SY-LAB Freezer Training 15M/L and 14S

Welcome toDr. Sompol and Dr. Heerklotz

to the annual

Scope of Training (Freezer)

1. IntroductionWhy do we need a programmable freezer?

2. Introduction to Freezer 15M/L, 14SMajor differences

3. Writing a ProgramTest Program to determine Temp. of CrystallizationModifying the Test Program (optimizing)

4. Training on the Freezer SoftwareProgram 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 PCSettings on PC, License Agreement, Hardlock (Dongle), TouchWare, Calibr. of Touch Monitor, Image Loader, Inserting YOUR Logo

8. TroubleshootingTaking 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 minimizesthe solute concentrationeffects as ice formsuniformly, but leads tomore intracellular ice.

• Slow cooling, on the otherhand, results in a greaterloss of water from the celland less internal ice, butincreases the solutioneffects.

• Cell permeability affectsthe rate of water loss;more permeable cells areable to tolerate rapidcooling better than lesspermeable cells.

•The ice crystal formation andsolution effects both play a role in cellinactivation, and that an optimumcooling rate minimizes the effect ofeach. Experience has shown that acontrolled cooling rate of 1-3°C perminute (with temp. compens. For thelatent release of heat) will provideimproved 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 SeedingPre-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

Profile of Test Curve

Corresponding Test Program

Position of Sensors in e.g. Ampoule

Sensor tip should be placed at the

CENTER of the sample (probe)

Chamber

TemperatureSensor

Sample

Test Program cont.

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

HeatSample temp.

Program and

Chamber temp.

Test Program cont.Full Curve

Heat

Test Curve ModificationNeed 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 -5°C which should not be harmful.

The goal to compensate for the heat given up by the sample during the crystallization has been reached.

From now on, this curve can be used for cryo-preservation of the sample-material in question.

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.

SeedingAnother Way of Freezing

E EE

E E

LN2

Random Freezing

Uniform Freezing due to Forcing Crystallization

Straw

Straw Good

Not Good

4. Training on the Freezer Software1. 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.

A minimum of 5 characters is needed for

User Name and

Password

2. User Administration

4. Training on the Freezer Software cont.3. Description of Program Tools and Buttons

Display Area Device Status Display

LogoArea

Sub-MenuArea

Instrument Panel

Operation Panel

4. Training on the Freezer Software cont.

4. Sub-Menu Area

4. Training on the Freezer Software cont.

This menu is only available to SY-LAB 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 FunctionsRestart