Application Microfluidic Cell Analysis Devices (I) Date: 2013/05/24. Dr. Yi-Chung Tung

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1 Application Microfluidic Cell Analysis Devices (I) Date: 2013/05/24 Dr. Yi-Chung Tung Mechanotransduction and the Study of Cellular Forces Mechanical forces play a critical role in nearly all aspects of cell biology, from cell migration to morphogenesis to cell proliferation. hese forces are ubiquitous to the interactions between cells and their substrates (such as shear stress in the vascular tree). Even in the absence of applied external forces, cells themselves apply forces against their surroundings by actively contracting their actin-myosin cytoskeletal networks.

2 Nature Protocol PDMS Micro-Post Array Characterization of micropost array. Deformation vs. Dimensions.

3 PDMS Micro-Post Array Replica molding of a micropost array master. Surface treatment and pattern transfer. Cells on Microposts Force measurement and beyond.

4 Introduction Flow Cytometer Rapid analysis of biological samples Disease diagnosis and monitoring Cell biology Toxicology Environmental monitoring Introduction Flow Cytometer Basic Operation of Flow Cytometer Scatter Signal ic System Fluorescence Signal Normalized Light Intensity (%) Absorption of Excitation Light Optical System Light Emission Enumeration & Characterization Hydrodynamic Focusing Wavelength, λ

5 PDMS Microfluidic Channel Hydrodynamic Focusing 2D Observation Area Focusing Outlet Coaxial Flow Experimental Results PDMS Microfluidic Channel Hydrodynamic Focusing 3D Observation Area Focusing Outlet Coaxial Flow Experimental Fabrication Results SU-8 Top Layer Bottom LayerTriangular Guides SU-8 Layer 1 Si Wafer 300 µm SU-8 Layer 2 Si Wafer 300 µm

6 Opto-ic Intergated System Optical Detection Experimental Setup 100 µm Grooves for Optical Fibers Emitted Fiber 1 2 Emitted Fiber Optical Fiber Integrated System 100 µm Interrogation 4 Zone 3 Experimental Results (I) High Frequency Noise Lock-in Measurement for Fluorescence Detection Using Silicon-Based PIN Photodiode Simultaneous PMT and PIN Photodiode Detection

7 Experimental Results (II) o 135 o 180 o Coefficient of Variation (%) Total Flow Rate = 15 ml/hr Flow Rate (ml/hr) Experimental Results (III) 60 ~96% Recovery o 135 o 180 o 40 Recovery (%) Total Flow Rate = 15 ml/hr Flow Rate (ml/hr)

8 Simple 3D Hydrodynamic Focusing PDMS Single Channel Micro Channel Layer, Minimum made by Soft Interconnections: Lithography Single Inlet and Single Flow Inlet Numerical Simulation PDMS 3D computational Micro Channel fluidic dynamics made by (CFD) model. Soft Lithography

9 Confocal Imaging PDMS Fluorescein Micro and Channel Confocal made Microscopy by Z-Stack Soft Imaging. Lithography Bead Characterization PDMS Time-Domain Micro Measurement. Channel made by Soft Histograms Lithography of Peak Fluorescence Intensities.

10 Air Flow Cytometry PDMS Minimize Micro instrument Channel footprint made by Soft Less Lithography contamination concern Promising for Point-of-Care (a) Inlet ports (b) Air Air Flow Chamber 300 µm Observation Channel Outlet Black: Air Grey: Hydrodynamic Focusing (a) Stably focused aqueous sample core flow along the microfluidic channel of µfc. (a) flow rate = 20 ml/h, (b) flow rate = 10 ml/h, (c) flow rate = 6 ml/h. The unit of size bar is µm. Syringe pump Water (a) (b) (c) Syringe pump Vacuum pump Vacuum pump (b)

11 Braille Flow Cytometer Efficient Loading without damaging cells Small Amount Handling Capability (tens of µl) Flow Simulation 3D Laminar Model in Fluent 6.2 (Steady State) Path Lines Static Pressure Velocity in X-Direction Velocity in Z-Direction

12 Loading & Hydrodynamic Focusing 1 : 1 1 : 1/2 1 : 1/4 1 : 1/8 Bead Calibration

13 Cell Viability after Loading C2C12 Myoblast Cells Stained Using LIVE/DEAD Viability/ Cytotoxicity Kit Calcein AM for LIVE Cell (ex/em 494 nm/517 nm) Ethidium homodimer-1 (EthD-1) for DEAD Cell (ex/em 528 nm/617 nm) Viability (%) % 95.96% 95.69% 1-HR Experiment Before Control After Cell Cycle Analysis Human promyelocytic leukemic (HL60) cells Hypotonic DNA Stain: Sodium citrate Triton X-100 Propidium iodide Ribonuclease A Distilled Water

14 Single Embryoid Body Cell Counting ES Cell: Hanging drop cell culture to form spheroid Dissociated in Trypsin mixed with Syto 9 (40 µl)