true bioprinting in 3d for the present and the future
TRANSCRIPT
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.