1. EARB MEETING 2012

2. Biofabrication by BioPrinting Advantages Precision in positioning cell types Scaffold “free” ( What are hydrogels?) “Mimics” Development (vs. expts in evolution) Potential for “scale up automation” Solves the vascularization problem of thick tissue constructs

3. Biofabrication by BioPrinting Disadvantages Explosion of interest but unsolved technology Few pioneers 2005+ – Boland, Nakamura, Forgacs and their respective groups Huvec cells survive printer and can be positioned 2D 3D tube printed in 2007 -- 1mm 2007 double layered (Huvec, aortic sm. Muscle) 1mm 2009 10 mm solid structures were printed but collapsed Technology of great potential looking for new ideas and solutions

4. NSF RII Thrusts Thrust Leaders are tactical leaders who lead teams Five Thrusts not Five Silos Shared Vision: build vascular constructs 2011-2012 milestone: 4mm X 27mm tubular prototype having 6,000 subunits of living tissue All participate in the three steps of biofabrication pre-processing  processing  postprocessing

5. TACTICAL APPROACHES Thrust I – Modeling and Computer-Aided-Design Mathematical Modeling, Software Design, Programming Leader: Qi Wang Members: Brian Canada, Thomas Trusk, William Mondy, Xiaofeng Yang, Xinfeng Liu, Feng Gu, Xigiang Zheng Thrust I – Modeling and Computer-Aided-Design Mathematical Modeling, Software Design, Programming Leader: Qi Wang Members: Brian Canada, Thomas Trusk, William Mondy, Xiaofeng Yang, Xinfeng Liu, Feng Gu, Xigiang Zheng Thrust II – BioInk, Approaches to the Building Blocks Aggregates, Stem Cells, Hydrogels, Differentiation, Endothelialization Leaders: Chris Drake and Richard Visconti Members: C. Bi, Agnes Nagy, Xuejun Wen Thrust II – BioInk, Approaches to the Building Blocks Aggregates, Stem Cells, Hydrogels, Differentiation, Endothelialization Leaders: Chris Drake and Richard Visconti Members: C. Bi, Agnes Nagy, Xuejun Wen Thrust III – Biomechanical Testing Natural Vessels, Collagen Tubes, BioPrinted Tubes Leader: Michael Sutton Members: Sue Lessner, Jay Potts, Mike Yost, Tarik Shazly, Esmail Jabbari Thrust III – Biomechanical Testing Natural Vessels, Collagen Tubes, BioPrinted Tubes Leader: Michael Sutton Members: Sue Lessner, Jay Potts, Mike Yost, Tarik Shazly, Esmail Jabbari Thrust IV – Processing / Printing / Assembly Manual templates, Izumi-ink jet printing, Laser-printing, Magnetic Assembly, Microfluidics Leader: Xuejun Wen Members: William Mondy, Frank Alexis, Scott Argraves, Yong Huang, Waleed Twal Thrust IV – Processing / Printing / Assembly Manual templates, Izumi-ink jet printing, Laser-printing, Magnetic Assembly, Microfluidics Leader: Xuejun Wen Members: William Mondy, Frank Alexis, Scott Argraves, Yong Huang, Waleed Twal Thrust V – Maturation ECM Synthesis, Perfusion, Bioreactors, Scaffolds, Small Molecules Leader: Scott Argraves Members: Chris Drake, Waleed Twal, Xuejun Wen, Gear Grantees Thrust V – Maturation ECM Synthesis, Perfusion, Bioreactors, Scaffolds, Small Molecules Leader: Scott Argraves Members: Chris Drake, Waleed Twal, Xuejun Wen, Gear Grantees

6. NSF RII Thrusts Thrust Leaders are tactical leaders who lead teams Five Thrusts not Five Silos Shared Vision: build vascular constructs 2011-2012 milestone: 4mm X 27mm tubular prototype having 6,000 subunits of living tissue

7. Major Milestone for Year 3 – bioprint 4.5mm x 27 mm tubular construct

8. NSF RII Thrusts Thrust Leaders are tactical leaders who lead teams Five Thrusts not Five Silos Shared Vision: build vascular constructs All participate in the three steps of biofabrication pre-processing  processing  post-processing

9. Modeling Natural Properties Thrusts I, III Modeling Natural Properties Thrusts I, III Spheroid-Based Vessel Design Parameters Spheroid-Based Vessel Design Parameters Computer Aided Design Virtual Blueprints Thrust I, IV, V Computer Aided Design Virtual Blueprints Thrust I, IV, V Spheroid Preparation Thrusts II, IV, V Spheroid Preparation Thrusts II, IV, V Bioprinter/Dispensor Thrust IV Bioprinter/Dispensor Thrust IV Hydrogels/biomaterials Thrusts II, IV, V Hydrogels/biomaterials Thrusts II, IV, V Directed Differentiation Thrusts II, V Directed Differentiation Thrusts II, V Perfusion Endothelialization Testing Thrusts I, II, III, IV, V Perfusion Endothelialization Testing Thrusts I, II, III, IV, V Maturogens Thrusts II, III, V Maturogens Thrusts II, III, V I. Pre-processing II. Processing III. Post-processing All Thrusts Participate in Multiple Steps of Bioprinting

10. Goals (Milestones) for Year 3 Develop by mathematical modeling numerical predictive tools for the formation of vascular constructs by the deposition of multicellular aggregates/spheroids in a designer fashion. – Spearheaded by Qi Wang/Thrust I leader

11. THRUST 1 PRESENTATION Leader: Qi Wang TACTICAL LEADER -------------------  QI Wang Members: Brian Canada, Thomas Trusk, William Mondy, Xiaofeng Yang, Xinfeng Liu, Feng Gu and Xigiang Zheng

12. Goals (Milestones) for Year 3 Prepare various types of cell spheroids +/- hydrogels Thrust I: Modeling of hydrogels that bind the cellular spheroids and enriches maturogens Thrust II : stem cells, ECM, hydrogels Thrust IV: enhance production (microfluidics) Thrust V: gelatin microcarrier spheroids: test bed Build Inkjet type bioprinter to dispense living spheroids *

13. Goals (Milestones) Yr 3 Design and Build an inkjet printer Designed by Xuejun Wen; assembled by Izumi Inc.

14. Bill Mondy with Jorge V. L. Silva, Chief of the Division of Three Dimensional Technology, Renato Archer Center for Information Technology, Campinas Brazil.

15. Beta testing (related to Thrust IV) Early result of printed feature resolution demonstrated by the surface penetration of cardboard on the right and clay on the left. The pliability of clay allowed for smaller feature representation. An eleven by ten array of physical impressions, with radii of 36 microns, created in a clay surface by bioprinter’s dispenser.

16. Beta Tested by Bill Mondy A. Series of 1 mm spheres printed during initial testing. B demonstrates 10 rows of 10 silicone spheres 1 mm in diameter printed in a z stack. C shows initial testing of bioprinter’s resolvable feature size. ABC

17. Goals (Milestones) for Year 3 Translate CAD into coordinates for the cellularized building blocks (spheroids) that can be used by the bioprinter to position (drop) the spheroids at specified locations – Thrust 1 Team Effort - Bill Mondy

19. Goals (Milestones) for Year 3 Dispense and assemble (“print”) living spheroids into variable- sized tubular constructs (6 to 6000) INK JET Izumi (Wen, Mondy et al.) Yong Huang (“drop on demand”) ** OTHER TISSUE ASSEMBLY APPROACHES magnetic particles (Frank Alexis, Xuejun Wen) laser assisted (Yong Huang) ** machined assisted devices/Argraves,Drake,Wen) microfluidics – Xuejun Wen presentation

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

22. Goals (Milestones) for Year 3 Dispense and assemble (“print”) living spheroids into variable-sized tubular constructs (6-10 to 6000 model) Izumi (Wen, Mondy et al.) Yong Huang (“drop on demand”) ** Other experimental approaches magnetic particles (Frank Alexis) laser assisted (Yong Huang) ** machined assisted

23. Background

24. Goals (Milestones) for Year 3 Dispense and assemble (“print”) living spheroids into variable-sized tubular constructs (6-10 to 6000 model) Izumi (Wen, Mondy et al.) Yong Huang (“drop on demand”) Other experimental approaches magnetic particles (Frank Alexis, Xuejun Wen) laser assisted (Yong Huang) machined assisted devices/Argraves,Wen)

25. Flow-through Bioreactor Template for 27mm x 4mm tube This template is designed to allow spheroids to fill a 4mm diameter tube-shaped space, which will allow the flow of media while the spheroids mature enough to be removed from the chamber.

26. Using a template to arrange spheroids into multi-layer tubes.

27. Goals (Milestones) for Year 3 Dispense and assemble (“print”) living spheroids into variable-sized tubular constructs (6-10 to 6000 model) Ink Jet Izumi (Wen, Mondy et al.) Yong Huang (“drop on demand”) Other experimental approaches magnetic particles (Frank Alexis, Xuejun Wen) laser assisted (Yong Huang) machined assisted devices/Argraves,Wen) Microfluidics: Xuejun Wen (Thrust IV leader)

28. Presentation by XUEJUN WEN THRUST IV: Microfluidic approach (Spheroid maker + Spheroid printer)

29. Goals (Milestones) for Year 3 Model and test for viability and stability of “printed” spheroids/constructs (thrusts I, II, III,V) Initiate post-processing differentiation and ways to “endothelialize” tubular constructs (thrust II, V) Develop post-processing mechanisms for accelerating stabilization and maturation (focus on ECM) (thrust II, V ) Biomechanical testing: cf.natural (authentic) blood vessels to engineered constructs (bioprinted, extruded collagen tubes, etc. (thrust III )

30. Presentations (sequentially)+ Thrust II - Chris Drake/Rick Visconti Thrust V - Scott Argraves Thrust III - Mike Sutton/Jay Potts