Letter pubs.acs.org/Langmuir
Directing and Boosting of Cell Migration by the Entropic Force Gradient in Polymer Solution Tatsuya Fukuyama,† Ariko Fuke,‡ Megumi Mochizuki,‡ Ken-ichiro Kamei,§ and Yusuke T. Maeda*,†,∥ †
Department of Physics, Faculty of Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, 812-8581 Fukuoka, Japan Department of Physics and Astronomy, Graduate School of Science, Kyoto University, Oiwake-cho, Kitashirakawa, Kyoto 606-8502, Japan § Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Ushinomiya-cho, Kyoto 606-8501, Japan ∥ PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan ‡
S Supporting Information *
ABSTRACT: Noncontact manipulation of nano/micromaterials presents a great challenge in fields ranging from biotechnology to nanotechnology. In this study we developed a new strategy for the manipulation of molecules and cells based on diffusiophoresis driven by a concentration gradient of a polymer solute. By using laser focusing in a microfluidic device, we created a sharp concentration gradient of poly(ethylene glycol) (PEG) in a solution of this polymer. Because diffusiophoresis essentially depends on solute gradients alone, PEG solute contrast resulted in trapping of DNA and eukaryotic cells with little material dependence. Furthermore, quantitative analysis revealed that the motility of migrating cells was enhanced with the PEG concentration, consistent with a theoretical model of boosted cell migration. Our results support that a solute contrast of polymer can exert an interfacial force gradient that physically propels objects and may have application for the manipulation of soft materials.
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INTRODUCTION The manipulation of biological materials and living cells is crucial to biochemical investigations involving single-cell analysis, force spectroscopy, tissue engineering, and many other procedures.1,2 In particular, contactless manipulation with light forms the foundation of optomicrofluidic devices, which in turn are used in a variety of optical tools for coarsening droplets or separating small objects for microanalysis and biosensing.3−5 These various devices and tools call for the efficient trapping of functionalized particles or the reliable separation of cells for genetic analysis and therapeutics. Some of the available techniques for manipulating small particles use distinct physical properties of the particles themselves, such as their electromagnetic physical properties.6−8 However, most types of contactless methods require strong light intensity to create a potential field and also rely on material properties such as dielectric or paramagnetic properties, which limit their application to objects attached to beads in magnetic tweezers. To overcome this difficulty, alternative principles that can control molecules and cells with contactless and low material dependence remain a challenging issue. One promising mechanism is diffusiophoresis, which is a transport phenomenon triggered by solute concentration gradients. In a concentration gradient of ion species, colloidal particles as another solute move due to interfacial osmotic pressure gradients occurring across the narrow surface layer. © 2015 American Chemical Society
Strikingly, diffusiophoresis is essentially driven by the interfacial entropic force gradient, which means that the drift of particles is less dependent on the electromagnetic characteristics of the material itself. Since diffusiophoresis may have potential as a clean and effective tool in nano/microengineering and microanalysis, it has been extensively studied for molecular manipulation9−19 and a miniaturized engine of self-propulsive particles.20−22 Despite the versatility of diffusiophoresis, the manipulation of eukaryotic cells is still a challenge: these cells have complicated membranes and spontaneous motility distinct from those of particles or enzymes. The manipulation of cells by a clean method such as this one using solute contrasts with little material dependence may help to resolve one of the great challenges in biotechnology and nanotechnology. However, it is not clear whether such solute contrasts exert sufficient force on complex living cells, even if they are motile. In this Letter, by using our newly developed microfluidic device to control diffusiophoresis, we realize the long-lasting manipulation of Dictyostelium discoideum of social amoeba cells with solute contrasts of polymer. We further show that the spontaneous cell migration can be boosted along the concentration gradient. Our approach could be used to control Received: July 12, 2015 Revised: October 23, 2015 Published: October 23, 2015 12567
DOI: 10.1021/acs.langmuir.5b02559 Langmuir 2015, 31, 12567−12572
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Figure 1. Experimental setup for the generation of a stable, sharp solute gradient. (A) Schematic graph of the poly(dimethylsiloxane) (PDMS) device containing a manipulation chamber and a pressure-regulated actuator. DNA or cells are manipulated in the chamber by the focusing of the infrared laser, and the PEG solution that diffuses in and out of the chamber avoids gradual water pervaporation. (B) Schematic protocol of the investigations into the diffusiophoresis effect. Infrared light locally heats an aqueous solution, and a concentration gradient of PEG arises due to thermophoresis. (C) Photograph of the microfluidic device. The scale bar is 10 mm. (D) Diffusiophoretic trapping of DNA in 3.0% PEG solution. The distribution is plotted at intervals of 30 min. The inset figure is the temperature distribution against the radial distance. Solid lines are fitted curves of the solution of the theoretical model at steady state. allowed us to visualize F-actin in the cells in order to precisely track the cells. For the preparation of nonmotile cells, we treated cells with 10 μM latrunculin B for 1 h and obtained round cells lacking visible actin meshworks. The round cells were less motile, exhibiting a small velocity of V = 0.6 ± 0.2 μm/min. Imaging and Cell Tracking. The time lapse of ABD-GFP expressing cells was recorded at a 30 s interval using an epifluorescence microscope equipped with a CMOS camera (Zyla, Andor) and a 75 W xenon lamp. Excitation light was selected from a 457 nm filter with an ND-25 filter, and emission light was from a 487 nm filter. The images were analyzed by MATLAB software, and then the center of mass was calculated so as to obtain the velocity and mean square displacement of cells.
an interfacial force gradient for a wide class of biomaterials, from DNA to living cells. This diffusiophoretic manipulation may be applicable to the assembly of biomaterials with living cells and the sorting of cells with regard to substratum adhesion.
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EXPERIMENTAL SECTION
Microfluidics. To examine the effectiveness of diffusiophoresis for cellular trapping, we built a microfluidic device in which the chamber was connected to a reservoir of the solution in order to improve the measurement duration. The microfluidic device was made of poly(dimethylsiloxane) (PDMS) elastomer (Sylgard 184 purchased from Dow Corning). The mold for the PDMS device was fabricated by a conventional photolithography technique (Supporting Information). Optical Setup. The local temperature and concentration gradients of a poly(ethylene glycol) polymer (PEG10000 purchased from WAKO chemicals) were built by infrared laser heating. An infrared laser of 1480 nm wavelength was focused by the objective lens at a magnification of 20× (NIKON).16 The height of the PDMS chamber was 30 μm, and this small thickness was sufficient to suppress thermal convection. The distribution of the temperature difference was measured through the temperature dependence of the fluorescence intensity of fluorescent dye 2′-7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF from Molecular Probes). The maximal temperature rise is 4.0 K from the peripheral temperature of 20 °C. The calibration curve of the fluorescence temperature was independently measured in a fluorescence spectrometer and was determined to be −1.3%/K. Cell Cultures and Chemical Treatment. We used the single-cell eukaryotic amoeba Dictyostelium discoideum to demonstrate the diffusiophoretic manipulation of living motile cells. The cells were grown in HL5 medium supplemented with folic acid and selective antibiotic G418. We used the AX-2 strain that constituently expresses ABD-GFP, which visualizes filamentous actin proteins and thus
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RESULTS AND DISCUSSION We first describe the theoretical framework of diffusiophoresis in an aqueous solution by considering single colloidal particles of large diameter immersed in a solution of small polymer molecules. At the solid surface under a no-slip condition, the polymer is repelled from the solid surface due to a steric effect, resulting in the formation of a depletion region of nanosized thickness. In the polymer concentration gradient ∇cPEG parallel to the solid surface, which leads to an osmotic pressure difference, the imbalance between hydrostatic pressure and osmotic interfacial stress occurred in the depletion layer near the solid surface. The excess hydrostatic pressure gradient is then balanced by the stress of shear flow along a polymer concentration gradient.23 One way to achieve this diffusiophoresis is through the cross effect of light-induced thermophoresis. The thermal diffusion of solutes, relative to fluid at rest, occurs under a temperature gradient.24,25 When the local temperature gradient ∇T is present, the steady-state concen12568
DOI: 10.1021/acs.langmuir.5b02559 Langmuir 2015, 31, 12567−12572
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Figure 2. Diffusiophoretic manipulation of eukaryotic amoeba cells. (A) Trajectory of directed motile Dictyostelium cells in various PEG gradients. The focused spot is the center of the boxes. (B) (Left) Representative images of single cells upon exposure to a concentration gradient in 5.0% PEG solution. Dictyostelium cells move toward the focal point of the infrared laser, which corresponds to the bottom of the PEG concentration gradient. The scale bar is 50 μm. (Right) Trapping of cells in 5.0% PEG solution and the absence of trapping in 0% PEG. We defined the trapping index as the relative increase in the number of cells within the circle area with a 60 μm radius (white dashed line in the left figure) from the focal point. We calculated this index by the number of cells at t = 50 min based on the number at t = 0 min. Cells are accumulated at the laser spot as the index becomes larger than 1. (C) The average time to travel 60 μm is inversely proportional to the PEG concentration. Each dot is obtained after averaging over n = 5 cells. The error bar is the standard deviation. The directed movement was also observed even in the actin-depolymerized cells.
tration profile is the gradient of solute concentration c(r) = c0exp[−STΔT(r)], where D is the diffusion coefficient of a solute, DT is the thermal diffusion coefficient, ST = DT/D is the Soret coefficient, and ΔT(r) = T(r) − T0 is the temperature difference at a radial distance r from a heated spot (r = 0). c0 and T0 are the solute concentration and temperature at infinity, respectively. We then consider the aqueous solution of three components: water, larger colloidal particles, and smaller polymer molecules (e.g., PEG) as solutes. Assuming that the concentration of the polymer whose Soret coefficient is SpT is denser than colloids but still in a dilute regime, one expects that its concentration profile at steady state cP(r) will follow the exponential function shown above. Single large particles are in turn subject to both temperature and polymer concentration gradients whose drive thermophoresis and diffusiophoresis, respectively, and the flow of large solute particles can be described by9,12,13 J = −D∇c − cDT∇T + cu. The velocity driven by diffusiophoresis is u, and for spherical colloids it is given as u = (kBT/3η)λ2(SpT − 1/T)cP∇T with the size of depletion layer of the polymer λ and the viscosity in bulk η. As a result, the particle moves, its transport free from material details. Low material dependence is advantageous for the manipulation of soft biological materials. However, in a closed chamber used in a previous study,16 the prevaporation of solvent sometimes disturbs the solute gradient. Toward stable diffusiophoretic trapping over hours, we built a microfluidic device in which the chamber was connected to a reservoir of the medium in order to improve the measurement duration. Figure 1A−C depicts the experimental setup. An infrared laser was focused into a square-shaped chamber, and a concentration gradient of polymer was generated. This
chamber was connected to a microchannel in which the PEG solution flowed so as to avoid the leaking of solvent. We also added a pressure-driven actuator in order to take (or eject) solutes into (or from) the chamber pneumatically. The mixing and repetitive intake of solution was carried out, and then a uniform distribution of PEG concentration was chosen as an initial condition just prior to laser irradiation. In our experimental setup where the maximal temperature rise was 4.0 K and the solute drop of PEG10000 was 21% with its Soret coefficient of SpT = 0.065 [1/K] (Supporting Information), linear DNA of 4.4 kbp that was a digested product of pBR322 by EcoR1 was trapped in the focusing center of the laser light (Figure 1D). We then investigate the effect of diffusiophoresis on living motile cells to determine whether a polymer concentration gradient can exert sufficient force to direct cell migration. For the sake of simplicity, we employed social amoeba cells, Dictyostelium discoideum, in the vegetative phase. The rationale underlying this choice was as follows. In the absence of external stimuli, the cells move slowly with a velocity of 3.8 ± 1.6 μm/ min, and their directional persistence is low enough to be treated as normal diffusion.26−28 In addition, chemotaxis and cell−cell signaling mediated by second messengers of cAMP can be neglected in this phase. 29 Thus, a vegetative Dictyostelium cell is a suitable model for this study. Figure 2A shows the trajectories of Dictyostelium cells that were placed in different concentration gradients of PEG10000 and the temperature gradient. At 5.0% PEG, the trajectory of cells was clearly directed toward the center with little angular fluctuation. The cells were trapped in the focused spot within 15 min (Figure 2B). As the PEG concentration in solution was 12569
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Figure 3. Analysis of the boosted migration of Dictyostelium cells. (A) Mean square displacement (MSD) of the cells under a concentration gradient of PEG. The MSD was calculated from the center of mass during forced cell migration. Red, 5.0% PEG; blue, 3.0% PEG; green, 0% PEG. Solid lines are curves fitted with eq 1. (B) Velocity of boosted cell transport for wild-type cells, which was obtained by fitting with eq 1, at various PEG concentrations. (C) Diffusion coefficient of boosted cell transport for wild-type cells at various PEG concentrations. It was obtained from curve fitting with eq 1. (D) MSD of the treating cells with latrunculin B in a gradient of PEG. Red, 5.0% PEG; blue, 3.0% PEG; green, 0% PEG. (E) Velocity of boosted cell transport for latrunculin B-treated cells under various PEG concentrations. Asterisk indicates significance (p = 0.05) by the student’s t test. (F) Diffusion coefficient of boosted cell transport for latrunculin B-treated cells.
transport? To quantitatively analyze this issue, we must take into account both random and forced motion: the directed motion observed in Figure 3 appears to be the sum of two distinct modes, forced ballistic motion and spontaneous random motion. The mode of spontaneous random motility can be assumed to be Brownian random motion. The mean square displacement (MSD), ⟨Δr(t)2⟩ = ⟨(r(t + t0) − r(t0))2⟩t0 where ⟨ ⟩t is the ensemble over time, follows as 4Dt in the random motion. In contrast, the forced ballistic motion has a different time dependence given by (2/π)2Vt2t2, where Vt is the transport velocity by diffusiophoresis. By taking into account these two modes, the generalized MSD of amoeba cells under an entropic force gradient can be written as (derivation in Supporting Information)
3.0%, the cells started to show fairly directed migration with wobbling toward the hot region. However, under the 0% PEG condition, cells show only spontaneous cell migration with an average velocity of 2 μm/min, which is comparable to the speed reported in a previous study.26 The cells at the 0% PEG were not accumulated in the focused spot (Figure 2B), meaning the absence of themotaxis or negligible thermophoresis compared to spontaneous motility. For detailed characterization of the observed motion, we tracked the cells that were 60 μm away from a laser focus (60 μm is comparable to the width of the trapped molecular distribution in Figure 1D) and then measured the time, τ, taken to reach the trapped locus. As shown in Figure 2C, τ was inversely proportional to the bulk PEG concentration, meaning that the directed migration was faster when a larger amount of PEG was present. Moreover, we also analyzed the trapping of nonmotile cells in order to evaluate the possibility of rheotaxis, but they were still trapped under PEG gradients along with the intact cells. One difference was that we required a longer duration of laser irradiation to fully trap the nonmotile cells. This result implies an asymmetry in spontaneous motion, namely, motile cells are given an additional boost forward as they approach the PEG gradient, but their motion appears to be reduced upon hitting the PEG gradient. The solute contrast of polymer can transport living eukaryotic cells, but the results shown in Figure 2 raise an important question: why is the time for trapping of nonmotile cells always longer than that for the trapping of motile cells? Does this difference reflect the change in diffusiophoretic
⟨Δr(t )2 ⟩ = 4Dt + (2/π )2 Vt 2t 2
(1)
. Figure 3A shows the MSD obtained from the experimental data and theoretical fitting curves of eq 1. We confirmed that nonlinear behavior dependent on t2 becomes more apparent as the PEG concentration is increased, meaning that the experimental results agreed well with the phenomenological expression. The obtained Vt and D are plotted in Figure 3B,C as a function of cPEG. The velocity displays a linear relation with cPEG, and this proportional relation is consistent with the phenomenological model discussed above while the diffusion coefficient is not affected, which indicates that this model describes enhanced and directed motility occurring on a shorter 12570
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Langmuir time scale. Although the accumulation of cells is shown in Figure 2, the trajectories were too short to detect the trapping of cells from the dynamics of MSD plots. Next, the MSD of the cells treated with latrunculin B was analyzed in order to determine if it has a detectable ballistic force mode (Figure 3D). We found that the transport velocity was lower than that of intact cells and directed motion was found in more than 4.0% PEG solution (Figure 3E). The diffusion coefficient of drug-treated cells is much reduced because both spontaneous deformation and motility are inhibited, but no tendency was found for the PEG concentration (Figure 3F). Diffusiophoresis can induce an enhanced motion of nonmotile cells in a large concentration of polymer solutes, but the reduction of Vt was observed. This may reflect the strengthened adhesion between cells and the PDMS substrate: the cells may not easily detach from the PDMS surface after F-actin inhibition and in turn adhered to the substrate.30 We also analyzed the aspect ratio of the cell shape, which is defined as the ratio of the major axis to the minor axis, and found that it was changed from 1.7 ± 0.4 to 1.3 ± 0.4 after actin inhibition (Supporting Information). This difference is not so large as to alter the diffusiophoresis. Although further investigation would be required, adhesion properties might be relevant to the reduced diffusiophoretic velocity.
ACKNOWLEDGMENTS
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REFERENCES
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OUTLOOK In this study we have shown that DNA and living cells of Dictyostelium were directly manipulated by exploiting a transport phenomenon called diffusiophoresis in sharp polymer gradients generated by local heating in a microfluidic device. We found that the diffusiophoretic manipulation ensures low material dependence as an exclusive property. In addition, the transport of the cells could be described in a phenomenological model with the transport velocity of Vt proportional to cPEG, which was qualitatively consistent with the theoretical model. The fact that, in the presence of a sufficient solute contrast, transport phenomena affect the direction of cell migration may have implications with regard to the role of the concentration gradient; solute gradients function not only as chemical cues7,8,29 but also physical couriers of cells. We envision that diffusiophoresis will ultimately provide the engine for clean nanotechnology, relying on solute contrast alone, from biosensing19 and the directed assembly of materials to the separation of drug-sensitive cells. ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02559.
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We thank K. Inoue and S. Sawai for their technical advice with regard to the Dictyostelium cells. Financial support was received in the form of a PRESTO grant from the Japan Science and Technology Agency and research grants from the Hakubi Center, WPI-iCeMS, Opto-Science Foundation, and Inamori Foundation.
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Letter
Experimental methods and theoretical model of boosted cell migration (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest. 12571
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