Cultrex Training

Gabriel Benton, Ph.D.

AACR Annual Meeting

New Orleans, LA

April 18, 2016

Goal: Improve Public Health by Treating Disease

• Drug development process is inefficient – Average cost ~ $5,000,000,000 (Forbes). – Average time ~ 10-15 years (PhRMA)

• Improve drug discover and preclinical assessment – Reduce cost – Increase throughput – Improve efficacy

Source: Pharmaceutical Research and Manufacturers of America, Drug Discovery and Development: Understanding the R&D Process, www.innovation.org

Why Are Preclinical Models Falling Short in Predicting Biological Response?

• Plastic is not a natural component of the human body

• Mice are not human

These models fail to recreate the complexity and specificity of living human tissues.

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Identify Factors that Are Necessary to Recreate In Vivo Structure and Function

• Tissue Resident Cells – Types – Quantities – Organization

• Extracellular Matrix – Composition – Organization – Compliance

• Soluble Factors – Growth Factors – Cytokines

Organoid Cultures Recreate In Vivo Structure and Function

• Organoid Progenitor Cells – Differentiate into tissue-

specific cells

• Extracellular Matrix – Basement Membrane

Extract (BME)

• Soluble Factors – Wnt – Noggin – EGF – R-Spondin-1 – Tissue-Specific Factors

Crypt base columnar cell

Paneth cells

Enterocytes

Goblet cells

Wnt

Organoids Possess Great Potential

• Disease Modeling • Drug Discovery • Drug Screening

iPSCs

Tissue Biopsy

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Regenerative Medicine

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Pancreas

Kidneys

Stomach

Heart

Retina

Salivary Glands

Many Organoid Models Exist

Prostate

Mammary

Long-term culture of Human Liver Organoids on BME 2 RGF. (Clonal cultures obtained by seeding sorted cells at one cell per well) (Image courtesy of Meritxell Huch, Gurdon Institute, University of Cambridge, UK)

Many Organoid Models are stable for Long Term Culture

Wash and mince at 4 °C

to 5 mm

Wash with cold PBS

until clear (10-20 times)

Incubate in cold 2 mM EDTA, PBS for 15-30

min on ice with gentle

shaking

Centrifuge and remove supernatant containing

cells

Crypt Isolation

Pipet up and down in cold

PBS to dissociate

crypts

Resuspend crypts in cold

culture medium and

count

Pass crypts through a 70 µm strainer

Isolate Tissue

Organoids from Crypts

Centrifuge crypts

Resuspend crypts in BME-2

• Advanced DMEM/F12 • Wnt • EGF • HA-R-spondin1-FC • Noggin • Tissue-Specific Factors

Crypt Culture Medium

Add 50 µl to a 24 well plate

Centrifuge crypts

Organoids are fragmented

Count organoids to determine split

for passaging

BME depolymerizes leaving intact

organoids

Add cold (4 °C) Organoid Harvesting

Solution – 30 min with gentle rocking

Pass organoids through a 20 ga needle

Aspirate Organoid Culture Medium and wash with cold (4 °C)

PBS

Resuspend crypts in BME

Add 50 µl to a 24 well plate

Add 500 µl Organoid Culture

Medium

5 – 14 Days Culture

Products for Organoid Culture Cycle

R-Spondin-1 Expressing Cell Line

• Roof plate-specific Spondin-1

• 27 kDa secreted protein

• positively regulate Wnt/beta-catenin signaling

– ligand for LGR4-6 receptors

– inhibitor for ZNRF3

• Essential medium component for most organoid culture models

R-Spondin-1

Production of R-Spondin1 for organoid culture. The 293T cell line is stably transfected to express murine Rspo1 with an N-terminal HA epitope tag and fused to a C-terminal murine IgG2a Fc fragment. A) The HA-R-Spondin1-Fc 293T cell line is cultured with zeocin to select for stably transfected cells. B) Production of HA-R-Spondin1-Fc is characterized using Western Blot for R-Spondin1 protein (arrow). C) HA-R-Spondin1-Fc induces activation of Wnt/ß-catenin response when evaluated using the Top-Flash Luciferase assay.

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R-Spondin-1 Expressing Cell Line

Catalog # Product Name Size Price

3710-001-01 Cultrex R-spondin1 (RSPO1) Cells 1 vial (0.5 ml),

1x10^6 cells

$490.00

Organoid Harvesting Solution

• Non-enzymatic chelating solution – Gentle for cells

– Preserves organoid morphology

• Uses – Organoid passaging

– Sample preparation • PCR

• Western Blot

• Immunohistochemistry

Count organoids to determine split for passaging

BME depolymerizes leaving intact organoids

Add cold (4 °C) Organoid Harvesting Solution – 30 min with gentle rocking

Aspirate Organoid Culture Medium and rinse with cold (4 °C) PBS

Organoid Harvesting Solution

Catalog # Product Name Size Price

3700-100-01 Cultrex Organoid Harvesting

Solution

100 ml $60.00

D0 D1 D2

D3 D4 D6

D8 D10 D11

BME-2 for Organoid Culture

• Each lot is validated for use in organoid culture

• Used extensively for organoid culture

– Little lot to lot variability

– Easy to work with

Cultrex BME-2

Catalog # Product Name Size Price

3533-001-02 Cultrex® Reduced Growth Factor Basement Membrane Extract, Type 2, PathClear®

1ml $35.00

3533-005-02 Cultrex® Reduced Growth Factor Basement Membrane Extract, Type 2, PathClear®

5ml $180.00

3533-010-02 Cultrex® Reduced Growth Factor Basement Membrane Extract, Type 2, PathClear®

2x5ml $275.00

Products Under Development

• Entactin Rich BME for Organoid Culture • Mouse and Human Enteric Organoid Progenitor

Cells – Gastric – Small Intestine – Colon – Pancreas

• Organoid Medium Supplements – R-Spondin-1 conditioned medium – Wnt conditioned medium

ECM Development: Entactin-Rich BME BME

+Entactin BME-2 B

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Entactin Rich BME BME-2

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Entactin is an integral component of the basement membrane

• Structural linkage for laminin, collagen-4, and perlecan

• Cell adhesion molecule for αvβ3 and α3β1 integrins

• Degraded by MMP-19 during basement membrane remodeling

Laminin

Entactin

Collagen-4

Perlecan

Cell Surface

Entactin Enrichment Improves Take for Human Gastric Organoids

100 µM

1 2 3 4 5 Days

Mouse Colon Organoids

3-D Culture: Where In Vivo Meets In Vitro™ We design, develop, and deliver matrices and kits for 3-D Culture.