bmbf go bio projectaxlr8.eu/workshops/marx.pdf · uwe marx, technische universität berlin, germany...
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BMBF Go‐Bio project: From single tissue culture to multi‐organ‐chipsFrom single tissue culture to multi organ chips
OUTLINE
the global substance testing dilemma
“human‐on‐a‐chip” – concepts
OUTLINE
project history
our multi‐organ‐chip approach scientific background
Summary & outlook
scientific background biological challenges & technical hurdles status quo
Dr. Uwe MarxHead of “Multi‐Organ Chip programInstitute of Biotechnology,
Summary & outlook
in sili‐tro ‐ synergies!?
AXLR8 satellite meeting / Berlin 22.05.2011
gy,Technische Universität BerlinGermanyuwe.marx@tu‐berlin.de
The dilemma of predictive substance testing
human cell culture test systems
animal models
Dynamic single organbioreactors
Static culture systems
„human on a chip“
&“virtual human”
Complexityphylogenic Proximity
systemic but not human
human
systemic but not humanAND not humane human but not systemic
Berlin 22.05.11 Uwe Marx AXRL8 satellite 2
Living systems on a chip – state of the art
Nature March 31st 2011: Technology feature: A living system on a chip. Vol 471 pp. 661‐665
miniaturized single‐organ‐chips
Huh D. et al Science 328, (2010) Guenther A. et al Lab Chip 10, (2010)
Hepregen
Hµrel
CellASIC
systemic multi‐organ‐chip concepts
Michael Shuler, Cornell University, U.S.A.S J H l L b Chi 11 389 392 (2011)Sung J.H. et al Lab Chip 11, 389‐392 (2011)Sung J.H., Kam C.; Shuler, M.L. Lab Chip 10 446‐455 (2010)Mahler G..J. et al. Biotechnol Bioeng. 104, 193‐205 (2009)
Kiichi Sato, University of Tokyo, JapanImura Y. et al Anal. Chem. 82, 9983‐9988 (2010)
Shuichi Takayama, University of Michigan, U.S.A.
Uwe Marx, Technische Universität Berlin, GermanySonntag F. et al , J. Biotechnol 148, 70‐75 (2010)
AXRL8 satellite 3Berlin 22.05.11 Uwe Marx
History …
… of the GoBio project, targeting for a systemic solution for long term substance testing ultimately predictive to human substance exposure
2007 U. Marx : Why is human tissue culture important to drug discovery? In: Drug Testing In Vitro – Breakthroughs and Trends in Cell Culture Technology, Eds. Marx and Sandig; Wiley‐VCH Verlag GmbH&Co.KgaA.
20112008 2009 2010
DM T‐chipA Eadsorption
01.03.10
Partnership:TU Berlin, BCRT BerlinFraunhofer IWS DresdenFraunhofer IGB StuttgartGESIM GmbH Dresden
Phase 1: ~ €3 Mio/3 years, distributionmetabolism
toxicityPhase 2: spinoff financing
adsorption
excretion
Partnership:
BMBF GO-BIO(multi-organ-chip)
GESIM GmbH Dresden
Routes i.v.. dermal oral
FP7 Health 2010
pTU Berlin, GermanyUniversitè de Geneve, SwitzerlandProBioGen GermanyInnsbruck Medical University, AustriaFraunhofer IGB IWS ITEM Germany
inhalation
AXRL8 satellite 4Berlin 22.05.11 Uwe Marx
Alternatives … Fraunhofer IGB , IWS, ITEM, Germany
Track record in dynamic bioreactor development
Human bone marrow
1991‐1994 Marx et al Nature's UK Product Review 569; 34, 1993
leukemia research
Human micro capillaries
1997‐2001 trans‐vascular leukocyte traffic tumor invasion mechanisms
Human artificial lymph node
2001‐2007 tumor invasion mechanisms
immunogenicity testing
Giese Ch et al. J. Artificial Organs (10):803‐808. 2006 and J. Biotechnology; 148, S. 38‐45; 2010
immune reactivity patternHuman artificial lymph node immunogenicity testing
Modular mini bioreactor
2006‐2008
high content tissue toxicity testing
Ski i
2007‐2009
ki l f i
Modular mini‐bioreactor high content tissue toxicity testing
Skin regeneration system skin transplant manufacturing
id lf bl th h i i i t ifi i hit t
Long term homeostasis at 100% viability?
Berlin 22.05.11 Uwe Marx 5AXRL8 satellite
organoid self assembly through angiogenesis into organ specific micro‐architectures
The crucial role of dynamic blood circulation
nutrient and oxygen transport through blood plasma and red blood cells
blood tissue barrier and neo angiogenesis through endothelial cells
interconnection of organs to create an organism
blood‐tissue barrier and neo angiogenesis through endothelial cells
tissue repair and immune response through white blood cells
kidney vasculaturekidney vasculature
Berlin 22.05.11 Uwe Marx AXRL8 satellite 6
Natures unique organ building blocks …
bibio-molecule cell
organs human
organism organoids
… are of µl‐scalei t lti l i li d ll t
7AXRL8 satellite Berlin 22.05.11 Uwe Marx
… consist multiple specialized cell types
Size, architecture & fluid dynamics matter!
A paradigm of stringent correlation between architecture, dynamic microenvironment and
skin segment withhair follicles
liver lobulus
, yfunctionality applies to all levels of biological
existence on earth
Berlin 22.05.11 Uwe Marx AXRL8 satellite 8
hair follicles
Biological challenges & technical hurdles
long term performancecapillary blood circulation
organoids of different functionality
performance
0 organoids of different functionality
micro‐pump28 days
control unit90 days
standardized chip platform1 year
OECD guidelines forrepeated dose oral toxicitytesting of chemicals: TG407 (28-days, rodents); TG408 and TG 409 (90 da s
AXRL8 satellite 9Berlin 22.05.11 Uwe Marx
408 and TG 409 (90-days,rodents and non-rodents)
Standardized chip fabrication platform establishedLayer Material Function
4 PDMS(2000 µm)
Channels and reservoirs
3 Glass (200 µm)
Closing layer with holes
2 Sili Ch l2 Silicon (400 µm)
Channels, organ growth segments & stem cell niches
1 Glass Sensors and heater
Sonntag et al Design and prototyping of a chip‐based multi‐micro‐organoid culture system for substance testing, predictive to human (substance) exposure J. Biotechnology 148, 70‐75, 2010
(200 µm)
Rapid prototyping of any sophisticated designThermal oxidation of Si‐Wafers
the size of a standard microscopic slide
organ culture spaces accessible for live tissue imaging
2 months
Anodic bounding
Wafer cutting and structuring PDMS‐layer
Laser structuring of silicon and glass organ culture spaces accessible for live tissue imaging
single use biocompatible materials
adequate micro‐systems fabrication procedures
Berlin 22.05.11 Uwe Marx 10AXRL8 satellite
Micro‐pump and control unit in place
peristaltic micro pump
t l it
Control of up to 8 pneumatic actors Free configurable e.g. 1 pump + 5 valves L 0 1 b Hi h 1 2 bcontrol unit Low‐pressure: 0 .. ‐1 bar; High‐pressure: 1 .. 2 bar Control via buttons and lamps Remote‐control via serial interface Platform independent PC control software(e g WINDOWS LINUX MAC)
Berlin 22.05.11 Uwe Marx 11AXRL8 satellite
(e.g. WINDOWS, LINUX, MAC)
Three pillars of the tissue engineering program
capillary blood circulationskin organoid researchchip basedblood circuit investigative dynamic
liver sinusoid model
investigative dynamic skin culture chip
liver organoid research
liver sinusoid model(technical perfusion)
p(technical perfusion)
vascularized liver and skin equivalent
epidermis / dermis equivalents Including hair follicles
O2
and skin equivalent
knowledge knowledge
Lee P.J. et al (2007)Biotechnol. Bioeng . 97,
Berlin 22.05.11 Uwe Marx 12AXRL8 satellite
Investigative dynamic skin culture chiphair follicle and skin self assembly in static culture
i i
hair follicle
in vitroneopapillae
in vivo
Lindner et al.; J. Biotech 152:108 (2011) pan‐cytokeratin trychohyalin
epidermis/dermis14,3 mm2 transwell
continuous medium perfusion(valves and reservoirs )
transwell insert for skin tissue culture
peristaltic micro pump
Dynamic instead of static human skin equivalent culture should support hair follicle ingrowths into a
Berlin 22.05.11 Uwe Marx 13AXRL8 satellite
Dynamic instead of static human skin equivalent culture should support hair follicle ingrowths into aepidermis‐dermis equivalent.
Investigative dynamic liver sinusoid model
Designed omocer® photo resist inserts in placepO2
Peri‐portal:60 mmHg
Peri‐venous:25 mmHg
500 µm
~400 hepatocytes
Zone IIIZone I
50 µm
glycolysislipogenesiscyt p450 baseddrug detoxification
ß‐oxydationgluconeogenesis
cholesterol synthesis
25
Disse space bile segregation stellate cell function d fl
zonal albumin secretion drug metabolism (CYP3A4) bile synthesis (CYP7A1)
AXRL8 satellite 14Berlin 22.05.11 Uwe Marx
adequate flow rates … static 7day culture of human fibroblast(2‐Photon microscope Z‐stack, 10µm steps)
bile synthesis (CYP7A1)
Summary
capillary blood circulation (under investigation)capillary blood circulation
organoids of different functionality
(under investigation)
vascularized liver and skin ‐multi‐organ chip (upcoming)vascularized liver and skin multi organ chip (upcoming)
investigative dynamic skin bioreactor investigative dynamic liver sinusoid bioractor (under investigation)
(in place)
micro‐pump (in place)
control unit (in place)
(i l )standardized chip platform (in place)
Berlin 22.05.11 Uwe Marx AXRL8 satellite 15
“In sili‐tro” synergies !?“virtual human”
in silico„human on a chip“
in vitro
be ‐ chip bio ‐ chip
knowledge basedData quality bio‐fit ‐ based
degree of emulationPrediction isrestricted to:
known mechanismsand their combinations
TGN 1412 – anti CD28 super‐agonistoverrides the basic immune control
Berlin 22.05.11 Uwe Marx 16AXRL8 satellite
mechanisms
Acknowledgement
Fraunhofer IWS, DresdenFrank SonntagNiels SchillingRoland Lauster
Institute of Biotechnology
R k H l d
Niels SchillingUdo Klotzbach
A ik J i kReyk HorlandGerd LindnerIlka WagnerEva Materne Funding
Annika JaenickeAgnes SchumacherLuzie Reiners‐SchrammDesiree Geßner
Katharina CierpkaKatharina SchimekSilke HoffmannS B i k
FundingBMBF: GO‐Bio
Karolina TykwinskaDevasena KanthiJennifer BinderShirin FatehiSven Brincker
Alexandra LorenzMatthias Gruchow
Shirin FatehiMark Rosowsky
Berlin 22.05.11 Uwe Marx 17AXRL8 satellite
Announcement
Berlin 22.05.11 Uwe Marx 18AXRL8 satellite