Organ Printing:How to print a human organ

Vladimir Mironov MD, PhDAssociate Professor & Director ofAdvanced Tissue Biofabrication Center (ATBC)Department of Regenerative Medicine & Cell BiologyMedical University of South CarolinaCharleston, SC 29425, USA

ATBC

Question 1:

How new technologiesevolve?

Answer: from preexistingtechnologies

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,

ut 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 engineeringscience 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

Biofabrication, 2009

From analytical to synthetic anatomy

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?

A d

OrganPrinting

$25 000 000market

only for TEkidney

$75 000annualcost ofdialysis

Huge Market

Question 7:

What is an organ printing?

Answer: Biomedicalapplication of RP or AM

Organ Printingis a computer-aided

robotic layer by layeradditive biofabrication

of functional livinghuman organ

constructs

Bioimaging

Blueprint

CAD

Bioink

Bioprinter

Biopaper

Maturogens

Bioreactor

Biomonitoring

I. Pre-processing

II. Processing

III. Post-processing

Components ofOrgan PrintingTechnology

3 Steps in Bioprinting

Question 8:What is a status

of biomedical applicationof rapid prototyping oradditive manufacturingin 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 modelingAuthor(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 29.112. Title: Fused deposition modeling of novel scaffold architectures for tissue engineering applicationsAuthor(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 27.753. Title: The design of scaffolds for use in tissue engineering. Part II. Rapid prototyping techniquesAuthor(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 21.124. Title: Solid freeform fabrication of three-dimensional scaffolds for engineering replacement tissues and organsAuthor(s): Leong KF, Cheah CM, Chua CKSource: BIOMATERIALS Volume: 24 Issue: 13 Pages: 2363-2378 Published: JUN 2003 18 27 31 50 27 161 23.005. Title: Organ printing: computer-aided jet-based 3D tissue engineeringAuthor(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 22.006. Title: Scaffold-based tissue engineering: rationale for computer-aided design andsolid free-form fabrication systemsAuthor(s): Hutmacher DW, Sittinger M, Risbud MVSource: TRENDS IN BIOTECHNOLOGY Volume: 22 Issue: 7 Pages: 354-362 Published: JUL 2004 13 25 34 35 27 136 22.677. Title: Bone tissue engineering using polycaprolactone scaffolds fabricated via selective laser sinteringAuthor(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 23.608. Title: Rapid prototyping of scaffolds derived from thermoreversible hydrogels and tailored for applications in tissue engineeringAuthor(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 14.629. Title: Fabrication of PLGA scaffolds using soft lithography and microsyringe depositionAuthor(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 15.4310. Title: Novel collagen scaffolds with predefined internal morphology made by solid freeform fabricationAuthor(s): Sachlos E, Reis N, Ainsley C, et al.Source: BIOMATERIALS Volume: 24 Issue: 8 Pages: 1487-1497 Published: APR 200313 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

Rounding

Fusion

Enveloping

Spreading

Evidence of Tissue Spheroids Fluidit

Sorting

Question 10:

What is a fundamental principle of organ

printing technology?

Answer: tissue fusion

‘Virtual and Physical Prototyping’ (2009)

Tissue spheroids fusion

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

ADSCs differentiate into SMCs in a developmentally appropriate

sequence

mRN

AW

este

rn a

naly

sis

Densinometric analysis

Quantitative Phenotypic Analysis

Proliferation

Differentiation

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 ofvascular 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 fusion without cell mixing

Tissue compaction during fusionof vascular tissue spheroids

Virtual and Physical Prototyping, 2009

Tissue Engineering, 2008

Bioengineering Vascular Tube UsingSelf-assembling Tissue Spheriods

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

Virtual and Physical Prototyping, 2009

‘Surface Evolver’, Ken Brakke

‘Blueprint’

A B C

D E F

G

How to print a human organ?

Stem cells

Tissue spheroids

Differentiated cells

Tissuespheroidsin hydrogel

Functionalmature macrotissue

Microfluidic minibioreactorsand cell sorters

Microfluidic mixers,droplet generators and encapsulators

Microfluidic dispensors

and bioprinters

Macrobioreactorwith 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 Spheroi

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-directedself-assembly

(a)

(c)

(b)

Digital Microfluidics: Cascade Droplet Generator

David Weitz, Science, 2005

Question 14:

What is a “bioprinter” ?

Answer: robotic dispenso& tissue bioassembler

Bioprinters

(a) (b)Microfluidics: Bioprinting

Continuous and digital (droplet) microfluidics-based bioprinting

Question 15:

What is a “bioreactor” ?

Answer: perfusion containerfor tissue maturation

Perfusion Bioreactor for Bioprinted Organs

Perfusion Bioreactor for Bioprinted Organs

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

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

12

3

Question 16:

What is a “maturogen” ?

Answer: factor acceleratingbioprinted tissue maturation

Methods of quantitative evaluation ofthe 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

EpMotion 5070 (Eppendorf)Acoustic excitator

(i) (k)

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

A B C

Robotic bioprinters

Perfusion Bioreactors for Bioprinted Organs

Lungs Kidney Liver

Organ Recovery Systems Inc.

nScript Inc.(Sciperio), USA

Kidney Biofabrication Plant

$1 billion?

$20 million NSF grant2025?

ATBC

Question 18:

What is a future of bioprintingtechnology?

Answer: in vivo bioprinter

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

“Face/Off” (1997)

Bioprinting in situ(a science fiction)

Bioprinting in situ (an emerging concept, 2003)

Clinical Bioprinter - Project “Michelangelo”?

(scientific reality, 2009)Bioprinting in situ

“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 SurgeryStanford 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 2010Organizer: Vladimir Mironov

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

We need multidisciplinaryNational Research CentersOn Biomedical Applicationof Rapid Prototyping or Bioprinting and Biofabricationin 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” similarto world famous & well respected “Wohlers Report”

We need annual conference:5th International Conference on Bioprinting, Biofabrication and Bioassembly (3B’10)Philadelphia, USA, October 2010Organizers: 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

Prototypehuman organbiofabricationassembly line

Tissue spheroidsrobotic biofabricator

Robotic bioprinter Perfusion bioreactorCell sorter

From blueprint to organ

ATBC

Development of new curriculum and new Bachelor Program inBiomedical Rapid Prototyping

SC RAPID PROTOTYPINGTRAINING CENTERS

ATBC

MUSC & NEATCO Inc.

ATBC

South Carolina Robotic Biofabrication Industry Cluster

Medical Devices & Implants, Cell, Tissue & Organ

Based Therapy

Tissue-Based BiotechnologyAnimal-Free Animal Products

(meat, leather, fur)

Business, financial & legal service

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

& High Content Assays

3D System UniversityYork Technical CollegeMUSC, USC, CU

South CarolinaGovernment:

Department ofCommerce,

South Carolina Research Authority

Blueprints, Softwares

SC Stem Cells Bank

Biomaterials

Risk capital:VC, Angel Groups

Advanced TissueBiofabrication Research

& Training CenterPerfusion Media

Bioreactors

Palmetto InstituteNew Carolina

Robotic Biofabrication Industry Companies:

Existing (3D Systems), Recruited & Start-up

Bioprinters

Acknowledgement:1. NSF FIBR grant2. NSF EPSCoR R-II3. MUSC Bioprinting

Research Center grant4. Tan Chin Tuan Fellowship

e-mail: mironovv@musc.edu

ATBC