Transcript Dr Frank Gesellchen Dr Theophile Dejardin Dr Mathis Riehle Dr Anne Bernassau Prof David Cumming School of Engineering • Methods to arrange cells in 3D – Bioprinting.

Dr Frank Gesellchen
Dr Theophile Dejardin
Dr Mathis Riehle
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Dr Anne Bernassau
Prof David Cumming
School of Engineering
• 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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with phase shift
Step 1 pattern cells
Step 2 shift phase
Step 3 seed other cells
Bernassau et al. 2012 Biomed Microdev. 14:559
3
Signal generator
Generating complex patterns
Fluorescent labelling
Device Setup
Mitotracker
green
Mitotracker
red
Hoechst DNA
stain
Culture medium
Glass cover slip
Agar layer
4
C2C12 mouse myoblasts
(a)
2
2
6
3
4
φ +120˚
φ +240˚
6
3
5
4
5
5
4
7
2
6
3
5
(b)
1
7
1
7
1
Scale bar – 100 µm
(a)
1
1
7
6
2
5
3
4
(b)
7
1
2
6
3
5
7
6
2
3
4
5
4
“lattice”
Scale bar – 100 µm
(a)
2
2
6
3
7
1
7
1
6
3
5
5
4
4
“tartan”
(b)
Cumming Tartan pattern….
• Sonotoweezing & peripheral nerve repair
• Aligned Schwann cells to guide regeneration*
o Schwann cells for peripheral nerve regeneration
o Isolation, purity
o Cell adhesion testing
o Cell migration?
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• 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)?
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Check Allodi et al. for original
figure on Wallerian Regeneration
Wiki: Wallerian de-regeneration
I. Allodi et al. Progress in Neurobiology 98
(2012) 16–37
≈ 4h
≈ 18h
2d
S100
Actin
1 mm
Scale bars 100 µm
•
•
•
•
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
* Rezakhaniha, Biomechanics and modeling in mechanobiology, SpringerLink
DOI: 10.1007/s10237-011-0325-z
http://bigwww.epfl.ch/demo/orientation/
Schwann
cell
seeding
RANDOM
US LINES
DRG
seeding +18h
4h
20
h
50
h
96
h
* Rezakhaniha, Biomechanics and modeling in mechanobiology, SpringerLink
DOI: 10.1007/s10237-011-0325-z
http://bigwww.epfl.ch/demo/orientation/
Analysis of directionality with OrientationJ (coherency 30, Energy1)
pattern1
pattern2
pattern3
arrows indicate direction of Schwann cell pattern at time of DRG seeding
random1
random2
random3
Raw data
Bin/Average
Correct for SC angle
Stats (n=6)
0.8
0.7
Coherency
0.6
0.5
0.4
0.3
0.2
0.1
0
-90
10
Orientation (degrees)
Raw data
Bin/Average
Correct for SC angle
Stats (n=6)
150
90
***
150
120
60
0.5
100
150
30
Pattern Avg
Random Avg
0.45
50
80
0.4
0
210
Relative frequency
0.35
330
240
300
270
90
250
120
60
200
150
150
30
100
150
180
0
210
330
300
270
0.25
0.2
0.15
0.1
0.05
50
240
0.3
0
-90 - <- -60 - <- -30 - <0 0 - <30
60
30
Orientation bins [º]
30 <60
60 <90
By topography
By Schwann cells
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•
•
•
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)