3D Scaffolds for Cancer Engineering

Three-‐Dimensional Cell Culture: 3D Insert for Cancer Engineering

Add Extra Dimension to Your Innovation™ © 3D BIOTEK, LLC

Cell Culture Evolu@on

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1838, Schleiden & Schwann

“cell theory”

1885, Wilhelm Roux

Cells can live outside the body

1907, Harrison

Inventor of Gssue culture

1952, Gey

HeLa cells

1955, Eagle defined medium

1965, Ham

Colonial growth of mammalian cells

1981, MarGn & Evans

Mouse ES cells

1998, Thomson & Gearheart Human

ES cells

3D

3D Cell Culture: Gels and Scaffolds

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1960’s 1900’s 2000’s

Glass-‐Based Tissue Culture

Polystyrene-‐Based Tissue Culture

Gel-‐Based Technology Matrigel

DissociaGon/Maintenance of Cells/Vaccine ProducGon

2D EvoluGon of Polymer-‐Based Scaffolds

2D

2D

3D

3D

MulGcellular Spheroids

Why 3D Cell Culture

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2D Does not Mimic in vivo Geometry

≠

Brain

Bone, CarGlage

Lung, Breast

Brain

Bone, Car+lage

Lung, Breast

Liver Liver

Petri Dish

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Limita@ons of 2D Cell Culture

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  Limited cell-‐cell interacGon   Disrupted cellular organizaGon and polarity   Inaccurate representaGon of the cellular environment experienced by cells in vivo   Disconnect between cellular behavior in vitro and in vivo

Debnath J, et al. 2003

Polarity

Fishbach C, et al. 2007

Disconnect to in vivo

Superior In Vivo Relevance of 3D

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Human 3D Cell Culture Mouse 2D Cell culture

Pearson Correla@on Coefficient

Human – Human = 0.45 3D –Human = 0.34 Mouse – Human = 0.28 2D – Human = 0.0

Most Relevant to Primary Tumor

Least Relevant

Ridki TW, et al., Invasive three-‐dimensional organoGpic neoplasia from mulGple normal human epithelia. Nature Medicine 16(12):1450-‐55, 2010

Increasing Funding (US)

© KIYATEC Inc

 DARPA BAA-‐11-‐73, “Microphysiological Systems” • “Three-‐dimensional constructs of one or more cell types are able to reproduce relaGvely authenGc human Gssue and organ physiology in an in vitro environment”...

•  “DARPA seeks in vitro placorms comprised of human Gssue constructs that will accurately assess efficacy, toxicity,

and pharmacokineGcs in a way that is relevant to humans”

Pharmaceu@cal Industry Challenges

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•  Frequent failures in Clinical Trials: Phase I, II, and III •  Waste of money and resources

New Strategies: •  Beher Early Screening, e.g. in vitro models. •  Fail early, save money and Gme.

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3D Cell Culture: What Flavor Please?

h>p://blog.wan+st.com

Gels Nano Polymers Animal ECM Spheroids

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3D Cell Culture: The Ideal Scaffold

3D Collagen Scaffold

3D OPLA Scaffold

3D Calcium Phosphate Scaffold

AlgiMatrix Matrigel / PuraMatrix / Coa@ngs

Alvatex®

Ready to use

100% interconnected

pores

High surface to

volume ratio

Variable configurations (customizable)

Easy cell recovery

Plate reader compatible

Transparency (direct observation with light microscope)

3D Insert™

Gel Matrices

PLA foam

CaP foam

Alginate Foam

Alvetex®

Compa@ble

Not Compa@ble

Polymer Deposi@on: Wider Biological Range

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3D Insert™

Enhanced Cell Growth

Enhanced Cell DifferenGaGon

Biological Range

3D Insert™

Core Technology: 3D Insert™ Pladorm

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3D Insert™ Pladorm

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Fiber Diameter Fiber-‐to-‐Fiber Spacing Scaffold Layering

Pore Size Scaffold Volume

3D Geometry

3D Insert™ Series

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3D Insert™-‐Polystyrene

3D Insert™-‐Poly-‐ε-‐Caprolactone

Poly-‐ε-‐Caprolactone (PCL) is a biodegradable polymer used in FDA approved medical devices

Polystyrene (PS) is a transparent polymer used in tradiGonal cell culture.

3D Insert™ Series

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3D Insert™-‐Polystyrene

3D Insert™-‐Poly-‐ε-‐Caprolactone

  ConfiguraGons: PS(1520) and PS(3040)  PS(1520) = 150-‐um Fiber/200-‐um Spacing  PS(3040) = 300-‐um Fiber/400-‐um Spacing  Total number of layers: 4

  ConfiguraGons: PCL(3030) and PS(3050)  PCL(3030) = 300-‐um Fiber/300-‐um Spacing  PCL(3050) = 300-‐um Fiber/500-‐um Spacing  Total number of layers: 6

3D Insert™ PS: Microscopy Advantage

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  4 layer configuraGon for real Gme imaging  Adaptable for Light and confocal Microscopy  Self-‐similar Structure

3D Insert™ Advantages in Cell Culture

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  Well-‐defined pore size and porous structure   100% Pore InterconnecGvity   Solvent-‐Free and Non-‐Toxic   CompaGble with Current 2D Assays   Batch-‐to-‐Batch Reproducibility   Free of Animal-‐derived Material   Custom Designed FabricaGon

3D Insert™-‐PS 3D Insert™-‐PCL

3D Insert™: Applica@ons

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3D Insert™: Biological Relevance

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Sta+c Cell Culture Models

Perfusion Models

Sta+c Co-‐culture Models

Perfusion Co-‐culture Models

Biological Relevance

Current Needs Cancer and Tissue Engineering +

Standard 3D Scaffold = Biological

Relevance

3D Insert™ Sta@c Seeding Protocol

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Immediately amer seeding: Cell droplet will cover ~80-‐90% of scaffold

3 h incuba+on 37º C, 5% CO2

Cell droplet will penetrate enGre scaffold

Pipehe the appropriate volume of cell suspension

Carefully place suspension droplet onto the center of

the scaffold surface

Gently add remaining media along well’s edge

For seeding volumes, please visit us at hfp://www.3dbiotek.com/web/3dProtocols.aspx

3 h

incuba+on

37 º C,

5% CO 2

12 h

incuba+on

37 º C,

5% CO 2

Rota+ng

Constant Rota@on

Standard Seeding in Sta@c Culture

Op@onal Dynamic Seeding for Co-‐culture

3D Insert™: Cell Morphology

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Fiber 2

Fiber 1 H. Astrocytes hMSCs

NIH 3T3 MCF-‐7

3D Insert™

Pore Structure

Fiber Curvature

3D Cancer Models: Drug Screening

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Advantages   RealisGc Signaling from Cellular Microenvironment   Beher RepresentaGon of in vivo Drug Resistance   Maintenance of True Cancer Phenotype

Applica@ons   3D Cellular Growth and OrganizaGon   3D Tissue-‐Drug InteracGon   3D Co-‐culture   3D Spheroid FormaGon

3D Cancer Models: Breast Cancer

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Absorbance (570 nm)

MTT assay

Absorbance (570/405 nm)

Alamar Blue

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MCF-‐7 cells imaged using a light microscope in real-‐Gme. (A-‐B: 100X, C: 200X)

p≤0.05

3D Cancer Models: 3D Drug Resistance

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Absorbance (570 nm)

con 10-‐6 M 10-‐5 M

Day

Effects of tamoxifen on MCF-‐7 cells grown in 2D and 3D. Cell viability amer tamoxifen treatment was measured by MTT assay.

MTT assay 2D 3D

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2D 3D

control + E2 + E2 + FUL

Enhanced MCF proliferaGon in 3D amer estrogen (E2) sGmulaGon. DNA assay was performed to determine proliferaGon response. Following with inhibiGon using fulvestran (Ful)

DNA assay

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© 3D BIOTEK, LLC Caicedo-‐Carvajal, CE., et al., Tissue Eng. ID 362326

Stroma Causes Lymphoma Aggrega+on in 2D and 3D

3D Co-‐culture Model: Blood Cancer

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3D Stroma Enhances Lymphoma Neoplas+c Growth

3D Co-‐culture Model: Neoplas@c Growth

Caicedo-‐Carvajal, CE., et al., Tissue Eng. ID 362326

3D Insert™: Take Home Message

© 3D BIOTEK, LCC

• The 3D Insert™ has shown opGmum performance under increasing complexity, an common trend in cell-‐based models

•  The 3D Insert™ is a versaGle 3D cell culture placorm for drug screening in cancer models to study drug resistance and the effect of stroma on cancer proliferaGon

3D Scaffolds for Cancer Engineering

Technology

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