Neuron Freezing Medium contains DMSO and is optimized for the cryopreservation of neurons (human, rat and mouse).
Required Supplies:1. Neural Differentiation Medium – also used to maintain neurons2. 0.05% Trypsin EDTA3. 0.25% Soybean Trypsin Inhibitor4. D-PBS with calcium and magnesium5. Neuron Freezing medium for NSCs
Preparing NSCs for Cryopreservation:1. For all volumes refer to Table 1 below
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. Remove the media from the plate and add trypsin4. Take the plates of cells immediately to the microscope so that you can observe the trypsin action
NOTE: Trypsinization of neurons takes less than 2 minutes. Extended time in trypsin decreases viability
5. 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 cabinet6. Neutralize the trypsin with an equal volume of Soybean Trypsin Inhibitor, pipetting up and down to ensure single cell suspension, then transfer cell suspension into appropriate size conical tube
7. Wash the plate with Neural Differentiation Medium (also used to maintain mature neurons) and add to the tube, pipetting up and down to ensure a homogenous cell suspension
8. 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 count Cell count X 10,000 X 2 X Volume = Total cell countNote: 10,000 is a hemacytometer constant and 2 is the Dilution factor9. Divide the total cell count by 4-5 x 10e6 to determine the number of cryovials needed for freezing down the neurons for long-term storage
10. Pellet the cells in all the tubes by centrifugation at 2000 RPM for 5 minutes
11. While the cells are in the centrifuge print the labels needed for each cryovial12. Place one label on each cryovial
13. After the centrifuge stops, resuspend the cell pellet in enough Neuron Freezing Medium to freeze 4-5 x 10e6 cells/ml as calculated in step 9 above
14. Transfer 1ml Neuron Freezing cell suspension into each cryovial15. Transfer the cryovials into a controlled rate freezer or a styrofoam container then place the container into a -80°C freezer over night16. Next day transfer the vials of cells into the LN2 freezer for long-term storage
