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

Abstract

The invention relates to a device for monitoring the temperature of a cryopreserved biological sample, comprising a sample container (1) having a receiving space (2) for receiving a biological sample (6) and a cover (3) for closing the receiving space. The device also comprises a chamber arranged inside the sample container, in particular in the receiving space and/or in the cover, the interior (12; 22; 32; 42) of which is not fluidically connected to the receiving space (2) and is only partially filled with at least one indicator substance (7) having a melting temperature in the region between 20 C. and 140 C. The invention also relates to a method for monitoring the temperature of cryopreserved samples, comprising the following steps: providing a device (10; 20; 30; 40) for 12b temperature monitoring; freezing the indicator sub stance/s, wherein the at least one chamber can be placed in a first position during the freezing of the indicator substance/s and in a second position after the freezing and when a temperature of the indicator substance/s is below the melting temperature, in which position a melting of the indicator substance/s leads to an at least partial shift and/or change in shape of the chamber filling, with the influence of the force of gravity.

Claims

1. A device for temperature monitoring of a cryoserved biological sample, comprising a) a sample container, having a receiving spaced for receiving a biological sample, and a cover for closing off the receiving space; and b) a chamber arranged in an interior of the sample container, in particular in the receiving space and/or in the cover, wherein an inner space of the chamber is not fluidically connected to the receiving space and is only partially filled with at least one 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 the sample container is a cryogenic tube.

3. The device according to claim 1, wherein the inner space of the chamber is divided into several sub-spaces which are separated from one another and which arc filled in each case only partially with an indicator substance, the melting temperature of which lies in a range from 20 C. to 140 C., wherein the indicator substances in the sub-spaces have different melting temperatures.

4. The device according to claim 1, wherein a) the cover has a shaft which is in engagement with an upper end region of the receiving spaced and b) the chamber is integrated into the shafts.

5. The device according to claim 1, wherein the chamber is a closed hollow body which is arranged in the receiving spaced of the sample container below the cover.

6. The device according to claim 5, wherein the closed hollow body is arranged loosely on a cryoserved biological sample present in the receiving space.

7. The device according to claim 1, wherein at least one solid body which has a higher density than the indicator substance is arranged loosely in the inner space of the chamber.

8. The device according to claim 7, wherein the melting temperature of the indicator substance lies below 100 C.

9. The device according to claim 7, wherein a solid body, a volume of which is greater than that of the indicator substance, is arranged loosely in the chamber.

10. The device according to claim 7, wherein a) at least two solid bodies are present in the inner space; and b) a volume of the solid bodies is in each case smaller than a volume of the indicator substance.

11. The device according to claim 1, wherein a) a chamber wall at at least one point is transparent or semi-transparent, and/or b) the indicator substance is colored.

12. The device according to claim 1, further comprising an optical, electric or optoelectric measuring device which is formed to detect a position of the indicator substance and/or the solid body in the chamber

13. 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 at least one indicator substance, wherein the at least one chamber is moved into a first position during freezing of the at least one indicator substance and, after freezing and at a temperature of the at least one indicator substance below the melting temperature, is moved into a second position in which a melting of the at least one indicator substance leads, as a result of an influence of gravity, to an at least partial displacement and or change in shape of the chamber filling.

14. The method according to claim 13, wherein a substance is selected as the indicator substance, the melting temperature of which or the threshold temperature of which, at which a viscosity of the melted indicator substance exceeds a determined setpoint value, corresponds to a predetermined threshold temperature, an exceeding of which is monitored.

15. The method according to claim 13, further comprising a) storing of a cryopreserved sample in the sample container, and b) determining whether an at least partial displacement and/or change in shape of the chamber filling performed by temporarily exceeding the melting temperature of the indicator substance has taken place.

16. 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.

17. The device according to claim 16, the dye is selected from the group consisting of triphenylmethane dyes, rhodamine dyes, azo dyes, phenazine dyes and phenothiazine dyes.

18. The device according to claim 16, wherein the indicator substance comprises at least two alcohol components which are 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

[0079] 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:

[0080] FIGS. 1-4 show schematic views of various exemplary embodiments of a device for temperature monitoring of a cryopreserved biological sample;

[0081] FIG. 5 shows a flow chart to illustrate an exemplary embodiment of a method for temperature monitoring of a cryopreserved biological sample;

[0082] FIGS. 6A, 6B, 7A show in each case a melting diagram of a liquid mixture;

[0083] FIG. 7B shows a table with melting points of a number of pure liquids; and

[0084] FIG. 8 shows a mixability matrix of solvents.

[0085] Identical elements or functionally equivalent elements are designated by the same reference numbers in all the figures and are partially not described separately.

[0086] Schematic sectional views A to E of FIG. 1 illustrate a first exemplary embodiment of the invention.

[0087] In this case, in FIG. 1A, a cylindrical receiving part 1 of a cryogenic tube is represented in section. Receiving cavity 2 formed by cylindrical receiving part 1 has already been filled here with biological sample 6. Biological sample 6 may be e.g. a cell suspension. In FIG. 1B, cover 3 which can be screwed on via a thread 8 for cryogenic tube 1, 3 is shown, which cover 3 is upside down, closes off receiving part 1 and optionally possesses at the top an engagement 4 via which cover 3 can be rotated with a tool (not shown) in the case of automation. The cover contains, in the screw-in part, i.e. in shaft 5, which engages in receiving volume 2 in the screwed state, a chamber 11 which forms a hollow volume 12. This 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 is selected in the range from 20 C. to 100 C. via the mixture ratio so that the melting point has the value of a temperature threshold to be monitored. This is also explained in greater detail below on the basis of FIGS. 5 to 8.

[0088] For storing such a biological sample 6, cryogenic tube 1 is deep-frozen to the storage temperature in the open state, as shown in FIG. 1A, and cover 3 in the upside-down position, as shown in FIG. 1B. Indicator substance 7 initially collects in the liquid state under the influence of gravity in sub-volume 12b of hollow volume 12 of chamber 11 and freezes there during cooling to the storage temperature or to a temperature which lies at least below the melting temperature of indicator substance 7.

[0089] At the storage temperature, at least, however, below the melting temperature of indicator substance 7, screw-on cover 3 is rotated by 180 and screwed in as in FIG. 1C and FIG. 1D in order to close off cylindrical receiving part 1 shown in FIG. 1A. Indicator substance 7 which is frozen solid in sub-volume 12b remains in upper sub-volume 12b of the chamber. Lower sub-volume 12a is substantially free of indicator substance 7.

[0090] Device 10 formed in this manner for temperature monitoring can thus be cryogenically stored. In this form, usually standing perpendicular in receptacles, device 10 is stored in the low-temperature containers.

[0091] If biological sample 6 is now brought above the melting point of indicator substance 7 in the event of any manipulation or damage situation in the storage tank, this becomes liquid, drips downwards, and the image represented in FIG. 1E is produced. If, however, the sample has been stored correctly, the indicator substance is still located in sub-region 12a of chamber 11 after a storage process. The state is represented in FIG. 1D. In this manner, inadmissible heating of sample 6 is easily apparent. The position of the indicator substance within chamber 11 may be optically detected by visual inspection. In the case of a transparent or semi-transparent embodiment of cryogenic tube 1, 3, the position of indicator substance 7 may also be determined in an automated visual manner via a horizontal detection, represented schematically by dashed line 100.

[0092] A further advantage of device 10 is the reusability of cover 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 recrystallization 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.

[0093] FIG. 2 shows a further exemplary embodiment of a device 20 for temperature monitoring of a cryopreserved biological sample. Device 20 in turn comprises a cryogenic tube as a sample container and an indicator apparatus 21 which can be arranged in the interior of the cryogenic tube in the form of a hollow cylinder form 21 or 21a which is filled partially with indicator substance 7.

[0094] FIG. 2A shows a cryogenic tube which can also, for example, be deep frozen. The cryogenic tube in turn comprises a cylindrical receiving part 1 which forms a receiving cavity 2 which has already been filled here with biological sample 6. The cryogenic tube further comprises a cover 3 for the cryogenic tube which closes off receiving part 1.

[0095] A closed hollow cylinder 21 which is partially filled with an indicator substance 7 and in this manner may be used as an indicator apparatus for temperature monitoring is used for future detection of the exceeding of a critical threshold temperature. Indicator substance 7 is again selected so that its melting point lies in the range from 20 C. to 100 C. and has the value of a temperature threshold to be monitored.

[0096] Hollow cylinder 21 is frozen in the position shown in FIG. 1B outside the cryogenic tube. The indicator substance collects in lower sub-volume 22b and freezes solid there.

[0097] Cryogenic tube 1, 23 shown in FIG. 1A is subsequently opened in that cover 23 is screwed off, hollow cylinder 21 is placed, rotated by 180, onto already frozen biological sample 6 and cryogenic tube 1, 23 is closed again so that the image represented in FIG. 1C is produced. Hollow cylinder 21 thus lies loosely on the biological sample, in an alignment in which the indicator substance is initially located in the frozen state at the top in cavity 22.

[0098] If the melting temperature of indicator substance 7 was subsequently reached, the liquid is once again located at the base of cylinder 21, in sub-volume 21a, from which it is apparent that a critical threshold temperature was exceeded.

[0099] A four-chamber hollow cylinder 21a which can be used in an analogous manner instead of hollow cylinder 21 and inserted in the same manner into a cryogenic tube 1, 23 is alternatively represented in FIG. 2D. In this case, the inner space of hollow cylinder 21 is divided by separating walls 25 into four fluidically separate sub-cavities which are filled in each case partially with an indicator substance. Indicator substances 7a, 7b, 7c, 7d in the sub-cavities are, however, different and have different melting points.

[0100] A graduation of the melting points of indicator substances 7a, 7b, 7c, 7d is expediently selected, e.g. 20 C. for indicator substance 7a, 50 C. for indicator substance 7b, 80 C. for indicator substance 7c and 110 C. for indicator substance 7d.

[0101] If, during an inspection, frozen indicator substances 7a to 7c are still located in upper region 22b of the hollow cylinder, but indicator substance 7d is still located at the base (sub-region 22a), a temperature of 110 C. would be exceeded in the case of sample 6. If indicator substance 7c is also located at the base, if 80 C. was exceeded, if all marker liquids 7a to 7d were located at the base, even 20 C. has been exceeded.

[0102] Hollow cylinder 21 or 21a incorporated in receiving space 2 should be sterile on its surface or otherwise made germ-free and contamination-free.

[0103] FIG. 3 shows a further exemplary embodiment of a device 30 for temperature monitoring of a cryopreserved biological sample. The particular feature of this device 30 in comparison with device 10 shown in FIG. 1 lies in the fact that chamber 31 integrated into cover 3 of the cryogenic tube is not only partially filled with an indicator substance 7, rather further contains small solid bodies 33 which are incorporated loosely in the inner space or cavity 32 of chamber 33.

[0104] FIG. 3A thus shows receiving part 1 of a cryogenic tube in section in an analogous manner to FIG. 1A. The chamber integrated into shaft 5 of upside-down cover 3 is, as shown in FIG. 3B, only filled at the base with an indicator substance 7. Small bodies 33 with comparatively higher density than the indicator substance and thus higher weight are located in this chamber. Bodies 33 may be, for example, metal balls. Receiving part 1 and upside-down cover 3 are now brought to the storage temperature. As a result of this, small bodies 33 freeze in indicator liquid 7. If the cover is now screwed on rotated by 180 as in FIG. 1, as represented in FIG. 3C, the chamber filling, comprising frozen indicator substance 7 with fixed small bodies 33, is then located at the top of cavity 32 of chamber 31.

[0105] If the melting temperature of indicator substance 7 is not exceeded during cryogenic storage, an image as shown in FIG. 3D is produced. The configuration of the chamber filling has not changed, both indicator substance 7 and small bodies 33 frozen solid therein are located at the top of cavity 32 of chamber 31.

[0106] If the melting temperature of indicator substance 7 is exceeded, at least weighted bodies 33 fall to the base of cavity 32 of chamber 31, as represented in FIG. 3E. This arrangement has the advantage that even liquids with a melting point far below 100 C. can be used which then usually possess a very high viscosity. Indicator substance 7 would then not flow on the wall to the base of cavity 32, but rather remain in the upper part. In contrast, weighted bodies 33 fall out of liquefied indicator substance 7 onto the base, as shown in FIG. 3E. This may be detected e.g. by a sensor (conductivity, optically, etc.). A visual determination of the state is also possible.

[0107] FIG. 4 shows a further exemplary embodiment of a device 40 for temperature monitoring of a cryopreserved biological sample. The particular feature of this device 40 in comparison with device 30 shown in FIG. 3 lies in the fact that, instead of several small bodies, a comparatively large and heavy body 43 is located in chamber 41 integrated into cover 3 next to the indicator substance.

[0108] FIG. 4A again shows receiving part 1 of a cryogenic tube for a biological sample 6 in section. FIG. 4B again shows an upside-down cover 3 of the cryogenic tube, in the interior of which a chamber 41 is again integrated which forms a cavity 42 which is partially filled with an indicator substance 7. Moreover, a larger and heavy body 43 is further incorporated loosely into cavity 42. The freezing of the indicator substance and storing of device 40 are performed in an analogous manner to that for device 30. In an analogous manner to the device shown in FIG. 3, here, the detachment of large body 43 is even less susceptible to faults in the event of the exceeding of the melting temperature of indicator substance 7, it simultaneously releases an optically transparent section or blocks it as a result of its sinking. The optically transparent section is represented schematically by dashed line 100 in FIG. 4E.

[0109] FIG. 5 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, 30 or 40. 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 may be set to a desired value, in particular in a range from 20 C. to 140 C.

[0111] By way of example, FIG. 6A 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. may be covered. Should e.g. a temperature threshold value of 118 C. be monitored, the ethanol ratio may 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. 6B 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. 7A. The table of FIG. 7B 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. 8.

[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 sub-cavities of the chamber.

[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 the first position and frozen. The first position corresponds in the case of the exemplary embodiments of FIGS. 1, 3 and 4 respectively to a position of cover 3, in the case of which it stands on its head, as is represented in FIG. 1B, 3B or 4B.

[0115] Thereafter, in step S3, the at least one chamber with the frozen indicator substance is moved into a second position and arranged inside the sample container. According to the exemplary embodiments shown in FIGS. 1 to 4, the second position is rotated by 180 with respect to the first position. According to the exemplary embodiments shown in FIGS. 1, 3 and 4, the chamber is moved into the second position by screwing cover 3 to receiving part 1. In the case of the embodiment variant of FIG. 2, the hollow cylinder is placed in receiving space 2 in a manner rotated by 180 with respect to frozen biological sample 6.

[0116] In this state, the device may 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 examining the state of the chamber filling at any desired point in time during the storage process whether an undesirable, if only temporary heating of the cryosample has taken place (step S5). To this end, a check is made as to whether an at least partial displacement and/or change in form of the chamber filling caused by a melting process has taken place, as explained above in the case of FIGS. 1 to 4. 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 encompass 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.