Appendix: User's Equipment Guide to the Cryomed Preprogrammed FreezingController

November 13,1990

Rosalie Veile

Introduction:

The Model 700 Controller is a precision electronic device used to control the cooling rate and freezing rate of biological specimens. The controller connects with two other pieces of equipment:

a freezing chamber, containing a samplerack, a permanently mounted chamber thermocouple and a sample thermocouple, and a strip chart recorder, calibrated in degrees centrigrade to display the chamber and/or sample temperature.

The freeze ampule program requires approximately one hour to freeze 1.2-2.0 ml vials with 7.5-12.5% cryoprotectant. The Model 700 automatically controls the injection of liquid nitrogen into the freezing chamber to provide 1 degrees C/minute freezing rate from +4 degrees C to -45deg.C (with automatic heat of fusion compensation), then a 10 degrees C/minute freezing rate to -90deg.C.

Warnings:

Asphyxiation. Assure adequate ventilation to prevent liquid nitrogen build up.

Frost Bite. Liquid nitrogen is extremely cold. Cryoprotectant gloves should be worn to protect skin from exposure.

Explosion. Protective eyeglasses should be worn to protect eyes from the explosion of a cryotube. Extreme changes in temperature or faulty sealing can induce a cryotube to burst.

Time required:

approximately 1 hour

Operating Instructions:

The Model 700 controller regulates the flow of liquid nitrogen to the freezing chamber. Additional information on the sample and/or chamber temperature can be provided by the strip chart recorder. Refer to figure 3.

The cryomed freezing sequence consists of the following steps and instructions:

Initial preparation:

The initial temperature of the biological specimens prior to loading in the freezing chamber is +4 degrees C to +10deg.C. Typically, biological specimens are mixed with a solution of cryoprotectant agent (7.5-12.5%), and are stored in 1.2 or 2.0 ml cryotubes. The controller start temperature is at +4 degrees C. Before the freezing chamber is activated to cool, it is necessary to prepare the strip chart recorder as follows:

1. Place the power toggle switch in the ON position.
2. Lower the pen to the strip chart recorder paper.
3. Calibrate the recorder by switching CAL switch on the controller to 0 degrees C and adjust the recorder control knob to read 0 degrees C on the paper. Switch to -90 degrees C and verify the -90deg.C reading on paper.
4. Turn the end temperature alarm switch to the ON position..
5. Turn the TC scanner switch to the SCAN position. This will record both sample and chamber temperatures.

Freezing the samples:

1. Place a control sample (freezing media in 2.0 ml cryotube) into the freezing chamber with the thermocouple probe placed equidistant from side to bottom. Refer to figure 1.

2. Turn the control switch from the OFF position on the preprogrammed controller to START. Refer to figure 2. It will take approximately 6 minutes for the sample temperature to reach the start temperature.

3. After the chart drive recorder paper reads +4 degrees C, load biological samples into the chamber. When the chamber temperature is +4 degrees C again, turn the control switch from the START position to the FREEZE AMPULE position. More liquid nitrogen will begin pumping into the chamber and the liquid nitrogen tank will build up pressure. The liquid nitrogen supply tank pressure should be between 10-25 PSIG for the best performance between controller and freezing chamber. To maintain less pressure than 25 PSIG, it is necessary to vent the tank a little. Try to maintain a pressure of 20 PSIG.

   The controller will automatically cycle through the freezing program until the end temperature is reached, as described below. Unloading instructions follow.





As the samples are processed, the Controller takes them through a series of steps:
1. Liquid Phase Cooling: This is the rate at which the liquid phase is cooled. The rate of cooling is 1 degrees C/minute. A regulated flow of liquid nitrogen into the freezing chamber is circulated by an internal electric fan to assure even sample cooling.

2. Super Cooling: This is the temperature drop achieved just prior to the liquid-to-solid phase change. It is generally 3-4 degrees below the freezing point.

3. Phase Change: The beginning of the liquid-to-solid phase change process is characterized by a rapid increase in temperature from supercooled temperature to freezing point temperature. To minimize temperature rise during phase change, temperature within the freezing chamber is rapidly dropped to -40 degrees C. This procedure is automatic to the Model 700 Controller in the freeze sample mode. This rapid cooling absorbs latent heat of fusion.

4. Solid Phase Cooling: The Model 700 Controller will automatically switch to a solid phase cooling rate of 10 degrees C/minute when the freezing chamber reaches -45 degrees C in both the freeze ampule and freeze tray modes.

5. End Temperature: This is the final temperature is -90 degrees C, achieved in the solid phase cooling sequence.

When the end freeze temperature is reached, and alarm will sound and the end freeze LED will illuminate. The alarm condition will exist until the controller is manually switched from the FREEZE AMPULE position to the OFF position.

Silence the alarm by placing the alarm switch to the MUTE position.

Unloading instructions:

1. Lift the pen off the strip chart recorder and turn chart drive toggle switch to OFF.
2. Open the freezing chamber door, remove samples and transfer quickly to a permanent storage container. Failure to follow this procedure will result in unnecessary and potentially harmful warming, thawing and sample deterioration.
3. After samples have been removed, close the chamber door.
4. Turn the selector switch to the WARM position. This activates the heater circuit of the freezing chamber. The chamber will warm to the start temperature and will be ready for another run. Liquid nitrogen will cycle on and off to maintain the start temperature.
5. Shut the chamber down, unless setting up for another run: The toggle switch should be in OFF position on the recorder, and the pen should be recapped and removed from the paper. The selector switch should be in the OFF position on the controller. Close the vent on the liquid nitrogen container.

References:

Cryomed technical manual for Model 700 preprogrammed freezing controller, 1985.