Proceedures For Freezing Human Cells In Research

1. Purpose- To describe the procedures utilized in freezing cells using the programmable Cryo-Med Freezing Chamber.

2. Responsibility- Laboratory technicians are responsible for conducting the freezing of cells and comply with this SOP.

3. Materials and Equipment-

3.1. Cryo-Med Model 2700 Freezing Chamber

3.2. Model 1010 Programmable Freezing Controller

3.3. Chart Recorder

3.4. Liquid Nitrogen Tank

3.5. Chart paper, Type 4003-6522 (Cryo-Med, 51529 Birch St., New Baltimore, Michigan 48047, ph: 313-725-4614)

4. Definitions/Abbreviations- N/A

5. Warning and Precautions-

5.1. Safety

5.1.1. Treat all specimens and waste as infectious. Use all safety precautions for handling infectious biological materials and waste as described in AFRIMS safety SOPs (CMD-LS-000, CMD-GS-002, CMD-LS-003, and CMD-LS-004).

5.1.2. Use all safety precautions when handling any of the chemicals used and chemical waste generated as described AFRIMS safety SOPs (CMD-GS-001, CMD-LS-001 and CMD-LS-002) as well as by the product inserts or MSDS provided by the chemical supplier.

6. Procedure-

6.1. Attach liquid nitrogen supply: Attach the freezing chamber hose to the "LIQUID" outlet on the liquid nitrogen tank. Turn the valve on the tank counter-clockwise to open it.

6.2. Zero the chart recorder

6.2.2. Chart recorder: Make sure that the chart recorder is loaded with chart paper. Each run takes approximately 4 feet of chart paper, provided that the chart drive is not turned on until cells are placed inside the freezing chamber.

6.2.3. Remove the recorder pen cap. Flip the red toggle switch on the chart recorder to the "ON" position. Do not turn the chart drive (silver switch) on; this wastes chart paper.

6.2.4. Freezing controller: Depress the power switch located on the rear panel of the controller in the upper left corner to turn on the power.

6.2.5. Calibrate the freezing controller and the chart recorder by first pressing the "CHAM" button in the TC SCANNER section. This should cause the recorder pen to move to "0oC" on the strip chart recorder. If the zero point is off, adjust it back to zero by turning the black control knob located in the lower left corner of the recorder.

6.2.6. Next press the "SAMP" button in the TC SCANNER. This should cause the recorder pen to move to -180oC on the chart recorder. If it does not, calibrate by using the set screw (located next to the black zero knob) and dialing it until the recorder pen reaches -180oC.

6.2.7. Finally, recheck the zero calibration as described in 6.2.4.

6.3. Cool freezing chamber

6.3.2. Open the freezing chamber and make sure that it contains a sample vial connected to the freezing probe. This vial should contain the same medium and volume as the cell vials that are to be frozen. The probe must reach into the liquid to a point equidistant from the sides and bottom of the sample vial. Place the sample vial near the center of the middle freezing tray.

6.3.3. Close the chamber door and depress the "SCAN" button in the TC SCANNER section.

6.3.4. Using the up and down arrow keys in the PROG ADV section, locate the program and section numbers which are to be run. Program number 1 is typically used for freezing down most cells.

6.3.5. Turn on the chart drive switch on the recorder.

6.3.6. Depress the "RUN" button in the MODE SELECT section. This allows the liquid nitrogen to cycle into the chamber. Once the chamber has reached the target temperature in the first section of the program, the valves will cycle on and off to maintain this temperature.

6.4. Freeze