THE GROUND TISSUES EPITHELIAL TISSUES CONNECTIVE AND SUPPORTIVE TISSUES MUSCLE TISSUES
Automated fabrication of multi-cellular 3D human tissues ...
Transcript of Automated fabrication of multi-cellular 3D human tissues ...
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.
Automated fabrication of multi-cellular 3D human tissues for use in drug development and therapy
SAFE HARBOR STATEMENT
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NYSE: ONVO
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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.
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
Compiled from EvaluatePharma; PharmaProjects; and CDER
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10000
20000
30000
40000
50000
60000
Num
ber o
f Org
ans
2011 Data for Wait List and Transplant of Organs
Waiting
Transplant
REASONS FOR PHASE 3 FAILURES (1990-2010) N=126
Lack of Efficacy,
45% Safety / Toxicity,
28%
Other, 27%
Target Discovery Lead ID Lead Optimization ADMET Development Registration
and approval
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The use of living cells in drug development and tissue engineering
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2-3 years 0.5-1 year 1-3 years 1-2 years 5-6 years 1-2 years
CELL-BASED ASSAYS CELLS & TISSUES AS THERAPEUTIC AGENTS
From Zreiqat et al., Biomaterials 2010 31:3175
• Actively pursued in oncology and toxicology – Multicellular tumor spheroids
(MCTS) – Hepatocyte spheroids
• Demonstrated benefits – Extended cell survival and/or
function – Morphologic features (polarity,
intracellular organization) – Cell-cell interactions – Better prediction of drug effects
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Development of 3D cell-based systems: A growing trend with proven benefits
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From Schmeichel & Bissell. J Cell Sci. 2003 Jun 15;116(Pt 12):2377-88.
From No et al. PLoS ONE 2012 7:e50723.
DESIRED ATTRIBUTES • 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
APPROACHES • Spheroids • Cells + Scaffolds or
Hydrogels • Micropatterned cultures • Bioprinting
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Achieving 3D in vitro: the pursuit of in vivo-like form and function
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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
The dynamics of aggregate fusion are driven by cell-cell and cell-environment interactions
Adjacent aggregates of cardiac tissue merge over 24 hours. Scale bar = 100µm. Adapted from Tissue Engineering Part A , 14(3):413, 2008.
Aggregates of CHO cells in a hydrogel merge over 120 hours. Adapted from J Mat Chem, 17:2054, 2007.
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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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Peripheral Nerve
Lung
Bone
Blood Vessel
Skeletal Muscle
Liver
Cardiac
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3D tissues have been bioprinted From cells throughout the body
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Day 0
500 µm
500 µm
Day 7
Day 21
500 µm
Bioprinted vascular conduits Develop mechanical strength upon conditioning
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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)
• Fabricated directly into multi-well plates
Endothelium Smooth Muscle Fibroblast
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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 in layered 3D blood vessel and small airway analogues
CON
TRO
L +
TGF-β1
TGF-β1 Treatment ↑ collagen deposition in blood vessel wall
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CON
TRO
L +
IL-1
3
IL-13 Treatment ↑ proliferation of fibroblasts and microvasculature,
and ↑ collagen deposition
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Organovo’s NovoGen BioprinterTM platform
was utilized to generate 3D human liver
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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
• Demonstration of both histologic and functional features of liver
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Single Unit
3mm
3D human liver tissue key phenotypic features
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E-Cadherin DAPI nuclei
Automated Fabrication with NovoGen BioprinterTM
Tissue-Like cellular density (H&E)
Cell type-specific compartmentalization
Hepatic stellate cells Hepatocytes Endothelial cells
>500 microns in thickness
Formation of microvasculature
CD-31 DAPI nuclei
Formation of tight junctions
Hepatocyte Sourcing: • Primary human • HepaRG-derived • iPSC-derived
Bioprinted 3D human liver tissues produce liver-specific proteins and have inducible CYP450s
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135 H
Bioprinted liver tissue (48 hrs)
Successful substitution of iCells for primary hepatocytes and enhanced albumin production in 3D vs. 2D
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H&E / 20x 100 μm
CD31 / 20x 100 μm
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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
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Standard Corning TranswellTM Plates
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Partner Companies & Institutions
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.
Enabling tissue on demand
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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.