Cryopreservation and Recovery of Mature Differentiated Neural Cells

实验概要

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.

主要试剂

Neurobasal® Medium

B-27® Serum-Free Supplement

GlutaMAX™-I

Trypan Blue

Synth-a-Freeze® Cryopreservation Medium

主要设备

Cryogenic Vials

Isopropanol Chamber

Freezer, -80°C

Liquid nitrogen freezer

Water bath set to 37°C

实验材料

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

实验步骤

1. Cryopreservation

1) Isolate  and prepare a suspension of rat brain cells in Neurobasal® medium  supplemented with 2% B-27® 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.

2. Cell Recovery

Handle  cells gently, because they are extremely fragile upon recovery from  cryopreservation. It is important to rinse pipette tips and vials with  complete Neurobasal®/B-27® medium 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®/B-27® 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®/B-27® 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®/B-27® 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.