1. Yong Huang Department of Mechanical Engineering Clemson University, Clemson, SC Scaffold-free Alginate Tube Fabrication using Inkjet and Laser printing

2. Table of Contents  Introduction  Background  Objective  Research Tasks  Tube Inkjetting  Tube Laser Printing  Conclusions  Future Work 2

3. Table of Contents  Introduction  Background  Objective  Research Tasks  Tube Inkjetting  Tube Laser Printing  Conclusions  Future Work 3

4. Background ­ Inkjet-based 4 [Boland, 2007] Inkjetting of CaCl 2 into 2% alginic acid to make a branched structure [Nakamura, 2008] Inkjetting 0.8% sodium alginate into 2% CaCl 2 to make a tube with the help of gravity

5. 5 [Skardal, 2010] Printing of agarose and 3T3 cell-containing PEGTA filaments to make a tube [Norotte, 2009] Printing of agarose and smooth muscle cell (SMC) cylinders to make a tube Background ­ Filament-based

6. Background ­ Laser-based 6 Laser printing of alternating layers of fibroblasts (green) and keratinocytes (red) to form a grid structure (Scale bar = 500 µ m) [Koch, 2012]

7. Challenges 7  How to scale-up scaffold-free bioprint complex structures such as overhang structures?  How to evaluate and mitigate the post-printing cell damage?

8. Table of Contents  Introduction  Background  Objective  Research Tasks  Tube Inkjetting  Tube Laser Printing  Conclusions  Future Work 8

9. Objective 9 Needs in cellular tube bioprinting  Vascularization as a main technological barrier for building 3D organs  Tube printing - a logical initial step towards vascularization Objective To scaffold-free fabricate viable cellular tubes using inkjet and laser printing

10. Table of Contents  Introduction  Background  Objective  Research Tasks  Tube Inkjetting  Tube Laser Printing  Conclusions  Future Work 10

11. Table of Contents  Introduction  Background  Objective  Research Tasks  Tube Inkjetting  Tube Laser Printing  Conclusions  Future Work 11

12. 12 Liquid level First layerSecond layer 3T3 cell suspension Tube Inkjetting ­ Experimental setup

13. Tube Inkjetting - Zig-zag tube  Fabrication process: bottom part, overhang part and top part  Wall thickness: 150~200 µm  Tube diameter: 3 mm and length: 10 mm Zigzag tube fabrication 13

14. Tube Inkjetting - Cell viability test (a) Printed tube, (b) tube surface and (c) cell viability test after liquefying 14 (a) Cells (b) Live Cell Dead Cell After liquefying (c)

15.  Cell viability immediately after printing: > 85%  Cell viability after 3 days: > 80%  Cell viability comparable to that of the control Tube Inkjetting - Cell viability 15

16. Tube Inkjetting - Other structures 16 PyramidCone

17. Table of Contents  Introduction  Background  Objective  Research Tasks  Tube Inkjetting  Tube Laser Printing  Conclusions  Future Work 17

18. Tube laser printing - Experimental setup 18 ArF laser Pulsed laser beam Ribbon Forming jet/droplet Quartz support Coating Container Optical table Laser-induced vapor/plasma pocket 3-axis stages Moving platform

19. Tube laser printing - Experimental setup 19 Direct-writing height = 1 mm

20. Tube laser printing - results 20 Top viewSide view Φ=3 mm

21. Table of Contents  Introduction  Background  Objective  Research Tasks  Tube Inkjetting  Tube Laser Printing  Conclusions  Future Work 21

22.  Overhang structures can be scaffold-free fabricated  Viable cellular tubes (3T3) can be printed Conclusions Inkjetting 22  Tubes can be scaffold-free fabricated using LIFT-based laser printing technology  Highly viscous materials (alginate) can be laser printed into well-defined tubular structures Laser printing

23. Table of Contents  Introduction  Background  Objective  Research Tasks  Tube Inkjetting  Tube Laser Printing  Conclusions  Future Work 23

24.  Laser printing of 3D cellular tubes  Printing of adipose-derived stem cell tubes Future Work Fabrication 24  Apoptosis/necrosis test, cell proliferation capacity, and phenotype variation  Post-printing fusion of tubular tissue  Mechanical property measurement of fabricated tubes Post-printing evaluation

25. Thanks and questions? 25