Cell Patterning with a heptagon acoustic tweezer: applications in neurite guidance

Dr Frank GesellchenDr Theophile DejardinDr Mathis Riehle

Dr Anne BernassauProf David CummingSchool of Engineering

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Lab Chip, 2014, DOI: 10.1039/C4LC00436A

Cells arranged in 3D

• Methods to arrange cells in 3D–Bioprinting using a bioprinter

(“ink jet”)• Very fast, embedded in gel

– Very good 3D capabilities, relies on embedded cells

–Layer by layer deposition of sheets• Grown in sheets then assembled

– Pattern limited

–Host growth • Stem cell based growth of organs

in situ– Takes weeks/month to mature

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Step 1 pattern cellsStep 2 shift phase

Step 3 seed other cells

with phase shift

Pi

ez

o

Pi

ez

o

Signal generator3

Bernassau et al. 2012 Biomed Microdev. 14:559

Generating complex patterns

Mitotracker green

Mitotracker red

Hoechst DNA stain

C2C12 mouse myoblasts

Device Setup Fluorescent labelling

Culture medium

Glass cover slip

Agar layer4

φ +120˚ φ +240˚

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34

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71

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34

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(a)

(b)

Scale bar – 100 µm

Complex pattern with phase shifts

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(b)

Scale bar – 100 µm

“lattice”

Complex patterns by transducer switching

Combining phase shifts and transducer switching

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(b)

“tartan”

Cumming Tartan pattern….

Proof of Principle OK, ….

• Sonotoweezing & peripheral nerve repair

• Aligned Schwann cells to guide regeneration*

o Schwann cells for peripheral nerve regenerationo Isolation, purity o Cell adhesion testingo Cell migration?

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Schwann cells

• peripheral nerve glial cells that surround and myelinate axons (saltatory action potential propagation)

• play key role in (peripheral) nerve regeneration – axon guidance

• Can patterning SCs guide neurite outgrowth (from dorsal root ganglia)?

I. Allodi et al. Progress in Neurobiology 98 (2012) 16–379

Check Allodi et al. for original figure on Wallerian Regeneration

Wiki: Wallerian de-regeneration

Schwann cell patterning (heptagon device)

≈ 4h ≈ 18h

Scale bars 100 µm1 mm

2d

S100Actin

Schwann cell guided DRG outgrowth

• Random seeded• Aligned by sonotweezer stencil• DRG seeded at day 1 (18-24h)• Outgrowth analysed at day 4 (n=6)

– OrientationJ*:• Orientation within a moving box (250x250

pixel) analysed, binned, referenced to linear ST pattern (if), and used to assess network

http://bigwww.epfl.ch/demo/orientation/

* Rezakhaniha, Biomechanics and modeling in mechanobiology, SpringerLinkDOI: 10.1007/s10237-011-0325-z

Schwann cell seeding

DRG seeding +18h

4h 20h

50h

96h

RANDOM

US LINES

OrientationJ

http://bigwww.epfl.ch/demo/orientation/

* Rezakhaniha, Biomechanics and modeling in mechanobiology, SpringerLinkDOI: 10.1007/s10237-011-0325-z

pattern1 pattern2 pattern3

random1random2

random3

Analysis of directionality with OrientationJ (coherency 30, Energy1)

arrows indicate direction of Schwann cell pattern at time of DRG seeding

OrientationJ Macro

Raw data Bin/AverageCorrect for SC angleStats (n=6)

OrientationJ Macro

-90 -40 10 600

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Orientation (degrees)

Coh

ere

ncy

Raw data Bin/AverageCorrect for SC angleStats (n=6)

Orientation analysis

-90 - <-60

-60 - <-30

-30 - <0 0 - <30 30 - <60

60 - <90

0

0.05

0.1

0.15

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0.25

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0.35

0.4 Pattern Avg Random Avg

Orientation bins [º]

Rela

tive

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150

150

***

How does bias work?By topography

By Schwann cells

ACKNOWLEDGEMENTS• Staff at the James Watt Nanofabrication Centre• Carol-Anne Smith for technical support• The makers of OrientationJ• The funders:

– EPSRC funded Sonotweezer Grant (EP/G012067/1) (DC, MR, AB, FG)

– Steven Forrest Trust (TD, MR, DC)– The University of Glasgow (AB)