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 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  m filter 40  m 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 (2  g/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 (1mm 3 ) (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  l of cell suspension into a small tube containing 10  l trypan blue. Mix and apply approximately 10  l 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%O 2, 5% CO 2.

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