Primary NSC Culture

Dissection and dissociation of adult DG

Protocol adapted from (Walker and Kempermann 2014). P60 mice are culled by cervical dislocation and immediately processed. This protocol describes how to obtain a single cell suspension for generating adult hippocampal stem cell primary cell cultures. Please see the following protocol for the derivation of AH-NSC adherent cultures.

Materials:

• Cold PBS and dissection plates.
• HBSS with Calcium and Magnesium (HBSS+) / HBSS without Calcium and Magnesium (HBSS-).
• Fire-polished Pasteur Pipettes, with large and small end tip.
• Neural Tissue dissociation kit (P) (Milteny Biotec, 130-092-628).

Before starting the dissections:

• Set up a shaker at 37 degrees and 100rpm.
• Prepare Falcon tubes with HBSS- (around 10ml) and HBSS+ (10ml for each sample to be processed). 
• Prepare one 30mm plate with 2ml of HBSS- for each sample and keep it on ice.
• Mix the Enzyme P and Buffer X of the dissection kit to obtain the Enzymatic Mix 1 (EM1) and incubate in the water bath at 37 degrees (minimum incubation time = 15 minutes).
  ○ For each NSCs sample, mix 1.3ml of Buffer X with 33ul of Enzyme P.

Dissect the DG as described in: 

http://www.jove.com/video/1543/dissection-of-hippocampal-dentate-gyrus-from-adult-mouse

Animals can be processed separately or in pools of up to 5 mice (10 DG).

Dissociation:

• Cut the DG/DGs transversally in 8 to 10 small pieces each.
• Transfer the pieces to a 15ml Falcon and centrifuge at 0.2rcf for 2 minutes.
• Resuspend in 1.3ml of EM1 and incubate for 15 minutes in the shaker at 37 degrees and 100rpm.
• Add 20ul of EM2.
  ○ Enzymatic Mix 2 (EM2) per sample:
  • NSCs = 13.3ul Buffer Y + 6.7ul Solution 4 
• Dissociate mildly (around 7 times) with a wide opened fire-polished Pasteur Pipette previously wetted on HBSS- and incubate for 10 minutes in the shaker at 37 degrees and 100rpm.
• Dissociate until no chunks of tissue are seen with a narrow opened fire-polished Pasteur Pipette previously wetted on HBSS- and incubate for 5 minutes in the shaker at 37 degrees and 100rpm.
• Filter to a 50ml Falcon through a 40um Falcon filter and add 10ml of HBSS+ to stop the enzymatic digestion.
• Transfer to a 15ml Falcon and centrifuge 4 minutes at 900rpm. Stop here to start Derivation of AHNSCs protocol. 

Derivation of AH-NSC adherent cultures

This protocol immediately follows the Adult DG Dissection protocol.

Materials:

• DMEM:F12 +L-Glutamine +Sodium Bicarbonate (Gibco, 11320).
• KCl (Sigma, P5405).
• Heparin (Sigma, H3393).
• BSA (Sigma, A9056).
• Neurocult Supplement (Stem Cell Technologies, 05701).
• Penicillin/Streptomycin
• EGF and FGF (Protech, 315-09 and 450-33).
• Trypsin
• Trypsin inhibitor (Sigma, T7659)
• Non-adherent culture flasks for bulk cultures or 96 well plates for clonal cultures.

The day before the dissections and culture prepare:

• Supplemented Neurospheres Media.

Mix:

○ 1 bottle (500ml) of DMEM:F12

○ 559mg of KCl

○ 1 gram of BSA (add and heat at 37°C without stirring until dissolved)

Filter and keep in the fridge

•  Heparin 1000X stock (2mg/ml)
  • E.g. 20mg in 10ml of DMEM:F12 or PBS. Filter and aliquot.

On the day of the dissociation:

• Prepare complete neurospheres media before starting the dissociation and keep at 37°C degrees:
  • 45ml Supplemented Neurospheres Media
  • 5ml Neurocult Supplement
  • 500ul Pen/Strep
  • 50ul bFGF (final conc. 10ng/ml)
  • 100ul EGF (final conc. 20ng/ml)
  • 50ul Heparin (final conc. 2ug/ml)
• Follow the dissociation protocol until the last centrifugation in HBSS+.
• For bulk culture, re-suspend in 6ml and plate in a T25 flask. The cultures can grow from a single animal (best is 2DG, but even 1DG could be enough) but pooling different animals favors them growing faster (up to 6DG).
• Place in the incubator and leave as undisturbed as possible for 10 to 14 days.

Once neurospheres are ready to split / Passage 1:

• Neurospheres are ready to split when most of them are big enough or at the very early signs of adhering to the plate of between them.
• Collect the media containing the spheres in a 15ml Falcon tube.
• Centrifuge 6 minutes at 100 rpm.
• Discard supernatant and add 1ml of Trypsin-EDTA.
• Incubate for 3 minutes at room temperature.
• Add 1ml of Trypsin Inhibitor (DNAse might be needed)
• Centrifuge 6 minutes at 100 rpm.
• Discard supernatant and add 200ul of complete media
• Dissociate mechanically with a P200 tip (around 12 times)
• Count live cells (6ul of cell suspension + 6ul of trypan blue)
• Plate clonally in non-adherent plates or P96 well plates for clonal analysis at a concentration < 10,000 cells/cm².
• Place in the incubator and leave as undisturbed as possible for 10 to 14 days

Once neurospheres are ready to split / Passage 2:

Prepare complete neurospheres media and AHNSCs media, and pre-coat a few adherent flasks following the Protocol for splitting AHNSCs (and guessing the amount of cells that will be obtained).

• Follow exactly the same protocol for passage 1.
• If the number of cells is lower than 400,000, split again as neurospheres. For clonal analysis, continue plating in P96 with complete neurospheres media, also at a concentration < 10,000 cells/cm².
• If the number of cells is higher than 400,000, start the adherent cultures. Use the same cell numbers than for normal AHNSCs splitting:
  • 400,000cells/25cm² flask
  • 1.2 million cells/75cm² flask

Once the culture is established, check for mycoplasma. Freeze aliquots and keep in liquid nitrogen, as usual.

References

Walker, Tara L., and Gerd Kempermann. 2014. 'One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice', Journal of visualized experiments : JoVE: e51225-e25.