DE102011115467A1 - Apparatus and method for pressure-cryopreserving a biological sample - Google Patents

Abstract

A cryopreservation device (100), which is set up for the cryopreservation of a biological sample (1), comprises a pressure vessel (10) having a container wall (11) and an interior (12), which is adapted to receive the biological sample (1) the pressure vessel (10) is configured for cooling with a lowering of the temperature and an increase of the pressure in the pressure vessel (10) and for the cryopreservation of the biological sample (1) configured with an adjusting device (20) which is connected to the vessel wall (11) is connected and at least one of at least one Druckeinstellelement (21-23, 31), at least one cooling element (34) and at least one heat conducting element (35-38), wherein the adjusting device (20) for a time- and / or location-dependent adjustment of Temperature and pressure in the pressure vessel (10) is configured. Also described are methods for cryopreserving a biological sample (1) comprising biological cells (2) and a preservation medium (3), and methods for vitality-preserving heating of the biological sample (1).

Description

The invention relates to a device for the cryopreservation of a biological sample, comprising biological cells and an aqueous preservation medium, with a pressure vessel. Furthermore, the invention relates to a method for cryopreserving the biological sample and the use of a stabilizer substance, with which a glassy phase of water is stabilized, in the cryopreservation of biological samples. Furthermore, the invention relates to a method for heating the cryopreserved biological sample.

Cryopreservation of cells has so far been the only way to reversibly halt vital processes at the cellular level in such a way that they can restart after being heated to physiological temperatures. The cryopreservation finds no equivalent in nature. Although organisms, such. As fish, found in polar regions embedded in ice, which maintain their vitality for a limited duration. However, these organisms are not completely frozen through, but contain in their cells glycerol and other freezing point depressants, or they express proteins (so-called antifreeze proteins) which influence the ice structure. Permanent preservation is not possible in this state, since even at temperatures below 0 ° C, diffusion processes over weeks and months lead to the disintegration of the biological system. The permanent preservation would rather a cooling z. B. require to a temperature of liquid nitrogen, in which the liquid in the cells would freeze. But then the organisms can not be reanimated.

A particular problem of cryopreservation is the formation of crystals ice (crystalline phase of the water) in and outside the cells, which directly or indirectly lead to irreversible damage. Since ice formation is one of the primary causes of cell damage, so-called cryoprotectants (antifreeze additives, cryoadditiva) have been sought for decades and added to the obligate physiological media. Cryoprotectants typically include small molecules, such as. As dimethyl sulfoxide (DMSO), which penetrate into the cells, or higher molecular weight substances, such as. As sugar, which remain in the medium outside the cells or at the surface. Cryoprotectants are often effective only in high, largely unphysiological concentrations (10% to 50%). They can therefore be added only at lower than physiological temperatures (around 4 ° C), must penetrate quickly into the cells and immediately washed out after thawing.

• [1] Polge C, Smith AU, Parkes AS (1949), Nature 164: 666
• [2] Lovelock JE (1954), Biochemical Journal 56 (2): 265–270
• [3] Lovelock JE, Bishop MWH, 1959, Nature 183: 1394–1395

Previous developments in the cryopreservation of biological samples are aimed at replacing cryoprotectants by physiologically and osmotically less problematic substances, reducing their concentration or completely without them. As a result, apart from the antifreeze proteins of the organisms themselves, only a few groups of substances have been found since the discovery of the antifreeze properties of DMSO and glycerol as well as some sugars by Polge and Lovelock [1, 2, 3]. So far, it is believed that no cryopreservation of cells without cell stress and cell repair processes with gene expression etc. after thawing is possible because the formation of the crystalline phase under physiological conditions can not be completely avoided.

• [1] Polge C, Smith AU, Parkes AS (1949), Nature 164: 666
• [2] Lovelock JE (1954), Biochemical Journal 56 (2): 265-270
• [3] Lovelock JE, Bishop MWH, 1959, Nature 183: 1394-1395

The success of cryopreservation may be due to a vitality rate (survival rate), e.g. B. by the quotient of the number of living cells after and before the cryopreservation, characterized. It is known that the vitality rate depends on the cell type, the volume and various other, usually already empirically optimized, boundary conditions. Due to the need for rapid diffusion of the cryoprotectants into the cells and the orderly dissipation of heat to the outside, the conventional cryopreservation methods have so far failed without exception for macroscopic objects such as tissues, organs and whole organisms. Rather, conventional cryopreservation with the addition of cryoprotectants leads to practicable vitality rates only for suspended cells and minute pieces of tissue (<0.5 mm ³ ). Highly water-containing plant cells, as well as a variety of large animal cells, such. As the oocytes of cats and other species, so far elude cryopreservation completely. On the other hand, vitality rates of more than 90% are achieved with cryopreserved cancer cells.

The success of the cryopreservation depends in particular on the physical-biological boundary conditions, eg. As the properties of the water and those of the cryoprotectants from. So far, it has been assumed that cryoprotectants must pass through diffusion into all cells and the heat must be dissipated to the outside in a short time (ms to min), since only from there can be cooled. It is further assumed that these conditions are met due to the thermal conductivity of the dominating even in the cells of water and the low diffusion rate of cryoprotectants through the cell membranes only in very small objects (cells suspended in a nutrient solution and with the addition of cryoprotectant).

• [4] J. L. M. Leunissen und H. Yi (2009) Journal of Microscopy, 235: 25–35

In order to avoid the formation of the crystalline phase, it has been tried in practice to convert biological samples by rapid cooling into a glassy or amorphous phase (vitrified state), which, however, had only limited success, even with the use of cryoprotectants. Therefore, until a publication by JLM Leunissen et al. [4] the idea that vitrification (vitrification) of cellular cell suspensions without additives would not be achievable for physical reasons.

• [4] JLM Leunissen and H. Yi (2009) Journal of Microscopy, 235: 25-35

From JLM Leunissen et al. [4] For cryomicroscopy, a method is described which is expected to vitrification: a thin-walled copper tube with a diameter of <1 mm is filled with a cell suspension, closed at the ends by squeezing gas-free and then in a cooling fluid such as propane, nitrogen etc., frozen in cryoprecipitations of the tubes excellent cell structures are obtained which indicate a Vitrifizierung. However, heating the cryogenic cryosamples shows that the cells did not survive the cooling process. The paper by JLM Leunissen et al. [4] described method is unsuitable for vitality-preserving heating of the sample. So far, it is therefore assumed that methods for cryomicroscopy do not allow revitalization by reheating.

There is an urgent interest in reducing or overcoming the disadvantages of conventional cryopreservation in view of evolving regenerative medicine and biotechnology as well as environmental protection and conservation.

The object of the invention is to provide an improved cryopreservation device which overcomes the disadvantages of conventional techniques and in particular enables cryopreservation with an increased vitality rate and / or increased sample volume. The object of the invention is furthermore to provide an improved method for cryopreservation which overcomes the disadvantages of conventional techniques and which, in particular, makes it possible to adjust process conditions during cooling and / or thawing in such a way that an increased vitality rate is achieved. Furthermore, it is an object of the invention to provide an improved method for heating a cryopreserved biological sample, which overcomes the disadvantages of conventional techniques and in particular allows to suppress a vitality-limiting influence of the biological samples in the transition to a thawed state.

These objects are achieved by devices and methods having the features of the independent claims. Advantageous embodiments and applications of the invention will become apparent from the dependent claims.

According to a first aspect of the invention, there is provided a cryopreservation device adapted for cryopreserving a biological sample. The cryopreservation device comprises a pressure vessel having a container wall and an interior adapted to receive the biological sample. The pressure vessel is configured to be stored in a cooling bath of a cooling device up to a cryopreservation temperature, e.g. Below -138 ° C, to be cooled. Furthermore, the pressure vessel is configured such that the interior of the cooling tank can be pressurized to a pressure higher than the surrounding atmospheric pressure. The pressure vessel is designed for the permanent cryopreservation of the biological sample and storage at the cryopreservation temperature while maintaining the elevated pressure in the pressure vessel.

According to the invention, the pressure vessel comprises an adjusting device, which is set up for a time- and / or location-dependent adjustment of the temperature and the pressure in the pressure vessel. The adjusting device is connected to the container wall of the pressure vessel and suitable to influence the setting of the cryopreservation temperature and the pressure in the interior of the pressure vessel time and / or location-dependent. The adjusting device is set up to specifically control the cooling and / or heating of the biological sample in accordance with a predetermined temperature-time function. Furthermore, the adjusting device is suitable for influencing the temperature distribution in the pressure vessel. Furthermore, the adjusting device is adapted to control the pressure in the pressure vessel according to a predetermined pressure-time function. The temperature and pressure-time functions can be adjusted relative to each other. For example, the adjusting device allows targeted first to cool the biological sample and then with an increased pressure to apply the cooling and the pressure increase simultaneously or first to increase the pressure and then adjust the cryopreservation temperature. Finally, the actuator is suitable to control the location of pressure generation in the pressure vessel.

According to the invention, the adjusting device comprises at least one Druckeinstellelement, at least one cooling element and / or at least one heat conducting element. The pressure adjusting element is configured to adjust the pressure-time function, a location of a primary pressure input and / or the amount of pressure applied to the interior of the pressure vessel. Accordingly, the cooling element and / or the heat-conducting element, optionally in conjunction with the action of the cooling device, are arranged to control the temperature-time function, the spatial temperature distribution and the cryopreservation temperature.

The biological sample contains biological cells and an aqueous preservation medium. The biological cells comprise individual cells, such as. B. individual stem cells, progenitor cells, fibroblasts, germ cells, cell groups, in particular from the cell types mentioned, pieces of tissue or organs or parts thereof. The biological cells may even form complete organisms, especially microorganisms such as worms or insects. The preservation medium comprises an aqueous physiological medium (nutrient medium, culture medium) as known from the cultivation of biological cells.

According to a second aspect of the invention, there is provided a method of cryopreserving the biological sample by placing the biological sample in a pressure vessel and cooling the pressure vessel in a cooling device until the biological sample is transferred to the cryopreserved state in an at least partially glassy phase , According to the invention, a time- and / or location-dependent adjustment of the temperature and the pressure in the pressure vessel using an adjusting device with at least one Druckeinstellelement, at least one cooling element and / or at least one heat-conducting takes place. The cryopreservation device according to the first aspect of the invention is preferably used to carry out the method according to the invention for the cryopreservation of biological samples.

According to a third aspect of the invention there is provided a method of heating a biological sample comprising biological cells and a preservation medium in a frozen state disposed in a glassy phase in a pressure vessel. According to the invention, the temperature of the pressure vessel and the biological sample is increased and maintained during the temperature increase in the pressure vessel, an elevated pressure above the ambient atmospheric pressure. Preferably, the method of heating the biological sample is carried out using the cryopreservation device according to the first aspect of the invention.

According to a fourth aspect of the invention, it is proposed to use at least one stabilizer substance (ie a single substance or a mixture of several substances) in the cryopreservation of biological samples as a constituent of the preservation medium which is suitable for increasing the temperature of a biological sample biological cells and a preservation medium, preferably until the transition to the liquid state to obtain a glassy state of the supercooled melt without crystallization.

The invention is based on the recognition that when freezing aqueous systems, the formation of the crystalline phase can be avoided and instead the glassy phase (amorphous phase) can be produced by applying a pressure to the aqueous system. The transfer of the biological sample into the cryopreserved state, the cryopreservation and / or the heating of the cryopreserved samples take place in a region of the phase diagram of the aqueous system in which the glassy phase is preferably 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 enables an increased vitality rate of the cryopreservation.

In conventional cryomicroscopy using pressure-resistant sample tubes (see above, [4]), an increased pressure was already generated in the closed sample tube. In the conventional technique, however, there is no degree of freedom to influence the pressure or temperature course during freezing, during the cryopreservation or during heating specifically according to predetermined time functions. The inventors have found that, especially during thawing, inevitably the ice avoided during rapid cooling is produced with all its negative effects on the sample, whereby the cells are killed. Advantageously, with the provision of the adjusting device according to the invention it is made possible that the pressure-temperature curves are controlled temporally and / or spatially so that in the interior of the Pressure vessel primarily the glassy phase of the biological sample is generated and maintained. In contrast to the conventional technique, the adjusting device makes it possible to selectively vary pressure and temperature parameters during freezing and / or thawing in order to achieve a maximum vitality rate. Advantageously, according to the invention, a true cryopreservation with the possibility of re-thawing and revitalization of the cells in the biological sample is provided, while the art of JLM Leunissen et al. [4] represents only a cryopreparation for microscopic examinations.

According to the above-mentioned third aspect of the invention, it is particularly proposed to obtain the glassy state of the sample, particularly the preservation medium, while maintaining the pressure until the water contained in the sample is melted. Advantageously, this allows cryopreserved biological samples such. B. from JLM Leunissen et al. [4] described in extremely good structural preservation and vitrified cell suspensions to thaw again so that living cells are present. This has not yet been achieved in any freeze-pressure shock method as used for cryomicroscopy. In order to achieve said stabilization, the preservation medium is preferably stabilized with at least one stabilizer substance capable of stabilizing the glassy phase of the supercooled melt as the temperature of the biological sample increases, preferably to the melt. The cryopreservation device according to the invention advantageously makes it possible to influence the path of the sample conditions through the phase diagram past the solid-liquid phase transition.

According to a preferred embodiment of the invention, the adjusting device comprises at least one Druckeinstellelement which is connected to the container wall and / or the interior of the pressure vessel. Advantageously, various variants of Druckeinstellelements are possible, which can be selected individually or in combination depending on the preservation task and the spatial conditions of the cryopreservation. According to a first variant, at least one pressure screw can be provided in the container wall of the pressure vessel. The container wall contains a threaded opening for liquid-tight receiving the pressure screw. By screwing the pressure screw into the threaded opening, the free volume of the interior can be reduced in the pressure vessel and increased according to the pressure in the pressure vessel. According to a second variant, an expansion region can be provided, which communicates with the interior of the pressure vessel and is adapted to receive a liquid or gaseous expansion medium. If the expansion medium expands in the expansion area, the free volume in the interior is correspondingly reduced and the pressure in the pressure vessel is increased. According to a third variant, the Druckeinstellelement may comprise a pressure clamp, which acts from the outside on the container wall. In this case, the container wall is formed of a flexible material to transmit the pressure exerted by the pressure clamp mechanical pressure on the interior of the pressure vessel. Advantageously, the pressure clamp can be equipped 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 may be designed for mechanical or electrical actuation.

According to a particularly preferred embodiment of the invention, the expansion region comprises at least one hollow conduit for receiving the expansion medium, which communicates with the interior of the pressure vessel and which protrudes from the pressure vessel. The hollow line contains z. As water, an aqueous solution or parts of the biological sample. By protruding the hollow conduit from the pressure vessel, the expansion medium in the hollow conduit is spatially separated from the interior of the pressure vessel. This advantageously allows a cooling of the hollow conduit in the cooling bath of the cooling device, while the remaining pressure vessel is still at elevated temperature, for. B. is at room temperature. In the hollow conduit crystalline ice can be generated, which expands towards the interior, reduces the remaining volume and thus causes an increase in the pressure in the interior. According to a preferred variant, the hollow conduit has branches. Advantageously, this makes it possible to increase the volume of the expansion area in comparison to a single, unbranched hollow conduit. At the same time, the branched hollow line makes it possible to influence the time function of the pressure generation in the pressure vessel. Alternatively and additionally, a plurality of hollow conduits may be provided which protrude from the pressure vessel in different spatial directions. Advantageously, this makes it possible to influence the pressure generation as a function of the orientation of the pressure vessel relative to the cooling bath of the cooling device.

If the adjusting device provided according to the invention comprises a cooling element, then this is preferably formed by a cooling line, which is arranged in the pressure vessel. The cooling line runs through the interior of the container. It is adapted to with a coolant such. B. with liquid nitrogen to be flowed through. Advantageously, the cooling line allows adjustment of the beginning and the time function of the temperature reduction in the pressure vessel.

If the adjusting device provided according to the invention comprises a heat-conducting element, this is preferably formed by an outer profile on an outer side of the pressure vessel, an inner profile on an inner side of the pressure vessel and / or heat-conducting body in the interior of the pressure vessel. These variants of heat conducting elements make it possible to accelerate the transport of heat from the pressure vessel into the cooling bath during the cooling of the pressure vessel.

Another advantage of the cryopreservation device according to the invention is that the pressure vessel can be produced with a large number of geometric shapes. Particularly preferred variants of the invention in which the container wall of the pressure vessel, the shape of a tube, a sphere or a cylinder, for. B. a flat cylinder (box) has. A tubular pressure vessel may be straight or curved. In both cases, the spatial distribution of the temperature and pressure setting in the pressure vessel can each be influenced by its shape.

Further advantageous features of the cryopreservation device according to the invention relate to the design of the interior of the pressure vessel. According to a variant of the invention may be provided in the interior of an inner container, which is adapted to receive the biological sample. In this case, the biological. Sample in the inner container from the remaining volume of the interior materially separated. This allows influencing the glassy phase in the immediate vicinity of the biological sample. According to a further variant, a substrate can be arranged in the interior, which is set up for the adherent recording of biological cells which are part of the biological sample. The substrate may, for. B. be arranged in the inner container. As a substrate substrate materials are suitable, which are used for adherent cell cultures, such. As plastic or glass. According to a further variant, a segmentation of the interior can be provided in interior sections. The segmentation advantageously enables the targeted adjustment of different pressure-temperature conditions in each of the interior sections. According to a further variant, a sensor device may be arranged in the interior, which comprises at least one temperature sensor and / or at least one pressure sensor. The sensor device advantageously allows a measurement of the temperature and / or the pressure in the interior. Depending on the at least one signal of the sensor device, the cryopreservation can be controlled. Finally, with a further variant of the invention, a substance reservoir can be arranged in the interior, which is set up to release a substance into the interior. The substance reservoir comprises z. B. hollow spheres that can be destroyed under the effect of increased pressure in the interior to release a substance. The mentioned variants of the interior design can be provided individually or in combination.

According to a further advantageous embodiment of the invention, the container wall may be equipped with an optical unit. The optical unit comprises an imaging optics, which is set up for an optical observation of the interior of the pressure vessel. The optical unit allows optical monitoring of the state of the biological sample during cryopreservation.

Advantageously, there are different variants of the pressure and temperature setting in the pressure vessel in order to arrive in different ways in the pressure-temperature-state diagram of the biological sample to the desired Kryokonservierungsbedingungen. For example, according to a first variant, it is possible first to increase the pressure in the pressure vessel with the at least one pressure setting element and then to lower the temperature in the pressure vessel with the cooling device. The pressure increase and the temperature reduction are in each case according to predetermined, separate time functions. According to a second variant, the increase of the pressure and the lowering of the temperature can be adjusted so that the respective time functions overlap by starting the lowering of the temperature before reaching the final pressure or vice versa the increase of the pressure before reaching the Kryokonservierungstemperatur. Finally, according to a further variant, it is possible first to lower the temperature in the pressure vessel to the cryopreservation temperature and then to increase the pressure in the pressure vessel. Which of the mentioned variants is chosen depends on the conditions of the specific cryopreservation task, in particular on the structure of the cryopreservation device and the composition of the biological sample. The optimal variant can be selected empirically by tests in which the vitality rate of the biological sample for the different variants is tested under the specific conditions of use. In the same way it is possible, the pressure and temperature-time functions, in particular with respect to the speed of the pressure increase and the temperature reduction and / or the shape of the function, such. B. a step shape to choose.

If, according to a preferred embodiment of the invention, the pressure-setting element comprises an expansion region in the form of a hollow line projecting from the pressure vessel, the pressure Temperature setting simplified. The pressure can be increased in the pressure vessel by first immersing the at least one hollow conduit in the cooling bath of the cooling device. An expansion medium in the at least one hollow conduit expands, so that the pressure in the rest of the pressure vessel increases. Subsequently, the remaining pressure vessel is immersed in the cooling bath of the cooling device, so as to set the cryopreservation temperature for the biological sample in the interior of the pressure vessel.

Preferably, a permanent storage of the biological sample is provided while maintaining the elevated pressure, in particular in liquid nitrogen or in the vapor of the liquid nitrogen. Particularly preferably, the storage of the sample takes place in the pressure vessel.

According to a particularly preferred embodiment of the method for cryopreservation, the preservation medium contains at least one stabilizer substance which is suitable for stabilizing the glassy phase of the supercooled melt when the temperature of the biological sample is increased, preferably until it changes to the liquid state. , Advantageously, the stabilizer substance causes the glassy phase to be maintained at higher temperatures than would be the case without the stabilizer substance. The glassy phase is maintained longer on thawing, and the formation of the crystalline phase is avoided. Furthermore, the glass transition temperature of the preservation medium is increased by the stabilizer substance. The stabilizer substance results in a reduced number of nucleation nuclei in the preservation medium, thus reducing the likelihood of nucleation and minimizing ice formation on thawing.

According to this embodiment, the above objects are achieved, in which the biological sample, e.g. B. a cell suspension, which is added at least one stabilizer substance. The at least one stabilizer substance is used, as it has been used only conditionally or not at all for conventional cryopreservation or according to the invention develops its effect at much lower concentrations and this is different than that of the known cryoprotectants. As the stabilizer substance preferably substances are selected which do not penetrate into the cells at atmospheric pressure and therefore normally find no use in the conventional cryopreservation of cells and tissues. The remarkable thing about the use of the stabilizer substance is that experiments by the inventors showed that with an addition of z. B. Percoll or Ficoll in the range of a few percent mammalian cells, the Druckschockgefrier- and thawing procedure similar to publication [4] with high survival rate (> 97%) survived. This is all the more astonishing since it can not be assumed that the cells have penetrated the cells.

The surprising effect of the stabilizer substance is that, for the first time during the thawing of the sample, it allows the return path via the combination of high pressure / rapid freezing and pressure-controlled heating with virtually unaffected vitality of the cells. Thus, it is the combination of at least one substance dissolved in the preservation medium which allows the return from the nitrified region by influencing the water structure and the presence of nucleation germs.

The stabilizer substance differs materially, in its effect and / or in relation to the preferred concentration selected from the conventional cryoprotectants. In contrast to the stabilizer substance, the number of nucleation germs is deliberately increased by conventional cryoprotectants in order to promote the formation of the largest possible number of ice crystals during freezing. However, with the number of ice crystals, their chance of size growth also decreases, so that large crystals are prevented by conventional cryoprotectants. For this purpose, in conventional cryoprotectants a distribution and high mobility in the preservation medium is desired to the cells.

Preferably, the stabilizer substance is selected from at least one of the substance groups, the long-chain uncharged polymers having 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), especially with silver, gold, diamond and / or nanotube particles, dendrimers, polycations, and polyanions. Long-chain polysaccharides, in particular hydrophilic copolymers prepared from sucrose and epichlorohydrin (Ficoll, reg. Name), and / or Polivinylpyrrolidone, in particular polyvinylpyrrolidone-coated silica gel (Percoll, reg. Name), having a molecular mass of between 2,000 and 2,000, have proven particularly suitable several million g / times, nanoparticle dispersions, in particular silver, gold, diamond and / or nanotube particles, are particularly advantageous since they are suitable for reducing the thermal conductivity of the preservation medium. Advantageously, the stabilizer substance is biocompatible, so that the cells in the biological sample are not adversely affected by the stabilizer substance.

The concentration (% = vol.%) Of the stabilizer substance is preferably less than 30%, particularly preferably less than 20%, in particular less than 10%, such. B. less than 3% or less than 1%. A preferred minimum concentration is 0.1%.

According to a further preferred embodiment of the invention, the stabilizer substance is arranged in the preservation medium outside the biological cells. The cells remain free of the stabilizer substance, which has advantages for the vitality after thawing the sample.

A further advantage of the stabilizer substance may be that it changes its properties, in particular structure and / or molecular weight, under the effect of the increased pressure and the reduced temperature during the cryopreservation. For example, molecules of the stabilizer substance can be broken down into fragments so that they can diffuse into the interior of the cells in order to achieve an additional cryoprotective effect there.

It is emphasized that when the stabilizer substance is added to the preservation medium, it is not necessary to maintain an elevated pressure in the pressure vessel until the biological sample reaches the transition from the supercooled melt to the liquid state. In this case, the pressure may drop even before reaching the transition, in particular reduced to atmospheric pressure.

According to a preferred variant of the o. G. Thus, in a third aspect of the invention, there is provided a method of heating a biological sample comprising biological cells and a preservative medium in a vitrified state disposed in a glassy phase in a pressure vessel, wherein the temperature of the pressure vessel and the biological sample is increased; until the biological sample reaches a liquid state, and wherein the preservation medium contains the at least one stabilizer substance capable of stabilizing the glassy state upon elevation of the temperature of the biological sample, preferably until transition to the liquid state.

According to a preferred embodiment of the invention, upon reaching the transition from the glassy state of the supercooled melt to the liquid state, the pressure in the pressure vessel is reduced. This minimizes damage to the biological sample after reaching the liquid state. Particularly preferably, the pressure in the pressure vessel is instantaneous, d. H. in particular stepped and with negligible delay, reduced.

The pressure reduction can be achieved by the pressure vessel released, z. B. is opened, so that a pressure equalization is achieved with an external atmospheric pressure. Advantageously, however, a release of the pressure vessel is not mandatory. Rather, the pressure reduction can also be generated by a contraction of the biological sample at the transition from the supercooled melt to the liquid state. The volume of the sample may vary according to the below with reference to the 1 to 4 reduce the processes explained so that the pressure in the pressure vessel drops.

According to a further preferred embodiment of the invention, the elevated pressure above the atmospheric pressure is at least 100 MPa, in particular at least 150 MPa and / or at most 300 MPa, in particular at most 250 MPa. These pressure ranges have proven to be particularly advantageous for a rapid transition from the glassy state to the liquid state and vice versa.

Further details and advantages of the invention will be described below with reference to the accompanying drawings. Show it:

1 to 4 : Phase diagrams with illustrations of different phases of water, and

5 : Embodiments of the cryopreservation device according to the invention;

6 : graphical representations of different variants of the pressure and temperature-time functions;

7 to 11 experimental results illustrating the effect of a cryoprotectant or a stabilizer substance;

12 : schematic sectional views of various variants of a pressure vessel, which is equipped with Wärmeleit- and / or cooling elements;

13 to 19 : further embodiments of cryopreservation devices according to the invention and their use;

20 to 28 : further embodiments of cryopreservation devices according to the invention, in which the pressure vessel has the shape of a flat cylinder;

29 a schematic illustration of an optical unit in the container wall of a pressure vessel;

30 a further embodiment of the cryopreservation device according to the invention and its use;

31 to 35 : further embodiments of cryopreservation devices according to the invention, in which the pressure vessel has the shape of a sphere or a cylinder;

36 a further embodiment of the cryopreservation device according to the invention;

37 to 39 : Further embodiments of inventive cryopreservation devices, in which a substrate is arranged in the interior of the pressure vessel;

40 and 41 : Further embodiments of inventive cryopreservation devices with a segmentation of the pressure vessel interior; and

42 A further embodiment of the cryopreservation device according to the invention and its use.

The invention will be described in the following by an explanation of findings of the inventors and then by the details of the cryopreservation device and the process control. It is emphasized that the following theoretical considerations serve as an approach to explain the excellent vitality rates achieved with the cryopreservation of the present invention. However, the embodiment of the invention is not bound to the completeness and accuracy of the theoretical considerations.

Theoretical considerations of the phase diagrams of aqueous systems and the effect of stabilizer substances

Cryopreservation has so far been described empirically and under simplifying 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 variety of carbohydrates, ions of numerous elements, nanoscale systems such as membranes, organelles and structural elements, such as the cytoskeleton, and bound water on surfaces, phase diagrams of the water are of limited use. However, for illustrative purposes, the inventors' explanation is referred to phase diagrams of the water which are incorporated herein by reference 1 . 2 and 4 are shown.

The pressure-temperature-state diagram of the water ( 1 , Source: Jackson et al., J. Phys. Chem. (1997) quoted in www.wikipedia.de , Keyword water) shows that upon cooling under normal pressure (0.1 MPa = 0.0001 GPa, line on the ordinate axis) so-called Ih (hexagonal ice) is formed. This takes up a larger volume than the liquid phase (about 1/10), which leads to mechanical stresses (pressure) in circumscribed volumes. In addition, in the formation of ice, a separation of multicomponent solutions (ions and other molecular components are concentrated), which otherwise does not occur in the cytoplasm and should therefore be avoided.

Like the phase diagram of the 1 shows that there are other ice structures that are known not to exhibit the stated volume increase at higher pressures. However, these are pressures (eg> 0.1 GPa) below which biological objects in the unfrozen state would be destroyed.

• 1. Bis 200 MPa liegt sogenanntes LDL-(Low Density Liquid, unterkühlte Flüssigkeit) Wasser in seinen Aggregatszuständen vor, das all die bei Normaldruck auftretenden Anomalien aufweist.
• 2. Darüber entstehen HDL-(High Density Liquid) Zustände, die möglicherweise für die Kryokonservierung günstiger wären. Es handelt sich aber um unphysiologisch hohe Drücke (in der Tiefsee werden maximal 100 MPa erreicht).

2 shows in extended state diagrams of the water and its metastable states that there are opportunities to influence ice formation. By adding substances, the freezing point z. B. be lowered stable (colligative effects). That's the principle that organisms use in nature. They freeze so z. At -10 ° C or -20 ° C. Furthermore, under certain conditions, an aqueous solution can be subcooled. Ice melts with pure water and atmospheric pressure at 0 ° C, but it does not freeze at this temperature. This requires nucleation. In so-called homogeneous nucleation, freezing is a stochastic process that is triggered by the smallest disturbances. In both cases, the liquid phase extends down to -50 ° to -70 ° C. When considering the pressure range up to 300 MPa = 3,000 atm ( 2 , right side), it follows:

• 1. Up to 200 MPa, so-called LDL (Low Density Liquid) water is present in its aggregate states, which has all the anomalies occurring at atmospheric pressure.
• 2. This creates HDL (High Density Liquid) conditions that may be more favorable for cryopreservation. However, these are unphysiologically high pressures (in the deep sea a maximum of 100 MPa is reached).

In the metastable states, at a temperature of about 136 K = -137 ° C, a limit temperature is found below which a glassy state of the water could be taken even at physiological pressures. Water then solidifies like glass, namely amorphous, ie without crystal formation. The water molecules then stay where they are. This is a metastable state. Achieving it is the desired state one seeks when deep-freezing biological objects. It is called "vitrification". The vitrification requires a very high cooling rate (> 10 ⁶ ° / s) for the rapid fluctuation of the water molecules, which in the dimension of a cell (> 10 μm) is no longer achievable due to the limited thermal conductivity of the water (max ⁴ ° / s). As the size of the objects to be frozen increases, the cooling rate gap becomes more and more dramatic (many orders of magnitude), so that true vitrification (formation of a glassy phase without additives) has hitherto not been possible.

The inventors have found that due to the anomaly of the water in the LDL range, the freezing point, however, can also be lowered by increasing the pressure. This process reverses exactly upon the transition from LDL to HDL, so that about 200 MPa is the preferred pressure upper limit still effective for freezing point depression. Thereafter, the freezing point, as in any other liquid normally, increases again with increasing pressure.

The path to vitrification (vitreous phase) is the shortest at this pressure (cf. 2 , right diagram, vertical line in the middle at 0.2 GPa), only a little more than 100 degrees must be passed through quickly, which is why one applies the shock freezing. A long-standing paradox of cryopreservation is that in pressure-shock freezing, the structures of the cells are best preserved, but there are no surviving cells after thawing. On the other hand, it is known that with slow cooling with even microscopically visible Eisdomänenbildung and damage to the cells (membrane leaks) it can lead to survival rates of over 90%. The inventors have found that the reason for this is mainly in the thawing process. Here you go through again the critical range of -137 ° C to the melting point (maybe -5 to -15 ° C), so that the avoided during freezing or reduced Ih-ice still takes place. Even high-pressure ice quickly transforms into low-pressure ice at low pressure. In addition, according to the thermal shocks (k · T) from -100 ° C with increasing temperature, the probability of a change of place of a water molecule is also relevant in the frozen phase. This leads to the so-called "migratory growth" of large ice crystals, as they increase in size at the expense of smaller ones (due to surface tension differences). Therefore, long-term storage must be carried out below the glass transition temperature (-137 ° C).

But there is another way to achieve vitrification or at least a "pseudo-vitrification" by adding additives that lead to very small ice domains. It's not really a physiological path either, but it's been used for a long time in freezing and works well at normal pressure.

In principle, there is still the possibility of increasing the glass transition temperature by adding additives. By adding trehalose can be z. For example, move the glass transition temperature to near 0 ° C, which would be ideal for biological objects. However, the required concentrations of trehalose are above any physiological compatibility for living cells. In freezing point depression, profits are low ( 3 , Freezing point depression and shift of the glass transition temperature by addition of trehalose), which applies to almost all such substance additives.

4 makes it clear once again that in the LD range, during cooling and thawing, one critical area in each case, which is referred to in the diagrams as "no mans land", has to pass through. But to reach this area or to aim at complete vitrification would have been the solution to the problem.

According to a practical example, it is envisaged to prepare a biological sample in a manner known per se with biological cells and a preservation medium at room temperature and under atmospheric pressure. At least one stabilizer substance is preferably added to the preservation medium at a concentration of less than or equal to 5%, or this takes place during the cooling process or else only in the low-temperature state or before thawing. But it can also be provided higher concentrations.

Suitable substance groups for the stabilizer substance, in particular for use with the embodiments of the cryopreservation device described below and the methods for cooling or heating biological samples, are: material class Examples substance alcohols ethylene glycol monosaccharides glucose fructose mannose galactose ribose xylose arabinose as well as non-naturally occurring sugars Di- and oligosaccharides Sucrose (cane sugar) Lactose (milk sugar) Maltose (malt sugar) trehalose cellobiose polyether polyethylene glycol Molecular weight 1000 to 35,000 polysaccharides cellulose hemicellulose amylose glycogen pectins Dextrane (artificial) alginates Ficoll, in particular with a molecular weight of 2500 g / mol to 2.5 million g / mol

Other media that have achieved excellent experimental results using the cryopreserving device embodiments described below and the methods for cooling or heating biological samples are: dextran Cryomedium to Mazur Cryomedium to Matsumura 30% dextran Ethylene glycol 10% (3.2M) Ethylene glycol 20% (6.5 M) Acetamide 10.7% (3.27 M) e-poly-L - lysine 10% Ficoll 70 24% (3.5 mM) Sucrose 10.9% (0.4 M) Sucrose 25.7% (0.75 M) BSA & glucose (low concentrations) 325 mOsm 3.4 Osm 6.7 osm

In experiments of the inventors z. As the cryomedium to Mazur with an addition of ethylene glycol and dextran proved advantageous.

Embodiments of the cryopreservation device and methods for cooling or heating biological samples

The 5A to 5C show embodiments of the cryopreservation device according to the invention 100 holding a pressure vessel 10 and an actuator 20 having. The pressure vessel 10 has a container wall 11 in the form of a tube, the interior of which is inside 12 of the pressure vessel 10 forms. The adjusting device 20 includes pressure adjustment elements, each by pressure screws 21 at the axial ends of the pressure vessel 10 be formed. A third pressure screw 21 can be radially projecting on the container wall 11 z. B. along the half axial length of the pressure vessel 10 be provided ( 5B ).

The pressure vessel 10 preferably has an outer diameter of less than or equal to 5 mm, more preferably less than or equal to 2 mm or 1 mm, z. B. is 0.5 mm. The axial length of the pressure vessel 10 is z. B. in the range of 10 mm to 20 cm. The thickness of the container wall 11 is z. B. 1/4 to 1/10 of the outer diameter of the pressure vessel. The container wall 11 is z. B. made of stainless steel, aluminum, gold or silver. Alternatively, other metals or alloys can be used which have a high thermal conductivity for rapid cooling in the interior 12 and a pressure resistance for prints up to z. B. 200 MPa. Furthermore, the pressure vessel made of a plastic or a composite material, for. As plastic-metal composite, be made.

For holding the pressure screws 21 points the pressure vessel 10 at its axial ends internal thread on. For receiving the radially protruding pressure screw 21 ( 5B ) is the container wall 11 provided with a threaded neck. The pressure screws 21 can also be used as a bleeder screw. Deviating from the illustrations of the 5 can as a setting device a single pressure screw 21 be provided (see, for example, 14A ).

According to 5C can the interior 12 of the pressure vessel 10 in individual interior sections 15 be divided. The segmentation with interior sections 15 has been shown to be beneficial for the vitality rate of biological cells in the middle interior sections 15 is maximum.

For the cryopreservation of a biological sample 1 , comprising z. B. biological cells 2 in a preservative medium 3 , the sample will 1 in the interior 12 of the pressure vessel 10 filled (see partially open view of the container wall 11 in 5A ). The preservation medium contains one or more stabilizer substances, such as. B. Dextran at a concentration of 30% mixed with 10% ethylene glycol. If the interior 12 is completely filled, the pressure screws 21 so closed, that the interior 12 is bubble-free. To fill the interior bubble-free, the inside of the container wall must 11 from the biological sample 1 be completely wet. For this purpose, the inside of the container wall 11 be hydrophilic coated. Furthermore, a venting takes place via one of the pressure screws 21 , By penetration of the pressure screws 21 If necessary, even at room temperature, an increased pressure in the interior 11 of the pressure vessel 10 , z. B. be set in the range of 0.1 MPa to 300 MPa. The level of pressure can be determined by calibration tests or by using a sensor device 16 (see below). Optionally, in or after this phase by squeezing the container wall 11 the segmentation in interior sections 15 ( 5C ) be provided.

For cryopreservation of biological samples becomes the filled cryopreservation device 100 in the cooling bath of a cooling device (in 5 not shown, see e.g. B. 13 ) cooled. For this purpose, the pressure vessel is preferably immersed horizontally in the cooling bath, so that the cooling along the entire axial length of the pressure vessel 10 essentially simultaneously. The cooling bath comprises z. As liquid propane, liquid nitrogen or other liquefied gas.

When immersed in the cooling bath, ice is first formed on the inside of the tank wall 11 , Since the crystalline phase is first formed, an expansion takes place, which results in an increase in pressure in the interior up to a final pressure of 200 MPa. Above this pressure, further ice growth is ruled out, leaving the rest of the biological sample 1 into a glassy (vitrified) state or at least not having a hexagonal crystalline phase.

When the cryopreservation device 100 in the cooling bath, the cryopreservation temperature, z. B. -197 ° C, the further cryopreservation in the cooling bath or in a storage container (not shown) can be carried out by liquid nitrogen or by vapor of the liquid nitrogen to a temperature of z. B. -140 ° C is cooled.

For vitality-preserving warming of the biological sample 1 becomes the cryopreservation device 100 in a heating bath (liquid bath at a temperature above 0 ° C), comprising, for. As water, alcohol or an oil immersed. The immersion is also preferably carried out horizontally and at high speed, so that the increased pressure in the pressure vessel 10 retained until the crystalline ice formed melts. Subsequently, the pressure drops abruptly to z. B. 0.1 MPa.

The 6A to 6D show four typical temperature and pressure-time functions, such as in the cryopreservation (left part of the curves) or in the thawing (right part of the curves) with the cryopreservation device 100 can be realized according to the embodiments of the invention explained here. 6A shows, for example, how the freeze first increases the pressure. For this purpose, for. B. the pressure screws 21 according to 5 used. Only after reaching the pressure of 200 MPa, the temperature is lowered by the immersion (Einwerfen) of Kryokonservierungsvorrichtung 100 in the cooling bath. When thawing, conversely, first a pressure relief and then a heating of the biological sample to room temperature can be provided. 6B shows the opposite variant in which the cryopreservation first lowers the temperature and then the pressure is built up. In this case, the pressure screws 21 only after reaching the cryopreservation temperature of z. B. -200 ° C actuated. When thawing a pressure relief during the temperature rise can be provided. 6C shows a more complicated temperature-time function, which can be chosen depending on the concrete preservation conditions. With the cryopreservation device according to the invention 100 It is also possible to realize a pressure-temperature curve, as would be the case with a cryopreservation device without an adjusting device ( 6D ), wherein the pressure build-up immediately after the freezing point is started by the formation of the crystalline phase of ice in the biological sample. With the adjusting device used according to the invention, the shape of the time functions is influenced. According to a further variant of a temperature and pressure-time function (not shown), it can be provided that the increased pressure is continuously set throughout the entire preservation period.

7 shows a sequence of microscope images of a section of the sample 1 with cells 2 at room temperature, after the preservation medium 3 a cryoprotectant was added. The time (in seconds, s) shows the time dependence after addition of the stabilizer substance to the sample 1 , It becomes clear that a considerable shrinking process takes place in a short time. The inventors have found that this shrinkage can be beneficial for achieving high vitality rates.

The 8th and 9 The dramatic shrinkage of the cells (here HeLa cells) clearly indicates the residual osmotic volume, which is beneficial for high vitality rates. The round shape of the cells ( 9A to 9C , top left) is completely lost ( 9A to 9C , right and below). The decrease in the diameter of the cells was determined using an Invitrogen measuring system (diagram in FIG 8th ), but does not completely reflect the osmotic shrinkage because the amount of deviation from the spherical shape is not taken into account. Consequently, the cells shrink more than can be seen from the graph number value.

10 shows the shrunken cells in the electron microscopic section. It can be seen the extremely strong shrinkage, which is achieved in the cryogenic medium with one of the above compositions, so that the cell membrane system is strongly folded. In 11 the structural changes of adherent cells (HeLa) are shown. Again, the osmotic shrinkages occur very quickly (after seconds).

Fluorescence microscopy studies with a different representation of the cell nuclei and the Golgi apparatus (not shown here) have shown that shrinking only densifies the cytoplasmic components, but does not affect the cell nuclei and other important cell organelles, resulting in vitality-preserving freezing and thawing of large cells Advantage is.

12 shows variants according to the invention provided Wärmeleit- and / or cooling elements. For comparison purposes, first shows 12A the circular cross-section of the tubular pressure vessel 10 (please refer 5 ). The 12B to 12I show embodiments of the cryopreservation device according to the invention in a schematic sectional view of the pressure vessel 10 in which the adjusting device is set up for setting the temperature using at least one heat-conducting element or at least one cooling element. According to 12B Heat-conducting elements comprise external profiles 35 that on an outside of the container wall 11 are arranged radially projecting. The external profiles 35 accelerate the cooling or heating of the pressure vessel 10 when immersing in the cooling bath or the warm liquid bath. According to 12C are interior profiles 36 on an inside of the container wall 11 arranged. The inner profiles 36 also assist in the transport of heat from or to the biological sample in the pressure vessel 10 ,

The 12D to 12G illustrate a pressure vessel 10 with a hexagonal, hexagonal cross-section. The outside of the container wall 11 forms the outer profile 35 which offers a large surface area for wetting with a coolant or a thawing agent and thus is suitable for influencing the temperature-time function during cooling or thawing. According to the 12E . 12F and 12G are additionally in the interior of the pressure vessel 10 thermal conductors 37 in the form of partitions ( 12E ), inserted filaments or balls ( 12F ) or colloidal particles ( 12G ) intended. The colloidal particles 37 according to 12G are shown schematically enlarged, but may in practice have dimensions in the sub-micron range. With the outer profiles 35 , Interior profiles 36 and / or Wärmeleitkörpern 37 Advantageously, the cooling or thawing speeds can be increased. The heat-conducting elements are preferably made of silver or gold or other substances with high thermal conductivity. According to other variants, the heat conduction between the interior 12 and the external environment of the pressure vessel 10 by a coating of the container wall 11 on the inside and / or outside, z. B. with diamond or other substances with high thermal conductivity can be influenced.

12H shows a variant of the invention, in the interior 12 of the pressure vessel 10 as a cooling element a cooling line 34 runs. The cooling line 34 is connected to a cooling medium reservoir and arranged by a cooling medium, such as. As liquid nitrogen or a cooling gas to be flowed through. Alternatively, a cooling line 34 adjacent to the interior 12 run as shown schematically in 12I is shown.

13 shows a further embodiment of the cryopreservation device according to the invention 100 ( 13A ) and their use in cooling ( 13B ) and thawing ( 13C ) of the biological sample with cells 2 in the preservation medium 3 , In this embodiment of the invention, the adjusting device for time- and / or location-dependent adjustment of the temperature and the pressure in the pressure vessel 10 three pressure adjustment elements, including pressure screws 21 and an expansion area 22 , The pressure screws 21 are for generating the pressure and / or for venting the interior 12 of the pressure vessel 10 provided (see 5 ). The expansion area 22 includes a radial from the pressure vessel 10 protruding hollow conduit, which at one end via an opening in the container wall 12 with the interior 12 of the pressure vessel 10 communicates and their opposite ,. free end is closed. Between the interior 12 and the expansion area 22 can be a filter 29 be provided for the penetration of biological cells in the expansion area 22 to prevent.

The expansion area 22 is z. B. made of the same material as the container wall 12 of the pressure vessel 10 , The expansion area 22 is designed to receive a liquid expansion medium that expands when cooled. The expansion medium includes z. For example, the aqueous preservative medium of the biological sample or, alternatively, another aqueous liquid suitable for forming the crystalline phase of water. The dimensions (length, inside diameter, outside diameter) of the expansion area 22 can be selected by the user depending on the concrete preservation conditions.

The expansion area 22 allows rapid formation of the crystalline phase when the temperature of the expansion area 22 below the freezing point of water is lowered. Advantageously, the cooling of the expansion area 22 in time from the cooling of the remaining pressure vessel 10 be decoupled, as in 13B is illustrated. For the cooling is a schematically shown cooling device 200 with a cooling bath 210 intended. The cooling device 200 For example, it includes a vessel into which the cooling bath 210 , z. B. from liquid nitrogen, filled and which is connected to a cooling medium reservoir.

For cryopreservation of the biological sample 1 becomes the cryopreservation device 100 initially exclusively with the expansion area 22 in the cooling bath 210 Immersed (immersion depth D1), while the rest of the pressure vessel is still outside the cooling bath. At this stage will be in the expansion area 22 formed crystalline ice that expands, leaving in the interior 12 of the pressure vessel 10 the pressure increases. The duration of pressure generation with the expansion range 22 is z. In the range of milliseconds, seconds or minutes. Subsequently, the cryopreservation device 100 completely in the cooling bath 210 dipped so that the desired cryopreservation temperature of z. B. -195.7 ° C is reached. For this purpose, the cryopreservation device 100 lowered to a second immersion depth D2.

The heating for recovery of the biological sample is carried out according to 13C in the opposite way. To maintain the pressure in the pressure vessel 10 First, the interior with the biological sample is placed in a heating bath 310 a thawing device 300 immersed (immersion depth D1), during the expansion area 22 not cooled yet. After thawing the biological sample 1 in the pressure vessel 10 the lowering takes place up to the immersion depth D2, so that the pressure in the pressure vessel 10 is reduced.

14A illustrates an embodiment of the cryopreservation device according to the invention 100 in the interior 12 of the pressure vessel 10 an inner container 13 is arranged. The inner container 13 is to receive the biological sample 1 provided and includes a bubble-free filled tube made of a flexible material. The inner container 13 is z. B. made of the same material as the container wall 12 of the pressure vessel 10 , The provision of the inner container 13 has the advantage that the formation of the crystalline phase on the inside of the vessel wall 12 from the biological sample 1 is disconnected. Furthermore, in the interior 12 outside the inner container 13 on the one hand and in the inner container 13 On the other hand, various liquids may be provided. For example, outside the inner container 13 pure wet or an aqueous composition of water with a salt, glycerol and / or an alcohol. The aqueous composition has the advantage of a freezing point depression, so that it can be adjusted, at which temperature below the freezing point of pure water, the pressure in the pressure vessel 10 should be generated.

The inner container 13 does not extend over the entire length of the interior 12 , This will be at the closed end 12.1 of the pressure vessel 10 created a relatively large space in which no biological sample 1 and in which preferably the crystalline phase of water can be generated. This will be within the pressure vessel 10 created an expansion area as part of the control device used in the invention, which is advantageous to the cooling and heating of the Kryokonservierungsvorrichtung 100 effect (see in particular 14C ).

The outer shape of the inner container 13 can be equal to the inner shape of the pressure vessel 10 be. As an alternative to the cylindrical shape shown, other cross sections of the outer and / or inner molds may be provided, such. Square, hexagonal, octagonal or all combinations thereof. In particular, different cross-sectional shapes of the outer and inner molds can be provided.

14B schematically illustrates the cooling of the cryopreservation device 100 in a cooler 200 , in this case, two superimposed cooling baths 210 with liquid nitrogen and 220 containing liquid propane. The use of liquid propane has the advantage that this is the outside of the pressure vessel 10 wets more easily and thus accelerates the cooling.

To warm the biological sample 1 in the pressure vessel 10 this is according to 14C in a heating bath 310 dipped. The pressure vessel 10 Can be oriented so that the entire length of the pressure vessel 10 at the same time in the heating bath 310 dives (horizontal orientation). In this case, an increase in the temperature of the biological sample takes place first 1 and, as soon as the crystalline ice has thawed, also reducing the pressure in the pressure vessel 10 , Alternatively, first the closed end 12.1 with the expansion area in the water bath 310 be immersed so that first the crystalline phase melted and the pressure in the pressure vessel 10 reduced and then the remaining biological sample 1 is heated (vertical orientation of the pressure vessel 10 ).

15 schematically illustrates that the application of the cryopreservation device according to the invention 100 is not limited to the preservation of cell suspensions. Rather, the biological sample can 1 Cell groups, cell aggregates, organs 4 biological organisms or complete biological organisms 5 , such as As nematodes, worms or arthropods included. This is indicated by the interior 12 of the pressure vessel 10 an inner diameter in the range of at least 5 mm, preferably at least 10 mm. Preferably, the inner diameter of the inner space 11 less than 5 cm, more preferably less than 3 cm. As in 14 is an inner container 13 for receiving the biological sample 1 provided, which may optionally be divided into individual chambers.

The cryopreservation of the biological sample 1 also takes place in the embodiment according to 15 by removing the pressure vessel 10 in at least one liquid bath of the cooling device 200 is immersed. According to 15C First, a submergence of the pressure vessel 10 with horizontal orientation first in the liquid bath 220 from liquid propane and then into the liquid bath 210 with liquid nitrogen. With the choice of the orientation of the pressure screw 21 relative to the liquid bath 220 . 210 , the time function of the pressure generation can be adjusted relative to the time function of the cooling. The heating takes place according to 15D Corresponding in reverse order, by first the pressure vessel 10 in a heating bath 310 is immersed until the biological sample 1 thawed. Subsequently, the complete lowering of the cryopreservation device takes place 100 in the heating bath 310 ,

The 16 and 17 show further embodiments of the cryopreservation device 100 that with an expansion area 22 Is provided. The expansion area 22 includes as in 13 a hollow conduit having branches in the illustrated variants. According to 16A indicates the expansion area 22 a T-shape with lateral arms on. The expansion area 22 Can first in the cooling bath 210 be immersed (immersion depth D1), so that the liquid freezes inside, forming hexagonal ice and increasing the pressure. Only then the system is fully submerged (immersion depth D2) and completely frozen ( 16B ). When heating, the procedure is reversed, or you have the option in two layers (boom first in the warm phase or as shown here last) in the heating 310 to dive ( 16C ). According to the latter principle, the pressure is maintained by the existing ice until thawing at values of about 200 MPa.

17 shows a further embodiment of the expansion area 22 with branches forming a fan-shaped boom system. As a result, a larger internal volume and a larger surface than, for example, in 16 provided so that the pressure can be increased faster.

18 shows embodiments of the cryopreservation device 100 with a spherical pressure vessel 10 and a plurality of extending in different spatial axes (spatial directions) hollow lines expansion area 22 , The pressure vessel 10 comprises a hollow sphere for receiving the biological sample. The hollow sphere is z. B. made of stainless steel and has an inner diameter of 10 mm and an outer diameter of 12 mm. The hollow conduits form stub lines projecting from the hollow sphere. The number, geometric dimensioning and orientation of the hollow conduits can be selected depending on the specific conditions of use (see examples in FIGS 18A to 18C ).

The filling of the hollow ball is effected by a closable opening (not shown) in the container wall or by one of the hollow conduits, which is equipped in this case with a closure element. Also in these embodiments allows the geometric orientation of the pressure vessel 10 when immersed to immersion depths D1 and D2 in a cooling bath 210 ( 18D ) or in a heating bath 310 ( 18E ) a setting when the pressure in the pressure vessel 10 should rise and fall.

The 19A and 19B show embodiments of the cryopreservation device 100 in which the container wall 11 of the pressure vessel 10 the shape of a curved tube with a single curvature ( 19A ) or a multiple curvature ( 19B ) having. In both cases, the pressure vessel 10 curved in a plane of curvature, so that the local temperature distribution when immersed in a cooling bath depending on the orientation of the pressure vessel 10 can be adjusted relative to the cooling bath (see 19C . 19D ). 19A shows exemplary variants of Druckeinstellelementen comprising pressure screws 21 and an expansion area 22 , which may be provided individually or as shown in combination. The pressure screws 21 are formed as above with respect to 5 is described. The expansion area 22 includes a plurality of hollow conduits that are in the plane of curvature of the pressure vessel 10 and in each case with the interior of the pressure vessel 10 are arranged communicatively. The Druckeinstellelemente can also in the variant according to 19B be provided. The multiple curvature according to 19B For example, a waveform may be formed with more than the three extremes shown.

The local distributions and time functions of pressure generation and temperature reduction in the cryopreservation device 100 depend on the orientation of the pressure vessel 10 when immersing in the cooling bath 210 from. 19C shows by way of example immersion with the plane of curvature perpendicular or parallel to the surface of the cooling bath 210 , With the vertical orientation, the pressure generation and the temperature drop first in the extremes of the multiple curvature, which protrude into the cooling bath. With the parallel orientation, however, the pressure generation and the temperature reduction take place in the entire pressure vessel 10 simultaneously. By way of derogation from the illustration, immersion with other orientations into the fluids may be provided, followed by an extended variability of adjustment of the pressure and temperature-time functions.

The local distributions and time functions of pressure reduction and, the temperature increase in the cryopreservation device 100 also depend on the orientation of the pressure vessel 10 when immersed in a heating bath 310 off ( 19D ).

20 shows an embodiment of the cryopreservation device 100 in which the container wall 11 of the pressure vessel 10 has the shape of a flat or disc-shaped cylinder. This embodiment of the invention has the advantage that when immersed in a cooling bath, a relatively large area is cooled. Therefore, on the inside of the container wall 11 formed a thin layer of crystalline phase, sufficient to the desired pressure in the pressure vessel 10 while leaving a relatively large space for the glassy phase. The pressure vessel 10 according to 20A has a diameter of z. B. 1 cm to 10 cm and thickness of z. B. 1 mm to 1 cm. He is z. B. made of stainless steel, aluminum, gold or silver.

According to 20B comprises the adjusting device for the time and / or location-dependent adjustment of the temperature and the pressure in the pressure vessel 10 a pressure clamp 23 , The pressure clamp 23 includes two clamp plates 24 that have a hinge 25 (Axis hinge) are pivotable. With a clamp screw 26 (Adjusting screw), the distance between the clamp plates 24 be set. The pressure clamp 23 is suitable to the pressure vessel 10 with a pressure increased compared to the atmospheric pressure before the cryopreservation device 100 by dipping in a cooling bath (see 24 ) is cooled. Furthermore, the pressure clamp allows 23 advantageously a stabilization of the pressure vessel 10 ,

The 20C to 20D show variants of the cylindrical pressure vessel 10 reinforced with a reinforced peripheral area (perspective sectional view in 20C ) and / or a double-sided (sectional view 20D ) or one - sided (sectional view in 20E ) elastically deformable container wall 11 can be equipped. In the latter case, one-sided is a stable bottom wall 11.3 intended.

The 21 to 24 show further variants of the pressure clamp 23 in which the clamp plates 24 with holes 27 and on her to the pressure vessel 10 facing insides with profiles 26 are in the open state and in the closed state (assembled state) of the pressure bracket 23 , When closed, the pressure clamp 23 is about the clamp plates 24 mechanical pressure on the container wall 11 of the pressure vessel 10 exercised. 23 shows a plan view of the pressure clamp 23 with one of the clamp plates 24 , the hinge 25 , the clamp screw 26 and the holes 27 ,

The holes 27 allow direct contact of a cooling fluid, eg. B. of liquid nitrogen or propanol, or of a heating fluid, for. B. of water, with the pressure vessel 10 and thus an acceleration of the cooling or heating of the pressure vessel 10 , The profiles 28 are provided to the pressure vessel 10 locally produce different prints. Furthermore, it is shown that the cylindrical pressure vessel 10 with a vent pipe 30 can be equipped.

25 shows a variant of the invention, in which the cryopreservation device 100 only one-sided in a liquid (cooling bath 210 or heating bath) is immersed. The pressure clamp 23 in this case has only on the lower, pointing to the liquid clamping plate 24 holes 27 on. This is especially beneficial if adherent cells are on the lower side of the pressure vessel 10 grow or the cells sediment there.

26 schematically illustrates a variant of the invention, in which the venting of the pressure vessel 10 and the mechanical pressure generation with the clamp plates 24 with electric drive elements 32 . 33 to be controlled. This embodiment of the invention is particularly advantageous for automation of cryopreservation.

In a modified, in 27 shown embodiment of the cryopreservation device 100 in which the container wall 11 of the pressure vessel 10 has the shape of a cylinder is as Druckeinstellelement no clip but a pressure screw 21 intended. With the pressure screw 21 is the pressure in the interior 12 of the pressure vessel 10 provided before or after the beginning of the cooling or heating in the cooling or heating bath (not shown). In the interior 12 are several inner containers 13 each arranged gas-free with biological samples, eg. B. cell suspensions filled and z. B. stacked one above the other. The wall thickness of the inner container 13 can be chosen extremely thin, z. B. less than 400 microns, since they are not exposed to pressure or corresponding mechanical forces.

According to 27 is in the interior 12 of the pressure vessel 10 a sensor device comprising a pressure sensor 16.1 arranged. The pressure sensor 16.1 provides a sensor signal used to control the pressure using the pressure screw 21 , if necessary with an electrical control element (eg piezoelectric element, not shown) can be used. It can be generated in the interior, a predetermined external pressure 12 on the inner container 13 when freezing and thawing acts.

A modified variant of the pressure vessel 10 with pressure screw 21 is in 28 shown. In this case, the inner container 13 formed by a flexible bag containing the biological sample, e.g. B. a cell suspension or a blood sample absorbs. The inner container 13 includes z. B. a blood bag, as it is used for blood donation purposes.

29 schematically illustrates an enlarged section of the container wall 11 a pressure vessel 10 , The container wall 11 contains an optical unit 60 necessary for an optical, in particular microscopic observation of the interior 12 of the pressure vessel 10 is set up. It is Z. B. possible that cells 2 on an optical lens 61 grow adherently. The optical lens 61 can z. B. for a microscopic picture of the cells 2 be configured.

30 shows a further embodiment of the invention, in which the cryopreservation device 100 a tube assembly with external pressure generation (hydrostatic, see arrow) via a T-element comprises. The tube assembly is equipped with a pressure sensor 16.1 and a temperature sensor 16.2 equipped with their sensor signals both the pressure and the temperature-time functions are adjustable.

The 31 and 32 show embodiments of the cryopreservation device 100 in which the container wall 11 of the pressure vessel 10 has the shape of a sphere. The pressure vessel 10 is made of two hemispheres 11.2 assembled in the middle of the pressure vessel 10 screwed together. The ball has a diameter in the range of z. B. 5 mm to 10 cm. According to 31A is the pressure vessel 10 with a pressure screw 21 equipped for filling the pressure vessel 10 , Vent the pressure vessel 10 and generating the pressure in the interior of the pressure vessel 10 is used. 31B additionally illustrates optionally provided parts, such as a sensor device 16 and one from the pressure vessel 10 decoupled filling line 11.1 , The 31C and 31D illustrate, in analogy to the methods described above, the cooling and heating of the cryopreservation device 100 ,

The spherical pressure vessel 10 may alternatively or additionally with a Druckeinstellelement in the form of an expansion region 22 be equipped ( 32 ). The expansion area 22 includes a cylinder nozzle configured as described above for receiving an expansion medium and for generating pressure on cooling. The 32C and 32D illustrate, in analogy to the methods described above, the cooling and heating of the cryopreservation device 100 ,

The cryopreservation of biological samples according to the invention, the organs 4 or whole organisms 5 is schematic in the 33 to 35 illustrated. In particular, in these applications of the invention, the biological sample is preferably added to the stabilizer substance. According to 33A is as a pressure vessel 10 provided a cylindrical vessel, which is dimensioned in the example shown for the reception of a fish embryo. In the 33C and 33D are two immersion variants for pressure-temperature control during freezing of the cryopreservation device 100 illustrated. According to the 34A and 34B is accordingly as a pressure vessel 10 a spherical vessel shown for the uptake of an organ 4 , with an inner diameter of the pressure vessel 10 in the range of z. B. 10 cm to 30 cm, is dimensioned. According to 34B is the organ 4 in an inner container 13 arranged. 35 shows as a pressure vessel 10 a spherical vessel containing tissues, organisms 5 or organs in an inner container 13 , z. As a bag or a thin-walled vessel, so that the outer solution to the bag inner medium can be chosen differently (eg, outside oil, inside nutrient solution with the stabilizer substance).

36 shows an embodiment of the cryopreservation device 100 in which pressure is not caused by the expansion of a freezing expansion medium but by a vaporous expansion medium, e.g. B. evaporating liquid nitrogen is formed. The expansion area 22 containing the liquid nitrogen is via a pressure line 39 with the pressure vessel 10 connected. The evaporating liquid nitrogen is continuously or in portions of the expansion area 22 in the pressure vessel 10 injected.

The 37 to 39 show embodiments of the cryopreservation device 100 by providing at least one substrate 14 in the interior 12 of the pressure vessel 10 for the cryopreservation of adherent cells 2 are configured. According to the in the 37A to 37C shown Variants is the substrate 14 in the form of an elongate strip (tongue) in a tubular pressure vessel 10 , the one or both sides with a pressure screw 21 and / or with a radially projecting pressure screw 21 is closed. The cells 2 are in the adherent state on one or both sides of the substrate 14 , which may be suitably functionalized (eg by coating with fibronectin, polylysine and / or growth factors). Otherwise, the interior is 12 of the pressure vessel 10 with the preservation medium, optionally filled with the stabilizer substance.

37C additionally shows a substance reservoir 17 that in the interior 12 of the pressure vessel 10 is arranged. The substance reservoir 17 is for an introduction of at least one additional substance in the interior 12 set up. For example, from the substance reservoir 17 during the freezing or thawing process substances in the interior 12 diffuse. The substance reservoir 17 can one under the effect of pressure in the interior 12 destructible reservoir wall, e.g. B. from a plastic. Advantageously, this makes it possible that the additional substance only when reaching a certain pressure in the interior 12 is released.

Instead of the elongated strip, there are more complicated shapes of the substrate 14 usable as exemplified in 38 is shown. According to 36A is a substrate 14 illustrated with a cross-shaped cross section, wherein it can be provided that cells 2 on all or only some surfaces of the substrate 14 are arranged. According to 38B is a substrate 14 shown in the form of a hollow cylinder, inside which the cells 2 are located. The hollow cylinder can be made of z. Example of ceramic, plastic, cellulose or chitin with an extremely small wall thickness, in particular less than 250 microns, be made. Advantageously, like the use of the inner containers described above, this allows the cells 2 inside other constraints and solutions are exposed as outside the substrate 14 , According to further variants of the invention, the substrate 14 comprise a body with a nano- or microstructured surface, optionally equipped with growth or differentiation factors in gradients.

The 39A to 39E show by way of example that the substrate 14 can be configured as a separate component (boat), which can be inserted into the pressure vessel. The boats can be formed in particular with a closed or an open mold.

The 39A and 40 further illustrates a variant of an inventively provided adjusting device for controlling the pressure in the pressure vessel 10 , As Druckeinstellelement is in this variant at least one end of the tubular pressure vessel 10 a winding section 31 intended. By screwing in the winding section 31 on the gas-free filled with the biological sample pressure vessel 10 can be increased in this before lowering the temperature of the pressure.

In 40A is a tubular pressure vessel 10 with winding sections 31 shown. In the interior 12 of the pressure vessel 10 is the biological sample 1 with the preservation medium 3 , The preservative medium 3 in which there are cells 2 The stabilizer substance may be in the form of a gradient (eg Ficoll or Percoll of different concentration and / or molecular weight). According to 40B can in the interior 12 additional partitions 18 be provided, which is a rapid mixing of the biological sample 1 and Z. B. prevent degradation of said gradient and allow a separation of the cells in compartments. Optionally, as in 40C is shown, in the interior of additional substance reservoir in the form of hollow spheres 19 be provided in the preservation medium 3 are suspended and arranged to be destroyed at a predetermined pressure and release their contents. For example, additional antifreeze and vitrification substances or media that aid the cells in thawing may be released. In 40D It is illustrated that two immiscible solutions can be stacked in the pressure vessel. The first solution contains the cells 2 , About the freezing behavior of the solutions, the pressure conditions can be adjusted with temperature change.

41 illustrates a tubular pressure vessel 10 which is one-sided with a pressure screw 21 and one-sided with a winding section 31 is closed. Inside is the preservation medium 3 in the form of layered solutions (eg Ficoll as gradient or overlays of different molecular weights, ie solutions of different density), as used in the density gradient centrifugation of cells. The cells 2 are given unilaterally in the gradient ( 41A ). The pressure vessel 10 becomes in vertical form with the cells 2 centrifuged so that different cell types collect at the sealing interface ( 41B ). In this arrangement, the cryopreservation then takes place.

In the 42A and 42B is another embodiment of the cryopreservation device according to the invention 100 and their application illustrated schematically. The cryopreservation device 100 includes one-sided with a pressure screw 21 sealed pressure vessel 10 in whose interior 12 the substrate 14 for the adherence of biological cells 2 is arranged. The pressure screw 21 is further provided with a heat conducting element in the form of a cooling wire 38 equipped, the pressure-tight in the pressure screw 21 is integrated and different from the interior 12 into the environment of the pressure vessel 10 extends. The cooling wire 38 is a metal wire, z. B. of silver. Outside the pressure vessel 10 is the cooling wire 38 into a compact bundle, e.g. In the form of a spiral, a coil, a cylinder or a sphere. When freezing, the cooling wire can 38 first in the cooling bath 210 , z. B. are dipped from liquid nitrogen, which is not in the interior of the tube ice on the container wall 11 but the cooling wire 38 forms and leads to pressure increase. This is followed by the complete immersion of the system in the cooling bath 210 ,

The features of the invention disclosed in the above description, the drawings and the claims may be of importance individually or in combination for the realization of the invention in its various embodiments.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited non-patent literature

• Polge C, Smith AU, Parkes AS (1949), Nature 164: 666 [0004]
• Lovelock JE (1954), Biochemical Journal 56 (2): 265-270 [0004]
• Lovelock JE, Bishop MWH, 1959, Nature 183: 1394-1395 [0004]
• JLM Leunissen and H. Yi (2009) Journal of Microscopy, 235: 25-35 [0007]
• JLM Leunissen et al. [0008]
• JLM Leunissen et al. [0020]
• JLM Leunissen et al. [0021]
• Jackson et al., J. Phys. Chem. (1997) [0062]
• www.wikipedia.de [0062]

Claims (31)

Cryopreservation device ( 100 ) used for the cryopreservation of a biological sample ( 1 ), comprising: - a pressure vessel ( 10 ) with a container wall ( 11 ) and an interior ( 12 ) used to receive the biological sample ( 1 ), wherein - the pressure vessel ( 10 ) for a cooling with a lowering of the temperature and an increase of the pressure in the pressure vessel ( 10 ) and for the cryopreservation of the biological sample ( 1 ), characterized in that - the pressure vessel ( 10 ) with an adjusting device ( 20 ) fitted with the container wall ( 11 ) and at least one of at least one Druckeinstellelement ( 21 - 23 . 31 ), at least one cooling element ( 34 ) and at least one heat conducting element ( 35 - 38 ), and - the adjusting device ( 20 ) for a time- and / or location-dependent adjustment of the temperature and the pressure in the pressure vessel ( 10 ) is configured. A cryopreservation device according to claim 1, wherein - the pressure adjusting element is at least one of a pressure screw ( 21 ) in the container wall ( 11 ), an expansion area ( 22 ), which is adapted to receive a liquid or gaseous expansion medium and with the interior ( 12 ), a pressure clamp ( 23 ), which from the outside on the container wall ( 11 ), and a winding section ( 31 ) placed on one end of the pressure vessel ( 10 ) includes. A cryopreservation device according to claim 2, wherein - the expansion area ( 22 ) comprises at least one hollow conduit extending from the pressure vessel ( 10 ) protrudes. Cryoconservation device according to claim 3, wherein - the at least one hollow conduit has branches and / or in different spatial directions from the pressure vessel ( 10 ) protrudes. Cryopreservation device according to one of the preceding claims, in which - the cooling element is a cooling line ( 34 ) contained in the pressure vessel ( 10 ) is arranged. Cryopreservation device according to one of the preceding claims, in which - the heat-conducting element ( 35 - 38 ) at least one of a profile on an outer side of the pressure vessel ( 10 ), a profile on an inner side of the pressure vessel ( 10 ) and Wärmeleitkörpern inside the pressure vessel ( 10 ). Cryopreservation device according to one of the preceding claims, in which - the container wall ( 11 ) of the pressure vessel ( 10 ) has the shape of a tube, a ball or a flat cylinder. Cryopreservation device according to claim 7, in which - the container wall ( 11 ) of the pressure vessel ( 10 ) has the shape of a curved tube. Cryopreservation device according to one of the preceding claims, wherein in the interior ( 12 ) of the pressure vessel ( 10 ) is provided - an inner container ( 13 ) used to receive the biological sample ( 1 ), - a substrate ( 14 ) for the adherence of biological cells in the biological sample ( 1 ), - a segmentation of the interior ( 12 ) in interior sections ( 15 ), - a sensor device ( 16 ) with at least one of a pressure sensor ( 16.1 ) and a temperature sensor ( 16.2 ), and / or - a substance reservoir ( 17 . 19 ), the release of a substance in the interior ( 12 ) is set up. Cryopreservation device according to claim 9, in which - the substance reservoir is hollow spheres ( 19 ) of a pressure-sensitive material which is in the interior ( 12 ) are arranged distributed. Cryopreservation device according to one of the preceding claims, in which - the container wall ( 11 ) an optical unit ( 60 ) required for an optical observation of the interior ( 12 ) of the pressure vessel ( 10 ) is set up. Method for cryopreserving a biological sample ( 1 ) comprising biological cells ( 2 ) and a preservation medium ( 3 ), comprising the steps of: - providing the biological sample ( 1 ) in a pressure vessel ( 10 ) with a container wall ( 11 ) and an interior ( 12 ), and - cooling the pressure vessel ( 10 ) in a cooling device ( 200 ) with a lowering of the temperature and an increase of the pressure in the pressure vessel ( 10 ), the biological sample ( 1 ) is at least partially transferred to a cryopreserved state in a vitreous phase, characterized in that - the pressure vessel ( 10 ) with an adjusting device ( 20 ), which comprises at least one of at least one pressure setting element ( 21 - 23 . 31 ), at least one cooling element ( 34 ) and at least one heat conducting element ( 35 - 38 ), and - a time- and / or location-dependent adjustment of the temperature and the pressure in the pressure vessel ( 10 ) using the adjusting device ( 20 ) is provided. Process according to Claim 12, in which the adjustment of the temperature and of the pressure in the pressure vessel ( 10 ) comprises the steps of: - increasing the pressure in the pressure vessel ( 10 ) with the pressure adjustment element ( 21 - 23 . 31 ), and then - lowering the temperature in the pressure vessel ( 10 ) with the cooling device. Method according to claim 13, in which - the pressure in the pressure vessel ( 10 ) using a pressure screw ( 21 ) in the container wall ( 11 ), an expansion area ( 22 ), which is adapted to receive a liquid or gaseous expansion medium and with the interior ( 12 ), a pressure clamp ( 23 ), which from the outside on the container wall ( 11 ) acts, and / or a winding section ( 31 ) placed on one end of the pressure vessel ( 10 ), is increased. Method according to claim 14, in which - the expansion area ( 22 ) comprises at least one hollow conduit extending from the pressure vessel ( 10 ), and - the pressure in the pressure vessel ( 10 ) is increased by first the at least one hollow conduit and then the remaining pressure vessel ( 10 ) in a cooling bath ( 210 . 220 ) of the cooling device ( 200 ) is dipped. Method according to one of claims 12 to 15, wherein the adjustment of the temperature and the pressure in the pressure vessel ( 10 ) comprises the steps of: - lowering the temperature in the pressure vessel ( 10 ) with the cooling device, and then - increasing the pressure in the pressure vessel ( 10 ) with the pressure adjustment element ( 21 - 23 . 31 ). A method according to claim 16, wherein - the preservation medium contains a stabilizing substance which is suitable for increasing the temperature of the biological sample ( 1 ), preferably until a transition to a liquid state, to stabilize the glassy phase of the supercooled melt. A method according to claim 16 or 17, wherein the stabilizer substance is selected from at least one of the substance groups comprising: Long-chain uncharged polymers having a molecular weight greater than 500 g / mol, in particular greater than 1000 g / mol, - monosaccharides, Ethylene glycol, - di- and oligosaccharides, - polysaccharides, - starch derivatives, such as. B. 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. The method of claim 18, wherein the stabilizer substance is selected from at least one of the substances comprising: - sucrose-epichlorohydrin copolymer (Ficoll, reg. Name), and - Polyvinylpyrrolidone coated silica gel, such as. B. Percoll (reg. Name). Method according to one of claims 12 to 19, in which - The stabilizer substance in the preservation medium has a concentration which is less than 10%, in particular less than 8, in particular less than 5%, in particular less than 3%, in particular less than 1%. Process according to one of Claims 12 to 20, in which - the stabilizer substance in the preservation medium ( 3 ) outside the biological cells ( 2 ) is arranged. Method according to one of claims 12 to 21, in which - the cryopreservation device ( 100 ) according to one of claims 1 to 11 is used. Method according to one of claims 12 to 22, comprising the step: - storage of the biological sample ( 1 ) while maintaining the elevated pressure. Method for vitality-preserving heating of a biological sample ( 1 ) comprising biological cells ( 1 ) and a preservation medium ( 3 ) in a frozen state, which in a glassy phase in a pressure vessel ( 10 ), wherein the temperature of the pressure vessel ( 10 ) and the biological sample ( 1 ) and simultaneously in the pressure vessel ( 10 ) an elevated pressure above the atmospheric pressure is maintained. Process according to claim 24, in which - in the pressure vessel ( 10 ) the elevated pressure above the atmospheric pressure is maintained until the biological sample ( 1 ) reaches a transition from the frozen or vitrified to the liquid state. Process according to Claim 25, in which, in the transition from the frozen or vitrified to the liquid state, the pressure in the pressure vessel ( 10 ) is reduced. Process according to Claim 26, in which, in the transition from the frozen or vitrified to the liquid state, the pressure in the pressure vessel ( 10 ) is reduced instantaneously. Method according to claim 26 or 27, in which - the pressure in the pressure vessel ( 10 ) by a contraction of the biological sample ( 1 ) is reduced at the transition from the frozen or vitrified to the liquid state. Method according to one of claims 24 to 28, in which - The increased pressure is at least 100 MPa, in particular at least 150 MPa and / or at most 300 MPa, in particular at most 250 MPa. Method according to one of Claims 24 to 29, in which - the preservation medium ( 3 ) contains a stabilizer substance which is suitable for increasing the temperature of the biological sample ( 1 ), preferably until the transition to stabilize the glassy phase of the supercooled melt. Use of a stabilizer substance which is suitable for increasing the temperature of a biological sample ( 1 ), comprising biological cells and a preservation medium, to obtain a glassy phase until a transition to the liquid state, in the cryopreservation of biological samples ( 1 ).

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