Cryopreservation & Recovery of
Mature Neural Cells & Neural Stem Cells

Preserving neural stem cells (NSCs) and mature neural cells for long-term research use requires cryopreservation methods that maintain cell viability and function after thawing. This protocol describes a workflow for freezing and recovering neural cells—such as primary neurons from rat brain or human-derived neural cells—using optimized media and cryoprotectants.

• Neural cells
• KnockOut D-MEM/F-12
• StemPro NSC SFM
• FGF-basic (AA 10–155), Recombinant Human (bFGF)
• EGF, Recombinant Human
• TrypLE Select Enzyme (1X), no phenol red
• Dulbecco’s Phosphate-Buffered Saline (D-PBS)
• DMSO (Dimethylsulphoxide)
• Gibco B-27 Plus Neuronal Culture System

• Trypan Blue Solution, 0.4%
• Synth-a-Freeze Cryopreservation Medium
• Cryogenic vials
• Mr. Frosty Freezing Container
• Isopropanol chamber
• Sterile 15-mL conical tubes
• Tabletop centrifuge
• Syringe filter
• –80°C freezer
• Liquid nitrogen freezer
• Water bath set to 37°C

• Cryopreserve NSCs when they are 80–90% confluent (2–4 days after seeding).
• Freeze NSCs at a concentration of 2–2.4 × 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 Enzyme 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.

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 enzyme to the culture vessel and incubate at 37°C for 2 minutes. 
   Note: Do not incubate the NSCs in TrypLE enzyme for more than 2 minutes to avoid cell death. Neutralize TrypLE enzyme 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 enzyme 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 2–2.4 × 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.

1. Isolate and prepare a suspension of rat brain cells in Neurobasal Plus Medium supplemented with 2% B-27 Plus.
2. Count the cell number using a hemocytometer or automated cell counter.
3. Centrifuge the cells at 200 × g for 4 minutes. Aspirate the supernatant.
4. Resuspend the cell pellet in cold Synth-a-Freeze at a concentration of 2.0 × 10⁶ – 1.0 × 10⁷ cells/mL.
5. Make 1 mL aliquots of the cells in pre-labeled, pre-chilled cryovials and place the vials in an isopropanol chamber at 4°C for 10 minutes.
6. Transfer the isopropanol chamber to –80°C for overnight.
7. Transfer the frozen vials to the vapor phase of liquid nitrogen storage until use is required.

Remember to handle cells gently because they are extremely fragile upon recovery from cryopreservation. It is important to rinse pipette tips and vials with complete Gibco B-27 Plus Neuronal Culture System (Neurobasal/B-27 Plus Supplement) before using them for transferring cell suspensions to avoid the cells sticking to the plastic. Do not centrifuge cells upon recovery from cryopreservation.

1. Remove one vial of frozen cells from liquid nitrogen.
2. Thaw the vial in a 37°C water bath with gentle swirling.
3. Wipe down the vial with ethanol and tap gently on a surface so that all of the medium collects at the bottom of the tube.
4. Open the vial in a laminar flow hood.
5. Rinse a pipette tip with medium and very gently transfer the cells from the vial to a prerinsed 15-mL tube.
6. Rinse the vial with 1 mL of pre-warmed complete Neurobasal Plus/B-27 Plus medium and transfer the rinse to the 15-mL tube containing the cells at a rate of one drop per second. Mix by gentle swirling after each drop.
7. Slowly add 2 mL of complete Neurobasal Plus/B-27 Plus medium to the tube (for a total suspension volume of 4 mL).
8. Mix the suspension very gently with P-1000 pipette. Avoid creating any air bubbles.
9. Add 10 μL of cell suspension to a microcentrifuge tube containing 10 μL of 0.4% Trypan Blue using a pre-rinsed tip. Mix the cells by gently tapping the tube. Determine the viable cell density using a hemocytometer or automated cell counter.
10. Plate ~1 × 10⁵ cells per well in poly-D-lysine coated 48-well plate or an 8-chambered slide. Bring the cell suspension volume to 500 μL per well by adding complete Neurobasal Plus/B-27 Plus medium.
11. Incubate the cells at 37 °C in a humidified atmosphere of 5 % CO₂ in air.
12. Feed the cells every third day by aspirating half of the medium from each well and replacing it with fresh medium.