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There are numerous protocols available for cryopreserving neural stem cells (NSCs) derived from human embryonic stem cells; the primary objective of these methods are the recovery of the cells post-thaw and the retention of their multipotent properties. This page describes a standardized cryopreservation protocol that does not alter the viability and sublineage differentiation capacity of the preserved cells.
Required Materials
Cells
Neural Stem Cells (NSCs)
Media and Reagents
Tools and Equipment
Required Materials
Cells
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
- Cryovials
- Cryo 1°C Freezing Container (Nalgene, Cat. no. 5100-0001)
StemPro® NSC SFM Complete Medium
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.
Freezing Medium
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 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.
| Component | Final concentration | Amount |
|---|---|---|
| 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 |
Freezing Medium
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.
| Component | Final concentration | Amount |
|---|---|---|
| StemPro® NSC SFM Complete Medium without bFGF and EGF (see above) | 90% | 9 mL |
| DMSO | 10% | 1 mL |
Guidelines for Cryopreserving Neural Stem Cells
Freezing Neural Stem Cells
- 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.
LT146 17-Mar-2011