Lab on a Chip PAPER Monolithically integrated biophotonic lab-on-a-chip for cell culture and simultaneous pH monitoring3 Cite this: Lab Chip, 2013, 13, 4239
´s,c ´ ria Vigue Xavier Mun ˜ oz-Berbel,*a Rosalı´a Rodrı´guez-Rodrı´guez,b Nu d c d Stefanie Demming, Jordi Mas, Stephanus Bu ¨ ttgenbach, Elisabeth Verpoorte,e af ad Pedro Ortiz and Andreu Llobera A poly(dimethylsiloxane) biophotonic lab-on-a-chip (bioPhLoC) containing two chambers, an incubation chamber and a monitoring chamber for cell retention/proliferation and pH monitoring, respectively, is presented. The bioPhLoC monolithically integrates a filter with 3 mm high size-exclusion microchannels, capable of efficiently trapping cells in the incubation chamber, as well as optical elements for real-time interrogation of both chambers. The integrated optical elements made possible both absorption and dispersion measurements, which were comparable to those made in a commercially available cuvette. The size-exclusion filter also showed good and stable trapping capacity when using yeast cells of variable size (between 5 and 8 mm diameter). For cell culture applications, vascular smooth muscle cells (VSMC), with Received 21st June 2013, Accepted 5th August 2013
sizes between 8 and 10 mm diameter, were used as a mammalian cell model. These cells were efficiently trapped in the incubation chamber, where they proliferated with a classical spindle-shaped morphology and a traditional hill-and-valley phenotype. During cell proliferation, pH changes in the culture medium
DOI: 10.1039/c3lc50746g
due to cell metabolism were monitored in real time and with high precision in the monitoring chamber
www.rsc.org/loc
without interference of the measurement by cells and other (cell) debris.
Introduction Cell and tissue cultures are important in vitro analytical tools with a wide and varied range of applications. These include drug screening,1 diagnostics2 or biological science studies.3 In all cases, cells isolated from tissues are introduced into suitable culture recipients (e.g. flasks, petri dishes, 96-well plates) where they proliferate under optimal experimental conditions (temperature, pH, oxygen, etc.). Although these conventional cell culture formats are extensively used worldwide, lab-on-a-chip (LoC) systems appear to be a good alternative, as they provide additional capabilities which traditional culture formats do not.4–6 Additional advantages a
Centre Nacional de Microelectro`nica (IMB-CNM, CSIC), Campus UAB, 08193 Bellaterra, Barcelona, Spain. E-mail:
[email protected]; Tel: +34935947700 b Department of Pharmacology, University of Sevilla, Profesor Garcia Gonzalez 2, 41012 Sevilla, Spain c `ma de Barcelona, Department of Genetics and Microbiology, Universitat Autono 08193 Bellaterra, Barcelona, Spain d ¨r Mikrotechnik, Technische Universita ¨t Braunschweig, Alte Salzdahlumer Institut fu Straße 203, 38124 Braunschweig, Germany e Pharmaceutical Analysis, Groningen Research Institute of Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands f Oxford Nanopore Technologies Ltd., Edmund Cartwright House, 4 Robert Robinson Avenue, Oxford Science Park, OX4 4GA Oxford, United Kingdom 3 Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3lc50746g
This journal is ß The Royal Society of Chemistry 2013
include the possibility of controlling cell–cell and cell– extracellular matrix interactions in static cultures,7–9 performing microfluidic perfusion cultures4 and generation of in vitro microenvironments that better mimic the in vivo situation.10 However, the requirement for controlled environmental conditions (temperature and CO2) as well as external inspection/measurement instrumentation (commonly fluorescence or confocal microscopes11,12) limits the use of LoC cell culture systems as routine laboratory tools. In this sense, the integration of transduction and/or environmental control elements into the LoC structure appears to be critical. Some examples of integration of detection elements in cell culture LoC systems can be found in the literature, most of them based on electrochemical transduction.13,14 For example, the LoC system developed by Satoh et al. integrated iridium-oxide, gold and Ag/AgCl electrodes into a poly(dimethylsiloxane) (PDMS) substrate for the real-time monitoring of ammonia metabolism in hepatocyte cells.15 The PDMS structure presented by Li et al. contained platinum circular microelectrodes for the real-time monitoring of dopamine release from PC12 cells.16 Despite the significant progress in the integration of electrochemical detection elements into LoC systems for cell culture, there is a progressive shift towards optical detection.17,18 The main reasons for this are: (i) optical measurements are non-invasive, do not alter cells or the culture medium composition and do not compromise sterility; (ii)
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Paper optical transduction does not require metals or metal oxides, reducing the fabrication costs; and (iii) the integration of optical elements into a LoC may require fewer fabrication steps, depending on the technology used. These advantages have been particularly exploited in microbiology, where wellbased systems integrating optical elements have been reported for the real-time monitoring of bacteria and yeast proliferation.19,20 These well-based systems are capable of monitoring cell concentration, pH and dissolved oxygen with simple optical sensors integrated into the LoC structure. In the case of cell concentration, they benefit from the capacity of suspended cells to disperse light, which produces a decrease in the recorded absorbance proportional to the cell concentration. This aspect of cell–light interaction, although extremely useful for suspended cultures, may become a drawback in the case of proliferation of adherent mammalian cells. Since it is the supernatant medium above an adherent cell culture which is probed, cells which detach or otherwise find themselves in the light beam can conceivably cause scattering or other interference to the optical measurement.21 In an attempt to address this open issue, a monolithically integrated biophotonic LoC (bioPhLoC) containing microoptical and microfluidic elements for cell retention, proliferation and simultaneous pH monitoring is presented. The monitoring of pH without interference from cells in the supernatant or in the optical path was achieved by incorporating two independent chambers connected by a microfluidic channel. A size-exclusion filter surrounded the first chamber to trap the cells, where they also proliferated, and the pH was monitored in the second chamber in real time. It is important to note that the micro-optical elements (microlenses, mirrors, etc.) integrated into the bioPhLoC were exclusively composed of PDMS and air, and were formed at the same time as the rest of the device structures in the PDMS casting step. This feature has an important impact on the cost and the robustness of the bioPhLoC.
Experimental BioPhLoC design The bioPhLoC with an integrated filter containing sizeexclusion microchannels is shown schematically in Fig. 1. From a fluidic point of view (Fig. 1A), it consists of two circular chambers connected by fluidic channels. The first chamber (incubation chamber: 0.42 cm diameter, 230 mm high) is connected to two inlets, one for the inoculation of cells and the other for culture medium. The cell inoculation inlet is directly connected to the chamber by a narrow fluidic channel (100 mm wide, 230 mm high), while culture medium is infused into the chamber through a 3 mm high size-exclusion channel, which allows the filtering of the culture medium before reaching the incubation chamber. This size-exclusion channel is part of the filter monolithically integrated into the incubation chamber. This filter consists of a 230 mm high wall with 21 size-exclusion channels (3 mm deep) on top of it to allow passage of medium but not
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Lab on a Chip
Fig. 1 A) Drawing of the bioPhLoC indicating the fluidic elements integrated into its structure (in grey in the figure), including the two fluidic inlets (one for cells and another one for cell culture medium), the fluidic outlet, the incubation and the monitoring chamber and the fluidic channels connecting them. B) 3D representation and cross section of the size-exclusion microchannels of the filter (in blue in the figure) integrated into the incubation chamber. C) Details of the optical elements integrated into each chamber of the bioPhLoC. These include self-alignment elements, PDMS microlenses and air mirrors, all of them in red in the figure, and absorbance filters (in dark grey in the figure).
cells. The size-exclusion channels are homogeneously distributed around the chamber structure to favour the uniform distribution of the cells around it. The geometry and dimensions of the filter are illustrated in Fig. 1B. As shown, the filter structure is trapezoid, with a progressive enlargement of the width from 300 mm in the incubation chamber to 450 mm outside it. This enlargement should benefit the filling of these structures, which are only 3 mm high. At their widest end, the size-exclusion microchannels are connected to a fluidic channel (250 mm wide, 230 mm high) that surrounds the incubation chamber and connects it with the second chamber, the monitoring chamber (0.5 cm diameter, 230 mm high). This second chamber is finally connected to the outlet. In terms of optical transduction, both chambers contain monolithically integrated micro-optical elements for optimal interrogation of the chip (Fig. 1C). Concretely, each reactor incorporates: (i) self-alignment elements (undulating channels in the PDMS matrix for the clamping and precise alignment of the optical fibres), (ii) microlenses (biconvex PDMS lenses for
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light collimation),22 (iii) absorption filters (circular cavities of 0.8 mm diameter filled with dye-doped polymeric materials to selectively absorb the excitation light during fluorescence measurements)23 and (iv) two sets of air mirrors surrounding the chambers (the inner mirror with a smooth reflection surface, the outer with a saw-tooth patterned surface). The role of the air mirrors is twofold. First, they are designed to confine the light inside the chamber, enhancing the performance of the bioPhLoC. Second, external optical noise (stray light beams reaching the bioPhLoC) will also be partially reflected back, thus reducing the amount of stray light inside the chamber. The working principle of the air mirrors is detailed in Fig. S1, ESI.3 It is important to note that the inclusion of the micro-optical elements does not increase the technological complexity of the device, since no additional photolithographic steps are required in their fabrication, as explained below. Fabrication of the bioPhLoC The bioPhLoC was fabricated by conventional soft-lithography.24 Briefly, a two-level master was fabricated using the negative tone SU-8 polymer (MicroChem Corporation, Newton, MA, USA). The size-exclusion channels were defined in the first level of the master (SU-8 2005, total height = 3 mm) whereas the other elements (chambers and microfluidic and micro-optical elements) were patterned in a second layer (SU-8 2050, total height = 230 mm). The masters were developed by immersion in propylene glycol methyl ether acetate solution (PGMEA, MicroChem Corporation, Newton, MA, USA) and baked. PDMS (Sylgard 184 elastomer kit, Dow Corning, Midland, MI, USA) replicas of the master (prepared as indicated by the supplier) were cured for 20 min at 80 uC. The structured PDMS was irreversibly bonded to a soda-lime glass wafer after treatment of both structures with oxygen plasma (18 s, 500 W) and heat (20 min, 60 uC). An image of the final bioPhLoC filled with crystal violet (only for visualization) is shown in Fig. 2A. It is important to note that the size-exclusion channels in the filter were prone to collapse during bonding because of their low aspect ratio (3 mm high, 300–450 mm wide). Optimization of this step resulted in devices where 60% of the microchannels bonded correctly to the glass wafer (Fig. 2B), 30% partially collapsed though still maintained their functionality, and around 10% totally collapsed (Fig. S2, ESI3). Though bonding of the microchannels of the filter was not perfect, it was still possible to carry out the experiments described below. Experimental set-up In order to minimize the risk of clogging, the bioPhLoC was introduced upside-down on a custom chip housing module fabricated in-house (Fig. S3, ESI3). With this orientation, the size-exclusion channels of the filter were positioned on the top part of the chip and not on the bottom, where cells will sediment and proliferate. The housing module was composed of a lid and a base piece with fluidic inlets and outlets coinciding with those designed in the chip. Suitable openings for the simple insertion of fibre optics were also included (see Fig. S3, ESI3). More precisely, the base piece included a shallow groove (2 mm deep) corresponding to the chip footprint, suitable channels for fluid transport
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Fig. 2 (A) Photograph of the microchip filled with crystal violet dye (for validation purposes). (B) Magnification of a section of the incubation chamber demonstrating the correct bonding of the structure. The dark yellow colour of the pillars surrounding the microfilters indicates their irreversible bonding on the glass wafer. In opposition, the size-exclusion microfilters, with a light yellow colour, were either not bonded or collapsed on the glass surface.
between the chip and the fluidic connects, and guiding grooves for the fibre optics and screw threads. The lid piece contained two chip observation windows (one for each chamber) and screw holes. The top and base parts were fastened together using M3 screws, once the chip was in place and the fibre optics inserted. The chip housing was fabricated by PMMA micromilling (Step Four, basi540) followed by solvent-assisted bonding. High-pressure fittings (Upchurch Scientific) were used as fluidic interconnects and PEEK tubing (Upchurch Scientific) was used to connect the chip housing module with the NEMESYS syringe pump (Cetoni GmbH, Germany) on the one hand, and the waste recipient on the other. Cells, buffer and culture media were introduced at constant flow rates by using a 10 mL glass syringe (Hamilton, Switzerland).
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Paper Optical measurements Optical measurements were performed simultaneously in both chambers using the previously described micro-optical elements. In both cases, light from a broadband halogen lamp (HL-2000-FHSA, Ocean Optics, USA) was coupled to the chamber through a 230 mm diameter multimode optical fibre (Thorlabs, Dachau, Germany). Transmitted light was collected at the opposite side of the chamber (optical path = 0.42 cm or 0.50 cm, for the incubation or the monitoring chamber, respectively) using identical fiber optics directly connected to a microspectrometer (USB2000, Ocean Optics, USA). This microspectrometer has a spectral resolution of 3 nm. Experimental protocol BioPhLoCs were sterilized by irradiation with UV light for 30 min in a laminar flowhood. The PMMA chip housing, fluidic connections, tubing and syringes were sterilized with 70% ethanol and placed in the laminar flow hood, where the experimental set-up was assembled under sterile conditions. After that, the whole system was sequentially flushed with ethanol (5 min) and phosphate buffered saline (PBS, pH 7.4, Life Technologies) for 5 min to remove ethanol traces. After the pre-conditioning of the experimental set-up, the bioPhLoCs were infused with cell suspensions (cell trapping stage) at a constant flow of 100 mL min21 to avoid cell sedimentation. Afterwards, depending on the experiment, the trapped cells were either washed with buffer (PBS), at 100 mL min21 (rinsing stage), or infused with culture medium (incubation stage) at very low flow rates (0.5 mL min21) to avoid shear stress during cell proliferation. During the incubation stage, the experimental set-up was sterilely introduced into a cell culture incubator just after cell inoculation. In the incubator, the cells proliferated at optimal experimental conditions (37 uC, 5% CO2). The variation of the transmitted light between 400 and 1100 nm was monitored with time in both chambers. In all experiments, the integration time per measurement was fixed at 80 ms, recording the average of 10 consecutive scans. Cell culture Escherichia coli (E. coli, CGSC 5073 K12) were grown overnight at 37 uC in Luria-Bertani (LB) culture medium. Similarly, Saccharomyces cerevisiae (S. cerevisiae, ATCC 9763) were grown for two days (48 h) at 25 uC in Saboraud dextrose broth (Pronadisa). Cultures were centrifuged at 3500 g and resuspended in PBS, pH 7.4. Microbial concentrations were determined by plating on agar containing Saboraud or LB medium, depending on the case. Concentrations around 108– 109 colony forming units per mL (CFU mL21) were normally obtained. Vascular smooth muscle cells (VSMC) were obtained from rat aorta by enzymatic dissociation with collagenase (Sigma, Spain) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic–antimytotic solution (10 kU mL21 penicillin G sodium, 10 mg mL21 streptomycin sulfate and 25 mg mL21 amphotericin B) (Gibco, Invitrogen, Spain). When confluent, the VSMC were passaged with 0.05% trypsin–EDTA (Gibco,
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Lab on a Chip Invitrogen, Spain) and an aliquot was dyed with trypan blue for cell counting. Then, the VSMC were centrifuged and resuspended in DMEM in a concentration of 106 cell mL21. The cells used in the experiments were between the 4th to 6th passage. The protocol for animal handling and experimentation agreed with the European Community guidelines for the ethical treatment of animals (86/609/EEC) and was approved by the Ethical Committee for Animal Research of the University of Seville.
Results and discussion Characterization of the optical elements integrated into the bioPhLoC For the characterization of the optical elements integrated into the bioPhLoC, three different testing structures were fabricated using the technology previously described. These structures (illustrated in Fig. S4, ESI3) were identical to the bioPhLoC but contained single chambers with optical paths of 0.30, 0.38 and 0.50 cm, respectively. Absorption and dispersion measurements were performed in these structures and compared with a commercial cuvette (optical path = 1.00 cm). It is important to note that, according to the International Union of Pure and Applied Chemistry (IUPAC), absorption refers to the physical process of absorbing light and dispersion of the light scattered by dispersing particles.25 Absorption measurements were performed using methylorange (Sigma-Aldrich), a dye with an intense absorption band at 470 nm, as the target analyte. Nine solutions containing between 0 and 140 mM methylorange in PBS were prepared and measured (in triplicate) using the test structures and a standard 1.00 cm cuvette. Measurements with the cuvette were performed with the same light source and spectrometer as those performed with the test structures and under the same experimental conditions. As predicted by the Lambert–Beer law, absorbance at 470 nm (Abs470 nm) increased linearly (between 0 and 80 mM) with the methylorange concentration (Fig. S5, ESI3), showing good correlation with the concentration in all cases (Table 1). The performance of the chips was evaluated by determining the methylorange molar absorption coefficient (eMO) and comparing it, first with the value obtained with the 1.00 cm cuvette, and next with the values reported in the literature. eMO were obtained (according to the Lambert–Beer law) from the slopes of the calibration curves, by dividing the slope by the optical path of each structure. The eMO of each structure is given in Table 1. All test structures presented values around 2 6 104 cm21 M21, very similar to that obtained by the 1.00 cm cuvette (2.24 6 104 cm21 M21) and those reported in the literature (between 2 6 104 and 3 6 104 cm21 M21, depending on the experimental conditions).26 These results verified the optical elements integrated therein for absorbance measurements. Dispersion measurements were performed using the Gramnegative bacterium E. coli. Seven E. coli suspensions (in PBS) ranging from 0 to 108 CFU mL21 were prepared and the dispersion was measured at 600 nm (in triplicate). As above with absorbance of dye solutions, a good correlation was
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Table 1 The linear fit, R2, LoD and eMO of the absorption (methylorange, at 470 nm) and dispersion measurements (Escherichia coli, at 600 nm) performed with the test structures with optical paths of 0.30, 0.38 and 0.50 cm and a 1.00 cm cuvette. In the linear fit, methylorange and E. coli concentrations are respectively in mM and CFU mL-1
Methylorange (absoption at 470 nm) Optical path (cm)
Linear fit
0.30 0.38 0.50 1.00 (cuvette)
Abs Abs Abs Abs
= = = =
(5 ¡ 3) 6 1023 + (58.7 ¡ 0.8) 6 1024 C (21 ¡ 4) 6 1023 + (76.8 ¡ 1.4) 6 1024 C (15 ¡ 4) 6 1023 + (93.1 ¡ 1.5) 6 1024 C (13 ¡ 9) 6 1023 + (224 ¡ 6) 6 1024 C
R2
LoDa (mM)
0.999 0.998 0.999 0.996
0.511 0.391 0.322 0.134
¡ ¡ ¡ ¡
0.007 0.008 0.005 0.004
eMO (cm21 M21) (1.96 (2.02 (1.86 (2.24
¡ ¡ ¡ ¡
0.03) 0.04) 0.03) 0.06)
6 6 6 6
104 104 104 104
Escherichia coli (dispersion at 600 nm) Optical path (cm)
Linear fit
0.30 0.38 0.50 1.00 (cuvette)
Abs Abs Abs Abs
a
= = = =
(22 ¡ 3) 6 1024 (29 ¡ 4) 6 1024 (5 ¡ 3) 6 1024 + (27 ¡ 4) 6 1024
+ (25.4 ¡ 0.8) 6 1026 C + (37.6 ¡ 1.3) 6 1026 C (64.1 ¡ 1.2) 6 1026 C + (131 ¡ 3) 6 1026 C
R2
LoDa (CFU mL21)
0.994 0.993 0.997 0.997
(11.8 ¡ 0.4) 6 106 (8.0 ¡ 0.3) 6 106 (4.68 ¡ 0.09) 6 106 (2.30 ¡ 0.05) 6 106
According to 3s criterion.
obtained between the absorbance at 600 nm (Abs600 nm) and bacteria concentration for all test structures and the 1.00 cm cuvette (Fig. S5, ESI3 and Table 1). This also verified the optical elements integrated into the bioPhLoC for dispersion measurements.
absorbance increased to 0.8 A.U., whereas in the monitoring chamber, it reached an initial value of 0.5 A.U. but quickly dropped to around 0.4 A.U. These differences are associated
Characterization of the filter integrated into the incubation chamber From a microfluidics point of view, the performance of the filter integrated into the incubation chamber in cell trapping should be evaluated. To this effect, small (,3 mm) and large particles (.3 mm) were inoculated into the bioPhLoC at 100 mL min21 (seeding and trapping steps). After that, a constant PBS flow (100 mL min21) was infused into the chip to remove nontrapped particles (rinsing step). Dispersion measurements were performed as previously detailed for the duration of the experiment using the PBS spectrum as a reference. The Gram-negative rod-shaped bacillus E. coli (1.5–2 mm long, 0.5 mm wide) and the round yeast S. cerevisiae (5–8 mm diameter) were selected as small and large particle models, respectively. 108 CFU mL21 suspensions of these microorganisms (in PBS) were infused into the bioPhLoC and subsequently rinsed with sterile PBS as previously described. The variation of the Abs600 nm with time for the E. coli suspensions is shown in Fig. 3A. Initially, the absorbance had a negligible value (within the experimental error) during the seeding step in both the incubation and monitoring chambers. As soon as the bacteria reached the incubation chamber (trapping step), the Abs600 nm increased to around 0.30 A.U. Almost immediately, an identical increase was recorded in the monitoring chamber. This confirmed that bacteria, too small to be trapped in the incubation chamber, crossed the sizeexclusion channels of the filter, reaching the monitoring chamber without impediment. Finally, during the rinsing step, the Abs600 nm gradually decreased in both chambers due to a progressive washing out of the bacteria with sterile PBS flow. In the case of the S. cerevisiae suspensions (Fig. 3B), the initial Abs600 nm value rapidly increased during the trapping step in both chambers. In the incubation chamber, the
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Fig. 3 Representation of the variation of the absorbance magnitude at a wavelength of 600 nm (Abs600 nm) with time in both the incubation and monitoring chamber during the seeding, trapping and rinsing steps for (A) small size E. coli bacteria and (B) large size S. cerevisiae yeast cells.
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Lab on a Chip
with the retention of yeast cells by the size-exclusion channels of the filter monolithically integrated into the incubation chamber. That is, yeast cells .3 mm in diameter were stably trapped in the microfiltering structure whereas yeast cells ,3 mm in diameter, membrane fragments and debris could easily cross the filter, reaching the monitoring chamber and increasing the absorbance. During the rinsing step, two different behaviours were recorded. In the monitoring chamber, the Abs600 nm quickly decreased with time as previously observed in the case of small-size bacteria. On the other hand, the Abs600 nm did not change in the incubation chamber during the rinsing stage. Thus, during this stage, most of the yeast cells remained trapped in the incubation chamber and were not washed out by the PBS flow. This confirms that the filter integrated in the incubation chamber effectively retains cells larger than 3 mm. A video illustrating the trapping of yeast cells in the size-exclusion filters has been included in the ESI3 (Fig. S6). VSMC culture Finally, the bioPhLoC was applied to cell culture using VSMC from rat aorta as a mammalian cell model. VSMC are round in suspension (8–10 mm in diameter), but once adhered to a surface, they proliferate with a classical spindle-shaped morphology (80–100 mm wide, 2.5 mm high) and a typical hill-and-valley phenotype.27 Therefore, once introduced into the bioPhLoC, the VSMC should be trapped in the incubation chamber, where they would proliferate under optimal experimental conditions (37 uC, 5% CO2 and a low continuous flow of DMEM at 0.5 mL min21 to avoid shear stress). At the same time, pH changes due to cell metabolism would be recorded in real time in the monitoring chamber, without interference of cells, by following the changes in the absorption band corresponding to the pH indicator phenol red, already present in the culture medium. Phenol red presents two absorbing forms, one at 440 nm (protonated form) and one at 560 nm (deprotonated form), whose magnitudes vary with pH.28 In order to evaluate this variation, nine DMEM solutions (containing 0.04 mM phenol red) at pH values ranging from 8.15 to 6.20 (determined with a pH-meter) were inoculated into 0.50 cm test structures and measured as previously detailed. Fig. 4 shows the absorbance spectra of these DMEM solutions (intensity spectra in Fig. S7, ESI3). As shown, absorption bands at 440 nm and 560 nm were respectively decreasing and increasing with the pH. A good linear relationship between the magnitude of both peaks and the pH was found between 8.15 and 7.05 (Fig. 4 inset and table). Below pH 6.70–6.80 and above pH 8.20, the magnitude of both bands does not vary linearly with pH. In terms of sensitivity (slope of the calibration curve, Fig. 4 table), the 560 nm band was 5 times more sensitive to the pH changes than the 440 nm one, under the experimental conditions presented here. The magnitude of the molar absorption coefficient, which is much larger for the deprotonated form of phenol red, is the reason for that difference. The precision for a DMEM solution at pH 7 was 7.1 ¡ 0.2 and 7.00 ¡ 0.02 (average value ¡ confidence interval considering 95% significance, n = 3) for calibration curves at 440 nm and 560 nm, respectively. Thus,
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Fig. 4 Absorbance spectra at wavelengths ranging from 350 to 900 nm for DMEM solutions at pH values ranging from 8.15 to 6.20. The variation of the absorbance at wavelengths of 560 nm and 440 nm with the pH is represented in the inset (error bars show the standard deviation of three repetitive measurements). In the table, the linear fit and the R square of the calibration curve at each wavelength are included.
under these experimental conditions, pH monitoring at 560 nm should be more precise and sensitive than at 440 nm. For cell culture assays, 106 cell mL21 suspensions of VSMC in DMEM were infused at 100 mL min21 (to avoid cell sedimentation) into the bioPhLoC already containing DMEM medium, with a 2.5 mL syringe (trapping step). After that, a constant DMEM flow (0.5 mL min21) was infused into the chip to remove non-trapped particles and the trypsin (enzyme used to detach cells) traces (rinsing step). During cell and DMEM infusion, optical measurements were performed as already detailed in both the incubation and monitoring chambers. Absorbance variation with time at three wavelengths was acquired: 440 nm (protonated form of phenol red), 560 nm (deprotonated form of phenol red) and 650 nm (cell dispersion). This latter wavelength was chosen instead of 600 nm to avoid the influence of the 560 nm band in the measurement. Results are shown in Fig. 5, where Fig. 5A corresponds to the incubation chamber and Fig. 5B to the monitoring chamber. The variation of the Abs650 nm in both chambers was different. In the incubation chamber, the Abs650 nm increased during the trapping stage to 0.10 A.U. and then progressively decreased to 0.02 A.U. at the end of the rinsing stage. This may indicate that VSMC infused into the bioPhLoC during the trapping stage were concentrated in the incubation chamber, increasing absorbance. After that, cells started settling on the bottom of the chamber by gravity, producing a constant decrease in absorbance. These results were confirmed by optical micro-
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Lab on a Chip
Fig. 5 Absorbance variation with time during seeding, trapping and rinsing stages at wavelengths of (&) 440, (#) 560 and (m) 650 nm, in (A) the incubation and (B) the monitoring chamber. (C) pH variation (from 560 nm wavelength) with time during seeding, trapping and rinsing stages in the incubation and monitoring chambers.
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Paper scopy (Fig. S8 and S9, ESI3 for the trapping and rinsing stages, respectively). Cell sedimentation may be favoured by the decrease in the flow rate from 100 (trapping stage) to 0.5 mL min21 (rinsing stage). In contrast, the Abs650 nm in the monitoring chamber remained constant at around 0 for the duration of the experiment. This result suggested that the number of VSMC reaching the monitoring chamber was not significant and most of them were stably trapped in the incubation chamber. Additionally, even considering the high plasticity of mammalian cells (without the rigid external wall present in most bacteria and yeast cells), VSMC are too big to safely cross the filter integrated into the incubation chamber. This is illustrated in Fig. S8, ESI,3 where most of the VSMC crossing the size-exclusion filter are shown to degenerate and die. Cell membrane breakdown, membrane fragments and small lipid vacuoles are clearly seen in this figure. Thus, the size-exclusion channels of the filter produce a stressful environment for mammalian cells, which degenerate and die before reaching the monitoring chamber. The Abs560 nm, initially around 0.28 A.U. (incubation) and 0.23 (monitoring), rapidly increased during the trapping stage to 0.63 and 0.43 A.U. for the incubation and monitoring chamber, respectively (Fig. 5A and B). This increase was attributed to the trypsinization process. Trypsin, the enzyme used to detach adhered cells, requires a basic medium (between 7.5 and 8.5) to work efficiently. Thus, the pH of these detached VSMC may be quite basic. Afterwards, the Abs560 nm decreased in both chambers until almost recovering the initial value (0.39 and 0.27 A.U. for the incubation and monitoring chambers, respectively). Although the trend was similar, the absorbance increase in the incubation chamber was double the one recorded in the monitoring chamber and the decrease was also deeper. This difference was produced by the presence of cells in the incubation chamber that interfere with the correct monitoring of the pH. This interference was even clearer when considering pH. Abs560 nm values were converted into pH values by interpolation in the calibration curve plotted in Fig. 4, inset. Fig. 5C illustrates the pH variation during seeding, trapping and rinsing steps in the incubation and monitoring chambers. The initial pH values (7.25 and 7.09 respectively for incubation and monitoring) were quite close to the value recorded with a pH-meter before inoculation into the bioPhLoC (7.11). However, the differences were accentuated at the end of the trapping stage. At this time, the pH recorded in the monitoring chamber (7.70) was still close to the one obtained with the pH-meter (7.80, determined in the suspended VSMC suspension before inoculation into the bioPhLoC), but it was quite far from the one registered in the incubation chamber (8.21) that was clearly influenced by the light dispersion produced by suspended cells. Thus, light dispersion produced by cells interferes with the correct monitoring of pH during cell introduction. Since Abs440 nm and Abs560 nm respectively correspond to the protonated and deprotonated form of phenol red, they are linked and an increase in one of them should produce an immediate decrease in the other. This connection was only found in the monitoring chamber where cells were not interfering with pH monitoring. Unfortunately, when interpolating the Abs440 nm for the monitoring chamber into the
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Paper corresponding calibration curve (Fig. 4, inset), the pH values obtained did not coincide with the ones recorded with the pHmeter (Fig. S10, ESI3). The differences were attributed to the interference of an additional band appearing at 390 nm (Fig.
Lab on a Chip S11, ESI3). This band, corresponding to the adsorption of the fetal bovine serum included in the culture medium to improve cell proliferation, impeded the correct monitoring of the pH through the 440 nm band. After rinsing, the bioPhLoC was placed in an incubator, where the trapped cells proliferated for 26 h under optimal environmental conditions (37 uC, 5% CO2) with a low continuous flow of DMEM (0.5 mL min21). During cell proliferation, optical measurements were performed as already detailed in both the incubation and monitoring chambers. The variation of Abs440 nm, Abs560 nm and Abs650 nm in the incubation and monitoring chambers are represented in Fig. 6A and 6B, respectively. In both chambers, the Abs560 nm decreased with time until stabilization after around 22 h, as expected by the medium acidification produced by cell metabolism. Although the tendency was similar, cell dispersion influenced the Abs560 nm in the incubation chamber (Fig. 6C) since small increases in the Abs650 nm (probably due to cell detachment, Fig. S12, ESI3) also produced an increase in the Abs560 nm in the incubation chamber, whereas the Abs650 nm continued to follow the expected trend in the monitoring chamber. Additionally, during cell proliferation, the 440 nm band could not be used to monitor pH due to interference by fetal bovine serum. Thus, in the monitoring chamber, pH changes (indicative of cell metabolism) could be suitably monitored by using the 560 nm band corresponding to the deprotonated form of the pH indicator phenol red, in real-time and without the interference of cells. As a future perspective, although control and monitoring of pH is a critical issue for cell engineering, this bioPhLoC may also be used to monitor other small or large molecules assimilated or secreted by cells. One interesting application may be the analysis of proteins, carbohydrates or DNA/RNA secreted by cells. These molecules can only be measured using UV light at wavelengths below 300 nm (specifically 280 nm for proteins,29 between 240 and 195 nm, depending on the molecules added to the solution, for carbohydrates30 or 260 nm for DNA/RNA31), which are toxic to cells. With bioPhLoC, cell proliferation and monitoring can be performed in separated chambers and secreted molecules could be measured in the monitoring chamber with highly energetic UV light sources without affecting cell proliferation. Thus, bioPhLoC may be an ideal tool for secretomics, which is the analysis of the secreted proteins of a cell.
Conclusions
Fig. 6 Absorbance variation with time during the proliferation stage at wavelengths of (&) 440, (#) 560 and (m) 650 nm , in the (A) incubation and (B) monitoring chambers. (C) pH variation (from 560 nm wavelength) with time during the proliferation stage in the incubation and monitoring chambers. Black arrows indicate areas where cell detachment influenced absorbance measurements.
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This article presents a bioPhLoC with two separated chambers, incubation and monitoring, for independent cell retention/ proliferation and pH monitoring, respectively. The bioPhLoC included a filter containing 3 mm high size-exclusion microchannels (for cell trapping) and optical elements (for the realtime interrogation of the cells and culture medium) monolithically integrated into the bioPhLoC structure. Both the filter and optical elements were demonstrated to perform correctly in the bioPhLoC structure. For cell culture applica-
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Lab on a Chip tion, VSMC were efficiently trapped in the incubation chamber, where they proliferated with normal morphology and proliferation patterns. During cell proliferation, pH changes due to cell metabolism were monitored in real-time in the monitoring chamber without interference of cells. As future work, the bioPhLoC architecture may also be applied to monitor small or large molecules assimilated or secreted by cells.
Acknowledgements The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007– 2013)/ERC grant agreement nu 209 243 and the I-LINK project ˜oz-Berbel was supported by the (I-LINK0392). Dr Xavier Mun ´n y Cajal’’ program from the Spanish Government. One ‘‘Ramo of the authors (S. B.) gratefully acknowledges the financial support of the Volkswagen Foundation and the German Research Foundation (DFG) in the framework of the Collaborative Research Group mikroPART FOR 856 (Microsystems for particulate life-science products). This work has been partially funded by grant CTQ2009-14390-C02-02 from the Spanish government to JM. Cell culture was performed in the Biology Service of the Centro de ´n, Tecnologı´a e Innovacio ´n of the University of Investigacio Seville (CITIUS). Authors want to acknowledge Dr Modesto Carballo (CITIUS) for scientific support and facilities.
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