isolation and culture of adult n eural s tem c ells
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Isolation and Culture of Adult N eural S tem C ells. Chenyan Ma Lab of Neural Circuit Development 2012-04-07. Adult Neurogenesis. Chunmei Zhao. et al . Cell 132, 645–660, February 22, 2008. Why in vitro culture. Mechanism study independent of complicated factors. - PowerPoint PPT PresentationTRANSCRIPT
Isolation and Culture of Adult Neural Stem Cells
Chenyan Ma
Lab of Neural Circuit Development
2012-04-07
Adult Neurogenesis
Chunmei Zhao. et al. Cell 132, 645–660, February 22, 2008
Why in vitro culture
• Mechanism study independent of complicated
factors.
• Pharmacological and genetic manipulation.
• Identical replicates
• Time- course and dose- response study.
Difficulties of isolation
• Limited number
• Limited self-renewal ability
• Limited survival rate because of debris of mature
cells.
• Tend to differentiate.
Approaches to isolate neural stem cells
Reference
Flow of steps
*
Materials• Two adult mice• HibernateA * ( A for adult)• Papain • B27 minus retinyl acetate *• Glutamax or L-Gln• OptiPrep density 1.32 (Sigma): 60% (w/v) solution of iodixanol ( 碘克沙醇) in water (sterile)
*• NeurobasalA* ( A for adult)• Growth factors: bFGF, EGF, PDGFbb*• P/S (Penicillin streptomycin) or Gentamycin• Surgical equipment• 15 ml and 50 ml centrifuge tubes (new, better to be corning , NEST or BD).• Pasteur pipette (or common dropper)• 0.22 mm filter• 40 mm cell strainer• Swinging bucket centrifuge • Ultralow adhesion plastic culture dishes ( or common dishes made in China, but not corning
treated dishes) *• New plastic pipette tips• Trypan blue• Hemacytometer
Reagent and equipment setup
• HABG (40ml-60ml): HA, B27 minus retinyl acetate, 0.5mM
Glutamax
• Culture medium: NeurobasalA, B27 minus retinyl acetate, 0.5mM
Glutamax, P/S, bFGF (10ng/ml), EGF (10ng/ml), PDGFbb(10ng/ml),
heparin (2mg/ml, optional).
• Papain: >34U/ml stock (final concentration 34U/ml), 37 for 20–30 ℃min, filter- sterilize into tubes.
• Disinfect surgical equipment with 70% ethanol.
Step 1. Tissue dissection
• Anesthetize adult mice.
• Disinfect head with 70% ethanol, and expose the
brain.
• Transfer the brain to a dissection dish with
HibernateA (or PBS) at 4 . ℃• Dissect hippocampi into HABG at 4 .℃
Step 2. Digestion
• Cut hippocampi in HABG into small pieces (1mm3) (the
smaller the better).
• Add papain to a final concentration of 34U/ml.
• Incubate at 30 or 37 for 30 min (better to shake).℃ ℃• Transfer the tissue to a 15- ml tube and centrifuge at 200g
for 3 min.
• Discard the supernatant.
• Re-suspend the tissue with 2 ml HABG.
Step 3*. Release Cells from Tissue----determine your yield
• Trituration with pasteur pipette (or dropper) (*1- ml pipette tip is too
sharp), without air bubbles for several times (determined by
yourself).
• Allow the pieces to settle for 1 min and transfer the supernatant to
an empty 15-ml tube. *• Re-suspend the sediment from the first tube in 2 ml HABG, repeat
the last two steps twice more.
• Finally, you got 6- ml suspended cells.
• Filter the suspended cells with cell strainer.
Step 4*. Density Gradient Centrifugation
• Prepare density gradient. * ( Be careful)
• Carefully apply the cell suspension to the top of the prepared
OptiPrep density gradient.
• Cenfrifuge the gradient at 800g (1,900 r.p.m. in a swinging
bucket centrifuge) for 15 min at 22 (or at room tempreture).℃
• Collect fraction 3 into a new 15- ml tube.
• Wash out the gradient material with 4- to 5- ml HABG,
centrifuge at 200g for 2 min, and discard the
supernatant.
• Repeat washing.
• Re-suspend the cell pallet with 0.5- to 1- ml culture
medium.
Step 5. Cell Plating
• Aliquot 10 ml of cell suspension into a small tube containing 10 ml
trypan blue.
• Mix and apply approximately 10 ml to a hemacytometer.
• Count phase bright spherical cells.
• Plate cells at a low density of 4000 to 8000 cells/ ml for clones of
neurospheres in ultralow adhesion substrate.
• Culture at 37 , in a 5% CO2, ℃ 9% O2 gas (optional), humidified
incubator.
Critical factors
• Optimized protease digestion
• Control of osmolarity and pH outside the incubator
• Density gradient separation
• Low-adhesion plastic substrate
• B27 minus retinyl acetate
• Suitable growth factors
• 9%O2, 5% CO2.