METHOD AND DEVICE FOR THE TEMPERATURE MONITORING OF A CRYOPRESERVED BIOLOGICAL SAMPLE

Abstract

The invention relates to a device for the temperature monitoring of a cryopreserved biological sample, comprising a sample container (in particular a cryotube) having a holding space for holding a biological sample and comprising at least one chamber, the interior of which is not fluidically connected to the holding space and is filled only partially with an indicator substance, the melting temperature of which lies in a range of 20 C. to 140 C. In particular, the chamber can be formed by a container that is detachably or pivotably fastened to the sample container. Alternatively, the chamber is formed by a double-walled slide-on part or the holding space of the sample container is double-walled, wherein an intermediate space between the inner wall and the outer wall is partially filled with the indicator substance.

Claims

1. A device for temperature monitoring of a cryopreserved biological sample, comprising a) a sample container with a receiving space for receiving a biological sample; and b) at least one chamber, the inner space of which is not fluidically connected to the receiving space and is only partially filled with an indicator substance, the melting temperature of which lies in a range from 20 C. to 140 C.

2. The device according to claim 1, wherein there is a plurality of chambers which are filled in each case only partially with a respective indicator substance, the melting temperature of which lies in a range from 20 C. to 140 C., wherein the indicator substances in the chambers have different melting temperatures.

3. The device according to claim 1, wherein a chamber wall at at least one point is transparent or semi-transparent.

4. The device according to claim 1, further comprising a measuring apparatus which is configured to detect a position of the indicator substance in the at least one chamber.

5. The device according to claim 1, wherein the indicator substance comprises an indicator additive which increases detectability of a physical property of the indicator substance.

6. The device according to claim 5, wherein the at least one chamber is formed by a container with one or more cavities which is fastened detachably or pivotably to the sample container.

7. The device according to claim 6, wherein the container is fastened pivotably to a longitudinal end of the sample container.

8. The device according to claim 6, wherein the container is fastened pivotably to the sample container by a bendable part.

9. The device according to claim 6, wherein the container is fastened pivotably to the sample container about an axis of rotation, which is perpendicular to a longitudinal axis of the sample container.

10. The device according to claim 6, wherein the container a) is semi-annular or annular; and b) can be moved into a first pivot position, in which it is arranged coaxially with respect to a longitudinal axis of the sample container, and into a second pivot position which is rotated by at least 45 with respect to the first pivot position.

11. The device according to claim 6, wherein a) the container is an elongated hollow body which is fastened pivotably indirectly or directly to the sample container) at a longitudinal end of the sample container; and/or b) the container can be moved into a first pivot position, in which a longitudinal axis of the container runs parallel to a longitudinal axis of the sample container, and can be moved into a second pivot position, which is rotated by at least 45 in comparison with the first pivot position.

12. The device according to claim 6, wherein the at least one chamber is formed by a double-walled push-on part.

13. The device according to claim 12, wherein the double-walled push-on part is a double-walled cap which can be pushed onto the sample container at a longitudinal end of the sample container.

14. The device according to claim 6, wherein the sample container has a cover for closing off the receiving space and the container is fastened detachably or pivotably to the cover.

15. The device according to claim 1, wherein the receiving space of the sample container for the formation of the at least one chamber is double-walled with an inner wall and an outer wall, wherein an intermediate space between the inner wall and the outer wall is partially filled with the indicator substance.

16. The device according to claim 16, wherein the sample container is a cryogenic tube.

17. The device according to claim 16, wherein the double-walled push-on part can be pushed, glued or slid onto an outer shell surface of the cryogenic tube and at least partially engages around it in a pushed-on state.

18. A method for temperature monitoring of cryopreserved samples, comprising the steps: a) providing a device for temperature monitoring according to claim 1; b) freezing the indicator substance, wherein the at least one chamber is moved into a first position during freezing of the indicator substance and thereafter is moved into a second position in which a melting of the indicator substance leads, as a result of an influence of gravity, to an at least partial displacement and/or change in shape of the indicator substance in the at least one chamber.

19. The method according to claim 18, wherein a substance is selected as the indicator substance, a melting temperature of which or a threshold temperature of which, at which a viscosity of melted indicator substance exceeds a determined setpoint value, corresponds to a predetermined threshold temperature, the exceeding of which is be monitored.

20. The method according to claim 19, further comprising a) storing of the device with a cryopreserved sample in the sample container, wherein the at least one chamber is arranged in the second position on the sample container; and b) ascertaining whether an at least partial displacement and/or change in shape of the indicator substance performed by temporarily exceeding the melting temperature of the indicator substance has taken place.

21. The device according to claim 1, wherein the indicator substance comprises at least one alcohol selected from the group consisting of octan-1 -ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butane-1,3-diol, butane-2-ol, pentane-1,5-diol, pentan-1-ol, cyclopentanol and benzyl alcohol as well as optionally at least one dye.

22. The device according to claim 21, wherein the at least one dye is selected from the group consisting of triphenylmethane dyes, rhodamine dyes, azo dyes, phenazine dyes and phenothiazine dyes.

23. The device according to claim 21, wherein the indicator substance comprises at least two alcohol components selected from the group consisting of octan-1-ol, nonan-1-ol, propane-1,2-diol, propane-1,3-diol, butane-1,2-diol, butane-1,3-diol, butane-2-ol, pentane-1,5-diol, pentan-1-ol, cyclopentanol, and 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, and methylene blue.

Description

[0087] The preferred embodiments and features of the invention described above can be combined with one another. Further details and advantages of the invention are described below with reference to the enclosed drawings. In the drawings:

[0088] FIGS. 1-6 show schematic views of various exemplary embodiments of a device for temperature monitoring of a cryopreserved biological sample;

[0089] FIG. 7 shows a flow chart to illustrate an exemplary embodiment of a method for temperature monitoring of a cryopreserved biological sample;

[0090] FIGS. 8A, 8B, 9A show in each case a melting diagram of a liquid mixture;

[0091] FIG. 9B shows a table with melting points of a number of pure liquids; and

[0092] FIG. 10 shows a mixability matrix of solvents.

[0093] Identical elements or functionally equivalent elements are designated by the same reference numbers in all the figures and are partially not described separately.

[0094] FIG. 1A shows a sample container in the form of a typical cryogenic tube (tube) 1, as is used in cryogenic biobanks. It generally comprises a receiving volume 2 for the biosample in which the biomaterials are located. The biosample here is a cell suspension 6. The cryogenic tube further comprises a cover 3 which closes off the vessel and at the top has an engagement 4 via which cover 3 can be turned with a tool (not shown) in the case of automation. These cryogenic tubes 1 can also contain a base 5 into which a barcode square or another mark is optionally inserted. In this form, usually standing perpendicular in receptacles, cryogenic tubes 1 are stored in the low-temperature containers.

[0095] According to the embodiment shown in FIG. 1, a double-walled transparent cap 11 in the deep-cooled state is pushed onto cryogenic tube 1. Cap 11 is represented in FIG. 1B. Double-walled cap 11 has an inner wall 13 and an outer wall 12. The wall volume or intermediate space 14 between inner wall 13 and outer wall 12 is partially filled with an indicator substance 7 in the form of a liquid or a liquid mixture, the freezing point/melting point of which in the range from 20 C. to 100 C. is selected via the mixture ratio. This is also explained in greater detail below on the basis of FIGS. 8 to 10.

[0096] Cap 11 is, in a first position which is rotated by 180 with respect to the rotational position of cap 11 shown in FIG. 1B, cooled below this threshold temperature so that the liquid runs under the influence of gravity within intermediate space 14 into cap tip 15 and freezes there. Cap 11 can now be rotated and shows the image represented in FIG. 1B. Indicator substance 7 is solidly frozen in cap tip 15.

[0097] In the case of a temperature below the freezing point/melting point of indicator substance 7, cap 11 is then pushed from above onto cryogenic tube 1 as shown in FIG. 1C. Device 10 formed in this manner for temperature monitoring can thus be cryogenically stored, e.g. in a cryogenic tank.

[0098] Should sample 6 and thus also indicator substance 7 at some point reach a temperature range above the melting point of indicator substance 7, melted indicator substance 7 in wall volume 14 flows downwards and collects in a lower cap region 16. The image shown in FIG. 1D is produced. If sample 16 has been kept under the freezing point of indicator substance 6 at all times, instead the state as shown in FIG. 1E is produced. In this manner, an inadmissible heating of sample 6 is easily apparent. The position of the indicator substance within cap 11 can be optically detected optoelectrically and in an automated manner by appearance, but also by means of an expediently formed measuring apparatus. If indicator substance 7 is dyed, this facilitates determination of the position. The determination of the position of the indicator substance can also be carried out very easily in the cooling tanks at the storage temperature. A further advantage of device 10 is the reusability of caps 11 and the use of marker liquids used as indicator substance 7 with a freely selectable freezing point. For living racks, a melting temperature around 80 C. is recommended since here a clear recrystallisation of the ice in the cells and around these occurs which leads to a reduction in quality of the cryogenic sample. For biological liquids and storage of genetic material which is stored at 80 C., a melting point around 30 C. is to be recommended.

[0099] FIG. 2 shows views of a modified two-phase-variant in the form of a part 21 which can be pushed on and is composed of a transparent material, shown in FIG. 2B, which as shown in FIG. 2D can be pushed onto a cryogenic tube 1, as shown in FIG. 2A.

[0100] Push-on part 21 is in turn embodied to be double-walled with an inner wall 23 and an outer wall 22 for the formation of a wall volume 24. Wall volume 24 is divided by a separating wall 25 into two partial volumes 24a and 24b which are separate from one another. The push-on part thus forms two chambers 24a, 24b. Chamber 24a is partially filled with a first indicator substance 7, chamber 24b is partially filled with a second indicator substance 26. Indicator substances 7, 26 have different melting points. These are brought under their freezing points in a first position, shown in FIG. 1B, and solidify. Push-on part 21 rotated by 180 is then pushed onto frozen cryogenic tube 1. The device comprising cryogenic tube 1 and pushed-on push-on part 21 is designated by reference number 20. Push-on part 21 is adapted to the dimensions of cryogenic tube 1 so that it can be pushed onto a casing surface of the cryogenic tube and engages around it.

[0101] If a liquid with a freezing point around 80 C. was selected as first indicator substance 7 and a liquid with a freezing point around 60 C. was selected as second indicator substance 26 and cryogenic tube 1 together with push-on part 21 was taken to a temperature above 80 C., but below 60 C. in the course of its storage during one of the removal and renewed storage processes or in the tank, the image represented in FIG. 2D is produced. Indicator substance 26 was temporarily melted and flowed into the lower region of wall volume 24b, while indicator substance 7 is still located in the upper region of the wall volume 24a. In this manner, the change in the storage temperature can thus be restricted. Had device 20 been heated at least temporarily above 60 C., both indicator substances 7, 26 would be located in the lower volume of the push-on part (not represented). If the sample has been correctly stored, push-on part 21 exhibits the image represented in FIG. 1C.

[0102] FIG. 3 shows two variants of a partially liquid-filled double-walled transparent annular body 31, which forms an inner volume 34 for receiving an indicator substance 7 and which is fastened pivotably to a cryogenic tube 1. In the case of the variant shown in FIGS. 3A and 3B of device 30, annular body 31 is fastened pivotably to cover 3 of cryogenic tube 1. In the case of the variant shown in FIGS. 3C and 3D of device 30c, annular body 31 is fastened pivotably to base 5 of cryogenic tube 1. Pivot axis D runs perpendicular to a longitudinal axis of cryogenic tube 1. The annular body can be pivoted into a vertical position, as represented in FIGS. 1A and 1C, and into a horizontal position, as represented in FIGS. 1B and 1D.

[0103] Device 30 or 30c composed of cryogenic tube 1 and annular body 31 is frozen in each case with a mounted ring (vertical position), as is shown in FIG. 1A and FIG. 1C. Indicator liquid 7 solidifies in a partial volume 33 in the lower half of annular body 31. It is hereby emphasized that the indicator substance does not, as shown, have to occupy half the inner annular volume, but rather can lie above it or below it. After solidification of indicator liquid 7, annular body 31 is rotated at an adhesion part 36 into the horizontal position, as shown in FIG. 1B and FIG. 1D. As long as this image is maintained, device 30, 30c or sample 6 stored therein has not been heated to be warmer than the melting point of indicator liquid 7. Otherwise, the liquid has spread and is thus also located partially in partial volume 32, which can be ascertained by visual inspection or also by optical measurements.

[0104] FIG. 4 shows a further exemplary embodiment in the case of which an elongated container 41, inner volume 44 of which is filled partially with indicator substance 7, is fastened pivotably to a cryogenic tube 1 about axis of rotation D. Indicator substance 7 is frozen in the case of an oblique or also perpendicular position of container 41. Container 41 with frozen indicator substance 7, as shown in FIG. 4B, is subsequently bent downwards. To this end, the container is fastened via a bending part 46 to cryogenic tube 1. As long as indicator substance 7 remains frozen in upper partial volume 42, the melting temperature has not been exceeded. If device 40 is thus found in the state shown in FIG. 4B after cryogenic storage, it can be concluded that the melting temperature of the indicator substance was not exceeded. Said device 40 is particularly well suited to optical detection. It is thus possible to check with a measuring apparatus (not represented), measurement beam path 45 (dashed line) of which is directed at the lower partial volume 43 of transparent container 41, whether indicator substance 7 became liquid as a result of a melting process and as a result flowed into lower partial volume 43. In this case, measurement beam 45 is absorbed by the indicator substance, which can be detected by an interruption of measurement beam 45.

[0105] FIG. 5 shows schematic sectional views of a further exemplary embodiment of a device 50 for temperature monitoring of a cryopreserved biological sample. In the case of the exemplary embodiment, chamber 54 for receiving indicator substance 7 is integrated into cryogenic tube 51. Cryogenic tube 51 has in turn a receiving volume (receiving cylinder) 2, into which biosample 6 is inserted, as well as a cover 3. The cover has a shaft 8 which engages into receiving volume 2. The wall of receiving cylinder 2 is embodied to be double-walled, having an outer wall 52 and an inner wall 53, which forms a wall volume 54 in which indicator substance 7 is located. Device 50 with sample 6 is thus frozen as follows:

[0106] Freezing to a temperature below the freezing point of biosample 6 (generally 20 C.) is performed in the position of cryogenic tube 51, as shown in FIG. 5A. The state as shown in FIG. 5B is produced. Now frozen biological sample 6 with still liquid indicator substance 7 is moved out of the position in FIG. 5B by 180 rotation into the position as shown in FIG. 5C and further cooled below the freezing point of indicator substance 7. If the melting temperature of indicator substance 7 has been selected e.g. with 60 C., sample 6 can then be moved back into the starting position of FIG. 5A below this temperature and after solidification of both liquids, as a result of which the state according to FIG. 5D is produced. Frozen indicator substance 7 is now located at the top and frozen biosample 6 is located at the bottom in cryogenic tube 51. If the melting temperature of 60 C. is undershot, this is apparent from the fact that both liquids are again located as represented in FIG. 1A in the lower cylinder region. This can also be detected via an optical transmission or scattered light measurement, etc., which is illustrated on the basis of dashed beam path 45 in FIG. 5D. Coloring of indicator substance 7 is expedient for manual operation.

[0107] The views of FIG. 6 show schematic views of a further device 60 for temperature monitoring of a cryopreserved biological sample. One particular feature of this embodiment variant lies in the fact that the container, which is partially filled with an indicator substance 7, is embodied as a semi-annular hollow body 61. Semi-annular body 61 is fastened pivotably at its two ends to cover 3 of a cryogenic tube 1, and indeed about an axis of rotation D which runs perpendicular to a longitudinal axis of cryogenic tube 1. In the position of FIG. 6A, in which semi-annular body 61 is pivoted into a vertical position, device 60 is frozen to the storage temperature. Indicator substance 7 flows still in the liquid assembly state as a result of gravity to the two lowest points in upright semi-annular body 61 and is located to the right and left directly above axis of rotation D. Indicator substance 7 freezes there. In the deep-frozen state, semi-annular body 61 is then moved into a horizontal position as represented in FIG. 6B.

[0108] An optical measurement can very easily be made from above on this semi-circular body 61, which optical measurement shows whether indicator substance 7 is still located in the original position or, as shown in FIG. 6C, has spread in annular base 62. In this case, the melting point of indicator substance 7 has been exceeded at some point in time. A flat side of semi-annular body 61 can also be mirrored so that optical measurement beam 45 is reflected if indicator substance has not spread.

[0109] FIG. 7 illustrates on the basis of a flow chart a method for temperature monitoring of a cryopreserved biological sample. In step S1, a device for temperature monitoring is provided, for example, one of devices 10, 20, 30, 30c, 40, 50 or 60. In this case, depending on the temperature threshold value which is supposed to be monitored in the case of cryogenic storage, a suitable liquid or a liquid mixture is to be selected as indicator substance 7.

[0110] Via the selection of suitable liquids and the mixture ratio of liquids, their melting point can be set to a desired value in a range from 20 C. to 140 C.

[0111] By way of example, FIG. 8A indicates the profile of the melting point as a function of the mixture ratio of an alcohol and water, with which, in the case of a moderate increase in viscosity with falling temperature, a temperature range between 0 C. and 118 C. can be covered. Should e.g. a temperature threshold value of 118 C. be monitored, the ethanol ratio can be set at 93.5%. Melting points up to a value of slightly below 60 C. can also be set by adding potassium hydroxide (KOH) to water, which is shown in FIG. 8B on the basis of a melting diagram. A mixture of water and antifreeze can also be used as the indicator substance, which is illustrated by the melting diagram of FIG. 9A. The table of FIG. 9B lists freezing points/melting points of further pure liquids which can be used on their own or as a mixture with another liquid as the indicator substance. Further liquid mixtures which are suitable as the indicator substance include chloroform/cyclohexane mixtures or other mixable liquids which can be inferred e.g. from the mixability matrix of solvents of FIG. 10.

[0112] Liquids and plastic materials with good wettability and low viscosity at low temperatures are primarily selected in order to configure the change in position to be as extensive as possible and the additional compartment as small as possible.

[0113] If several temperature threshold values are supposed to be monitored during cryogenic storage or if the achieved temperature intervals which the sample reaches should be restricted more precisely, several different indicator substances with different melting points can correspondingly be used which are then arranged in different chambers in the sample container.

[0114] In step S2, the indicator substance in the chamber is then frozen, wherein the chamber is moved into a first position during freezing of the indicator substance. In the case of different indicator substances and several chambers, these are moved in an analogous manner in each case into a first position and frozen.

[0115] Thereafter, in step S3, the at least one chamber with the frozen indicator substance is moved into a second position and, if the chamber is not yet arranged on the sample container, arranged thereon. The second position changes the spatial position of the frozen indicator substance at least to such an extent that a melting after the change in position leads to a visible displacement of the liquid or its delimiting geometry in the chamber.

[0116] In this state, the device can be stored with a cryosample in the receiving space of the sample container in the case of a storage temperature below the melting temperature (step S4).

[0117] It is subsequently possible to check by means of the indicator substance at any desired point in time during the storage process whether an undesirable, if only temporary heating of the cryosample has taken place. To this end, a check is made as to whether an at least partial displacement and/or change in form of the indicator substance(s) caused by a melting process has taken place. If this is the case, an exceeding of the threshold temperature(s) to be monitored can be concluded.

[0118] Although the invention has been described with reference to specific exemplary embodiments, it is apparent for a person skilled in the art that various changes can be made and equivalents can be used as a replacement without departing from the scope of the invention. The invention should consequently not be restricted to the disclosed exemplary embodiments, but rather should enclose all the exemplary embodiments which fall into the scope of the enclosed claims. In particular, the invention also claims protection for the subject matter and the features of the subordinate claims independently of the claims referred to.

LIST OF REFERENCE NUMBERS

[0119] 1 Sample container, e.g. cryogenic tube [0120] 2 Receiving volume [0121] 3 Cover [0122] 4 Engagement [0123] 5 Base [0124] 6 Biosample, e.g. cell suspension [0125] 7 Indicator substance [0126] 8 Shaft [0127] 10 Device for temperature monitoring [0128] 11 Cap [0129] 12 Outer wall [0130] 13 Inner wall [0131] 14 Wall volume [0132] 15 Cap tip [0133] 16 Lower cap region [0134] 20 Device for temperature monitoring [0135] 21 Double-walled push-on part [0136] 22 Outer wall [0137] 23 Inner wall [0138] 24 Wall volume [0139] 24a Partial volume [0140] 24b Partial volume [0141] 25 Separating wall [0142] 26 Second indicator substance [0143] 30 Device for temperature monitoring [0144] 30c Device for temperature monitoring [0145] 31 Annular body [0146] 32 First partial volume [0147] 33 Second partial volume [0148] 34 Inner volume [0149] 36 Adhesion part [0150] 40 Device for temperature monitoring [0151] 41 Container [0152] 42 First partial volume [0153] 43 Second partial volume [0154] 44 Inner volume [0155] 45 Measurement beam [0156] 46 Bending part [0157] 50 Device for temperature monitoring [0158] 51 Container, e.g. cryogenic tube [0159] 52 Outer wall [0160] 53 Inner wall [0161] 54 Wall volume [0162] 60 Device for temperature monitoring [0163] 61 Semi-annular body [0164] 62 Semi-annular base [0165] 63 Axis of rotation [0166] 64 Inner volume [0167] D Axis of rotation