Patent ID: 12207650

DETAILED DESCRIPTION

The apparatus and methods presented herein improve control of ice formation during freezing of biological samples, compared with known techniques. In examples, a device is provided comprising a base and a receptacle holder, the receptacle holder comprising at least two parts each configured to withdraw heat energy from a different region of the sample at a different heat withdrawal rate. The different heat withdrawal rates allow a temperature gradient to be established within the sample during the freezing process such that ice nucleation occurs in one region of the sample without ice nucleation in a second region of the sample. As such, an extent of undercooling in the sample may be reduced, thereby increasing cell viability. The apparatus and methods presented in examples herein enable ice formation to be controlled in a repeatable and flexible manner. Controlling ice formation according to the apparatuses and methods presented in examples herein facilitates an improved control of the freezing process.

Specific details will be described in the following description, in accordance with certain examples of the apparatus and methods, to explain and expand on specific features thereof.

Ice formation is an important consideration in the freezing of biological samples. As a temperature in the sample descends below the equilibrium melting point of the sample, ice nucleation may occur (around so-called ice nucleus particles), followed by growth of ice crystals throughout the sample. As ice formation proceeds, biological material such as cells may concentrate in solute-dense channels between the ice crystals. Such channels may then solidify, for example (e.g.) through vitrification.

During freezing of relatively small samples, for example samples stored in cryovials, the whole sample if cooled uniformly may undercool to a temperature substantially below the equilibrium melting point of the sample. Undercooling, or supercooling or subcooling, refers to the process of lowering the temperature of a fluid beyond its melting point without the fluid solidifying. Some samples may be undercooled to more than 10° C. below the melting point of the sample. An extent of undercooling may vary with sample volume. In particular, smaller volumes may undercool to a greater extent than larger volumes. When ice nucleates in an undercooled sample, the temperature in the sample may increase to near the melting point of the sample due to energy released from the latent heat of crystallisation. The temperature may then decrease rapidly, e.g. at a much faster rate than desired, which may impact cell viability upon thawing. Viability and/or function after thawing may be dependent on the type of cell undergoing the freezing/thawing process.

FIG.1shows a representation100of an example data series of temperature values in a sample versus time during a freezing operation performed on the sample. Each of the three lines in the representation100corresponds to measured temperature data obtained by a thermocouple placed at a different location in the sample. In the example ofFIG.1, the entire sample undercools to between −15° C. and −10° C., until ice nucleation occurs at approximately 1500 seconds (s), causing a rapid jump in temperature throughout the sample. In the example ofFIG.1, the sample does not contain biological cells, although the presence of cells in a solution does not influence the nucleation behaviour in such a solution.

Following ice nucleation in one or more regions of an undercooled sample, a dendritic network of ice crystals coexists with a continuous phase of freeze concentrated material in which solutes and cells may be distributed. Ice nucleation may be spontaneous. Spontaneous nucleation may be a stochastic event and thus inherently difficult to control. For example, the temperature at which ice nucleation spontaneously occurs may vary between different samples. The structure of the ice network formed at nucleation and the corresponding freeze concentrated matrix may be dependent on the temperature at which nucleation occurs.

Alternatively, ice nucleation may be induced or triggered, in a process known as facilitated ice nucleation. Facilitated ice nucleation, or heterogeneous ice nucleation, may occur at temperatures that are higher than those at which homogeneous nucleation occurs, e.g. closer to the melting point of the sample. Facilitated ice nucleation may involve the use of a nucleation trigger. Examples of nucleation triggers include: generation of a cold spot on the outside of the cryocontainer; introduction of chemical ice nucleating catalysts into the sample; and the application of ultrasound. However, known methods of inducing ice nucleation may have shortcomings. For example, known methods may require user interference, use chemical nucleants which are not biocompatible, may not be repeatable in a standardised manner, or more may be prohibitively expensive and/or complicated to implement.

The devices, receptacles and methods presented in examples herein allow ice formation to be controlled in a repeatable and flexible manner during a freezing operation. Progressive solidification, which may otherwise be referred to as directional solidification, is used to control ice formation and reduce an extent of undercooling of samples. Progressive solidification refers to a process of causing a sample to solidify progressively, typically along an axis. For example, solidification may start in one region of a sample and proceed, along the axis, through the sample towards a second region of the sample. In progressive solidification, ice nucleation may be restricted to the region of the sample in which solidification commences, e.g. the first region. Progressive solidification may be implemented by establishing a temperature gradient within a sample, e.g. between a first, colder region and a second, warmer region, and allowing the sample to progressively solidify along the axis of the temperature gradient from the colder region to the warmer region. Reducing undercooling may increase a likelihood of cell survival after thawing of the sample, and controlling the incidence and/or location of ice nucleation in a standardised way may allow for a reduction in the stochastic variability of freezing procedures.

A device is provided herein in examples for use in freezing at least part of a biological sample in a receptacle. The device comprises a base and a receptacle holder. The base and the receptacle holder may be integrally formed or may be separate bodies. The receptacle holder is configured to hold one or more receptacles, containers, cryocontainers or the like. The base is configured to be cooled by a cooler device. The cooler device may be part of a cryocooler such as a Sterling cryocooler, although other examples of cooler devices that may be used are described below. The base of the device may be contactable by a cooling surface. Such a cooling surface may be part of a heat sink. A heat sink may absorb or withdraw heat energy from a sample during a cooling operation performed on the sample. As such, the base of the device may be cooled by conduction via the cooling surface.

The receptacle holder comprises a first part configured to, with a receptacle held by the receptacle holder during cooling of the base using the cooler device, withdraw heat energy from a first portion of the receptacle at a first heat withdrawal rate. As such, heat energy may be withdrawn from a first region of a sample via the first part of the receptacle holder, the first region of the sample being in contact with the first portion of the receptacle. In examples, the first portion of the receptacle is placed in conductive contact with the first part of the receptacle holder to allow heat energy to be withdrawn via the first part of the receptacle holder. In examples, the first part is configured to at least partially surround the first portion of the receptacle.

The receptacle holder also comprises a second part configured such that, with the receptacle held by the receptacle holder during cooling of the base using the cooler device, a second heat withdrawal rate of heat energy withdrawal from a second portion of the receptacle via the second part is less than the first heat withdrawal rate. As such, a second region of the sample may lose heat energy via the second part of the receptacle holder at a lower rate than a rate at which the first region of the sample loses heat via the first part of the receptacle holder, the second region of the sample being in contact with the second portion of the receptacle. In some examples, the second portion of the receptacle is placed in conductive contact with the second part of the receptacle holder. In other examples, the second portion of the receptacle is not placed in conductive contact with the second part of the receptacle holder. For example, the second portion of the receptacle may be spaced apart with respect to the second part of the receptacle holder. In examples, the second part is configured to at least partially surround the second portion of the receptacle.

In some examples, the first part of the receptacle holder is closer to the base than the second part of the receptacle holder. In other words, the second part may be further from the base than the first part. Therefore, the device may be configured to withdraw heat from the sample at a higher rate closer to the base of the device and at a lower rate further from the base of the device.

In some examples, the second part of the receptacle holder is configured to withdraw heat energy from the second portion of the receptacle at the second heat withdrawal rate. In other words, the second part of the receptacle holder may actively withdraw heat from the second portion of the receptacle. In some cases, the second part of the receptacle holder is configured to reduce, inhibit or prevent heat loss from the second portion of the receptacle.

In some examples, the first part of the receptacle holder comprises a first material having a first value of a heat flow characteristic and the second part of the receptacle holder comprises a second material having a second, different value of the heat flow characteristic. The heat flow characteristic may be considered as a heat transfer characteristic, heat withdrawal characteristic, cooling rate characteristic or the like. The heat flow characteristic of a given receptacle holder part may in some examples be considered a characteristic or parameter of the material of which the given receptacle holder part is comprised, which influences a flow of heat energy from a portion of the receptacle to the given receptacle holder part. The heat flow characteristic of a given receptacle holder part may determine the heat withdrawal rate from the receptacle via that given receptacle holder part.

A difference between the first heat withdrawal rate and the second heat withdrawal rate may be at least sufficient to establish a temperature gradient within a sample in the receptacle such that, during a freezing operation using the cooler device, ice nucleation occurs in the first region of the sample, without ice nucleation in the second region of the sample. In some examples, the first and second heat withdrawal rates are determined and/or configured based on a desired temperature gradient within the sample, where the desired temperature gradient is to restrict ice nucleation in the sample to the first region of the sample and/or prevent ice nucleation in the second region of the sample. The temperature gradient may have an axis substantially perpendicular to the base of the device.

In some examples, a difference between the first heat withdrawal rate and the second heat withdrawal rate is such that, for a sample having a volume of 5 millilitres (ml), a temperature difference between a first region of the sample in contact with the first portion of the receptacle and a second region of the sample in contact with the second portion of the receptacle during cooling of the base is at least 15° C.

Inducing ice nucleation in the first region of the sample and preventing ice nucleation in the second region of the sample may reduce an exposure of biological material, e.g. cells, to sudden thermal changes associated with nucleation. For example, only cells in the first region may be exposed to such thermal changes. Therefore, cell viability in a sample may be increased compared to a case in which ice nucleation is allowed to occur throughout the sample.

Further, nucleation in the first region may be induced without user intervention or a separate, manual nucleation step, which may involve, for example, touching an outer surface of the receptacle with cold forceps to generate a cold spot on the outer surface. A repeatability of the freezing process may therefore be enhanced. In examples described herein, inducement of ice nucleation may be performed by the receptacle holder itself as part of the cooling process, e.g. by cooling of the receptacle holder whilst at least a part of the receptacle holder is in contact with the receptacle. The receptacle holder may therefore perform both cooling of the sample and triggering of ice nucleation, without the need for separate nucleation trigger means. As such, there may be no need for chemical catalysts to be added to the sample to induce nucleation, where such chemical catalysts may not be biocompatible and/or may require an extra step of washing the biological material after thawing to remove the chemical catalysts. The measures provided herein may also be less complex than systems in which ice nucleation is induced using a separate ice nucleation means.

Moreover, the first receptacle part and the second receptacle part may each have heat transfer characteristics which can be individually optimised or tuned. For example, the material and/or geometric properties of both the first receptacle part and the second receptacle part may be optimised such that a desired temperature gradient within the sample is achieved. As such, multiple degrees of freedom are provided for modifying the heat flow characteristics from a sample to a receptacle holder during freezing of the sample. Different properties for the first and/or second receptacle parts may be used for different types of sample, different types of receptacle, different freezing modes, different types of cooling apparatus, or different sample volumes. A flexible and adaptable mechanism for optimising temperature gradients in biological samples and achieving directional solidification is therefore provided.

Examples of devices and features thereof will now be described in detail.

FIG.2shows a device200according to examples for use in freezing at least part of a biological sample in a receptacle (not shown). In this example, the device200is for freezing biological samples in cryopreservation bags, or cryobags, it being understood that other types of receptacle can be used in other examples. A cryobag is for example a specialist flexible bag suitable for storing biological samples during cryopreservation. Cryobags may be configured to store relatively large sample volumes, e.g. between 5 millilitres (ml) and 1000 ml.

A sample may include a fluid or liquid, e.g. an aqueous solution, to be cooled or frozen. A biological sample may be considered a sample that contains biological material (or matter, substance, or media). Biological material may be considered material that is produced in, or present in, a living organism. Examples of biological material include, but are not limited to, cells, organelles, viruses, vaccines, organs, matrices, microorganisms and tissue. A sample may be contained within a container or receptacle, e.g. a cryocontainer or a cryobag. The receptacle may be a vessel, a test tube, a vial, a straw, or a bag in examples.

The device200comprises a base210and a plurality of walls220extending from the base210. The base210may be considered a bottom or lower part of the device200, a surface of which the device200may rest upon. For example, with the device resting on its base, a non-bag type receptacle held by the device and holding a sample may be oriented in an upright position. The base210may have a generally planar shape, for example if it is a plate. The base may have a planar surface, for example on which the device may rest. The base210may be mounted on or otherwise contacted by a cooler device, e.g. a cryocooler, for example via a planar surface of the base. The plurality of walls220includes a wall225. The device200may be configured to hold or retain a plurality of receptacles, e.g. cryobags, each receptacle being placed substantially upright between a pair of walls. As such, the device200may be considered to comprise a receptacle holder. The walls220may be substantially perpendicular (within acceptable measuring tolerances) to the base210or angled by a non-perpendicular angle with respect to the base210. In this example, the walls220are substantially parallel (for example within acceptable measuring tolerances) with respect to each other. In other examples, the walls220may be non-parallel with respect to each other.

In this example, the base210comprises a plate. The plate210may comprise a heat conductive plate. The plate210, or panel, may act as a heat sink. The plate210may be cooled by a cooler device such as a cryocooler. In some examples, the plate210is part of a cryocooler. In this example, a first receptacle holder part230of the device200is part of a surface of the plate210. The surface may be an upper surface of the plate210. The surface may be a heat conductive surface. The first receptacle holder part230is configured to withdraw heat energy from a first portion of a receptacle at a first heat withdrawal rate, when the base210is cooled by a cooler device. In this example, the first receptacle holder part230is configured to contact the lowermost portion of the receptacle, e.g. the base of the receptacle, during cooling of the sample.

In this example, a second receptacle holder part240is part of the wall225extending from the plate210, e.g. one of the plurality of walls220. In some examples, the second receptacle holder part240is the entirety of the wall225. In some examples, the second receptacle holder part240includes part of multiple walls in the plurality of walls220. The second receptacle holder part240is configured such that a second heat withdrawal rate of heat energy withdrawal from a second portion of the receptacle via the second receptacle holder part240is lower than the first heat withdrawal rate. For example, with a receptacle standing substantially upright between a pair of walls, heat may be transferred to one or both of the pair of walls from the receptacle at a lower rate than the rate at which heat is transferred from the base of the receptacle to the upper surface of place210.

The second receptacle holder part240may have a different heat flow characteristic than the first receptacle holder part230. For example, the first and second receptacle holder parts may comprise materials having different thermal conductivities. The difference between the heat flow characteristics of the first and second receptacle holder parts allows a temperature gradient to be established in the sample during cooling of the sample that is sufficient to induce ice nucleation in a first region of the sample that is in contact with the first portion of the receptacle, but not in a second region of the sample that is in contact with the second portion of the sample.

FIG.3shows an example device300for use in freezing at least part of a biological sample in a receptacle (not shown). Some items depicted inFIG.3are similar to items shown inFIG.2. Corresponding reference signs, incremented by 100, are therefore used for similar items, and corresponding descriptions should be taken to apply also.

In this example, a base portion of the wall325is wider than a non-base portion of the wall325, the base portion of the wall325being closer to the base310than the non-base portion of the wall325. In this example, the first part of the receptacle holder330is a first wall part of the wall325, e.g. the base portion of the wall325, and the second part of the receptacle holder340is a second wall part of the wall325, e.g. the non-base portion of the wall325. The first part of the receptacle holder330may additionally include the upper surface of the plate310in some examples.

Since the base portion of the wall325is wider than the non-base portion of the wall325, a contact area between the base portion of the wall325and a receptacle held by the device300may be greater than a contact area between the non-base portion of the wall325and the receptacle. The size of the contact area between a given receptacle holder part and the receptacle is an example of a heat transfer characteristic of the given receptacle holder part. The relatively small contact area between the non-base portion of the wall325and the receptacle allows a relatively low rate of heat transference from the receptacle via the non-base portion of the wall325during cooling of the base310, compared with the rate of heat transference from the receptacle via the base portion of the wall325. As such, a temperature gradient may be established in the sample to enable progressive solidification to occur in the sample. The relative widths of the different portions of the wall325may be adapted and/or optimised in order to control or modify a temperature gradient for different sample types, volumes, or cooling rates.

In this example, each of the plurality of walls320has a relatively wide base portion and a relatively narrow non-base portion. In some examples, one or more of the plurality of walls320have a varying width as they extend from the base310and one or more others of the plurality of walls320do not have a varying width as they extend from the base310.

FIG.4shows an example device400for use in freezing at least part of a biological sample in a receptacle (not shown). Some items depicted inFIG.4are similar to items shown inFIG.3. Corresponding reference signs, incremented by 100, are therefore used for similar items, and corresponding descriptions should be taken to apply also.

In this example, the wall425comprises an opening450through the wall425. The opening450, or hole, may be produced by cutting out a portion of the wall425, for example. A first width456of the opening450is narrower than a second width455of the opening450, the first width456closer to the base410than the second width455. As such, the width of the opening450may vary along an axis perpendicular to the base410. When a receptacle is placed between the wall425and an adjacent wall of the plurality of walls420, there may be a relatively low contact area between the wall425and the receptacle where the opening450has the second width456, compared to the contact area between the wall425and the receptacle where the opening450has the first width456. As such, a lower rate of heat transference from the receptacle to the region of the wall425having the second width455of the opening450may be established compared to a rate of heat transference from the receptacle to the region of the wall425having the first width456of the opening450. The varying width of the opening450along a height of the wall425may facilitate the establishment of a temperature gradient in the sample sufficient to achieve progressive solidification of the sample. The relative widths of the opening450in different portions of the wall325may be adapted and/or optimised in order to control or modify a temperature gradient for different sample types, volumes, or cooling rates.

In this example, the wall425comprises a relatively wide base portion and a relative narrow non-base portion, where only the non-base portion comprises the opening450. In other examples, both the base portion and the non-base portion may comprise openings, which may be similarly or differently sized.

Although in this example the wall425comprises a relatively wide base portion and a relatively narrow non-base portion with respect to the base portion, in other examples walls may be used that have base and non-base portions of similar or the same width, where the non-base portion may comprise a wider opening than that of the base portion, or where only the non-base portion comprises an opening.

FIG.5shows an example device500for use in freezing at least part of a biological sample in a receptacle (not shown). Some items depicted inFIG.5are similar to items shown inFIG.4. Corresponding reference signs, incremented by 100, are therefore used for similar items, and corresponding descriptions should be taken to apply also.

In this example, a first wall525comprises an opening550, such as that described with reference toFIG.4above, and a second wall526does not comprise an opening. As such, a contact area between the first wall525and a receptacle may be smaller than a contact area between the second wall526and the receptacle, due to the presence of the opening550in the first wall525and an absence of an opening in the second wall526. A first width of the opening550in the first wall525is greater than a second width of the opening550, the first width further from the base510than the second width.

The first wall525and the second wall526may be adjacent walls in the plurality of walls520. As such, a given receptacle when held by the device500may be placed in contact with both the first wall525and the second wall526. In such a case, there may be a greater rate of heat energy withdrawal from the receptacle via the second wall526than that from the receptacle via the first wall525, due to the presence of the opening550in the first wall525.

In some examples, the second wall526also comprises an opening. The opening in the second wall526may be differently sized and/or shaped than the opening550in the first wall525.

The inclusion of openings, or holes, in one or both of a pair of adjacent walls provides an additional degree of freedom for optimising the temperature gradient in a sample during freezing of the sample. The presence, position, size and shape of openings, as well as whether such openings are present on one or both of the pair of walls between which the receptacle is to be received, are all parameters which may be separately optimisable. For example, different types and sizes of openings may be provided for different sample types and/or volumes, in order to achieve progressive solidification of samples in a repeatable and flexible manner.

FIGS.6and7show an example device600for use in freezing at least part of a biological sample in a receptacle (not shown). Some items depicted inFIGS.6and7are similar to items shown inFIG.2. Corresponding reference signs, incremented by 400, are therefore used for similar items, and corresponding descriptions should be taken to apply also.

In the example shown inFIGS.6and7, the device600comprises a slot650, or track. The slot650is part of the base610of the device600. The plurality of walls620may be slidably mounted on the base610via the slot650. As such, the slot650enables the separation between adjacent walls in the plurality of walls620to be adjusted. Having slidably mounted walls may allow the device600to accommodate receptacles of different sizes, for example. Additionally or alternatively, the slidably mounted walls may ensure that the walls are in contact with the receptacle during the freezing operation. For example, the slidably mounted walls may be pressed securely against the sides of the receptacle to make thermal contact with the receptacle, and then pulled away from the sides of the receptacle after the freezing operation is completed.

FIG.8shows an example device800for use in freezing at least part of a biological sample in a receptacle (not shown). Some items depicted inFIG.8are similar to items shown inFIG.6. Corresponding reference signs, incremented by 200, are therefore used for similar items, and corresponding descriptions should be taken to apply also.

In this example, the wall825comprises a base portion and a non-base portion. The first receptacle holder part830comprises the base portion of the wall825. The second receptacle part840comprises the non-base portion of the wall825. The first receptacle holder part830may additionally comprise an upper surface of the base810.

In this example, the base portion of the wall825comprises a first material, or substance, and the non-base portion of the wall825comprises a second, different material. In some examples, the first material comprises a metal. For example, the first material may comprise aluminium, silver, copper or brass. In other examples, the first material comprises a non-metal. For example, the first material may comprise sapphire. The second material may comprise a polymer. For example, the second material may comprise polypropylene, polystyrene or polytetrafluoroethylene (PTFE). The first material may have a first thermal conductivity and the second material may have a second thermal conductivity. A thermal conductivity is for example a rate at which heat energy is transmitted through a particular material. In an example, the first thermal conductivity is at least 10 Watts per meter-Kelvin (W/mK). In an example, the second thermal conductivity is at most 1 W/mK. The first material and/or the second material may be selected such that a predetermined temperature gradient is established in the sample during a freezing operation. For example, the first material may be selected in order to achieve a first heat transfer rate from the sample to the base portion of the wall825, and the second material may be selected in order to achieve a second, lower heat transfer rate from the sample to the non-base portion of the wall825. The first and/or second materials may be chosen based on their heat conducting properties. Using different materials for the first and second receptacle holder parts830,840provides an additional degree of freedom for optimising the temperature gradient in a sample during freezing of the sample. The materials, and material characteristics, of each of the receptacle holder parts830,840may be separately optimisable. For example, one or both of the materials used for the first and second receptacle holder parts830,840may be adapted or changed for different sample types, volumes, or cooling rates, in order to achieve progressive solidification of samples in a repeatable and flexible manner.

In some examples, the base810and the base portion of the wall825are comprised of the same material. For example, the first part of the receptacle holder830may include both the upper surface of the base810and the base portion of the wall825. In some examples, the base810is comprised of a different material than that of the base portion of the wall825.

In this example, the wall825is slidably mounted on the base810via slot850. In other examples, the wall825is fixed to the base810. In this example, the base portion of the wall825has the same width to the non-base portion of the wall825, within acceptable manufacturing tolerances. In other examples, the base portion of the wall825is wider than the non-base portion of the wall825, such that a contact area between the base portion of the wall825and the receptacle is greater than a contact area between the non-base portion of the wall825and the receptacle. In some examples, the base portion of the wall825and/or the non-base portion of the wall825comprise one or more openings, as described in more detail above.

FIGS.9to11show a device900in accordance with examples for use in freezing at least part of a biological sample in a receptacle905. In this example, the receptacle905comprises a cryovial, it being understood that other types of receptacle can be used in other examples. A cryovial may be a specialist tube or vial that is suitable for storing biological samples during cryopreservation. Cryovials may be configured to store relatively small sample volumes, e.g. between 0.5 and 50 ml.

The device900comprises a base910. The base910is for example a bottom part of the device900, configured to support the device900or on which the device900may rest. The base910may be contactable with a cooling surface of a cooler device such as a cryocooler or heat conductive plate. In this example, the device900comprises one or more receptacle holders for holding, respectively, one or more receptacles such as the receptacle905. The receptacle holder may be considered part of a sample plate for freezing samples in vials. In other examples, the device comprises a single receptacle holder configured to hold only a single receptacle, e.g. the receptacle905.

In this example, the receptacle holder comprises a recess to receive the receptacle905. The recess, or cavity, may be produced by boring or drilling a hole into a solid plate. The recess may be configured to have similar dimensions, within acceptable measurement tolerances, to the receptacle which is to be placed therein, such that the walls of the receptacle are flush with the walls of the recess.

The receptacle holder comprises a first receptacle holder part930and a second receptacle holder part940. The first receptacle holder part930may be considered a base portion and the second receptacle holder part940may be considered a non-base portion, since the first receptacle holder part930is closer than the second receptacle holder part940to the base910. The first receptacle holder part930may be configured to contact a first portion of the receptacle905, e.g. a lower portion of the receptacle905. The second receptacle holder part930may be configured to contact or otherwise hold a second portion of the receptacle905, e.g. an upper portion of the receptacle905. In this example, the first receptacle holder part930extends partway up the walls of the recess in which the receptacle905is received. In some examples, the first receptacle holder part is part of the base upon which the receptacle stands, but is not part of the walls of the recess. In some other examples, the first receptacle holder part930includes part of the base910and part of the walls of the recess.

In this and other examples, the first receptacle holder part930is configured to at least partly surround the first portion of the receptacle905and the second receptacle holder part940is configured to at least partly surround the second portion of the receptacle905. Surrounding a given portion of the receptacle may involve a surface of the respective receptacle holder part extending at least partly around the given portion of the receptacle, e.g. such that more than half of the surface of the given receptacle portion faces the surface of the respective receptacle holder part. The surface of the given receptacle portion may be in contact with the surrounding receptacle holder part or may be spaced apart from it, as described in examples below.

In this example, the first receptacle holder part930is comprised of a first material and the second receptacle holder part940is comprised of a second material, different from the first material. The first material may have a relatively high thermal conductivity compared to the second material, and the second material may have a relatively low thermal conductivity compared to the first material. The second receptacle holder part940may be comprised of a thermally insulating material.

In some examples, the first receptacle holder part930and the second receptacle holder part940have different porosities. For example, the second receptacle holder part940may be comprised of a material having a relatively high porosity compared to the material of the first receptacle holder part930, and the first receptacle holder part930may be comprised of a material having a relatively low porosity compared to the material of the second receptacle holder part940. The greater porosity of the second receptacle holder part940relative to the first receptacle holder part930may result in a lower thermal conductivity of the second receptacle holder part940relative to the first receptacle holder part930. In some examples, the materials of the first receptacle holder part930and the second receptacle holder part940may differ only in their relative porosities. Although materials having different porosities are described in relation to the device900, it will be understood that materials having different porosities may also be used in other examples devices, such as the device800described with reference toFIG.8above. In an example, the second receptacle holder part940may be comprised of a material with a hollow therein and the first receptacle holder part930may be comprised of a solid material. The material with the hollow may provide a layer of insulating gas, e.g. air in the hollow, within the second receptacle holder part940, thereby reducing the effective thermal conductivity of the second receptacle holder part940. Due to the different materials and/or material properties used, the first receptacle holder part930and the second receptacle holder part940have different heat transfer characteristics to enable heat energy to be withdrawn from the sample at different rates during a freezing operation.

The device900is configured to hold the receptacle905along a freezing operation temperature gradient axis950extending away from the base910. The temperature gradient axis950may be substantially perpendicular (within acceptable measurement tolerances) to the plane of the base910in some examples. The temperature gradient axis950may define a direction in which the sample progressively solidifies during a freezing operation. For example, solidification may commence in a base region of the sample and proceed upwards towards an upper region of the sample. The base region of the sample may be the region in which ice nucleation occurs, and the region to which ice nucleation is restricted. The temperature gradient in the sample having temperature gradient axis950may be implemented via the different heat withdrawal rates of the first and second receptacle holder parts930,940. Although the temperature gradient axis950is shown inFIGS.9to11, it will be understood that temperature gradients having temperature gradient axes may also be implemented in the other examples described herein.

FIGS.12to14show an example device1200for use in freezing at least part of a biological sample in a receptacle1205. In this example, the receptacle1205comprises a cryovial. Some items depicted inFIGS.12to14are similar to items shown inFIGS.9to11. Corresponding reference signs, incremented by 300, are therefore used for similar items, and corresponding descriptions should be taken to apply also.

The receptacle holder comprises a first receptacle holder part1230and a second receptacle holder part1240. The receptacle holder is configured to hold the receptacle1205along a freezing operation temperature gradient axis1250extending away from the base1210. The temperature gradient axis1250may be substantially perpendicular to the plane of the base1210in some examples, e.g. within an acceptable measurement tolerance. The first receptacle holder part1230comprises a first surface1232. The second receptacle holder part1240comprises a second surface1242. The first and second surfaces1232,1242may be inner surfaces of the recess. The first surface1232may abut the receptacle1205when the receptacle1205is received by the device1200. The second surface1242is further from the base1210than the first surface1232. In this example, the second surface1242is further from the temperature gradient axis than the first surface1232. In this example, the second surface1242does not abut the receptacle1205when the receptacle1205is received by the device1200. The second surface1242is therefore spaced from the receptacle1205by a predetermined amount when the receptacle1205is received by the device1200. In some examples, the first surface1232is substantially parallel to the temperature gradient axis1250, e.g. within an acceptable tolerance. In some examples, the second surface1242is angled with respect to the temperature gradient axis1250by a non-parallel angle, though in other examples, the second surface1242is substantially parallel (within acceptable measurement tolerances) to the temperature gradient axis1250.

As such, with the receptacle1205held by the receptacle holder, a first volume of ambient gas between the first surface1232and a first portion of the receptacle1205may be less than a second volume of ambient gas between the second surface1242and a second portion of the receptacle1205. Typically, an ambient gas is a gaseous medium surrounding the receptacle, which may be air or another gas or gas mixture. During a cooling operation, for example cooling of the base1210with a cooler device, the temperature of the ambient gas may be higher than the temperature of the base1210. The first volume of ambient gas may be zero in some examples, for example where the first surface1232is flush with the first portion of the receptacle1205. The ambient gas may provide an insulting layer, for example between the receptacle1205and a given surface of the receptacle holder. The presence and/or thickness of such an insulating gas layer may enable heat energy to be withdrawn from the second portion of the receptacle via the second surface1242at a lower rate than a rate of heat energy withdrawal from the first portion of the receptacle via the first surface1232.

In this example, the first receptacle holder part1230also comprises a third surface1234. The third surface1234is opposed from the first surface1232across the recess. The second receptacle holder part1240comprises a fourth surface1244, opposed from the second surface1242across the recess. A first width of the recess taken between the first surface1232and the third surface1234is less than a second width of the recess taken between the second surface1242and the fourth surface1244in this example. As such, the recess may be wider in a plane further from the base1210than in a parallel plane closer to the base1210.

In this example, the recess tapers towards the base1210. In other examples, the width of the recess increases along an axis perpendicular to the base1210in a non-continuous manner. For example, the width of the recess may vary according to a step function, wherein a base portion of the recess abuts the receptacle1205and a non-base portion of the recess is spaced from the receptacle1205by a predetermined amount, the base portion and non-base portion being substantially parallel with respect to one another, e.g. within an acceptable manufacturing tolerance.

In the example shown inFIGS.12to14, the first receptacle holder part1230and the second receptacle holder part1240have different values of a geometric property. For example, the geometric property may comprise a width of a recess taken between two opposing surfaces of a respective receptacle holder part, and/or a distance between a surface of the respective receptacle holder part and a corresponding surface of the receptacle during a freezing operation. Such a geometric property is an example of a heat flow characteristic of a given receptacle holder part, since the geometric property affects how heat energy may be transferred from the receptacle to the given receptacle holder part. For example, the thickness of an air layer between the receptacle and a given receptacle holder part may be determined according to a desired withdrawal rate of heat energy from the receptacle via the given receptacle holder part.

The devices described herein may be cooled using a variety of cooling equipment.

In some known systems, a fluid in a container, for example a fluid at room temperature, may be cooled by placing the container onto a cold surface that is maintained at a very low temperature, for example below −130° C. Placing the container on such a cold surface that is isothermal, that is having a fixed temperature, may cause a rapid change of temperature in the fluid.

Biological samples such as cells, however, are at risk of being damaged, or injured, during cooling, if such cooling is not controlled in an adequate manner. For example, as ice nucleation occurs and ice crystals form during cooling, cells may suffer direct damage from the ice crystals and also damage caused by an increase in concentration of solutes in the sample as progressively more ice is formed. Damaged cells may be less likely to recover and/or function when the sample is thawed.

Cooling a biological sample in a controlled manner may reduce the damage to the biological material caused by such effects and may thereby help to retain cell viability and function after thawing. For example, the biological sample may be cooled at a controlled rate which may be constant or may vary with time, according to a cooling protocol.

The cooling protocol may be implemented by a cooling apparatus, e.g. comprised as part of a cooling system such as the VIA Freeze™ equipment manufactured by Asymptote Limited. Some cooling systems blow cold gas over the sample which can cause an uneven cooling rate, or cooling profile (e.g. representing the change in cooling rate over time or temperature) across the sample or between samples where a plurality of samples are to be cooled by the cooling system. The VIA Freeze™ cooling system cools a sample by conduction on the underside of the sample and therefore every sample may experience the same cooling profile.

The cooling rate can depend on the nature of the sample being frozen. The sample quality, e.g. cell viability or function, on thawing may be severely reduced if the cooling rate after freezing is excessively fast or slow. Once frozen to, for example −80° C. or −100° C., the sample may be removed from the VIA Freeze™ system and placed in long term frozen storage.

FIG.15shows an example cooling apparatus1500for cooling a sample. The cooling apparatus1500may comprise a refrigerator or freezer device, for example a controlled rate freezer. In some examples, the cooling apparatus1500comprises and/or uses a cryocooler usable to cool the sample to a cryogenic temperature. A cryogenic temperature may be considered to be a temperature below −50° C., or below −180° C., as described above. For example, the cooling apparatus1500may be useable to cool the sample to −196° C. Examples of cryocoolers that may be used as the cooling apparatus1500include a Stirling cryocooler, an acoustic Stirling cryocooler, a Kleemenco cycle cryocooler, a pulse tube cryocooler, and a Joule-Thompson cryocooler.

The cooling apparatus1500comprises a cooler device1510. In this example, the cooler device1510comprises a cold finger of the cooling apparatus1500, which is a localised part of the cooling apparatus1500that is to be cooled during operation of the cooling apparatus1500. The cooling apparatus1500may further comprise one or more compressors, pistons, heat exchangers or the like, for withdrawing heat from the cold finger1510.

The cold finger1510may cool a heat conductive plate1520, for example through direct contact with the heat conductive plate1520. A device comprising a receptacle holder may be placed in contact with the heat conductive plate1520. In some examples, the heat conductive plate1520is part of the device. The device may be one of the devices200,300,400,500,600,800,900,1200described above. As such, the cooler device1510may be used to cool the devices200,300,400,500,600,800,900,1200.

Different types of receptacle holder may be used, for example to accommodate different types and/or sizes of receptacle. The receptacle holder, as part of a device described previously, for example, may be removable from the cooling apparatus1500to allow samples to be added or removed easily. The heat conductive plate1520and/or the cooler device1510may be considered to be a heat sink, in other words an area or part of the cooling apparatus1500that is to absorb or withdraw heat energy from the sample during a cooling operation performed on the sample.

The cooling apparatus1500may perform a cooling operation on the sample, and the cooling operation may be associated with a cooling profile. The cooling profile, or temperature profile, may be a dataset that comprises objective temperatures at time intervals of the cooling process. For example, the cooling profile describes how a measured temperature, e.g. of the sample, should change with time during the cooling operation. An objective cooling rate, e.g. change of temperature with time, may be set at different stages of the cooling profile. In some examples, a constant objective cooling rate may be set in the cooling profile.

The cooling profile may be pre-programmed into a control module configured to control operation of the cooling apparatus1500. The control module may comprise control circuitry, e.g. including one or more processors and computer-readable storage with appropriate instructions executable by the one or more processors to control the cooling apparatus. For example, the control module may receive temperature data from one or more temperature sensors, or probes, representing a measured temperature of the sample, and/or cooling chamber in some examples, at a given time during the cooling operation. The control module may compare the received temperature data to an objective temperature value according to the particular cooling profile at a corresponding time, and may adjust the cooling operation based on the outcome of the comparison. For example, if the measured temperature at a given time were higher than a target temperature at a corresponding time according to the temperature profile, the control module may control the cooling operation to decrease the temperature of the entity being measured (i.e. the sample and/or chamber). Such comparisons and possible resulting adjustments may occur at predetermined times during the cooling operation, and/or may occur at a regular time interval. In this way, the control module may track the cooling profile in real time during the cooling operation, adjusting the cooling operation to follow the set cooling profile.

FIG.16shows an example cooling apparatus1600for cooling a sample.

In this example, a device comprising a receptacle holder may be cooled by contact with a high conductivity heat sink1620. The heat sink1620may comprise a heat conductive plate. The heat sink1620may be cooled by the internal flow of cryogenic gas such as liquid nitrogen in a chamber1630. The temperature of the heat sink1620may be isothermal throughout a cooling process or may be varied. The temperature of the heat sink1620may be controlled by modifying the flow of the gas through the chamber1630, or by placing a heater on the top surface of the heat sink1620and controlling its temperature. The cooling process using the cooling apparatus1600may be a batch process allowing for the processing of single samples. The heat sink1620and/or the chamber1610may be considered a cooler device that is useable to cool a device such as the devices200,300,400,500,600,800,900,1200described above.

FIG.17shows an example cooling apparatus1700for cooling a sample.

The cooling apparatus1700comprises a plurality of heat sinks1720and a chamber1730. The plurality of heat sinks1720may be cooled by the flow of cryogenic gas through the chamber. Each of the plurality of heat sinks1720may be configured to receive a respective device, each device comprising a respective receptacle holder. Different heat sinks may be configured to receive devices having different properties. For example, a first device that has a first characteristic temperature differential between first and second respective receptacle holder parts may be received by a first heat sink of the cooling apparatus1700, and a second device having a second characteristic temperature differential between first and second respective receptacle holder parts may be received by a second heat sink of the cooling apparatus1700. As such, different devices having different heat withdrawal properties may be cooled simultaneously by the cooling apparatus1700. The cooling process using the cooling apparatus1700may allow cooling of multiple different samples simultaneously. The different samples may be of different sample types, different volumes, or be contained in different types of receptacle.

FIG.18shows an example cooling apparatus1800for cooling a sample. Some items depicted inFIG.18are similar to items shown inFIG.17. Corresponding reference signs, incremented by 100, are therefore used for similar items, and corresponding descriptions should be taken to apply also.

The cooling apparatus1800comprises a heat sink1820and a chamber1830. The heat sink1820may be cooled by the flow of cryogenic gas through the chamber1830. A plurality of devices, each comprising a respective receptacle holder, may be placed on the heat sink1820. Different devices having different heat withdrawal properties may be cooled simultaneously by the cooling apparatus1800.

FIG.19shows an example cooling apparatus1900for cooling a sample.

The cooling apparatus1900comprises a heat sink1920and a freezer1930. The freezer1930may be a laboratory freezer, for example a −80° C. freezer. The heat sink1920may be placed inside the freezer1930to passively cool the heat sink1920. A device comprising a receptacle holder may be placed on the heat sink1920and may in turn be cooled by contact with the heat sink1920.

Examples described above involve using a receptacle holder, of a device, having different parts with different heat withdrawal properties to establish a temperature gradient in a sample during cooling of the sample, in order to induce ice nucleation in a first region of the sample and prevent ice nucleation in a second region of the sample. Additionally and/or alternatively, such an effect may be achieved through use of a receptacle adapted to have different parts with different heat transfer properties, as described in the examples below. Such a receptacle may be used with one of the example devices described above, or may be used with a known receptacle holder.

FIG.20shows a receptacle2000for use in freezing a biological sample contained therein. In this example, the receptacle2000is a cryovial, it being understood that the receptacle2000could be a different type of container or cryocontainer in other examples.

The receptacle2000comprises a first portion2010. The first portion2010comprises a first inner surface2012and a first outer surface2014. The first inner surface2012is an inner surface of the receptacle2000and the first outer surface2014is an outer surface of the receptacle. The first portion2010is configured to transfer heat energy from the first inner surface2012of the first portion2010to the first outer surface2014of the first portion2010at a first heat transfer rate.

The receptacle2000also comprises a second portion2020. The second portion2020comprises a second inner surface2022and a second outer surface2024. The second inner surface2022is an inner surface of the receptacle2000. The second outer surface2024is an outer surface of the receptacle2000. The second portion2020is configured to transfer heat energy from the second inner surface2022of the second portion2020to the second outer surface2024of the second portion2020at a second heat transfer rate. The second heat transfer rate is less than the first heat transfer rate. In some examples, the second heat transfer rate is greater than the first heat transfer rate.

In some examples, the first portion2010comprises a first material having a first value of a heat flow characteristic and the second portion2020comprises a second material having a second value of the heat flow characteristic. The second value is different from the first value. The heat flow characteristic may be a thermal conductivity. In some examples, a wall of the first portion2010and a wall of the second portion2020may have different thicknesses. For example, a distance between the first inner surface2012and the first outer surface2014taken in a plane substantially perpendicular with the major axis2030of the receptacle2000may be different than a distance between the second inner surface2022and the second outer surface2016taken in a plane substantially perpendicular with the major axis2030of the receptacle2000. The major axis is for example a longitudinal axis of the receptacle.

In some examples, the heat flow characteristic is a geometric property. For example, the first portion2010may be wider than the second portion2020. The width of a given receptacle portion may be taken perpendicular to the major axis2030of the receptacle2000. The width of a given receptacle portion may correspond to the cross-section of the receptacle as a whole. As such, the first portion2010may be configured to have a greater contact area with a receptacle holder than the second portion2020. In some examples, for example where the receptacle2000is cylindrical, the circumference of the first portion2010may be greater than that of the second portion2020. In some examples, the radius of the first portion2010may be greater than that of the second portion2020.

In some examples, the receptacle2000comprises an internal cavity, or volume, for receiving a biological sample. The first portion2010may comprise a third inner surface2016opposing the first inner surface2012across the cavity. The third inner surface2016opposes the first inner surface2012in a plane perpendicular to the major axis2030of the receptacle2000. The second portion2020may comprise a fourth inner surface2026opposing the second inner surface2022across the cavity. The fourth inner surface2026opposes the second inner surface2022in a plane perpendicular to the major axis2030of the receptacle2000. The plane between the second inner surface2022and the fourth inner surface2026is parallel to the plane between the first inner surface2012and the third inner surface2016. A distance2032between the first inner surface2012and the third inner surface2016may be greater than a distance2034between the second inner surface2022and the fourth inner surface2026. Therefore, the first portion2010may be wider than the second portion2020. As such, the first portion2010may be configured to have greater heat flow properties than the second portion2020, e.g. due to having a higher greater surface area than the second portion2020.

In examples, a difference between the first heat transfer rate and the second heat transfer rate is at least sufficient to establish a temperature gradient within a sample in the receptacle2000such that, during a freezing operation with heat transfer via the first portion2010and the second portion2020, ice nucleation occurs in a first region of the sample, the first region in contact with the first portion2010, without ice nucleation in a second region of the sample, the second region in contact with the second portion2020. Ice formation in the sample may therefore be controlled during cooling of the sample.

FIG.21shows a flow diagram illustrating a method2100of freezing part of a biological sample stored in a receptacle.

At block2110, the receptacle containing the sample is arranged in a receptacle holder. The receptacle holder may be part of a device such as one of the devices200,300,400,500,600,800,900,1200described above. In some examples, the receptacle holder is a different receptacle holder from that which is described as part of the devices200,300,400,500,600,800,900,1200. The receptacle may be the receptacle2000described above, in some examples.

At block2120, the receptacle holder is cooled using a cooler device. For example, a base of the receptacle holder may be placed in contact with a cooling surface of the cooler device. The temperature of the cooling surface may vary during a cooling operation performed by the cooler device. The cooler device may comprise a cryocooler. In some examples, the receptacle holder is cooled at a controlled rate. For example, the receptacle holder may be cooled by a controlled rate freezer.

At block2130, during cooling of the receptacle holder using the cooler device, heat energy is withdrawn from a first portion of the receptacle via a first receptacle holder part of the receptacle holder. The heat energy is withdrawn from the first portion of the receptacle via the first receptacle holder part at a first heat withdrawal rate. For example, the first portion of the receptacle may be arranged in conductive contact with the first receptacle holder part.

At block2140, during cooling of the receptacle holder using the cooler device, a second heat withdrawal rate of heat energy withdrawal from a second portion of the receptacle via a second receptacle holder part of the receptacle holder is caused to be less than the first heat withdrawal rate. The second receptacle holder part may be further from the base of the receptacle holder than the first receptacle holder part. In some examples, heat energy is withdrawn from the second portion of the receptacle via the second receptacle holder part at the second heat withdrawal rate. A difference between the first heat withdrawal rate and the second heat withdrawal rate may be at least sufficient to establish a temperature gradient within the biological sample such that, during a freezing operation using the cooler device, nucleation of the sample occurs in a first region of the sample, the first region in contact with the first portion of the receptacle, without nucleation of the sample in a second region of the sample, the second region in contact with the second portion of the receptacle.

FIG.22shows an example representation2200of temperature versus time in a sample undergoing a freezing operation with progressive solidification.

InFIG.22, a first line2210corresponds to measured data obtained by a first thermocouple in a base region of the sample, a second line2220corresponds to measured data obtained by a second thermocouple in a middle region of the sample, and a third line2230corresponds to measured data obtained by a third thermocouple in an upper region of the sample. The sample was cooled using the methods as described herein.

As shown inFIG.22, the base region of the sample undercools to around −10° C., and upon ice nucleation in the base region the temperature in the base region rapidly increases. However, there is no corresponding temperature jump in the middle or upper regions of the sample, nor is there undercooling in those regions. Therefore, apart from the region in which nucleation occurs, the remainder of the sample may freeze without undercooling, thus improving cell viability at least in regions of the sample other than the base region. Following ice nucleation in the base region, ice may form across the whole sample. As an illustrative example, the post-thawing viability of induced pluripotent stem cell (iPS)-derived neurones and mammalian embryos may be improved through use of measures described herein. The post-thawing viability of cells of other types, and of other types of biological material, may also be improved by the measures described herein.

The above examples are to be understood as illustrative. Further examples are envisaged.

It is to be understood that any feature described in relation to any one example may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the examples, or any combination of any other of the examples. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope defined in the accompanying claims.