BD Biosciences

February 16, 2011

3D Cell Culture SystemsMarshall J. Kosovsky, Ph.D.

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Topics for Discussion

• Overview of Cell Culture Systems: 2D vs. 3D

• Extracellular Matrix (ECM) Proteins– Epithelial cell differentiation– Endothelial cell tube formation

• Animal-Free Matrix: BD™ PuraMatrix™ Peptide Hydrogel– Primary hepatocytes differentiation– Neuronal cell differentiation– Endothelial cell tube formation

• Cell Culture Insert Platform– Tumor cell migration/invasion– Endothelial cell migration/invasion– General cell differentiation (3D co-culture)

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Cell culture systems: 2D 3D

2D2DNon-treated (hydrophobic)

BD Falcon™ Tissue Culture (TC)-treated [net (-) charge]BD Primaria™ [mixture of (-) and (+) charge]

BD BioCoat™ (large variety of ECMs, poly-lysine) BD PureCoat™ [carboxyl (-), amine (+); animal free]

3D3DECM coatings (BD Matrigel™ matrix, BD™ Laminin/Entactin, Collagens)

Rigid 3D Scaffolds (e.g., PLA, PGA)Animal-free hydrogels (BD™ PuraMatrix™ Peptide Hydrogel)

Cell culture inserts (e.g., co-culture models such as BBB)Animal models

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2D vs. 3D Cell Culture

YesNoCell Encapsulation

Slow; biochemical gradients regulate cell-cell communication and signaling

RapidGrowth Factor Diffusion

‘Physiological’; promotes close interactions between cells, ECMs, growth factors

Not physiological; cells partially interact

Architecture

Mimics natural tissue environment

Rigid; inertGrowth Substrate

3D2D

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Why Culture Cells in 3D?

• Tissue and organs exist in 3D

• Studying cells in 3D enables researchers to ‘mimic’ or approximate physiological conditions that exist in vivo

• Strong historical evidence that 3D cell culture works for establishing differentiated cell morphology and function__________________________________________________________________

Feature Articles in Nature, August 2003Goodbye, Flat Biology?".... biologists starting to explore the merits of culturing cells in 3D have been stunned by the difference it makes to the way cells behave, which is much closer to their behavior in vivo."

Biology’s New Dimension (highlights BD Matrigel™ matrix and BD™ PuraMatrix™)“In 10 years, anyone trying to use 2D analyses to get relevant and novel biological information will find it difficult to get funded.” – Mihael Polymeropoulos, CSO, Vanda Pharmaceuticals (former Head of Pharmacogenetics, Novartis)

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Cell Culture Models Support or Promote Cell Behavior

Cell behaviors (differentiation, functionality) influenced by cues in the microenvironment:

• Cell morphology (structure, phenotype)

• Polarity (functional directionality)

• Growth (proliferation)

• Cell motility (migration, invasion)

• Neurite outgrowth

• Signal transduction (surface receptor function)

• Gene and protein expression (different cell types can express different genes/proteins; liver vs. heart vs. brain)

• Biochemical activities (proteins, enzymes)

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The Extracellular Matrix

• Complex mixture containing glycoproteins, collagens, and proteoglycans

• Forms structural framework that stabilizes tissues and provides mechanical support for cell attachment

• Plays important role in cell functionality and differentiation– Receptor-mediated signaling

– Regulation of gene expression

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basal lamina = basement membrane

BD Matrigel™ matrix = reconstituted basement membrane

Figure: Molecular Biology of the Cell (3rd Edition)

The Basal Lamina: A Thin ‘Mat’ that Underlies Epithelial Cell Sheets and Tubes

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epithelial cells

basal lamina

collagen fibrils

Figure: Molecular Biology of the Cell (3rd Edition)

Basal Lamina in Chick Embryo Cornea

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ECM Contributes to Intracellular Signaling Pathways

• ECM-based growth substrates provide a physiological environment that supports and promotes key cell functions

• ECM molecules interact with cell surface receptors (e.g., regulation of integrin signaling by fibronectin:integrin interactions)

• ECM appears to function in the storage and presentation of growth factors

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Composition

Laminin ~ 60%

Collagen IV ~ 30%

Entactin ~ 8%

Heparan sulfate proteoglycan (perlecan)

Growth factors (e.g., PDGF, EGF, TGF-β)

Matrix metalloproteinases

BD Matrigel™ matrix: Reconstituted Basement Membrane

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BD Matrigel Matrix High Concentration

• Protein concentration: ~ 18-22 mg/ml

• ApplicationsHuman tumor xenograft in immunodeficient nude mice

− Subcutaneous injection of Matrigel mixture containing human tumor cells (e.g., lung, prostate, breast, colon)

‘BD Matrigel™ matrix Plug Assay’ for in vivo angiogenesis

− Subcutaneous injection of BD Matrigel matrix mixture containing test substances (e.g., antibodies, growth factors, synthetic peptides) and/or tumor cells

− Removal of plug and analysis of new vessel formation

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High Concentration ECM Proteins for 3D Cell Culture

High Concentration Laminin/Entactin Complex• Major component of basement membrane in Engelbreth-Holm-Swarm (EHS)

mouse tumors• Protein concentration: ≥ 10 mg/ml• Purity >90% by SDS-PAGE• Forms 3D gel that models tissue microenvironment in vivo• Supports cell differentiation (e.g., mouse submandibular cells, endothelial cell

tube formation)

High Concentration Collagen I• Source: rat tail tendon• Protein concentration: 8-11 mg/ml• Full-length protein (not treated with pepsin)• Purity >90% by SDS-PAGE• Forms sturdy gel that provides maximal 3D support matrix

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Factors that Influence Cell Differentiation and Functionality

• Biological composition of the culture environment(e.g., cell types, ECMs, growth factors)

• Molecular interactions and cell adhesion (cell:cell, cell:ECM, ECM:ECM, cell:growth factor, and ECM:growth factor)

• Mechanical strength and structural properties(degree of rigidity, 3D architecture)

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Collagen I (2D thin coat) Collagen I (3D gel) BD Matrigel™ matrix (3D)

Primary Hepatocytes Exhibit Differentiated Morphology on 3D Growth Substrates

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Activated caspase 3Laminin-5

DAPI

8 days

15 days

20 days

Phospho-ERM (ezrin/radixin/moesin)

hDlg (human disk large)

DAPI

Data kindly provided by Dr. Joan Brugge and originally described in Debnath J, et al. (2003) Methods 30:256-268.

Mammary Epithelial Cell Differentiation in BD Matrigel™ Matrix using 3D ‘Overlay’ Method

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Human Microvascular Endothelial Cells Form Tubules on BD Matrigel Matrix

BD Matrigel™ matrix

3D

Bright Field

BD Matrigel matrix

3D

Collagen I

2D

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Non-confocal single plane Confocal single plane Confocal collapsed stack

BD™ HUVEC-2 cells, stained with Calcein AM, 4X confocal images on BD Pathway™ Bioimager

Endothelial Cell Tubulogenesis: High Resolution Detection Using Confocal Collapsed Stack Imaging

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0 160 µM

Concentration-Dependent Inhibition of Endothelial Cell Tube Formation by Suramin

Calcein AM staining, 4x confocal

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1.0×10-7 1.0×10-6 1.0×10-5 1.0×10-4 1.0×10-30

10002000300040005000600070008000

Log [M] Suramin

Tota

l Tub

e Le

ngth

(pix

els)

1.0×10-7 1.0×10-6 1.0×10-5 1.0×10-4 1.0×10-30

10000

20000

30000

40000

50000

60000

70000

Log [M] Suramin

Tota

l Tub

e A

rea

(pix

els)

1.0×10-7 1.0×10-6 1.0×10-5 1.0×10-4 1.0×10-30

50

100

150

200

250

300

350

Log [M] Suramin

Tube

Com

plex

ity(t

otal

# o

f seg

men

ts)

IC50 = 2.93E-05

IC50 = 2.94E-05

IC50 = 2.97E-05

Different Analysis Parameters - Similar Results

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Potential Limitations of Current Systems

• Composition of the environment not optimal for key cell type(s)

• Growth substrate or material not well defined

• Complex materials often exhibit lot-to-lot variability

• Presence of animal-derived components

• Synthetic materials may generate acidic breakdown products that are cytotoxic

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PuraMatrix is a trademark of 3DM, Inc., Cambridge, MA

BD™ PuraMatrix™ Peptide Hydrogel

A Synthetic Biomaterial for Optimizing 3D Cell Culture Environments

• Developed at Massachusetts Institute of Technology, Cambridge, MA

• Composed of synthetic peptide (1% w/v) and 99% water

• Salt-mediated assembly into 3D hydrogel with average pore size of 50-200 nm

• Supports attachment & differentiation of cell types such as primary hepatocytes, neurons, endothelial cells, chondrocytes, neural & mesenchymal stem cells

• Suitable for in vivo studies of tissue regeneration and repair

• Biocompatible, devoid of animal-derived materials & pathogens

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Images of molecular models kindly provided by Dr. Shuguang Zhang, Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, MA

• 16 amino acid sequence; 1% peptide solution (w/v)

• Alternating hydrophilic and hydrophobic side chains

• Promotes cell attachment, but does not require integrin-based adhesion

Synthetic Peptide Composition

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nanofiberssingle peptide

• Ionic and hydrogen bonding

• Hydrophobic and van der Waals interactions

• Nanofibers exhibit β-sheet bilayer structure

Salt-Mediated Molecular Self-Assembly into Nanofiber Structure

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nanofiber scaffold

AFM image provided by Dr. Shuguang Zhang, MIT

Nanofiber β-Sheets Further Organize into Macroscopic Scaffolds

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Images of BD™ PuraMatrix™ Peptide Hydrogel at increasing magnification

• At lower magnification, the scaffold exhibits felt-like appearance (a).

• Higher magnification reveals interwoven peptide fibers approximately 10-20 nm in diameter (f).

Originally described in Holmes, T.C., et al. (2000) PNAS, Vol 97, pp 6728-6733.

SEM Images of Macroscopic Structure

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SEM Analysis of Macroscopic Structure: BD PuraMatrix vs. BD Matrigel Matrix

BD Matrigel™ matrix BD™ PuraMatrix™

Data kindly provided by Dr. Shuguang Zhang and originally described in Gelain, F, et al. (2006) PLoS ONE 1(1):e119.

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• Hydrated ‘hydrogel’ environment promotes effective diffusion of nutrients and macromolecules

• Samples readily visualized using standard staining and microscopy

Images provided by Dr. Shuguang Zhang, MIT

The Assembled Hydrogel is Comprised of≥ 99% water

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Spheroids attached on the surface and embedded within the hydrogel(white arrows)

High magnification view of a spheriod

Data kindly provided by Sihong Wang and originally described in Wang, S, et al. (2008) Tissue Engineering Part A 14(2):227.

Scanning Electron Microscopy of Rat Primary HepatocyteSpheroids on BD™ PuraMatrix™ Peptide Hydrogel

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Rat PC12 cell neurite outgrowth.The image is a merged stack of multiple confocal optical sections.

BD™ PuraMatrix™ Peptide Hydrogel: Neuronal Cell Differentiation

Primary rat hippocampal neurons form active synapses. Confocalimage of synaptically active neuronal membranes.

Data provided by 3DM, Inc. and originally described in Holmes, TC, et al. (2000) PNAS USA 97:6728-6733.

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BD PuraMatrix Peptide Hydrogel: Human Umbilical Vein Endothelial Cells

A B

• HUVECs encapsulated within BD™ PuraMatrix™ elongate and form interconnected microvascular networks

• Cells cultured for 2 days in endothelial cell growth medium supplemented with 50 ng/ml VEGF and 50 ng/ml PMA. (A) 0.74 mg/ml BD PuraMatrix; (B) 1.3 mg/ml BD PuraMatrix

Data kindly provided by Dr. Alisha Sieminski

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Representative ReferencesBD™ PuraMatrix™ Peptide Hydrogel

Misawa, H., et al. (2006) PuraMatrix facilitates bone regeneration in bone defects of calvaria in mice. Cell Transplant. 15(10):903.

Bone

Sieminski, AL, et al. (2008) Primary sequence of ionic self-assembling peptide gels affects endothelial cell adhesion and capillary morphogenesis. J Biomed Mater Res A 87:494.

Endothelial

Wang, S., et al. (2008) Three-dimensional primary hepatocyte culture in synthetic self-assembling peptide hydrogel. Tissue Engineering Part A 14(2):227.

Hepatocyte

Thonhoff, J.R., et al. (2008) Compatibility of human fetal neural stem cells with hydrogel biomaterials in vitro. Brain Res. 1187:42.

Neuronal

Yamaoka, H., et al. (2006) Cartilage tissue engineering using human auricular chondrocytes embedded in different hydrogel materials. J. Biomed. Mater Res A. 78(1):1.

Cartilage

ReferenceCell/Tissue Type

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Cell Culture Inserts: Membrane Supports for Complex Cellular Assays

• Cell culture inserts provide a two compartment culture system suitable for a variety of complex cellular assays

• Insert wells contain a microporous membrane ‘floor’ composed of polyethylene terephthalate (PET) available with different pore diameters

• Pores allow exchange of media, nutrients, molecules and the passage of cells (pore size dependent)

– Small pore diameters (0.4 and 1.0 µm) prevent cell passage

– Large pore diameters (3.0 and 8.0 µm) allow cell passage

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Cell Culture Inserts:BD FluoroBlok™ Platform

A unique fluorescence-blocking membrane ideal for analysis of cellmigration and invasion

• Angiogenesis– Endothelial Cell Migration/Invasion

• Tumor Cell Biology– Tumor Cell Migration/Invasion– 3D models for metastasis

• Inflammation– Monocyte, Leukocyte Chemotaxis– Transendothelial Cell Migration

• Drug Discovery (single parameter or multiplexing)

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8 µm pores – visible light

BD FluoroBlok™ PET Membrane

• Treated with proprietary blue dye

• Unique ‘fluorescence blocking’ membrane blocks light transmission from 490–700 nm

• Quantitative analysis using fluorescence detection

• Increases productivity and assay throughput

– No need to dismantle, wash and manually count cells

– Eliminates multiple handling steps: just add cells, label and read

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The blue dyed membrane physically and visually separates cells above the membrane from those below the membrane.

3 and 8 µm pore diameters24-well Individual, 24-Multiwell,

and 96-Multiwell formats

Cross section(not to scale)

Track-Etched Pores

Basal ChamberBase plate

Apical Chamber

Insert (individual or multiwell)

BD Falcon™ FluoroBlok™ Cell Culture Inserts

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BD FluoroBlok™ Assays are Analyzed using Fluorescent Dyes

Appropriate fluorescent dyes exhibit emission wavelength between 490-700 nm [e.g., BD Calcein AM (green) and BD DiIC12(3) (red)]

Cell Labeling Methods

• Pre-Labeling (label cells prior to the assay)

• Post-Labeling (label cells on underside of membrane after assay)

• Transfected cells (e.g, transient or permanent expression of GFP)

See BD Biosciences Technical Bulletin #451 - Compatible Fluorophores and Dyes for BD Falcon™ FluoroBlok™ Inserts and Insert Systems

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Pseudopodial Endothelial Vacuole Vacuoles join to extension cell division formation create lumen

Figure: Adapted from Molecular Biology of the Cell (3rd Edition)

• Release of pro-angiogenic factors

• Receptor activation

• MMP release

• Cell migration, invasion, and proliferation

The Angiogenesis Pathway

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BD BioCoat™ Angiogenesis Systems

Endothelial Cell Migration • 24- or 96-Multiwell FluoroBlok™ Insert (3 µm pore size)

• Coated with Human Fibronectin

Endothelial Cell Invasion • 24-Multiwell FluoroBlok™ Insert (3 µm pore size)

• Coated with BD Matrigel™ matrix

Endothelial Cell Tube Formation • 96-well black/clear microplate coated with BD Matrigel™ matrix

(non-insert system)

BD Human Umbilical Vein Endothelial Cells

(BD™ HUVEC-2)• Pre-qualified for VEGF responsiveness and

for use with cell migration assay; also suitable

for cell invasion and tube formation assays

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Analysis of Endothelial Cell Migration and Invasion Using BD FluoroBlok Membrane Inserts

BD FluoroBlok™PET Membrane

(3 μm pores)

Excitation(485 nm)

Attractant

Emission(530 nm)

Fibronectin (migration)

or

BD Matrigel™matrix (invasion)

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3-4 fold stimulation

0.000 1.000 3.100 6.200 12.5000.0

0.5

1.0

1.5

2.0

mea

n+

se (n

=4)

Cell migration assessed using the BD BioCoat™ Angiogenesis System: Endothelial Cell Migration(Fibronectin-coated BD FluoroBlok™; 96-well format).

Endothelial Cells Exhibit Concentration-Dependent Migration Towards VEGF

8-10 fold stimulation

0.000 1.000 3.125 6.250 12.500 25.0000

2

4

6

8

10

12

VEGF (ng/ml)Fo

ld in

crea

se o

ver c

ontr

olm

ean

+se

(n=4

)

BD HUVEC-2 Cells HAEC Cells

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• Combined Benefits of BD FluoroBlok™ and BD Matrigel™ matrix

• Optimized Protocols

• 24- and 96-well Formats (8.0 µm pores)

BD BioCoat™ Tumor Invasion Systems

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Analysis of Tumor Cell Invasion Using BD FluoroBlok Membrane Inserts

BD FluoroBlok™

PET Membrane(8 µm pores)

Excitation(485 nm)

Attractant

Emission(530 nm)

BD Matrigel™matrix (invasion)

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Fluorescently labeled cells on underside of BD FluoroBlok™ membrane post-invasion

• No need to disrupt sample for manual counting

• Automated quantitation using bottom-reading fluorescence plate reader

• Option to perform real-time kinetic analysis

MDA-MB-231 Human Breast AdenocarcinomaCell Invasion Through BD Matrigel™ Matrix

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Inhibition of MDA-MB-231 Cell Invasion Through BD Matrigel™ Matrix by Doxycycline

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Use of Cell Culture Inserts to Establish a 3D Co-Culture Model

Top of insert membrane may be coated with 2D or 3D matrix

Cell type Yseeded on

underside of membrane (e.g., BBB); cells may

physically interact through

pores

Well floor may be coated with 2D or 3D matrix

Cell type Xseeded on top of membrane

Clear or BD FluoroBlok™PET Membrane

Cell type Z seeded on floor of well, physically separated from other cell type(s); potential for

paracrine regulation

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Representative Cell Culture Insert References

Daneman, R, et al. (2009) PNAS, Vol. 106, pp. 641-646.

Fibronectin(BD BioCoat™ Angio Migration System)

Endothelial

Li, Y, et al. (2010) Cancer Research, Vol. 70, pp. 1486-1495.

BD Matrigel™ matrix (BD BioCoat Tumor Invasion System)

Pancreatic cancer cells

Ramirez, SH, et al. (2008) Journal of Immunology, Vol. 180, pp. 1854-1865.

Col I-coatedBD FluoroBlok (migration)

Monocytes

Kong, D, et al. (2008) Cancer Research, Vol. 68, pp. 1927-1934.

BD Matrigel matrix (BD BioCoat Tumor Invasion System)

PC3 prostate carcinoma

Cote, MC, et al. (2010) Journal of Biological Chemistry, Vol. 285, pp. 8013-8021.

Gelatin-coated BD FluoroBlok™ (migration)

Endothelial

ReferenceSurfaceCell Type

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Questions?

Contact information:Marshall Kosovsky, PhDe-mail: marshall_kosovsky@bd.com

Technical Support:tel: 877.232.8995e-mail: labware@bd.combdbiosciences.com/webinarsFor research use only. Not intended for use in diagnostic or therapeutic procedures. BD, BD Logo, and all other trademarks are property of Becton, Dickinson and Company. ©2011 BD