Automated fabrication of multi-cellular
3D human tissues for use in drug
development and therapy
Sharon Presnell, Ph.D.
Chief Technology Officer
August 27, 2013
CDI User’s Group Meeting
(Madison, WI)
© Copyright 2013, Organovo Holdings, Inc. This report is solely for the use of intended audience. No part
of it may be circulated, quoted, or reproduced for distribution outside the organization without prior
written approval from Organovo Holdings, Inc.
SAFE HARBOR STATEMENT
Any statements contained in this presentation that do not describe historical facts may
constitute forward-looking statements as that term is defined in the Private Securities
Litigation Reform Act of 1995. Any forward-looking statements contained herein are based on
current expectations, but are subject to a number of risks and uncertainties. The factors that
could cause actual future results to differ materially from current expectations include, but
are not limited to, risks and uncertainties relating to the Company's ability to develop, market
and sell products based on its technology; the expected benefits and efficacy of the
Company’s products and technology; the availability of substantial additional funding for the
Company to continue its operations and to conduct research and development, clinical
studies and future product commercialization; and, the Company's business, research,
product development, regulatory approval, marketing and distribution plans and strategies.
These and other factors are identified and described in more detail in our filings with the SEC,
including our annual report for the period ended December 31, 2012 on Form 10-K and our
current reports filed on Form 8K. We do not undertake to update these forward-looking
statements made by us.
NYSE: ONVO
©Copyright 2013 Organovo, Inc.
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New treatment options are needed
that restore or replace critical tissue functions
• Shortage of donor organs for transplantation
• Failure of drugs in late-stage clinical trials
2011 Data for Wait List and Transplant of
Organs
REASONS FOR PHASE 3 FAILURES (1990-2010)
N=126
Safety /
Toxicity,
28%
60000
Number of Organs
Other, 27%
Lack of
Efficacy,
45%
50000
Waiting
Transplant
40000
30000
20000
10000
0
Compiled from EvaluatePharma; PharmaProjects; and CDER
©Copyright 2013 Organovo, Inc.
The use of living cells
in drug development and tissue engineering
2-3 years
Target Discovery
0.5-1 year
Lead ID
CELL-BASED ASSAYS
1-3 years
Lead
Optimization
1-2 years
ADMET
5-6 years
Development
1-2 years
Registration
and approval
CELLS & TISSUES AS THERAPEUTIC AGENTS
From Zreiqat et al., Biomaterials
2010 31:3175
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Development of 3D cell-based systems:
A growing trend with proven benefits
• Actively pursued in oncology and
toxicology
– Multicellular tumor spheroids
(MCTS)
– Hepatocyte spheroids
From Schmeichel & Bissell. J Cell Sci. 2003 Jun 15;116(Pt 12):2377-88.
• Demonstrated benefits
– Extended cell survival and/or
function
– Morphologic features (polarity,
intracellular organization)
– Cell-cell interactions
– Better prediction of drug effects
From No et al. PLoS ONE 2012 7:e50723.
©Copyright 2013 Organovo, Inc.
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Achieving 3D in vitro:
the pursuit of in vivo-like form and function
DESIRED ATTRIBUTES
APPROACHES
• Tissue-like cellular density
• True 3D; >200 μm in x, y, and
z axes
• Multiple tissue-specific cell
types present
• Spatially-controlled cell
compartments
• Reproducible; compatible with
automated fabrication
• Spheroids
• Cells + Scaffolds or
Hydrogels
• Micropatterned cultures
• Bioprinting
©Copyright 2013 Organovo, Inc.
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3D tissue formation and maturation are driven by
principles of tissue liquidity and cell adhesion
Multicellular Building Blocks have
liquid-like properties that allow
them to merge into a unified
structure
Adjacent aggregates of cardiac tissue merge
over 24 hours. Scale bar = 100µm. Adapted
from Tissue Engineering Part A , 14(3):413,
2008.
The dynamics of aggregate
fusion are driven by cellcell and cell-environment
interactions
Aggregates of CHO cells in a hydrogel
merge over 120 hours. Adapted from
J Mat Chem, 17:2054, 2007.
©Copyright 2013 Organovo, Inc.
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Overview of the
Bioprinting Process
Multicellular Building
Blocks are generated
from human cells
Building Blocks = Bio-Ink loaded onto the
NovoGen MMX BioprinterTM
(~103- 10 4 cells per droplet)
Bioprinter builds 3D structures
layer-by-layer with 20µm precision
Adjacent bio-ink cell
aggregates fuse to yield
contiguous 3D tissues
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3D tissues have been bioprinted
From cells throughout the body
Cardiac
Lung
Liver
Blood Vessel
Bone
Skeletal Muscle
Peripheral Nerve
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Bioprinted vascular conduits
Develop mechanical strength upon conditioning
Day 0
500 µm
Day 7
500 µm
Day
21
500 µm
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Human tissue analogues:
generation of tissues with laminar architecture
REPRODUCING THE VESSEL WALL ARCHITECTURE:
• Smooth muscle-comprising wall (representing media)
– Aligned smooth muscle cells
– Collagen deposition
• Endothelial covering on one surface (representing intima)
– Complete coverage
• Optional fibroblast layer on opposing surface (representing adventitia)
Endothelium
Smooth Muscle
Fibroblast
•
Fabricated directly into multi-well plates
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Vascular wall analogues
are viable and dynamic with stable architecture
Smooth Muscle Cells (SMA)
TUNEL-positive cells
Endothelial Cells (CD31)
Ki67-positive cells
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Multicellular response to pathogenic stimuli
+
TGF-β1
+
IL-13
CONTROL
CONTROL
in layered 3D blood vessel and small airway analogues
TGF-β1 Treatment ↑ collagen
deposition in blood vessel wall
IL-13 Treatment ↑ proliferation of
fibroblasts and microvasculature,
and ↑ collagen deposition
©Copyright 2012 Organovo, Inc.
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Organovo’s NovoGen BioprinterTM platform
was utilized to generate 3D human liver
Design concepts for 3D human liver
focused on the following key features:
•
•
•
True three-dimensionality, reaching at
least 250 microns in the smallest
dimension
Incorporation of multiple cell types with
spatially-controlled placement in x, y,
and z axes
3m
m
Single Unit
Demonstration of both histologic and
functional features of liver
© Copyright 2013 Organovo Holdings, Inc.
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Tissue-Like cellular density (H&E)
3D human liver tissue
key phenotypic features
Automated Fabrication
with NovoGen BioprinterTM
>500 microns in
thickness
Formation of
microvasculature
CD-31
DAPI nuclei
Cell type-specific
compartmentalization
Hepatic stellate cells
Hepatocytes
Endothelial cells
Formation of
tight junctions
E-Cadherin
DAPI nuclei
Hepatocyte Sourcing:
• Primary human
• HepaRG-derived
• iPSC-derived
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Bioprinted 3D human liver tissues
produce liver-specific proteins and have inducible CYP450s
135 H
Bioprinted liver tissue (48 hrs)
© Copyright 2013 Organovo, Inc.
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Successful substitution of iCells for primary hepatocytes
and enhanced albumin production in 3D vs. 2D
100 μm
H&E / 20x
-
100 μm
CD31 / 20x
© Copyright 2013 Organovo Holdings, Inc.
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Next Steps: 3D Liver
•
Extend functional characterization
and development program for
hepatocyte-containing 3D tissues
•
Continue exploring potential for
stem cell-derived hepatocytes in
product portfolio
Standard Corning TranswellTM Plates
©Copyright 2013 Organovo, Inc.
Partner Companies & Institutions
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Enabling tissue on demand
Technical Team:
Systems Engineering:
Vivian Gorgen
Frank Lin
Stephen Pentoney, Ph.D., MBA
Tissue Applications:
Justin Robbins, Ph.D.
Albert J. Evinger, M.S.
Shelby King, Ph.D.
Benjamin Shepherd, Ph.D.
Therapeutic Tissues:
Vaidehi Joshi
Samir Damle
Jamie Brugnano, Ph.D.
Scott Rapoport, Ph.D.
Bioprocess & Cell Production:
Melissa Romero
Alex Le
Susan Lin
Krystal Moon
Chirag Khatiwala, Ph.D.
Craig Halberstadt, Ph.D.
©Copyright 2013 Organovo, Inc.
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