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.

THANK YOU FOR YOUR ATTENTION!

Room 504, ION building

cyma@ion.ac.cn