WO1991002458A1

WO1991002458A1 - Temperature maintenance of biological or other samples at a selected temperature

Info

Publication number: WO1991002458A1
Application number: PCT/AU1990/000347
Authority: WO
Inventors: Charles Marvin Herr
Original assignee: A.B. Technology Pty. Limited
Priority date: 1989-08-15
Filing date: 1990-08-14
Publication date: 1991-03-07

Abstract

A method of maintaining a biological or other sample at a selected temperature which comprises bringing the sample into an intimate relationship with one or more containers which contain a substance whose melting temperature is at or near the selected temperature and wherein the one or more containers are comprised of material capable of transmitting heat to or from the biological or other thermolabile sample.

Description

TEMPERATURE MAINTENANCE OF BIOLOGICAL OR OTHER SAMPLES AT A SELECTED TE.ΦERATURE

This invention relates to temperature maintenance, and is particularly concerned with methods and apparatus which may be used to maintain biological samples or other samples at a selected temperature- Many biological materials are thermolabile. For example, semen is a thermolabile biological material and becomes inactive, that is, not capable of achieving fertilisation, if it is not stored or maintained at appropriate temperatures. It has been found that the storage of semen from both animal and human species is best achieved at a temperature at or near 17°C. Under these conditions, semen may be stored for many days and possibly weeks.

Bovine semen may be frozen at low temperature such as in liquid nitrogen and may be conveniently stored and transported in the frozen state. However, a high proportion of sperm is damaged by the freezing and thawing process such that far higher numbers of <

- 2 - frozen/thawed sperm are required to obtain a fertilisation rate comparable to that attainable with fresh or unfrozen semen.

Further, in bovine embryo transfer programmes, where donor cows are super-ovulated before artificial insemination, the use of fresh or unfrozen rather than frozen/thawed semen results in higher rates of fertilisation because sperm in fresh or unfrozen semen survive longer after being transferred into the uterus. This is a particularly important factor because the donor cows' oocytes (eggs) are ovulated (released from the ovarian follicles) over a prolonged period of time.

Similar considerations apply to the use of fresh ram sperm, with the addition that techniques for freezing ram semen are particularly inconvenient.

Embryos, particularly livestock embryos, require temperature maintenance around 38°C in order remain viable.

Highly labile biological materials such as cell culture media may require low temperature storage, such as at around -65°C to avoid inactivation.

The invention is based on the observation that substances are capable of absorbing large amounts of heat, or of giving up large amounts of heat, at the temperature at which they melt or solidify, respectively, without a significant rise or fall in temperature. This is due to the principle of latent heat of melting/fusion, which is characteristic of a particular substance.

According to one aspect of the present invention there is provided a method for maintaining a biological or other sample at a selected temperature which comprises bringing said sample into an intimate relationship with one or more containers which contain a substance whose melting temperature is at or near said selected temperature and wherein said one or more containers are comprised of material capable of transmitting heat to or from the biological or other sample; and wherein if ambient temperature is higher than the melting temperature of the substance, the substance contained within said one or more containers is first frozen such that the latent heat of melting of the substance serves to maintain the one or more containers and sample at a substantially constant temperature until all of said substance has undergone a phase transition from solid to liquid; or wherein if the ambient temperature is lower than the melting temperature of the substance, the substance contained within said one or more containers is first melted such that the latent heat of fusion of the substance serves to maintain the one or more containers and sample at a substantially constant temperature until all of said substance has undergone a phase transition from liquid to solid; or regardless of ambient temperature, the substance within said one or more containers is first melted and the substance within one or more additional containers is first solidified such that the latent heat of fusion and the latent heat of melting of the substance serve to maintain the containers and sample at a substantially constant temperature until all of said substance is either solid or liquid. By this aspect of the invention, a frozen or solid substance having a melting temperature below ambient temperature melts gradually, whilst maintaining the temperature within the container at the melting temperature of the substance. By virtue of heat transfer between the container and the biological or other sample, the sample is maintained at a desired temperature. An appreciable temperature change does not occur until all of the frozen or solid material has melted. Large amounts of heat may thus be absorbed by the substance without appreciably raising the temperature of the substance, or of biological or other material in intimate contact with a container which contains the frozen or c

- 4 - solid substance.

Similarly, a liquid substance having a melting temperature above ambient temperature gradually solidifies or fuses without an appreciable temperature shift from the melting temperature of the substance. The temperature of the substance does not appreciably decrease until all of the material has solidified. Large amounts of heat may thus be emitted without appreciably lowering the temperature of the substance, or the biological or other thermolabile sample, by virtue of heat transfer between the container holding the substance and the sample.

Biological samples may, for example, be human or animal reproductive material such as animal or human semen, sperm or ova; fertilised eggs; embryos; organs such as heart, liver, spleen and kidneys; blood; living cells; etc.

Other samples may be cultured media or reagents required for assays of various types or for the manipulation and culture of biological materials. The term "sample" is used herein to denote any material which is desired to be maintained at a selected temperature according to the methods of this invention.

The term "intimate relationship" as used herein refers to contact, juxtaposition, or close proximity of a container to a biological or other sample, such that heat transfer is able to take place.

Said one or more containers may be comprised of impervious material such as impervious plastics, metal foils or sheets, or other materials which do not prevent heat flow into or out of the said one or more containers. By "impervious" is meant that the containers do not leak appreciable amounts of substance contained therein. Heat flow or transfer between the substance within a container and the biological or other sample acts to maintain the sample at a selected temperature.

A container may be of any desired shape and may, for example, be shaped to accommodate a cryogenic liquid such as liquid nitrogen to facilitate freezing of the substance within the container, allowing its rapid and convenient "recharging". A container may be constructed in the shape of a box or similar vessel, wherein the sides of the container are filled with the substance and the external walls and base of the container are coated with a thermal insulating material, and/or the box-like container is placed in a close-fitting thermal insulating vessel and fitted with an insulated lid.

A container may contain depressions in its outer surface, or suitably shaped portions, or be shaped in its entirety to receive biological or other thermolabile samples. Such portions may closely surround the samples, and may contact the samples at one or more locations to provide effective heat transfer. For example, a container may contain depressions which are a close fit with test tubes, vials, flasks, straws or bottles containing a biological or other sample. Preferably, one or more containers may be placed in an insulating vessel, such as a plastic "esky" comprised of styrofoam, or other insulating vessel, to insulate against heat exchange with the external environment. For example, containers in the form of freezer blocks, that is, rectangular plastic blocks containing a frozen substance, may be placed in an insulating vessel together with a biological or other sample, such as semen in a storage receptacle. The storage receptacle may contact the freezer blocks, or may be in close or intimate relationship therewith, that is, at a distance which enables the transfer of heat from the sample to the freezer blocks and vice versa. An insulating vessel may contain rectangular or other shaped containers, some of which contain a frozen substance, and others which contain the substance in a liquid state. The use of an insulating vessel is desirable to maintain a constant temperature environmen . It is particularly preferred in an aspect of this invention to maintain biologically active material at about 17°C or 38°C. Where a temperature of about 17°C is to be maintained, a substance having a melting temperature at or near 17°C such as dimethylsulphoxide (DMSO) or glacial acetic acid is utilised, that is, is sealed within the impervious container(s). Where a temperature at or near 38°C is to be maintained, a substance having a melting temperature at or near 38°C, such as myristyl alcohol or n-Eicosane, is used, n-

Eicosane has a melting point of about 36.8°C and a latent heat of fusion of about 59.11 calories/g. The term "about 38°C" is to be taken to mean temperature maintenance within the range of about 36.5°C to 39.5°C. It is preferable that in each case the substance has a high heat capacity and a high latent heat of fusion\melting, such as about 40 calories/g or more.

Specific applications for the maintenance of temperature at or near 17°C include the storage and transport of livestock semen or sperm.

Specific applications for the maintenance of temperature at or near 38°C include the storage and transport of livestock embryos, the prevention of post- thaw shock of frozen semen, and the provision of a cheap convenient alternative to warm rooms and constant temperature water baths or heating blocks in research, clinical and production facilities. Myristyl alcohol is particularly suited for maintaining samples at temperatures between 38°C to about 39.5°C, such temperatures are particularly suited for the temperature maintenance of animal tissues (particularly domestic species) and embryos. n-Eicosane is particularly suited for the temperature maintenance of human tissues at around 36.5°C to 38°C. In accordance with another aspect of this invention there is provided a method for maintaining a sample of animal or human semen, or other sample at or about 17°C which method comprises bringing said sample into an intimate relationship with frozen or liquid DMSO or one or more containers containing said DMSO, wherein said one or more containers are comprised of material capable of transmitting heat across the container wall; and wherein at ambient temperatures greater than 17°C the frozen DMSO maintains a temperature at or near 17°C until substantially all of the DMSO has undergone a phase transition to liquid phase, and by virtue of heat transfer between said DMSO or containers containing DMSO and said sample, said sample is maintained at a temperature of about 17°C; and wherein at ambient temperatures less than 17^CC liquid DMSO maintains a temperature at or near 17°C until substantially all of the DMSO has frozen, and by virtue of heat transfer between said DMSO or containers containing said DMSO and said sample, said sample is maintained at a temperature of about 17°C.

This aspect of the invention is based in part on the surprising findings that semen may be maintained at or about 17°C for an extended time period by bringing the semen into contact with a container or containers of DMSO in the liquid and/or solid state as hereinbefore described. The melting temperature of DMSO is at or near 17°C at atmospheric pressure. As liquid DMSO freezes or solidifies it gives up large amounts of heat without a significant fall in temperature below 17°C, due to its high latent heat of fusion and its higher heat capacity. Similarly, frozen DMSO absorbs large amounts of heat during the melting process without a significant rise in temperature above 17°C, due to its high latent heat of melting and its high heat capacity.

Furthermore, we have determined experimentally that DMSO does not supercool, that is, it does not remain in the liquid state at atmospheric pressure as the ambient temperature falls below 17°C, unlike other simple, inexpensive and commonly available substances whose freezing/melting points are at or near 17°C (e.g. acetic acid). This property is most advantageous but not essential to this invention, particularly for temperature maintenance at about 17°C. As DMSO does not supercool, it begins to solidify almost immediately the temperature drops below its freezing point of about 17°C. This solidification generates heat which in-turn maintains a sample at around 17°C. The DMSO may therefore be regarded as acting as a heating and cooling device.

Accordingly, bringing a semen sample into contact with DMSO in either a solid or a liquid state when the ambient temperature is above or below 17°C, respectively, enables the semen sample to be maintained at a temperature at or about 17°C for a prolonged time, particularly when both the semen and the DMSO are enclosed within a thermally insulated vessel. Bringing a semen sample into contact with DMSO in both a solid and a liquid state, regardless of the ambient temperature, enables the semen sample to be maintained at a temperature at or about 17°C for a prolonged time when both the semen and DMSO are enclosed within^'a thermally insulated vessel.

In the above method, DMSO may be substituted with acetic acid.

This aspect of the invention is not only applicable to the maintenance and storage of semen, but applies also to the maintenance of any other biological or other sample at around 17°C. In yet another aspect of this invention there is provided a method for maintaining embryos or other thermolabile sample at or about 38°C or for preventing post-thaw shock of a frozen semen sample, which method comprises bringing said sample into an intimate relationship with frozen or liquid myristyl alcohol or one or more containers containing said myristyl alcohol, wherein said one or more containers are comprised of material capable of transmitting heat across the container wall; ^' . and wherein at ambient temperature greater than 38°C the frozen myristyl alcohol maintains a temperature at or near 38°C until substantially all of the myristyl alcohol has undergone a phase transition to liquid phase, and by virtue of heat transfer between said myristyl alcohol or containers containing myristyl alcohol and said sample, said sample is maintained at a temperature of about 38°C; and wherein at ambient temperatures less than 38^CC liquid myristyl alcohol maintains a temperature at or near 38°C until substantially all of the myristyl alcohol has frozen, and by virtue of heat transfer between said alcohol or containers containing myristyl alcohol and said sample, said sample is maintained at a temperature of about 38°C.

As previously mentioned, myristyl alcohol is particularly suited at about 38°C to 39.5°C. Where temperatures from around 36.5°C to 38°C are to be maintained n-Eicosane is used in place of myristyl alcohol.

Temperature maintenance is particularly effected when containers which contain DMSO, acetic acid, myristyl alcohol or n-Eicosane are housed within an insulating vessel; such as a vessel having a floor, one or more walls, and a removable lid.

It is to be understood that this invention has broad applicability. Any biological or other sample may be maintained at a selected temperature for substantial time periods (many days) by choosing a container filling (previously referred to as a "substance") having a melting temperature at or about said selected temperature. The material is either frozen or melted, depending upon the conditions in question, and then brought into contact with the sample for temperature maintenance.

According to yet another aspect of the present invention, there is provided an apparatus for temperature maintenance which comprises one or more containers within an insulating vessel, said one or more containers being impermeable and containing a substance having a melting temperature at or about a temperature desired to be maintained; such that a biological or other thermolabile sample brought into intimate relationship with said one or more containers maintains a substantially constant temperature by virtue of heat transfer between the sample, and the substance within the container, and optionally, said one or more containers are shaped or have one or more depressions or recesses formed in the surface thereof which may receive a biological or other thermolabile sample for intimate contact, and other recesses in the surface thereof which may be filled with a cryogenic liquid such as liquid nitrogen for convenient "recharging" (rapid freezing of said substance). Preferably, the substance used to fill the containers is selected from DMSO, acetic acid, myristyl alcohol, and n-Eicosane.

Such apparatus may be used for the storage of biological samples as previously mentioned or, for example, as a constant temperature environment to replace water baths, heating blocks or freezers in research, clinical and production facilities.

An example of an insulated vessel for the storage of semen is set forth below.

An insulated vessel which may be used to enclose the semen and DMSO is a device known commonly as a "six-pack cooler" (hereinafter "cooler") which is composed of high density polyurethane and has a tightly fitting lid. The contents of the vessel are thermally insulated from the environment by polystyrene or polyurethane included within the walls and lid of the vessel. The DMSO is contained within the cooler in either freezable drink containers or freezer blocks, both of which are commonly available for use in a "six pack cooler" or similar thermal insulating vessel.

The temperature within the cooler is monitored by a thermocouple or other temperature sensitive device such as a dial thermometer whose reading can be viewed externally without opening the lid of the vessel. This allows the operator to determine whether the cooler requires "recharging" prior to and during a period of use. The unit, comprising the insulated cooler and its included containers of DMSO, is used to transport and/or store fresh semen by placing the semen held in straws against containers within the cooler.

The unit is maintained and "recharged" as follows: (a) Each unit includes within the thermally insulated vessel (cooler) two or more containers of DMSO; (b) On cold days when the ambient temperature is and is anticipated to remain substantially below 17°C, the DMSO in the two or more containers is melted prior to use of the unit but the resultant liquid DMSO is not substantially above 17°C when the containers of melted DMSO are placed into the insulated vessel;

(c) On warm days when the ambient temperature is anticipated to remain substantially above 17°C, the DMSO in the two or more containers is frozen by placing the containers of DMSO in a refrigerator or freezer or in liquid nitrogen vapour. Subsequent to freezing but prior to use the blocks are allowed to stand at a temperature about 17°C until it can be observed that the DMSO starts to thaw. Treating the blocks in this manner ensures that when the blocks are placed into the insulated vessel the temperature within the unit is similar at the outset to the melting temperature of DMSO, i.e., 17°C, rather than being below that temperature initially;

(d) On days when the temperature is anticipated to vary above and below 17°C, the DMSO in one or some but not all of the blocks is in the liquid state and the DMSO in one or some but not all of the blocks is in the solid state, those states being attained as hereinbefore i

- 12 - described;

(e) Information and instructions for the maintenance of the unit and for correct use of the unit in the transport and/or storage of fresh semen are included with the unit in written information and instructions affixed to the cooler.

This procedure may be adapted for other temperature maintenance operations described herein with appropriate temperature modifications. For example, containers may be charged with myristyl alcohol and if ambient temperature is to remain substantially below 38°C (i.e. more than several degrees) the myristyl alcohol is melted prior to use (by simple immersion in hot water) but the resultant myristyl alcohol is not substantially above 38°C when the containers of melted myristyl alcohol are placed into the insulated vessel. Under ambient conditions (around 20-25°C) containers charged with myristyl alcohol may only need to be recharged (that is melted) about every fifth day of use. Using the aforementioned unit pig embryos have been stored for transport at 17°C, bovine embryos stored at 39°C (containers charged with DMSO), and human embryos and mammalian cell cultures stored at 37°C (containers charged with n-Eicosane).

Claims

CLAIMS :

1. A method for maintaining a biological or other sample at a selected temperature which comprises bringing said sample into an intimate relationship with one or more containers which contain a substance whose melting temperature is at or near said selected temperature and wherein said one or more containers are comprised of material capable of transmitting heat to or from the biological or other sample; and wherein if ambient temperature is higher than the melting temperature of the substance, the substance contained within said one or more containers is first frozen such that the latent heat of melting of the substance serves to maintain the one or more containers and sample at a substantially constant temperature until all of said substance has undergone a phase transition from solid to liquid; or wherein if the ambient temperature is lower than the melting temperature of the substance, the substance contained within said one or more containers is first melted such that the latent heat of fusion of the substance serves to maintain the one or more containers and sample at a substantially constant temperature until all of said substance has undergone a phase transition from liquid to solid; or regardless of ambient temperature, the substance within said one or more containers is first melted and the substance within one or more additional containers is first solidified such that the latent heat of fusion and the latent heat of melting of the substance serve to maintain the containers and sample at a substantially constant temperature until all of said substance is either solid or liquid.

2. A method according to claim 1, wherein the biological or other sample is selected from sperm, ova, fertilised eggs, embryos, human or animal organs, blood, wine or food. .

3. A method according to claim, 2, wherein said organs are selected from heart, liver, spleen and kidney.

4. A method according to claim 1, wherein said substance is selected from DMSO, acetic acid, myristyl alcohol and n-Eicosane.

5. A method for maintaining a sample of animal or human semen or other sample at or about 17°C which method comprises bringing said sample into an intimate relationship with frozen or liquid DMSO or one or more containers containing said DMSO, wherein said one or more containers are comprised of material capable of transmitting heat across the container wall; and wherein at ambient temperatures greater than 17°C the frozen DMSO maintains a temperature at or near 17°C until substantially all of the DMSO has undergone a phase transition to liquid phase, and by virtue of heat transfer between said DMSO or containers containing DMSO and said sample, said sample is maintained at a temperature of about 17°C; and wherein at ambient temperatures less than 17°C liquid DMSO maintains a temperature at or near 17^CC until substantially all of the DMSO has frozen, and by virtue of heat transfer between said DMSO or containers containing said DMSO and said sample, said sample is maintained at a temperature of about 17°C.

6. A method according to claim 5, wherein said DMSO is substituted with acetic acid.

7. A method for maintaining embryos or other sample at or about 38°C or for preventing post-thaw shock of a frozen semen sample, which method comprises bringing said sample into an intimate relationship with frozen or liquid myristyl alcohol or o or more containers containing said myristyl alcohol, wherein said one or more containers are comprised of material capable of transmitting heat across the container wall; and wherein at ambient temperature greater than 38°C the frozen myristyl alcohol maintains a temperature at or near 38°C until substantially all of the myristyl alcohol has undergone a phase transition to liquid phase, and by virtue of heat transfer between said myristyl alcohol or containers containing myristyl alcohol and said sample, said sample is maintained at a temperature of about 38°C; and wherein at ambient temperatures less than 38°C liquid myristyl alcohol maintains a temperature at or near 38°C until substantially all of the myristyl alcohol has frozen, and by virtue of heat transfer between said alcohol or containers containing myristyl alcohol, and said sample, said sample is maintained at a temperature of about 38°C.

8. A method according to claim 7, wherein myristyl alcohol is substituted with n-Eicosane such that temperature is maintained between about 36.5°C and 38^CC

9. A method according to any one of claims 1 to 8, wherein said one or more containers and said sample are housed within a thermally insulated vessel.

10. An apparatus for temperature maintenance which comprises one or more containers within an insulating vessel, said one or more containers being impermeable and containing a substance having a melting temperature at or about a temperature desired to be maintained, such that a biological or other thermolabile sample brought into intimate relationship with said one or more containers maintains a substantially constant temperature by virtue of heat transfer between the sample, and the substance within the container.

11. An apparatus according to claim 10, wherein said substance is selected from frozen or liquid DMSO; and wherein at ambient temperatures greater than 17°C the frozen DMSO maintains a temperature at or near 17°C until substantially all of the DMSO has undergone a phase transition to liquid phase, and by virtue of heat transfer between said DMSO or containers containing DMSO and said sample, said sample is maintained at a temperature of about 17°C; and wherein at ambient temperatures less than 17°C liquid DMSO maintains a temperature at or near 17°C until substantially all of the DMSO has frozen, and by virtue of heat transfer between said DMSO or containers containing said DMSO and said sample, said sample is maintained at a temperature of about 17°C.

12. An apparatus according to claim 11, wherein said DMSO is replaced with acetic acid.

13. An apparatus according to claim 10, wherein said substance is selected from frozen or liquid myristyl alcohol; and wherein at ambient temperature greater than 38°C the frozen myristyl alcohol maintains a temperature at or near 38°C until substantially all of the myristyl alcohol has undergone a phase transition to liquid phase, and by virtue of heat transfer between said myristyl alcohol or containers containing myristyl alcohol and said sample, said sample is maintained at a temperature of about 38°C; and wherein at ambient temperatures less than 38°C liquid myristyl alcohol maintains a temperature at or near 38°C until substantially all of the myristyl alcohol has frozen, and by virtue of heat transfer between said alcohol or containers containing myristyl alcohol, and said sample, said sample is maintained at a temperature of about 38 °C.

14. An apparatus according to claim 13, wherein said myristyl alcohol is replaced with n-Eicosane, for temperature maintenance between about 36.5°C and 38°C.

15. An apparatus according to claim 10, wherein said one or more containers are shaped or have one or more depressions or recesses formed in the surface thereof to receive a biological or other sample for intimate contact.

16. An apparatus according to any one of claims 10 to 15, which additionally comprises a thermocouple or other temperature sensitive device which can be viewed externally without opening of the vessel.