6. END SOLID PHASE I FREEZING. The final temper-
ature achieved in Solid Phase I freezing cycle. It is typi-
cally set between -30°C and -50°C, as required by the
operator.
7. SOLID PHASE II FREEZING. The freezing rate is
independent of the Solid Phase I freezing rate. At -30°C
to -50°C, the sample is considered safe. This permits the
temperature to be dropped at a much faster rate, such as
10°C to 20°C per minute.
Note:
Researchers generally find that a Liquid Phase cooling
rate of 1°C/min, a solid Phase I freezing rate of 1°C/min, and a
Solid Phase II rate of 10°C/min will provide maximum recov-
ery of viable cells, while minimizing freezing cycle time.
8. END FREEZE TEMPERATURE. The final temperature
achieved at Solid Phase II freezing cycle. It is typically
set at -90°C, and maintains freezing chamber tempera-
ture until sample is ready to be transferred to long term
storage.
The following chart shows the relationship between the
freezing point temperature of biological samples and the per-
centage of cryoprotective agent added.
Chart 4-1
% Cryoprotective Agent
Freezing Point Temperature
(DMSO, Glycerol, Etc.)
0 0°C
5%
-2°C
7.5% -3°C
10%
-4°C
12.5% -5°C
15%
-6°C
20% -8°C
30% -12°C
4.4 Initiating a Freezing Run
After the freezing system has been connected as illustrated
in Figure 3-1, proceed as follows:
1. Turn on the controller.
Note:
Upon start-up, the controller will run through a complete
lamp test, and return to the “Idle Mode” (no lights are illumi-
nated in the “Mode Select” section of the front panel).
2. Press the power switch on the strip chart recorder, and
permit the system to warm-up for 20 minutes.
3. Uncap and lower the pen to the graph paper.
4. Calibrate the recorder as follows:
a. Press “CHAM” in the TC Scanner section of the
controller. The recorder pen should move to zero on
the strip chart recorder. If it does not, dial it to zero,
using the black knob on the recorder.
b. Press “SAMP” in the TC Scanner section of the
controller. The recorder pen should move to -180°C.
If it is out of calibration, adjust using the set screw
adjacent to the zero knob until the pen reaches
-180°C.
c. Recheck zero calibration as directed in step 4A.
5. To select program:
User Program - Press the program number, the decimal
point, (and section number, if required).
Factory-set Program - Press the program number (and
section number, if required).
Note
: Program and Section of desired program may be select-
ed by using the up/down arrow keys to locate the program
number and section number that you wish to run. (Up arrow
means increase in numbers; down arrow means decrease in
numbers.)
6. Depress “RUN” to start.
4.5 Factory Pre-set Programs
PROGRAM #1
Standard 1.2/2.0ml w/7% to 12% Cryoprotectant Agent
Section 1
Wait @ 4° Cool+
Section 2
1 degree/minute to -4° Sample
Section 3
25 degrees/minute to -40° Cool+
Section 4
15 degrees/minute to -12° Cool+
Section 5
1 degree/minute to -40° Cool+
Section 6
10 degrees/minute to -90° Cool+
Section 7
End
PROGRAM #2 Standard Microtest Tray Freeze
Section 1
Wait @ 4° Cool+
Section 2
1 degree/minute to -40° Sample
Section 3
10 degrees/minute to -90° Cool+
Section 4
End
PROGRAM #3 Standard Embryo Freeze (0.25 to 0.50ml
straw)
Section 1
Wait @ +20° Cool+
Section 2
1 degree/minute to -4°
Section 3
0.5 degree/minute to -7°
Section 4
5 minute hold time at -7°
Section 5
0.3 degrees/minute to -35°
Section 6
0.1 degrees/minute to -37°
Section 7
End
Models 1010 and 1011__________________________________________________________________________Operation
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