Cryopreservation and Recovery of Mature Differentiated Neural Cells

Primary neuronal cultures are indispensable in the field of neurobiology and pharmacology. Many researchers favor freshly isolated neuronal cells as they maintain their functional viability, but for convenience, an alternate route is to cryopreserve fresh cells for later use. This chapter describes the generation of cryopreserved stocks from the freshly isolated neural cells, and thawing procedures for recovering the stocks.

Rat Brain Cells

• Homogenous cell preparation from E18 rat brain tissue as described in Isolation, Culture, and Characterization of Cortical and Hippocampal Neurons.

Media and Reagents

• Gibco B-27 Plus Neuronal Culture System (A3653401)
  • Includes  Neurobasal Plus Medium (A3582901) and B-27 Plus Supplement (A3582801)    
• Trypan Blue (Cat. no. 15250-061)
• Synth-a-Freeze Cryopreservation Medium (Cat. no. A12542-01)

Equipment

• Cryogenic Vials
• Isopropanol Chamber
• Freezer, -80°C
• Liquid nitrogen freezer
• Water bath set to 37°C

Freezing Neural Cells

1. Isolate and prepare a suspension of rat brain cells in Neurobasal Plus medium supplemented with 2% B-27 Plus as described in Isolation, Culture, and Characterization of Cortical and Hippocampal Neurons.
2. Count the cell number using a hemocytometer.
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.

Recovering Frozen Neural Cells

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.
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.