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

INVESTIGATING THE MECHANICAL PROPERTIES OF LIVING HUMAN CELLS

Mark Murphy:GERI & BML

Catherine Randall:GERI

Alexis Guillaume:Université Claude Bernard, Lyon


Investigating the mechanical properties of living human cells

PROJECT BREAKDOWN

  • Mark Murphy

  • Cell Biology

  • Atomic Force Microscopy

  • Fluorescence microscopy

  • Catherine Randall

  • Force Data Analysis

  • Image Processing

  • Alexis Guillaume

  • Computer Modelling

  • Simulation


Investigating the mechanical properties of living human cells

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


Investigating the mechanical properties of living human cells

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


Investigating the mechanical properties of living human cells

WHY SHOULD WE STUDY CELL MECHANICS?


Investigating the mechanical properties of living human cells

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’


Investigating the mechanical properties of living human cells

HOW DO WE MEASURE MECHANICAL PROPERTIES OF CELLS?


Investigating the mechanical properties of living human cells

Laser

Photo Detector

Z-piezo

Sample

X-Y Stage

Objective Lens

THE ATOMIC FORCE MICROSCOPE


Investigating the mechanical properties of living human cells

CANTILEVER WITH HAIR

25 µM


Investigating the mechanical properties of living human cells

Indentation (nm)

Deflection (nm)

2

1

Non-contact region

0

0

Z-Movement (µM)

1

2

AFM FORCE CURVES


Investigating the mechanical properties of living human cells

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


The hertz model1

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

RESULTS USING THE HERTZ MODEL

Parabolic tip

Conical tip


Problems with the hertz model

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

VISCOELASTIC CELLS

  • Constitutive equation

  • Linear Elastic

  • Non-Linear Viscous


Strain hardening

STRAIN HARDENING

  • Cells get stiffer with increased applied force

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


Investigating the mechanical properties of living human cells

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

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


Investigating the mechanical properties of living human cells

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

HOW A SIMULATION CAN HELP

  • Similar experiments without bias

  • New experiments

  • Observe what you can not measure

  • Generally : test some hypothesis


What is simulated

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

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

RESULTS

  • Fairly reproduce experimental conditions ;

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

  • The future : from continuous materials to living materials


Investigating the mechanical properties of living human cells

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

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 work1

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


Investigating the mechanical properties of living human cells

THANK YOU FOR YOUR TIME!

QUESTIONS ?


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