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

1. Cell Patterning with a heptagon acoustic tweezer: applications in neurite guidance Lab Chip, 2014, DOI: 10.1039/C4LC00436A • Dr Frank Gesellchen • Dr TheophileDejardin • Dr Mathis Riehle • Dr Anne Bernassau • Prof David Cumming • School of Engineering

2. 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

3. with phase shift Step 2 shift phase Step 3 seed other cells Step 1 pattern cells Piezo Piezo Bernassau et al. 2012 Biomed Microdev. 14:559 Signal generator

4. Generating complex patterns Fluorescent labelling Device Setup Mitotracker green Mitotracker red Hoechst DNA stain Culture medium C2C12 mouse myoblasts Glass cover slip Agar layer

5. 7 1 7 1 Complex pattern with phase shifts 2 6 2 6 (a) 3 5 3 5 4 4 φ +240˚ φ +120˚ (b) 1 7 6 2 3 5 4 Scale bar – 100 µm

6. Complex patterns by transducer switching (a) 1 7 1 7 1 7 6 2 6 2 6 2 5 3 5 3 3 5 (b) 4 4 4 “lattice” Scale bar – 100 µm

7. Combining phase shifts and transducer switching 7 1 7 1 2 6 2 6 (a) 3 5 4 3 5 4 “tartan” (b) Cumming Tartan pattern….

8. Proof of Principle OK, …. • Sonotoweezing & peripheral nerve repair • Aligned Schwann cells to guide regeneration* • Schwann cells for peripheral nerve regeneration • Isolation, purity • Cell adhesion testing • Cell migration?

9. Schwann cells Check Allodi et al. for original figure on Wallerian Regeneration Wiki: Wallerian de-regeneration • 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) 16–37

10. Schwann cell patterning (heptagon device) ≈ 4h ≈ 18h 2d S100 Actin 1 mm Scale bars 100 µm

11. 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 * Rezakhaniha, Biomechanics and modeling in mechanobiology, SpringerLink DOI: 10.1007/s10237-011-0325-z http://bigwww.epfl.ch/demo/orientation/

12. Schwann cell seeding DRG seeding +18h 4h 20h 50h 96h RANDOM US LINES

13. OrientationJ * Rezakhaniha, Biomechanics and modeling in mechanobiology, SpringerLink DOI: 10.1007/s10237-011-0325-z http://bigwww.epfl.ch/demo/orientation/

14. 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

15. OrientationJ Macro Raw data Bin/Average Correct for SC angle Stats (n=6)

16. OrientationJ Macro Raw data Bin/Average Correct for SC angle Stats (n=6)

17. Orientation analysis *** 150 150

18. How does bias work? By topography By Schwann cells

19. 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)