challenges for 3d cancer cell culture on basement membrane

Management of Disease Through DNA Repair

Challenges for 3D Cancer Cell Culture On Basement Membrane


AACR, 2009Presented by Hynda K. Kleinman, PhD

ams biotechnology (europe) ltd – [email protected] www.amsbio.com (UK) +44(0)1235 828200 (CH) +41(0)91 604 55 22 (DE) +49(0)69 779099


What Is 3D Culture?• In vitro cell culture model where cells can

grow and assemble in three dimensions• Cells mimic tissues of origin

– Structure– Function

• Recapitulate in vivo environment

Tumor cells are generally epithelial in originAlbini et al, Cancer Research, 1987 first person to put

tumor cells on basement membrane matrix


Why Use Extracellular Matrix (ECM) Proteins?

• Provide complete cellular microenvironment– Structure

• gel formation• malleable matrix

– Cellular signaling• Alternatives

– Suspension systems• promote aggregate formation• lack structure/signaling

– Artificial scaffold• lack signaling


Variables Associated with 3D Models

• ECM Environment• Cell Type/Lines• Cell Culture Medium• Cell “Health”• Cell Seeding Density (cells/cm2)• Time of Culture (days)


ECM Environment

• Extracellular protein(s)– Basement Membrane Extract (BME): a

reconstituted basement membrane• Thickness of gel (density/stiffness)

– Thinner, denser, or stiffer gels generally promote cell spreading and elongated morphology


Thickness of basement membrane gel density/stiffness affects cell spreading


Cell Type/Lines• Normal

– Structure and function of tissue of origin• Carcinoma

– Tumorigenic – grow tumors– Malignant - metastasize

• Stem Cells– Progenitor cells– May differentiate (hierarchy to lineage)


Cell Types


Cell Culture Medium

• Serum• Growth Factors• Hormones• Cytokines• Notes

– Each model may have specific requirements– Work synergistically with ECM proteins


Cell “Health”• Primary cells lose tissue specific

characteristics after several passages• Cells must be allowed to recover from

cryogenic freezing– A percentage of all cells undergo freezing-

associated cell death– Cell must re-enter cell cycle– Subsequent passages select for healthy,

dividing cells (usually 2 or three)


Cell Passage Number After Thawing Affects Acinar Formation


Seeding Density

• Number of cells per unit surface area (cells/cm2)

• Affects paracrine interactions– Survival– Alignment– Migration– Formation cell-to-cell bonds


Seeding Density Affects Morphology


Time of Culture• Certain cellular events occur as a function of

time– Alignment– Migration– Growth arrest– Luminal apoptosis– Protein production– Invasion of matrix

• Cells that lack growth control may exceed capacity of environment = dead cells


Time of Culture


Summary• 3D cultures are in vitro models where the

in tissue microenvironment is recapitulated to promote in vivo structure and function

• Variables affecting 3D culture– ECM Environment (thickness)– Cell Type/Lines– Cell Culture Medium– Cell Health (passage number after thawing)– Cell Seeding Density (cells/cm2)– Time of Culture (days)


Ordering Information:

Proliferation (96 wells)Cultrex 3D Culture Cell Proliferation Assay Core Kit 3445-096-CKCultrex 3D Culture BME Cell Proliferation Assay 3445-096-KCultrex 3D Culture Laminin I Cell Proliferation Assay 3446-096-KCultrex 3D Culture Collagen I Cell Proliferation Assay 3447-096-K

HarvestingCultrex 3D Culture Cell Harvesting Kit (20 tests) 3448-020-K

Matrices:Cultrex 3D Culture Matrix™ BME (15 ml) 3445-048-01Cultrex 3D Culture Matrix Collagen I (100 mg) 3447-020-01Cultrex 3D Culture Matrix Laminin I (5 ml) 3446-005-01

©2009 Trevigen, Inc. 3D Culture Matrix is a trademark and Trevigen, Cultrex and CultreCoat are registered trademarks of Trevigen, Inc.

ams biotechnology (europe) ltd – [email protected] www.amsbio.com (UK) +44(0)1235 828200 (CH) +41(0)91 604 55 22 (DE) +49(0)69 779099

challenges for 3d cancer cell culture on basement membrane

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