True Bioprinting in 3D for the Present and the Future

Malcolm Willson

UK Manager

Digilab

Introduction

It is now commonly accepted that 2D culture conditions cannot efficiently represent the complex in vivo microenvironment Cells cultured in 2D monolayers were found to display different gene expression and functionality compared to cells in native tissues or 3D culture conditions Development of 3D bioprinting technologies can enable novel biomedical researches by creating 3D structures resembling in vivo microenvironment and tissue structures

Biological Patterns Are Ubiquitous

Differences between 2D and 3D

Differences between 2D and 3D

2 D 3D

3D Bioprinting Market – Global Industry Forecast

3D bioprinting is a process of creating spatially-controlled cell patterns in 3D, where viability and cell function are conserved within printed construct. The 3D bioprinting industry that is currently at the embryonic stage of generating replacement human tissue has been forecast to be worth billion dollars by 2019. 3D bioprinting at present largely involves the creation of simple tissue structures in lab settings, but is estimated to be scaled up to involve the creation of complete organs for transplants. This technology is expected to be used for more speedy and accurate drug testing, as potential drug compounds could be tested on bioprinted tissue before human trials commenced.

Persistence Market Research

3D in Academia 3D bioprinting is steadily emerging as an area that is gathering attention from a lot of academics. Some of the researchers have recently opened start-up firms with aim of commercializing the technology in coming years. A number of start-ups have recently sprung up to build up products based on bioprinting. Some are spin outs from university research. Examples:- Aspect Biosystems focused on printing tissue models for toxicity testing TeViDo BioDevices focused on printing breast tissue SkinPrint focused on developing human skin.

Commercial Market The market of 3D bioprinting particularly focuses on the commercial bioprinters and those under development, their applications and the expected future evolution. It is widely predictable that the 3D Bioprinting market has great potential.

It requires :- Biocompatible materials (bio-ink and bio-paper) Software (CAD) Hardware (bioprinters) and each has the capability to grow into separate niche industries The commercial companies in bioprinting market include SkinPrint that is developing a replacement skin for the burns patients or for those suffering from skin disorders. Aspect Biosystems that is developing printed tissue for drug testing Organonovo working with L’Oreal for toxicity testing

Safety screening

Drug discovery Drug discovery is a highly expensive process which in most cases will end in failure to gain regulatory clearance .The reason for this high failure rate is related to the lack of sufficiently accurate pre-clinical (prior to human volunteer) testing methodologies which have to date been limited to 2-dimensional human cell assays together with animal testing. Differences between animal models and human tissues have contributed to approximately one dozen failures of late-stage drugs in 2012 alone

Large Pharma interest

•Recently announced ”World's largest consumer goods company Procter & Gamble has become the first company in the world to explore 3D bioprinting” •P&G has banned animal testing on all its wide range of products, except the 20% that require animal testing by law. It has engaged in the development of new techniques for testing products. 3D printed organ tissues would allow P&G to research how their cleaning and healthcare products might affect the organ systems of their customers.

Due to European restrictions on animal testing on cosmetics L'Oreal the world's largest cosmetic company is exploring the use of 3D bioprinting on liver cells collaborating with Organonovo for cosmetic safety testing, specifically skin care products. Merck also announced they are working with Organonovo

Cellomics(3D) Detection Imaging Function Data

The ‘last mile’ of ‘omics

Protein Proteomics Metabolomics

RNA

DNA Genomics

Why print in 3D?

• Differences between animal

models and human tissues

contribute to late-stage drug

failures.

• Alternative is in vitro testing

for drug discovery .

• 3D supports coalescence and

proliferation.

• X-section of bio-printed liver (above) with stains for

viability and density. Hepatocytes (blue) endothelial cells

(red) and hepatic stellate cells (green).

3D cell culture revenue growth

Estimated Annual Growth rate 30%

$586.1 Million $ 2.2 Billion

2014 2019

BCC data

Other estimates range to $7Billion by 2021

Cell culture commercializes across a complex 3D landscape

Major benefits of 3D

2D Issues

• Cells can’t grow in 3rd dimension (surface, gravity)

• Limited communication/contact with other cells

• Result: not relevant to human physiology

3D Benefits

• Cells can grow in 3rd dimension >on all sides, layers can function

• Communication with other cells >interplay (co-cultures)

• More ‘phenotypic’ assays >human primary and stem cells

The Challenge:

• Engineer tissue that emulates patterns of biological structure

• Visualize complex 3D structural patterns in biology

• Understand the physical, chemical, and biological basis of such biological patterns.

• Fabricate 3D tissue constructs which emulate such biological patterns.

3D cell culture provides physiological

environment to assess biological response

• Cells are cultivated in a

micro-environment that

allows them to grow and

interact with their

surrounding in all

dimensions.

• 3D cell culture can be used

to mimic acinar structures

• Organs on chip take

advantage of microfluidics

and microfabrication to

portray dynamic properties

of the living organ.

3D cell culture: Organ on chip model for

pharmaceutical testing

Embryoid body formation Bioprinting method

• Schematic of the EB formation process using bioprinting approach. Droplets of

cell-medium suspension were bioprinted onto the lid of a Petri dish and were hung up for 24 h to allow for EB aggregation. The formed EBs were transferred to a 96-well plate for additional culture up to 96 h.

Aspirations of 3D live cell printing

Patterned 3D cell deposition

Biological “blueprint”

Clinical Need Technology Applications

Simple structure

Complex structure

•Regeneration of muscle

following trauma or

dystrophic degeneration.

•Regeneration of heart

muscle post-myocardial

infarction.

•Regeneration of skin

and subcutaneous tissue

in burn patients.

•Regeneration of neural

connectivity patterns.

•3D tissue constructs for

pharmaceutical testing

Use of scaffolds for 3D spacing

• new technologies are being developed to fabricate 3D constructs to explore the cellular behaviors in the 3D condition.

• i.e. cell interaction • stem cell differentiation • vascularization • ossification • with the potential application in drug screening and

regenerative medicine • Scaffold-based strategy is a commonly used tissue

engineering approach to create 3D structure

Pittsburgh-Based Team Engineers Muscle, Bone Cell Differentiation With Aid Of Ink-Jet Printer

Pitsburg group at Carnegie Mellon's Robotics Institute -"Controlling what types of cells differentiate from stem cells and

gaining spatial control of stem cell differentiation are important capabilities if researchers are to engineer replacement tissues that might be used in treating disease, trauma or genetic abnormalities,"

They developed a method to deposit and immobilize growth factors in virtually any design, pattern or concentration, laying down patterns on native extracellular matrix-coated slides (such as fibrin).

These slides are then placed in culture dishes and topped with muscle-derived stem cells (MDSCs). Based on pattern, dose or factor printed by the ink-jet, the MDSCs can be directed to differentiate down various cell-fate differentiation pathways (e.g. bone- or muscle-like).

Customisable BioInks

Researchers at Michigan Technological University are in the process of developing bio links, or printable tissue, using a 3D bio printer in order to make synthesized nerve tissue that could help regenerate damaged nerves in patients with spinal cord injuries. They have incorporated Graphene bioflakes into a collagen hydrogel which they hope to use for nerve regeneration

Bioprinting: 3D fabrication of tissues

using biological materials

Young-Joon Seol et al. Eur J Cardiothorac Surg 2014;46:342-348

Structural complexity and efficacy of 3D

bioprinting

Murphy and Atala, Nature Biotechnology, 32: 773-785, 2014

• 2D organs have already

been fabricated and will

be one of the first types

of bio-printed tissues to

be transplanted.

• Hollow tubes, including

blood vessels & tracheas

in development.

• Hollow organs: GI tract

• Solid organs complex:

heart, tongue, liver.

Top-down versus bottom-up approaches

to tissue engineering

• A top-down approach, wherein cells are seeded on pre-

fabricated biodegradable scaffolds, and are expected to

generate biomimetic tissue environments.

• A bottom-up approach, also known as modular tissue

engineering, creates smaller building blocks or modules

made of cells and/or scaffolding material having the

microarchitecture of native tissue, which can then be

assembled to create larger functional tissues.

Bioprinting is in principle a bottom-up approach.

Components of inkjet, micro-extrusion

and laser-assisted bio-printers

(a)Thermal inkjet printers electrically heat print-head to produce air-

pressure pulses that force droplets from the nozzle

(b)Micro-extrusion printers use pneumatic dispensing systems to

extrude continuous beads of material and/or cells

(c)Laser-assisted printers use lasers focused on an absorbing

substrate to generate pressures that propel cell-containing

materials onto a collecting substrate.

Malda, J. Adv. Mater.25, 5011–5028 (2013)

Principal limitation of current bio-printing systems is cell viability.

The CellJet incorporates

liquid dispensing, on-the-

fly, and/or drop-by-drop

non-contact cell printing

while maintaining

viability.

BIO-PRINTING VALIDATION

PRINTING CELLS

• Viability

• Functionality

• Concentration consistency

PRINTING HYDROGELS

• Drop dispense at high viscosity

• 2D Shapes

• 3D Layering

3D CONSTRUCT: Cells in hydrogel architecture

On the fly dispensing

Stop and dispense

Cell Dispensing -On the fly

U-937: on the FLY

Plate images from MIAS 2 of U 937 cells

For less robust cells, where

the momentum of a fast

dispense can disrupt the cell

controlling the height and

speed of dispense enables

better cell viability

e.g with U937 cells

Comparing 3 syringe

dispense speeds:-

5ul/sec

10ul/sec

20ul/sec

Higher impact by Combining syringe speed with momentum of dispense ie on the fly at 20ul/sec affects viability

Carrier: U bottom; dispense height: top of plate; dispense speed: default; cell density: 50,000 cells/ml; medium added immediately; 3 days after seeding

20 µl/sec 10 µl/sec 5 µl/sec

Cell Dispensing -stop and Dispense

5 µl/sec

U-937: Drop by Drop

Carrier: U bottom; dispense height: top of plate; dispense speed: default; cell density: 50,000 cells/ml; medium added immediately; 3 days after seeding

Plate images from MIAS 2 of U937 cells

For less robust cells, momentum of a

fast dispense can disrupt the cell

controlling the height and speed of

dispense enables better cell viability

e.g with U937 cells

Comparing 3 syringe dispense speeds:-

5ul/sec

10ul/sec

20ul/sec

10 µl/sec 20 µl/sec

No loss of cells at all three dispense speeds

Valve determines the dispense timing of the drop.

Syringe stepper motor controls the volume dispensed.

The valve, syringe and stage are all synchronised

syringe stepper motor

stage stepper motor

synQUAD valve

master controller

Digilab dispense technology (Synquad):

high speed micro-solenoid valve based

dispensing

Nano-dispensing fluidic control

• System incorporates non-contact

hydraulic dispensing under steady

state pressure of nanoliter volumes in

arrays of up to 16 channels.

• Specific attributes include

aspirate/dispense and continuous

dispensation modes.

• Ceramic Tip size: 100um, 190um,

250um, and 500um

• Syringe size: 100ul, 250ul, 1ml, 2.5ml

• Temperature and humidity control

Physical attributes of CellJet system

FAST: Fills 1536 wells < 1 min

FLEXIBLE:

• Multi-Channel

• 20 nL – 4 µL range

ACCURATE: 10 µm spatial

GENTLE: Non-contact fluid

MODULARITY OF SYSTEM: – Number of Channels: Varies depending on system 1-16 +

– Ceramic Tip size: 100um, 190um, 250um, and 500um

– Syringe size: 100ul, 250ul, 1ml, 2.5ml - depends on volume

Viability of bio-printed mesenchymal

stem cells

• hMSC-suspension dispensed

using either the CellJet or manual

pipetting into 96-plate wells.

• All wells were stained with Live-

Dead stain (Invitrogen)

3D Using Hydrogels

• The in vitro 3D cellular environments simulate the complexity of an in vivo environment and natural extracellular matrices (ECM).

• Bioprinting utilizing hydrogels as 3D scaffolds are advantageous for cell culture as they are highly permeable to cell culture media, nutrients, and waste products generated during metabolic cell processes.

• They have the ability to be fabricated in customized shapes with various material properties with dimensions at the micron scale

Hydrogels – Drop dispense

O,5% w/w Sodium Alginate

• Viscosity: 58.5 cP

• Volume Range: 100 nL – 4 µL

1.0% w/w Sodium Alginate

• Viscosity: 269.5 cP

• Volume Range: 125 nL – 4 µL

1.5% w/w Sodium Alginate

• Viscosity: 1180.2 cP

• Volume Range: 250 nL – 4 µL

Hydrogels / Scaffold assembly

• Hydrogels – Alginate

– Collagen

– Matrigel*

– Agar

– etc

Solid skeletons -Plastics - Sugars -etc

Some groups have reported improved cell survival and growth after being mixed in a hydrogel and dispensed into a 3d lattice vs 2d culture

Hydrogels – 2D Shapes

Sodium Alginate gel printed to a glass slide Trial

Lines Average Line Thickness

Hydrogels – 2D Shapes

Sodium Alginate gel printed in 10 mm circle Trial

Printed Geometry – controlled by Dispense Volume, Height and Speed

Circles Average Line Thickness

Live Cell Printing • Live Cell Dispensing – mammalian, bacterial & other cells

• Embryonic Stem Cells with >95% viability

• Viscous Solutions – Protein/DNA suspensions, Hydrogels

• Layering in 2D Structure in 3D

• Print Functional Tissue - Biofabrication

Construction of complex shapes with

various fluids

Bio-printed hMSCs in 0.5% Sodium

Alginate in simple geometrical patterns

hMSC suspension in 0.5% Alginate dispensed using the

CellJet to form continuous patterns in 6-well plate

No. of channels 1-16+

Size of syringe

Size of tip

Length of tip – shallow and deep well

Orientation of dispensing head

Optional Humidity Chambers

– PixSys

– MicroSys

PreSys – automation option

PixSys- larger deck, more channels

ALTERNATIVE CONFIGURATIONS

Synopsis of CellJet

• Prints live-cells with >95% viability with sterile conditions

• Performs 3D bioprinting utilizing both Aspiration-

Followed-by-Printing and Continuous-flow-printing

• Handles simultaneously up to 16 different Bio-inks having

the viscosities as thin as acetone or as thick as glycerol

• Can use any standard lab-ware, or a customized bio-

reactor chamber, both for a reservoir source and for

destination giving tremendous freedom in terms of the

protocol that can be used for bioprinting

Bioengineered tissue models

-The future

• Image the initial tissue

• Design engineered tissue from template

• Fabricate tissue model with 3D bioprinter

Helically aligned myofibers of the heart:

3D contraction with rotation Image Design

Heart conceived as crossing

helical structures, whose

crossing angles vary as a

function of transmural depth

“Designing” tumors from their

underlying architecture Image Design

“Pseudopalisading” necrosis in

GBM: Morphologic feature linking

vascular pathology, hypoxia, and

angiogenesis Rong et al, J Neuropath Exp Neurology, 2006

Summary

• It is now commonly accepted that 2D culture conditions cannot efficiently represent the complex invivo micro-environment

• Cells cultured in 2D monolayers were found to display different gene expression and functionality compared to cells in native tissues or 3D culture conditions

• Development of 3D bioprinting technologies can enable novel biomedical researches by creating 3D structures resembling in vivo microenvironment and tissue structures

• new technologies are being developed to fabricate 3D constructs to explore the

cellular behaviors in the 3D condition i.e. cell interaction, stem cell differentiation, vascularization, ossification -with potential applications in drug screening and regenerative medicine

• Scaffold-based strategy is a commonly used tissue engineering approach to create

3D structure.

Conclusions

• 3D bioprinting in the future would enable the design and

fabrication of tissue constructs with complex architectures

derived from structural images.

• Current capabilities enable smaller scale /partial models

using a variety of Bioinks and printing methods

• The CellJet apparatus provides a mechanism to control in

time and space the dispensation of multiple cell types

while retaining viability.

• Bioprinting of tissue provides a mechanism for testing of

pharmaceutical substances in the setting of functional

tissue, as well as the fabrication of tissues for therapeutic

implantation.