols described in our previous work [17]. The microsystem consists of four microstructures with a network of microchannels (a width of 100 ␮m, a height of 50 ␮m) which are arranged in V-shaped configuration. Moreover, each V-shaped microstructure includes: (1) three pairs of microchambers (with a diameter of 1 mm, a height of 50 ␮m) without any additional (connecting) microchannels; (2) five pairs of microchambers connected with connecting microchannels having a length of: 1000, 800, 600, 400 and 200 ␮m, respectively; (3) a common microchamber. This Vshaped microstructure was designed in such a way to provide various types of cell cultures: monoculture, coculture and mixed culture. Three pairs of the microchambers, without connecting microchannels, were dedicated to obtaining cell monoculture of two different cell lines. The next five microchambers could provide coculture of cells and an analysis of the migration of cells along the connecting microchannels. In contrast to the culture obtained in the above described microchambers, cell coculture could be derived automatically in the common microchamber. Two cell lines could be loaded in the same microchamber side by side, therefore we called this kind of culture a mixed culture. In order to automate and improve the experiment procedure, the geometry of the microsystem with a concentration gradient generator (CGG) was enriched. Thanks to this, in V-shaped structures four different concentrations of a tested photosensitizer in a single step could be received. The microsystem for PDT procedures was fabricated in PDMS (Sylgard 184, Dow Corning) and a sodium glass plate (Helmand, 75 mm × 55 mm × 0.5 mm). Microstructures in PDMS were produced using a soft lithography process according to the protocols described in our previous work [34]. In short, a replication stamp was fabricated using a capillary film (Pro/Cap 50, Chromaline), which was deposited on a sodium glass plate and next patterned using standard photolithography. Afterwards, PDMS was prepared by mixing the prepolymer with a curing agent with a weight ratio of 10:1. After the degassing, the liquid mixture was poured over the stamp and cured for 1 h at 60 ◦ C to form a 4 mm thick PDMS layer. The PDMS replica was peeled off from the stamp and holes for the tubings were drilled. In this way, the PDMS top layer of the microsystem was obtained. PDMS layer was bonded with the glass plate using surface plasma activation (Plasma Preen System Inc. II 973). The fabricated microsystem is presented in Fig. 1(b). 2.2. Fluid flow modeling To verify the geometry of the V-shaped microstructure, which should enable separate introduction of two cell lines, fluid flow in the designed V-shaped microstructure was examined. For this purpose, simulation by computer modeling using the MEMS Module of COMSOL MULTIPHYSICS software was performed. Moreover, a flow analysis of fluorescein solution (Fluka) with the concentration 1 × 10−5 M and culture medium (MEME, Sigma Aldrich) was performed. These solutions, with a flow rate of 5 ␮L min−1 each were introduced simultaneously through two inlets of Vshaped structure. Next, fluorescence intensity was analyzed in each microchamber. Therefore, after introduction of an aqueous solution, images were taken using an inverted microscope (Olympus IX-71).

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Fig. 1. (a) The scheme of a microfluidic system used for evaluation of the efficiency of PDT procedures. Red color is corresponding to irradiation area. (b) The fabricated microsystem. (c) Scheme of the cells introduction into the microchambers through V-shaped inlets. (d) Simulation of a flow rate of two introduced substances through microchambers (COMSOL MULTIPHYSICS). (e) Tests of solution flow in the microsystem during the introduction of fluorescein solution and MEME. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

2.3. Cell culture in the microsystem The A549 (human lung carcinoma cell line), MRC-5 (human fetal lung fibroblast cells) and Balb/c 3T3 (mouse embryonic fibroblast cell line) were used in experiments. The cells were purchased from American Type Culture Collection and European Type Culture Collection. Cells were cultured in MEME medium (Sigma Aldrich) supplemented with 10%v/v fetal bovine serum (Gibco), 1%v/v streptomycin and penicillin (Sigma Aldrich), and 2 mM l-glutamine (Sigma Aldrich). The cells were cultured at 37 ◦ C in a humidified atmosphere including 5% CO2 (HeraCell 150, Thermo Scientific). Before the cell culture, the microsystem was sterilized by flushing with 70%v/v ethyl alcohol (POCh, Poland) for 20 min and exposing to UV light (Black Ray). After that, the microsystem was filled with a culture medium and placed in an incubator for 3 h. This procedure was made to improve cell attachment. The peristaltic pumps (Ismatec Reglo-Digital MS-4/12) were used for introduction of all fluids and cells at each stage of the experiment. The suspensions of 2 × 106 cells mL−1 were prepared and seeded into the sterilized microchambers of the microsystem. The carcinoma (A549) and non-malignant (Balb/c 3T3 or MRC5) cells were introduced through two V-shaped inlets of each V-shaped microstructure (a flow rate of 5 ␮L min−1 ). The cell suspension density and a flow rate through the microchannels were optimized in order to obtain a proper growth rate of the cells and minimal

hydrodynamic stress. To assure cell attachment to the substrate the microfluidic device was sealed and placed in a CO2 incubator. Monitoring of the cell culture was carried out using an inverted microscope coupled with a CCD camera (Olympus IX-71). During the experiments the temperature in the microsystem was monitored by the usage of a heated microscope table (Olympus). 2.4. Cell tracer test As was described above, the proposed microsystem should allow the culture of cells in monoculture, coculture and mixed culture simultaneously. The V-shaped geometry of microstructures should ensure cells load in appropriate microchambers. The proper seeding of carcinoma and non-malignant cells was confirmed in tests with long-term trackers. Therefore prior cell seeding, MRC5 and A549 cells were marked with two different long-term trackers. CellTracker Red CMTPX and CellTracker Green CMFDA (Invitrogen) were used to mark carcinoma (A549) cells and non-malignant (MRC-5), respectively. Cell tracker solutions were prepared according to the manufacturer protocol. At the beginning, the 10 mM stock solutions of these two kinds of CellTracker in dimethyl sulfoxide—DMSO (Sigma Aldrich) were prepared. Afterwards, the stock solutions were diluted to a final working concentration of 10 ␮M in a serum-free medium. These solutions were incubated for 30 min. with the adherent cells, cultured in the culture flask.

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After that time, CellTracker solutions were removed and A549 and MRC5 were detached using Tryple Express solution (Gibco). Cell suspensions were introduced into the appropriate microchambers through V-shaped inlets, according to the protocol described in the previous section. 2.5. PDT assay in the microsystem 5-aminolevulinic acid (ALA) (Sigma Aldrich) was adopted as a precursor of photosensitizer–protoporphyrin IX (PPIX). PDT efficiency on the carcinoma (A549) and non-malignant (Balb/c 3T3 or MRC5) cells was investigated. For this purpose, the microsystems with A549-Balb/c 3T3 and A549-MRC5 cells, cultured 48 h in V-shaped structures, were utilized. Previously prepared solutions of 0.75 mM ALA in a serum-free medium (MEME) and a serumfree medium (as a control sample) were introduced through two CGG inlets with a flow rate 5 ␮L min−1 for 10 min. These two solutions were used to generate four different concentration levels: 0; 0.25, 0.5 and 0.75 mM. Next, the microsystem was sealed and placed for 4 h in the CO2 incubator. After that time, the cells were irradiated through the PDMS using a high power LED (a distance of 10 mm, time 60 s,  = 640 nm, energy dose 30 J cm−2 ). The viability of the cells was determined 24 h after the PDT procedures. To evaluate efficiency of PDT the staining with calcein AM (CAM, Sigma Aldrich) and propidium iodide (PI. Sigma Aldrich) (Sigma Aldrich) was made. The viability test was performed according to the previously described method [17]. 2.6. Data analysis The cell viability was determined by counting the number of green (corresponding to live cells) and red (corresponding to dead cells) objects. The cellSens Dimension image analysis software (Olympus) was used for data acquisition and analysis. Experimental data are expressed as mean ± standard deviation (SD) from at least four independent experiments. Statistical analysis was performed using T-test. Values of p < 0.05 were considered to be statistically significant (asterisks indicate p < 0.05). 3. Results and discussion 3.1. The design of microchannels and microchambers As shown in Fig. 1(a) and (b), the proposed microsystem provides four V-shaped microstructures dedicated to cell culture. Such a geometry of the microsystem was designed for a few reasons. First of all, the V-shaped design allows for separate introduction of two cell lines: non-malignant and carcinoma cells. PDT therapy is effective, when it have toxic effect only on the carcinoma cells. Thanks to culturing of two kinds of cells in one microsystem, evaluation of an efficiency of PDT procedures (i.e. photosensitizer concentration, incubation time, energy light dose) on both cell lines was possible. The arrangement of microchambers and connecting microchannels enables to provide three kinds of cell culture (mono-, co- and mixed culture), simultaneously (Fig. 1(c)). This is the next advantage of the designed microsystem. The dimensions of the microchamber 1 mm in diameter and 50 ␮m in depth let us to introduce the number of cells, which was sufficient for cell–cell interaction and cell proliferation studies. Three pairs of microchambers, without a connecting microchannel, were used for the monoculture of two cell lines. The next five pairs of microchambers were connected with connecting microchannels with different lengths. Such configuration provided cell coculture. It means that the non-malignant and carcinoma cells can be cultured in strictly definite distances. The common microchamber was designed to obtain an automatic coculture (called mixed culture) of two cell

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lines side by side. In the coculture microchambers the medium was exchanged along the connecting microchannels. Thereby, the influence of carcinoma cells presence on viability of non-malignant cells could be monitored. Cocultures and mixed cultures could mimic in vivo conditions, where tumor cells proliferate with various distances around the non-malignant cells. Four V-shaped structures in one plate were located in order to analyze few concentrations of photosensitizers. Moreover, these structures were connected with a concentration gradient generator (CGG), which generates four different concentrations of a photosensitizer in a V-shaped network in a single step. The geometry of the microsystem includes two kinds of inlets: (1) V-shaped inlets used to introduce of two different cell lines; (2) CGG inlets used to introduce of medium and photosensitizer solutions. Thanks to the above described geometry of the microsystem evaluation of an efficiency of PDT procedures could be easier to perform. We assume that during the introduction of the non-malignant and carcinoma cells through V-shaped inlets, these cells will be introduced separately. Then, they will be loaded in the appropriate microchambers and will attach to the surface in the next hours as is shown in Fig. 1(c). To verify the fluid flow in the designed microsystem during the cell seeding a numerical simulation was made. For this purpose, computer modeling using MEMS Module of Comsol Multiphysics software was used. Fig. 1(d) shows simulation of a flow of two solutions, which were introduced in the V-shaped structure. As can be seen in figure, the solutions were introduced separately, they did not mix. Microchambers dedicated to monoculture contain only one kind of solution (red or blue color). Similarly, in the microchambers for coculture only one kind of solution was introduced. Solutions contact only in connecting microchannels, where diffusion occurs. In turn, common microchamber contains both introduced solutions. It means that both kinds of the introduced cells will be loaded in this area. Besides the numerical simulation, we also investigated a fluid flow in the V-shaped structure using 1 × 10−5 M fluorescein solution. For this purpose, the aqueous solution of fluorescein and medium MEME were introduced through V-shaped inlets. Fluorescence intensity in each pair of the microchamber was monitored. As is shown in Fig. 1(e), solutions were introduced without mixing along connecting microchannels. Based on COMSOL simulation and image analysis, we concluded that the designed microsystem can be useful for our application (separate introduction of non-malignant and carcinoma cells into microchambers). Analysis of a flow rate through CGG was reported in our previous work [34]. 3.2. A549-Balb/c 3T3 and A549-MRC5 cell cultures Carcinoma (A549) and non-malignant (Balb/c 3T3 and MRC5) cells were used in experiments. PDT procedure is most often used as an anticancer therapy for skin tumors. However, it could also be used to treat cancer of other organs such a breast, lung, stomach successfully. Due to the fact that lung cancer is one of the most common cancers in the world as well as leading cause of cancer death in men and women, we decided to investigate this type of cells. To evaluate the effectiveness of PDT procedures, two nonmalignant cell lines were chosen as a control (1) Balb/c 3T3 cells represent non-malignant tissue, this seems to be a good model for analysis of photoactivity of the tested photosensitizer (2) MRC-5 cells represent non-malignant lung tissue. Prior to the evaluation of PDT procedures on two pairs of cell cultures (1) A549-Balb/c 3T3 and (1) A549-MRC5 a verification of cell seeding was examined. Thanks to the usage of long-term trackers, analysis of cell seeding in the appropriate microchambers, analysis of mixing of non-malignant and carcinoma cells, detection and distinction between cell lines was possible. Cell trackers (Red CMTPX and Green CMFDA) used in our experiments, are fluorescent dyes

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Fig. 2. Microchambers with A549 and MRC5 cells introduced through V-shaped inlets (a) directly after cells introduction, (b) 24 h after cells introduction. A549 (red color) and MRC5 (green color) cells were marked with Red CMTPX and CellTracker Green CMFDA, respectively. Single microchamber corresponds to monoculture, microchambers with a different length of a connecting microchannel correspond to coculture, common microchamber corresponds to the mixed culture. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

applicable in monitoring cell location and migration. These trackers freely move through cell membranes into cells, where they can remain for a few generations. Therefore, they could be used to monitor cell seeding and mixing in the presented microsystem, successfully. The A549 and MRC5 cells, marked with trackers, were introduced into the microsystem (sterilized and filled with a medium culture) through V-shaped inlets. As is shown in Fig. 2(a), after the introduction A549 cells were seeded in the left part and MRC5 cells in the right part of the microsystem, respectively. Both single microchambers (corresponding to monoculture) and microchambers with the connecting microchannels (corresponding to coculture) contain only one kind of cell. There was no cell in the connecting microchannels. This confirmed, that the cells were

seeded in the appropriate microchamber without their mixing. In turn, the common microchamber was filled with both A549 and MRC5 cells. In Fig. 2(b) microchambers 24 h after cell seeding are shown. It was observed that in the next hours cells attached to the surface and proliferated very well. This proved, that the seeded cell density allowed maintenance of cellular interaction (of the same type of cells) to ensure their proper growth. The morphology of non-malignant and carcinoma cells was different. Based on the morphology, distinction of the two cells’ types was enabled in the common microchamber. For PDT assays the A549-Balb/c 3T3 and A549-MRC5 cells were seeded in the microsystem without labeling them with long-term trackers. Cell adhesion and proliferation was observed within 24 h after cell introduction. Microchambers with A549-Balb/c 3T3 cells

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Fig. 3. Microchambers with (a) A549-Balb/c 3T3 cells (b) A549-MRC5 cells cultured as a monoculture, coculture and mixed culture.

and A549-MRC5 cells, cultured in the microsystem, are shown in Fig. 3. A monoculture of A549, Balb/c 3T3 and MRC5 cells is presented in the first line of microchambers. The proposed geometry of the microsystem and a medium flow rate ensure proper conditions for cell proliferation. Hydrodynamic stress on cells cultured in the microchamber was not noticed. Cells were attached very well to the glass surface. Each microchambers with coculture of A549-Balb/c 3T3 and A549-MRC5 cells contains only one kind of cell line. Cell attachment and moving within microchambers was only observed. The connecting micochannels could constitute a place for cell–cell migration in a long-term cell culture. In the experiments described in this paper, they provide an area for medium exchanging between non-malignant and carcinoma cells before and after PDT procedures. In turn, the common microchamber was filled with both cell types: A549 and Balb/c 3T3 or A549 and MRC5. However, to distinguish the cultures performed in the five pairs of microchambers connected with different lengths of microchannels, this culture is called a mixed culture. In the common microchambers non-malignant and carcinoma cells directly contact each other. We assumed that in these microchambers the cells will have the strongest interaction with each other after PDT assays. The presented results proved that the microsystem could be applicable for (1) introducing two types of cells without mixing; (2) creation of mono-, co- and mixed cultures in one step (3) culture and analysis of cells (4) evaluation of influence carcinoma cells on viability of non-malignant cells (thanks to diffusion along the connecting microchannels). 3.3. PDT procedures efficacy analysis Effective PDT therapy should decrease the viability of carcinoma cells, which leads the reduction in tumors in the body. Anticancer therapy is effective when non-malignant cells, next to which carcinoma cells grow, should stay alive after treatment. Therefore, to optimize PDT procedures, the investigation of the photocytotoxicity of photosensitizers on carcinoma and non-malignant cells

is important, in both mono- and coculture. In the microsystem, presented in this article, in vivo conditions could be simulated, when carcinoma cells, are located close to non-malignant cells. Two cultures of carcinoma and non-malignant cells: A549Balb/c 3T3 and A549-MRC5 were used to evaluate an efficiency of the PDT procedures. PDT assays were executed according to the protocol described in previous section. Thanks to the work of CGG, in each V-shaped structure a different concentrations of the photosensitizer was achieved. Photocytotoxicity (cells incubation with photosensitizer and light irradiation) of four concentrations of ALA: 0, 0.25, 0.5 and 0.75 mM were examined. The serum-free medium (0 mM ALA) was used as a control sample. It was found that the viability of A549, Balb/c 3T3 and MRC5 cells, which were incubated with 0 mM ALA and light irradiated, was extremely high. After cell staining with CAM and PI, they gave only the green fluorescence. Therefore, for quantitative analysis of the cell viability after PDT procedures (for other ALA concentrations) the data were calculated as a percent of control. 3.3.1. A549-Balb/c 3T3 cell culture The results of the cell viability for A549-Balb/c 3T3 culture are shown in Fig. 4(a)–(c). First, we analyzed the viability of cells after the PDT procedures for monoculture (in the single chambers) and coculture—in the microchambers connected with additional microchannels. We found that the usage of 0.25 mM ALA caused death 39.3% of carcinoma A549 cells, whereas only 5.7% of nonmalignant Balb/c 3T3 cells in monoculture. A comparison of the cell viability in the monoculture shows that it is dependent on the cell line. The number of live A549 (carcinoma) cells was dependent on ALA concentration—it decreases with an increase of photosensitizer concentration. The viability of A549 cells equals 27.7% for 0.50 mM and 6.7% for 0.75 mM of ALA. In turn, the viability of Balb/c 3T3 (non-malignant) cells was on a similar level (91.5–94.3%) for each tested concentration. The above results confirm the effectiveness of the PDT procedures, because it is selective only in carcinoma

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Fig. 4. The viability of Balb/c 3T3 and A549 cells after PDT procedures for (a) 0.25 mM (b) 0.50 mM (c) 0.75 mM of ALA. (d) Microchambers of A549 and Balb/c 3T3 cells cultured in monoculture and coculture (a length of connecting microchannel—400 ␮m). Cells were stained with calcein AM and propidium iodide (green color—live cells, red color—dead cells). Asterisks indicate p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

cells. As was mentioned before, carcinoma and non-malignant were cultured in the separate microchambers in monoculture. The cells did not influence each other. A comparison of the A549 cell viability for each tested ALA concentration in coculture shows the same tendency as in the monoculture. The number of live A549 cells (within the same concentration) in microchambers with a different length of connecting microchannels was approximate. We expected, that in the microchambers with connecting microchannels (coculture) the Balb/c 3T3 cell viability will be lower than in a single microchamber (monoculture). Connecting microchannels could provide area for diffusion of reactive oxygen species (ROS) or death factors released by carcinoma cells. However, we did not observe any dependence and change in Balb/c 3T3 cell viability. Even for the shortest microchannel (200 ␮m) the Balb/c 3T3 cell viability was close to the value obtained in the monoculture. The statistical analysis showed that the difference between the experimental group of Balb/c 3T3 cells and the control samples was not statistically significant for mono- and cocultures. In turn, the viability of A549 cells after PDT procedures was significantly different from the control samples. Most probably, the connecting microchannels are too long, therefore death factors do not diffuse to Balb/c 3T3 cells. Moreover, these kinds of non-malignant cells (Balb/c 3T3) could be resistant to death factors, which could be released by carcinoma cells. In Fig. 4(d) microchambers with A549-Balb/c 3T3 cells monocultures and cocultures (a length of the connecting microchannel—400 ␮m) after the PDT procedures are shown. CAM and PI viability tests present that Balb/c 3T3 cells are still alive (green objects) in almost all microchambers, for different ALA concentrations. Whereas, the number of the dead A549 cells increases (red objects) with the higher concentration of photosensitizer.

3.3.2. A549-MRC5 cell culture The results of cell viability for A549-MRC5 culture are shown in Fig. 5(a)–(c). Analysis of the cell viability in the monoculture shows that the number of live A549 cells after the PDT procedures decreases with an increase of ALA concentration. The viability of A549 cells equals 59.8, 41.9 and 13.5% for 0.25, 0.50 and 0.75 mM of ALA, respectively. Whereas, the viability of MRC5 cells was high for each photosensitizer concentration and it equals 92.2, 103.1, 84.0% for 0.25, 0.50 and 0.75 mM of ALA, respectively. The statistical analysis showed that the difference between the experimental group of MRC5 cells and the control samples was statistically significant for A549 cells (for each tested ALA concentration) and for MRC5 cells for 0.75 mM concentration of ALA. As was seen, for both A549-Balb/c 3T3 and A549-MRC5 cultures similar tendency of the cell viability in the monoculture was noticed. In monoculture, carcinoma and non-malignant cells are cultured in single/separated chambers, where medium exchanging is not possible. Therefore, any factors can influence on the viability of the non-malignant cells. The A549 cell viability for each tested ALA concentration in coculture shows the same tendency as in the monoculture. In contrast to the A549-Balb/c 3T3 culture, here the dependence on the viability of MRC5 cells to the length of connecting microchannels in the coculture was observed. The number of live MRC5 cells in the coculture decreases with the length of connecting microchannels. For example in a V-shaped structure incubated with 0.25 mM ALA, the viability of MRC5 cells for a microchannels with a length of 1000 ␮m equals 97.4%, whereas for microchannels with a length of 200 ␮m is 85.2%. However, the observed differences were not statistically significant. A larger decrease of the cell viability was noticed for other ALA concentrations: from 103.1% to 58.1% and from 84.0% to 54.8% for 0.50 and 0.75 mM, respectively. Based on T-test it was examined that the difference of the viability of MRC5 cells was statistically significant for both 0.5 mM of ALA (for

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Fig. 5. The viability of MRC5 and A549 cells after PDT procedures for (a) 0.25 mM (b) 0.50 mM (c) 0.75 mM of ALA. (d) Microchambers of A549 and MRC5 cells cultured in monoculture and coculture (a length of connecting microchannel—400 ␮m). Cells were stained with calcein AM and propidium iodide (red color—dead cells, green color—live cells). Asterisks indicate p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

cocultures with a length of connecting microchannels of 200, 400 and 600 ␮m) and 0.75 mM of ALA (for each coculture). Based on these results, we conclude that the highest photocytotoxicity effect was observed in the microchambers, which were connected with the shortest microchannel. Moreover, the obtained results indicate, that the number of alive MRC5 cells decreased for higher ALA concentration. In contrast to monoculture of MRC5 cells, in coculture carcinoma cells could influence on the viability of the non-malignant cells. This dependence was especially observed for the highest (0.75 mM) ALA concentration. Most probably, the death factors or ROS diffuse from carcinoma to non-malignant cells. A549MRC5 cells staining with CAM and PI is shown in Fig. 5(d). The highest number of live cells (green objects) was observed in the microchamber with MRC5 cells cultured in a monoculture, whereas the number with red objects (dead cells) increased in coculture. In turn, the number of the dead A549 cells was the same within the same ALA concentration in mono- and coculture. More red objects were detected with an increase of concentration of photosensitizer.

3.3.3. Mixed cultures The viability of A549-Balb/c 3T3 and A549-MRC5 cells in the mixed culture is shown in Fig. 6. 50% of carcinoma and 50% of non-malignant cells represented the cell population in the common microchambers. Comparison of the viability in this kind of culture shows that the total number of the dead cells is higher than in the number of non-malignant cells in the monoculture. The dependence of ALA concentration on the cell viability was also proven for both A549-Balb/c 3T3 and A549-MRC5 cultures. In the common microchamber, carcinoma and non-malignant cells proliferate side by side. Therefore, the interactions between these cells should be the strongest. Based on T-test, it was examined that the difference of the viability of both A549-Balb/c 3T3 and A549-MRC5 cell

Fig. 6. The viability of A549-Balb/c 3T3 and A549-MRC5 cells after PDT procedures for (a) 0.25 mM (b) 0.50 mM (c) 0.75 mM of ALA in the common microchamber (the mixed culture). Asterisks indicate p < 0.05.

cultures from control samples was statistically significant. The type of the cells was not distinguished in the common microchamber after PDT procedures. Therefore, an effect of A549 cells on nonmalignant cells could be also supposed based on discussion of Figs. 4 and 5. We concluded, that the total number of the dead cells in the mixed culture of A549-Balb/c3T3 cells could indicate mainly A549 cells. In turn, the total number of the dead cells in A549-MRC5 cell mixed culture could be a combination of both cell lines. Balb/c 3T3 cells could be more resistant to the death A549 cells factors than MRC5 cells. Probably, the death factors which were produced

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by the A549 cells also have a higher toxic effect on MRC5 cells than Balb/c3T3 cells cultured in close proximity. 3.3.4. Discussion The above experiments proved that PDT procedures are toxic for carcinoma (A549) cells. For both A549-Balb/c 3T3 and A549-MRC5 cultures the viability of A549 cells was dependent on the concentration of photosensitizer. In turn, non-malignant cells (Balb/c 3T3 and MRC5) after PDT procedures in monoculture were still alive. It suggests that the investigated PDT procedures are effective. Analysis of this phototoxicity in the monoculture can be performed both in a macroscale and a microsystem. Therefore, we propose the microsystem in which, we expect monoculture, coculture and mixed culture can be obtained. Thanks to this the influence of PDT procedures on non-malignant cells, cultured in different distances with carcinoma cells, could be examined. An influence of the carcinoma (A549) cells presence on the non-malignant cell viability was noticed for A549-MRC5 cocultures for high concentrations of the tested ALA. For mixed cultures (A549-Balb/c 3T3 and A549-MRC5) the total number of the dead cells was higher than in the number of non-malignant cells in the monoculture. It could also indicate an influence of the A549 cells on the viability of non-malignant cells. However, we suppose that A549 cells have a higher toxic effect on MRC5 than on Balb/c3T3 cells. It was proved that Balb/c 3T3 are more resistant than MRC5 cells. Decrease of the viability of non-malignant cells can be caused by death signals from the dying carcinoma cells (for example TNF-␣ factor). This may lead mainly to the initial damage to mitochondria in the cancerous cells and as well as to the destruction of the vascular wall in the tumor tissue [37]. We suppose, that death factors (i.e. TNF-␣), which are released by A549 could diffuse through connection microchannels and influence the viability of non-malignant. It could be the most likely cause of mortality in non-malignant cells after PDT procedures. Besides this, ROS produced by A549 could have a toxic effect on Balb/c 3T3 and MRC5 in the mixed culture. ROS lifetime is limited, the diffusion distance causing the photodegradation was estimated to be on the order of 0.01–0.02 ␮m [38,39]. The cells cultured in the common microchamber proliferated side by side, therefore the ROS live time could be sufficient to diffuse to the non-malignant cells. In literature, the evaluation efficiency of PDT procedures is most often investigated using conventional methods, i.e. 96-well plates [40–42]. For example, the mechanisms of human biliary cancer cell death after PDT by in vitro and in vivo conditions were analyzed [42]. The cell viability after incubation with Photofrin and irradiation with energy density range: 4.0 to 16.0 J cm−2 was assessed using MTT tests. It was investigated that Photofrin in this human biliary cancer cell line has antitumor effect and induces apoptotic cell death after PDT. As we mentioned before, the effectiveness of PDT procedures in the Lab-on-a-chip systems was investigated by only a few research groups [34–36]. Lou et al. presented a high-throughput microplatform that enables the evaluation of the efficacy of PDT over multiple factors (drug concentration, light energy dose and oxygen level) [35]. In this microsystem photocytotoxicity of methylene blue on C6 was examined. The other group presented a microsystem for PDT analysis using 3D cell culture [36]. Photocytotoxicity of 5-ALA and gold nanoparticles under various light energy doses was investigated on MCF-7 cells. Moreover, a lot of microsystems for chemotaxis or electrotaxis research have been presented [18,19]. Analysis of migration and interactions for different cell types were also investigated in microfluidic systems [21,22,19]. These works present similar trends of evaluation efficiency of PDT procedures or interaction of two cell types, however none of them demonstrated the microsystem such we present in this manuscript—for analysis of PDT procedures on monoculture, coculture and mixed culture (which are created in one step).

Moreover, different value of factors (i.e. concentration of photosentitizers) used in PDT procedures could be investigated. The presented microchip is applicable for optimization of PDT procedure on the various kinds of cells. Thanks to that the non-malignant and carcinoma cells can be seeded and cultured in strictly defined distances, in vivo condition is mimicked. This is not possible in a conventional method. 4. Conclusions In this study, we have demonstrated a microsystem for evaluation efficiency of PDT procedures on three kinds of cell cultures. The biggest advantage of the developed microsystem is that the arrangement of the microchannels’ network on the plate creates a V-shaped structure in which mono-, coculture and mixed culture of cells can be performed, simultaneously. Such geometry of the microsystem enables examination of how intercellular signals which can be released by either cell type and can diffuse along a connecting microchannel influence on the viability of cells. The non-malignant and carcinoma cells can be seeded and cultured in strictly defined distances, which enables the evaluation of the efficiency of PDT in a conditions similar to in vivo. Two cultures of carcinoma and non-malignant cells: A549-Balb/c 3T3 and A549MRC5 were investigated. The results confirm that the microsystem is useful for PDT procedures analysis. Moreover, our investigations show that it is important to analyze the viability of cells not only in monoculture (like in standard laboratories) but also in coculture and mixed culture. Tests in monoculture suggest that PDT procedures are effective, because only the number of dead carcinoma cells increases, whereas, in coculture the viability of non-malignant cells also decreases. It was proven that the effectiveness of PDT procedures is dependent on the kind of the tested cells. We observed (in the coculutre) that the presence of A549 cells influence on the MRC5 cells viability, whereas they did not influence on Balb/c 3T3 cells. Using the fabricated microsystem, we demonstrated PDT assay on A549-Balb/c 3T3 and A549-MRC5 cultures for different conditions on a single chip. In the future, the microsystem is planned to be used for optimization PDT procedures conditions for cells delivered from various organs in mono-, co- and mixed culture. Acknowledgements ´ The authors would like to thank Marlena Rybinska for CellTracker staining. This work was financially supported by National Centre for Research and Development within a frame of LIDER program No LIDER/026/573/L-4/12/NCBR/2013 and National Science Centre—project No UMO-2012/07/B/ST5/02753. References [1] A. Khademhosseini, R. Langer, J. Borenstein, J.P. Vacanti, Proc. Natl. Acad. Sci. U.S.A. 103 (2006) 2480–2487. [2] A. Rios, M. Zougagh, M. Avila, Anal. Chim. Acta 740 (2012) 1–11. [3] I. Wagner, E.M. Materne, S. Brincker, U. Süßbier, C. Frädrich, M. Busek, F. Sonntag, D.A. Sakharov, E.V. Trushkin, A.G. Tonevitsky, R. Lauster, U. Marx, Lab Chip 13 (2013) 3538–3547. [4] J.M. Prot, E. Leclerc, Ann. Biomed. Eng. 40 (2012) 1228–1243. [5] G.M. Whitesides, Nature 442 (2006) 368–373. [6] P.M. Valencia, O.C. Farokhzad, R. Karnik, R. Langer, Nat. Nanotechnol. 7 (2012) 623–629. [7] Z. Tang, Y. Akiyama, K. Itoga, J. Kobayashi, M. Yamato, T. Okano, Biomaterials 33 (2012) 7405–7411. [8] K. Hattori, Y. Munehira, H. Kobayashi, T. Satoh, S. Sugiura, T. Kanamori, J. Biosci. Bioeng. 118 (2014) 327–332. [9] J. Nilsson, M. Evander, B. Hammarström, T. Laurell, Anal. Chim. Acta 649 (2009) 141–157. [10] W. Chena, R.H.W. Lama, J. Fu, Lab Chip 12 (2012) 391–395. [11] C.W. Beha, W. Zhoua, T.H. Wang, Lab Chip 12 (2012) 4120–4127.

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Biographies Elzbieta Jastrzebska 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, and PhD in chemistry from the same faculty in 2012. Currently she has been an assistant professor at the Department of Microbioanalytics, where he is a member of Chemical Sensors Research Group. Her current research interests are: designing and manufacturing of microfluidic Lab-on-a-Chip systems for cell culture and analysis i.e. heart-o-a-chip, photodynamic therapy analysis. Magdalena Bulka was born in Poland in 1991. She received her Eng. in biotechnology from the Department of Chemistry, Warsaw University of Technology (WUT), Poland in 2014. Currently she has been an master student 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 dedicated for photodynamic therapy analysis. Natalia Rybicka was born in Poland in 1989. She received her MSc. Eng. in chemical technology from the Department of Chemistry, Warsaw University of Technology (WUT), Poland in 2013. Her research interests were focused on Lab-on-a-Chip systems for photodynamic therapy analysis. Kamil Zukowski was born in Poland in 1983. He received his MSc in chemical technology from the Faculty of Chemistry, Warsaw University of Technology, Poland in 2002 and PhD in chemistry from the same faculty in 2015. Currently he has been an PhD student at the Department of Microbioanalytics, where he is a member of Chemical Sensors Research Group. His current research interests are: designing and manufacturing of microfluidic Lab-on-a-Chip systems, developing new analytical methods for medical and bioanalytical application.