Fichas
Cell-Biomaterial Surface Interaction (wk. 6)
Fichas del conjunto (49)
- 1objectives- components cell
- types receptor-ligand interaction
- c&c ECM major components
- important paramenters:
cell adhesion
cell spreading
cell migration
basic characterization methods to observe cell-surface interaction - 2protein-cell interactionsspecific
usually occur via receptor-ligand interaction - 3cell functions
- 4cell typesdifferentiated
non-differentiated (progenitor) - 5cellular structure
- 6Cell Plasma Membranephospholipid bilayer
proteins present which give specific functions
eg. channels- transmembrane, receptors-project primarily to the extracellular environment) - 7Phospholipidspolar hydrophillic heads
nonpolar hydrophobic tails (fatty acids)
- 8Lipid bilayer
- 9Transmembrane Proteins- hydrophillic and hdyrophobic domains
- span entire membrane
- mostly channels or pumps:
"small pores" allowing Na+ K+ Ca2+ Cl- to pass through (not binding) according to ion concentration gradient
-some ion pumps: physically bind to molecule transported, usually require energy to transport - 10Selectively permeable (depends on...)depends on:
size
electrical charges
molecular shape
lipid solubility - 11"outside-in" signallingcell-cell
cell-environment
cell spreading, migration, communication differentiation - 12"inside-out" signallingcell secrete molecules/rearrange contacts, change extracellular environment
- 13Protein-based Receptorson membrane
enable cell-cell
cell-ECM interactions - 14Adhesion receptorsallow cells to attach to other cells/ECM
bind to specific peptide sequences of other proteins
each is specific to a small range of target molecules (ligands) - 15Common Receptor Molecules1. Cadherins (cell-cell)
2. Integrins (Cell-ECM)
3. other cell adhesion molecules (CAMs) - 16Cell Adhesion Forcecell adhesion receptors+ligands = weak
- a lot of weak interactions make a strong bond
- cell adhesion too weak to support permanent contact between cells
- following adhesion connections have to be stabilized by cytoskeleton to form cell junctions - 17Types of cell contacts1. cell-cell interaction
2. Cell-ECM
3. cell-surface - 18Cell-cell interactions (3)tight junctions; adjacent cell membranes adhere, prevent even small molecules from passing between the cells
gap junctions: small, hydrophilic channel created by a plaque-like structure that connects two different cell membranes
Desmosomes: mechanical attachments of two cells, either broad bands or specific spots. Caused by association of cadherin receptors of each cell - 19Cell-ECMHemidesmosomes
focal adhesions
(receptor is integrin) - 20Cadherinscell-cell adhesion
Ca2+ dependent binding
attach via His-Ala-Val - 21Integrinscell-ECM adhesion
important signal tranduction receptors for regulation of cell growth
Ca2+/Mg2_ dependent binding
attach to Arg-Gly-Asp (RGD), Leu-Asp-Val (LDV) & others
composed of alpha and beta subunits, which have 18 and 8 types respectively. form different combinations
ligand binding site composed of both subunits - 22Cell surface interactionsproteins + receptors
ligand-receptor (integrin) binding:
adhesion, contraction , motility, secretion, proliferation - 23Cell surface receptorsmembrane-embedded proteins/glycoproteins that control signal tranduction & cell adhesion
- 24Extracellular Matrix (ECM)structural support, network of proteins & carbohydrates.
1. supports and surrounds cells
2. regulates cells activities
3. lattice for cell movement
- 25ECM types1) Fiber-forming elements (insoluble): collagen, elastin
2) space-filling molecules (soluble) = glycoproteins: fibronectin, laminin, proteoglycans
collagen most abundant protein in mammals, primarily for tissue tensile strength
elastin forms fibres, allows large elastic deformation at low stress - 26Ex. 6.1 proteins often adsorb to biomaterial surfaces implanted in vivo or exposed to serum-containing media in vitro. Many biomaterials do notsupport cell adhesion prior to adsorption of a protein layer. How might proteins facilitate adhesion of cells to a biomaterial? Hint: consider to coat ECM proteins of biomaterial surface.First a protein layer forms on the biomaterial surface
protein-cell interactions can occur because of ligand-receptor interactions. The receptors are proteins, and these will react with ligands to facilitate adhesion of cells to a biomaterial. - 27basic interactionssPASM without the first s
Protein adsorption
Adhesion: recognition of cell-binding domains ECM
Spreading: receptor-ligand interactions, cytoskeletal modulation
Migration: gene modulation (proliferation, synthesis) - 28Cell spreadingcells extend pseudopodia along substrate surface
integrin receptors in ceell membrane interact with ligands on material surface
generate contractile force, concomitant release of rear receptors for forward movement/migration - 29in vivo vs. in vitro
- 30Types of cell culturesprimary cultures: directly from living organism
- reflect real function
- but die off, some cell types are poor dividers (nerve)
cell lines: derived form primary cells (not clones)
- altered genes - immortality
- isolated spontaneous mutation
- tumor cells
- viral oncogen transfection. - 31characterization methodsmorphology-based techniques
analytical techniques - 32upright vs inverted microscopehigh school one = upright
inverted = light and condensor on top, above sample, lenses pointing up - 33bright field vs dark field microscopefield describes the BACKGROUND
brightfield = background bright
dark field = background dark
bright field magnification = ocular lens + objectives lenses combined
dark field used for looking at living cells without stains - 34Phase-Contrast microscopeenhances contrast intracellular structures with slight differences in refractive index (n)
excellent way to observe living cells - 35Fluorescence microscopyoptical microscope that uses fluoresence/phosphorescence instead of/in addition to reflection & absorption
uses wavelength that excites fluorescence in sample
fluoresced light at longer wavelength than illumination
1. exposes specimen to UV, V, or blue
light
2. specimens usually stained w fluorochromes
3. bright image from fluorescent light - 36Fluorescence microscopy sample preparationsample must be fluorescent: label or expression of fluorescent protein
common stains:
Nuclaic Acid stains like DAPI
drugs/toxins w fluorescent reporter (fluorescently labelled phalloidin, to stain actin fibers in mammalian cells) - 37Confocal fluorescence microscopyimproves optical resolution and contrast using point illumination and spatial pinhole
enables reconstruction of 3D structures from obtained images
- 38confocal fluorescence microscopy common charactersisticslaser beam used to illuminates spots on specimen
computer compiles images created from each point, generates a 3D image - 39Cell Adhesion Assaysimportance: cell adhesion necessary for many other cell functions, provides physical and biochemical stimulation
Sedimentation assay
1. cell type on surface at given DENSITY
for specified time, in vitro
2. Surface watched, cells counted via optical microscopy/coulter counter (cells detached, suspended and measured by electrical resistance thru narrow channel)
=> sedimentation assay gives info on strength of adhesion
# ADHERED - 40Cell Spreading Assays1. cell type on surface at given DENSITY
for specified time
2. Mesaure: INCREASE projected surface area (optical microscopy)
spreading indicates focal contact formation, metabolic activity
# SPREAD - 41Centrifugation Assay (Normal Force)1. cell seeded 24 well plate, w surface coating, specified time
2. plate inverted in centrifuge
MEASURES: cells attached vs. applied Force - 42Flow Chamber Assay (Shear Force)1. cells seeded into parallel plate chamber
2. Fluid flow vvelocity gradiant results in shear force on cell-surface bonds - 43Cell Migration Assaytissue organization (embryonic)
immune and inflammatory response (chemotaxis of white blood cells)
angiogenesis, - endothelial cell migration to form vasculature
wound healing - fibroblast migration to form connective tissue
tumor metastasis - 44—Cell adhesion
# cells adhered
- 45—Centrifugation
- 46—Flow Chamber
Shear force
- 47flow chamber vs centrifuge
- 48—Cell Spreading
- 49—Cell migration
