Isolation and Culture of Adult Neural Stem Cells

Isolation and Culture of Adult Neural Stem Cells
Chenyan Ma
Lab of Neural Circuit Development
Adult Neurogenesis
Chunmei Zhao. et al. Cell 132, 645–660, February 22, 2008
Why in vitro culture
• Mechanism study independent of complicated
• 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
• Tend to differentiate.
Approaches to isolate neural stem cells
Flow of steps
Two adult mice
HibernateA * ( A for adult)
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
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
• 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
• 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
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
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.
Room 504, ION building
[email protected]

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