Week 7 - Tools for Mechanotransduction + Lab on a Chip

Tools for testing ECM

1. Hydrophobicity = Wettability

2. Topography = SEM

3. Stiffness = micro-indentation, AFM

Tools for testing Integrin

adhesion rupture force (AFM)

Tools for testing FAC

Fluorescence Resonance Energy Transfer

Tools for testing Actin-myosin traction force

Micropillar/microneedle

Traction Force Microscopy

Tools for testing Nuclear lamina

protein expression = immunostaining

Tools for testing DNA

trancription into mRNA (Polymerase Chain Reaction)

Wettability

measures hydrophobicity

put water drop on surface and measure angle

larger angle = more hydrophobic

Scaning electron microscopy (SEM) for ECM

measures topography

can view surface to see structure of ECM

Testing stiffness of ECM (3 types of tests)

Macro scale tests = compression, tensile, rheology tests

Micro scale tests = microindentation

Micro/nano scale tests = atomic force microscop (AFM)

Microindentation (ECM testing)

measures stiffness of ECM

compresses surface to test stiffness

micro scale

Atomic force microscope (ECM testing)

measure stiffness of ECM

compresses surface and measures how much surface bent with laser (to test stiffness)

nano (atomic) scale

5 things an Atomic force microscope measure?

force

stiffness

height of contact (at each point = roughness)

adhesion force

rupture force

[and more]

AFM graph interpretation

slope of curve (indentation vs force → top graph) shows stiffness of material

steeper = stiffer

Area between line for Approach and Retraction represents the adhesion force

• the closer the lines are the less adhesions (bottom graph)

Atomic Force microscope (integrin measurments)

Rupture of integrin-mediated focal adhesion can be measured (rupture length + force)

Measured by detecting degree of conformational change (change in stiffness)

Fluorescence Resonance Engergy transfer (FRET) for testing FAC

Conformational changes in Vinculin and Talin

• they stretch based activation from ECM forces

Fluorophores attached to Focal Adhesion Complex emit light when activated

• when protein is unfolded

emitted light can cause nearby fluorophores to emit light

= determines how close fluorophores are (if no other fluorophores are emitted = FAC is spread out)

Measuring traction force (micropillar/microneedle)

Put cells on array of pillars

Force = k(spring constant) * displacement

large displacement of cell = large traction force

Variable (in spring constant) = stiffness, diameter, length

Traction force microscopy

Measure forces applied by cells (on hydroge) based on displacement of fluorescent beads

Micropillar pros and cons (vs TFM)

Pros:

- no need for FEM

- no need for beads

- high resolution

Cons:

- no z axis movement

- discontinued focal adhesion

Traction Force Microscopy pros and cons (vs micropillars)

Pros:

- x/y/z axis data

- continuous focal adhesion

Cons:

- required equipment

- FEM

FEM

finite element method (analysis)

• simulating the effects of mechanical stimuli on cells

• uses computer

Lab on a Chip

devices that integrate laboratory functions on a single chip

capable of handlign extremely small fluid volumes

Organ on a chip

3d multichannel and microfluidic cell structure device on microchip

mimics human organ (liver/lungs)

Organ on a chip (in relation to animal study)

usually used before animal/human study

see if drug is efficacious, before testing on animal/humans to see how it interacts with the system

Human on a chip

system of several organ on a chips

better mimics human system

Pros of Lab on a chip (7)

low fluid volume = low cost

more specific/defined

faster analysis = more analysis per vol

better control over process

easy to modify conditions

inter-connectivity with chips (more tissue types)

data supports human clinical trial

Cons of Lab on a chip (4)

not perfect as animal study for whole system

not fully developed yet

never reaching complexity of real tissue

detection principles may not always scale down positively (signal:noise ratio)

Lung on a chip (how does it mimic human lungs)

Attach side chambers to vacuum = stretch tissue

(mimics alveoli stretching)

can measure stretch by viewing movement of pores on the membrane (under microscope)

Mimicing cancer growth

put cancer cell in hydrogel (mimic stiffness of ECM)

let cancer grow = view tumour cell composition

view which cells are present in tumour (can be influenced by stiffness of hydrogel)

What can we determine/measure when Mimicing cancer growth?

proportion and cell types in tumour

tumour composition (cells in center vs periphery)

epithelial to mesenchymal transition

Cancer tissue mechanics (metastasis)

Local invasion:

- cells migrate through collagen fibres

- requires aggressive phenotype

Intravasation (and extravasation):

- cells squeeze through cell-cell adhesions

- ruptures nuclear envelope

Circulating tumour cells