Questionsconcerns

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Questions/concerns

• Why so little media? Why not fill up flask all
  the way?
• Cleanliness and
• more importantly, gas diffusion
• Why are my counts low?
• Very normal for beginners!
• Check website for hints
• http//www.bioen.uiuc.edu/courses/498jm/cell-cultu
  re/troubleshooting.html
• 99 of time, it is operator error

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Adhesion of Fibroblasts
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This Weeks Objectives

• Understand the role of the ECM components/macromol
  ecules in cell adhesion
• Be able to infer the relative adhesion
  strengths of the molecules we use in todays
  cell adhesion assay.
• Expand your knowledge of extracellular matrix
  proteins, fibrous proteins, adhesive proteins,
  fibronectin, laminin, glycosaminoglycans, cell
  surface receptors, and integrins
• Get a glimpse of why this is important to tissue
  engineers
• Learn characteristics of "abnormal" cells

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Parameters that define the Tissue Culture
Environment

• Matrix interactions
• Should match native tissue
• Physical factors
• Extracellular matrix geometry
• Use proper 3D structure
• Appropriate physical forces

• Cell-cell interactions
• Seed at appropriate density
• Co-culture if necessary
• Soluble factors
• Hormones
• Growth factors
• neurotransmitters

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• Unfortunately, no one can be told what the Matrix
  is. You have to see it for yourself.

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What is the Extracellular Matrix (ECM)?

• Intervening spaces between connective tissue
  cells occupied by material produced by the cells.
• Complex weaves, glues, struts and gels that
  interconnect cells in a tissue with their
  cytoskeletal components.
• For epithelial cells, the ECM is the basement
  membrane
• Three layers of various molecules produced by
  both the epithelial cells and underlying
  connective tissue cells
• More on this later

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What is the Extracellular Matrix good for?

• Provides physical support, flexibility,
  resistance to crushing/tearing, joint
  articulation
• Provides components for signal transduction,
  which can influence cell behavior
• Hyaluronan needed for cell migration
• Laminin required for tissue regeneration

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ECM density

• Affects
• Cell adhesion
• Cell spreading
• Cell shape
• Cell migration
• polarity
• Apoptosis

• All of those factors affect cell fate and
  function
• Ability to regenerate
• Specialized for particular functions in tissues
• Strength (tendon)
• Filtration (kidney glomerulus
• adhesion

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ECM affects cell shape
Chondrocytes on plastic dish
Chondrocytes in vivo
Chondrocytes on collagen gel
Cell shape change usually affects cell secretions
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Structural Proteins of ECM

• Fibrous proteins
• Collagen
• Elastin
• Reticular fibers
• Adhesive proteins
• small glycoproteins
• Fibronectin
• connects cell to extracellular structures
• Laminin
• major component of basal lamina

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Protein/polysaccharide complexes

• attract water
• Glycosaminoglycans (GAGs)
• Hyaluronan
• a complex of proteoglycans
• Proteoglycans
• Chondroitan sulfate
• keratan sulfate

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Other terminology

• Ligand
• The molecule that a receptor binds to
• ECM components, molecules on membranes of other
  cells, signaling molecules (neurotransmitters),
  etc.
• Integrins
• Transmembrane protein, receptor helps link ECM to
  cytoskeleton

• Receptors
• Proteins expressed on cell membrane that
  specifically bind to molecules in their
  environment
• Fibroblasts have receptors for several molecules
  and if the fibroblasts attach to a surface coated
  with a specific molecule, you can conclude that
  the cell has a receptor for that molecule

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Outside the cell
Plasma membrane
Inside the cell
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From Tissue Engineering By Minuth, Strehl and
Schumacher. 2005
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From Tissue Engineering By Minuth, Strehl and
Schumacher. 2005
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RGD motifs

• Arginine-Glycine-Aspartic Acid
• A consistent amino acid sequence for molecules
  that help cells bind to the ECM
• Where cell receptors anchor
• Found in fibronectin, laminin, entactin,
  thrombin, tenascin, fibrinogen, vitronectin, type
  I and VI collagen, bone sialoprotein, osteopontin

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Cell Behavior of Transformed Cells

• Growth and Division Abnormalities
• Grow to high density
• (no __________________)
• Lowered requirement for growth factors
• Less anchorage dependence
• Immortality
• (unlimited ______________)
• Can cause tumors in when injected in susceptible
  animals
• (__________)

• Adherence Abnormalities
• Diminished adhesion to surfaces
• (missing _______________)
• Failure of actin fibers to organize into stress
  fibers
• Reduced external coat of fibronectin
• (needed for ___________)
• High production of plasminogen activators,
  causing increased extracellular proteolysis,
  cleaving laminin
• (tumor is ____________)

Fibronectin Receptors
Contact Inhibition
Adhesion
Passage Number
Pervasive
Invasive
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Our 3T3-1 Cells

• Are immortal (by spontaneous mutation)
• Are anchorage dependent
• Given time, nutrients, ions, growth factors, and
  space will sit down on untreated plastic, secrete
  ECM and multiply.
• Factors to consider
• ECM components are created by fibroblasts, but
  not immediately.
• The presence of these proteins will facilitate
  adhesion, but only if your fibroblasts have
  receptors for them!
• We want to test to see which substrates will
  promote the strongest or weakest adherence of our
  cells.

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Why analyze this?

• If cells began to behave differently while
  managing a culture in a lab.
• Import nanoparticles, new scaffold/substrate
• If we wanted to quantify a behavior change should
  cells undergo a mutation.
• In trying to transform a cell line, could test
  for a change in cell behavior, like adhesion, to
  see if transformation had taken place.

Thus if we prepare several substrates and test
for cells ability to adhere, the results could
answer those questions.
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Adhesion in Tissue Engineering

• Surface chemistry of biomaterials can affect
  adhesion
• Teflon, titanium, dacron, silk, plastics,
  ceramics
• Surface topography of biomaterials can affect
  adhesion
• ECM can act as a regeneration template
• Skin, nerve
• Usually proteins adsorbed to surface of
  biomaterials, then cells attach to the protein
  surface.
• So, if you wanted to implant something that cells
  would be attracted to, knowing adherence
  properties of material and cells would be helpful.

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Components Tested this Week

• NCS (newborn calf serum)
• Liquid left after blood has clotted and clot
  removed.
• Has large, sticky proteins, but not ECM, some
  growth factors (mitogens).
• DMEM
• The media we use for cell culture, with serum,
  pen/strep and sodium pyruvate.

• DMEM-
• The media without added serum.
• Conditioned Media
• Media from culture dish with growing cells.
• Nutrients may be expended, but contains
  additional growth factors secreted by cells, some
  ECM molecules.

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More components

• Albumin or BSA
• Large carrier protein in the blood stream,
  produced by the ___________.
• Sticky but not an ECM protein.
• Used as a control
• When cells release from albumin, can assume that
  only receptor bound cells are left in the other
  wells

• PBS-CMF
• Negative control.
• Fibronectin
• ECM protein secreted by fibroblast cells.
• Collagen
• ECM fiber in many tissues,
• dissolved in acetic acid, residual will cause
  media to change color to yellow

Liver
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Protocol-TAs

• TA will incubate substrates on multi-well strip
  (1hr, 37C)
• trypsinize cells from last week and resuspend in
  media 2 BSA
• Calculate cells/ml. Determine volume of cells so
  there are 2x104/well
• If more than 100,000 cells/ml, dilute
• Wash excess solutions from wells, add 2x104
  cells/well, incubate in 37oC incubator for 30min.

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Protocol continued

• Terminate assay by shaking cells on orbital
  shaker, wash cells with DMEM- (no serum or BSA).
• Cells still there? Wash, shake, look again until
  Albumin well is free of cells.
• Under the microscope, count the number of cells
  in each well. Are cells spread out or flat or
  rounded?
• Add WST-1 assay reagents
• Wait appropriate time and read on microplate
  reader
• Clean up! Dump strip wells in basin, discard
  strip wells in trash, put holders away, bleach
  kill cells and clean basins.

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How WST-1 works

• Similar to a MTT assay
• Cells have mitochondrial enzymes
• We will be looking for succinate tetrazolium
  reductase (a mitochondrial dehydrogenase)
• When cells cant be counted directly, one can
  lyse the cells and assay the amount of
  mitochondrial enzyme present
• Colorimetric agent reacts with mit. enzyme.
  Absorbance of 450nm light corresponds directly
  to the number of cells.

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Microplate Reader

• A specialized spectrophotometer for reading
  multiple samples usually on a 96 well plate

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Spectrophotometry

• Transmitted light passed through a solute
• Can measure light that has been transmitted or
  absorbed (by the sample)
• Darker reactions will absorb more light
• Ax will be higher
• A blank is used to eliminate any absorbance
  caused by solvent or buffer
• Helps determine if there is a contamination or
  cross-reaction in your buffer
• A blank was used in the creation of the standard
  curve in the lab manual.
• You do not have a blank, although PBS is your
  negative control

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Nomenclature

• Absorbance is often reported as Al.
• Transmittance is reported as T
• Optical Density (OD) is the relationship between
  transmittance and the concentration of the
  colored compound
• We will only be using Absorbance
• Compounds created by sample and reagent have
  optimal wavelengths where absorbance occurs.
• WST is read at A 450

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TAs Calculating cells needed

• Calculate cell concentration as usual
• Cells counted x DF (usually 2) x 10,000
• 10
• Total cells needed in your flask to successfully
  complete this exercise
• 2x104 x 8 wells 1.6x105
• Use the following formula to calculate volume of
  cell suspension to add to well
• 20,000 (cells desired) x ml/cells (inverse of
  cells/ml from above)

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Expected Results

• FBS _____________
• DMEM _____________
• Conditioned Media ______________
• DMEM- _____________
• Albumin ____________
• Fibronectin ______________
• Collagen ____________
• PBS-CMF ______________

Minimal
Minimal
Minimal
None
More
Yes ( Control)
Yes (lt Fibronectin)
No (- Control)
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Other exercises this week

• Coat glass coverslips with fibronectin
• Will seed these coverslips on your return day for
  use next lab period
• Seed two new flasks for return day