EARB MEETING 2012

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

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

NSF RII Thrusts Thrust Leaders are tactical leaders who lead teams Five Thrusts not Five Silos Shared Vision: build vascular constructs 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

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

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

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

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

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 Computer Aided Design Virtual Blueprints Thrust I, IV Spheroid Preparation Thrusts II, IV, V Spheroid Preparation Thrusts II, IV, V Bioprinter/Dispensor Thrust IV team Bioprinter/Dispensor Thrust IV team 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, II, III, IV, V Perfusion Endothelialization Testing Thrusts, 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

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

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

Goals (Milestones) for Year 3 Prepare various types of cell spheroids +/- hydrogels (“BioInk”) Thrust I: Modeling of hydrogels that bind the cellular spheroids and enrich maturogens Thrust II : stem cells, ECM, hydrogels (X.Bi, Chas So Univ) Thrust IV: enhance production (microfluidics, laser assisted ) Thrust V: gelatin microcarrier spheroids: test bed for maturogens etc. Build Inkjet type bioprinter to dispense living spheroids *

Goals (Milestones) Yr 3 Design and Build an inkjet printer Designed by Xuejun Wen; assembled by Izumi Inc. 2 years in the making

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

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.

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

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

Goals (Milestones) for Year 3 Dispense and assemble (“print”) living spheroids into variable- sized tubular constructs (6 to 6000) INK JET Izumi (Wen, Mondy, Dr. Waleed Twal) 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

4 mm x 17mm

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

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 devices

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)

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.

Using a machined construct to arrange “carrier” spheroids into multi-layer tubes (test bed)

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)

Presentation by Xuejun Wen THRUST IV: Microfluidic approach (Spheroid maker* + Spheroid printer)*

Goals (Milestones) for Year 3 Model and test for viability* and stability of “printed” spheroids/constructs ( all thrusts) *grant opportunities 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, III V ) Biomechanical testing: cf.natural (authentic) blood vessels to engineered constructs (bioprinted, extruded collagen tubes, etc. (thrust III )

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

Preliminary analysis demonstrates that: the measured in vivo longitudinal strain differed between the main renal artery and the first branch by a factor of 2 (25% for main renal artery, 12% for first branch) the measured in-vivo circumferential strain differed between the main renal artery and the first branch by a factor of 2 (12% for main renal artery, 25% for first branch).”

Mondy-CAD “Blueprint” Next level

Goals to reach the next level Higher resolution CADs Translation of CADs into programming for directing printheads (dispensors) Modeling & testing the biology of spheroid formation and fusion Hydrogel or ECM properties of Spheroids Integration of printing (assembly) approaches Endothelialization, Differentiation,Maturogens