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INVESTIGATING THE MECHANICAL PROPERTIES OF LIVING HUMAN CELLS

INVESTIGATING THE MECHANICAL PROPERTIES OF LIVING HUMAN CELLS. Mark Murphy: GERI & BML Catherine Randall: GERI Alexis Guillaume: Université Claude Bernard, Lyon. PROJECT BREAKDOWN. Mark Murphy Cell Biology Atomic Force Microscopy Fluorescence microscopy. Catherine Randall

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INVESTIGATING THE MECHANICAL PROPERTIES OF LIVING HUMAN CELLS

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  1. INVESTIGATING THE MECHANICAL PROPERTIES OF LIVING HUMAN CELLS Mark Murphy: GERI & BML Catherine Randall: GERI Alexis Guillaume: Université Claude Bernard, Lyon

  2. PROJECT BREAKDOWN • Mark Murphy • Cell Biology • Atomic Force Microscopy • Fluorescence microscopy • Catherine Randall • Force Data Analysis • Image Processing • Alexis Guillaume • Computer Modelling • Simulation

  3. WHAT DO WE ALREADY KNOW ABOUT CELL MECHANICS? • Very Little! • Cytoskeleton • Microtubules, Actin Filaments, Intermediate Filaments & Associated Proteins • Changes in the cytoskeleton occur during most normal cellular processes

  4. ACTIN (green) & TUBULIN (red) CYTOSKELETON OF HUMAN LUNG FIBROBLAST CELL (LL24)

  5. WHY SHOULD WE STUDY CELL MECHANICS?

  6. WHY SHOULD WE STUDY CELLMECHANICS? • Changes in the cytoskeleton are associated with many human diseases, such as: • Cancer • Heart Disease • Premature Aging • Skin Fragility • Liver Disease • Such pathologies were first interpreted as ‘Mechanical Weakness Disorders’

  7. HOW DO WE MEASURE MECHANICAL PROPERTIES OF CELLS?

  8. Laser Photo Detector Z-piezo Sample X-Y Stage Objective Lens THE ATOMIC FORCE MICROSCOPE

  9. CANTILEVER WITH HAIR 25 µM

  10. Indentation (nm) Deflection (nm) 2 1 Non-contact region 0 0 Z-Movement (µM) 1 2 AFM FORCE CURVES

  11. LIVE HUMAN LUNG FIBROBLAST CELL AFM Deflection Image of Human Lung Cell 3-D Image of Human Lung Cell (Reconstructed From Height Image)

  12. THE HERTZ MODEL

  13. THE HERTZ MODEL • Describes simple elastic deformations for perfectly homogeneous smooth surfaces Fcone = 2/π · E / (1-v2) · tan(α) · δ2 Fparabola = 4/3 · E / (1-v2) · √R · δ3/2 • Two Unknown Values • Powell's minimization method

  14. RESULTS USING THE HERTZ MODEL Parabolic tip Conical tip

  15. PROBLEMS WITH THE HERTZ MODEL • Cells are not planar and do not extend infinitely in all directions • The cantilever is not infinitely stiff • Data is not linear • Cells are not perfectly elastic Must consider other possible solutions

  16. VISCOELASTIC CELLS • Constitutive equation • Linear Elastic • Non-Linear Viscous

  17. STRAIN HARDENING • Cells get stiffer with increased applied force • Possibly a similar mathematical model to those used in materials science

  18. CELL INDENTATION OVER TIME WITHOUT SPHERE (n=4) Applied force = Approx 1.5 nN Time = 8.5 min Voltage conversion 1 V/65 nm • The tip indents the cell (roughly 400 nm) and does not seem to push back but reaches a plateau • This trend is consistent and repeatable

  19. CELL INDENTATION OVER TIME USING ATTACHED SPHERE (n =4) Applied force = Approx 1.5 nN Time = 8.5 min Voltage conversion 1 V/65 nm

  20. ADAPTIVE EVIDENCE! • The sphere indents the cell and after a couple of minutes the cell seems to be pushing back • This trend is consistent and repeatable when using an attached sphere

  21. HOW A SIMULATION CAN HELP • Similar experiments without bias • New experiments • Observe what you can not measure • Generally : test some hypothesis

  22. WHAT IS SIMULATED? • Adhesion with other cells • Lipidic bilayer • Actin cortex • Cytoskeleton • Nucleus • Cytoskeleton • Actin cortex • Lipidic bilayer • Focal adhesion complex • Substrate

  23. HOW IS THE CELL SIMULATED? • Continuum Mechanics : • Equations describing the behaviour of the smallest amount of matter that can be seen as continous. • Finite Elements Method : • A node = an equation • Huge system to solve • Well-known mechanical materials • Realism • Slow

  24. RESULTS • Fairly reproduce experimental conditions ; • Displacement, speed and acceleration for each node of the simulation • The future : from continuous materials to living materials

  25. CONCLUSIONS • The cells in this study exhibit viscoelastic properties and exhibit strain hardening • They Show adaptive behaviour over time when an external force is applied • The cell behaves differently when a global force is applied compared to a local force

  26. CURRENT & FUTURE WORK • Develop a model to analyse the force data • Disrupt cytoskeleton to determine contribution of each filament type • Disrupt cytoskeleton to see how the cell behaves over time

  27. CURRENT & FUTURE WORK • Compare mechanical properties of normal and pathological cells (cancer) • Determine mechanical properties of other cellular components • Compare mechanical properties of cells growing on different substrates

  28. THANK YOU FOR YOUR TIME! QUESTIONS ?

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