Cryopreserving Neural Stem Cells

实验概要

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.

主要试剂

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) 

主要设备

Sterile 15-mL conical tubes

Tabletop centrifuge

Syringe filter

Cryovials

Cryo 1°C Freezing Container

实验步骤

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.