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Isolation and Culture of Adult N eural S tem C ells

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.

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Isolation and Culture of Adult N eural S tem C ells

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  1. Isolation and Culture of Adult Neural Stem Cells ChenyanMa Lab of Neural Circuit Development 2012-04-07

  2. Adult Neurogenesis ChunmeiZhao. et al. Cell 132, 645–660, February 22, 2008

  3. Why in vitro culture • Mechanism study independent of complicated factors. • Pharmacological and genetic manipulation. • Identical replicates • Time- course and dose- response study.

  4. Difficulties of isolation • Limited number • Limited self-renewal ability • Limited survival rate because of debris of mature cells. • Tend to differentiate.

  5. Approaches to isolate neural stem cells

  6. Reference

  7. Flow of steps *

  8. Materials • Two adult mice • HibernateA* ( A for adult) • Papain • B27 minus retinylacetate * • 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

  9. 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.

  10. 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 hippocampiinto HABG at 4 ℃.

  11. 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.

  12. 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.

  13. 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).

  14. 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.

  15. 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.

  16. 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.

  17. Thank you for your attention! Room 504, ION building cyma@ion.ac.cn

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