Welcome to Dr. Sompol and Dr. Heerklotz to the annual
SY-LAB Freezer Training 15M/L and 14S
Scope of Training (Freezer) 1.
Introduction Why do we need a programmable freezer?
2.
Introduction to Freezer 15M/L, 14S Major differences
3.
Writing a Program Test Program to determine Temp. of Crystallization Modifying the Test Program (optimizing)
4.
Training on the Freezer Software Program Log-In, Program Tools and Buttons, Menu Area
5.
Special Functions (Auto)-seeding, Trigger, Supply Pressure, Restart-Level
6.
Maintenance (hands on the equipment) Leak Check, Cleaning, Temperature Sensors, Ice Water Calibr.,
7.
Installation of Programs from CD to PC Settings on PC, License Agreement, Hardlock (Dongle), TouchWare, Calibr. of Touch Monitor, Image Loader, Inserting YOUR Logo
8.
Troubleshooting Taking the Freezers apart – Parts description, etc.
9.
Furniture
1. Why do we need a Programmable Freezer? • • • • • • • • •
Freezing processes involves complex phenomena Water is the major component of all living cells and must be available for the chemical processes of life to occur. Cellular metabolism stops when all water in the system is converted to ice. Ice forms at different rates during the cooling process. During slow cooling, freezing occurs external to the cell before intracellular ice begins to form. As ice forms, water is removed from the extracellular environment and an osmotic imbalance occurs across the cell membrane due to changes in the salt concentration and the dehydration. This leads to migration of water out of the cell. If too much water remains inside the cell, damage due to ice crystal formation and re-crystallization during warming can occur. It has been found that the rate of cooling has a dramatic effect on these phenomena.
Why do we need a Programmable Freezer? cont., •
•
•
Rapid cooling minimizes the solute concentration effects as ice forms uniformly, but leads to more intracellular ice. Slow cooling, on the other hand, results in a greater loss of water from the cell and less internal ice, but increases the solution effects. Cell permeability affects the rate of water loss; more permeable cells are able to tolerate rapid cooling better than less permeable cells.
•The ice crystal formation and solution effects both play a role in cell inactivation, and that an optimum cooling rate minimizes the effect of each. Experience has shown that a controlled cooling rate of 1-3°C per minute (with temp. compens. For the latent release of heat) will provide improved post – freezing viability.
Why do we need a Programmable Freezer? cont,.
•
Using cryoprotective additives or chemicals that protect the cells during freezing can also minimize the detrimental effects of increased solute concentration and ice crystal formation. The most commonly used cryoprotective agents are dimethylsulfoxide (DMSO) and various concentrations of glycerol. Additionally, maintaining frozen cells at the proper storage temperature and using an appropriate warming rate will minimize damage to frozen cells.
Please Note •
Different Organic Matter has different freezing behaviour
•
Have to develop specific freezing profile for each TYPE of sample material.
•
Do not change parameters for a specific “working” freezing profile (e.g. changing to different type of ampoule, bag, freezing protectant, supply vessel, etc.).
2. Introduction to Freezer 15M/L, 14S Major Differences 15M
14S
A
B
C
D
Major Differences cont. 15M
14S
Price
(check Price List)
(check Price List)
Size – Freezing Chamber (LxWxH in mm)
297 x 317 x 378
217 x 217 x 348
Size – Frezzer Dimension (LxWxH in mm)
845 x 540 x 785
540 x 420 x 520
Size – Required Area (LxWxH in mm) with open Lid and LN2 Vessel
1420 x 650 x 1100
850 x 550 x820 (without Vessel)
Weight
69 kg / 150 lbs
29 kg / 63 lbs.
Trigger
Yes
No
LN2 – Supply Pressure
Yes
No
Heating Rates
1000W
750W
Available Models
2 (M and L)
4 (Variant A to D)
Common Features •
Range: +40 bis -180° Celsius (+104 bis -292° Fahrenheit)
•
Manual and automatic control of heating and cooling
•
Pt-100 Temperature measurement with 2 (standard) and/or 3rd or 4th optional resistance-thermometers using 4 – wire technique, 1.5 mm, 65 mm standard length or 100 mm (optional)
•
Electrical power-turn-OFF to motor, fan, heater and magnetic valves when lid is opened
•
Build-in safety valve – opens at approx. 3,4 bar
•
Variable LN2 conn. from either side of the freezer with original tubing
•
Maximum working pressure: 1.5 bar (0.5 bar recommended)
•
Autom. / Manual Seeding Pre-Prepared for optional AutoSeeding installation
•
Stainless Steel Chamber
Common Features cont. •
Hinged transparent glass cover
•
Additional sample sensors possible
•
Thermo fuse over-heat protection
•
Unlimited number of programs
•
12.1“ TFT Touch Monitor (not on 14S/Variant A), program menu driven via Touch Monitor
•
Hold/Continue and Next Ramp function
•
Free adjustable start temperature
•
Temperature sensor calibration routine
•
Zoom function
•
Date- and Time integrated Filename Convention
•
Various User–Level Assignments
3. How do I find an appropriate freezing profile? How to Write a Program? •
Define in which containment your sample will be placed (ampoule, bag, straw, etc.)
•
How much of material will normally be handled at one time? A few samples or a large amount?
•
Which cryo-protectant will be used?
•
What is the initial temperature of the sample / protectant? Refrigerator or ambient temperature?
If all questions are answered, set up a test curve to determine the heat of crystallization.
One of such a test curve could look like this:
Test Program
Corresponding Test Program
Profile of Test Curve
Position of Sensors in e.g. Ampoule
Chamber Temperature Sensor Sample
Sensor tip should be placed at the CENTER of the sample (probe)
Test Program cont.
Sample temp. Heat Program and Chamber temp.
White line on right graph shows the start of the crystallization process on all three curves. The blue curve represents the sample temperature. The red/green curve the program/chamber temperature
Test Program cont. Full Curve
Heat
Test Curve Modification Need to remove the Heat of Crystallization 1. Try The compensation of “Heat� is accomplished by establishing a cold environment (chamber) around the sample. Just an instant before the sample temperature is rising, we have to lower the temperature in the chamber rapidly. Thereafter, the chamber must be brought back to meet the sample temperature curve again. This experiment will have to be repeated several times before an appropriate freezing curve is established.
Test Curve Modification cont. Need to remove the Heat of Crystallization 2. Try We have started the rapid drop of temperature somewhat earlier. Still, we can see that the chamber was not fully at its lowest programmed temperature at the time of crystallization (see white line). Also, there is still a lot of heat in the sample compartment (e.g. Ampoule).
So, let’s try another run.
Test Curve Modification cont. Need to remove the Heat of Crystallization 3. Try As we can see, the Heat of Crystallization is already somewhat compensated and is now much less than before. Nevertheless, there is still some “Heat” which we would like to remove by “Optimizing” the freezing curve.
Once more, let’s try another run.
Test Curve Modification cont. Need to remove the Heat of Crystallization 4. Try Looking at the complete curve, we now see that the “Heat of Crystallization� is vastly compensated by the low temperature in the chamber But anyhow, we can give it another try and see what happens.
Test Curve Modification cont. Need to remove the Heat of Crystallization 5. Try
Spreading out our test curve, we can see that on this one, there is only a minute increase of heat around -5째C which should not be harmful. The goal to compensate for the heat given up by the sample during the crystallization has been reached.
Please remember, if any parameters such as sample containment (e.g. Ampoule), cryo-protectant, supply vessel, etc. is altered, the freezing curve must be tested again and if necessary, be modified.
From now on, this curve can be used for cryopreservation of the sample-material in question.
Seeding Another Way of Freezing
Random Freezing Straw
E
E
Not Good
Uniform Freezing due to Forcing Crystallization E
Straw
LN2
E
Good
4. Training on the Freezer Software 1. Program Log-In
4. Training on the Freezer Software cont. At initial delivery, a specific Username and Password has the be entered in order to open the freezer program. Once the program is opened, individual users with selected user rights can be assigned. This is done in
Main Menu – Options – User Administration
4. Training on the Freezer Software cont. 2. User Administration
A minimum of 5 characters is needed for User Name and Password
4. Training on the Freezer Software cont. 3. Description of Program Tools and Buttons
Display Area
Operation Panel
Device Status Display
Instrument Panel
Sub-Menu Area Logo Area
4. Training on the Freezer Software cont. 4. Sub-Menu Area
4. Training on the Freezer Software cont.
This menu is only available to SYLAB Production Area
4. Training on the Freezer Software cont. Main Menu – File – Edit Description
Part of the Sample/Patient name
4. Training on the Freezer Software cont. Main Menu – File – Open Historical Data
Here, we will be able to view and edit finished freezing processes. Events can be looked at and additional Windows be created or removed.
Note: If no previous freezing run was selected, this page will initially be blank
Press Menu to continue
4. Training on the Freezer Software cont. Main Menu – File – Open Historical Data
4. Training on the Freezer Software cont. List of Events
4. Training on the Freezer Software cont. Diagram List
4. Training on the Freezer Software cont. Printout of historical file Once all graphs (enlargements) are selected, a hard copy can be produced. The user has the choice of printing the diagram, program & events or both of the above.
4. Training on the Freezer Software cont. Printout of historical file (cont.)
4. Training on the Freezer Software cont. Main Menu – File – Freezing Program
Create New
4. Training on the Freezer Software cont. Create New
4. Training on the Freezer Software cont. Create New (cont.)
4. Training on the Freezer Software cont. Main Menu – File – Set Data (Program) Directory
4. Training on the Freezer Software cont. Main Menu - Display
4. Training on the Freezer Software cont. Main Menu – Options – Device Settings
4. Training on the Freezer Software cont. Main Menu – Options – User Administr.
4. Training on the Freezer Software cont. Main Menu – Options – Language
4. Training on the Freezer Software cont. Main – Menu – Version Info
4. Training on the Freezer Software cont. Main Menu – Maintenance – Ice Water Calibration
5. Special Functions Autoseeding
5. Special Functions Trigger
5. Special Functions Restart