- Methodology article
- Open Access
An easy-to-build and re-usable microfluidic system for live-cell imaging
© The Author(s). 2018
- Received: 24 November 2017
- Accepted: 11 June 2018
- Published: 20 June 2018
Real-time monitoring of cellular responses to dynamic changes in their environment or to specific treatments has become central to cell biology. However, when coupled to live-cell imaging, such strategies are difficult to implement with precision and high time resolution, and the simultaneous alteration of multiple parameters is a major challenge. Recently, microfluidics has provided powerful solutions for such analyses, bringing an unprecedented level of control over the conditions and the medium in which cells under microscopic observation are grown. However, such technologies have remained under-exploited, largely as a result of the complexity associated with microfabrication procedures.
In this study, we have developed simple but powerful microfluidic devices dedicated to live-cell imaging. These microsystems take advantage of a robust elastomer that is readily available to researchers and that presents excellent bonding properties, in particular to microscopy-grade glass coverslips. Importantly, the chips are easy-to-build without sophisticated equipment, and they are compatible with the integration of complex, customized fluidic networks as well as with the multiplexing of independent assays on a single device. We show that the chips are re-usable, a significant advantage for the popularization of microfluidics in cell biology. Moreover, we demonstrate that they allow for the dynamic, accurate and simultaneous control of multiple parameters of the cellular environment.
While they do not possess all the features of the microdevices that are built using complex and costly procedures, the simplicity and versatility of the chips that we have developed make them an attractive alternative for a range of applications. The emergence of such devices, which can be fabricated and used by any laboratory, will provide the possibility for a larger number of research teams to take full advantage of these new methods for investigating cell biology.
- Live-cell imaging
- Control of the cellular environment
Deciphering cell biological processes and understanding the dynamics that underlie fundamental functions in living cells has greatly benefited from the continual development of advanced methods in microscopy. It has now become clear that the investigation of cellular events in a wide range of time scales is integral to our understanding of cell biology, elucidating immediate responses to various stimuli as well as long-term behaviors. However, while complex time-lapse acquisitions based on state-of-the-art imaging procedures have become a standard approach in the life sciences, the real-time observation of cellular responses to changes in the environment or to specific treatments has not progressed at the same pace. Indeed, most of the strategies that are adapted to such experiments still rely on basic perfusion systems that lack versatility and time resolution. Recently, microfluidic technologies coupled with high-resolution microscopy have brought a number of solutions to the limitations that are intrinsic to standard perfusion setups . For instance, they rely on microvolumes , improve the time resolution of the assays , are compatible with non-adherent cells [4–6], and allow for multiplexing assays in a single experiment . Importantly, taking advantage of the well-established properties of fluid flow within microchannels [8–10], microfluidic systems also provide the capacity to simultaneously alter several parameters of the cellular environment with unprecedented flexibility, accuracy and dynamics [11–13]. Finally, their emergence has been supported by the ongoing development of advanced chip structures, opening new doors of investigation for researchers [2, 14, 15]. From ever more complex fluidic circuits [16–18] to the use of microsystems for assessing the physical properties of living cells , the advantages of microfluidics for the life sciences are undisputed, and the design of novel microdevices has become a dedicated area of research.
Nevertheless, the impact of microfluidics and its popularization in biological research laboratories have not been as significant as may have been predicted . One of the major obstacles is the complexity of the fabrication and usage of these devices. Indeed, the building of microfluidic chips adapted to live-cell imaging is a time consuming process that requires costly equipment, dedicated and controlled clean rooms, as well as specific expertise in microfabrication [20–22]. Furthermore, modulating the flow and composition of the growth medium in a chip while monitoring cells by microscopy is challenging. It relies on a clear understanding of specific concepts including the impact of the fluidic resistance at different points in the network and the relationship between flow rate and fluid velocity, which can generate shear stress in the cells. In addition, flows in microchannels are generally laminar, and the mixing of solutions is limited to diffusion: additional microstructures are therefore often required to enhance mixing efficiency . All together, these different aspects of the use of microfluidic systems have hampered their integration in most biology research groups.
A number of commercial systems have recently tried to circumvent these issues, offering high-end and expensive plug-and-play devices compatible with most standard microscopy setups. However, they do not allow any customization of the fluidic networks, a property that is an integral part of what microfluidics can offer to research in the life sciences. More versatile “home-made” systems have been described, including low-cost paper-based devices , wax microchips  or easy-to-pattern materials . Simpler channel designs and fluid handling methods have also been used for biological assays [27, 28]. However, the fabrication and utilization of these devices are still time-consuming and rely on equipment that is not always available to non-specialized research labs. Furthermore, most of these systems are not re-usable. Implementing microfluidic technologies in a laboratory therefore remains an endeavor that is challenging and costly. Paradoxically, the race toward complexity and ever more powerful microfluidic devices has lost sight of the needs of a majority of research teams. Indeed, while the ideas of lab-on-chips [14, 29, 30] or even organ-on-chips [31, 32] are promising in the long-run, they do not reflect what is currently critical for a large number of investigations in cell biology. It is therefore crucial to establish methods and protocols for the fabrication and use of simple customizable microsystems by non-experts. This is an essential step for fully taking advantage of these revolutionary tools.
As discussed above, one of the major barriers to the integration of microfluidic approaches in biological research laboratories is the complexity of the microfabrication process. In this study, we have addressed this issue and developed a novel microfluidic system dedicated to live-cell imaging that is easy-to-build, customizable and cost-effective, while possessing many of the advantages associated with microfluidics. This device employs a commercially available elastomer in which different types of microfluidic networks can be easily engineered without high-end equipment. We show that the transparent elastomer layer can be bonded to microscopy-grade glass coverslips and re-used several times, a significant advantage for the implementation of these chips in research laboratories. We then provide evidence that it can be coupled to precise controllers, allowing for rapid, accurate and simultaneous control of various parameters of the cellular environment such as the temperature of the sample and the composition of the growth medium. Importantly, we present proof-of-concept experiments that demonstrate the biocompatibility of such microdevices with both yeast and mammalian model systems. Finally, we show that when applying a constant flow of medium through the chips, these systems are well-adapted for the real-time monitoring of cellular responses to dynamic perturbations of their environment. Taken together, our results describe an affordable, easy-to-build and re-usable microfluidic system that will be an asset for research teams in cell biology and contribute to the expansion of microfluidics in the life sciences.
A new material for the simple fabrication of complex microfluidic chips
Bonding strength of the elastomer
Relationship between the flow rate and the applied pressure
Flow rate (μL/min)
Finally, we investigated the possibility to design elastomer chips that would allow for performing multiple simultaneous assays (e.g. experiment and controls, different treatments of a single cell line). This would involve the fabrication and independent use of distinct channels within a single chip. To test this, we built two channels separated by only 300 μm (Fig. 1f) and intercalated this elastomer layer between two glass coverslips. Water was then injected in one of the channels, while the other was perfused with a solution of the fluorescent dye Rhodamine B. Identical and constant flow rates in both channels were maintained for 8 h (20 μL/min; as a reference, the total volume of liquid in each of these channels is ~ 6 μL), and the fluorescence intensity was measured at different positions (Fig. 1f). Strikingly, we observed no cross-contamination between the channels even at the narrowest point of the network, demonstrating the compatibility of this material with the design and use of multi-channel microsystems.
Taken together, these results demonstrate that the LSR elastomer is a promising candidate for generating microfluidic devices for live-cell imaging, which is compatible with the fabrication of complex channel networks and with experimental multiplexing on single chips.
Precise and simultaneous control of the environment in the perfusion channel
As border effects and parabolic flow profiles are often observed in microfluidic devices [8, 10], we set out to assess the homogeneity of the media switches along an axis perpendicular to the flow. We performed the same assay as above (flow rate = 20 μL/min) but determined the fluorescence intensity over time at various distances from the edge of the channel (Fig. 3c). This showed that the position within the channel had only a limited influence on the dynamics of medium exchange. The border effect is therefore likely to be negligible for most applications and only a concern when it is essential to have a high level of homogeneity in the channel. Finally, we demonstrated the reproducibility of the media switch dynamics by performing successive renewals within the same microdevice using the same Rhodamine B assay (Experiments 1 and 2 in Fig. 3d). Importantly, although all the experiments presented above were made with a proof-of-concept design, our conclusions are applicable to other networks as well, with only minor additional characterizations and optimizations. Furthermore, simple gravity-based flows and manual valves can easily be used to renew and switch the fluids in the chips, although they do not provide the degree of precision and reproducibility of more high-end controllers.
While the LSR elastomer cannot be compared to demanding photolithography processes, as it does not allow the fabrication of extremely small channels and multilayered structures, our results show that it represents an ideal material for the fabrication of simpler but powerful microsystems. In addition, the possibility to re-use the elastomer layer multiple times is an important advantage, making these microdevices cost-effective, easy to use, and more likely to become integrated in biology laboratories.
Live-cell imaging using the elastomer chips
Autofluorescence of the elastomer
100.7 ± 1.8
100.9 ± 1.8
102.3 ± 2.2
102.8 ± 2.3
104.5 ± 2.7
104.9 ± 2.7
101.6 ± 1.9
101.6 ± 2.1
101.5 ± 2.0
101.6 ± 2.0
Finally, we tested the use of the elastomer chips with adherent mammalian cell lines. To this end, we seeded HeLa cells in either our microsystems or standard cell culture-grade 12-well plates. While the plates were subsequently kept in a controlled CO2 incubator without medium renewal, a constant flow of fresh medium (5 μL/min) was applied to the chips after cells had adhered to a fibronectin-coated glass coverslip (~ 3 h after their inoculation). For ease of manipulation in this assay, a temperature of 37 °C was ensured in the microchannels using a high precision hot plate. Cells were then allowed to grow for 28 h in these conditions. As was the case for fission yeast, we did not observe any detectable difference in cell growth between the chips and the plates, with similar increases in cell density, no apparent defects in cell morphology and no increase in cell death (Fig. 4c). Again, not only did this experiment show the compatibility of our devices with the use of mammalian cells, but they also suggested that the shear stress resulting from the maintenance of a constant flow of medium in the chips does not affect these cells.
Collectively, these results demonstrate that our microsystems can be used to perform live-cell imaging experiments with different types of cells without any apparent defects or alterations to their proliferation potential.
Dynamic modulation of the cellular environment and real-time monitoring of cellular responses
To fully establish our chips as easy-to-build but powerful systems for live-cell imaging experiments, in particular when dynamic modulation of the medium composition is required, we set out to perform different media switches and monitor the responses of cells to these alterations.
Finally, we performed a similar synchronization experiment using HeLa cells and the CDK1 inhibitor RO-3306 . Cells resuspended after trypsin treatment were injected in the chips or in standard culture dishes and allowed to re-adhere to the surfaces for 3 h at 37 °C. 6 μM RO-3306 was then added to the control cultures or perfused to the cells in the microdevices for 20 h. In the latter case, the flow was triggered for 10 min every hour at a flow rate of 13 μL/min. This periodicity was implemented as a constant flow of fresh inhibitor over such a long period of time was toxic to the cells (our unpublished observations); this effect is not seen in the control cultures, in which the inhibitor is not constantly renewed. Cells were then released in inhibitor-free medium for 15 min at 20 μL/min and then maintained at a constant flow rate of 5 μL/min. Controls were subjected to a single medium switch. The percentages of both mitotic cells (rounded) and post-mitotic cells (duplets) were then determined. We observed similar profiles in both the chips and the controls, however with somewhat different fine kinetics (Fig. 6b). This is likely due to the major differences in culture conditions between the two approaches. In addition, cells may still be affected by some toxicity of the RO-3306, despite our protocol of periodic rather than constant renewal. Nevertheless, together with our biocompatibility assays in the presence of a constant flow (Fig. 4c), this demonstrates that media switches and the analyses of the responses of cells to such treatments in real-time are also possible for mammalian cells in our microsystems.
Microfluidic technologies represent a powerful approach for investigating cell biology, in particular when coupled to high-resolution fluorescence microscopy. However, they are not used to their full potential in research laboratories due to the complexity of the procedures for microdevice fabrication as well as to the requirement for expensive equipment and specific expertise. We have developed a system that is easy to fabricate and use, taking advantage of cost-effective solutions in its simplest version while still providing a number of advantages associated with microfluidics. These chips are re-usable, biocompatible, easily coupled with high-end photonic microscopy and allow for a dynamic control of the cellular environment during live-cell imaging assays. Furthermore, in contrast to the commercially available systems, they remain customizable and permit the implementation of a range of fluidic networks, including the multiplexing of several experiments on a single chip.
While simple to implement, this methodology has certain limitations. Specifically, it does not allow the fabrication of microsystems with the complexity and resolution obtained when using high-end clean room equipment. For instance, multi-layered chips that require advanced photolithographic processes cannot be constructed using our method. In addition, as we found that the LSR material absorbs small hydrophobic molecules, a well-known issue when using PDMS, our devices are especially suited when such treatments are not necessary. However, as discussed above, experiments involving small molecules are possible, but particular attention must then be paid to testing the interference of the LSR with each compound before use and adapting the experimental conditions accordingly. In addition, the scoring area must be restricted in order to limit the influence of the material’s absorptive properties.
Nevertheless, while these microsystems do not cover the full spectrum of microfluidic-based experiments for cell biology, they remain well-adapted to a broad range of assays that require the real-time monitoring of living cells in optimal and highly controlled growing environments. Furthermore, the thickness of the material makes these devices suited for the use of more complex multicellular systems, from organoids to embryos. Together with the simplicity, limited associated costs and compatibility of the described chips with live-cell imaging approaches, these features are likely to promote the expansion of microfluidics to laboratories that do not have expertise in the development of microdevices.
Microfluidic systems provide an unprecedented level of dynamic control over the cellular environment in a variety of experimental contexts, in particular for challenging live-cell imaging approaches. However, their complexity of fabrication and usage has been an obstacle to the integration of these technologies in a majority of research laboratories. Through the identification of a novel material, we propose a simple approach for the implementation of basic yet powerful microfluidic chips that can be achieved without extensive technical expertise and microfabrication equipment. These systems will allow a larger community of researchers to benefit from the use of microfluidics for cell biology.
Fission yeast strains and methods
Standard media and methods were used [44, 45]. The strain used in this study was DC450 (leu1Δ:Pcdc13::cdc13-L-cdc2as::cdc13–3’UTR::ura4 + cdc2Δ::kanMX6 cdc13Δ::natMX6 cig1Δ::hphMX6 cig2Δ::kanMX6 puc1Δ::leu2 + ura4-D18 h + ) . The inhibitor-sensitive Cdc13-L-Cdc2as fusion protein was previously described . All experiments were carried out in minimal medium plus supplements (EMM6S) at 32 °C. The 3-MBPP1 inhibitor (A603003, Toronto Research Chemical Inc.) was dissolved in DMSO at a stock concentration of 10 mM and added to the medium at the indicated concentrations. Size at division was determined from DIC images using Fiji (National Institutes of Health). Generation time of cells grown in the chips was determined by live-cell imaging as the time between two successive septation events.
Mammalian cell lines and methods
All experiments were performed using HeLa Kyoto cell lines stably expressing an H2B::mCherry fusion protein (from plasmid 21,045, Addgene; Gregory Eot-Houllier, unpublished data). Cells were grown in DMEM GlutaMax medium (Thermo Fisher Scientific) supplemented with 10% Fetal Calf Serum, 10 mg/L streptomycin (Life Technologies), 10 units/L penicillin (Life Technologies) and 25 mM HEPES (Sigma Aldrich). The CDK inhibitor RO-3306 (Selleckchem) was used at a final concentration of 6 μM. All control experiments were performed in 12-well polystyrene dishes. For injection in the microdevices, cells cultures in polystyrene dishes were washed with PBS, treated with trypsin and resuspended in culture medium.
Imaging for the results presented in Figs. 2c-e, 3b-d, 4a-b, 5a-b and 6a was performed using an inverted Zeiss Axio Observer (Carl Zeiss Inc.) equipped with a Lumencor Spectra X illumination system and a Hamamatsu Orca Flash 4.0 V2 sCMOS camera. The data for Figs. 1f and 3c were obtained using a similar setup that is equipped with a spinning disc confocal head and a laser bench (Visitron GmbH). For both systems, images were acquired using Visiview (Visitron GmbH). The images displayed in Figs. 1c-e (bottom panel) and 4c were acquired using a Zeiss Axio Vert. A1 (Carl Zeiss Inc.) equipped with a Nikon 1 J3 camera and a PlasDIC system. All image analyses were performed using Fiji (National Institutes of Health).
6 mm-thick extruded Perspex® PMMA (poly methyl methacrylate) for the chip manifolds was purchased from Vink-France. The double-sided adhesive tape (142 μm thick) used for the temperature control layer is ARcare 90,880 (Adhesives Research Inc.). The 250 μm-thick elastomer used for fabricating all the perfusion channels is Liquid Silicone Rubber (LSR) HT6240 40SH from Silex Silicones Ltd. UK. Glass coverslips (170 μm thick) were purchased from Menzel-Gläser, and plastic coverslips (Rinzel 72,261–60, 280 μm thick) are from Electron Microscopy Sciences. Bonding of the plastic coverslips to the PMMA manifolds for Figs. 4c and 6b was achieved using epoxy adhesive (Sader).
The designs and dimensions of the different layers constituting the chips described in this study are presented in Additional file 4.
Fabrication of chips for short-term experiments
The PMMA manifolds, temperature control layers and perfusion channels were obtained in the appropriate materials using a CO2 laser cutter (Speedy 100, Trotec, Austria). All designs were made using Corel Draw. The cutting parameters (laser power and speed) must be optimized for the laser machine used. To prevent deterioration when exposed to solvents, PMMA blocks were cured for 2 h at 90 °C. To assemble the chips, the manifolds were first bonded to the temperature control layers (fabricated in double-sided adhesive) and then to glass coverslips containing connecting holes generated using the CO2 laser cutting machine. The elastomer perfusion channels were then manually mounted (slowly depositing the LSR sheet onto the manifold layer from one side to prevent the formation of bubbles), sealing the PMMA/adhesive top layers with microscopy-grade glass coverslips. Rigid Teflon tubing (Cluzeau) was used to connect the chips to the temperature control machine as well as to the flow control system; the connections were sealed using epoxy glue. For the perfusion layers, the inner diameters of the tubings were 0.5 mm (upstream) and 1 mm (downstream), thereby limiting the impact of the chip fluidic resistance on the fluidic networks.
Fabrication of chips for long-term experiments
For long-term experiments, the glass coverslips separating the temperature and perfusion layers were replaced by plastic coverslips (Electron Microscopy Sciences, Rinzel 72,261–60, 280 μm thick). This was due to the relative weakness of the glass coverslips after laser cutting of the connecting holes. In this case, the temperature control layers were directly engraved within the PMMA manifolds using the CO2 laser machine, and bonding to the plastic coverslips was achieved by stamping of epoxy glue. Connecting holes in the plastic coverslips were made using a drilling machine. The other layers of the chips were identical to those of the devices fabricated for short-term experiments. For simplicity, all the proof-of-concept experiments using mammalian cells were performed using similar chip structures but without the temperature control channels (direct bonding of the plastic coverslip to the manifold). The temperature of the samples was then maintained using high precision hot plates.
Chip preparation and cell loading
All mounted chips were sterilized by treatment with UV for 15 min prior to use. For experiments with non-adherent fission yeast cells, the bottom glass coverslip was coated with lectin (Sigma Aldrich). This was achieved by injecting lectin (0.5 mg/mL) in the chips using manual syringes. After a 30 min incubation, chips were thoroughly rinsed with deionized water, and cells were manually injected in the system. For experiments with adherent mammalian cells (Fig. 6b), glass coverslips were similarly coated with fibronectin (0.5 mg/mL) and rinsed with culture medium prior to cell injection. Resuspended cells were then injected and allowed to re-adhere to the glass substrate in a wet chamber placed at 37 °C in a CO2 incubator. Chips were kept in these conditions for a minimum of 3 h prior to applying any flow of medium.
Control of the cellular environment in the microfluidic chips
Control of the temperature within the chip was calibrated and performed as previously described [13, 46], taking advantage of fine temperature regulation by Peltier elements (Cherry Biotech). The precise dimensions of the temperature control channels for the calibration were slightly different than for the layers used in the chips (see Additional file 4): while the thickness was the same, the length and width were altered to facilitate the connection of the metal electrodes to the measurement device (model 34972A from Agilent Technologies). Importantly, using this microfluidic system, the temperature of the sample relies on the thickness of the perfusion channel as well as the flow rate in the temperature channel (≥5 mL/min according to manufacturer’s instruction, which was validated in all our chips). Thus, the calibration performed using the modified temperature channel is valid for all chips described in this study. Control of the medium flow rates was achieved using flow sensors that regulate pressure controllers (Elvesys), and a valve matrix permitted rapid media switches (Elvesys). To limit the presence of bubbles within the microfluidic networks, bubble traps (Elvesys) connected to vacuum pumps (KNF) were mounted upstream of the chips. See Fig. 3a for a schematic of the full system. The application of a constant flow or frequent renewal of medium is necessary to maintain optimal growing conditions. In this context, the environment of the cells is imposed by the medium injected rather than by exchanges through the LSR material.
Cleaning of the chips for reusability
The chips developed in this study can be re-used several times, with the bottom glass coverslip being the only single-use component of the entire system. The LSR layer can easily be peeled off manually from both bottom and top layers. Prior to re-use, all chips were dismantled, and all tubings were thoroughly washed with deionized water, 70% ethanol and dried using pressurized air. The elastomer layer was cleaned in an ultrasound waterbath at room temperature for 15 min, subsequently washed with 70% ethanol and dried using pressurized air.
We thank Pei-Yun Jenny Wu for critically reading the manuscript and members of the SyntheCell and Genome Duplication and Maintenance teams for the useful discussions. We are grateful to Grégory Eot-Houllier and Laura Magnaghi-Jaulin for their help with the use of HeLa cells, Damien Debuisson from Cherry Biotech for his support with the temperature control system, and Michael O’Brien from Adhesives Research Ireland for his advice and for generously providing us with samples of adhesive tapes.
JB was co-funded by a grant to DC from the Direction Générale des Armées (DGA), France and by Cherry Biotech, France. This work was supported by a collaborative grant from the Région Bretagne, France. The DGA and Région Bretagne did not participate in any aspect of the research project and preparation of the manuscript. Cherry Biotech helped with the temperature calibration of the chips.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
JB designed and performed all the experiments. LG provided support with the microfabrication. JC financially supported JB and helped with the temperature calibration. DC supervised the study, designed the experiments and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
LG and DC declare that they have no competing interests. JB was co-funded by Cherry Biotech, France. JC is the president of Cherry Biotech, France, which commercializes the temperature control system integrated in the microdevices.
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