1. Introduction Abstract Results and Analysis Conclusions Materials and Methods Acknowledgements Microfluidic devices allow for high throughput experimentation and complex biological analysis. There was a need for classification of various trap and device designs which inspired three new design concepts of (1) increasing trap density, (2) wingback traps, and (3) curved-side arrays. Quantification of trapping characteristics and informal, yet careful observations led to trap and device characterization. To this, a novel multi-perspective imaging system was added to further integrate the strengths of the old system with cutting edge techniques in cellular imaging. Materials and Methods Design of internal cell trapping and imaging systems are central to development of lab- on-a-chip technology. These products combine to give our final design, a high efficient internal system design for lab-on-a-chip technology. Optimized Cell Trapping and Multi-Perspective Imaging Using Microfluidic Devices Jeff Chamberlain, Matt Houston, Eric Kim Advisors:Kevin Seale, John Wikswo Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE), Department of Biomedical Engineering at Vanderbilt University The benefits of using microfluidics for cellular studies are well documented. Microelectromechanical systems (MEMS) devices are constructed using batch fabrication techniques, such as soft lithography, which allow rapid fabrication of multiple devices. The devices used in this project contain arrays of barriers which passively trap cells. These arrays allow the experimenter to perform high-throughput experimentation. Using only microliters of reagent, complex biochemical analyses of hundreds of individual cells can be accomplished in each experiment. There was a need in the VIIBRE laboratories for characterization of the trapping efficiency of various trap designs. There were many trap designs already in use, and no classifications existed for the different designs. When maximizing efficiency, one must first define what efficiency is for each experiment. For some experiments, efficiency is considered to be capturing the greatest percentage of loaded cells possible, minimizing waste. For other experiments, the experimenter may desire control over the number of cells captured by each trap. Computational fluid dynamics were used to characterize flow around the traps, connecting theory and application. Design Specifications and Constraints for MPW / Microfluidic Device Action/Solution (1) Mirrored Pyramidal Wells (MPWs) (a) [100 ]Silicon etches at fixed angle of 54.7 o (b) Side lengths of bottom of wells must be 40% larger than diameter of specimen (c) Incident light ray reflected off top of well goes clears the top of the specimen (1) Calculations and photolithography mask design (a) Accounted for in calculations (see Figure 3) (b) Accounted for in calculations (see Figure 3) (c) Accounted for in calculations (see Figure 3) and time of etching in KOH (2) Cell Traps (a) Improve trapping efficiency (b) Allow multi-perspective imaging (2) Changes to trap design (see Figure 2) (a) Added gap to increase flow through trap, wings to direct cells into traps (b) No part of the well is covered by the trap (3) Microfluidics (a) Minimize fluidic resistance (b) Prevent clogging of device (c) Provide uniform flow throughout device (3) Changes to microfluidic design (a) Eliminate extraneous channel lengths, use wide channels (b) Density of traps low in front, increases toward the back (c) Device width in trapping region is uniform (4) Assembled Device (a) Prevent leakage (b) Allow high magnification ( > 50x) imaging (c) Traps must align with wells with maximum error of + 3 micromoeters (4) Assembled device design (see Figure 4) (a) Minimize fluidic resistance, ensure strong passive bonding by maintaining clean surfaces - careful fabrication (b) Thin PDMS layer to keep total device thickness less than working distance of high-power objectives (WD<1mm) done by spinning the polymer onto the master (c) Device aligned under microscope during fabrication and slow-cured at room temperature to prevent shrinkage The MPWs are fabricated using well-documented processes for etching silicon and metal film deposition. Briefly, the array is defined using photolithographraphy on a silicon wafer with an oxide layer as a mask. The silicon is etched using KOH. Once the wells are etched and the oxide mask is removed, the entire wafer is coated with aluminum using electron beam evaporation. The result is an array of MPWs with dimensions appropriate for the specimen. Fluidic delivery and cell loading was done using the same equipment and techniques as previously described for the trapping efficiency experiments. The second portion of our project was to design a microfluidic network to interface with a novel microscopic imaging technology, known as Mirrored Pyramidal Wells (MPWs), developed by one of our advisors* (Figure 1). Using the original techniques to load and analyze the specimen in the MPWs, data acquisition is limited to only a single well for short periods of time. Our design (a) greatly increases the experimental throughput by passively and rapidly guiding the specimen into many wells, (b) allows for long-term analyses by providing continuous and controllable perfusion, and (c) compounds the experimental potential of the MPWs by maintaining the specimen in a microfluidic environment. A special thanks to our advisors John Wikswo and Kevin Seale, as well as Igor Ges, Dmitry Markov, Don Berry, and the rest of the members of the VIIBRE laboratory. D Figure 1. MPWs allow simultaneous multi-perspective imaging using inverted square pyramids with reflective walls, with each wall providing a different view of the specimen. MPWs can provide three- dimensional spatial information and increased signal-to-noise ratios not possible with conventional microscopy. A) SEM micrograph of MPW. B) SolidWorks rendering illustrating MPW concept. C) Autofluorescent Helianthus Annuus pollen grain in MPW. D) Demonstration of scale. *Technology and images developed by Kevin Seale* 40,000-well array Device Fabrication: 1) Our devices were made of polydimethylsiloxane (PDMS), a silicon elastomer, 2) SU-8 masters from photolithography techniques, and PDMS fluidics from soft lithography practices, and 3) Fluid was pumped by gas-tight syringes via PEEK Polymer Tubing (ID 50 µm). Flow was controlled by Harvard Apparatus Syringe Pumps. Cell loading: 1) cells aspirated from a pellet, 2) into inlet B, 3) captured inside the square trapping array. Analysis of loading: Different trap designs were tested. Media was continually flowed into the device for viability and modeling. Images were taken using an inverted microscope and Metamorph©. The images were analyzed using ImageJ. Three new trap designs were created and tested (Figure 2): A) Curved-side arrays: Cell flow weaves between traps. Non-linear boundaries to force diagonal flow, yet maintain constant flow velocities throughout B) Wingback traps: attempt to guide flow into downstream traps. C) Gradient-Trap-Density arrays: High density trap arrays tended to clog due to cell clumping. Density of traps was varied from low in front to high in back of the array. Low density traps helped break up the clumps. ABC Figure 2. Schematics to elucidate reasons for changed trap and device designs. A) Curved-side arrays. B) Wingback traps. C) Trap density increases with axial position in device. Figure 11. Different trap types represented here are graphed according to the percent of traps filled and percent of traps filled with only one cell. Calculation of percentages are based upon sums of all traps in each column, each column representing N=1. N values are as follows: 1) SFLD N=24, 2) TSLD N=16, 3) SSLD N=8, 4) TSHD N=30, 5) TFHD N=30, and 6) Curve- Sided N = 84. The standard deviations represented in graph B show the relatively even trapping in the Curve- Sided array. This is also elucidated by the front to back line graphs in C and D. These line graphs show the % filled for each column in the array from front to back. C represents low density older traps, and D represents the Curve-Sided traps. AB DC S = Square T = Triangular S = Split-Back F = Full-Back LD = Low Density HD = High Density Flow Si Wafer Cross Section of One Well PDMS Cell Light Source and Imaging Exit Port Trap Array Figure 4. AutoCAD image of general cell trapping device. Inset shows DIC image of trapped cells within a device. Minimum Etch Depth h = d + w/2 * tan(19.4 o ) LIGHT RAY 19.4 O h d w b h - d 54.7 O h h / tan(54.7 o ) Figure 3. Schematic of a cell inside of a well and equations used to determine well dimensions Well Bottom Size b = w – 2*h / tan(54.7 o ) Loading Ports GoalsSuccesses Cell trapping system must fill at least 90% of traps; 10% of those traps, at least, must hold single cells only (20% for high-success). Necessary for MPW operation Gradient density, straight, square, split-back traps achieved a total trapping of ~90% and a total single cell trapping of ~20%. This makes them optimal for MPW integration. Imaging phenomena on the cellular scale from multiple, and potentially simultaneous, viewpoints of the specimen with MPW's. Microfluidics successfully coupled with MPW arrays, and images of cells in the wells have been acquired. Prototypes with both thick and thin PDMS. Accurately generate computer fluid dynamics profiles for our traps for use to support theory of cell loading and allow for future research and development. Vector velocity approximation of single and array of traps for two different trap types obtained. Also velocity contours for same. Figure 9. Velocity vector simulation of wing back (left) and triangular split back (right) individual traps. Vectors display velocity magnitude with both color and length. Symmetry was utilized. Figure 10. Velocity vector simulation of wing back (left) and triangular split back (right) trapping arrays. Vectors display velocity magnitude with both color and length. Cell momentum is not accounted for in simulation. Figure 6. Microfluidic PDMS coupled with MPW. Figure 5. 3D representaions of single coupled trap and well (top) and array of coupled microfluidics and MPWs. Figure 7. Actual image taken (10X magnification) during experiment showing issues with air bubbles and cell clumping. Figure 8. Actual image taken to display proper alignment of trapping array with MPW array.