DEVICE AND METHOD FOR PRESSURIZED CRYOPRESERVATION OF A BIOLOGICAL SAMPLE
A cryopreservation device (100) which is arranged for cryopreservation of a biological sample (1) comprises a pressure vessel (10) with a vessel wall (11) and an internal space (12) which is arranged to receive the biological sample (1), wherein the pressure vessel (10) is equipped with an actuating device (20) for cooling by lowering the temperature and raising the pressure in the pressure vessel (10) and configured for the cryopreservation of the biological sample (1). Said actuating device (20) is connected to the vessel wall (11) and comprises at least one pressure setting element (21-23, 31) and at least one of at least one cooling element (34) and at least one heat conducting element (35-38), wherein the actuating device (20) is configured for a time-dependent and/or location-dependent setting of the temperature and of the pressure in the pressure vessel (10). Methods are also described for cryopreservation of a biological sample (1), comprising biological cells (2) and a preservation medium (3), and methods for heating the biological sample (1) to maintain vitality.
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The invention relates to a device for cryopreservation of a biological sample, comprising biological cells and an aqueous preservation medium, with a pressure vessel. The invention furthermore relates to a method for cryo-preservation of the biological sample and the use of a stabiliser substance with which a vitreous phase of water can be stabilised in the cryopreservation of biological samples. Furthermore, the invention refers to a method to heat the cryopreserved biological sample.
The low temperature preservation (cryopreservation) of cells is so far the only possibility of suspending vital processes reversibly at a cellular level (to maintain vitality) such that they revive after heating to physiological temperatures. Cryopreservation has no correspondence in nature. Whilst organisms, such as fish, are found embedded in ice in polar regions which maintain their vitality for a limited duration, these organisms are not completely frozen through but contain glycerine and other substances in their cells which serve to lower the freezing point, or they express proteins (so-called anti-freezing proteins) which influence the ice structure. Permanent preservation is not possible in this state because even at temperatures below 0° C., diffusion processes over weeks and months lead to the disintegration of the biological system. Permanent preservation would moreover require cooling, e.g. to a temperature of liquid nitrogen, at which the fluid in the cells would also freeze. In this case, however, the organisms can no longer be reanimated.
A special problem of cryopreservation is the formation of crystal ice (crystalline phase of water) inside and outside the cells which leads directly or indirectly to irreversible damage. Since the formation of ice is one of the primary reasons for cell damage, so-called cryoprotectants (anti-freeze additives, cryoadditives) have been sought for decades and added to the obligate physiological media.
Cryoprotectants typically comprise small molecules such as dimethyl sulfoxide (DMSO) which penetrate into the cells, or higher molecular substances such as sugar which remain in the medium outside the cells or on their surface. Cryoprotectants are frequently only effective in high, largely non-physiological concentrations (10% to 50%). Therefore they can only be added at lower than physiological temperatures (around 4° C.), must quickly penetrate into the cells and be washed out immediately after thawing.
Former developments in the cryopreservation of biological samples have been aimed at replacing cryoprotectants by physiologically and osmotically less problematical substances, reducing their concentration or foregoing them completely. With the exception of anti-freeze proteins of the organisms themselves, only few substance groups have been found since the discovery of the anti-freeze properties of DMSO and glycerine as well as a few sugars by Polge and Lovelock [1, 2, 3]. It has so far been assumed that the cryopreservation of cells is not possible without cell stress and cell repair processes with gene expressions etc. after thawing because the formation of the crystalline phase under physiological conditions cannot quite be avoided.
- [1] Polge C, Smith A U, Parkes A S (1949), Nature 164: 666
- [2] Lovelock J E (1954), Biochemical Journal 56(2):265-270
- [3] Lovelock J E, Bishop M W H, 1959, Nature 183: 1394-1395
The success of cryopreservation can be characterised by a vitality rate (survival rate), e.g. by the quotients of the number of living cells after and before cryopreservation. It is known that the vitality rate depends on the type of cell, the volume and other different boundary conditions which have usually already been empirically optimised. In view of the necessity of a fast diffusion of the cryoprotectants into the cells and the regulated outward dissipation of heat, conventional cryopreservation methods have so far failed in microscopic objects such as tissues, organs and entire organisms without exception. Rather, conventional cryopreservation with the addition of cryoprotectants leads to practicable vitality rates only in suspended cells and very small tissue pieces (<0.5 mm3). It has not so far been possible to subject highly aqueous plant cells as well as a large number of animal cells, such as the oocytes of cats and other species, to cryopreservation at all. On the other hand, vitality rates in excess of 90% have been achieved with cryopreserved cancer cells.
The success of cryopreservation depends, in particular, on the physical-biological boundary conditions, e.g. on the properties of the water and those of the cryoprotectants. It has so far been assumed that cryoprotectants must penetrate into all cells by diffusion and that the heat must dissipate outwards in a short period (ms to min) because cooling can only take place from here. It is furthermore assumed that these conditions are satisfied only in very small objects (cell suspended in a nutrient solution with the addition of anti-freeze agents) due to the heat conductivity of the water which also dominates in the cells and due to the low diffusion speed of cryoprotectants through the cell membranes.
To avoid the creation of the crystalline phase, it has been attempted in practice to transfer biological samples to a vitreous or amorphous phase (vitrified state) through fast cooling. However, success has been limited even when using cryoprotectants. Up to a publication by J. L. M. Leunissen et al. [4] the general view was therefore that a vitrification of cellular cell suspensions could not be achieved without additives for physical reasons.
- [4] J. L. M. Leunissen and H. Yi (2009) Journal of Microscopy, 235: 25-35
A method for cryomicroscopy is described by J. L. M. Leunissen et al. [4] which would lead vitrification to be expected: if a thin-walled copper tube with a diameter of <1 mm is filled with a cell suspension, closed gas-free at the ends by pressing them together and then frozen in a cooling fluid such as propane, nitrogen etc., very well maintained structures are found in deep temperature cryosections of the tubes which demonstrate vitrification. However, once the deep temperature cryosamples are heated, it becomes apparent that the cells did not survive the cooling process. The method described by J. L. M. Leunissen et al. [4] is not suitable for a heating of the sample such as to maintain vitality. It has therefore been assumed up to now that methods for cryomicroscopy do not permit revitalisation through reheating.
In view of the developing regenerative medicine and biotechnology and of environment protection and species preservation, there is an urgent interest in reducing or overcoming the disadvantages of conventional cryopreservation.
The objective of the invention is to provide an improved cryopreservation device using which the disadvantages of conventional techniques are overcome and which, in particular, permits a cryopreservation with a higher vitality rate and/or enlarged sample volumes. It is furthermore the objective of the invention to provide an improved method of cryopreservation in which the disadvantages of conventional techniques are overcome and which, in particular, permits the method conditions on cooling and/or thawing to be adjusted such that an improved vitality rate is achieved. Furthermore, it is an objective of the invention to provide an improved method to heat a cryopreserved biological sample with which the disadvantages of conventional techniques are overcome and which, in particular, permits any vitality-restricting influence of biological samples in the transition to a thawed state to be suppressed.
These objectives are solved by devices and methods with the features of the independent claims. Advantageous embodiments and applications of the invention are provided in the dependent claims.
In accordance with a first aspect of the invention, a cryopreservation device is provided which is adapted for the cryopreservation of a biological sample. The cryopreservation device comprises a pressure vessel with a vessel wall and an internal space which is arranged to receive the biological sample. The pressure vessel is configured to be cooled in a cooling bath of a cooling device up to a cryopreservation temperature, e.g. below −138° C. The pressure vessel is furthermore configured such that pressure can be applied to the internal space of the cooling vessel which is higher than the ambient atmospheric pressure. The pressure vessel is adapted for the permanent cryopreservation of the biological sample and storage at the cryopreservation temperature whilst maintaining the higher pressure in the pressure vessel.
In accordance with the invention, the pressure vessel has an actuating device which is suitable for a time and/or location-dependent setting of the temperature and of the pressure in the pressure vessel. The actuating device is connected to the vessel wall of the pressure vessel and is able to influence the setting of the cryopreservation temperature and of the pressure in the internal space of the pressure vessel depending on time and location. The actuating device is suitable to selectively control the cooling and/or heating of the biological sample in accordance with a predetermined temperature-time function. Furthermore, the actuating device is suitable to influence the temperature distribution in the pressure vessel. Furthermore, the actuating device is suitable to control the pressure in the pressure vessel in accordance with the predetermined pressure-time function. The temperature and pressure-time functions can be set relatively to each other. For example, the actuating device enables the biological sample to firstly be cooled and then a higher pressure applied to it, the cooling and increase in pressure to be conducted simultaneously or the pressure to firstly be increased and then the cryopreservation temperature set. Finally, the actuating device is suitable to control the place of pressure generation in the pressure vessel.
In accordance with the invention, the actuating device comprises at least one pressure setting element, at least one cooling element and/or at least one heat conducting element. The pressure setting element is configured such as to set the pressure-time function, a location of a primary pressure input and/or the amount of the pressure which is applied to the internal space of the pressure vessel. Accordingly, the cooling element and/or the heat conducting element, possibly in connection with the effect of the cooling device, are configured so as to control the temperature-time function, the spatial temperature distribution and the cryopreservation temperature.
The biological sample contains biological cells and an aqueous preservation medium. The biological cells comprise individual cells, such as individual stem cells, precursor cells, fibroblasts, gametes, groups of cells, in particular from the named cell types, pieces of tissue or organs or their parts. The biological cells can even form complete organisms, in particular small organisms, such as worms or insects. The preservation medium comprises an aqueous physiological medium (culture medium, cultivation medium) as known in the cultivation of biological cells.
In accordance with a second aspect of the invention, a method for the cryopreservation of the biological sample is provided in which the biological sample is arranged in a pressure vessel and the pressure vessel is cooled down in a cooling device until the biological sample in the cryopreserved state has been transferred to an at least partially vitreous phase. In accordance with the invention, a time and/or location dependent setting of the temperature and of the pressure in the pressure vessel takes place using an actuating device with at least one pressure setting element, at least one cooling element and/or at least one heat conducting element. The cryopreservation device in accordance with the first aspect of the invention is used by preference to conduct the inventive method of cryopreservation of biological samples.
In accordance with a third aspect of the invention, a method is provided to heat a biological sample, comprising biological cells and a preservation medium in a frozen state which is located in a vitreous phase in a pressure vessel. According to the invention, the temperature of the pressure vessel and of the biological sample is increased and during the temperature increase in the pressure vessel a higher pressure is maintained above the ambient atmospheric pressure. The method is preferably executed to heat the biological sample using the cryopreservation device in accordance with the first aspect of the invention.
In accordance with a fourth aspect of the invention, it is proposed to use at least one stabiliser substance (i.e. an individual substance or a mixture of several substances) in the cryopreservation of biological samples as a component of the preservation medium which is suitable to maintain a vitreous state of the supercooled melt without crystallisation preferably up to the transition to the liquid state whilst increasing the temperature of the biological sample comprising biological cells and a preservation medium.
The invention is based on the recognition that on freezing aqueous systems the formation of the crystalline phase is avoided and instead the vitreous phase (amorphous phase) can be generated by applying pressure to the aqueous system. The transition of the biological sample to the cryopreserved state, the cryopreservation and/or the heating of the cryopreserved samples are conducted in an area of the phase diagram of the aqueous system in which preferably the vitreous phase is formed. Since the survival rate of the biological cells in the biological sample in the vitreous phase is considerably higher than in the crystalline phase, the invention facilitates a higher vitality rate of the cryo-preservation.
In conventional cryomicroscopy using pressure-tight sample tubes (see above, [4]) an increased pressure was already generated in the closed sample tubes. However, with the conventional technique there is no degree of freedom to influence the pressure or temperature characteristic during freezing, during cryopreservation or during selective heating in accordance with the set time function. The inventors have determined that ice avoided during rapid cooling is inevitably created—particularly during thawing—with all its negative effects on the sample, thereby destroying the cells. The inventive provision of the actuating device advantageously permits the pressure-temperature processes to be controlled in terms of time and/or space such that the vitreous phase of the biological sample is primarily generated and maintained in the internal space of the pressure vessel. Contrary to the conventional technique, the actuating device permits the pressure and temperature parameters to be varied selectively during freezing and/or thawing in order to achieve a maximum vitality rate. This advantageously provides a real cryopreservation with the possibility of rethawing and revitalisation of the cells in the biological sample whilst the technique of J. M. L. Leunissen et al. [4] merely represents cryopreparation for microscopic examinations.
In accordance with the above-mentioned third aspect of the invention, it is suggested in particular to maintain the vitreous state of the sample, in particular of the preservation medium, maintaining pressure until the water contained in the sample melts. This is advantageously facilitated by thawing cryopreserved biological samples, as those described by J. M. L. Leunissen et al. [4], with an extremely good maintenance of structure and vitrified cell suspensions such that living cells exist. This has not so far been successful in any freeze-pressure shock method as used for cryomicroscopy. In order to achieve the above-mentioned stabilisation, preferably one stabiliser substance is added to the preservation medium which is suitable to stabilise the vitreous phase of the supercooled melt on increasing the temperature of the biological sample preferably up to the melt. The inventive cryopreservation device advantageously facilitates the influencing of the path of the sample conditions through the phase diagram past a solid/fluid phase transition.
In accordance with a preferred embodiment of the invention, the actuating device comprises at least one pressure setting element which is connected to the vessel wall and/or the internal space of the pressure vessel. Advantageously, different variants of the pressure setting element are possible which, depending on the preservation task and the spatial conditions of the cryopreservation, can be selected individually or in combination. In accordance with a first variant, at least one pressure screw can be provided in the vessel wall of the pressure vessel. The vessel wall contains a threaded opening for the fluid-tight reception of the pressure screw. Screwing the pressure screw into the threaded opening serves to reduce the free volume of the internal space in the pressure vessel and to permit the pressure in the pressure vessel to be increased accordingly. According to a second variant, an expansion area can be provided which is connected with the internal space of the pressure vessel and is adapted to receive a fluid or gaseous expansion medium. When the expansion medium expands in the expansion area the free volume in the internal space is reduced accordingly and the pressure in the pressure vessel increased. In accordance with a third variant, the pressure setting element can comprise a pressure clamp which acts on the vessel wall from outside. In this case, the vessel wall is formed of a flexible material in order to transfer the mechanical pressure exercised by the pressure clamp to the internal space of the pressure vessel. Advantageously, the pressure clamp can be adapted with cooling openings in order to accelerate the cooling of the pressure vessel in the cooling bath of the cooling device. Furthermore, the pressure clamp can be designed for a mechanical or electrical actuation.
According to a specially preferred embodiment of the invention, the expansion area comprises at least one hollow duct to accommodate the expansion medium which communicates with the internal space of the pressure vessel and which protrudes from the pressure vessel. The duct contains water, for example, an aqueous solution or parts of the biological sample. The protrusion of the duct from the pressure vessel causes the expansion medium in the duct to be spatially separated from the internal space of the pressure vessel. This advantageously facilitates a cooling of the duct in the cooling bath of the cooling device whilst the remaining pressure vessel is still at a higher temperature, e.g. at room temperature. Crystalline ice can be generated in the duct which extends through to the internal space, decreasing the remaining volume and therefore causing an increase in the pressure in the internal space. In accordance with a preferred variant, the duct has branches. Advantageously, this facilitates an enlargement of the volume of the expansion area compared with an individual duct without branches. At the same time the branched duct permits the time function of the pressure generation in the pressure vessel to be influenced. Alternatively and additionally, several ducts can be provided which protrude from the pressure vessel in different directions. Advantageously, this permits the pressure generation to be influenced, depending on the alignment of the pressure vessel relative to the cooling bath of the cooling device.
If the actuating device provided by the invention comprises a cooling element, this is preferably formed by a cooling line which is arranged in the pressure vessel. The cooling line runs through the internal space of the vessel. It is designed to have a cooling agent, such as liquid nitrogen, run through it. Advantageously, the cooling line facilitates a setting of the start and of the time function of the temperature reduction in the pressure vessel.
If the actuating device provided by the invention comprises a heat conducting element, this is preferably formed by an outside profile on the outer side of the pressure vessel, an inside profile on the inner side of the pressure vessel and/or heat conducting bodies in the inside of the pressure vessel. These variants of heat conducting elements permit the heat transport from the pressure vessel to the cooling bath to be accelerated during the cooling of the pressure vessel.
A further advantage of the inventive cryopreservation device is that the pressure vessel can be manufactured in a large number of geometric shapes. Variants of the invention are given particular preference in which the vessel wall of the pressure vessel has the shape of a tube, a sphere or a cylinder, e.g. of a flat cylinder (box). A tubular pressure vessel can be straight or curved. In both cases the spatial distribution of the temperature and pressure setting in the pressure vessel can be influenced by its shape.
Further advantageous features of the inventive cryopreservation device refer to the shape of the internal space of the pressure vessel. According to a variant of the invention, an inner vessel may be provided in the internal space which is arranged to receive the biological sample. In this case, the biological sample in the inner vessel is separated in terms of the substance from the remaining volume of the internal space. This facilitates the influencing of the vitreous phase in the direct vicinity of the biological sample. In accordance with a further variant, a substrate can be arranged in the internal space which is suitable to adherent receive biological cells which are part of the biological sample. The substrate can, for example, be arranged in the inner vessel. Substrate materials are suitable as substrates which are used for adherent cell cultures such as plastic or glass. In accordance with a further variant, a segmentation of the internal space can be provided in internal space sections. Advantageously, the segmentation facilitates the selective setting of different pressure-temperature conditions in each of the internal space sections. In accordance with a further variant, a sensor device can be arranged in the internal space which comprises at least one temperature sensor and/or at least one pressure sensor. Advantageously, the sensor device facilitates a measurement of the temperature and/or of the pressure in the internal space. The cryopreservation can be controlled depending on the at least one signal of the sensor device. Finally, in a further variant of the invention a substance reservoir can be arranged in the internal space which is suitable to release a substance into the internal space. The substance reservoir comprises, for example, hollow spheres which may be destroyed under the effect of a higher pressure in the internal space in order to release a substance. The variants specified for the internal space design can be provided individually or in combination.
In accordance with a further advantageous embodiment of the invention, the vessel wall can be provided with an optical unit. The optical unit comprises an imaging optic which is set up for a visual observation of the internal space of the pressure vessel. The optical unit facilitates a visual monitoring of the state of the biological sample during cryopreservation.
Advantageously, different variants of the pressure and temperature setting exist in the pressure vessel in order to achieve the required cryopreservation conditions in different ways in the pressure-temperature phase diagram of the biological sample. For example, in accordance with the first variant it is possible to first increase the pressure in the pressure vessel with the at least one pressure setting element and then to reduce the pressure in the pressure vessel with the cooling device. The pressure increase and the temperature reduction are conducted in accordance with predetermined separate time functions. In accordance with the second variant, the increase in the temperature and the reduction in temperature can be set such that the respective time functions overlap by starting the lowering of the temperature before achieving the final pressure or vice versa by starting the increase in pressure before achieving the cryopreservation temperature. Finally, in accordance with a further variant it is possible to firstly reduce the temperature in the pressure vessel up to the cryopreservation temperature and then to increase the pressure in the pressure vessel. Which of the variants stated is selected will depend on the conditions of the specific cryopreservation task, in particular on the design of the cryopreservation device and the composition of the biological sample. The ideal variant can be selected empirically by tests in which the vitality rate of the biological sample is tested for the different variants under specific conditions of application. It is possible in the same way to select the pressure and temperature time function, particularly with respect to the speed of pressure increase and temperature reduction and/or the shape of the function, such as a stepped shape.
The pressure-temperature setting is simplified if the pressure setting element in accordance with a preferred embodiment of the invention comprises an expansion area in the form of a hollow duct protruding from the pressure vessel. The pressure can be increased in the pressure vessel by immersing the at least one duct in the cooling bath of the cooling device. An expansion medium in the at least one duct expands so that the pressure in the remaining pressure vessel increases. Finally, the remaining pressure vessel is immersed in the cooling bath of the cooling device in order to set the cryopreservation temperature for the biological sample in the internal space of the pressure vessel.
Preferably, a permanent storage of the biological sample is provided whilst maintaining the increased pressure, in particular in liquid nitrogen or in the vapour of the liquid nitrogen. The sample is stored by special preference in the pressure vessel.
In accordance with a specially preferred embodiment of the cryopreservation method, the preservation medium contains at least one stabiliser substance which is suitable to stabilise (maintain) the vitreous phase of the supercooled melt preferably up to the transition to the fluid state whilst increasing the temperature of the biological sample.
Advantageously, the stabiliser substance brings about a situation in which the vitreous phase remains up to higher temperatures than would be the case without the stabiliser substance. The vitreous phase is maintained for longer during thawing and the formation of the crystalline phase is avoided. Furthermore, the glass transition temperature of the preservation medium is increased by the stabiliser substance. The stabiliser substance produces a lower number of nucleation sources in the preservation medium so that the nucleation probability is reduced and the formation of ice during thawing minimised.
In accordance with this embodiment, the above objectives are solved by at least one stabiliser substance being added to the biological sample, e.g. a cell suspension. The at least one stabiliser substance is used as was used only under certain conditions or not at all for the conventional cryopreservation or, in accordance with the invention, develops its effect at far lower concentrations and this is a different effect than that of the known cryoprotectants. Substances are preferably selected as stabiliser substance which do not penetrate the cells at normal pressure and which therefore are not normally used in conventional cryopreservation of cells and tissues. The noteworthy aspect of using the stabiliser substance is that experiments of the inventors have shown that with an addition of Percoll or Ficoll, for example, in the range of a few percent mammalian cells survived the pressure shock freezing and thawing procedure analogue to publication [4] with a high survival rate (>97%). This is all the more surprising in view of the fact that a penetration into the cells is not to be assumed.
The surprising effect of the stabiliser substance is that for the first time during the thawing of the sample it permits the return path via the combination of high pressurefast deep cooling and pressure-controlled heating virtually without influencing the vitality of the cells. It is therefore the combination of at least one substance dissolved in the preservation medium which permits the return from the vitrified phase by influencing the water structure and the existence of nucleation sources.
The stabiliser substance differs in terms of substance, in terms of its effect and/or with relation to the preferred selected concentration of conventional cryopreservation. Unlike the stabiliser substance, conventional cryoprotectants selectively increase the number of nucleation sources in order to promote the formation of a large as possible number of ice crystals during freezing. However, as the number of ice crystals increase, their chance of growing in size reduces so that large crystals are prevented by conventional cryoprotectants. A distribution and high motility in the preservation medium through to the cells is required in conventional cryoprotectants.
The stabiliser substance is preferably selected from at least one of the substance groups which comprise long-chain uncharged polymers with a molecular weight greater than 500 g/mol, in particular greater than 1000 g/mol, monosaccharides, di- and oligosaccharides, polysaccharides, starch derivatives such as starch hydrolysis products, sugar alcohols, water-soluble polymers, colloids (nanoparticle dispersions), in particular with silver, gold, diamond and/or nanotube particles, dendrimers, polycations and polyanions. Long-chain polysaccharides proved to be particularly suitable, in particular hydrophilic copolymerisates made from saccharose and epichlorohydrin (Ficoll, reg. name), and/or polyvinylpyrrolidone, in particular silica gel coated with polyvinylpyrrolidone (Percoll, reg. name) with a molecular mass of between 2,000 and several million g/mol. Nanoparticle dispersions, in particular silver, gold, diamond and/or nanotube particles are particularly advantageous because they are suitable to reduce the heat conductivity of the preservation medium. Advantageously, the stabiliser substance is biocompatible so that the cells in the biological sample are not unfavourably influenced by the stabiliser substance.
The concentration (%=vol. %) of the stabiliser substance is preferably smaller than 30%, in particular preferably less than 20%, in particular smaller than 10%, such as for example smaller than 3% or smaller than 1%. A preferred minimum concentration is 0.1%.
In accordance with a further preferred embodiment of the invention, the stabiliser substance in the preservation medium is positioned outside the biological cells. The cells remain free from the stabiliser substance which has advantages for the vitality after thawing of the sample.
A further advantage of the stabiliser substance can be that it alters its properties, in particular structure and/or molecular weight, under the impact of the increased pressure and the reduced temperature during cryopreservation. For example, molecules of the stabiliser substance can be fragmented so that they can diffuse into the inside of the cells in order to achieve an additional cryoprotective effect here.
It is emphasised that with the addition of the stabiliser substance to the preservation medium it is not necessary for an increased pressure to be maintained in the pressure vessel until the biological sample has achieved the transition from the supercooled melt to the liquid state. In this case, the pressure can drop even before achieving the transition, in particular down to atmospheric pressure.
According to a preferred variant of the above-mentioned third aspect of the invention, a method is thereby provided to heat a biological sample, comprising biological cells and a preservation medium in a vitrified state, which with a vitreous phase is located in a pressure vessel, wherein the temperature of the pressure vessel and of the biological sample is increased until the biological sample reaches the liquid state and wherein the preservation medium contains the at least one stabiliser substance which is suitable to stabilise the vitreous state on increasing the temperature of the biological probe preferably up to the transition to the liquid state.
In accordance with a preferred embodiment of the invention, the pressure in the pressure vessel is reduced on reaching the transition from vitreous state of the supercooled melt through to liquid state. Damage to the biological sample after reaching the liquid state is minimised here. Special preference is given to the reduction of the pressure in the pressure vessel instantaneously, i.e. in particular in steps and with negligible delay.
The pressure reduction can be achieved by releasing the pressure vessel, e.g. opening the pressure vessel such that a pressure balance with the outer atmospheric pressure is achieved. Advantageously, a release of the pressure vessel is not absolutely necessary, however. Rather, the pressure reduction can also be achieved by a contraction of the biological sample at the transition from the supercooled melt to the liquid state. The volume of the sample can reduce in accordance with the processes explained with reference to the
In accordance with a further preferred embodiment of the invention, the increased pressure above atmospheric pressure is at least 100 MPa, in particular at least 150 MPa and/or at the most 300 MPa, in particular 250 MPa at the most. These pressure areas have proven to be particularly advantageous for a fast transition from the vitreous to the liquid state and vice versa.
Further details and advantages of the invention are described in the following, making reference to the attached drawings, which show in:
The invention will firstly be described in the following by explanation of findings of the inventors and then by specifying details of the cryopreservation device and the method. It is emphasised that the following theoretical considerations serve as an approach to explain the outstanding vitality rates achieved with the inventive cryopreservation. However, the implementation of the invention is not bound by the completeness and correctness of the theoretical considerations.
Theoretical Considerations of the Phase Diagrams of Aqueous Systems and the Effect of Stabiliser Substances
Cryopreservation has so far been described empirically and using simplified assumptions. The empirical approach results from the complexity of the composition of the cytoplasm of the biological cells. Since the cytoplasm contains hundreds of proteins, nucleotides and a large number of carbohydrates, ions of numerous elements, nano-scale systems such as membranes, organelles and structure elements such as cytoskeleton and water bonded to the surfaces, phase diagrams of water can be used to only a restricted extent. Nevertheless, reference is made to phase diagrams of water as shown in
The pressure-temperature phase diagram of water (
As the phase diagram in
In extended phase diagrams of water and of its metastable states,
-
- 1. Up to 200 MPa so-called LDL (Low Density Liquid, supercooled liquid) water exists in its aggregate states with all the anomalies occurring at normal pressure.
- 2. In addition HDL (High Density Liquid) states arise which may possibly be more favourable for cryopreservation. However, these are unphysiologically high pressures (in the deep sea a maximum of 100 MPa is achieved).
In metastable states a limit temperature below that which a vitreous state of water could be assumed even with physiological pressures is to be found at temperatures of around 136 K=−137° C. Water then freezes like glass, namely amorphously, i.e. without the formation of crystals. The water molecules then remain where they were. This is a metastable state, the desirable state which is aspired to as found when deep-freezing biological objects. It is called “vitrification”. The vitrification requires a very high cooling rate (>106°/s) with the rapid fluctuation of the water molecules which, given the dimension of a cell (>10 μm), cannot be reached due to the neighbouring heat conductivity of the water (max. a few 104°/s). With increasing size of the objects to be frozen, the cooling rate interval becomes increasingly dramatic (many powers of ten) so that a true vitrification (formation of a vitreous phase without additives) has not so far been possible.
The inventors have determined that in view of the anomaly of water in the LDL range the freezing point can be lowered by increasing pressure. In the transition from LDL to HDL this process is exactly reversed so that a pressure of around 200 MPa is the preferred pressure upper limit which is still effective to lower the freezing point. Thereafter the freezing point increases as is normal in any other liquid as the pressure increases.
The path to vitrification (vitreous phase) is the shortest at this pressure (see
However, there is another way of achieving vitrification or at least a “pseudo vitrification” by adding additives which lead to very small ice domains. This is not a truly physiological way but it has been used for freezing for a very long time and also functions at normal pressure.
The possibility exists in principle to increase the glass transition temperature through additives. Adding Trehalose permits the glass transition temperature, for example, to be shifted to almost 0° C., which would be ideal for biological objects. The requisite concentrations of Trehalose are, however, above any physiological compatibility for living cells. The gains achieved by lowering the freezing point are small (
In accordance with a practical example, it is intended to prepare a biological sample in a known manner with biological cells and a preservation medium at room temperature and under atmospheric pressure. At least one stabiliser substance is added to the preservation medium, preferably with a concentration smaller than or equal to 5% or this is done during the cooling process or in the deep-frozen state or before thawing. Higher concentrations can also be used, however.
Suitable substance groups for the stabiliser substance, in particular for use with the embodiments of the cryopreservation device and the methods to cool or heat biological samples described below are as follows:
Further media with which excellent experimental results have been achieved using the embodiments of the cryopreservation device described below and methods to cool and heat biological samples are as follows:
In the experiments of the inventors the cryomedium according to Mazur with an addition of ethylene glycol and dextran proved to be advantageous.
Embodiments of the Cryopreservation Device and the Methods to Cool or Heat Biological Samples
The pressure vessel 10 preferably has an outer diameter which is smaller than or equal to 5 mm and by special preference smaller than or equal to 2 mm or 1 mm, e.g. 0.5 mm. The axial length of the pressure vessel 10 has been selected, for example, in the range of 10 mm to 20 cm. The thickness of the pressure wall 11 is, for example, ¼ to 1/10 of the outer diameter of the pressure vessel. The pressure wall 11 is made, for example, from stainless steel, aluminium, gold or silver. Alternatively, further metals or alloys can be used which have a high heat conductivity for fast cooling in the internal space 12 and a pressure resistance for pressures of up to 100 MPa, for example. Furthermore, the pressure vessel can be made of a plastic or a composite material, e.g. plastic-metal composite.
The pressure vessel 10 has internal threads at its axial ends to receive the pressure screws 21. The vessel wall 11 has a threaded piece to accommodate the radially protruding pressure screw 21 (
In accordance with
For the cryopreservation of a biological sample 1 comprising, for example, biological cells 2 in a preservation medium 3, the sample 1 is filled into the internal space 12 of the pressure vessel 10 (see partly cross-sectioned view of the vessel wall 11 in
For the cryopreservation of the biological samples, the filled cryopreservation device 100 is cooled in the cooling bath of a cooling device (not shown in
On immersion in the cooling bath ice firstly forms on the inner side of the vessel wall 11. Since the crystalline phase is firstly formed, an expansion takes place which leads to a pressure increase in the internal space up to a final pressure of 200 MPa. Above this pressure further ice growth is ruled out so that the remainder of the biological sample 1 moves to a vitreous (vitrified) state or at least does not exhibit a hexagonally crystalline phase.
Once the cryopreservation device 100 in the cooling bath has reached the cryopreservation temperature, e.g. −197° C., the further cryopreservation can take place in the cooling bath or in storage vessel (not shown) which has been cooled to a temperature of, for example, −140° C. through liquid nitrogen or through vapour of the liquid nitrogen.
For the heating of the biological sample 1 so as to maintain vitality, the cryopreservation device 100 is immersed in a heating bath (liquid bath with a temperature above 0° C.) comprising, for example, water, alcohol or an oil. Immersion is similarly conducted horizontally by preference and at a high speed so that the increased pressure in the pressure vessel 10 is maintained until the crystalline ice which is formed melts. Finally, the pressure drops suddenly to 0.1 MPa, for example.
Fluorescence-microscope investigations with a different presentation of the cell nucleus and of the Golgi apparatus (not shown here) have shown that the shrinking merely compacts the cytoplasma components but does not influence the cell nuclei and other important cell organelles; this is of great advantage to freezing and thawing such as to maintain vitality.
The expansion area 22 is made, for example, from the same material as the vessel wall 12 of the pressure vessel 10. The expansion area 22 is adapted to receive a liquid expansion medium that expands during cooling. The expansion medium comprises, for example, the aqueous preservation medium of the biological sample or alternatively a different aqueous liquid which is suitable to form the crystalline phase of water. The dimensions (length, inner diameter, outer diameter) of the expansion area 22 can be selected by the user depending on the specific preservation conditions.
The expansion area 22 permits a fast formation of the crystalline phase once the temperature of the expansion area 22 is reduced to below the freezing point of water. Advantageously, the cooling of the expansion area 22 can be decoupled in terms of time from the cooling of the remaining pressure vessel 10, as shown in
For cryopreservation of the biological sample 1 the cryopreservation device 100 is firstly immersed exclusively with the expansion area 22 into the cooling bath 210 (immersed depth D1) whilst the remaining pressure vessel is still above the cooling bath. In this phase, crystalline ice is formed in the expansion area 22 which expands such that the pressure increases in the internal space 12 of the pressure vessel 10. The duration of pressure generation with the expansion area 22 is selected, for example, in the range of milliseconds, seconds or minutes. Finally, the cryopreservation device 100 is completely immersed in the cooling bath 210 so that the desired cryopreservation temperature of −195.7° C., for example, is achieved. For this purpose, the cryopreservation device 100 is lowered to a second immersed depth D2.
The heating to recover the biological sample is conducted in reverse in accordance with
The inner vessel 13 does not extend over the entire length of the internal space 12. This creates a relatively large space at the closed end 12.1 of the pressure vessel 10 in which no biological sample 1 is located and in which the crystalline phase of water can preferably be generated. This creates an expansion area within the pressure vessel 10 as part of the inventive actuating device which advantageously can have an effect on the cooling and on the heating of the cryopreservation device 100 (see in particular
The outer shape of the inner vessel 13 can be the same as the inner shape of the pressure vessel 10. By way of alternative to the cylindrical shape shown, other cross-sections of the outer or inner shapes can be provided such as quadratic, hexagonal, octagonal or all combinations thereof. In particular, different cross-section shapes of the outer and inner shapes can be provided.
To heat the biological sample 1 in the pressure vessel 10 it is immersed in a heating bath 310 in accordance with
The cryopreservation of the biological sample 1 is also conducted in the embodiment in accordance with
Heating is conducted in accordance with
The local distributions and time functions of the pressure generation and the temperature reduction in the cryopreservation device 100 depend on the alignment of the pressure vessel 10 on immersion in the cooling bath 210.
The local distributions and time functions of the pressure reduction and of the temperature increase in the cryopreservation device 100 similarly depend on the alignment of the pressure vessel 10 on immersion into a heating bath 310 (
In accordance with
The holes 27 permit direct contact of a cooling liquid, e.g. of liquid nitrogen or propanol, or of a heating fluid, e.g. of water, with the pressure vessel 10 and therefore an acceleration of cooling or heating of the pressure vessel 10. The profiles 28 are provided to generate different pressures at the pressure vessel 10 locally. It is furthermore shown that the cylindrical pressure vessel 10 can be adapted with a bleed connector 30.
In an altered embodiment of the cryopreservation device 100 shown in
According to
An altered variant of the pressure vessel 10 with pressure screw 21 is shown in
The spherical pressure vessel 10 can be alternatively or additionally provided with a pressure setting element in the shape of an expansion area 22 (
The inventive cryopreservation of biological samples comprising organs 4 or entire organisms 5 is illustrated diagrammatically in
Instead of the stretched strip, complicated shapes of the substrate 14 can be used as shown, for example, in
The features of the invention disclosed in the above description, the drawings and the claims can be of importance individually and also in combination for the realisation of the invention in its different embodiments.
Claims
1. Cryopreservation device which is adapted for cryopreservation of a biological sample, comprising: wherein
- a pressure vessel with a vessel wall and an internal space which is adapted to receive the biological sample, wherein
- the pressure vessel is configured for cooling with a lowering of a temperature and an increase of a pressure in the pressure vessel and for the cryopreservation of the biological sample,
- the pressure vessel is provided with an actuating device which is connected with the vessel wall and comprises at least one pressure-setting element and at least one of at least one cooling element and at least one heat conducting element, and
- the actuating device is configured for a time and/or location dependent setting of the temperature and of the pressure in the pressure vessel,
- the pressure setting element comprises at least one of a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous expansion medium and communicates with the internal space, and a pressure clamp which acts from outside on the vessel wall.
2. Cryopreservation device in accordance with claim 1 in which
- the pressure setting element comprises a coiled section which acts on one end of the pressure vessel.
3. Cryopreservation device in accordance with claim 2 in which
- the expansion area comprises at least one hollow duct which protrudes from the pressure vessel.
4. Cryopreservation device in accordance with claim 3 in which
- the at least one hollow duct has at least one of branches and protrusions to different directions from the pressure vessel.
5. Cryopreservation device in accordance with claim 1 in which
- the cooling element comprises a cooling line which is arranged in the pressure vessel.
6. Cryopreservation device in accordance with claim 1 in which
- the heat conducting element comprises at least one of a profile on an outer side of the pressure vessel, a profile on an inner side of the pressure vessel and heat conducting bodies in the inside of the pressure vessel.
7. Cryopreservation device in accordance with claim 1 in which
- the vessel wall of the pressure vessel has a shape of a tube, a sphere or a flat cylinder.
8. Cryopreservation device in accordance with claim 7 in which
- the vessel wall of the pressure vessel has the shape of a bent tube.
9. Cryopreservation device in accordance with claim 1 in which the at least one of the following is provided in the internal space of the pressure vessel:
- an inner vessel adapted to receive the biological sample,
- a substrate which is adapted for adherent receipt of biological cells in the biological sample,
- a segmentation of the internal space into internal space sections,
- a sensor device with a least one pressure sensor and a temperature sensor, and
- a substance reservoir which is adapted to release a substance into the internal space.
10. Cryopreservation device in accordance with claim 9 in which
- the substance reservoir comprises hollow spheres made of a pressure sensitive material which are arranged distributed throughout the internal space.
11. Cryopreservation device in accordance with claim 1 in which
- the vessel wall includes an optical unit which is adapted for a visual observation of the internal space of the pressure vessel.
12. Method for the cryopreservation of a biological sample, comprising biological cells and a cryopreservation medium, with the steps: wherein
- provision of the biological sample in a pressure vessel with a vessel wall and an internal space, and
- cooling of the pressure vessel in a cooling device with lowering of a temperature and increasing of a pressure in the pressure vessel, wherein the biological sample is transferred at least partly into a cryopreserved state in a vitreous phase,
- the pressure vessel is provided with an actuating device which comprises at least one pressure setting element and at least one of at least one cooling element and at least one heat conducting element, and
- a time and/or location dependent setting of the temperature and of the pressure in the pressure vessel using the actuating device,
- the pressure in the pressure vessel is adjusted using a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous extension medium and communicates with the internal space, and/or a pressure clamp which acts from outside on the vessel wall.
13. Method in accordance with claim 12 in which the setting of the temperature and of the pressure in the pressure vessel comprises the following steps:
- increase in pressure in the pressure vessel with the pressure setting element, and then
- lowering of the temperature in the pressure vessel with the cooling device.
14. Method in accordance with claim 13 in which
- the pressure in the pressure vessel is increased using a coiled section which acts on one end of the pressure vessel.
15. Method in accordance with claim 12 in which
- the expansion area comprises at least one hollow duct which protrudes from the pressure vessel, and
- the pressure is increased in the pressure vessel by first the at least one hollow duct being immersed into a cooling bath of the cooling device followed by a remainder of the pressure vessel.
16. Method in accordance with claim 12 in which the setting of the temperature and of the pressure in the pressure vessel comprises the following steps:
- lowering of the temperature in the pressure vessel with the cooling device, and subsequently
- increasing of the pressure in the pressure vessel with the pressure setting element.
17. Method in accordance with claim 16 in which
- the cryopreservation medium includes a stabiliser substance which is suitable to stabilise the vitreous phase of a supercooled melt preferably up to a transition to a liquid state, on increasing the temperature of the biological sample.
18. Method in accordance with claim 17 in which the stabiliser substance is at least one member selected from the group consisting of:
- long-chain uncharged polymers with a molecular weight greater than 500 g/mol,
- monosaccharides,
- ethylene glycol,
- di- and oligo saccharides
- polysaccharides,
- starch derivatives,
- sugar alcohols;
- water-soluble polymers
- colloids comprising nano particle dispersions,
- dendrimers,
- polycations, and
- polyanions.
19. Method in accordance with claim 18 in which the stabiliser substance is at least one member selected from the group consisting of:
- saccharose epichlorhydrin copolymer, and
- silica gel coated with polyvinyl pyrrolidone.
20. Method in accordance with claim 17, wherein
- the stabiliser substance in the cryopreservation medium has a concentration which is lower than 10%.
21. Method in accordance with claim 17, wherein
- the stabiliser substance in the cryopreservation medium is arranged outside the biological cells.
22. Method for the cryopreservation of a biological sample, comprising biological cells and a cryopreservation medium, comprising the steps:
- providing a cryopreservation device of claim 1,
- providing the biological sample in the pressure vessel of the cryopreservation device, and
- cooling of the pressure vessel with lowering of the temperature and increasing of the pressure in the pressure vessel, wherein the biological sample is transferred at least partly into a cryopreserved state in a vitreous phase,
- wherein the pressure in the pressure vessel is adjusted using a pressure screw in the vessel wall, an expansion area which is adapted to receive a liquid or gaseous extension medium and communicates with the internal space, and/or a pressure clamp which acts from outside on the vessel wall.
23. Method in accordance with claim 12 with the following step:
- storage of the biological sample whilst maintaining the increased pressure.
24. Method for the heating of a biological sample such as to maintain vitality, comprising biological cells and a cryopreservation medium in a frozen state which is arranged in a vitreous phase in a pressure vessel, wherein a temperature of the pressure vessel and of the biological sample is increased and simultaneously an increased pressure above atmospheric pressure is maintained in the pressure vessel.
25. Method in accordance with claim 24 in which
- the increased pressure above the atmospheric pressure is maintained in the pressure vessel until the biological sample achieves a transition from the frozen or vitrified state to a liquid state.
26. Method in accordance with claim 25 in which
- on transition from the frozen or vitrified state to the liquid state the pressure in the pressure vessel is reduced.
27. Method in accordance with claim 26 in which
- on transition from the frozen or vitrified state to the liquid state the pressure in the pressure vessel is instantaneously reduced.
28. Method in accordance with claim 26 in which
- the pressure in the pressure vessel is reduced by a contraction of the biological sample at the transition from the frozen or vitrified state to the liquid state.
29. Method in accordance with claim 24 in which
- the increased pressure is at least one of at least 100 MPa and maximum 300 MPa.
30. Method in accordance with claim 24 in which
- the cryopreservation medium contains a stabiliser substance which is suitable to stabilise the vitreous phase of the supercooled melt on increasing the temperature of the biological sample, preferably up to the transition.
31. Method of using a stabiliser substance for the cryopreservation of biological samples, including the steps of
- increasing the temperature of a biological sample, comprising biological cells and a cryopreservation medium, and
- maintaining a vitreous phase of the biological sample by an effect of the stabiliser substance up to a transition to a liquid state.
Type: Application
Filed: Oct 10, 2012
Publication Date: Sep 18, 2014
Applicants: Fraunhofer-Gesellschaft zur Foerderung der angewandten Forschung e.V. (Muenchen), Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V. (Muenchen)
Inventors: Guenter R. Fuhr (Berlin), Heiko Zimmermann (Frankfurt am Main), Frank Stracke (Saarbruecken), Markus Grabenbauer (Dielheim), Jan Huebinger (Duesseldorf), Philippe Bastiaens (Wuppertal), Frank Wehner (Dortmund)
Application Number: 14/351,136
International Classification: F25D 31/00 (20060101);