Engineering H193A - Team Project Gateway Engineering Education Coalition P. 1Spring Quarter 2004 Engineering H193A Basics of Microscope Use (Guest Lecture Introduction to Cell Adhesion Cell Adhesion Measurement using Lab on a Chip Week 3 Day 2

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 2Spring Quarter 2004 Introduction to Cell Adhesion All types of cells will adhere to each other, to surfaces, and to other components in the ECM (ExtraCellular Matrix)

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 3Spring Quarter 2004 Forces Involved in Adhesion Biochemical Interactions –Membrane proteins can recognize specific molecules outside the cell “Receptor-Ligand” interaction Combination of molecular structure and functionality Physical or Topographical Interactions –Micro- and Nanostructured surfaces guide or direct protein adsorption the helps control cell orientation and migration. –Physical cues may also guide the production of ECM or exoskeleton –The size of the cell and the surface topology dictate how many interactions occur between the cell and the surface.

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 4Spring Quarter 2004 Biological response to surface interaction If the interactions are highly attractive, the cell may deform to maximize those interactions – it is not a rigid body. –Cells may orient or move Cell-surface interactions do play a role in the biology of the cell. The transfer of information from outside to the inside the cell via membrane proteins is called “signaling”. These complex pathways affect: –Differentiation as cell divides –Proliferation or growth –Expression of genes or proteins

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 5Spring Quarter 2004 “Biomimetic” Surface Structures The ability to control surface features on the same scale as molecules (I.e. nanoscale) has opened new areas of research: –Do nanoscale structural features impact cell adhesion? –How many ligands are necessary to bind a cell and initiate a biochemical signal? –Can we create synthetic surfaces to elicit a certain biological response?

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 6Spring Quarter 2004 Lotus-effect™ An example of physical surface structure affecting interactions Biomimetic micro/nanoscale surface texture based on a lotus leaf Combines with hydrophobic surface molecules to repel water and dirt Super-hydrophobic 20μm 10 μm 200nm W. Barthlott, Planta, 1997 (surface of lotus leaf) Feng et. al, Angew. Chem., 2003 (PVA extrusion through membrane) Xie et. al, Advanced Materials, 2004 (Phase separation of PMMA and PFU mixture in DMF)

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 7Spring Quarter 2004 Design Idea: Super-Hydrophobic Valve Typical PMMA Surface, θ~ 70 º (Water drop: 10 μl) Fluorine Plasma Treated Smooth PMMA Surface, θ ~ 95 º (Medium: CHF 3 ; Power: 300w; Flow rate: 50 SCCM; Time: 2 min) Super-Hydrophobic PMMA Surface, θ > 160 º (Fluorine plasma treated surface with microstructures) Water flow

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 8Spring Quarter 2004 A Potential Pattern for Study We are investigating the feasibility of creating these “nanopillars” on PDMS through a plasma treatment. –Dr. John Lannutti - MSE You may choose this type of surface as one of your three surface structures. –May delay initiation of experiments by one lab period because of the extra processing step –Have a back-up in case it doesn’t work on PDMS

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 9Spring Quarter 2004 Yeast Shearing Procedure Several Phases: 1.Yeast Setup and Incubation in Chip 2.Preparing Microfluidics System 3.Low Pressure Flush 4.Yeast Shearing at gradually increasing water column heights 5.Cleanup and Repeat as necessary

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 10Spring Quarter 2004 Procedure Notes Things to be aware of: Differences in chips designs call for different low pressure flush approaches Chips with auxiliary yeast ports will require tape to seal third unused port during different phases Be careful not to over-pressure when inserting syringe directly into PDMS chip port. Keen observation is a must! Proper cleanup is a must! Iteration is a must!

Engineering H193A - Team Project Gateway Engineering Education Coalition P. 11Spring Quarter 2004 Cell Adhesion Experiment – Overview We have 3 microscopes available for use Follow procedures closely –Flow is similar to microfluidics experiment –Some will have chips that can be innoculated with a separate feed well Those not working on a microscope can prepare your chips by priming them with water and getting rid of air bubbles –Tomorrow is also set aside for this experiment. Worksheet #2 should be completed while not working on the microscope