Method: Preparation of Lymphoblastoid Cell Lines for Long Term Storage

Rosalie Veile

Purpose:

To store cell lines in a form that will insure recovery with high viability.A culture in logarithmic phase of growth with a total volume of 80-100 ml/T-75 flask should yield enough cells to freeze 10 ampules (1.0 ml/ampule). Cells should have a count of 4 X 106 cells/ampule to 9 X 106 cells/ampule. Too high or too low a cell count lowers recovery viability. Cell are frozen in RPMI-1640 with 15% Fetal Bovine Serum + 10% DMSO. Cultures are frozen slowly using a Model 700 Controller freezing chamber. This precision electronic device automatically controls the injection of liquid nitrogen into the freezing chamber to provide a 1 degrees C/minute freezing rate from +4 degrees C to -45 degrees C (with automatic heat of fusion compensation), then a 10 degrees C per minute freezing rate to -90 degrees C. Frozen ampules should be stored in liquid nitrogen for long term storage or in a -135 degrees C Cryopreservation System. Note: Cryotubes should be labelled with cell line number anddate prior to beginning this procedure.

Time required:

2.5-3.0 hours to freeze 10 aliquots from each of 6 cell lines. Only 6 cell lines or 60 cryotubes should be frozen at one time. It is essential to keep the time the cells are exposed to the DMSO at a minimum. The freezing chamber can hold up to 120 tubes so two people can freeze samples at the same time to save liquid nitrogen.

Procedure:

1. Aspirate media from the T-75 flask down to the 50 ml mark.

2. Resuspend cells by shaking gently and transfer 40 ml of the cell suspension to a 50 ml centrifuge tube.

3. Add 10 ml of fresh media to the culture flask and reincubate at 37 degrees C. Keep the culture flask growing until a test thaw is done on one cryotube (done to determine if the cells were successfully frozen. Refer to reactivating cell line for DNA growth and extraction procedure. The cell line will begin growing within days if the freezing conditions were correct). Greater than 99% of the cell lines are successfully frozen using this procedure.

4. Remove 200 µl of the cell suspension from centrifuge tube for a cell count. (Refer to cell counting procedure.) Use the cell count to adjust the cell concentration to between 4 X 106 and 9 X 106 cells/ampule. Too high or too low a cell concentration decreases the viability of the cell line when the cryotube is thawed for growth.

5. Centrifuge the 50 ml tube for 10 minutes at 1200 rpm, no brake, room temperature, in the TJ-6 centrifuge.

6. Aspirate supernatant down to 1/4 inch above the cell pellet.
7. Place a control sample (freezing media in 1.0 ml cryotube) into the freezing chamber in a central location with the thermocouple probe placed equidistant from side to bottom. It will take approximately 6 minutes for the sample temperature to reach start temperature of 4 degrees C on the chart drive.

8. Resuspend cell pellet with 10 ml of freezing media. Pipette 1.0 ml into each of 10 cryotubes on ice. DMSO is toxic to cells, therefore begin freezing immediately after transferring the cells to cryotubes.

9. Load the cryotubes into the chamber when the sample temperature is +4 degrees C on the chart drive paper.
10. Again allow the chamber and cells to cool to the start temperature of + 4 degrees C.

11. Place the selector switch to the freeze ampule position. The controller will automatically cycle through the freezing program until the end temperature is reached. This takes approximately 55 minutes.

12. Remove samples after the recorder has reached -90 degrees C and transfer to a permanent storage container. Samples should be moved quickly to prevent thawing or warming and sample deterioration.

    Warning: Wear cryoprotective gloves when working with the freezing chamber and other permanent storage containers. Also, protective eyeglasses are necessary in case of explosion of a cryotube.

Solutions:

• Freezing media: (1 liter)

        Prepare a 1 liter volume and divide into 25-50 ml      centrifuge tubes containing 40 ml each.      Store the tubes at -80 degrees C for up to one year.  	700 ml RPMI-1640 with 2mM L-Glutamine 	200 ml fetal bovine serum (FBS) 	100 ml dimethyl sulfoxide (DMSO, Sigma) 	------------ 	1000 ml total volume  	   Note: Filter sterilize media and FBS with          a 0.22 µm cellulose acetate filter.          Do NOT filter DMSO, it will dissolve          the cellulose acetate membrane.  

References:

Cryomed Technical Manual for Model 700 Preprogrammed Freezing Controller, 1985.

Sigma catalog, (1988), "Commonly used tissue culture techniques" page 1435.