Organ Printing:

ATBC

How to print a human organ Vladimir Mironov MD, PhD Associate Professor & Director of Advanced Tissue Biofabrication Center (ATBC) Department of Regenerative Medicine & Cell Biology Medical University of South Carolina Charleston, SC 29425, USA

Question 1: How new technologies evolve? Answer: from preexisting technologies

1. Francis Bacon (1607) (old linear model) 2. Terence Kealey (1996) (radical parallel model) 3. Brian Arthur (2009) (new integrated model) New technologies are emerging from preexisting technologies and not from basic science, but emerging technologies must explore and incorporate new phenomena discovered by basic scientists

Question 2: Is South Carolina an emerging high tech manufacturing state? Answer: yes

South Carolina is a High Tech State

North Charleston, SC

South Carolina is a High Tech State

Spartanburg, SC

South Carolina is a High Tech State Rock Hill, SC

South Carolina is a High Tech State

Charleston, SC

Question 3: Is tissue engineering science or technology? Answer: it is both science and technology

"Scientists discover the world that exists; engineers create the world that never was.” Theodore Von Kármán Tissue engineers are trying to “recreate” human tissues & organs that exist

Question 4: What is Synthetic Anatomy? Answer: Anatomy of XXI century

From analytical to synthetic anatomy

Biofabrication, 2009

Question 5: What is a status of industrial tissue engineering today? Answer: it could be better

Organogenesis, USA

Genzyme, USA

Cytograft Tissue Engineering, USA

Tengion, USA

Question 6: Why do we need bioprinted functional human organ constructs? Answer: donor shortage

Organ Printing

Huge Market

$75 000 annual cost of dialysis

$25 000 000 market only for TE kidney

Question 7: What is an organ printing? Answer: Biomedical application of RP or AM

Organ Printing is a computer-aided robotic layer by layer additive biofabrication of functional living human organ constructs

CAD

Bioimaging

I. Pre-processing

Blueprint

Bioink

II. Processing

Components of Organ Printing Technology

Biopaper Bioprinter

Maturogens

Biomonitoring Bioreactor

III. Post-processing

3 Steps in Bioprinting

Question 8: What is a status of biomedical application of rapid prototyping or additive manufacturing in tissue engineering?

Answer: it is growing

RP

RP in TE

RP

RP in TE

1. Title: Mechanical properties and cell cultural response of polycaprolactone scaffolds designed and fabricated via fused deposition modeling Author(s): Hutmacher DW, Schantz T, Zein I, et al. Source: JOURNAL OF BIOMEDICAL MATERIALS RESEARCH Volume: 55 Issue: 2 Pages: 203-216 Published: MAY 2001 36 44 35 53 35 262 29.11 2. Title: Fused deposition modeling of novel scaffold architectures for tissue engineering applications Author(s): Zein I, Hutmacher DW, Tan KC, et al. Source: BIOMATERIALS Volume: 23 Issue: 4 Pages: 1169-1185 Published: FEB 2002 32 42 41 42 20 222 27.75 3. Title: The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniques Author(s): Yang SF, Leong KF, Du ZH, et al. Source: TISSUE ENGINEERING Volume: 8 Issue: 1 Pages: 1-11 Published: FEB 2002 29 31 22 37 14 169 21.12 4. Title: Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organs Author(s): Leong KF, Cheah CM, Chua CK Source: BIOMATERIALS Volume: 24 Issue: 13 Pages: 2363-2378 Published: JUN 2003 18 27 31 50 27 161 23.00 5. Title: Organ printing: computer-aided jet-based 3D tissue engineering Author(s): Mironov V, Boland T, Trusk T, et al. Source: TRENDS IN BIOTECHNOLOGY Volume: 21 Issue: 4 Pages: 157-161 Published: APR 2003 18 25 18 40 28 154 22.00 6. Title: Scaffold-based tissue engineering: rationale for computer-aided design and solid free-form fabrication systems Author(s): Hutmacher DW, Sittinger M, Risbud MV Source: TRENDS IN BIOTECHNOLOGY Volume: 22 Issue: 7 Pages: 354-362 Published: JUL 2004 13 25 34 35 27 136 22.67 7. Title: Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sintering Author(s): Williams JM, Adewunmi A, Schek RM, et al. Source: BIOMATERIALS Volume: 26 Issue: 23 Pages: 4817-4827 Published: AUG 2005 2 18 34 42 22 118 23.60 8. Title: Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineering Author(s): Landers R, Hubner U, Schmelzeisen R, et al. Source: BIOMATERIALS Volume: 23 Issue: 23 Pages: 4437-4447 Published: DEC 2002 20 22 13 25 17 117 14.62 9. Title: Fabrication of PLGA scaffolds using soft lithography and microsyringe deposition Author(s): Vozzi G, Flaim C, Ahluwalia A, et al. Source: BIOMATERIALS Volume: 24 Issue: 14 Pages: 2533-2540 Published: JUN 2003 17 23 16 27 10 108 15.43 10. Title: Novel collagen scaffolds with predefined internal morphology made by solid freeform fabrication Author(s): Sachlos E, Reis N, Ainsley C, et al. Source: BIOMATERIALS Volume: 24 Issue: 8 Pages: 1487-1497 Published: APR 2003 13 16 24 15 15 92 13.14

TOP 10

Research clusters (RP in TE)

Question 9: What is a “bioink” ? Answer: self-assembling “fluidic” tissue spheroids

Evidence of Tissue Spheroids Fluidity Rounding

Fusion

Enveloping

Sorting

Spreading

Question 10: What is a fundamental principle of organ printing technology? Answer: tissue fusion

Tissue spheroids fusion

‘Virtual and Physical Prototyping’ (2009)

Question 11: What is a clinically relevant cell source for organ printing? Answer: iPS cells or ADSC

Adipose Tissue Derived Mesenchymal Stem Cell (ADSC) is a clinically relevant stem cell source

“David after two years staying in USA” (two photos on left side) “Celution System” (Cytori Therapeutics Inc. & GE Health Care)

Immunofluorescence analysis

Western analysis

mRNA

ADSCs differentiate into SMCs in a developmentally appropriate sequence

Densinometric analysis

Quantitative Phenotypic Analysis Differentiation

Proliferation

3-dimensional constructs of ADSC-derived SMCs contract in response to Angiotensin administration

Real time RT-PCR analysis of vascular ECM gene expression

Matrigel vasculogenesis assay

anti‐hPECAM1

anti‐αSMA

Co‐incident fluorescence 

Question 12: How to print an intraorgan branched vascular tree ? Answer: using 3 types of vascular tissue spheroids

Basic Kidney Anatomy

Design principles of biofabrication of intraorgan branched vascular tree from self-assembling vascular tissue spheroids

Three types of vascular tissue spheroids

Road Map & Timeline for Organ Printing

2003 2009 2020?

Tissue compaction during fusion of vascular tissue spheroids

Tissue fusion without cell mixing Virtual and Physical Prototyping, 2009

Bioengineering Vascular Tube Using Self-assembling Tissue Spheriods

Tissue Engineering, 2008

Bioprinting of Vascular Tree

‘Tissue Engineering’ (2008), featured in ‘Nature News’

Dev. Dynamics (submittted)

Tissue engineered branched segments of vascular tree

Regenerative Medicine, 2008 & Biomaterials, 2009

Mechanism of Lumen Formation

Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE, Weinstein BM. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature. 2006 Jul 27;442(7101):453-6

Bioprinting of Vascular Tree

‘Surface Evolver’, Ken Brakke

‘Blueprint’

Virtual and Physical Prototyping, 2009

A

B

C

D

E

F

G

How to print a human organ?

Stem cells Differentiated cells

Tissue spheroids

Tissue spheroids in hydrogel

Functional mature macrotissue

Microfluidic minibioreactors and cell sorters

Microfluidic mixers, droplet generators and encapsulators

Microfluidic dispensors and bioprinters

Macrobioreactor with removable porous tubes

“Trends in Biotechnology’ (under review process)

Question 13: What is a tissue spheroids “biofabricator” ? Answer: robotic tissue spheroids bioassembler

Scalable Robotic Biofabrication of Tissue Spheroids

EpMotion 5070 (Eppendorf)

Digital (Droplets) Microfluidics

Acoustic excitation based droplet generator in collaboration with Dr. Wen and Dr. Huang (Clemson University)

Digital Microfluidics: Droplet Generator

Jennifer Lewis & David Weitz

Self-directed self-assembly

Digital Microfluidics: Cascade Droplet Generator (a)

David Weitz, Science, 2005 (b)

(c)

Question 14: What is a “bioprinter” ? Answer: robotic dispensor & tissue bioassembler

Bioprinters

(a)

(b)

Microfluidics: Bioprinting

Continuous and digital (droplet) microfluidics-based bioprinting

Question 15: What is a “bioreactor” ? Answer: perfusion container for tissue maturation

Perfusion Bioreactor for Bioprinted Organs

Perfusion Bioreactor for Bioprinted Organs

Microfluidics-based irrigation dripping tripled perfusion bioreactor with removable microporous tubes 3

2 1

In collaboration with Dr. Kasyanov, RSU, Latvia, EU & Prof. Chua Chee Kai, NTU, Singapore

Question 16: What is a “maturogen” ? Answer: factor accelerating bioprinted tissue maturation

Methods of quantitative evaluation of the material properties of tissue spheroids

Tensiometry

Aspiration

Centrifugation

Tissue Spheroids Enveloping Assay

Question 17: How robotic automated industrial production of human organs will look like?

Answer: like assembly line

Clinical cell sorters

TGI 1200

Robotic Biofabrication of Tissue Spheroids

Acoustic excitator

EpMotion 5070 (Eppendorf)

Robotic bioprinters A(i)

B

(k)

C

Neatco-1(A), Neatco-2(B), Sciperio/nScript(C)

Perfusion Bioreactors for Bioprinted Organs Lungs

Kidney

Organ Recovery Systems Inc.

Liver

Kidney Biofabrication Plant

$1 billion?

ATBC

2025?

$20 million NSF grant nScript Inc.(Sciperio), USA

Question 18: What is a future of bioprinting technology? Answer: in vivo bioprinter

“Da Vinci” - First Generation of Robotic Surgery Tool (Intuitive Surgery Inc, CA)

Bioprinting in situ (a science fiction)

“Face/Off” (1997)

Bioprinting in situ (an emerging concept, 2003)

Clinical Bioprinter - Project “Michelangelo”?

Bioprinting in situ (scientific reality, 2009) “In Vivo High-Throughput Biological Laser Printing of Nano-Hydroxyapatite in Mice Calvaria Critical Size Defect: Preliminary Results”

Virginie Keriquel et al. (best poster) Presented in Bordeaux 6-8 July 2009 at The International Conference

Bioprinting and Biofabrication in Bordeaux (3B’09)

“There is no such thing as a science fiction. There is only science eventuality.” Prof. Krummel Chair of Department of Surgery Stanford University, CA, USA

“The best way to predict the future is to invent it” Alan C. Kay fellow at Apple Computer Inc., a visionary

Question 19: What must be done? Answer: a lot

We need training course “Biomedical Applications of Rapid Prototyping” Charleston, USA 2010 Organizer: Vladimir Mironov

We need to build necessary infrastructure for emerging field a) research society, b) new journal “Biofabrication” Editor: Wei Sun, c) e-textbook “Biofabrication” Editor: Vladimir Mironov

We need multidisciplinary National Research Centers On Biomedical Application of Rapid Prototyping or Bioprinting and Biofabrication in every developed nation and large developing and emerging nations

ATBC

We need virtual conferences on “Virtual Tissues” and “Digital Human” enabled by modern telecommunication center as well as virtual global community and networks and interactive website

We need Industrial Report on: “Biomedical Applications of Rapid Prototyping” similar to world famous & well respected “Wohlers Report”

We need annual conference: 5th International Conference on Bioprinting, Biofabrication and Bioassembly (3B’10) Philadelphia, USA, October 2010 Organizers: Wei Sun & Gabor Forgacs Welcome in City of Brotherhood Love and Motherland of US democracy

Question 20: How to build a new biofabrication industry in South Carolina? Answer: joined focused & integrated efforts

ATBC

Advanced Tissue Biofabrication Center ATBC

ATBC

ATBC

From blueprint to organ

Cell sorter

Tissue spheroids robotic biofabricator

Robotic bioprinter

Prototype human organ biofabrication assembly line

Perfusion bioreactor

SC RAPID PROTOTYPING TRAINING CENTERS Development of new curriculum and new Bachelor Program in Biomedical Rapid Prototyping

ATBC

ATBC

MUSC & NEATCO Inc.

South Carolina Robotic Biofabrication Industry Cluster Medical Devices & Implants, Cell, Tissue & Organ Based Therapy

Tissue-Based In Vitro Diagnostic, Drug Discovery & Toxicology High Throughput & High Content Assays

Tissue-Based Biotechnology Animal-Free Animal Products (meat, leather, fur)

Blueprints, Softwares

Bioprinters SC Stem Cells Bank

Robotic Biofabrication Industry Companies: Existing (3D Systems), Recruited & Start-up

South Carolina Government: Department of Commerce, South Carolina Research Authority

Biomaterials Perfusion Media

Advanced Tissue Biofabrication Research & Training Center

Business, financial & legal service Risk capital: VC, Angel Groups

Bioreactors MUSC, USC, CU

3D System University York Technical College

Palmetto Institute New Carolina

Acknowledgement: 1. NSF FIBR grant ATBC 2. NSF EPSCoR R-II 3. MUSC Bioprinting Research Center grant 4. Tan Chin Tuan Fellowship

e-mail: [email protected]