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    Cryopreservation and Recovery of Mature Differentiated Neural Cells

    互联网

    573

    实验试剂

     

    Neurobasal® Medium

    B-27® Serum-Free Supplement

    GlutaMAX™-I

    Trypan Blue

    Synth-a-Freeze® Cryopreservation Medium

    实验设备

     

    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.

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