Management of Disease Through DNA Repair Challenges for 3D Cancer Cell Culture On Basement Membrane AACR, 2009 Presented by Hynda K. Kleinman, PhD ams biotechnology (europe) ltd info@amsbio.com 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 origin Albini 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/cm 2 ) 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
Normal Cell Type/Lines Structure and function of tissue of origin Carcinoma Tumorigenic grow tumors Malignant - metastasize Stem Cells Progenitor cells May differentiate (hierarchy to lineage)
Cell Types Management of Disease Through DNA Repair
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 freezingassociated cell death Cell must re-enter cell cycle Subsequent passages select for healthy, dividing cells (usually 2 or three)
Management of Disease Through DNA Repair Cell Passage Number After Thawing Affects Acinar Formation
Seeding Density Number of cells per unit surface area (cells/cm 2 ) Affects paracrine interactions Survival Alignment Migration Formation cell-to-cell bonds
Seeding Density Affects Morphology
Seeding Density Affects Morphology
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 Management of Disease Through DNA Repair
Time of Culture Management of Disease Through DNA Repair
Time of Culture Management of Disease Through DNA Repair
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/cm 2 ) Time of Culture (days)
Ordering Information: Proliferation (96 wells) Cultrex 3D Culture Cell Proliferation Assay Core Kit 3445-096-CK Cultrex 3D Culture BME Cell Proliferation Assay 3445-096-K Cultrex 3D Culture Laminin I Cell Proliferation Assay 3446-096-K Cultrex 3D Culture Collagen I Cell Proliferation Assay 3447-096-K Harvesting Cultrex 3D Culture Cell Harvesting Kit (20 tests) 3448-020-K Matrices: Cultrex 3D Culture Matrix BME (15 ml) 3445-048-01 Cultrex 3D Culture Matrix Collagen I (100 mg) 3447-020-01 Cultrex 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 info@amsbio.com www.amsbio.com (UK) +44(0)1235 828200 (CH) +41(0)91 604 55 22 (DE) +49(0)69 779099