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
2
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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2
3
4
5
6
7
1
2
34
5
6
71
2
34
5
6
7
(a)
(b)
Scale bar – 100 µm
Complex pattern with phase shifts
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1
2
34
5
6
71
2
34
5
6
7 1
2
34
5
6
7(a)
(b)
Scale bar – 100 µm
“lattice”
Complex patterns by transducer switching
Combining phase shifts and transducer switching
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2
34
5
6
7 1
2
34
5
6
7
(a)
(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
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
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
0.2
0.25
0.3
0.35
0.4 Pattern Avg Random Avg
Orientation bins [º]
Rela
tive
freq
uenc
y
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)