Bladder reconstruction

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Bladder reconstruction

• To make a bladder, you need
• Transitional epithelia
• Connective tissue
• Three layers of muscle
• Smooth and skeletal

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Urothelium/transitional epithelium

• Scalloped when relaxed
• Flattened when distended
• Many intermediate filaments
• Can see plaques
• Intramembranous proteins that cytoskeletal
  proteins attach to

relaxed
distended
Plaques indicated by arrows
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Neuronal Patterning
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A Neuron Primer
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Nerve cell morphology
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Morphology examples
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Perikaryon or soma

• Large nucleus
• Prominent nucleolus
• Nissl (ribosomes and rER)

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Neuronal Processes

• Axons
• impulses travel away from the cell body
• Only one per cell
• diameter constant along length
• Dendrites
• impulses travel toward cell body
• Taper gradually

Neuropil
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Gray matter of the CNS

• Nerve cell bodies
• Protoplasmic astroglia
• (oligodendroglia?)
• Neuropil
• Between neuron cell bodies
• Axons, dendrites and glial cells

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Classification of Cell Renewal

• Nonrenewing or Permanent or Static Cells
• Permanent G0 phase
• Potentially renewing or Expanding or Stable Cells
• Long G0 phase
• Continuously renewing or Labile Cells
• Short G0 phase
• Division and Interconversion

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Nonrenewing or Permanent cells

• No proliferative capacity whatsoever
• Never seem to divide
• Irreplaceable
• Have a long life span and live in protected
  environments
• Scientists now finding exceptions

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Examples

• Nerve cells
• nerve fibers have regenerative potential
• Cardiac muscle cells
• Auditory hair cells of the ear
• Lens cells of the eye

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Our Neurons

• Primary culture from neonatal rat brain (Brain
  Bits)
• From the cortex
• Contains maybe about 10 astrocytes
• Using serum would select for astrocytes
• Use Neurobasal medium and B27 instead

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Practical issues in handling E18 cortical cells

• Kept cold but not frozen
• Cells do not multiply
• Passaging them does not make sense
• Can be cultured for up to one (1) month
• Use Neurobasal media
• B27-a serum free supplement
• Glutamine
• Change only ½ media at a time
• Must seed at a low density to see the pattern
• Usually require a feeder layer of glial cells
• But we wont use one

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Interesting side notes

• If you want to study neuron response to free
  radicals, buy B-27 without antioxidants
• minus vitamin E, vitamin E acetate, superoxide
  dismutase, catalase and glutathione
• If you want to prevent neural differentiation of
  stem cells, buy B-27 without vitamin A
• Retinoic acid is a common differentiation inducer
• Q can you name another common inducer of
  differentiation?
• Dexamethasone (DMSO, hydrocortisone too)
• In what lab was that inducer important?
• Adipocyte differentiation

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Cell Response to Feature Size

• Container walls can dictate where cells grow
• 5mm channels encourage cells to grow inside the
  channel
• gt5mm cells spread like they are in a culture dish
• lt5mm cells grow over the feature
• Optimal sized patterns for neuron growth
• 5mm wide
• 120mm x 80mm long

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Protein Microstamping
PDMS Microstamp
Stamp substrate
Soak in Polylysine
Soak in SDS
http//soma.npa.uiuc.edu/labs/wheeler/current20pr
oject/sld001.htm
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The Role of SDS

• Pretreatment with SDS
• Ease of protein release from stamp
• Creates well defined patterns
• Extends stamp life

Chang et al. Biomaterials 2003 24(17) 2863-70
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The Role of Poly-D-Lysine

• Using as an ink for our microstamps
• Free amine groups offer a hydrophilic surface for
  preferential cell attachment
• Neurons will migrate to areas of higher
  poly-D-lysine concentration

O H H
( C C N )n
CH2 CH2 CH2 CH2 NH3
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Microelectrode Array
60 metal electrodes integrated micrometer size
(single neuron) Multi-site monitoring
Extracellular recording and stimulation Bio-Electr
onic Hybrid system Integration with electronic
device We are NOT using this!!
http//soma.npa.uiuc.edu/labs/wheeler/current20pr
oject/sld001.htm
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Neuronal Network Activity
http//soma.npa.uiuc.edu/labs/wheeler/current20pr
oject/sld001.htm
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Possible Applications

• Allows for investigation into neural development
• Allows for probing of neural learning and memory
• Development of neural tissue for tissue
  engineering
• Cell-based biosensors
• Biocomputing

• Acknowledgements
• Betty Ujhelyi
• Dr. Bruce Wheeler and his laboratory

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Other Tasks This Week

• Feed 2 and 3 week osteoblasts
• Seed 1wk diff and 1wk undiff D1 OR UVAs
• Observe ORS colonies on feeder plate--photograph

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Other practical considerations

• Acetone dissolves plastic!
• Use glass pipets
• Dont rinse stamp in the 6cm dish

• Save the glass pipets
• Return to the side bench
• Dont throw in trash can or sharps box

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What your colonies should look like
10x
20x