EP3448563A1

EP3448563A1 - Sample container for a cryogenically preserved biological sample, method for producing the sample container, method for monitoring the temperature of a cryogenically preserved sample

Info

Publication number: EP3448563A1
Application number: EP17717624.5A
Authority: EP
Inventors: Günter R. Fuhr; Heiko Zimmermann
Original assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Current assignee: Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
Priority date: 2016-04-27
Filing date: 2017-03-31
Publication date: 2019-03-06
Also published as: JP2019515284A; WO2017186333A1; US20190193075A1; CN109153018A; KR20190003609A; DE102016005077A1

Abstract

The invention relates to a sample container that is designed to accommodate a cryogenically preserved biological sample, and to a method for producing the sample container. The invention further relates to a method for monitoring the temperature of a cryogenically preserved biological sample. The sample container that is designed to accommodate a cryogenically preserved biological sample has, on a region of its outer surface, a frozen-solid indicator substance whose melting point is in a range from -20°C to -140°C.

Description

SAMPLE CONTAINER FOR A CRYOKON-SAMPLEED BIOLOGICAL SAMPLE, METHOD FOR PRODUCING THE SAMPLE CONTAINER, METHOD FOR THE TEMPERATURE MONITORING OF A CRYOKON-SAMPLEED SAMPLE

DESCRIPTION

The invention relates to a sample container, which is designed to receive a cryopreserved biological sample and a method for producing the sample container. The invention further relates to a method for monitoring the temperature of a 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. Cryopreservation has become an indispensable element for clinics, pharmaceutical companies, species conservation, environmental protection and health care through large biobanks in recent decades. Biological material is stored in low-temperature compatible sample containers (cryobanks), eg. As tubes, straws and bags, different sizes. During cryopreservation, the stored biomaterials are frozen while maintaining the vitality of the sample material, mostly at temperatures below -80 ° C, for live collections below -140 ° C to the temperature of the liquid nitrogen. For a cryopreserved sample or a sample intended for cryopreservation, the term "cryoprobe" is also used below.

For macroscopic samples, such. As blood or tissue, numerous techniques for sample storage at low temperatures have been developed. In modern medicine, genetic engineering and biology, there is a tendency to undergo increasingly small samples of cryopreservation. For example, small volumes of suspension (milliliter or below) are frozen with suspended cells or cell groups. The cryocon- Serving cells from in vitro cultures predominantly takes place in a suspension. However, most of the biomedically relevant cells require substrate contact for propagation and orderly development. Therefore, samples may be frozen after culturing in the substrate-bound state.

The quality of the samples is crucial as they are used for cell therapies in clinics, the development of pharmaceuticals and biotechnology products, as national resources and much more. The storage time is several days to decades, with a tendency for long-term storage. The samples are stored in refrigerated containers, mostly in metal drawers and racks, with which they are subject to temperature fluctuations in new deposits or withdrawals. In the case of live deposits (cells, cell suspensions and tissue parts) not only the uninterrupted cold chain plays a decisive role, but also the avoidance of large temperature jumps in the frozen phase. Since it is not uncommon for cryo containers to warm to temperatures of -80 ° C to -20 ° C, even though they are still frozen, quality reductions, which not only reduce the value of the sample, are unrecognized but can also lead to life-threatening situations when used in the clinical field. Even when samples are thawed for a short time, when they are frozen they do not show that they no longer correspond to the original state. However, it is not only a question of detecting a thawing of the biomaterials, but of documenting the exceeding of a limit temperature in the range between -140 ° C and -20 ° C. Temperature control and documentation for each sample is the requirement and so far only rarely - and if, then with great technical effort - to meet. In addition, there are extensive laboratory tests after thawing, which also consume valuable sample material and generate costs even in the case of now worthless cryoprobes.

It is therefore an object of the invention to provide a sample container for a cryopreserved sample which is suitable for temperature monitoring of a cryopreserved biological sample. Another object is to provide a method for producing such a sample container. Another object is to provide a method for monitoring the temperature of a cryopreserved biological sample, with the disadvantages of conventional techniques can be avoided and which is characterized by a simplified process management.

A further object is to provide a possibility for being able to recognize at the simplest possible marker or mark whether a cryoprobe has heated above a definable limit temperature, and even if only for a short time. The limit temperature must be determinable between -20 ° C and -140 ° C before freezing. This should be possible quickly and easily recognizable on every single cryoprobe and therefore with millions of samples, must not alter the biomaterials and should already take place in the deep-frozen state. If possible, the condition of the sample should also be detectable in the storage container, as each storage and retrieval brings with it the risk of sample change of a variety of samples in the stored goods, as a rule, whole racks are raised. The device or the method should be easy to handle, low temperature tolerant and adjustable. It may consume little or no energy and possibly cause only the least cost, since the storage of a bioprobe in a refrigerated state should cost only a few Euros in its total expenditure. This requirement must also meet the applicable materials.

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 and are explained in more detail in the following description with partial reference to the figures.

According to a first aspect of the invention, said objects are achieved by a sample container which is designed to receive a cryopreserved biological sample and which bears on a region of its outer surface a solidified indicator substance whose melting temperature at normal pressure, ie at 1013.25 hPa, in range from -20 ° C to -140 ° C. The melting temperature may also be in a range of -20 ° C to -100 ° C. This provides a sample container designed for temperature monitoring of a cryopreserved biological sample. The sample container is a container suitable for cryopreservation, for example a tube, a straw (also referred to as seminal tube), a bag for storing blood or stem cells, a box or another container suitable for cryopreservation. Such containers are also referred to as cryotubes, Kryostraw, cryobags, cryobox or generally as a cryocontainer. The sample container has a receiving space for receiving the biological samples. The receiving cavity may contain a cryopreserved sample.

Cryogenic tubes are also referred to as biobank or cryobank tubes. Cryotubes have a receiving space that forms an internal cavity for receiving a biological sample. The cryovial usually also has a lid for closing the receiving space. The lid may have an engagement over which the lid can be rotated with a tool. The cryotube may also include a bottom member having an identifier, e.g. B. in the form of a machine-readable code.

When the melting temperature is exceeded, the solidified indicator substance becomes liquid. Thereupon, a configuration state of the indicator substance on the outer surface of the sample container, z. B. due to gravity and / or the surface tension. For example, if its melting point is exceeded, the indicator substance can change its position on the sample container and / or its surface shape, which can be determined visually or by a measuring device. The change in the configuration state is retained even when the indicator substance is re-frozen. The indicator substance frozen to a region of the outer surface of the sample container thus preferably has a configuration whose shape and / or arrangement changes when a melting temperature of the indicator substance is exceeded.

A sample container carrying a frozen indicator substance at a region of its outer surface whose melting temperature is in a range from -20 ° C. to -140 ° C. can thus be advantageously used for temperature monitoring of a cryopreserved biological sample. The sample container according to the invention is Furthermore, inexpensive to produce and claimed in comparison to a conventional sample container little additional space.

The indicator substance can thus be applied directly to an outer surface of a sample container. The indicator substance may be fixed to an outer surface of the sample container exclusively by freezing, i. h., the indicator substance is not supported by other fasteners, such as an additional vessel, etc. on the sample container. The indicator substance can be frozen frozen on the container. For better visibility, the indicator substance may contain an indicator additive which increases a detectability of a physical property of the indicator substance. The indicator additive can be, for example, a dye, so that the indicator substance colored or colored, d. H. is not transparent, and so their shape and / or location is better visually recognizable.

In principle, any dye which satisfies at least the following conditions is suitable as the dye:

- intensive dyeing ability even in small amounts and concentrations (eg starting from a saturated dye solution addition in the range <1% by volume, usually in the per mil or subpromille range).

- frost-tolerant

- Lightfast at the shipping as well as the relevant low temperatures

- soluble in all components of the indicator substance

- no segregation when freezing

no reaction with plastic materials which come into contact with the indicator substance.

Preferably, the dye is selected from the group consisting of triphenylmethane dyes, rhodamine dyes, especially xanthenes, azo dyes and phenazine and phenothiazine dyes. In more specific embodiments, the dye is selected from the group comprising Oil Red, Methyl Red, Brilliant Green, Rhodamine B, Neutral Red, Methylene Blue, or other dyes used to stain cells in cytology. The indicator additive may be particles, in particular nanoparticles, which increase a scattering effect and / or polarization effect of the indicator substance for electromagnetic radiation impinging on the indicator substance. As a result, a configuration change of the indicator substance can be detected more reliably by means of an optical transmission measurement, scattering measurement and / or polarization measurement. The indicator additive may be conductive particles. By adding conductive particles, the conductivity or impedance of the indicator substance can be influenced. In this way, a configuration change of the indicator substance can be detected by means of a conductivity measurement or impedance measurement. As an indicator substance, a substance can be selected whose melting temperature corresponds to a predetermined limit temperature whose exceeding is to be monitored. The indicator substance is a liquid or a mixture of different liquids whose melting point corresponds to the desired limit temperature. By way of example only, a mixture of water (H ₂ O) and ethanol (C ₂ H 6 O), a mixture of water (H ₂ O) and potassium hydroxide (KOH) or a mixture of water and an antifreeze can be selected as the indicator substance. The mixing ratio is adjusted according to the respective melting diagram, which indicates the course of the melting point as a function of the mixing ratio, so that the melting point of the liquid mixture has the desired value, namely the limit temperature to be monitored.

In a preferred embodiment, the indicator substance comprises at least one alcohol selected from the group consisting of octan-1-ol, nonan-1-ol, propan-1, 2-diol, propane-1,3-diol, butane-1,2 -diol, butane-l, 3-diol, butan-2-ol, pentane-l, 5-diol, pentan-l-ol, cyclopentanol, benzyl alcohol. The at least one alcohol is particularly preferably selected from propan-1, 3-diol, propan-1, 2-diol and butan-2-ol. In another preferred embodiment, the indicator substance comprises at least two different alcohol components:

a) an alcohol selected from the group consisting of octan-1-ol, nonan-1-ol, propan-1, 2-diol, propan-1, 3-diol, butane-l, 2-diol, butane-l 3-diol, butan-2-ol, pentane-l, 5-diol, pentan-1-ol, cyclopentanol, benzyl alcohol;

b) an alcohol selected from the group consisting of octan-1-ol, nonan-1-ol, propan-1, 2-diol, propane-1,3-diol, butane-1,2-diol, butane-1 3-diol, butan-2-ol, pentane-l, 5-diol, pentan-1-ol, cyclopentanol, benzyl alcohol, having a lower melting point than the alcohol of component a);

wherein the mixing ratio of the components a) and b) is set so that the melting temperature of the mixture within a temperature range of -20 ° C to -160 ° C, especially from -25 ° C to -160 ° C or -50 ° C to -150 ° C, lies.

More specific embodiments are characterized in that the indicator substance comprises one of the following combinations of components a) and b):

Octan-1-ol and butan-2-ol in a mixing ratio of 5 to 95% by volume;

Octan-1-ol and pentan-1-ol in a mixing ratio of 5 to 95% by volume;

- Octan-l-ol and propane-l, 2-diol in a mixing ratio of 5 to 95 vol .-%;

- Nonan-1-ol and butan-2-ol in a mixing ratio of 5 to 95 vol .-%;

- Nonan-l-ol and propane-l, 2-diol in a mixing ratio of 5 to 95 vol .-%;

- Nonan-l-ol and pentane-l-ol in a mixing ratio of 5 to 95 vol .-%;

Propan-1, 2-diol and butan-2-ol in a mixing ratio of 5 to 95 vol .-%;

- propane-l, 2-diol and propane-l, 3-diol in a mixing ratio of 5 to 95 vol .-%;

- propane-l, 2-diol and butane-l, 2-diol in a mixing ratio of 5 to 95 vol .-%; Propan-1, 3-diol and butan-2-ol in a mixing ratio of 5 to 95 vol .-%;

- propane-l, 3-diol and butane-l, 2-diol in a mixing ratio of 5 to 95 vol .-%;

- pentane-l, 5-diol and 'butan-2-ol in a mixing ratio of 5 to 95 vol .-%;

- Benzyl alcohol and butan-2-ol in a mixing ratio of 5 to 95 vol .-%;

- Pentan-l-ol and butan-2-ol in a mixing ratio of 5 to 95 vol .-%;

- Pentan-l-ol and methanol in a mixing ratio of 5 to 95 vol .-%;

- Cyclopentanol and butan-2-ol in a mixing ratio of 5 to 95 vol .-%;

- Cyclopentanol and propane-l, 2-diol in a mixing ratio of 5 to 95 vol .-%; - Cyclopentanol and pentane-l-ol in a mixing ratio of 5 to 95 vol .-%;

- Cyclopentanol and butane-l, 2-diol in a mixing ratio of 5 to 95 vol .-%; wherein the indicated value of the mixing ratio refers in each case to the proportion of the first-mentioned component in the mixture of the two components.

In particularly preferred embodiments, this indicator mixture comprises, for example, propane-1,2-diol and butan-2-ol in a mixing ratio of 40 to 60% by volume (gives a melting temperature of about -90 ° C.), propane-1,2 -diol and propane-1,3-diol in a mixing ratio of 30 to 70 vol .-%, or propane-l, 3-diol and butan-2-ol in a mixing ratio of 30 to 70 vol .-%.

In addition to the at least one alcohol, the indicator substance preferably also comprises at least one dye as described above. Most preferably, this dye is selected from the group comprising Oil Red, Methyl Red, Brilliant Green and Rhodamine B.

An even more specific embodiment is characterized in that the indicator substance two alcohols a) and b), which are selected from propan-l, 3-diol, propane-l, 2-diol and butan-2-ol, preferably in a mixing ratio as above, and a dye selected from the group consisting of Oil Red, Methyl Red, Brilliant Green and Rhodamine B.

The concentration of the dye in the alcohol component can vary widely depending on the dye and alcohol. As a rule, the concentration should be kept as low as possible during intensive staining, so that the color molecules do not change or increase the viscosity of the freezing and melting behavior of the alcohols in which they are dissolved. The dye concentration is typically in a range of <10% by volume, in particular <1% or <0.1%, ie in the percent or per thousand or subpromute range.

In a variant of the present invention, the limit temperature to be monitored does not correspond directly to the melting temperature of the indicator substance, but rather to the temperature above the melting temperature at which the viscosity of the molten substance has decreased so much that the required liquid transport can take place. This temperature is also referred to herein as a threshold temperature and is typically in a temperature range of 3-30 ° C or 5-30 ° C, for example 3-10 ° C, 3-20 ° C, 5-10 ° C or 5-20 ° C, above the nominal melting temperature.

In an advantageous embodiment, the indicator substance is therefore characterized in that the liquid mixture in a temperature range of 3-30 ° C or 5-30 ° C above the melting temperature has a viscosity in a range of 10 to 10 ⁶ mPa * s, preferably 10 to 10 ⁴ mPa * s.

According to a preferred embodiment, the area carrying the frozen indicator substance has a coating, roughening and / or structuring. For example, the area bearing the solidified indicator substance may have an adhesion-promoting texture coating. This improves the adhesion of the frozen indicator substance. According to a further aspect, the area bearing the frozen indicator substance may have a mirror coating. According to this variant, the indicator substance is thus frozen on a mirrored area of the outer wall of the sample container. In this embodiment, a configuration change of the indicator substance by means of a measuring device or purely visually to detect particularly reliable.

In another aspect, the region bearing the frozen indicator substance may comprise an electrode assembly. The electrode arrangement can be designed, for example, as gold or platinum electrodes. According to this variant, the indicator substance is thus frozen onto the electrode arrangement, in particular in such a way that a resistance measurable via the electrode arrangement or an impedance depends on whether or not the indicator substance is located on the electrode arrangement. Thus it can be determined on the basis of the measured resistance whether the indica- The substance of the substance is still present in the initially attached frozen state on the electrode arrangement, whether the indicator substance has flowed out of the region of the electrode arrangement when its melting point has been exceeded due to the liquid becoming liquid.

In addition to the sample container, a measuring device can also be provided, which is designed to detect the resistance or the impedance of the electrode arrangement.

It is particularly advantageous if the indicator substance is applied to the outer surface of the sample container in a predetermined arrangement. The indicator substance firmly frozen on a region of the outer surface of the sample container can thus have a specific arrangement which changes when a melting temperature of the indicator substance is exceeded, for example. B. under the influence of gravity. The arrangement may represent a number, a letter, a symbol, an identifier and / or another structure that is visually easily recognizable to a user. If the arrangement after a cryogenic storage is still recognizable, the melting temperature of the indicator substance was not exceeded during the cryogenic storage. If the arrangement has changed or has disappeared, it can be determined that the melting temperature and thus a critical limit temperature has been exceeded. A user can thus easily determine by a visual visual inspection, whether an undesirable increase in temperature has occurred above the melting temperature or above the limit temperature to be monitored.

According to a further aspect, the indicator substance applied on the outer surface can be obtained by firmly frozen drops of the indicator substance. This allows accurate metering of the applied indicator substance and a precise arrangement of the indicator substance, for. B. using a drop-shot device. According to another preferred embodiment, a plurality of different indicator substances whose different melting temperatures are each in a range of -20 ° C to -140 ° C, at different areas of the outer surface be applied to the sample container. This offers the advantage that several temperature limits can be monitored during the cryogenic storage or that the achieved temperature intervals, in which the sample has arrived, can be narrowed down more precisely. In an advantageous variant of this embodiment, the sample container is a cryotube, and a plurality of different indicator substances, which differ in their melting temperatures, are each applied in the form of tightly packed fixed drops on a receiving cylinder of the cryotube. In particular, the various indicator substances can each be arranged in the form of band-shaped, for example ring-shaped, frozen drops fixed to one another on the outer surface of the receiving cylinder of the cryotube, wherein the various indicator substances are arranged offset to one another in the axial direction of the cryotube. This arrangement is visually easy to check for changes. The axial direction corresponds to the longitudinal direction of the cryotube.

For example, the lower the melting point of the indicator substance in the axial direction compared to the melting points of the other indicator substances, the lower the value of the indicator substance. In other words, the various indicator substances may be sorted by their melting temperatures in descending or ascending order in the axial direction. This allows a visually particularly simple to be carried out limitation of the temperature intervals in which the sample container stored in the sample has arrived.

In accordance with a further embodiment of the invention, the frozen-in indicator substance may have a pattern and / or a surface structure, for example a molded or embossed pattern, at least on a partial area of its surface. A pattern or a surface structure disappears or at least changes when the indicator substance becomes liquid, so that it can be checked on the basis of the presence or absence of the pattern whether the melting temperature has been exceeded at least temporarily. According to an advantageous variant of this embodiment may be on the pattern and / or the surface structure of a transparent or semi-transparent

Be arranged protective cover. This protects the pattern from external mechanical damage.

For example, the pattern or surface texture may be an embossed pattern. An embossed pattern may be obtained, for example, by shaping the indicator substance in a liquid state in a mold and then freezing the molded indicator substance.

According to a further aspect of the invention, there is provided a device for monitoring the temperature of a cryopreserved biological sample, comprising a sample container according to the invention, as described in this document, and a measuring device that is configured, a configuration change, in particular a change of shape, arrangement and / or position to detect the frozen indicator substance.

The measuring device can be designed appropriately depending on the embodiment. In the embodiment in which the solid-frozen indicator substance-carrying region has an electrode arrangement, the measuring device can be designed to measure a resistance or an impedance of the electrode arrangement, as already described above.

In the embodiment in which the area bearing the frozen indicator substance has a reflective coating, the measuring device can be designed, for example, to generate a measuring beam, eg a measuring beam. B. an electromagnetic beam, to judge on the Verspiegelung frozen indicator substance and to detect a reflected measuring beam, if it is reflected. When the indicator substance is exceeded, it will flow down the sample container in the liquid state of aggregation, so that the measuring beam now directly strikes the mirrored surface, which was previously covered by the solidified indicator substance. The measuring beam is now reflected on the mirrored surface, which can be detected by the measuring device. As soon as the trade fair tion thus detects a reflected measuring beam, it can be concluded that the melting temperature has been exceeded at least for a short time. For example, in the embodiment in which the frozen indicator substance has a pattern or a surface texture, the measuring device may be configured to optically detect whether the pattern or the surface pattern is still present or not. In an analogous manner, the measuring device can be designed, for example, to detect whether the arrangement of the indicator substance is still present or not, etc.

The term sample container refers in particular to a container designed for cryopreservation. The sample container is preferably produced using low-temperature-compatible plastic material for temperatures below -140.degree. The plastic material can tolerate repeated temperature changes without change and without damage. A plastic material is preferably used whose water absorption capacity is <1% of the intrinsic mass, in particular <0.1% of the intrinsic mass. Cryogenic storage elements according to the invention are based, for example, on polyurethane or polyethylene.

The term "biological sample" refers to biological material such as cells, tissue, cell constituents, biological macromolecules, etc. which is subjected to cryopreservation in the sample container, possibly in a suspension and / or composite with a substrate material In accordance with another aspect of the invention, there is provided a method of making a sample container designed for temperature monitoring of a cryopreserved biological sample comprises the provision of a sample container designed to receive a biological sample The sample container is preferably a cryoprobe container.

The method further comprises applying an indicator substance whose melting temperature is in a range of -20 ° C to -140 ° C to an area of the outside Surface of the sample container in liquid state and the freezing of the applied indicator substance.

According to a first embodiment of the method, the sample container is cooled to a temperature below the melting temperature of the indicator substance prior to application of the indicator substance in the liquid state of aggregation. As a result, a rapid freezing of the applied indicator substance can be achieved.

For example, the indicator substance in the liquid state of aggregation drop-shaped by means of a drop deposition device, for. As a drop-shot device, are applied to the outer surface of the frozen sample container. Drop guns, z. B. executed as a piezo-pressure nozzle or piezo-printhead, are known per se from the prior art and not described in detail here. According to a second embodiment of the method, the application of the liquid indicator substance and the subsequent freezing take place according to the following steps:

First, the sample container is partially immersed in a container filled with an indicator substance in the liquid state of aggregation, so that at one point the indicator substance adheres to an outside of the sample container. Subsequently, the sample container is positioned on a hollow mold, such that the indicator substance adhering to the sample container fills an embossing in the interior of the hollow mold. After the indicator substance has been frozen in the mold, the mold is removed from the indicator substance solidified on the sample container. As a result, the introduced through the mold into a surface of the indicator substance imprinting is exposed.

According to another aspect of the invention, there is provided a method of monitoring the temperature of a cryopreserved biological sample. The method comprises providing a sample container designed to receive a cryopreserved biological sample and carrying at a portion of its outer surface a solid frozen indicator substance whose melting temperature is within a range from -20 ° C to -140 ° C. The sample container may also be designed according to the embodiments and variants described in this document. To avoid repetition, features disclosed purely in accordance with the device should also be disclosed as being in accordance with the method and be able to be claimed. The sample container may include a cryopreserved biological sample in its receiving space. The sample container can be stored at storage temperature below the melting temperature of the indicator substance for cryopreserved storage of the biological sample.

The method further comprises determining whether a configuration change of the indicator substance has taken place due to a temporary exceeding of the melting temperature of the indicator substance, in particular whether a change of shape or arrangement, in particular position, of the indicator substance has taken place. This change can be detected immediately by looking or technically automated, whether the limit temperature to be monitored has been exceeded.

The preferred embodiments and features of the invention described above can be combined with one another. Further details and advantages of the invention will be described below with reference to the accompanying drawings. In the drawings: FIG. 1-4 are schematic views of various embodiments of a sample container designed for temperature monitoring of a cryopreserved biological sample;

FIG. 5A, 5B, 6A are each a melting diagram of a liquid mixture;

FIG. 6B is a table with melting points of some pure liquids; and

FIG. 7 shows a miscibility matrix of solvents. Identical or functionally equivalent elements are denoted by the same reference numerals in all figures and are in part not described separately. FIG. 1A shows a first exemplary embodiment of a sample container 10, which is designed for temperature monitoring of a cryopreserved biological sample. FIG. 1A further illustrates in a highly schematic manner the production of such a sample container 10.

The sample container 10 is a cryotube, which is shown in Figure 1A in the fully screwed state. The cryotube comprises a cylindrical receiving part 1, which forms a receiving space 2 in which a biological sample (bioprobe) 6 is stored. The biological sample may be a cell suspension. The cylindrical receiving part 1 is closed with a lid 3. The cryovial also has a bottom part 4.

The sample container 10 is already in Figure 1A on the storage temperature, z. B. at -140 ° C, but at least below the melting point of the indicator substance 8. The cryotube carries at a portion 11 of its outer surface a frozen indicator substance 12 whose melting temperature is in a range of -20 ° C to -140 ° C.

In this case, depending on the temperature limit value which is to be monitored during the cryopreservation, a suitable liquid or a liquid mixture is selected as the indicator substance 12.

Through the selection of suitable liquids and the mixing ratio of liquids, their melting point can be set to a desired value, in particular in a range from -20 ° C to -140 ° C.

By way of example, FIG. 5A shows the course of the melting point as a function of the mixing ratio of an alcohol and water, with which a temperature range between 0 ° C. and -118 ° C. can be covered with a moderate increase in viscosity with decreasing temperature. Should z. B. a temperature limit of -118 ° C are monitored, the ethanol content can be set to 93.5%. Melting points up to a value of just below -60 ° C can also be adjusted by admixing potassium hydroxide (KOH) to water, which is shown in FIG. 5B by means of a melting diagram is shown. A mixture of water and cryoprotectant may also be used as the indicator substance, as illustrated by the melt diagram of Figure 6A. The table of FIG. 6B lists freezing points / melting points of further pure liquids which can be used alone or as a mixture with another liquid as an indicator substance. Other suitable as indicator substance liquid mixtures are chloroform-cyclohexane mixtures or other miscible liquids, the z. B. from the miscibility matrix of solvents of Figure 7 can be removed. If several temperature limit values are to be monitored in the case of cryogenic storage or if the temperature intervals reached by the sample are to be narrowed down more precisely, a plurality of different indicator substances with different melting points can be used accordingly, which will be described below with reference to FIG.

The region 11 of the cryovial shown in FIG. 1A carries the indicator substance 12 in the form of frozen drops 13 arranged in rows in the axial direction A (represented by the vertical arrow in FIG. 1). The drops 13 of the indicator substance 12 are applied to the Cryotubes applied: On the cold surface of the region 11 of the cryotube, the warm or pre-cooled indicator substance 8 is dropped dropwise in the liquid state of aggregation via a dropping device 7, z. B. via piezo-pressure nozzles. The drops 8 freeze on the cryogenic surface, as shown by the reference numeral 13 in Figure 1A.

To improve the adhesion, the surface area 11, which is provided for holding the indicator substance, be changed in its properties so that a good wetting and adhesion of the drops takes place, for. By roughening the region 11 and / or by applying a structure or chemical coating to the region

11th The drops 13 solidify on the surface, as shown in Figure 1A. When the cryoprobe exceeds the melting temperature of the indicator substance 8 at any time during storage, the frozen drops 13 liquefy and flow together completely or partially. This results in an image that looks something like the one shown in FIG. 1B.

If the indicator substance 12 is no longer in the state shown in FIG. 1A after a cryogenic storage, but in a state in which the drops have at least partially fused together, as illustrated schematically in FIG. 1B, then it can be concluded that the melting temperature and so that a critical temperature limit has been exceeded. If, on the other hand, an unchanged arrangement of the indicator substance is found after cryopreservation, the sample 6 has been properly stored continuously below the melting temperature. A user can thus easily determine by a visual visual inspection, whether an undesirable increase in temperature has occurred above the melting temperature or above the limit temperature to be monitored.

The possibility of using piezo systems to shoot very fine droplet systems means that larger droplet areas can also be located on the surface of the sample container

Generate letters, patterns and quickly recognizable structures through to barcodes and other identifiers. These structures are lost when the critical temperature of the indicator substance is exceeded. Exemplary arrangements are shown in Figure 1C and Figure 1D.

FIG. 1C shows a sample container 10a which carries at a region 11 of its outer surface a frozen indicator substance 12a, which is formed from a larger drop area and / or a plurality of indicator substances which are arranged regularly one above the other and next to each other. FIG. 1D shows a sample container 10a, which carries at a region 11 of its outer surface a frozen indicator substance 12b, which is applied in the form of a letter. Does the arrangement Loss of indicator substance 12a or 12b lost in the cryogenic storage, in turn, can be closed to an at least temporary exceeding of the melting temperature.

FIG. 2A again shows a cryotube 20, which is at the storage temperature, and a droplet gun 7.

Annular in this example, as shown in Figure 1A, a series of indicator substance drops 8 are banded onto an area 21 of the outer cryogenic surface of the cryotube 1 where they freeze.

In the embodiment of FIG. 2, various indicator substances 23a, 23b and 23c whose melting temperatures are different are used. For example only, the indicator substance 23a may have a melting temperature of -60 ° C, the indicator substance 23b may have a melting temperature of -70 ° C, and the indicator substance 23c may have a melting temperature of -80 ° C. If the various indicator substances are applied to the cryotube as shown in FIG. 2A, their melting temperatures decreasing from top to bottom, it is likewise possible to detect when one or more melting temperatures on the structure have been exceeded that an inadmissible temperature increase has taken place.

FIG. 2B shows by way of example the case that only the melting temperature of the indicator substance 23a (upper droplet ring in FIG. 2A) has been exceeded, so that only the upper droplet ring has flowed downwards in FIG. 2B, which in turn can be easily detected from the outside and The bioprobe inside is not contaminated.

FIG. 3A shows in the left part of the figure a cryotube analogous to FIGS. 1 and 2, in which there is still an electrode arrangement 33, 34 at the region 31 of the outer surface, on which the indicator substance 32 is applied, e.g. B. in the form of miniaturized gold or platinum electro-den.

If the melting temperature of the indicator substance 32 is exceeded, this flows out of the electrode region, whereby the resistance or the impedance changes, which can be detected by scanning the electrodes 33, 34. A state of the cryotube 30 at which the melting temperature of the indicator substance 32 has been exceeded is shown in the right-hand sub-figure in FIG. 3A. FIG. 3B shows an embodiment of the cryotube 30a in which there is a mirrored surface 35a, 35b on two regions 31 of the outer surface of the cryotube, to which the indicator substance 32a, 32b is applied. It can be determined optically, visually or via a measuring beam 100 whether the indicator substance 32a, 32b is still located on these original position fields on the mirrored surface 35a, 35b. If this is the case, the melting temperature of the indicator substance has not been exceeded. On the other hand, a state in which the indicator substance 32a, 32b has left this region by melting and draining off is shown in the right part of FIG. 3B. Here can thus be concluded that an intermediate exceeded the melting temperature and thus the monitored limit temperature.

Further, different indicator substances may be selected so that the indicator substance 32a on the first mirrored surface 35a has a melting temperature corresponding to a first temperature limit to be monitored and that the indicator substance 32b on the second mirrored surface 35b has a melting temperature corresponding to a second temperature limit to be monitored equivalent.

FIG. 4 schematically illustrates in the time sequence of FIGS. 4A, 4B, 4C and 4D the production of a further cryotube 40, which is designed for temperature monitoring of a cryopreserved biological sample.

In FIG. 4A, a sample container in the form of a typical sealed cryovial (tube) 1, as used in cryobiobanks, is shown in a sectional view. It usually includes a recording volume 2 for the bioprobe in which the biomaterials are located. The bioprobe is z. The cryotube further comprises a lid 3, which closes the vessel and has an engagement 4 above, over which the lid 3 can be rotated with a tool (not shown) in the case of automation. These cryotubes 1 can also contain a bottom 4 in the optional a barcode rectangle or other identifier is inserted. In this form, usually standing vertically in recordings, the cryovials 1 are stored in the cryogenic containers. The cryotube may be at a temperature between room temperature and just above the melting point of the indicator substance 42. This is in liquid form in a container 46, in which the bottom 4 of the cryotube, as shown in Figure 4B, is immersed. As a result, a part 42a of the indicator substance 42 adheres to the bottom 4 in liquid form. The cryotube with the indicator substance amount 42a is now pressed into a structured mold 44 and brought to storage temperature. As a result, the indicator substance 42b inversely takes the surface structure and embossment 45 of the interior of the mold 44 in an inverse manner.

The cryotube 40 shown in FIG. 4C thus carries on its underside a frozen indicator substance which has an embossed pattern 43 or a surface structure at a portion of its surface. For protection during further storage, this solidified structure is covered with a cap 47, the z. B. optically transparent, so that an automatic identification of the structure via a camera system or an optical measuring beam 101 can be controlled.

If the pattern 43 or the introduced structure impressed into the indicator substance 42b is lost or changed, the cryoprobe has again been brought above the melting temperature of the indicator substance 42b at some point in time. The cryotube 40 is thus designed to monitor the temperature of a cryopreserved biological sample. Subsequently, it can be checked by means of the frozen indicator substance 42b at any time during the storage process whether undesired, if only temporary heating of the cryoprobe has taken place. For this purpose, it is checked whether the pattern 43 embossed in the indicator substance 42b has been lost or changed. If this is the case, it can be concluded that the limit temperature (s) to be monitored have been exceeded.

If a structure is produced in which a flowing or group-wise transition from very fine to coarser structural elements is realized, a very short-term excess of the melting point can also be recognized via the changes in the structure. The geometrically smallest and finest structures change first.

Although the invention has been described with reference to particular embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for without departing from the scope of the invention. Accordingly, the invention should not be limited to the disclosed embodiments, but should include all embodiments which fall within the scope of the appended claims. In particular, the invention also claims protection of the subject matter and the features of the subclaims independently of the claims referred to.

Claims

Claims 1. A sample container (10; 20; 30; 30a; 40) adapted to receive a cryopreserved biological sample located at a portion (11; 31,41) of its outer surface

Surface carries a frozen indicator substance (12, 22, 32, 42b) whose

Melting temperature in a range of -20 ° C to -140 ° C.

2. Sample container according to claim 1, characterized in that the

solidified indicator substance (12; 22; 32; 42b) carrying region (11; 21; 31, 41) has a coating, roughening and / or structuring.

3. Sample container according to claim 1 or 2, characterized in that the solid-frozen indicator substance (12; 22; 32; 42b) carrying region has a reflective coating (35).

4. Sample container according to claim 1 or 2, characterized in that the solid-frozen indicator substance-carrying region has an electrode arrangement (33, 34).

5. Sample container according to one of the preceding claims, characterized

characterized in that the indicator substance is applied in a predetermined arrangement to the outer surface of the sample container (10; 20; 30; 30a; 40), the arrangement comprising a number, a letter (12b), a symbol, an identifier and / or a Structure (12; 12a; 22).

6. Sample container according to one of the preceding claims, characterized

in that the indicator substance (12; 12a; 12b) applied to the outer surface is obtained by frozen drops (8) of the indicator substance.

7. Sample container according to one of the preceding claims, characterized in that a plurality of different indicator substances (23a, 23b, 23c: 32a; 32b), whose different melting temperatures are in a range from -20 ° C to -140 ° C, in different areas the outer surface of the sample container (20; 30a) are applied.

8. Sample container according to one of the preceding claims, characterized

characterized in that the sample container (10; 20; 30; 30a; 40) is a cryotube.

9. Sample container according to claim 8, characterized in that

a) that several different indicator substances are present in their

Distinguish melting temperatures, each in the form of juxtaposed

frozen drops (23a, 23b, 23c) are applied to a receiving cylinder of the cryotube (20), in particular in the form of band-shaped, for example annular, juxtaposed drops, and

b) that the various indicator substances in the axial direction (A) of the cryotube (20) are arranged offset to one another.

10. Sample container according to one of claims 1 to 9, characterized in that the solid frozen indicator substance has a pattern or a surface structure at least at a portion of its surface.

A sample container according to claim 10, characterized in that the pattern or the surface structure is an embossed pattern (43), preferably obtained by shaping the indicator substance in liquid state in a mold (44) and subsequently freezing the shaped indicator substance (42b). ,

12. Apparatus for monitoring the temperature of a cryopreserved biological sample comprising

a sample container (10; 20; 30; 30a; 40) according to any preceding one

Claims; and a measuring device which is designed to detect a configuration change, in particular a change in the shape, arrangement and / or position, of the frozen indicator substance.

13. A method of manufacturing a sample container (10; 20; 30; 30a; 40) adapted for temperature monitoring of a cryopreserved biological sample

Providing a sample container for receiving a biological sample; and applying an indicator substance whose melting temperature is in a range of -20 ° C to -140 ° C at a portion of the outer surface of the sample container in the liquid state; and

Freezing of the applied indicator substance.

14. The method according to claim 13, characterized in that is cooled to a temperature below the melting temperature of the indicator substance prior to application of the indicator substance in the liquid state of aggregation of the sample container.

15. The method according to claim 13 or 14, characterized in that the

Indicator substance is applied dropwise by means of a drop deposition device (7) on the outer surface of the sample container.

16. The method according to claim 13, characterized by

a) partial immersion of the sample container (1) in a filled with an indicator substance (42) in the liquid state of aggregation state container (46), so that at one point the

Indicator substance (42a) adheres to an outside of the sample container (1);

b) positioning the sample container (1) on a hollow mold (44) such that the indicator substance (42a) adhering to the sample container (1) fills an embossment (45) in the interior of the hollow mold (44); and

c) after the indicator substance has frozen, removing the mold cavity (44) from the indicator substance (42b) frozen to the sample container.

17. A method of monitoring the temperature of a cryopreserved biological sample, comprising the steps of:

a) providing a sample container (10; 20; 30; 30a; 40) according to any one of claims 1 to 12;

b) determining whether a change in the shape or arrangement, in particular position, of the indicator substance has taken place due to a temporary exceeding of the melting temperature of the indicator substance or the threshold temperature at which the viscosity of the molten indicator substance falls below a certain target value.

18. Sample container, apparatus or method according to one of the preceding

Claims, characterized in that the indicator substance comprises at least one alcohol selected from the group consisting of octan-1-ol, nonan-1-ol, propan-1, 2-diol, propan-1, 3-diol, butane-1, 2-diol, butane-l, 3-diol, butan-2-ol, pentane-l, 5-diol, pentan-l-ol, cyclopentanol, benzyl alcohol, is selected, and optionally comprises at least one dye.

19. Sample container, apparatus or method according to claim 18, characterized

characterized in that the dye is selected from the group consisting of

Triphenylmethane, rhodamine, especially xanthene, azo dyes and phenazine and Phenothiazinfarbstoffe includes.

20. A sample container, apparatus or method according to claim 18 or 19, characterized in that the indicator substance at least two alcohol components, which from the group, the octan-1-ol, nonan-1-ol, propane-l, 2-diol, propane -l, 3-diol, butane-1,2-diol, butane-l, 3-diol, butan-2-ol, pentane-l, 5-diol, pentan-1-ol, cyclopentanol, benzyl alcohol , and / or the indicator substance comprises at least one dye selected from the group consisting of Oil Red, Methyl Red,

Brilliant green, rhodamine B, neutral red, methylene blue or other dyes that are used to

Staining of cells used in cytology includes.

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