U.S. patent application number 14/351136 was filed with the patent office on 2014-09-18 for device and method for pressurized cryopreservation of a biological sample.
This patent application is currently assigned to Fraunhofer-Gesellschaft zur Foerderung der angewandten Forschung e.V.. The applicant listed for this patent is Fraunhofer-Gesellschaft zur Foerderung der angewandten Forschung e.V., Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V.. Invention is credited to Philippe Bastiaens, Guenter R. Fuhr, Markus Grabenbauer, Jan Huebinger, Frank Stracke, Frank Wehner, Heiko Zimmermann.
Application Number | 20140260346 14/351136 |
Document ID | / |
Family ID | 47022625 |
Filed Date | 2014-09-18 |
United States Patent
Application |
20140260346 |
Kind Code |
A1 |
Fuhr; Guenter R. ; et
al. |
September 18, 2014 |
DEVICE AND METHOD FOR PRESSURIZED CRYOPRESERVATION OF A BIOLOGICAL
SAMPLE
Abstract
A cryopreservation device (100) which is arranged for
cryopreservation of a biological sample (1) comprises a pressure
vessel (10) with a vessel wall (11) and an internal space (12)
which is arranged to receive the biological sample (1), wherein the
pressure vessel (10) is equipped with an actuating device (20) for
cooling by lowering the temperature and raising the pressure in the
pressure vessel (10) and configured for the cryopreservation of the
biological sample (1). Said actuating device (20) is connected to
the vessel wall (11) and comprises at least one pressure setting
element (21-23, 31) and at least one of at least one cooling
element (34) and at least one heat conducting element (35-38),
wherein the actuating device (20) is configured for a
time-dependent and/or location-dependent setting of the temperature
and of the pressure in the pressure vessel (10). Methods are also
described for cryopreservation of a biological sample (1),
comprising biological cells (2) and a preservation medium (3), and
methods for heating the biological sample (1) to maintain
vitality.
Inventors: |
Fuhr; Guenter R.; (Berlin,
DE) ; Zimmermann; Heiko; (Frankfurt am Main, DE)
; Stracke; Frank; (Saarbruecken, DE) ;
Grabenbauer; Markus; (Dielheim, DE) ; Huebinger;
Jan; (Duesseldorf, DE) ; Bastiaens; Philippe;
(Wuppertal, DE) ; Wehner; Frank; (Dortmund,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Fraunhofer-Gesellschaft zur Foerderung der angewandten Forschung
e.V.
Max-Planck-Gesellschaft zur Foerderung der Wissenschaften
e.V. |
Muenchen
Muenchen |
|
DE
DE |
|
|
Assignee: |
Fraunhofer-Gesellschaft zur
Foerderung der angewandten Forschung e.V.
Muenchen
DE
Max-Planck-Gesellschaft zur Foerderung der Wissenschaften
e.V.
Muenchen
DE
|
Family ID: |
47022625 |
Appl. No.: |
14/351136 |
Filed: |
October 10, 2012 |
PCT Filed: |
October 10, 2012 |
PCT NO: |
PCT/EP2012/004248 |
371 Date: |
April 10, 2014 |
Current U.S.
Class: |
62/62 ;
62/440 |
Current CPC
Class: |
A01N 1/0284 20130101;
A01N 1/0268 20130101; F25D 31/00 20130101; A01N 1/0289
20130101 |
Class at
Publication: |
62/62 ;
62/440 |
International
Class: |
F25D 31/00 20060101
F25D031/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 10, 2011 |
DE |
102011115467.5 |
Claims
1. Cryopreservation device which is adapted for cryopreservation of
a biological sample, comprising: a pressure vessel with a vessel
wall and an internal space which is adapted to receive the
biological sample, wherein the pressure vessel is configured for
cooling with a lowering of a temperature and an increase of a
pressure in the pressure vessel and for the cryopreservation of the
biological sample, the pressure vessel is provided with an
actuating device which is connected with the vessel wall and
comprises at least one pressure-setting element and at least one of
at least one cooling element and at least one heat conducting
element, and the actuating device is configured for a time and/or
location dependent setting of the temperature and of the pressure
in the pressure vessel, wherein the pressure setting element
comprises at least one of a pressure screw in the vessel wall, an
expansion area which is adapted to receive a liquid or gaseous
expansion medium and communicates with the internal space, and a
pressure clamp which acts from outside on the vessel wall.
2. Cryopreservation device in accordance with claim 1 in which the
pressure setting element comprises a coiled section which acts on
one end of the pressure vessel.
3. Cryopreservation device in accordance with claim 2 in which the
expansion area comprises at least one hollow duct which protrudes
from the pressure vessel.
4. Cryopreservation device in accordance with claim 3 in which the
at least one hollow duct has at least one of branches and
protrusions to different directions from the pressure vessel.
5. Cryopreservation device in accordance with claim 1 in which the
cooling element comprises a cooling line which is arranged in the
pressure vessel.
6. Cryopreservation device in accordance with claim 1 in which the
heat conducting element comprises at least one of a profile on an
outer side of the pressure vessel, a profile on an inner side of
the pressure vessel and heat conducting bodies in the inside of the
pressure vessel.
7. Cryopreservation device in accordance with claim 1 in which the
vessel wall of the pressure vessel has a shape of a tube, a sphere
or a flat cylinder.
8. Cryopreservation device in accordance with claim 7 in which the
vessel wall of the pressure vessel has the shape of a bent
tube.
9. Cryopreservation device in accordance with claim 1 in which the
at least one of the following is provided in the internal space of
the pressure vessel: an inner vessel adapted to receive the
biological sample, a substrate which is adapted for adherent
receipt of biological cells in the biological sample, a
segmentation of the internal space into internal space sections, a
sensor device with a least one pressure sensor and a temperature
sensor, and a substance reservoir which is adapted to release a
substance into the internal space.
10. Cryopreservation device in accordance with claim 9 in which the
substance reservoir comprises hollow spheres made of a pressure
sensitive material which are arranged distributed throughout the
internal space.
11. Cryopreservation device in accordance with claim 1 in which the
vessel wall includes an optical unit which is adapted for a visual
observation of the internal space of the pressure vessel.
12. Method for the cryopreservation of a biological sample,
comprising biological cells and a cryopreservation medium, with the
steps: provision of the biological sample in a pressure vessel with
a vessel wall and an internal space, and cooling of the pressure
vessel in a cooling device with lowering of a temperature and
increasing of a pressure in the pressure vessel, wherein the
biological sample is transferred at least partly into a
cryopreserved state in a vitreous phase, the pressure vessel is
provided with an actuating device which comprises at least one
pressure setting element and at least one of at least one cooling
element and at least one heat conducting element, and a time and/or
location dependent setting of the temperature and of the pressure
in the pressure vessel using the actuating device, wherein the
pressure in the pressure vessel is adjusted using a pressure screw
in the vessel wall, an expansion area which is adapted to receive a
liquid or gaseous extension medium and communicates with the
internal space, and/or a pressure clamp which acts from outside on
the vessel wall.
13. Method in accordance with claim 12 in which the setting of the
temperature and of the pressure in the pressure vessel comprises
the following steps: increase in pressure in the pressure vessel
with the pressure setting element, and then lowering of the
temperature in the pressure vessel with the cooling device.
14. Method in accordance with claim 13 in which the pressure in the
pressure vessel is increased using a coiled section which acts on
one end of the pressure vessel.
15. Method in accordance with claim 12 in which the expansion area
comprises at least one hollow duct which protrudes from the
pressure vessel, and the pressure is increased in the pressure
vessel by first the at least one hollow duct being immersed into a
cooling bath of the cooling device followed by a remainder of the
pressure vessel.
16. Method in accordance with claim 12 in which the setting of the
temperature and of the pressure in the pressure vessel comprises
the following steps: lowering of the temperature in the pressure
vessel with the cooling device, and subsequently increasing of the
pressure in the pressure vessel with the pressure setting
element.
17. Method in accordance with claim 16 in which the
cryopreservation medium includes a stabiliser substance which is
suitable to stabilise the vitreous phase of a supercooled melt
preferably up to a transition to a liquid state, on increasing the
temperature of the biological sample.
18. Method in accordance with claim 17 in which the stabiliser
substance is at least one member selected from the group consisting
of: long-chain uncharged polymers with a molecular weight greater
than 500 g/mol, monosaccharides, ethylene glycol, di- and oligo
saccharides polysaccharides, starch derivatives, sugar alcohols;
water-soluble polymers colloids comprising nano particle
dispersions, dendrimers, polycations, and polyanions.
19. Method in accordance with claim 18 in which the stabiliser
substance is at least one member selected from the group consisting
of: saccharose epichlorhydrin copolymer, and silica gel coated with
polyvinyl pyrrolidone.
20. Method in accordance with claim 17, wherein the stabiliser
substance in the cryopreservation medium has a concentration which
is lower than 10%.
21. Method in accordance with claim 17, wherein the stabiliser
substance in the cryopreservation medium is arranged outside the
biological cells.
22. Method for the cryopreservation of a biological sample,
comprising biological cells and a cryopreservation medium,
comprising the steps: providing a cryopreservation device of claim
1, providing the biological sample in the pressure vessel of the
cryopreservation device, and cooling of the pressure vessel with
lowering of the temperature and increasing of the pressure in the
pressure vessel, wherein the biological sample is transferred at
least partly into a cryopreserved state in a vitreous phase,
wherein the pressure in the pressure vessel is adjusted using a
pressure screw in the vessel wall, an expansion area which is
adapted to receive a liquid or gaseous extension medium and
communicates with the internal space, and/or a pressure clamp which
acts from outside on the vessel wall.
23. Method in accordance with claim 12 with the following step:
storage of the biological sample whilst maintaining the increased
pressure.
24. Method for the heating of a biological sample such as to
maintain vitality, comprising biological cells and a
cryopreservation medium in a frozen state which is arranged in a
vitreous phase in a pressure vessel, wherein a temperature of the
pressure vessel and of the biological sample is increased and
simultaneously an increased pressure above atmospheric pressure is
maintained in the pressure vessel.
25. Method in accordance with claim 24 in which the increased
pressure above the atmospheric pressure is maintained in the
pressure vessel until the biological sample achieves a transition
from the frozen or vitrified state to a liquid state.
26. Method in accordance with claim 25 in which on transition from
the frozen or vitrified state to the liquid state the pressure in
the pressure vessel is reduced.
27. Method in accordance with claim 26 in which on transition from
the frozen or vitrified state to the liquid state the pressure in
the pressure vessel is instantaneously reduced.
28. Method in accordance with claim 26 in which the pressure in the
pressure vessel is reduced by a contraction of the biological
sample at the transition from the frozen or vitrified state to the
liquid state.
29. Method in accordance with claim 24 in which the increased
pressure is at least one of at least 100 MPa and maximum 300
MPa.
30. Method in accordance with claim 24 in which the
cryopreservation medium contains a stabiliser substance which is
suitable to stabilise the vitreous phase of the supercooled melt on
increasing the temperature of the biological sample, preferably up
to the transition.
31. Method of using a stabiliser substance for the cryopreservation
of biological samples, including the steps of increasing the
temperature of a biological sample, comprising biological cells and
a cryopreservation medium, and maintaining a vitreous phase of the
biological sample by an effect of the stabiliser substance up to a
transition to a liquid state.
Description
[0001] The invention relates to a device for cryopreservation of a
biological sample, comprising biological cells and an aqueous
preservation medium, with a pressure vessel. The invention
furthermore relates to a method for cryo-preservation of the
biological sample and the use of a stabiliser substance with which
a vitreous phase of water can be stabilised in the cryopreservation
of biological samples. Furthermore, the invention refers to a
method to heat the cryopreserved biological sample.
[0002] The low temperature preservation (cryopreservation) of cells
is so far the only possibility of suspending vital processes
reversibly at a cellular level (to maintain vitality) such that
they revive after heating to physiological temperatures.
Cryopreservation has no correspondence in nature. Whilst organisms,
such as fish, are found embedded in ice in polar regions which
maintain their vitality for a limited duration, these organisms are
not completely frozen through but contain glycerine and other
substances in their cells which serve to lower the freezing point,
or they express proteins (so-called anti-freezing proteins) which
influence the ice structure. Permanent preservation is not possible
in this state because even at temperatures below 0.degree. C.,
diffusion processes over weeks and months lead to the
disintegration of the biological system. Permanent preservation
would moreover require cooling, e.g. to a temperature of liquid
nitrogen, at which the fluid in the cells would also freeze. In
this case, however, the organisms can no longer be reanimated.
[0003] A special problem of cryopreservation is the formation of
crystal ice (crystalline phase of water) inside and outside the
cells which leads directly or indirectly to irreversible damage.
Since the formation of ice is one of the primary reasons for cell
damage, so-called cryoprotectants (anti-freeze additives,
cryoadditives) have been sought for decades and added to the
obligate physiological media.
[0004] Cryoprotectants typically comprise small molecules such as
dimethyl sulfoxide (DMSO) which penetrate into the cells, or higher
molecular substances such as sugar which remain in the medium
outside the cells or on their surface. Cryoprotectants are
frequently only effective in high, largely non-physiological
concentrations (10% to 50%). Therefore they can only be added at
lower than physiological temperatures (around 4.degree. C.), must
quickly penetrate into the cells and be washed out immediately
after thawing.
[0005] Former developments in the cryopreservation of biological
samples have been aimed at replacing cryoprotectants by
physiologically and osmotically less problematical substances,
reducing their concentration or foregoing them completely. With the
exception of anti-freeze proteins of the organisms themselves, only
few substance groups have been found since the discovery of the
anti-freeze properties of DMSO and glycerine as well as a few
sugars by Polge and Lovelock [1, 2, 3]. It has so far been assumed
that the cryopreservation of cells is not possible without cell
stress and cell repair processes with gene expressions etc. after
thawing because the formation of the crystalline phase under
physiological conditions cannot quite be avoided. [0006] [1] Polge
C, Smith A U, Parkes A S (1949), Nature 164: 666 [0007] [2]
Lovelock J E (1954), Biochemical Journal 56(2):265-270 [0008] [3]
Lovelock J E, Bishop M W H, 1959, Nature 183: 1394-1395
[0009] The success of cryopreservation can be characterised by a
vitality rate (survival rate), e.g. by the quotients of the number
of living cells after and before cryopreservation. It is known that
the vitality rate depends on the type of cell, the volume and other
different boundary conditions which have usually already been
empirically optimised. In view of the necessity of a fast diffusion
of the cryoprotectants into the cells and the regulated outward
dissipation of heat, conventional cryopreservation methods have so
far failed in microscopic objects such as tissues, organs and
entire organisms without exception. Rather, conventional
cryopreservation with the addition of cryoprotectants leads to
practicable vitality rates only in suspended cells and very small
tissue pieces (<0.5 mm.sup.3). It has not so far been possible
to subject highly aqueous plant cells as well as a large number of
animal cells, such as the oocytes of cats and other species, to
cryopreservation at all. On the other hand, vitality rates in
excess of 90% have been achieved with cryopreserved cancer
cells.
[0010] The success of cryopreservation depends, in particular, on
the physical-biological boundary conditions, e.g. on the properties
of the water and those of the cryoprotectants. It has so far been
assumed that cryoprotectants must penetrate into all cells by
diffusion and that the heat must dissipate outwards in a short
period (ms to min) because cooling can only take place from here.
It is furthermore assumed that these conditions are satisfied only
in very small objects (cell suspended in a nutrient solution with
the addition of anti-freeze agents) due to the heat conductivity of
the water which also dominates in the cells and due to the low
diffusion speed of cryoprotectants through the cell membranes.
[0011] To avoid the creation of the crystalline phase, it has been
attempted in practice to transfer biological samples to a vitreous
or amorphous phase (vitrified state) through fast cooling. However,
success has been limited even when using cryoprotectants. Up to a
publication by J. L. M. Leunissen et al. [4] the general view was
therefore that a vitrification of cellular cell suspensions could
not be achieved without additives for physical reasons. [0012] [4]
J. L. M. Leunissen and H. Yi (2009) Journal of Microscopy, 235:
25-35
[0013] A method for cryomicroscopy is described by J. L. M.
Leunissen et al. [4] which would lead vitrification to be expected:
if a thin-walled copper tube with a diameter of <1 mm is filled
with a cell suspension, closed gas-free at the ends by pressing
them together and then frozen in a cooling fluid such as propane,
nitrogen etc., very well maintained structures are found in deep
temperature cryosections of the tubes which demonstrate
vitrification. However, once the deep temperature cryosamples are
heated, it becomes apparent that the cells did not survive the
cooling process. The method described by J. L. M. Leunissen et al.
[4] is not suitable for a heating of the sample such as to maintain
vitality. It has therefore been assumed up to now that methods for
cryomicroscopy do not permit revitalisation through reheating.
[0014] In view of the developing regenerative medicine and
biotechnology and of environment protection and species
preservation, there is an urgent interest in reducing or overcoming
the disadvantages of conventional cryopreservation.
[0015] The objective of the invention is to provide an improved
cryopreservation device using which the disadvantages of
conventional techniques are overcome and which, in particular,
permits a cryopreservation with a higher vitality rate and/or
enlarged sample volumes. It is furthermore the objective of the
invention to provide an improved method of cryopreservation in
which the disadvantages of conventional techniques are overcome and
which, in particular, permits the method conditions on cooling
and/or thawing to be adjusted such that an improved vitality rate
is achieved. Furthermore, it is an objective of the invention to
provide an improved method to heat a cryopreserved biological
sample with which the disadvantages of conventional techniques are
overcome and which, in particular, permits any vitality-restricting
influence of biological samples in the transition to a thawed state
to be suppressed.
[0016] These objectives are solved by devices and methods with the
features of the independent claims. Advantageous embodiments and
applications of the invention are provided in the dependent
claims.
[0017] In accordance with a first aspect of the invention, a
cryopreservation device is provided which is adapted for the
cryopreservation of a biological sample. The cryopreservation
device comprises a pressure vessel with a vessel wall and an
internal space which is arranged to receive the biological sample.
The pressure vessel is configured to be cooled in a cooling bath of
a cooling device up to a cryopreservation temperature, e.g. below
-138.degree. C. The pressure vessel is furthermore configured such
that pressure can be applied to the internal space of the cooling
vessel which is higher than the ambient atmospheric pressure. The
pressure vessel is adapted for the permanent cryopreservation of
the biological sample and storage at the cryopreservation
temperature whilst maintaining the higher pressure in the pressure
vessel.
[0018] In accordance with the invention, the pressure vessel has an
actuating device which is suitable for a time and/or
location-dependent setting of the temperature and of the pressure
in the pressure vessel. The actuating device is connected to the
vessel wall of the pressure vessel and is able to influence the
setting of the cryopreservation temperature and of the pressure in
the internal space of the pressure vessel depending on time and
location. The actuating device is suitable to selectively control
the cooling and/or heating of the biological sample in accordance
with a predetermined temperature-time function. Furthermore, the
actuating device is suitable to influence the temperature
distribution in the pressure vessel. Furthermore, the actuating
device is suitable to control the pressure in the pressure vessel
in accordance with the predetermined pressure-time function. The
temperature and pressure-time functions can be set relatively to
each other. For example, the actuating device enables the
biological sample to firstly be cooled and then a higher pressure
applied to it, the cooling and increase in pressure to be conducted
simultaneously or the pressure to firstly be increased and then the
cryopreservation temperature set. Finally, the actuating device is
suitable to control the place of pressure generation in the
pressure vessel.
[0019] In accordance with the invention, the actuating device
comprises at least one pressure setting element, at least one
cooling element and/or at least one heat conducting element. The
pressure setting element is configured such as to set the
pressure-time function, a location of a primary pressure input
and/or the amount of the pressure which is applied to the internal
space of the pressure vessel. Accordingly, the cooling element
and/or the heat conducting element, possibly in connection with the
effect of the cooling device, are configured so as to control the
temperature-time function, the spatial temperature distribution and
the cryopreservation temperature.
[0020] The biological sample contains biological cells and an
aqueous preservation medium. The biological cells comprise
individual cells, such as individual stem cells, precursor cells,
fibroblasts, gametes, groups of cells, in particular from the named
cell types, pieces of tissue or organs or their parts. The
biological cells can even form complete organisms, in particular
small organisms, such as worms or insects. The preservation medium
comprises an aqueous physiological medium (culture medium,
cultivation medium) as known in the cultivation of biological
cells.
[0021] In accordance with a second aspect of the invention, a
method for the cryopreservation of the biological sample is
provided in which the biological sample is arranged in a pressure
vessel and the pressure vessel is cooled down in a cooling device
until the biological sample in the cryopreserved state has been
transferred to an at least partially vitreous phase. In accordance
with the invention, a time and/or location dependent setting of the
temperature and of the pressure in the pressure vessel takes place
using an actuating device with at least one pressure setting
element, at least one cooling element and/or at least one heat
conducting element. The cryopreservation device in accordance with
the first aspect of the invention is used by preference to conduct
the inventive method of cryopreservation of biological samples.
[0022] In accordance with a third aspect of the invention, a method
is provided to heat a biological sample, comprising biological
cells and a preservation medium in a frozen state which is located
in a vitreous phase in a pressure vessel. According to the
invention, the temperature of the pressure vessel and of the
biological sample is increased and during the temperature increase
in the pressure vessel a higher pressure is maintained above the
ambient atmospheric pressure. The method is preferably executed to
heat the biological sample using the cryopreservation device in
accordance with the first aspect of the invention.
[0023] In accordance with a fourth aspect of the invention, it is
proposed to use at least one stabiliser substance (i.e. an
individual substance or a mixture of several substances) in the
cryopreservation of biological samples as a component of the
preservation medium which is suitable to maintain a vitreous state
of the supercooled melt without crystallisation preferably up to
the transition to the liquid state whilst increasing the
temperature of the biological sample comprising biological cells
and a preservation medium.
[0024] The invention is based on the recognition that on freezing
aqueous systems the formation of the crystalline phase is avoided
and instead the vitreous phase (amorphous phase) can be generated
by applying pressure to the aqueous system. The transition of the
biological sample to the cryopreserved state, the cryopreservation
and/or the heating of the cryopreserved samples are conducted in an
area of the phase diagram of the aqueous system in which preferably
the vitreous phase is formed. Since the survival rate of the
biological cells in the biological sample in the vitreous phase is
considerably higher than in the crystalline phase, the invention
facilitates a higher vitality rate of the cryo-preservation.
[0025] In conventional cryomicroscopy using pressure-tight sample
tubes (see above, [4]) an increased pressure was already generated
in the closed sample tubes. However, with the conventional
technique there is no degree of freedom to influence the pressure
or temperature characteristic during freezing, during
cryopreservation or during selective heating in accordance with the
set time function. The inventors have determined that ice avoided
during rapid cooling is inevitably created--particularly during
thawing--with all its negative effects on the sample, thereby
destroying the cells. The inventive provision of the actuating
device advantageously permits the pressure-temperature processes to
be controlled in terms of time and/or space such that the vitreous
phase of the biological sample is primarily generated and
maintained in the internal space of the pressure vessel. Contrary
to the conventional technique, the actuating device permits the
pressure and temperature parameters to be varied selectively during
freezing and/or thawing in order to achieve a maximum vitality
rate. This advantageously provides a real cryopreservation with the
possibility of rethawing and revitalisation of the cells in the
biological sample whilst the technique of J. M. L. Leunissen et al.
[4] merely represents cryopreparation for microscopic
examinations.
[0026] In accordance with the above-mentioned third aspect of the
invention, it is suggested in particular to maintain the vitreous
state of the sample, in particular of the preservation medium,
maintaining pressure until the water contained in the sample melts.
This is advantageously facilitated by thawing cryopreserved
biological samples, as those described by J. M. L. Leunissen et al.
[4], with an extremely good maintenance of structure and vitrified
cell suspensions such that living cells exist. This has not so far
been successful in any freeze-pressure shock method as used for
cryomicroscopy. In order to achieve the above-mentioned
stabilisation, preferably one stabiliser substance is added to the
preservation medium which is suitable to stabilise the vitreous
phase of the supercooled melt on increasing the temperature of the
biological sample preferably up to the melt. The inventive
cryopreservation device advantageously facilitates the influencing
of the path of the sample conditions through the phase diagram past
a solid/fluid phase transition.
[0027] In accordance with a preferred embodiment of the invention,
the actuating device comprises at least one pressure setting
element which is connected to the vessel wall and/or the internal
space of the pressure vessel. Advantageously, different variants of
the pressure setting element are possible which, depending on the
preservation task and the spatial conditions of the
cryopreservation, can be selected individually or in combination.
In accordance with a first variant, at least one pressure screw can
be provided in the vessel wall of the pressure vessel. The vessel
wall contains a threaded opening for the fluid-tight reception of
the pressure screw. Screwing the pressure screw into the threaded
opening serves to reduce the free volume of the internal space in
the pressure vessel and to permit the pressure in the pressure
vessel to be increased accordingly. According to a second variant,
an expansion area can be provided which is connected with the
internal space of the pressure vessel and is adapted to receive a
fluid or gaseous expansion medium. When the expansion medium
expands in the expansion area the free volume in the internal space
is reduced accordingly and the pressure in the pressure vessel
increased. In accordance with a third variant, the pressure setting
element can comprise a pressure clamp which acts on the vessel wall
from outside. In this case, the vessel wall is formed of a flexible
material in order to transfer the mechanical pressure exercised by
the pressure clamp to the internal space of the pressure vessel.
Advantageously, the pressure clamp can be adapted with cooling
openings in order to accelerate the cooling of the pressure vessel
in the cooling bath of the cooling device. Furthermore, the
pressure clamp can be designed for a mechanical or electrical
actuation.
[0028] According to a specially preferred embodiment of the
invention, the expansion area comprises at least one hollow duct to
accommodate the expansion medium which communicates with the
internal space of the pressure vessel and which protrudes from the
pressure vessel. The duct contains water, for example, an aqueous
solution or parts of the biological sample. The protrusion of the
duct from the pressure vessel causes the expansion medium in the
duct to be spatially separated from the internal space of the
pressure vessel. This advantageously facilitates a cooling of the
duct in the cooling bath of the cooling device whilst the remaining
pressure vessel is still at a higher temperature, e.g. at room
temperature. Crystalline ice can be generated in the duct which
extends through to the internal space, decreasing the remaining
volume and therefore causing an increase in the pressure in the
internal space. In accordance with a preferred variant, the duct
has branches. Advantageously, this facilitates an enlargement of
the volume of the expansion area compared with an individual duct
without branches. At the same time the branched duct permits the
time function of the pressure generation in the pressure vessel to
be influenced. Alternatively and additionally, several ducts can be
provided which protrude from the pressure vessel in different
directions. Advantageously, this permits the pressure generation to
be influenced, depending on the alignment of the pressure vessel
relative to the cooling bath of the cooling device.
[0029] If the actuating device provided by the invention comprises
a cooling element, this is preferably formed by a cooling line
which is arranged in the pressure vessel. The cooling line runs
through the internal space of the vessel. It is designed to have a
cooling agent, such as liquid nitrogen, run through it.
Advantageously, the cooling line facilitates a setting of the start
and of the time function of the temperature reduction in the
pressure vessel.
[0030] If the actuating device provided by the invention comprises
a heat conducting element, this is preferably formed by an outside
profile on the outer side of the pressure vessel, an inside profile
on the inner side of the pressure vessel and/or heat conducting
bodies in the inside of the pressure vessel. These variants of heat
conducting elements permit the heat transport from the pressure
vessel to the cooling bath to be accelerated during the cooling of
the pressure vessel.
[0031] A further advantage of the inventive cryopreservation device
is that the pressure vessel can be manufactured in a large number
of geometric shapes. Variants of the invention are given particular
preference in which the vessel wall of the pressure vessel has the
shape of a tube, a sphere or a cylinder, e.g. of a flat cylinder
(box). A tubular pressure vessel can be straight or curved. In both
cases the spatial distribution of the temperature and pressure
setting in the pressure vessel can be influenced by its shape.
[0032] Further advantageous features of the inventive
cryopreservation device refer to the shape of the internal space of
the pressure vessel. According to a variant of the invention, an
inner vessel may be provided in the internal space which is
arranged to receive the biological sample. In this case, the
biological sample in the inner vessel is separated in terms of the
substance from the remaining volume of the internal space. This
facilitates the influencing of the vitreous phase in the direct
vicinity of the biological sample. In accordance with a further
variant, a substrate can be arranged in the internal space which is
suitable to adherent receive biological cells which are part of the
biological sample. The substrate can, for example, be arranged in
the inner vessel. Substrate materials are suitable as substrates
which are used for adherent cell cultures such as plastic or glass.
In accordance with a further variant, a segmentation of the
internal space can be provided in internal space sections.
Advantageously, the segmentation facilitates the selective setting
of different pressure-temperature conditions in each of the
internal space sections. In accordance with a further variant, a
sensor device can be arranged in the internal space which comprises
at least one temperature sensor and/or at least one pressure
sensor. Advantageously, the sensor device facilitates a measurement
of the temperature and/or of the pressure in the internal space.
The cryopreservation can be controlled depending on the at least
one signal of the sensor device. Finally, in a further variant of
the invention a substance reservoir can be arranged in the internal
space which is suitable to release a substance into the internal
space. The substance reservoir comprises, for example, hollow
spheres which may be destroyed under the effect of a higher
pressure in the internal space in order to release a substance. The
variants specified for the internal space design can be provided
individually or in combination.
[0033] In accordance with a further advantageous embodiment of the
invention, the vessel wall can be provided with an optical unit.
The optical unit comprises an imaging optic which is set up for a
visual observation of the internal space of the pressure vessel.
The optical unit facilitates a visual monitoring of the state of
the biological sample during cryopreservation.
[0034] Advantageously, different variants of the pressure and
temperature setting exist in the pressure vessel in order to
achieve the required cryopreservation conditions in different ways
in the pressure-temperature phase diagram of the biological sample.
For example, in accordance with the first variant it is possible to
first increase the pressure in the pressure vessel with the at
least one pressure setting element and then to reduce the pressure
in the pressure vessel with the cooling device. The pressure
increase and the temperature reduction are conducted in accordance
with predetermined separate time functions. In accordance with the
second variant, the increase in the temperature and the reduction
in temperature can be set such that the respective time functions
overlap by starting the lowering of the temperature before
achieving the final pressure or vice versa by starting the increase
in pressure before achieving the cryopreservation temperature.
Finally, in accordance with a further variant it is possible to
firstly reduce the temperature in the pressure vessel up to the
cryopreservation temperature and then to increase the pressure in
the pressure vessel. Which of the variants stated is selected will
depend on the conditions of the specific cryopreservation task, in
particular on the design of the cryopreservation device and the
composition of the biological sample. The ideal variant can be
selected empirically by tests in which the vitality rate of the
biological sample is tested for the different variants under
specific conditions of application. It is possible in the same way
to select the pressure and temperature time function, particularly
with respect to the speed of pressure increase and temperature
reduction and/or the shape of the function, such as a stepped
shape.
[0035] The pressure-temperature setting is simplified if the
pressure setting element in accordance with a preferred embodiment
of the invention comprises an expansion area in the form of a
hollow duct protruding from the pressure vessel. The pressure can
be increased in the pressure vessel by immersing the at least one
duct in the cooling bath of the cooling device. An expansion medium
in the at least one duct expands so that the pressure in the
remaining pressure vessel increases. Finally, the remaining
pressure vessel is immersed in the cooling bath of the cooling
device in order to set the cryopreservation temperature for the
biological sample in the internal space of the pressure vessel.
[0036] Preferably, a permanent storage of the biological sample is
provided whilst maintaining the increased pressure, in particular
in liquid nitrogen or in the vapour of the liquid nitrogen. The
sample is stored by special preference in the pressure vessel.
[0037] In accordance with a specially preferred embodiment of the
cryopreservation method, the preservation medium contains at least
one stabiliser substance which is suitable to stabilise (maintain)
the vitreous phase of the supercooled melt preferably up to the
transition to the fluid state whilst increasing the temperature of
the biological sample.
[0038] Advantageously, the stabiliser substance brings about a
situation in which the vitreous phase remains up to higher
temperatures than would be the case without the stabiliser
substance. The vitreous phase is maintained for longer during
thawing and the formation of the crystalline phase is avoided.
Furthermore, the glass transition temperature of the preservation
medium is increased by the stabiliser substance. The stabiliser
substance produces a lower number of nucleation sources in the
preservation medium so that the nucleation probability is reduced
and the formation of ice during thawing minimised.
[0039] In accordance with this embodiment, the above objectives are
solved by at least one stabiliser substance being added to the
biological sample, e.g. a cell suspension. The at least one
stabiliser substance is used as was used only under certain
conditions or not at all for the conventional cryopreservation or,
in accordance with the invention, develops its effect at far lower
concentrations and this is a different effect than that of the
known cryoprotectants. Substances are preferably selected as
stabiliser substance which do not penetrate the cells at normal
pressure and which therefore are not normally used in conventional
cryopreservation of cells and tissues. The noteworthy aspect of
using the stabiliser substance is that experiments of the inventors
have shown that with an addition of Percoll or Ficoll, for example,
in the range of a few percent mammalian cells survived the pressure
shock freezing and thawing procedure analogue to publication [4]
with a high survival rate (>97%). This is all the more
surprising in view of the fact that a penetration into the cells is
not to be assumed.
[0040] The surprising effect of the stabiliser substance is that
for the first time during the thawing of the sample it permits the
return path via the combination of high pressurefast deep cooling
and pressure-controlled heating virtually without influencing the
vitality of the cells. It is therefore the combination of at least
one substance dissolved in the preservation medium which permits
the return from the vitrified phase by influencing the water
structure and the existence of nucleation sources.
[0041] The stabiliser substance differs in terms of substance, in
terms of its effect and/or with relation to the preferred selected
concentration of conventional cryopreservation. Unlike the
stabiliser substance, conventional cryoprotectants selectively
increase the number of nucleation sources in order to promote the
formation of a large as possible number of ice crystals during
freezing. However, as the number of ice crystals increase, their
chance of growing in size reduces so that large crystals are
prevented by conventional cryoprotectants. A distribution and high
motility in the preservation medium through to the cells is
required in conventional cryoprotectants.
[0042] The stabiliser substance is preferably selected from at
least one of the substance groups which comprise long-chain
uncharged polymers with a molecular weight greater than 500 g/mol,
in particular greater than 1000 g/mol, monosaccharides, di- and
oligosaccharides, polysaccharides, starch derivatives such as
starch hydrolysis products, sugar alcohols, water-soluble polymers,
colloids (nanoparticle dispersions), in particular with silver,
gold, diamond and/or nanotube particles, dendrimers, polycations
and polyanions. Long-chain polysaccharides proved to be
particularly suitable, in particular hydrophilic copolymerisates
made from saccharose and epichlorohydrin (Ficoll, reg. name),
and/or polyvinylpyrrolidone, in particular silica gel coated with
polyvinylpyrrolidone (Percoll, reg. name) with a molecular mass of
between 2,000 and several million g/mol. Nanoparticle dispersions,
in particular silver, gold, diamond and/or nanotube particles are
particularly advantageous because they are suitable to reduce the
heat conductivity of the preservation medium. Advantageously, the
stabiliser substance is biocompatible so that the cells in the
biological sample are not unfavourably influenced by the stabiliser
substance.
[0043] The concentration (%=vol. %) of the stabiliser substance is
preferably smaller than 30%, in particular preferably less than
20%, in particular smaller than 10%, such as for example smaller
than 3% or smaller than 1%. A preferred minimum concentration is
0.1%.
[0044] In accordance with a further preferred embodiment of the
invention, the stabiliser substance in the preservation medium is
positioned outside the biological cells. The cells remain free from
the stabiliser substance which has advantages for the vitality
after thawing of the sample.
[0045] A further advantage of the stabiliser substance can be that
it alters its properties, in particular structure and/or molecular
weight, under the impact of the increased pressure and the reduced
temperature during cryopreservation. For example, molecules of the
stabiliser substance can be fragmented so that they can diffuse
into the inside of the cells in order to achieve an additional
cryoprotective effect here.
[0046] It is emphasised that with the addition of the stabiliser
substance to the preservation medium it is not necessary for an
increased pressure to be maintained in the pressure vessel until
the biological sample has achieved the transition from the
supercooled melt to the liquid state. In this case, the pressure
can drop even before achieving the transition, in particular down
to atmospheric pressure.
[0047] According to a preferred variant of the above-mentioned
third aspect of the invention, a method is thereby provided to heat
a biological sample, comprising biological cells and a preservation
medium in a vitrified state, which with a vitreous phase is located
in a pressure vessel, wherein the temperature of the pressure
vessel and of the biological sample is increased until the
biological sample reaches the liquid state and wherein the
preservation medium contains the at least one stabiliser substance
which is suitable to stabilise the vitreous state on increasing the
temperature of the biological probe preferably up to the transition
to the liquid state.
[0048] In accordance with a preferred embodiment of the invention,
the pressure in the pressure vessel is reduced on reaching the
transition from vitreous state of the supercooled melt through to
liquid state. Damage to the biological sample after reaching the
liquid state is minimised here. Special preference is given to the
reduction of the pressure in the pressure vessel instantaneously,
i.e. in particular in steps and with negligible delay.
[0049] The pressure reduction can be achieved by releasing the
pressure vessel, e.g. opening the pressure vessel such that a
pressure balance with the outer atmospheric pressure is achieved.
Advantageously, a release of the pressure vessel is not absolutely
necessary, however. Rather, the pressure reduction can also be
achieved by a contraction of the biological sample at the
transition from the supercooled melt to the liquid state. The
volume of the sample can reduce in accordance with the processes
explained with reference to the FIGS. 1 to 4 so that the pressure
in the pressure vessel drops.
[0050] In accordance with a further preferred embodiment of the
invention, the increased pressure above atmospheric pressure is at
least 100 MPa, in particular at least 150 MPa and/or at the most
300 MPa, in particular 250 MPa at the most. These pressure areas
have proven to be particularly advantageous for a fast transition
from the vitreous to the liquid state and vice versa.
[0051] Further details and advantages of the invention are
described in the following, making reference to the attached
drawings, which show in:
[0052] FIGS. 1 to 4: phase diagrams with illustrations of different
phases of water; and
[0053] FIG. 5: embodiments of the inventive cryopreservation
device;
[0054] FIG. 6: graphic representations of different variants of the
pressure and temperature-time functions;
[0055] FIGS. 7 to 11: experimental results which illustrate the
effect of a cryoprotectant or of a stabiliser substance;
[0056] FIG. 12: diagrammatic sections of different variants of a
pressure vessel which is provided with heat conducting and/or
cooling elements;
[0057] FIGS. 13 to 19: further embodiments of inventive
cryopreservation devices and their use;
[0058] FIGS. 20 to 28: further embodiments of inventive
cryopreservation devices in which the pressure vessel has the shape
of a flat cylinder;
[0059] FIG. 29: a diagrammatic illustration of an optical unit in
the vessel wall of a pressure vessel;
[0060] FIG. 30: a further embodiment of the inventive
cryopreservation device and its use;
[0061] FIGS. 31 to 35: further embodiments of inventive
cryopreservation devices in which the pressure vessel has the shape
of a sphere or of a cylinder;
[0062] FIG. 36: a further embodiment of the inventive
cryopreservation device;
[0063] FIGS. 37 to 39: further embodiments of inventive
cryopreservation devices in which a substrate is arranged in the
internal space of the pressure vessel;
[0064] FIGS. 40 and 41: further embodiments of inventive
cryopreservation devices with a segmentation for the internal space
of the pressure vessel; and
[0065] FIG. 42: a further embodiment of the inventive
cryopreservation device and its use.
[0066] The invention will firstly be described in the following by
explanation of findings of the inventors and then by specifying
details of the cryopreservation device and the method. It is
emphasised that the following theoretical considerations serve as
an approach to explain the outstanding vitality rates achieved with
the inventive cryopreservation. However, the implementation of the
invention is not bound by the completeness and correctness of the
theoretical considerations.
[0067] Theoretical Considerations of the Phase Diagrams of Aqueous
Systems and the Effect of Stabiliser Substances
[0068] Cryopreservation has so far been described empirically and
using simplified assumptions. The empirical approach results from
the complexity of the composition of the cytoplasm of the
biological cells. Since the cytoplasm contains hundreds of
proteins, nucleotides and a large number of carbohydrates, ions of
numerous elements, nano-scale systems such as membranes, organelles
and structure elements such as cytoskeleton and water bonded to the
surfaces, phase diagrams of water can be used to only a restricted
extent. Nevertheless, reference is made to phase diagrams of water
as shown in FIGS. 1, 2 and 4 to illustrate the explanation of the
findings of the inventors.
[0069] The pressure-temperature phase diagram of water (FIG. 1,
source: Jackson et al., J. Phys. Chem. (1997), cited in
www.wikipedia.de, keyword water) shows that so-called Ih (hexagonal
ice) is formed on cooling under normal pressure (0.1 MPa=0.0001
GPa, line on the ordinate axis). This assumes a greater volume
compared to the liquid phase (approx. 110) leading to mechanical
tensions (pressure) in delimited volumes. During formation of ice,
multi-component solutions (ions and other molecular constituents
are concentrated) decombine as would not otherwise occur in the
cytoplasm and this should therefore be avoided.
[0070] As the phase diagram in FIG. 1 shows, further ice structures
exist which are known not to exhibit the specified volume increase
at higher pressure. However, biological objects in unfrozen state
would be destroyed under these pressures (e.g. >0.1 GPa).
[0071] In extended phase diagrams of water and of its metastable
states, FIG. 2 shows that possibilities exist to influence the
formation of ice. By adding substances, the freezing point can, for
example, be lowered in a stable manner (colligative effects). This
is the principle used by organisms in nature. They then freeze only
at -10.degree. C. or -20.degree. C. for example. Furthermore, an
aqueous solution can be supercooled under certain conditions. Ice
melts with pure water and at normal pressure at 0.degree. C. but it
does not freeze at this temperature. Nucleation is necessary for
this. In a so-called homogenous nucleation, freezing is a
stochastic process which is triggered by the smallest disturbances.
In both cases, the range of the liquid phase is extended down to
below -50.degree. C. to -70.degree. C. Under consideration of the
pressure range up to 300 MPa=3,000 atm (FIG. 2, right hand side)
the following results: [0072] 1. Up to 200 MPa so-called LDL (Low
Density Liquid, supercooled liquid) water exists in its aggregate
states with all the anomalies occurring at normal pressure. [0073]
2. In addition HDL (High Density Liquid) states arise which may
possibly be more favourable for cryopreservation. However, these
are unphysiologically high pressures (in the deep sea a maximum of
100 MPa is achieved).
[0074] In metastable states a limit temperature below that which a
vitreous state of water could be assumed even with physiological
pressures is to be found at temperatures of around 136
K=-137.degree. C. Water then freezes like glass, namely
amorphously, i.e. without the formation of crystals. The water
molecules then remain where they were. This is a metastable state,
the desirable state which is aspired to as found when deep-freezing
biological objects. It is called "vitrification". The vitrification
requires a very high cooling rate (>10.sup.6.degree./s) with the
rapid fluctuation of the water molecules which, given the dimension
of a cell (>10 .mu.m), cannot be reached due to the neighbouring
heat conductivity of the water (max. a few 10.sup.4.degree./s).
With increasing size of the objects to be frozen, the cooling rate
interval becomes increasingly dramatic (many powers of ten) so that
a true vitrification (formation of a vitreous phase without
additives) has not so far been possible.
[0075] The inventors have determined that in view of the anomaly of
water in the LDL range the freezing point can be lowered by
increasing pressure. In the transition from LDL to HDL this process
is exactly reversed so that a pressure of around 200 MPa is the
preferred pressure upper limit which is still effective to lower
the freezing point. Thereafter the freezing point increases as is
normal in any other liquid as the pressure increases.
[0076] The path to vitrification (vitreous phase) is the shortest
at this pressure (see FIG. 2, right hand side diagram, vertical
line in the middle at 0.2 GPa); only a little more than 100 degrees
must be passed through rapidly, which is why shock freezing is
used. A long-standing paradox of cryopreservation is that the
structures of the cells are best maintained with pressure-shock
freezing but after thawing no cells survive. On the other hand, it
is known that survival rates of over 90% can be achieved with slow
cooling with even microscopically visible ice domain formation and
damage of the cells (membrane leaks). The inventors have determined
that the reason for this is primarily to be found in the thawing
process. The critical range from -137.degree. C. to melting point
(perhaps -5 to -15.degree. C.) is run through once again so that
the avoided or reduced Ih ice formation during freezing does occur.
At low pressure even HD ice quickly changes into the LD ice form.
Added to this is that in accordance with the thermal impulses (k*T)
starting from -100.degree. C. with rising temperature the
probability of a water molecule changing place is relevant also in
the frozen phase. This leads to the so-called "migratory growth" of
large ice crystals because they gain size at the expense of the
smaller ones (due to the surface tension differences). Therefore,
long-term storage must be conducted below the glass transition
temperature (-137.degree. C.).
[0077] However, there is another way of achieving vitrification or
at least a "pseudo vitrification" by adding additives which lead to
very small ice domains. This is not a truly physiological way but
it has been used for freezing for a very long time and also
functions at normal pressure.
[0078] The possibility exists in principle to increase the glass
transition temperature through additives. Adding Trehalose permits
the glass transition temperature, for example, to be shifted to
almost 0.degree. C., which would be ideal for biological objects.
The requisite concentrations of Trehalose are, however, above any
physiological compatibility for living cells. The gains achieved by
lowering the freezing point are small (FIG. 3, lowering the
freezing point and shifting the glass transition temperature by
adding Trehalose) and this applies to virtually all similar
substance additives.
[0079] FIG. 4 shows once again that in the LD range it is necessary
to pass through a critical range described as "no man's land" in
the diagrams during cooling and thawing. However, the solution to
the problem would be precisely to reach this area or to aspire to
complete vitrification.
[0080] In accordance with a practical example, it is intended to
prepare a biological sample in a known manner with biological cells
and a preservation medium at room temperature and under atmospheric
pressure. At least one stabiliser substance is added to the
preservation medium, preferably with a concentration smaller than
or equal to 5% or this is done during the cooling process or in the
deep-frozen state or before thawing. Higher concentrations can also
be used, however.
[0081] Suitable substance groups for the stabiliser substance, in
particular for use with the embodiments of the cryopreservation
device and the methods to cool or heat biological samples described
below are as follows:
TABLE-US-00001 Substance class Substance examples Alcohols Ethyl
glycol Monosaccharides Glucose Fructose Mannose Galactose Ribose
Xylose Arabinose as well as non-naturally occurring sugars Di- and
oligo-saccharides Sacchrarose (sugar cane) Lactose Maltose
Trehalose Cellobiose Polyether Polyethylene glycol Molecular mass
1000 to 35,000 Polysaccharides Cellulose Hemicellulose Amylose
Glycogen Pectins Dextran (artificial) Alginates Ficoll, in
particular with a molecular weight of 2500 g/mol to 2.5 million
g/mol
[0082] Further media with which excellent experimental results have
been achieved using the embodiments of the cryopreservation device
described below and methods to cool and heat biological samples are
as follows:
TABLE-US-00002 Cryomedium according Cryomedium according Dextran to
Mazur to Matsumura 30% dextran Ethylene glycol 10% Ethylene glycol
20% (3.23M) (6.5M) Acetamide 10.7% e-poly-L-Lysine 10% (3.27M)
Ficoll 70 24% (3.5 mM) Sucrose 10.9% Sucrose 25.7% (0.4M) (0.75M)
BSA & glucose (low concentrations) 325 Mosm 3.4 Osm 6.7 Osm
[0083] In the experiments of the inventors the cryomedium according
to Mazur with an addition of ethylene glycol and dextran proved to
be advantageous.
[0084] Embodiments of the Cryopreservation Device and the Methods
to Cool or Heat Biological Samples
[0085] FIGS. 5A to 5C show embodiments of the inventive
cryopreservation device 100 which has a pressure vessel 10 and an
actuating device 20. The pressure device 10 has a vessel wall 11 in
the shape of a tube (small tube) the inside of which forms the
internal space 12 of the pressure vessel 10. The actuating device
20 comprises pressure setting elements which are each created by
pressure screws 21 at the axial ends of the pressure vessel 10. A
third pressure screw 21 can be provided protruding radially at the
vessel wall 11, for example along the half axial length of the
pressure vessel 10 (FIG. 5B).
[0086] The pressure vessel 10 preferably has an outer diameter
which is smaller than or equal to 5 mm and by special preference
smaller than or equal to 2 mm or 1 mm, e.g. 0.5 mm. The axial
length of the pressure vessel 10 has been selected, for example, in
the range of 10 mm to 20 cm. The thickness of the pressure wall 11
is, for example, 1/4 to 1/10 of the outer diameter of the pressure
vessel. The pressure wall 11 is made, for example, from stainless
steel, aluminium, gold or silver. Alternatively, further metals or
alloys can be used which have a high heat conductivity for fast
cooling in the internal space 12 and a pressure resistance for
pressures of up to 100 MPa, for example. Furthermore, the pressure
vessel can be made of a plastic or a composite material, e.g.
plastic-metal composite.
[0087] The pressure vessel 10 has internal threads at its axial
ends to receive the pressure screws 21. The vessel wall 11 has a
threaded piece to accommodate the radially protruding pressure
screw 21 (FIG. 5B). The pressure screws 21 can be used additionally
as bleed screw. Unlike the illustrations in FIG. 5, a single
pressure screw 21 can be provided as actuating device (see for
example FIG. 14A).
[0088] In accordance with FIG. 5C, the internal space 12 of the
pressure vessel 10 can be subdivided into individual internal space
segments 15. The segmentation with internal space segments 15 has
proven to be advantageous for the vitality rate of biological cells
which is a maximum in the middle internal space segments 15.
[0089] For the cryopreservation of a biological sample 1
comprising, for example, biological cells 2 in a preservation
medium 3, the sample 1 is filled into the internal space 12 of the
pressure vessel 10 (see partly cross-sectioned view of the vessel
wall 11 in FIG. 5A). The preservation medium contains one or
several stabiliser substances such as dextran with a concentration
of 30% mixed with 10% ethylene glycol. If the inner space 12 is
completely filled, the pressure screws 21 are closed such that the
internal space 12 is free from bubbles. In order to fill the
internal space free from bubbles, the inner side of the vessel wall
11 must be completely wetted by the biological sample 1. For this
purpose, the inner side of the vessel wall 11 can be coated in a
hydrophilic manner. Furthermore, one of the pressure screws 21 can
be used to bleed the system. As a result of penetration of the
pressure screws 21 an increased pressure can be set in the internal
space 11 of the pressure vessel 10, e.g. in the range from 0.1 MPa
to 300 MPa even at room temperature. The amount of pressure can be
determined by calibration tests or using a sensor device 16 (see
below). As an option, the segmentation into internal space segments
15 (FIG. 5C) can be provided in or after this phase by squashing
the vessel wall together.
[0090] For the cryopreservation of the biological samples, the
filled cryopreservation device 100 is cooled in the cooling bath of
a cooling device (not shown in FIG. 5, see FIG. 13, for example).
For this purpose, the pressure vessel is preferably immersed
horizontally into the cooling bath so that the cooling essentially
takes place simultaneously along the entire axial length of the
pressure vessel 10. The cooling bath comprises, for example, liquid
propane, liquid nitrogen or another liquid gas.
[0091] On immersion in the cooling bath ice firstly forms on the
inner side of the vessel wall 11. Since the crystalline phase is
firstly formed, an expansion takes place which leads to a pressure
increase in the internal space up to a final pressure of 200 MPa.
Above this pressure further ice growth is ruled out so that the
remainder of the biological sample 1 moves to a vitreous
(vitrified) state or at least does not exhibit a hexagonally
crystalline phase.
[0092] Once the cryopreservation device 100 in the cooling bath has
reached the cryopreservation temperature, e.g. -197.degree. C., the
further cryopreservation can take place in the cooling bath or in
storage vessel (not shown) which has been cooled to a temperature
of, for example, -140.degree. C. through liquid nitrogen or through
vapour of the liquid nitrogen.
[0093] For the heating of the biological sample 1 so as to maintain
vitality, the cryopreservation device 100 is immersed in a heating
bath (liquid bath with a temperature above 0.degree. C.)
comprising, for example, water, alcohol or an oil. Immersion is
similarly conducted horizontally by preference and at a high speed
so that the increased pressure in the pressure vessel 10 is
maintained until the crystalline ice which is formed melts.
Finally, the pressure drops suddenly to 0.1 MPa, for example.
[0094] FIGS. 6A to 6D show four typical temperature and
pressure-time functions as may be realised in cryopreservation
(left part of the curves) or during thawing (right part of the
curves) with the cryopreservation device 100 in accordance with the
embodiments of the invention explained here. FIG. 6A shows, for
example, how during freezing the pressure is initially increased.
For this purpose, pressure screws 21 as shown in FIG. 5 are used.
Only after the pressure of 200 MPa is reached is the temperature
reduced by immersion (dipping) of the cryopreservation device 100
into the cooling bath. During thawing pressure can be released and
then the biological sample heated to room temperature. FIG. 6B
shows the opposite variant in which during cryopreservation the
temperature is firstly reduced and then the pressure increased. In
this case the pressure screws 21 are actuated only after reaching
the cryopreservation temperature of, for example, -200.degree. C.
During thawing, pressure can be released as the temperature rises.
FIG. 6C shows a more complicated temperature-time function which
can be selected as dependent on the specific preservation
conditions. Using the inventive cryopreservation device 100 a
pressure temperature curve can also be realised as would exist with
a cryopreservation device without actuating device (FIG. 6D),
whereby the pressure build-up is started directly after falling
below the freezing point by the creation of the crystalline phase
of ice in the biological sample. The shape of the time functions is
influenced by the actuating device used in the invention. In
accordance with a further variant of a temperature and
pressure-time function (not shown) the increased pressure can be
set throughout the entire preservation time.
[0095] FIG. 7 shows a sequence of microscopic images of a section
of sample 1 with cells 2 at room temperature after a cryoprotectant
has been added to the preservation medium 3. The time (in seconds,
s) shows the dependence on time after adding the stabiliser
substance to sample 1. It becomes clear that a considerable
shrinking process takes place in a short period of time. The
inventors have found that this shrinking can be advantageous for
the achievement of high vitality rates.
[0096] FIGS. 8 and 9 show the dramatic shrinking of the cells (here
HeLa cells) virtually to the osmotic residual volume which is
advantageous for high vitality rates. The round form of the cells
(FIGS. 9A to 9C, top left) is lost completely (FIGS. 9A to 9C,
right and bottom). The reduction in the diameter of the cells was
determined with an invitrogenic measurement system (diagram in FIG.
8), but does not completely reflect the osmotic shrinking because
the part of deviation from the spherical form is not considered.
The cells consequently shrink more greatly than shown by the
diagram figures.
[0097] FIG. 10 shows the shrunken cells in an electron-microscopic
section. The extremely strong shrinking can be recognised which is
achieved in the cryomedium with one of the above-mentioned
compositions so that the cell membrane system is greatly folded.
FIG. 11 shows the structural changes of adherent cells (HeLa).
Here, too, the osmotic shrinking occurs very quickly (after
seconds).
[0098] Fluorescence-microscope investigations with a different
presentation of the cell nucleus and of the Golgi apparatus (not
shown here) have shown that the shrinking merely compacts the
cytoplasma components but does not influence the cell nuclei and
other important cell organelles; this is of great advantage to
freezing and thawing such as to maintain vitality.
[0099] FIG. 12 shows variants of the inventive heat conducting
and/or cooling elements intended. For purposes of comparison, FIG.
12A firstly shows the circular cross-section of the tubular
pressure vessel 10 (see FIG. 5). FIGS. 12B to 12I show embodiments
of the inventive cryopreservation device as diagrammatic sectional
views of the pressure vessel 10 in which the actuating device is
suitable for a setting of the temperature using at least one heat
conducting element or at least one cooling element. In accordance
with FIG. 12B, heat conducting elements comprise outer profiles 35
which are arranged radially protruding on an outer side of the
vessel wall 11. The outer profiles 35 accelerate the cooling or
heating of the pressure vessel 10 on immersion in the cooling bath
or the warm liquid bath. In accordance with FIG. 12C, the inner
profiles 36 are arranged on an inner side of the vessel wall 11.
The inner profiles 36 similarly support the heat transport from or
to the biological sample in the pressure vessel 10.
[0100] FIGS. 12D to 12G illustrate the pressure vessel 10 with a
hexagonal cross-section. The outer side of the vessel wall 11 forms
the outer profile 35 which provides a large surface for the wetting
with a cooling agent or thawing agent and is therefore suitable to
influence the temperature-time function during cooling or thawing.
In accordance with FIGS. 12E, 12F and 12G, heat conducting bodies
37 in the shape of partition walls (FIG. 12E), inserted filaments
or spheres (FIG. 12F) or colloidal particles (FIG. 12G) are
provided additionally in the internal space of the pressure vessel
10. The colloidal particles 37 in accordance with FIG. 12G are
shown diagrammatically enlarged but in practice can have dimensions
in the sub-micrometre range. The cooling or thawing speeds can be
increased advantageously with the outer profiles 35, inner profiles
36 and/or heat conducting bodies 37. The heat conducting elements
are preferably made from silver or gold or other substances with
high heat conductivity. In accordance with further variants, the
heat conductivity between the internal space 12 and the outer
environment of the pressure vessel 10 can be influenced by a
coating of the vessel wall 11 on its inner or outer side, e.g. with
diamond or other substances with high heat conductivity.
[0101] FIG. 12H shows a variant of the invention in which a cooling
line 34 runs through the inner space 12 of the pressure vessel 10
as cooling element. The cooling line 34 is connected with a cooling
medium reservoir, and it is adapted in order to be supplied with a
cooling medium such as liquid nitrogen or a cooling gas.
Alternatively, a cooling line 34 can run next to the internal space
12 as shown diagrammatically in FIG. 12I.
[0102] FIG. 13 shows a further embodiment of the inventive
cryopreservation device 100 (FIG. 13A) and its use in cooling (FIG.
13B) and thawing (FIG. 13C) of the biological sample with cells 2
in the preservation medium 3. In this embodiment of the invention
the actuating device comprises three pressure setting elements
comprising pressure screws 21 and an expansion area 22 for the time
and/or location-dependent setting of the temperature and of the
pressure in the pressure vessel 10. The pressure screws 21 are
provided for the generation of pressure and/or to bleed the
internal space 12 of the pressure vessel 10 (see FIG. 5). The
expansion area 22 comprises a duct which protrudes radially from
the pressure vessel 10 which at one end is connected to the
internal space 12 of the pressure vessel 10 via an opening in the
vessel wall 12 and whose opposite free end is closed. A filter 29
can be provided between the internal space 12 and the expansion
area 22 in order to prevent the penetration of biological cells
into the expansion area 22.
[0103] The expansion area 22 is made, for example, from the same
material as the vessel wall 12 of the pressure vessel 10. The
expansion area 22 is adapted to receive a liquid expansion medium
that expands during cooling. The expansion medium comprises, for
example, the aqueous preservation medium of the biological sample
or alternatively a different aqueous liquid which is suitable to
form the crystalline phase of water. The dimensions (length, inner
diameter, outer diameter) of the expansion area 22 can be selected
by the user depending on the specific preservation conditions.
[0104] The expansion area 22 permits a fast formation of the
crystalline phase once the temperature of the expansion area 22 is
reduced to below the freezing point of water. Advantageously, the
cooling of the expansion area 22 can be decoupled in terms of time
from the cooling of the remaining pressure vessel 10, as shown in
FIG. 13B. A cooling device 200 as shown in the diagram with a
cooling bath 210 is provided for cooling. The cooling device 200
comprises, for example, a vessel into which the cooling bath 210,
e.g. of liquid nitrogen, is filled and which is connected with a
cooling medium reservoir.
[0105] For cryopreservation of the biological sample 1 the
cryopreservation device 100 is firstly immersed exclusively with
the expansion area 22 into the cooling bath 210 (immersed depth D1)
whilst the remaining pressure vessel is still above the cooling
bath. In this phase, crystalline ice is formed in the expansion
area 22 which expands such that the pressure increases in the
internal space 12 of the pressure vessel 10. The duration of
pressure generation with the expansion area 22 is selected, for
example, in the range of milliseconds, seconds or minutes. Finally,
the cryopreservation device 100 is completely immersed in the
cooling bath 210 so that the desired cryopreservation temperature
of -195.7.degree. C., for example, is achieved. For this purpose,
the cryopreservation device 100 is lowered to a second immersed
depth D2.
[0106] The heating to recover the biological sample is conducted in
reverse in accordance with FIG. 13C. To maintain the pressure in
the pressure vessel 10, the internal space with the biological
sample is firstly immersed in a heating bath 310 of a thawing
device 300 (immersed depth D1) whilst the expansion area 22 is not
yet cooled. After thawing of the biological sample 1 in the
pressure vessel 10, the device is lowered to the immersed depth D2
so that the pressure in the pressure vessel 10 is also reduced.
[0107] FIG. 14A illustrates an embodiment of the inventive
cryopreservation device 100 in which an inner vessel 13 is arranged
in the internal space 12 of the pressure vessel 10. The inner
vessel 13 is intended to receive the biological sample 1 and
comprises a bubble-free filled tube of a flexible material. The
inner vessel 13 is made, for example, of the same material as the
vessel wall 12 of the pressure vessel 10. The provision of the
inner vessel 13 has the advantage that the formation of the
crystalline phase on the inner side of the vessel wall 12 is
separated from the biological sample 1. Furthermore, different
fluids can be provided in the internal space 12 outside the inner
vessel 13 on the one hand and in the inner vessel 13 on the other.
For example, pure water or an aqueous composition from water with a
salt, glycerine and/or an alcohol can be provided outside the inner
vessel 13. The aqueous composition has the advantage of reducing
the freezing point so that the temperature at which the pressure in
the pressure vessel 10 is to be generated can be determined below
the freezing point of pure water.
[0108] The inner vessel 13 does not extend over the entire length
of the internal space 12. This creates a relatively large space at
the closed end 12.1 of the pressure vessel 10 in which no
biological sample 1 is located and in which the crystalline phase
of water can preferably be generated. This creates an expansion
area within the pressure vessel 10 as part of the inventive
actuating device which advantageously can have an effect on the
cooling and on the heating of the cryopreservation device 100 (see
in particular FIG. 14C).
[0109] The outer shape of the inner vessel 13 can be the same as
the inner shape of the pressure vessel 10. By way of alternative to
the cylindrical shape shown, other cross-sections of the outer or
inner shapes can be provided such as quadratic, hexagonal,
octagonal or all combinations thereof. In particular, different
cross-section shapes of the outer and inner shapes can be
provided.
[0110] FIG. 14B illustrates diagrammatically the cooling of the
cryopreservation device 100 in a cooling device 200 which in this
case contains two cooling baths 210 on top of one and other with
liquid nitrogen and 220 with liquid propane. The use of liquid
propane has the advantage that this wets the outer side of the
pressure vessel 10 more easily and therefore accelerates
cooling.
[0111] To heat the biological sample 1 in the pressure vessel 10 it
is immersed in a heating bath 310 in accordance with FIG. 14C. The
pressure vessel 10 can be oriented such that the entire length of
the pressure vessel 10 is immersed simultaneously into the heating
bath 310 (horizontal alignment). In this case, the temperature of
the biological sample 1 is firstly increased and, as soon as the
crystalline ice has melted, the pressure in the pressure vessel 10
is reduced. Alternatively, the closed end 12.1 with the expansion
area can firstly be immersed in the water bath 310 so that the
crystalline phase is firstly melted and the pressure in the
pressure vessel 10 reduced and finally the remaining biological
sample 1 heated (vertical alignment of the pressure vessel 10).
[0112] FIG. 15 illustrates diagrammatically that the use of the
inventive cryopreservation device 100 is not restricted to the
preservation of cell suspensions. Rather, the biological sample 1
can contain cell groups, cell aggregates, organs 4 of biological
organisms or complete biological organisms 5 such as nematodes,
worms or arthropods. For this purpose, the internal space 12 of the
pressure vessel 10 has an inner diameter in the range of at least 5
mm, preferably at least 10 mm. The inner diameter of the internal
space 11 is preferably smaller than 5 cm, in particular preferably
less than 3 cm. As in FIG. 14, an inner vessel 13 is provided to
receive the biological sample 1 which can be optionally subdivided
into individual chambers.
[0113] The cryopreservation of the biological sample 1 is also
conducted in the embodiment in accordance with FIG. 15 by the
pressure vessel 10 being immersed in at least one liquid bath of
the cooling device 200. In accordance with FIG. 15C, the pressure
vessel 10 is first immersed with horizontal alignment in the liquid
bath 220 with liquid propane and then in the liquid bath 210 with
liquid nitrogen. By choosing the alignment of the pressure screw 21
relative to the liquid bath 220, 210, the time function of the
pressure generation can be set relative to the time function of the
cooling.
[0114] Heating is conducted in accordance with FIG. 15D in reverse
order by firstly immersing the pressure vessel 10 in the heating
bath 310 until the biological sample 1 has thawed. The
cryopreservation device 100 is then completely lowered into the
heating bath 310.
[0115] FIGS. 16 and 17 show further embodiments of the
cryopreservation device 100 which is provided with an expansion
area 22. As in FIG. 13, the expansion area 22 comprises a duct
which has branches in the illustrated variants. In accordance with
FIG. 16A, the expansion area 22 has a T-shape with lateral arms.
The expansion area 22 can first be immersed in the cooling bath 210
(immersed depth D1) so that the liquid inside freezes, hexagonal
ice is formed and the pressure increased. Only then is the system
lowered entirely (immersed depth D2) and completely deep frozen
(FIG. 16B). The reverse process is applied for heating or the
possibility is provided to immerse into the heating bath 310 in two
layers (arms first into the warm phase or last as shown here) (FIG.
16C). According to the latter principle, the pressure is maintained
by the existing ice up to thawing at values of around 200 MPa.
[0116] FIG. 17 shows a further embodiment of the expansion area 22
with branches which form a fanned arm system. A larger inner volume
and a larger surface than provided, for example in FIG. 16, results
so that the pressure can be increased more quickly.
[0117] FIG. 18 shows embodiments of the cryopreservation device 100
with a spherical pressure vessel 10 and one expansion area 22
comprising several hollow ducts protruding in different axes
(directions). The pressure vessel 10 comprises a hollow sphere to
receive the biological sample. The hollow sphere is made of
stainless steel, for example, and has an inner diameter of 10 mm
and an outer diameter of 12 mm. The ducts form stubs protruding
from the hollow sphere. The number, geometrical dimensioning and
alignment of the ducts can be selected as dependent on the specific
conditions of use (see examples in FIG. 18A to 18C). The hollow
sphere is filled through a closable opening (not shown) in the
vessel wall or through one of the ducts, which in this case is
adapted with a closing element. In these embodiments too, the
geometric alignment of the pressure vessel 10 permits a setting as
to when the pressure in the pressure vessel 10 is to increase and
drop on immersion to the immersed depth D1 and D2 in a cooling bath
210 (FIG. 18D) or in a heating bath 310 (FIG. 18E).
[0118] FIGS. 19A and 19B show embodiments of the cryo-preservation
device 100 in which the vessel wall 11 of the pressure vessel 10
have the shape of a curved tube with simple curvature (FIG. 19A) or
multiple curvature (FIG. 19B). In both cases, the pressure vessel
10 is bent in one bending level so that the local temperature
distribution on immersion in the cooling bath can be set relative
to the cooling bath depending on the alignment of the pressure
vessel 10 (see FIG. 19C, 19D). FIG. 19A shows by way of example
variants of pressure setting elements comprising pressure screws 21
and an expansion area 22 which can be provided individually or in
combination as shown. The pressure screws 21 are formed as
described above with reference to FIG. 5. The expansion area 22
comprises a large number of ducts which are arranged in the bending
level of the pressure vessel 10 and which each communicate with the
internal space of pressure vessel 10. Pressure setting elements can
also be provided in the variant shown in FIG. 19B. The multiple
bending in accordance with FIG. 19B can be shaped as a wave with
more than the three extremes shown.
[0119] The local distributions and time functions of the pressure
generation and the temperature reduction in the cryopreservation
device 100 depend on the alignment of the pressure vessel 10 on
immersion in the cooling bath 210. FIG. 19C shows, for example, an
immersion with the bending level vertically or parallel to the
surface of the cooling bath 210. Pressure generation and
temperature lowering with the vertical alignment is conducted
firstly in the extremes of the multiple bending which protrude into
the cooling bath. By contrast, with the parallel alignment,
pressure generation and temperature reduction is conducted
simultaneously in the entire pressure vessel 10. Contrary to the
illustrations, immersion with other alignments into the liquids is
possible, leading to an extended variability of the settings of the
pressure and temperature-time functions.
[0120] The local distributions and time functions of the pressure
reduction and of the temperature increase in the cryopreservation
device 100 similarly depend on the alignment of the pressure vessel
10 on immersion into a heating bath 310 (FIG. 19D).
[0121] FIG. 20 shows an embodiment of the cryopreservation device
100 in which the vessel wall 11 of the pressure vessel 10 has the
shape of a flat or disc-like cylinder. This embodiment of the
invention has the advantage that on immersion in a cooling bath a
relatively large area is cooled. Therefore, a thin layer of the
crystalline phase is formed on the inside of the vessel wall 11
which is sufficient to generate the required pressure in the
pressure vessel 10 and simultaneously leave a relatively large
amount of space for the vitreous phase. The pressure vessel 10 in
accordance with FIG. 20A has a diameter of, for example, 1 cm to 10
cm and a thickness of, for example, 1 mm to 1 cm. It is made, for
example, of stainless steel, aluminium, gold or silver.
[0122] In accordance with FIG. 20B, the actuating device for the
time and/or location dependent setting of the temperature and of
the pressure in the pressure vessel 10 comprises a pressure clamp
23. The pressure clamp 23 comprises two clamp plates 24 which can
be pivoted via a hinge 25 (axial hinge). The distance between the
clamp plates 24 can be set using a clamp screw 26 (setting screw).
The pressure clamp 23 is suitable to apply pressure to the pressure
vessel 10 which is higher than atmospheric pressure before the
cryopreservation device 100 is cooled by immersion in a cooling
bath (see FIG. 24). Furthermore, the pressure clamp 23
advantageously permits a stabilisation of the pressure vessel
10.
[0123] FIGS. 20C to 20D show variants of the cylindrically shaped
pressure vessel 10 which can be adapted with a reinforced
circumference area (perspective section in FIG. 20C) and/or a
vessel wall 11 which can be deformed elastically on both sides
(section in FIG. 20D) or on one side (section in FIG. 20E). In the
latter case, a stable floor wall 11.3 is provided on one side.
[0124] FIGS. 21 to 24 show further variants of the pressure clamp
23 in which the clamp plates 24 are adapted with holes 27 and with
profiles 28 on the inner side facing the pressure vessel 10 in open
state and in closed state (assembled state) of the pressure clamp
23. In closed state of the pressure clamp 23 mechanical pressure is
exerted on the vessel wall 11 of the pressure vessel 10 via the
clamp plates 24. FIG. 23 shows a top view of the pressure clamps 23
with one of the clamp plates 24, the hinge 25, the clamp screw 26
and the holes 27.
[0125] The holes 27 permit direct contact of a cooling liquid, e.g.
of liquid nitrogen or propanol, or of a heating fluid, e.g. of
water, with the pressure vessel 10 and therefore an acceleration of
cooling or heating of the pressure vessel 10. The profiles 28 are
provided to generate different pressures at the pressure vessel 10
locally. It is furthermore shown that the cylindrical pressure
vessel 10 can be adapted with a bleed connector 30.
[0126] FIG. 25 shows a variant of the invention in which the
cryopreservation device 100 is immersed only on one side into a
liquid (cooling bath 210 or heating bath). In this case the
pressure clamp 23 has holes 27 only on the lower clamp plate 24
facing the liquid. This is particularly advantageous if adherent
cells grow on the lower side of the pressure vessel 10 or the cells
sediment down to it.
[0127] FIG. 26 illustrates diagrammatically a variant of the
invention in which the bleeding of the pressure vessel 10 and the
mechanical pressure generation can be controlled with the clamp
plates 24 with electrical drive elements 32, 33. This embodiment of
the invention is particularly advantageous for an automation of
cryopreservation.
[0128] In an altered embodiment of the cryopreservation device 100
shown in FIG. 27 in which the vessel wall 11 of the pressure vessel
10 has the shape of a cylinder, no clamp but rather a pressure
screw 21 is provided as pressure setting element. Using the
pressure screw 21 the pressure in the internal space 12 of the
pressure vessel 10 can be set before or after the start of cooling
or heating in the cooling or heating bath (not shown). Several
inner vessels 13 are arranged in the internal space 12 which are
filled gas-free with biological samples, e.g. cell suspensions and
are arranged, for example, stacked on top of one another. The wall
thickness of the inner vessel 13 can be extremely thin, e.g.
thinner than 400 .mu.m because they are not exposed to pressure or
corresponding mechanical forces.
[0129] According to FIG. 27, a sensor device comprising a pressure
sensor 16.1 is arranged in the internal space 12 of the pressure
vessel 10. The pressure sensor 16.1 provides a sensor signal which
can be used to control the pressure using the pressure screw 21,
possibly with an electrical control element (e.g. piezo element,
not shown). A predetermined outer pressure can be generated which
acts on the inner vessel 13 in the internal space 12 during
freezing and thawing.
[0130] An altered variant of the pressure vessel 10 with pressure
screw 21 is shown in FIG. 28. In this case the inner vessel 13 is
formed by a flexible bag which receives the biological sample, e.g.
a cell suspension or blood sample. The inner vessel 13 comprises,
for example, a blood bag as used for blood donation purposes.
[0131] FIG. 29 illustrates diagrammatically an enlarged section of
the vessel wall 11 of a pressure vessel 10. The vessel wall 11
contains an optical unit 60 which is adapted for a visual, in
particular microscopic, observation of the internal space 12 of the
pressure vessel 10. It is possible, for example, for cells 2 to
adherently grow on an optical lens 61. The optical lens 61 can be
configured, for example, for a microscopic image of the cells
2.
[0132] FIG. 30 shows a further embodiment of the invention in which
the cryopreservation device 100 comprises a tube arrangement with
external pressure generation (hydrostatic, see arrow) via a T
element. The tube arrangement is adapted with a pressure sensor
16.1 and a temperature sensor 16.2 using the sensor signals of
which the pressure and the temperature-time functions can be
regulated.
[0133] FIGS. 31 and 32 show embodiments of the cryopreservation
device 100 in which the vessel wall 11 of the pressure vessel 10
has the shape of a sphere. The pressure vessel 10 is composed of
two semi spheres 11.2 which are screwed together in the middle of
the pressure vessel 10. The spheres have a diameter in the range
of, for example, 5 mm to 10 cm. According to FIG. 31A, the pressure
vessel 10 is adapted with a pressure screw 21 which is used to fill
the pressure vessel 10, bleed the pressure vessel 10 and generate
the pressure in the internal space of the pressure vessel 10. FIG.
31B illustrates additionally optional parts such as a sensor device
16 and a filling line 11.1 which is decoupled from the pressure
vessel 10. In analogous application of the above-described
procedures, FIGS. 31C and 31D illustrate the cooling and heating of
the cryopreservation device 100.
[0134] The spherical pressure vessel 10 can be alternatively or
additionally provided with a pressure setting element in the shape
of an expansion area 22 (FIG. 32). The expansion area 22 comprises
a cylinder connection which, as explained above, is configured to
receive an expansion medium or to generate pressure during cooling.
In analogous application of the above-described procedures, FIGS.
32C and 32D illustrate the cooling and heating of the
cryopreservation device 100.
[0135] The inventive cryopreservation of biological samples
comprising organs 4 or entire organisms 5 is illustrated
diagrammatically in FIGS. 33 to 35. Particularly in these
applications of the invention, the biological sample is preferably
combined with the stabiliser substance. According to FIG. 33A, a
cylindrical vessel is provided as pressure vessel 10 which, in the
example shown, is dimensioned to receive a fish embryo. Two
immersion variants for pressure-temperature control are shown in
FIGS. 33C and 33D for the freezing of the cryopreservation device
100. A spherical vessel is shown as pressure vessel 10 in
accordance with FIGS. 34A and 34B which is dimensioned to receive
an organ 4 with an inner diameter of the pressure vessel 10 in the
range of, for example, 10 cm to 30 cm. According to FIG. 34B, the
organ 4 is arranged in an inner vessel 13. FIG. 35 shows a
spherical vessel as pressure vessel 10 in which tissue, organisms 5
or organs in an inner vessel 13, for example a bag or a thin-walled
vessel, are located so that the outer solution to the inner bag
medium can be different (e.g. outside oil, inside nutrient with a
stabiliser substance).
[0136] FIG. 36 shows an embodiment of the cryopreservation device
100 in which the pressure is formed not by the expansion of a
freezing expansion medium but by a vaporous expansion medium, e.g.
vaporising liquid nitrogen. The expansion area 22 which contains
the liquid nitrogen is connected to the pressure vessel 10 via a
pressure line 39. The vaporising liquid nitrogen is injected
continuously or in portions into the pressure vessel 10 from the
expansion area 22.
[0137] FIGS. 37 to 39 show embodiments of the cryopreservation
device 100 which are configured by the provision of at least one
substrate 14 in the internal space 12 of the pressure vessel 10 for
the cryopreservation of adherent cells 2. According to the variants
shown in FIGS. 37A to 37C, the substrate 14 is located in the form
of a long extended strip (tongue) in a tubular pressure vessel 10
which is closed on one or both sides by way of a pressure screw 21
and/or with a radially protruding pressure screw 21. The cells 2
are in an adherent state on one or both sides on the substrate 14
which can be functionalised in a suitable manner (e.g. by coating
with fibronectin, polylysine and/or growth factors). Furthermore,
the internal space 12 of the pressure vessel 10 is filled with a
preservation medium, possibly with the stabiliser substance.
[0138] FIG. 37C also shows a substance reservoir 17 which is
arranged in the internal space 12 of the pressure vessel 10.
Substance reservoir 17 is adapted for an introduction of at least
one additional substance into the internal space 12. For example,
substances can diffuse into the internal space 12 from the
substance reservoir 17 during the freezing or thawing procedure.
The substance reservoir 17 can have a reservoir wall, for example
made of plastic, which can be destroyed under the effect of
pressure in the internal space 12. Advantageously, this permits the
additional substance to be released in the internal space 12 only
once a specific pressure has been reached.
[0139] Instead of the stretched strip, complicated shapes of the
substrate 14 can be used as shown, for example, in FIG. 38. In
accordance with FIG. 38A, a substrate 14 is illustrated with a
cross-like cross-section whereby it can be possible for the cells 2
to be arranged on all or only on some surfaces of the substrate 14.
According to FIG. 38B, a substrate 14 is shown in the shape of a
hollow cylinder in the inside of which the cells 2 are located. The
hollow cylinder can be made, for example, from ceramics, plastic,
cellulose or chitin with an extremely thin wall, in particular
thinner than 250 .mu.m. As for the use of the above described inner
vessel, this advantageously enables the cells 2 in the inside to be
subjected to other peripheral conditions and solutions as outside
the substrate 14. According to further variants of the invention,
the substrate 14 can comprise a body with a nanostructured or
microstructured surface which is adapted optionally with growth or
differentiation factors in gradients.
[0140] FIGS. 39A to 39E show by way of example that the substrate
14 can be configured as a separate component (shuttle) which can be
pushed into the pressure vessel. The shuttle can, in particular, be
formed with a closed or open shape.
[0141] FIGS. 39A and 40 furthermore illustrate a variant of an
inventive actuating device to control the pressure in the pressure
vessel 10. In this variant, a coiled section 31 is provided as
pressure-setting element at at least one end of the tubular
pressure vessel 10. By turning the coiled section 31 at the
pressure vessel 10 filled gas free with the biological sample the
pressure can be increased here before lowering the temperature.
[0142] FIG. 40A shows a tubular pressure vessel 10 with coiled
sections 31. The biological sample 1 with the preservation medium 3
is located in the internal space 12 of the pressure vessel 10. The
preservation medium 3 in which the cells 2 are located can contain
the stabiliser substance in the form of a gradient (e.g. Ficoll or
Percoll of different concentrations and/or molecular weights).
According to FIG. 40B, additional partition walls 18 can be
provided in the internal space 12 which prevent a rapid mixing of
the biological sample 1 and, for example, a disintegration of the
gradient and permit a separation of the cells into compartments.
Optionally, as shown in FIG. 40C, additional substance reservoirs
in the shape of hollow spheres 19 can be provided in the internal
space which are suspended and set out in the preservation medium 3,
can be destroyed at a certain pre-set temperature and release their
content. For example, further anti-freeze and vitrification
substances or media which support the cells during thawing can be
released. It is illustrated in FIG. 40D that two non-miscible
solutions can be arranged layered on top of one another in the
pressure vessel. The cells 2 are located in the first solution. The
pressure relationships on temperature change can be set via the
freeze behaviour of the solutions.
[0143] FIG. 41 illustrates a tubular pressure vessel 10 which is
closed on one side with a pressure screw 21 and on one side with a
coiled section 31. The preservation medium 3 is located in the
internal space in the form of layered solutions (e.g. Ficoll as
gradient or top layers of different molecular weights, i.e.
solutions with different density) as used in the density gradient
centrifusion of cells. The cells 2 are added unilaterally to the
gradient (FIG. 41A). The pressure vessel 10 is centrifuged in
vertical form with the cells 2 so that different cell types collect
on the border areas (FIG. 41B). The cryopreservation then takes
place in this arrangement.
[0144] FIGS. 42A and 42B diagrammatically illustrate a further
embodiment of the inventive cryopreservation device 100 and its
use. The cryopreservation device 100 comprises a pressure vessel 10
closed on one side with a pressure screw 21 in whose internal space
12 the substrate 14 is arranged for the adherent reception of
biological samples 2. The pressure screw 21 is furthermore adapted
with a heat conducting element in the form of a cooling wire 38
which is integrated pressure-tight into the pressure screw 21 and
extends from the internal space 12 to the environment of the
pressure vessel 10. The cooling wire 38 is a metal wire, for
example, made of silver. Outside the pressure vessel 10, a cooling
wire 38 is concentrated into a compact bundle, e.g. in the form of
a spiral, a ball, a cylinder or a sphere. On freezing the cooling
wire 38 can first be immersed in the cooling bath 210, e.g. of
liquid nitrogen whereby ice does not form on the vessel wall 11 on
the inside of the tube but on the cooling wire 38 leading to an
increase in pressure. Thereafter the system is completely immersed
into the cooling bath 210.
[0145] The features of the invention disclosed in the above
description, the drawings and the claims can be of importance
individually and also in combination for the realisation of the
invention in its different embodiments.
* * * * *
References