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

yszhang@research.bwh.harvard.edu

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