PROBING DYNAMIC CELL RESPONSES USING A ROBUST …
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PROBING DYNAMIC CELL RESPONSES USING A ROBUST DIFFUSIVE GRADIENT GENERATOR J. Atencia * , G.A. Cooksey and L.E. Locascio National Institute of Standards and Technology, USA ABSTRACT This abstract describes a diffusive gradient generator that leverages the properties of laminar flow to control the diffusive microenvironment in a cell culture chamber with unprecedented robustness and simplicity in design and operation. Using this device, a population of mammalian cells is exposed to different concentrations of a toxin within a concentration gradient. Cell response is probed dynamically by cycling the gradient on and off. KEYWORDS: Diffusive gradients, chemical gradients, dynamic cell assays INTRODUCTION Engineered diffusive gradients preserve cell-cell communication and are shear free, but are difficult to create. Current de- signs rely on using membranes, gels or resistive pressure balance between diffusive sources and sinks to prevent convection. These approaches result in long gradient formation transients, compromise the robustness of the gradient, or require complex geometries that are difficult to operate [1-5]. THEORY Our approach leverages the properties of laminar flow in a unique way, to passively decouple convection from diffusion. The design has two layers. In the top layer, two fluids of different solute concentration flow through a microchannel with a ‘T’ configuration (Fig.1A). Vias (through-holes) located at the same ‘x’ and different ‘y’ position, connect the microchannel in the top layer with a microfluidic chamber in the bottom layer (Fig.1B). In fully developed laminar flow, the velocity field is purely axial and the pressure varies only in the ‘x’ direction [6]. Thus, the pressure at the vias is inherently balanced and mass transport between them through the chamber is purely diffusive. Figure 1: In a straight channel under fully developed laminar flow, the pressure is constant at each cross-section. (A) Vias located at the same cross section in the main channel (A) serve as connection to a microfluidic chamber in the bot- tom layer (B). Thus, the vias are subjected to the same pressure, which ensures purely diffusive mass transport inside the chamber. (C) Macrograph showing a microfluidic gradient generator with four diffusive microfluidic chambers. 978-0-9798064-4-5/μTAS 2011/$20©11CBMS-0001 39 15th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 2-6, 2011, Seattle, Washington, USA
Transcript of PROBING DYNAMIC CELL RESPONSES USING A ROBUST …
PROBING DYNAMIC CELL RESPONSES USING A ROBUST DIFFUSIVE GRADIENT GENERATOR
J. Atencia* , G.A. Cooksey and L.E. Locascio National Institute of Standards and Technology, USA
ABSTRACT
This abstract describes a diffusive gradient generator that leverages the properties of laminar flow to control the diffusive microenvironment in a cell culture chamber with unprecedented robustness and simplicity in design and operation. Using this device, a population of mammalian cells is exposed to different concentrations of a toxin within a concentration gradient. Cell response is probed dynamically by cycling the gradient on and off. KEYWORDS: Diffusive gradients, chemical gradients, dynamic cell assays
INTRODUCTION
Engineered diffusive gradients preserve cell-cell communication and are shear free, but are difficult to create. Current de-signs rely on using membranes, gels or resistive pressure balance between diffusive sources and sinks to prevent convection. These approaches result in long gradient formation transients, compromise the robustness of the gradient, or require complex geometries that are difficult to operate [1-5].
THEORY
Our approach leverages the properties of laminar flow in a unique way, to passively decouple convection from diffusion. The design has two layers. In the top layer, two fluids of different solute concentration flow through a microchannel with a ‘T’ configuration (Fig.1A). Vias (through-holes) located at the same ‘x’ and different ‘y’ position, connect the microchannel in the top layer with a microfluidic chamber in the bottom layer (Fig.1B). In fully developed laminar flow, the velocity field is purely axial and the pressure varies only in the ‘x’ direction [6]. Thus, the pressure at the vias is inherently balanced and mass transport between them through the chamber is purely diffusive.
Figure 1: In a straight channel under fully developed laminar flow, the pressure is constant at each cross-section.
(A) Vias located at the same cross section in the main channel (A) serve as connection to a microfluidic chamber in the bot-tom layer (B). Thus, the vias are subjected to the same pressure, which ensures purely diffusive mass transport inside the chamber. (C) Macrograph showing a microfluidic gradient generator with four diffusive microfluidic chambers.
978-0-9798064-4-5/µTAS 2011/$20©11CBMS-0001 39 15th International Conference onMiniaturized Systems for Chemistry and Life Sciences
October 2-6, 2011, Seattle, Washington, USA
RESULTS AND DISCUSSION Concept validation A chip was fabricated using plastic laminates, double-sided tape and a razor cutter. To validate that the diffusion chamber is free from advection, a fluorescein solution and water were flowed into the main channel. Fluorescence intensity was moni-tored in three diffusive chambers over time. Concentration gradients of fluorescein were observed to form gradually and reached steady state after ≈ 30 min. Gradients in the 3 replicate chambers were linear, stable, and identical. Dynamic cell assays. Vero cells, transfected to produce destabilized green fluorescence protein (GFP) to indicate ribosomal activity [7], were seeded in three chambers and exposed to an intermittent chemical gradient of cycloheximide (CHX), a ribosomal inhibitor (Fig. 2). Incorporation of a proteasome targeting sequence in the GPF results in reduction in cell fluorescence during CHX exposure. Within the 43 h duration of the experiment, the gradient was switched on and off three times. Fig. 3 shows the temporal evolution of the GFP intensity averaged over three diffusion chambers. Each trace corresponds to a binned region within the diffusion chamber, and thus, to GFP levels at a different concentration of toxin. We also compared the reproduci-bility of GFP decay and accumulation in each region over the different cycles. To this end, the GFP values were normalized at the beginning of each cycle. We found that GFP accumulation followed the same trends across cycles.
Figure 2. (A) A population of GFP-Vero cells was exposed to an intermittent gradient of the toxin CHX. For analysis, the
diffusive chamber was binned into 12 regions of similar CHX concentration ranges (linear gradient across the chamber). (B-D) Micrographs showing the GFP intensity evolution inside the diffusive chamber: (B) at the beginning of the experiment, (C) 12 h after exposure to CHX, and (D) 7 h after recovering cells with CHX-free media. Scale bar is 200 µm.
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Figure 3. Schematic showing the timeline of the experiment to probe cells over three consecutive cycles of toxin and re-
covery. Traces show the evolution of mean GFP intensity in each binned region of the CHX gradient. During exposure to toxin, GFP intensity decayed in all except region 12 (the region without toxin), which increased linearly over the duration of the experiment.
CONCLUSIONS
The ability to create arbitrary perturbations on the cell diffusive microenvironment and monitor protein activity in time will open the wide repertoire of mathematical tools and assays from control engineering to study cell dynamics, such as time-domain and frequency-domain based assays.
ACKNOWLEDGEMENTS We thank John Elliott, Michael Halter, Anne Plant and Daniel Sisan for helpful discussions. We also thank Connie Rog-ers-Newcome from Adhesive research. Inc for providing tape samples. REFERENCES [1] V. V. Abhyankar, M. A. Lokuta, A. Huttenlocher and D. J. Beebe, Lab Chip, 2006, 6, 389-393. [2] D. Kim, M. A. Lokuta, A. Huttenlocher and D. J. Beebe, Lab Chip, 2009, 9, 1797-1800. [3] B. Mosadegh, C. Huang, J. W. Park, H. S. Shin, B. G. Chung, S. K. Hwang, K. H. Lee, H. J. Kim, J. Brody and N. L.
Jeon, Langmuir, 2007, 23, 10910-10912. [4] D. Irimia, G. Charras, N. Agrawal, T. Mitchison and M. Toner, Lab Chip, 2007, 7, 1783-1790. [5] J. Atencia, J. Morrow and L. E. Locascio, Lab Chip, 2009, 9, 2707-2714. [6] F. M. White, Viscous fluid flow, McGraw-Hill Higher Education, New York, NY, 2006. [7] M. Halter, J. L. Almeida, A. Tona, K. D. Cole, A. L. Plant and J. T. Elliott, Assay Drug Dev Techn, 2009, 7, 1-9. CONTACT *J. Atencia, tel: +1-301-9753589; [email protected]
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