Neural Freezing Medium contains DMSO and is optimized for the cryopreservation of neural stem cells (human, rat and mouse).
Required Supplies:1. Neural StemCell Growth medium2. 0.05% Trypsin EDTA3. 0.25% Soybean Trypsin Inhibitor4. D-PBS without calcium and magnesium5. D-PBS with calcium and magnesium6. Neural Freezing medium for NSCs
Preparing hNSCs for Cryopreservation:1. For all volumes refer to Table 1
2. Working with multiple platesa. Only work with a stack of 4 x 100mm plates at a timeb. Minimize time out of the incubator during the processc. Work swiftly but carefully to minimize cell loss3. PBS Wash: Remove old medium and add D-PBS w/o calcium and magnesium4. Remove the PBS wash and add trypsin5. Take the plates of cells immediately to the microscope so that you can observe thetrypsin action
Note: Trypsinization of hNSCs takes less than a minute. Extended time in trypsin decreases viability. 6. Tap the dishes against the palm of your hand to dislodge the cells and when the cells are free floating, return the plates to the safety cabinet7. Neutralize the trypsin with Soybean Trypsin Inhibitor, pipetting up and down to ensure single cell suspension, then transfer cell suspension into appropriate size conical tube
8. Wash the plate with Neural StemCell Growth medium and add to the tube, pipetting up and down to ensure a homogenous cell suspension9. Take an aliquot for cell countinga. Take 50ul cell suspension and add it to 50ul Trypan Blueb. Pipette up and downc. Load 10ul of cell suspension to both counting chambers of a hemacytometerd. Count the cells within the center gride. Calculate the total cell countCell count X 10,000 X 2 X Volume = Total cell countNote: 10,000 is a hemacytometer constant and 2 is the Dilution factor10. Divide the total cell count by 1.5 x 10e6 to determine the number of cryovials needed for freezing down the cells for long-term storage
11. Pellet the cells in all the tubes by centrifugation at 2000 RPM for 5 minutes
12. While the cells are in the centrifuge print the labels needed for each cryovial13. Place one label on each cryovial
14. After the centrifuge stops, resuspend the cell pellet in enough Neural Freezing Medium to freeze 1.5x106/ml as calculated in step 10 above
15. Transfer 1ml Neural Freezing cell suspension into each cryovial16. Transfer the cryovials into a controlled rate freezer or a styrofoam container then place the container into a -80°C freezer over night17. Next day transfer the vials of cells into the LN2 freezer for long-term storage
