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

1

NYSE: ONVO

©Copyright 2013 Organovo, Inc.

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


0

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

3

The use of living cells in drug development and tissue engineering


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

4

Development of 3D cell-based systems: A growing trend with proven benefits


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

5

Achieving 3D in vitro: the pursuit of in vivo-like form and function


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.

6 ©Copyright 2013 Organovo, Inc.

7

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


Peripheral Nerve

Lung

Bone

Blood Vessel

Skeletal Muscle

Liver

Cardiac

8

3D tissues have been bioprinted From cells throughout the body


Day 0

500 µm

500 µm

Day 7

Day 21

500 µm

Bioprinted vascular conduits Develop mechanical strength upon conditioning


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


Vascular wall analogues are viable and dynamic with stable architecture

Smooth Muscle Cells (SMA) TUNEL-positive cells

Endothelial Cells (CD31) Ki67-positive cells


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

12

CON

TRO

L +

IL-1

3

IL-13 Treatment ↑ proliferation of fibroblasts and microvasculature,

and ↑ collagen deposition


Organovo’s NovoGen BioprinterTM platform

was utilized to generate 3D human liver

13

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

© Copyright 2013 Organovo Holdings, Inc.

Single Unit

3mm

3D human liver tissue key phenotypic features

14

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

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


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


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.

automated fabrication of multi-cellular 3d human tissues

Documents