Investigating the mechanical properties of living human cells
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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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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

  • Force Data Analysis

  • Image Processing

  • Alexis Guillaume

  • Computer Modelling

  • Simulation


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


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


WHY SHOULD WE STUDY CELL MECHANICS?


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’


HOW DO WE MEASURE MECHANICAL PROPERTIES OF CELLS?


Laser

Photo Detector

Z-piezo

Sample

X-Y Stage

Objective Lens

THE ATOMIC FORCE MICROSCOPE


CANTILEVER WITH HAIR

25 µM


Indentation (nm)

Deflection (nm)

2

1

Non-contact region

0

0

Z-Movement (µM)

1

2

AFM FORCE CURVES


LIVE HUMAN LUNG FIBROBLAST CELL

AFM Deflection Image of Human Lung Cell

3-D Image of Human Lung Cell

(Reconstructed From Height Image)


THE HERTZ MODEL


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


RESULTS USING THE HERTZ MODEL

Parabolic tip

Conical tip


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


VISCOELASTIC CELLS

  • Constitutive equation

  • Linear Elastic

  • Non-Linear Viscous


STRAIN HARDENING

  • Cells get stiffer with increased applied force

  • Possibly a similar mathematical model to those used in materials science


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


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


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


HOW A SIMULATION CAN HELP

  • Similar experiments without bias

  • New experiments

  • Observe what you can not measure

  • Generally : test some hypothesis


WHAT IS SIMULATED?

  • Adhesion with other cells

  • Lipidic bilayer

  • Actin cortex

  • Cytoskeleton

  • Nucleus

  • Cytoskeleton

  • Actin cortex

  • Lipidic bilayer

  • Focal adhesion complex

  • Substrate


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


RESULTS

  • Fairly reproduce experimental conditions ;

  • Displacement, speed and acceleration for each node of the simulation

  • The future : from continuous materials to living materials


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


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


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


THANK YOU FOR YOUR TIME!

QUESTIONS ?


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