Alternative Methods for Predictive Safety Testing:
3D Bioprinted Tissue Models
April 9, 2019
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
Conflict of Interest Statement
⚫ Scientific Advisory Board Member, Allevi Inc.
⚫ Co-Founder, Scientific Advisory Board Member, Aprex Biochips
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
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
Complex Tissue Microarchitectures
Macroscale functional tissue/organ
Microscale tissue-building units
VectorStock; Smart Servier Medical Art
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
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
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
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
Multi-Material Bioprinting of Complex 3D Patterns
Adv Mater, 2017, 1604630; patent pending
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
Multi-Material Bioprinting of Bone-Muscle Interface
Data publication pending at time of presentation
Multi-Material Bioprinting of Bone-Muscle Interface
Data publication pending at time of presentation
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
Multi-Material Bioprinting of Glioblastoma Model
Adv Mater, 2019, 31, 1806590 (J. Prakash, U Twente)
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
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
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
Functions in Sacrificially Bioprinted Vessels
Biomicrofluidics, 2017, 11, 044109 (S.R. Shin, BWH); unpublished data, 2019
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
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
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
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
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
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
Blood/Lymphatic Pair on Anti-Cancer Drug Effects
Adv Funct Mater, 2019, in press
GelM
A +
Tumor cells
Blood flow
Lymphatic drain
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
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
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
Slide Title in this Font and Color
Laboratory of Engineered Living Systems (www.shrikezhang.com)
Acknowledgements
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