Neural Stem Cells (NSCs)
Media and Reagents
- KnockOut™ D-MEM/F-12 (Cat. no. 12660)
- StemPro® NSC SFM (Cat. no. A10509-01)
- FGF-basic (AA 10–155), Recombinant Human (bFGF) (Cat. no. PHG0024)
- EGF, Recombinant Human (Cat. no. PHG0314)
- TrypLE™ Select (1X) (Cat. no. 12563-029)
- Dulbecco’s Phosphate-Buffered Saline (D-PBS) (Cat. no. 14190-144)
- DMSO (Dimethylsulphoxide) (Sigma, Cat. no. D2650)
Tools and Equipment
- Sterile 15-mL conical tubes
- Tabletop centrifuge
- Syringe filter
- Cryo 1°C Freezing Container (Nalgene, Cat. no. 5100-0001)
StemPro® NSC SFM complete medium consists of KnockOut™ D-MEM/F-12 with StemPro® Neural Supplement, EGF, bFGF, and GlutaMAX™-I. Complete medium is stable for 4 weeks when stored in the dark at 2–8°C.
To prepare 50 mL of StemPro® NSC SFM complete medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. If desired, add 0.5 mL of Antibiotic-Antimycotic solution per 50 mL of complete medium.
|KnockOut™ D-MEM/F-12||1X||48.5 mL|
|GlutaMAX™-I Supplement||2 mM||0.5 mL|
|bFGF||20 ng/mL||1 μg|
|EGF||20 ng/mL||1 μg|
|StemPro® Neural Supplement||2%||1 mL|
To prepare 10 mL of freezing medium, aseptically mix the following components. For larger volumes, increase the component amounts proportionally. Filter sterilize the freezing medium and store at 2–8°C until use. You may observe a white precipitate when thawing StemPro® Neural Supplement; this precipitate will disappear when the supplement is completely thawed or dissolved.
|StemPro® NSC SFM Complete Medium
without bFGF and EGF (see above)
- Cryopreserve NSCs when they are 80–90% confluent (2–4 days after seeding).
- Freeze NSCs at a concentration of 2 × 106– 2.4 × 106 viable cells/mL and a volume of 1 mL/vial.
- Use a freezing medium composed of 90% complete StemPro® NSC SFM without the growth factors (i.e., bFGF and EGF) and 10% DMSO.
- Do not incubate the NSCs in TrypLE™ Select for more than 2 minutes to avoid cell death.
- Pre-label all cryovials with the following information: cell line, passage number, concentration, date of freezing, and your initials.
Freezing Neural Stem Cells
- When NSCs are 80–90% confluent (2–4 days after seeding), aspirate the complete StemPro® NSC SFM from the culture vessel.
- Wash the cells twice with D-PBS. Aspirate the D-PBS and discard.
- Add 1 mL of pre-warmed TrypLE™ Select to the culture vessel and incubate at 37°C for 2 minutes. Note: Do not incubate the NSCs in TrypLE™ Select for more than 2 minutes to avoid cell death. Neutralize TrypLE™ Select by adding complete StemPro® NSC SFM immediately after the incubation period (see below).
- Detach the NSCs from the culture vessel by pipetting off the cells or by tapping the culture vessel against the heel of your hand.
- Stop the TrypLE™ Select treatment by adding 5 mL of complete StemPro® NSC SFM.
- Gently pipet the NSCs up and down to get a single cell suspension and transfer the cell suspension into a sterile 15-mL conical tube.
- Centrifuge the NSCs at 200 × g for 5 minutes. Aspirate the supernatant and discard.
- Resuspend the cell pellet in a minimal volume of pre-warmed complete StemPro® NSC SFM and remove a sample for counting.
- Determine the total number of cells using your method of choice.
- Gently aspirate the medium from the conical tube and drop-wise add pre-chilled (4°C) freezing medium to resuspend the cells at a concentration of 2 × 106– 2.4 × 106 viable cells/mL.
- Transfer 1 mL of the NSC suspension in freezing medium into each pre-labeled, prechilled (4°C) cryovial.
- Transfer the cryovials to the Cryo 1°C Freezing Container and place the container into a –80°C freezer. This procedure ensures that the cells freeze slowly.
- The next day, transfer the cells into a liquid nitrogen.