實驗概要
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.
主要試劑
KnockOut? D-MEM/F-12
StemPro? NSC SFM
FGF-basic (AA 10–155), Recombinant Human (bFGF)
EGF, Recombinant Human
TrypLE? Select (1X)
Dulbecco’s Phosphate-Buffered Saline (D-PBS)
DMSO (Dimethylsulphoxide)
主要設備
Sterile 15-mL conical tubes
Tabletop centrifuge
Syringe filter
Cryovials
Cryo 1°C Freezing Container
實驗步驟
1. When NSCs are 80–90% confluent (2–4 days after seeding), aspirate the complete StemPro? NSC SFM from the culture vessel.
2. Wash the cells twice with D-PBS. Aspirate the D-PBS and discard.
3. 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).
4. Detach the NSCs from the culture vessel by pipetting off the cells or by tapping the culture vessel against the heel of your hand.
5. Stop the TrypLE? Select treatment by adding 5 mL of complete StemPro? NSC SFM.
6. 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.
7. Centrifuge the NSCs at 200 × g for 5 minutes. Aspirate the supernatant and discard.
8. Resuspend the cell pellet in a minimal volume of pre-warmed complete StemPro? NSC SFM and remove a sample for counting.
9. Determine the total number of cells using your method of choice.
10. 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.
11. Transfer 1 mL of the NSC suspension in freezing medium into each pre-labeled, prechilled (4°C) cryovial.
12. 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.
13. The next day, transfer the cells into a liquid nitrogen.
