Alternative Methods for Predictive Safety Testing: 3D ...€¦ · 150 m control control control 30...
Transcript of Alternative Methods for Predictive Safety Testing: 3D ...€¦ · 150 m control control control 30...
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Alternative Methods for Predictive Safety Testing:
3D Bioprinted Tissue Models
April 9, 2019
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Putting 3D Bioprinting to the Use of Tissue Model Fabrication
Y. Shrike Zhang
Department of Medicine, Harvard Medical School
Division of Engineering in Medicine, Brigham and Women’s Hospital
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Conflict of Interest Statement
⚫ Scientific Advisory Board Member, Allevi Inc.
⚫ Co-Founder, Scientific Advisory Board Member, Aprex Biochips
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3D Organ/Tumor Models & Human-on-a-Chip
Drug development
Personalized medicine
Drug Discov Today, 2017, 22, 1392; Microphysiol Sys, 2017, 1, 1; ufluidix, Jan 31, 2018; Adv Biosys, 2018, 2, 1800109
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Streamlined Multi-Organ-on-Chip System
AutomatedMulti-Organ-on-Chips
Automated Valve Controller
Organs-on-a-Chip Platform
Electronics for Physical/Chemical Sensors
N2 Gas Pressure Source
Electrochemical Station
Real-time Data Display
Real-time Data Display
III.InsituBioanalysisI.MicrofluidicAutomation&DeviceDesigns
II.3DBioprinting/Biofabrication
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Complex Tissue Microarchitectures
Macroscale functional tissue/organ
Microscale tissue-building units
VectorStock; Smart Servier Medical Art
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Photopatterned Microtissues
Time (s)
0 5 10 15 2010 mm
50 mm
fibrin
100 mm 50 mm
200 mm
400 mm
hγ
100 mm
200 mm
Day 1 Day 7
Proc Natl Acad Sci USA, 2017, 114, E2293
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3D Bioprinting for Tissue/Tissue Model Fabrication
Extrusion Bioprinting Stereolithography Inkjet BioprintingLaser-Assisted
Forward Transfer
Near-Field
Electrospinning
-Structural flexibility. -High spatial control -Large-scale fabrication -Automation
Small, 2019, in press; Lab Chip, 2019, in press
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Digitally Tunable Rapid Multi-Material Bioprinting
400 µm
Open/Close
Pressure
Software
Valve 1 Reservoir 1
Valve 2 Reservoir 2
Valve 3 Reservoir 3
Valve 4 Reservoir 4
Valve 5 Reservoir 5
Valve 6 Reservoir 6
Valve 7 Reservoir 7
Nozzle
Adv Mater, 2017, 1604630; patent pending
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Rapid Multimaterial Bioprinting System
At least one order of magnitude faster Conventional multi-nozzle platform
Adv Mater, 2017, 1604630; patent pending; Adv Mater, 2014, 26, 3124
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Multi-Material Bioprinting of Complex 3D Patterns
Adv Mater, 2017, 1604630; patent pending
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Multi-Material Bioprinting of Tissues
Alizarin RedHAp Gradient
0 2051.25
Da
y 1
Da
y 3
HAp Conc.
Time (Day)
1 2 3 4A
bso
rption
(a.u
.)
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Construct 1
Construct 2
Construct 3
HDF HUVEC
HepG2 hMSC GelMA
HDFs
HUVECs
HepG2
hMSCs
Via
bili
ty (
%)
0
20
40
60
80
100
120Day 0
Day 1
Day 7
Adv Mater, 2017, 1604630; patent pending
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Multi-Material Bioprinting of Bone-Muscle Interface
Data publication pending at time of presentation
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Multi-Material Bioprinting of Bone-Muscle Interface
Data publication pending at time of presentation
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Multi-Material Bioprinting of Bioelectronics
Adv Mater, 2016, 28, 3280 (S.R. Shin, BWH); Adv Mater, 2017, 1604630; Adv Mater, 2018, 30, 1704189 (S.R. Shin, BWH)
CNT Concentration (mg mL-1
)
0 1 2 3 4 5 6 7
Re
sis
tan
ce
(W
mm
-1)
0
1
2
3
4
5
5 mm
100 μm
F-actin Nuclei
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Multi-Material Bioprinting of Glioblastoma Model
Adv Mater, 2019, 31, 1806590 (J. Prakash, U Twente)
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Multi-Material Stereolithographic Bioprinting
LED lamp
Biconvexlens
Plano convexlens
Arduinoboard
Fluidic chip
Bioinks
N2
CAD file
Pneumatic valves
Unpublished, 2019; Adv Mater, 2018, 30, 1800242; provisional patent
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Multi-Material Stereolithographic Bioprinting
(i) (ii)
(i) (ii)
5 mm
(i) (ii)
Crosslinking
UV
Wash
Coated Glass
Membrane1
3
2
4
Unpublished, 2019; Adv Mater, 2018, 30, 1800242; provisional patent
Multi-material stereolithographic bioprinting of tissues
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Building Blood Vessels Using Sacrificial Bioprinting
Front
Agarose fiber
Side
Remove fiber
GelMA
GelMA
Crosslink GelMA using UV light
Seed cells
Other possible shapes
Printing & hydrogel filling
Fiber removal
Cell seeding & perfusion
1 mm
Fiber removal
i ii
iii
iv
v
vi
vii
i) ii)
iii) iv)
v)
vi)
Agarose template Pluronic template
Science Kids; Lab Chip, 2016, 16, 4097; Biotechnol J, 2019, in press
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Functions in Sacrificially Bioprinted Vessels
Biomicrofluidics, 2017, 11, 044109 (S.R. Shin, BWH); unpublished data, 2019
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Modeling Vascularized Liver for Drug Testing
***
**
**
***
***
***
100 µm
DAPI/GFP/F-actin DAPI/GFP DAPI/GFP
DAPI/F-actin
DAPI/GFP/F-actin
b c
DAPI/F-actin
DAPI/GFP/F-actin
150 µm
control
control
control
30 mM APAP
30 mM APAP
30 mM APAP
d
PMMA plate
HepG2/C3A cells in GelMA
HUVEC
Drug perfusion
Drug diffusion
Drug diffusion
Top view Cross-sectional view Bioreactor
a
e
ECviability
Hepatocyteviability
Biomicrofluidics, 2017, 11, 044109 (S.R. Shin, BWH); unpublished data, 2019
15%5% 10% Low-MAMedium-MA
High-MA
~3 mm
~1 mm~250 µm
500 µm
GelMA Medium-MA 5%, 30 s GelMA Medium-MA 10%, 30 s GelMA Medium-MA 15%, 30 s
L: Characterization length D: Diffusion coefficient
t: Crosslinking time X, Y: Constants (from measurements)
15%
5% 10% 15%
5% 10%
5% 10% 15%
Low-MAMedium-MA
High-MA
Low-MAMedium-MA
High-MA
Low-MAMedium-MA
High-MA
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Disease Modeling: Thrombosis
Deep vein thrombosis
Incidence: 1/500 in US
Healthcare: >$10 billion
Model Humanthrombus
J Vasc Interv Radiol, 2014; Lab Chip, 2016, 16, 4097
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Modeling Mammary Ductal Carcinoma (in space)
Time (Day)
0 5 10 15 20 25 30
Pe
rce
nta
ge
Co
ve
rage
(%
)
0
20
40
60
80
100
120
Time (Day)
0 5 10 15 20 25 30
Ave
rag
e I
nva
sio
n D
ista
nce
(µ
m)
-10
0
10
20
30
40
Day 3 Day 7
Day 11 Day 13
Day 19 Day 24
200 μm
Live Dead
E-cadherin N-cadherin Nuclei Superimposed
Launch
Recycle
Cancer Res 2017;77(13 Suppl):Abstract nr 4828; Biotechnol J, 2019, in press; provisional patent; unpublished, 2019
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Paper-Based Vascularized Tissue Models
Remove
ink
Endothelial
cells
InkBacterial
Cellulose
MCF-7 Endothelial cells BC nanofibers
Paper-based tissue model
Cross-sectional view of microchannel
Lateral view of microchannel
Nano Lett, 2019, under revision
Live Dead10 µM Tamoxifen
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Direct Bioprinting of Perfusable Cannular Structures
Biomaterials, 2016, 106, 58
0
500
1000
1500
2000
1 2 3 4 5 6
Size(mm)
Samples
Outerdiameter
Innerdiameter
Wallthickness
400 µm
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Tuning the Permeability of Bioprinted Hollow Tubes
PE
GD
A-2
0k
Da
PE
GO
A-2
0k
Da
A
B
500 m
Phase constrast BSA-FITC-0.5h BSA-FITC-1h
m
PE
GD
A-2
0k
Da
PE
GO
A-2
0k
Da
A
B
500 m
Phase constrast BSA-FITC-0.5h BSA-FITC-1h
m
Adv Funct Mater, 2019, in press; provisional patent
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Blood/Lymphatic Pair on Anti-Cancer Drug Effects
Adv Funct Mater, 2019, in press
GelM
A +
Tumor cells
Blood flow
Lymphatic drain
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200µm200µm
i ii
200µm200µm
i ii
3
2
1
Tunable Multi-layer Hollow Fiber
1cm
1mm 500µm
500µm
500µm
ZO-1 DAPI Merged
100 µm
ZO-1
α-SMA DAPI Mergedα-SMA
100 µm
20 µm
E-Cad
20 µm
20 µm
α-SMA DAPI α-SMA
CD31 DAPI Merged
CD31 VE-Cad
Merged
100 µm
100 µm
20 µm 20 µm
20 µm
Urothelialcells/Smoothmusclecells
Endothelialcells/Smoothmusclecells
Ureters
Urothelialcells
Smoothmusclecells
Bloodvessels
Endothelialcells
Smoothmusclecells
Emulating Multi-Layered Cannular Tissues
Adv Mater, 2018, 30, 1706913
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Vascularized Microtissues for Drug Testing
Engineered
endothelialized
myocardium
Cardiomyocytes
Blood
vessels
Native
myocardium
10 µm
GFP (HUVECs) / sarcomeric α-actinin / nuclei
ECs/Cardiomyocytes /Nuclei
5 µm10 mm
10 mm
Time (day)
0 1 2 3 4 5 6
Re
lative
be
atin
g r
ate
(%
)
0
20
40
60
80
100
120
0 µM
10 µM
100 µM
Time (day)
0 1 2 3 4 5 6
Re
lative
ch
an
ge
vW
F le
ve
l (%
)
0
20
40
60
80
100
120
0 µM
10 µM
100 µM
Time (day)
0 1 2 3 4 5 6
Re
lative
be
atin
g r
ate
(%
)
0
20
40
60
80
100
120
0 µM
10 µM
100 µM
Time (day)
0 1 2 3 4 5 6
Re
lative
ch
an
ge
vW
F le
ve
l (%
)
0
20
40
60
80
100
120
0 µM
10 µM
100 µM
Neonatalratcardiomyocytes/HUVECs
HumaniPSC-cardiomyocytes/HUVECs
Biomaterials, 2016, 110, 45
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Bioink Developments
Biomacromolecule network- Chemical/physical crosslinking
of biomacromolecule- Removal of alginate through
monovalent ion exchange or
using Ca2+-chelator
Templated network- Physical crosslinking
of alginate by Ca2+
Composite bioink- Alginate
- Biomacromolecule
Bioprinting
Wash & Culture
CaCl2Bioink
Shear-thinning bioinks Microfluidic bioprinting of composite bioinks
CaCl2+GelMA+Cells
Cell-laden constructs
Bioprinting
Alginate
Cell culture
UV crosslinking
Pore-forming bioinks
In-fiber bioprinting of
low-stiffness bioinks
Biomaterials, 2016, 110, 45; Adv Funct Mater, 2017, 27, 1605352; JoVE, 2017, 126, e55957; Adv Healthcare Mater, 2017, 6, 1601451
Biofabrication, 2018, 10, 024102; Macromol Biosci, 2018, 18, 1800127; Adv Mater, 2018, 30, 1805460; provisional patent
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Slide Title in this Font and Color
Laboratory of Engineered Living Systems (www.shrikezhang.com)
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Acknowledgements