1. ASKION GmbH
Gewerbepark Keplerstraße 17-19
D-07549 Gera
Maximum storage temperatures
Permanent cooling below the threshold of recrystallization
White Paper
created on 08 May 2012
White Paper page 1 of 7
42-03-R01

2. ASKION GmbH
Gewerbepark Keplerstraße 17-19
D-07549 Gera
Contents:
1. Background
2. Biological fundamentals
3. Reasons for temperature requirements – current state of the art
4. Implementation by the Askion C-line® hermetic storage
5. Summary
6. Sources
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3. ASKION GmbH
Gewerbepark Keplerstraße 17-19
D-07549 Gera
1. Background:
The method of cryo-preservation is used for the long-term storage of biological material
and for the possibility to maintain its viability over decades. Therefore, specific
cryoprotectants in the gas phase of liquid nitrogen are used to freeze and store the
samples in.
According to the SOPs of hospitals/customers, and to the flow charts created by the
Paul-Ehrlich Institute (PEI), a maximum storage temperature of -140°C is defined.
The Swiss Guidelines for cord blood banks (2) even require a storage temperature
-150°C. Askion GmbH has made it its business to develop a system that keeps the
required temperatures and, as a consequence, contributes significantly to the safety of
the biological samples.
2. Biological fundamentals:
When freezing biological sample, the following processes take place in the cell: At a
temperature of 10°C to 0°C, only a reduced cellular metabolism is maintained and the
signal transfer has ceased already. Extracellular ice formation and dehydration of the
cell occur at a temperature between –2°C and –15°C. At –15°C to –25°C, intracellular
ice formation and the segregation of the cytoplasm take place. The migratory growth of
ice crystals starts at –25°C. Finally from < –130°C on, the cell exists as a solid body
without considerable changes over a long period of time (9).
In order to keep living cells stable over several decades, a temperature below -130°C is
mandatory. Many cells stored at higher temperatures are no longer stable possibly due
to unfrozen parts of solution (4). They will die as fast as several percent per hour up to
several percent per year depending on the temperature, the nature and type of the cell,
and the composition of the medium they were frozen in (5). Below -130°C there are no
longer any unbound water molecules. The only existing physical states of water at these
temperatures are crystalline or glassy. At both states, the viscosity is so high
(>1013poise) that the diffusion over few geological periods is negligible (6,7).
Above -130°C, there are still measurable recrystallization processes.
In the phase diagram of water by Wowk et al. (8) it is very nicely shown at what
temperature the state of aggregation of water will change depending on the
concentration of the freezing media. Note that all crystallization processes have
completed at -130°C, regardless of the content of cryoprotectants.
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4. ASKION GmbH
Gewerbepark Keplerstraße 17-19
D-07549 Gera
Wowk et al. 2000
3. Reasons for the temperature requirements – current state of the art
The Paul-Ehrlich Institut obtains the requirements for the storage temperature from the
European Pharmacopoeia, which demands the storage at -140°C. In case the samples
are stored at higher temperatures, the functionality and the duration of the storage have
to validate (1).
In principle, storage in the gas phase of liquid nitrogen (according to PEI) is to be
guaranteed as well as temperatures of -140 to -150°C inside the storage tanks should be
maintained. The Swiss Guidelines for cord blood banks specify a storage temperature of
-150°C that must not be exceeded. The reason given is that, at -135°C, the sample
comes too close to the glass transition (2). The risk of the ambient temperature of the
samples exceeds these -130°C during the storage, especially in the event of further
storage and retrieval processes would be too big.
It has to be considered that for cryostorage in conventional tanks, the racks have to be
lifted from deep-cold temperatures into room temperature. During the storage and
retrieval process of single samples, all samples are exposed to a temperature leap of
about 170K (from -150°C in the tank up to the room temperature). Thereby, heat is
inserted into the system and the storage temperature increases for a short time. This
temperature increase depends on the duration and frequency of the storage and retrieval
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5. ASKION GmbH
Gewerbepark Keplerstraße 17-19
D-07549 Gera
processes. In particular, small sample volumes such as straws as well as such samples
positioned at the edge of the rack, are strongly affected.
4. Implementation by the Askion C-line® hermetic storage
Due to the special concept of the Askion C-line® hermetic storage with a lid precooled
to -100°C, the temperature increase during the storage and retrieval of single small
tubes is only max. 30K at the top position of the sample storage in the tank.
Figure 1 shows the Askion C-line® hermetic storage type 102. Note the lid and its
precooled working space in which the racks are lifted by means of a motor, and where
the samples are protected against the temperature increase caused by the room
temperature. Figure 2 describes the temperature curve in the storage tank during several
storage and retrieval processes in a row over the period of a working day in the Askion
C-line® hermetic storage.
Fig.1 Askion C-line® hermetic storage 102
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Gewerbepark Keplerstraße 17-19
D-07549 Gera
resting phase storage and retrieval processes
Fig.2 Temperature layering in the tank of the Askion C-line® hermetic storage in several rack levels during
1. resting phase and 2. storage and retrieval processes. In the storage tank, five different sensors (CH1-CH5)
were mounted 30mm apart, and the temperature curve was recorded. The storage temperature in the tank does
not exceed -140°C in the resting phase. During the storage and retrieval, the temperature increases for a short
time up to max. -128°C at the highest position in the storage tank (Referenz).
5. Summary:
According to the temperatures specified by the Paul-Ehrlich Institute for the permanent
storage of stem cell products in the gas phase of liquid nitrogen at maximum
temperatures of -140°C, Askion has set itself the task not to exceed the glass transition
temperature of water during the storage and retrieval processes. Conventional systems
have to compensate a temperature difference of about 170K for the period of the storage
and retrieval processes because the storage racks have to be brought to room
temperature for a short time. Askion, on the other hand, has developed a system in
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7. ASKION GmbH
Gewerbepark Keplerstraße 17-19
D-07549 Gera
which the racks are lifted into a cooled working space at temperatures of -100°C. A
maximum gradient of 30K is reached. Therefore in the storage tank, the samples
themselves no longer get to temperatures above the glass transition
6. Sources:
[1] European Pharmacopoeia 5.6 01/2007 Human haematopoietic stem cells
[2] Cord Blood Accreditation Manual
International Standards for Cord Blood Collection, Banking and Release for Administration
Accreditation Manual
[3] PEI
Fließschema der Herstellung von Stammzellzubereitungen aus Nabelschnurblut
110207-Fließschema-NSBSC.doc
(flow sheet of the production of stem cells out of cord blood)
Fließschema der Herstellung von Stammzellzubereitungen aus peripherem Blut
110207-Fließschema-PBSC-autolog+allogen.doc
(flow sheet of the production of stem cells out of peripheral blood)
Fließschema der Herstellung von Stammzellzubereitungen aus Knochenmark
110207-Fließschema-KMSC.doc
(flow sheet of the production of stem cells out of bone marrow)
[4] Mazur 1970, Cryobiology: the freezing of biological systems, Science
[5] Mazur 1984, Freezing of living cells: mechanisms and implications
[6] Mazur 1985, Basic concepts in freezing cells; aus Deep Freezing of Boar Semen, Uppsala, Sweden,
Proceedings First International Conference on Deep Freezing of Boar Seemen, 1985
[7] Özkavukcu et al. 2002, Cryopreservation: basic knowledge and biophysical effects
[8] Wowk et al. 2000, Vitrification Enhancement by Synthetic Ice Blocking Agents, Cryobiology 40,
228-236.
[9] IBMT Kryobankbroschüre, Februar 2005
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