driving cellular communication - bd ex. bd falcon non-treated surface. synthetic surfaces: surface...
TRANSCRIPT
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Driving Cellular Communication –Effects of Culture Environments on Cell Growth and Differentiation
Amy Laws, Ph.D.BD Biosciences – Discovery Labware
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Outline
• Culture surfaces– Synthetic– Biological – extracellular matrix (ECM)
• Introduction to 2D vs 3D culture• Cell type-specific examples
– Epithelial cells– Endothelial cells– Neurons– Human embryonic stem cells
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How Surfaces Affect Cell Culture
• Surface properties– Surface charge, protein motifs
• A misconception: more hydrophilic the better (Poly-lysine coated surface is more hydrophobic than Tissue Culture (TC)-treated surface)
– 2D vs 3D structure• Effect of surface is dependent on the
culture environment– Cell type– Media composition– Incubation time, etc.
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Culture Surfaces
• Vertebrate cells have negative surface charge– Cells spread using the ECM they lay down as well as serum or media
derived attachment factors
• Synthetic surfaces– Negative: glass, TC-treated polystyrene– Positive and negative: BD Primaria™ Cultureware– Positive: poly-lysine
• ECM components (2D or 3D)– Fibronectin is a key attachment factor derived from serum– BD BioCoat™ Cultureware (ex., Collagen I, Collagen IV or Fibronectin)– Reconstituted basement membrane (ex., BD Matrigel™ Matrix)
• Microporous membranes (cell culture inserts)
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Synthetic Surfaces: Surface Chemistry
• Non-treated polystyrene– Ex. BD Falcon Non-treated Surface
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Synthetic Surfaces: Surface Chemistry
• TC-treatment– Rendered hydrophilic by a process that adds a variety of
negatively charged functional groups to the surface– Ex. BD Falcon™ TC-treated Surface
• Vacuum-gas plasma treatment – oxygen• Specific conditions of pressure and temperature
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Synthetic Surfaces: Surface Chemistry
• BD Primaria™ Surface– Vacuum-gas plasma treatment – oxygen + ammonia– Integrates amine and amide functional groups with
the traditional hydroxyl/carboxyl TC-surface chemistry
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Non-treated Polystyrene
BD Falcon™ TC BD Primaria™
Synthetic Surfaces: Cell Growth on Polystyrene Substrates
Chinese hamster ovary (CHO) cells, 72 hours after seeding, 10% serum
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Non-treated Polystyrene
BD Falcon™ TC BD Primaria
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Synthetic Surfaces: Cell Growth on Polystyrene Substrates
LNCaP (human carcinoma) cell, 24 hours after seeding, 10% serum
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Non-treated Polystyrene
BD Falcon™ TC BD Primaria
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3T3 (mouse fibroblast) cells, 24 hours after seeding, serum-free media
Synthetic Surfaces: Cell Growth on Polystyrene Substrates
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Synthetic vs. Biological Surfaces
• Synthetic– Covalent modification of polystyrene
• TC-treated• BD Primaria™ Cultureware
– Coated polystyrene surface• Poly-lysine
• Biological– Coated polystyrene surface
• ECM protein(s)• Thin layer (2D)• Gel (3D)
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Biological Surfaces: ECM Proteins
• 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
• ECM components– ex., Fibronectin, Laminin, Collagen
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Biological Surfaces: ECM Proteins
• Fibronectin (FN)– Large dimeric protein (multiple isoforms)– Contributes to matrix organization– Promotes cell adhesion via interaction between
FN ‘RGD motif’ and integrin receptors– Promotes cell differentiation and functionality (e.g.,
cell migration, integrin signaling, gene expression)
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Biological Surfaces: ECM Proteins
• Laminin (LM)– Large heterotrimeric proteins– Primarily found in basal lamina– Major structural component of basal lamina– Promotes cell adhesion via integrin and
non-integrin receptors– Promotes cell differentiation and functionality
(e.g., neurite outgrowth, receptor signaling, gene expression)
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Biological Surfaces: ECM Proteins
• Collagen– Most ubiquitous ECM molecules– Fibrous proteins that provide structure and
resiliency to tissues– Major component of skin
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Biological Surfaces:Reconstituted Basement Membrane
• BD Matrigel™ Matrix– Purified preparation from EHS mouse tumors– Composition
• Laminin ~60%• Collagen IV ~30%• Entactin ~8%• Heparan sulfate proteoglycan (perlecan)• Growth factors (e.g., PDGF, EGF, TGF-β)• Matrix metalloproteinases
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2D vs. 3D Cell Culture:Cell Differentiation and Function
• Biological composition of the culture environment– Cell type(s)– ECMs– Growth factors
• Molecular interactions and cell adhesion– Cell:cell, cell:ECM, cell:growth factor– ECM:ECM, ECM:growth factor
• Mechanical strength and structural properties– Degree of rigidity– 3D architecture
• Size scale– Pore or fiber size relative to cell size (microfibers vs. nanofibers)
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YesNoCell Encapsulation
Slow; chemical and biological gradients regulate signaling, cell-cell communication
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
2D vs. 3D Cell Culture
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2D vs. 3D Cell Culture: Surfaces
• 2D culture surfaces– Synthetic
• Ex. TC, BD Primaria™, Poly-lysine
– Biological• Ex. Fibronectin, Collagen, Laminin
• 3D culture surfaces– Synthetic
• Ex. BD™ PuraMatrix™ Hydrogel
– Biological• Ex. BD Matrigel™ Matrix, Collagen, Laminin/Entactin
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MCF-7 (breast cancer cell line) labeled with Hoechst (blue), Mitotracker green (green) and wheat germ agglutinin (red). BD Pathway™ 855 High-Content Bioimager.
Factors in Surface Selection
• Assay– Undifferentiated cells– Differentiation
• Media• Fast attachment• Strong attachment• Cell type
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Factors in Surface Choice: Media
• Serum containing media– Culture surface may be coated with serum
proteins – partially or entirely screen out underlying surface properties
– Surface properties may affect cell attachment through the selective binding of proteins
• Reduced serum or serum-free media– Underlying surface properties may become more
important
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Non-treated Polystyrene
BD Falcon™ TC-treated
5% serum 1% serum
Factors in Surface Choice: Media
MRC5 (human fibroblast) cells
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BHK21 (hamster fibroblast) cells, 1 hour after seeding
TC-treated BD BioCoat™ Fibronectin
Factors in Surface Choice:Rapid Attachment
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Before wash After washAfter wash,
Calcein AM staining
BD Falcon™ TC
BD BioCoat™ PDL
Transfected HEK-293 (human kidney epithelial) cells, 24 hours in serum-free media
Samples were washed using a Skatron Washer (Molecular Devices)
Factors in Surface Selection:Strength of Attachment
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Factors in Surface Selection:Cell Type
• Epithelial cells• Endothelial cells• Neurons• Human embryonic stem cells
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Epithelial Cells
• Line all cavities and free surfaces of the body• Tightly bound into sheets ‘epithelia’• Epithelia are barriers to water, solutes, cells• ECM that underlies epithelia: basal lamina• Examples
– Liver (hepatocytes)– Skin (e.g. keratinocytes)– Gut, Lung– Exocrine glands (e.g., mammary, sweat)
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Epithelial Cells: Hepatocytes
• Applications– Drug metabolism studies, toxicity assays– Liver regeneration, tissue engineering– Liver-specific gene expression and signaling
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TC BD BioCoat™ Collagen I
Surface treatment required for hepatocyte attachment.
Epithelial Cells: Hepatocytes
Cryopreserved human hepatocytes,thawed and plated for 4 hours
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Primary Rat Hepatocytes
Col I (2D thin coat) Col I (3D gel) BD Matrigel™ Matrix
Hepatocyte morphology and function altered by surface treatment.
Epithelial Cells: Hepatocytes
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Uncoated Collagen I PDL
Integrin αv expression is affected by the growth surface.
EcoPack™ 2-293 Cells (derived from HEK-293)
Epithelial Cells: EcoPack™ 2-293 Cells
EcoPack trademark is the property of Clontech Laboratories, Inc., a Takara Bio Company.
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TC-treated BD BioCoat™ Collagen I
Epithelial Cells: Caco-2
Caco-2 cells, thawed and cultured for 24 hours
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Caco-2 Pore Papp PappLine Size A -B B-A Ratio
10% FBS 1 uM mean: 2.9 9.7 3.6SD: 0.8 1.8
%CV: 27 19(n=4)
20% FBS 1 uM mean: 1.3 17.3 14SD: 0.24 2.50
%CV: 19 14(n=8)
Digoxin Permeability Comparison
Initial growth conditions affect cell Caco-2 function.
Epithelial Cells: Caco-2
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Endothelial Cells
• Line closed internal body cavities (e.g. blood vessels)• Angiogenesis (e.g., neovascularization during wound
healing and tumorigenesis)– Formation (sprouting) of capillaries from existing small blood
vessels)– Distinct from vasculogenesis, which is de novo synthesis of
vessels from angioblasts (endothelial progenitor cells)– Required for tumor growth and survival
• Promote blood cell adhesion during inflammatory response
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TC-treated BD BioCoat™ Collagen I
Human Umbilical Vein Endothelial Cells (HUVEC)
Endothelial Cells: HUVEC
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TC-treated BD BioCoat™ Collagen I
Fetal Bovine Heart Endothelial (FBHE) Cells
Endothelial Cells: FBHE cells
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Human Microvascular Endothelial Cells
Collagen I BD Matrigel™ Matrix
Endothelial Cells: Differentiation
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• Endothelial Cell Migration– 24- or 96-Multiwell BD FluoroBlok™ Insert (3 μm pore size)– Coated with Human Fibronectin
• Endothelial Cell Invasion– 24-Multiwell BD FluoroBlok Insert (3 μm pore size)– Coated with BD Matrigel™ Matrix
• Endothelial Cell Tube Formation– Comprised of a BD Falcon™ 96-well black/clear plate 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 migration assay; also suitable for usewith invasion and tube formation assays
Endothelial Cells:BD BioCoat™ Angiogenesis Systems
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Neurons
• Primary Neurons– Central nervous system/brain– Peripheral neurons (e.g., sensory, motor)
• Neurons– Axons conduct and transmit signals– Dendrites receive signals– Neuronal communication mediated by
neurotransmitters (e.g., glutamate, dopamine, serotonin, GABA)
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Saline Control 100 μM Glutamate
Glutamate receptor activity on BD BioCoat™ Laminin/Fibronectin Cultureware.
Neurons: Rat Cortical Neurons
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BD Falcon™ TC
BD BioCoat™
Poly-D-Lysine
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PC-12 (rat pheochromocytoma) cells, 24 hours after seeding, 10% serum
Neurons: PC-12
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Control 200 ng/ml NGF
Neurite outgrowth after 10-day treatment with NGF. Visualized with anti-β-tubulin and Hoechst using BD Pathway™ Bioimager.
PC-12 cells, grown on Collagen I
Neurons: PC-12
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Human Embryonic Stem Cells
• Generated from the inner cell mass of blastocyst
• Pluripotent cells• Maintaining pluripotency requires specific
culture conditions– BD Matrigel™ Matrix– BD Laminin/Entactin
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MEF-CM
mTeSR™1
BD Matrigel™ hESC-qualified MatrixBD™ Laminin/Entactin Complex High Concentration
Human Embryonic Stem Cells:Surfaces
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MEF-CM
mTeSR™1
BD Matrigel™ hESC-qualified MatrixBD™ Laminin/Entactin Complex High Concentration
Pluripotency marker, OCT-4, expression in H9 cells.
Human Embryonic Stem Cells:Surfaces
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Summary
• Cell / surface interactions are very complex• Currently, our main approach is empirical• Ultimate goal – predict the surface properties
needed for certain cell types and culture conditions
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Technical SupportIn the US
e-mail: [email protected]: 877.232.8995
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