WO2016016886A1

WO2016016886A1 - Devices and methods for cryopreserving a biological sample by liquid nitrogen

Info

Publication number: WO2016016886A1
Application number: PCT/IL2015/050771
Authority: WO
Inventors: Amir Arav
Original assignee: Fertilesafe Ltd
Priority date: 2014-07-27
Filing date: 2015-07-27
Publication date: 2016-02-04

Abstract

Devices and methods for cryopreserving a biological sample by liquid nitrogen, the device comprising: a first container comprising an internal space, the first container is configured to be submerged in liquid nitrogen stored in a cryopreservation space inside a liquid nitrogen container and to hold the biological sample in the internal space while preventing contact of the biological sample with the liquid nitrogen; and an airway comprising a first end and a second end, the airway is coupled to the first container at its first end and configured to couple the internal space to an outer environment, external to the liquid nitrogen container, so as to balance pressure changes inside the internal space.

Description

DEVICES AND METHODS FOR CRYOPRESERVING A BIOLOGICAL SAMPLE BY LIQUID NITROGEN

FIELD OF THE INVENTION

The invention relates to cryopreservation. More specifically, it relates to devices for cryopreservation of biological samples.

BACKGROUND OF THE INVENTION

Preservation of biological samples, for example oocytes and embryos, at very low temperatures, is known as cryopreservation. One common method of cryopreservation is utilizing liquid nitrogen ("LN") stored in a liquid nitrogen container ("LN container"), a method referred to herein as "liquid nitrogen cryopreservation" ("LN cryopreservation").

There are different methods for cryopreservation of biological samples. One such method, for example, is known as vitrification. There are several protocols for vitrification known in the art. For example, the article "Vitrification of oocytes and embryos" (Amir Arav, in "Embryonic development and manipulation in animal development", edited by A. Lauria and F. Gandolfi, Portland Press, London, U.K., 1992), presents a method of vitrifying cells enclosed in small drops sufficient to keep them in physiological conditions. Another example is the article "New trends in gamete's cryopreservation" (Amir Arav, Saar Yavin, Yoel Zeron, Dity Natan, Izik Dekel, and Haim Gacitua. Molecular and Cellular Endocrinology, 187: 77-81, 2002) that presents techniques to improve freezing and vitrification of sperm, oocytes and embryos, based on 'Multi-Thermal-Gradient' (MTG) freezing.

It is known in the art that crucial stages in vitrification, and in cryopreservation in general, are preparation, freezing and thawing of a biological sample. For example, the article "Measurement of essential physical properties of vitrification solutions" (S. Yavin and A. Arav. Theriogenology, 67(1): 81-9, 2007) examines the principal parameters associated with successful vitrification, and composes guidelines to aspects of the vitrification process. In the article, Yavin and Arav teach that the warming rate should be as rapid as possible to avoid devitrification and recrystallization which damage the cells and tissue upon warming.

One of the challenges of liquid nitrogen cryopreservation is to prevent contamination of the biological sample by biological contaminants that are either preserved in the liquid nitrogen or can be transferred from a contaminated biological sample to a non-contaminated biological sample stored in the same liquid nitrogen container, i.e. cross-contamination.

Efforts have been invested, therefore, in trying to isolate the sample from the liquid nitrogen, trying to hermetically seal containers used for cryopreservation of biological samples. For example, in a letter to the editor (Parmegiani Lodovico and Rienzi Laura, "Hermetical goblets for cryostorage of human vitrified specimens", Human Reproduction Journal, Advanced Access publication on September 9, 2011) Parmegiani and Rienzi attend to the risk of contamination of culture medium if the liquid nitrogen used for vitrification/storage is accidently contaminated. They recognize the need for an "open systems" with which an aseptic vitrification can be performed by sterilizing liquid nitrogen for the vitrification procedure and subsequently cryostoring the carriers inside hermetical containers in a sterile nitrogen-vapor-phase environment.

Technical memorandum 102190 published by NASA (Salerno Louis J. et al, "Performance of All-Metal Demountable Cryogenic Seals at Superfluid Helium Temperatures", April 1989), discusses sealing by all-metal seals.

SUMMARY

In one aspect, the present disclosure includes a device for cryopreserving a biological sample by liquid nitrogen, the device comprising:

a first container comprising an internal space, the first container is configured to be submerged in liquid nitrogen stored in a cryopreservation space inside a liquid nitrogen container and to hold the biological sample in the internal space while preventing contact of the biological sample with the liquid nitrogen; and

an airway comprising a first end and a second end, the airway is coupled to the first container at its first end and configured to couple the internal space to an outer environment, external to the liquid nitrogen container, so as to balance pressure changes inside the internal space.

The above device wherein the first container is sealable with reference to the a cryopreservation space used for storing the liquid nitrogen so as to prevent contaminants carried by the liquid nitrogen from penetrating into the internal space.

The above device, wherein the airway is coupleable to a humidity blocking member the second end, the humidity blocking member is configured to maintain humidity inside the airway below a threshold so as to prevent blocking the airway by frozen humidity.

Any one of the above devices, wherein the first container comprises a cover; Any one of the above devices, further comprising:

a holding element configured to enable placing the first container inside the liquid nitrogen container.

The above device, wherein the holding element is configured to hold the first container submerged in the liquid nitrogen. The above device, wherein the holding element is configured to hold the first container fully submerged in the liquid nitrogen.

The above device, wherein the holding element is configured to hold the first container in nitrogen vapors.

The above device, wherein the holding element is coupled to the first container. The above device, wherein the holding element is coupled to the cover.

The above device, wherein the holding element is coupled to the airway.

The above device, wherein the holding element and the airway are the same.

Any one of the above devices, wherein the first container comprises metal elements, characterized by high thermal conductivity. In another aspect, the present disclosure includes a device for storing a biological sample in a liquid nitrogen container, the device comprising: a first container comprising an internal space, the first container is configured to seal the internal space from nitrogen while submerged in the liquid nitrogen container; and an airway configured to couple the internal space to an environment external to the liquid nitrogen container;

wherein the device is configured to hold the sample in the internal space, and to allow extracting the first container from said liquid nitrogen container.

The above device, wherein the device is configured to allow opening the first container.

The above device, wherein the device is configured to allow re-sealing the first container. In yet another aspect, the present disclosure includes a method for storing a biological sample in liquid nitrogen, the device comprising:

Inserting the biological sample into a first container;

Verifying that the first container has an open airway coupling the first container and the second end of the airway is susceptible to dry air characterized by a humidity level which is below a threshold.

In yet another aspect, the present disclosure includes a system for sealing a biological sample in a container while cryopreserving the biological sample by a cryoliquid, the system comprising: a sealable container configured to hold the biological sample in an internal space of the container, the sealable container comprises an opening configured to allow inserting and removing elements to and from the container, respectively; a cover configured to close the opening, wherein the sealable container and the cover are configured together to form a closed sealable container leaving sealable openings; and a sealing device configured to seal a sealable opening which is a gap between the cover and the sealable container in the closed sealable container so as to prevent penetration of molecules comprised in the cryoliquid via the sealable openings; wherein the system is configured to grip the sealable opening from the outside while contracting around the sealable closed container.

The above system, wherein gripping the sealable opening from the outside leaves a gap of 40nm between the sealing device and walls of the sealable closed container. The above system, wherein gripping the sealable opening from the outside leaves a gap of less than 40nm between the sealing device and walls of the sealable closed container.

The above system, wherein the sealable container and the cover are made of a material having weaker contraction characteristics compared to contraction characteristics of sealing device.

In yet another aspect, the present disclosure includes a system for sealing a biological sample in a container while cryopreserving the biological sample by a cryoliquid, the system comprising: a sealable container configured to hold the biological sample in an internal space of the container, the sealable container comprises walls made of a first shrinkable material configured to shrink while exposed to ultra-cold temperature; and a sealing device made of a second shrinkable material that is characterized by stronger contraction while exposed to the ultra-cold temperature compared to the first shrinkable materials, thereby the sealing device is configured to shrink and grip the sealable container thereby sealing openings in the sealable container. The above system, wherein the second shrinkable material is Polypropylene (PP).

The above system, wherein the second shrinkable material is silicon.

The above system, wherein the second shrinkable material is Polytetrafluoroethylene (PTFE).

The above system, wherein the second shrinkable material is poly amide (PA). The above system, wherein the sealable container is a closed sealable container. The above system,, further comprising: an airway configured to couple the internal space to an external environment, for balancing abrupt pressure changes in the internal space.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

FIG. 1A schematically illustrates a liquid nitrogen cryopreservation system for cryopreserving a biological sample;

FIG. IB schematically illustrates a liquid nitrogen cryopreservation system, for cryopreserving a biological sample;

FIG. 2 schematically describes an all-metal seal for cryogenic applications;

FIG. 3A schematically illustrates an open sealable container and its cover;

FIG. 3B illustrates the sealable container of FIG. 3A when closed with the cover of FIG. 3A;

FIG. 3C schematically illustrates a sealing device sealing the sealable closed container of FIG. 3B ;

FIG. 3D { illustrates the sealing device of FIG. 3C;

FIG. 4A illustrates embodiments of a sealable closed container and a respective sealing device;

FIG 4B presents embodiments of an alternative sealable closed container;

FIG 4C presents an alternative sealable closed container with a cover and an alternative sealing device;

FIG. 5 presents embodiments of a sealing device alternative to the sealing device of FIG. 4A;

FIG. 6 presents other embodiments of a sealing device;

FIG. 7 illustrates embodiments of a sealing device;

FIG. 8A illustrates a sealing device with a releasing member;

FIG. 8B illustrates a releasing handle coupled to the releasing member;

FIG. 8C illustrated a sealing device whose releasing member is broken;

FIG. 9 presents a sealable closed container with an airway;

FIG. 10 presents an alternative configuration of an airway; and FIG. 11 illustrates hanging a first container submerged within a liquid nitrogen container.

DETAILED DESCRIPTION

In the following description some components are "common components", i.e. components that embodiments thereof are illustrated in more than one figure. For example, a first container is a common component whose embodiments are illustrated in FIGs 1A-1B, 3A, 6, etc. Generally, if there is a common component referred to as "X", then embodiments of X illustrated in more than one figure may be similar or different non-limiting embodiments thereof. For example, the embodiments of the first container illustrated, e.g., in FIGs 1A-1B, 3A and 6 are considered to be non-limiting examples of a first container. Furthermore, an embodiment of the first container illustrated in any one of these figures (out of FIGs 1A-1B, 3A, 6, in this example) may be similar to or different from an embodiment of the first container illustrated in any other of these figures (out of FIGs 1A-1B, 3A, 6).

In some cases, an embodiment of a common component may be denoted in different figures by the same reference numeral. In other cases, an embodiment of a common component may be denoted in different figures by the different reference numerals. For example, a sealable container that is denoted 302 in FIGs 3A-3C, is denoted 402 in FIG. 4A. In some cases, a different reference numeral is utilized in order to indicate that one or more structural and/or functional aspects of an embodiment of a common component described with reference to a figure do not necessary apply to embodiments of the common component described with reference to other figures. This is non-limiting though and in other cases different reference numerals are used to denote common components, e.g., due to considerations such as clarity and readability of the description.

In addition, unless specifically noted otherwise, embodiments described or referenced in the present description can be additional and/or alternative to any other embodiment described or referenced therein.

FIG. 1A schematically illustrates a liquid nitrogen cryopreservation system 100, for cryopreserving a biological sample 102, according to embodiments of the invention. The illustrated system comprises a liquid nitrogen container 104. The space delimited inside liquid nitrogen container 104 constitutes a cryopreservation space 105. In the figure the cryopreservation space is depicted as if it is almost completely filled with liquid nitrogen 106, accordingly, a liquid nitrogen level 107 is depicted inside cryopreservation space 105. This is non-limiting and liquid nitrogen container 104 may include more or less liquid nitrogen compared to the illustration, as applicable to the case. If applicable it can even be completely full.

Inside the liquid nitrogen container illustrated is a first container 108, which is configured to hold biological sample 102. First container 108 is submerged in liquid nitrogen 106. It is noted that liquid nitrogen 106 that fills liquid nitrogen container 104 and is external to first container 108 constitutes "external liquid nitrogen", while the external liquid nitrogen occupies an "external volume". In those cases when liquid nitrogen occupies at least part of an internal space 109 of first container 108, this liquid nitrogen (internal to the first container) constitutes "internal liquid nitrogen" while the internal liquid nitrogen occupies an "internal volume".

FIG. IB schematically illustrates a liquid nitrogen cryopreservation system 110, additional or alternative to system 100 of FIG. 1A, for cryopreserving a biological sample 102, according to embodiments of the invention. While in system 100 of FIG. 1A biological sample 102 is stored directly within first container 108, in FIG. IB sample 102 is stored in a storage container 112, while first container 108 may be configured to hold any applicable number, i.e., one or more, of storage containers 112. More precisely, first container 108 of FIG. IB is configured to store in an internal space 109 thereof one or more storage containers 112. Indeed, in the example of FIG. IB, first container 108 is depicted while holding two storage containers 112, while each storage container 112 stores one biological sample 102. It is noted that the illustrated example is non-limiting and, if applicable, a storage container 112 may store more than one biological sample, the number of storage containers 112 stored in first container 108 may be other than two, liquid nitrogen container 104 may be configured to hold one or more first containers 108, etc. A storage container 112 may be of any applicable type and size, for example, a vial (such as V5007 SIGMA®, Nalgene® cryogenic vials and others), a goblet, etc. A storage container may be configured to directly store one or more samples and/or it may be configured to hold one or more additional containers, wherein a sample may be stored. While there are more than one storage containers 112 in first container 108, each one of the more than one storage containers may be of different type. Moreover, if applicable, first container 108 may even be configured to hold one or more storage containers 112 alongside one or more directly stored biological sample, such as sample 102 illustrated in FIG 1A.

Herein, the description refers to liquid nitrogen (LN) as a cryogenic fluid (shortly "cryoliquid") used for cryopreservation. However, the invention is not limited thereto. For example, embodiments of the invention may be used also when the cryoliquid is liquid air, which may be referred to also as "liquefied air", or others.

The term "liquid air" refers to a cryoliquid obtained by liquefaction of atmospheric air and/or of derivatives thereof. It is appreciated that cryoliquid may evaporate. Liquid remnants of an evaporating liquid air constitutes "derivatives" as long as they comprise at least two of nitrogen, oxygen, and argon. Similarly, derivatives are obtainable also by re-liquefaction of vapors, as long as the liquid obtained from the re- liquefaction comprises at least two of nitrogen, oxygen, and argon.

Herein "atmospheric air" refers to air derived from the atmosphere or to a composition of artificially combined gasses that resembles atmospheric air by constituting at least two of nitrogen, oxygen and argon.

Hereinafter, the description relates to systems resembling system 110 of FIG. IB, i.e., wherein the biological samples are stored within storage containers unlike directly within the first container. However, it is noted that should there be a case in which the sample is stored directly within the first container the invention should be applicable thereto as well.

In the background of the invention it is mentioned that Parmegiani and Rienzi, suggested sealing the storage containers and suggested a method for doing so. In Parmegiani and Rienzi' s case the storage containers are always goblets. Embodiments of sealing devices, according to the present invention, usable for sealing a first container are presented herein. At least some of these embodiments may be applicable for sealing storage containers that are goblets, as an alternative solution to Parmegiani and Rienzi. However, the present invention allows sealing also storage containers that are not goblets. Moreover, in certain cases it may be possible to seal both the first container and the storage container with sealing devices according to the invention, thus increasing the safety level for preventing contamination. Hereinafter, the term "a sealing device" is sometimes shortly drafted as "a seal".

Herein, "sealing" refers to the ability to block penetration of molecules comprised in the cryoliquid. Accordingly, when the cryoliquid is liquid nitrogen, "sealing" refers to the ability to block penetration of liquid nitrogen through the sealing device or through a gap between the sealing device and the sealed container. When the cryoliquid is liquid air, "sealing" refers to the ability to block penetration of any of the gas constituents comprising the liquid air.

FIG. 2 schematically describes an all-metal seal 202 for cryogenic applications, as illustrated, e.g., by NASA's technical memorandum (Salerno Louis J. et al. ,) mentioned in the background. Seal 202 is adapted for sealing containers having a round cross section at their opening ("round shaped opening"). Seal 202 has an annular-shaped form, which fits into the round shaped opening from the inside, while the walls 204 around the opening surround the seal.

FIG. 3A schematically illustrates an open sealable container 302 and a cover 304 suitable for usage therewith, according to embodiments of the invention. The sealable container comprises an internal space that constitutes a "sealable space" 303. A sealable container serve as a first container, such as first container 108 in FIG. 1A or IB. It may be a storage container such as any one of storage containers 112 in FIG. IB, or it may be any other sealable container utilizable for cryopreservation. In the figure, an opening 306 of the sealable container is illustrated, while the opening is delimited by the sealable container's walls 308. In this specific case the opening cross section is round, though this is non-limiting and other forms of openings are allowed. Moreover, in the figure the opening is configured for inserting elements into the sealable container and for removing elements from the sealable container. Elements may be, for example, biological sample 102, storage container 112 if the sealable container is a first container, or generally, any other applicable element that can be cryopreserved within the sealable container (including, e.g., other, smaller sealable containers). Hence, opening 306 is a "wide open" passageway. It is noted that other openings, which are not "wide open" may exist. For example, under certain circumstances a slit in the sealable container's walls may form an opening used, e.g., for gas exchange between the interior of the container and its exterior. Other elements cannot pass through this slit, and hence the slit is not considered as being wide open. A sealable container is a container configured for cryopreservation whose openings are sealable.

Any opening that can be sealed in accordance with embodiments of the invention is referred to herein as a "sealable opening". In addition, it is noted that the term "wall" or "walls", such as in "container's walls", is not limited to the edge of the wall around openings. The term "walls" may be used for describing the walls delimiting the container from any of its applicable sides.

Cover 304 (which may be referred to also as a "lid", or a "closer") suits sealable container 302. In this case the cover may close opening 306 by sliding down over a sealable container's neck 310. In the figure this is represented by the little arrows appearing below the cover. The shape and mode of operation of the sliding cover is non-limiting. For example, in other embodiments the cover may be screwable to the sealable container, such as to its neck, or it may connect and close the opening via any other way applicable to the case.

FIG. 3B illustrates sealable container 302 of FIG. 3 A when closed with cover

304, according to embodiments of the invention. In embodiments such as this specific example, the walls of the sealable container and the walls of the cover are coupleable so as to form one level, i.e., the outer shape and dimensions (such as radius in a round cross-sectioned container) of the cover is similar to the outer shape and dimensions (such as radius in a round cross-sectioned container) of the sealable container. However, this is non-limiting. In other embodiments the outer radius of the cover may be smaller or larger compared to the outer radius of the sealable container, the cover's side walls and/or the sealable container's side walls should not necessarily be perpendicular etc. In the figure, the coupling of the sealable container and the cover is illustrated as a slit 312 in the closed container's wall.

A closed container delimits an internal space from all around though it is not necessarily sealed. Cryoliquid molecules, such as nitrogen molecules, and/or contaminants may penetrate through the closure into the sealable space and vice versa: they may leak from the sealable space into the external liquid nitrogen.

In FIG. 3B, slit 312 demonstrates that the sealable container, at the state illustrated in the figure, is closed, though not necessarily sealed. It is noted that a closed, yet un-sealed container may be susceptible to pressure balancing via the closed opening, i.e., via slit 312 in the present example. Therefore, if closed container 314 is submerged in a cryoliquid in a cryopreservation space, such as cryopreservation space 105, the pressure in the closed container would tend to equalize with the pressure around slit 312 inside the cryopreservation space. Moreover, if the closed container is submerged in a cryoliquid whose level (such as liquid nitrogen level 107 in FIG. 1A or IB) is below the slit, the slit would be most probably susceptible to cryoliquid vapors and the internal pressure therein would tend to equalize with the vapors pressure around the slit.

It should be appreciated that a container may be closed with reference to one environment while open with reference to another environment at the same time. For example, when looking at container 108 in FIG. 11 described below, it can be appreciated that depicted container 108 is closed with reference to the cryopreservation space of container 104. At the same time, the container is open with reference to atmospheric air via airway 1102, i.e., the container is open with reference to the environment external to container 104.

When sealable container 302 is coupled to cover 304, they form together a

"sealable closed container" 314. In the figure, a sealable closed container 314 is comprised of sealable container 302 and cover 304. As explained above, a sealable closed container is not necessarily sealed. Moreover, in the case of sealable closed container 314, the closing is not sealed and additional means are required in order to prevent contaminant from penetrating into the sealable space, forming contact with a biological sample that may be cryopreserved therein, or from leaking from the sealable container into the external liquid nitrogen, should a sample stored therein is contaminated, or should the sealable container confine contaminated liquid nitrogen.

Prior to introducing sealing devices and embodiments thereof, it is noted that a closed sealable container is a sealable container. The opposite is not necessarily true and a sealable container must not be a closed sealable container. For example, sealable container 302 is open and therefore it is not a closed sealable container.

FIG 3C schematically illustrates a sealing device 316 sealing sealable closed container 314 of FIG. 3B, according to embodiments of the invention. In the illustrated embodiment sealing device 316 is an O-ring gripping the closed device while being placed around it and while covering slit 312. In order to be able to apply and properly place the sealing device on the sealable closed container, so it completely covers the slit, the sealing device must be either elastic, whereupon placing it tight on the sealable closed container may require an applicator, or it may be loosely placed around the sealable closed container. A combination (elastic and loose) is also allowed. However, in order to seal slit 312, sealing device 316 must grip the container as tight as possible while covering the slit, until the sealing device 316 effectively nearly merges into the sealable closed container' s walls around the slit, leaving a gap that prevents penetration of liquid nitrogen molecules at -196°C, such as 40nm or less, between the sealing device and the sealable closed container's walls.

Similar to a closed container, a sealed container can be sealed with reference to one environment while unsealed with reference to another. Returning to FIG. 11 , it can be appreciated that container 108 may be sealed with reference to the interior of container 104, while it may still be open, or closed yet unsealed, to atmospheric air via airway 1102.

Unlike a closed yet unsealed container, the sealing device prevents pressure balancing. Therefore, the pressure in the internal space of container 108 cannot equalize with the pressure around slit 312 inside the cryopreservation space. On the other hand, airway 1102, which couples the inner space of container 108 to the environment external to container 104, allows the internal pressure in container 108 to equalize with the environmental pressure external to container 104, balancing thereby abrupt and/or massive pressure changes in the internal space of container 108.

According to the invention, seal 316 is configured for sealing a sealable opening and thereby for sealing the sealable closed container. The container, in turn, is configured for being suspended in an ultra-cold temperature. Therefore, should sealing device 316 be made of a second shrinkable material, configured to shrink while exposed to ultra-cold temperature, it can be appreciated that upon submerging sealable closed container 314 with sealing device 316 attached thereto, e.g., in liquid nitrogen (whose temperature is -196°C) or in any other ultra-cold environment, the sealing device, which has a circular structure, would shrink towards the center and thereby it will potentially tighten the grip. Such shrinkable materials are, for example, Polypropylene (PP), silicon, Teflon® (Polytetrafluoroethylene, shortly known as PTFE), nylon (aliphatic polyamides referred to as polyamide (PA)) and others.

However, a shrinkable seal shrinking around the sealable closed container does not necessarily mean sealing the slit. If the sealable closed container is made of a material having contraction characteristics similar as those of the shrinkable seal, or contraction characteristics that allow even better shrinking compared to the seal, while the sealable closed container is submerged in the ultra-cold environment it will shrink as well, and the shrinkable seal might not achieve sealing. It is therefore important that the sealable closed container, e.g., sealable container 302 and cover 304, be made of a first shrinkable material (or materials) having contraction characteristics weaker than those of the seal, so as to allow the seal stronger contraction compared to the sealable closed container.

On the other hand, it should be appreciated that if the seals' contraction is much higher than the contraction of the sealable closed container, the seal may break upon being submerged, when coupled to a sealable closed container, in an ultra-cold environment.

FIG. 3D illustrates sealing device 316 of FIG. 3C, according to embodiments of the invention. In this embodiment the sealing device is an O-ring with a predetermined width 317 and a radius 318. Radius 318 is pre-designed so as to enable placing the O- ring over the sealable opening, such as over slit 312, and yet to allow the sealing device to shrink enough so as to grip the sealable closed container tight enough in order to seal the sealable opening. Width 317 is pre-designed to fully cover the sealable opening, which in this example is slit 312.

Apart of the sealing device's shape, it is also the material it is made of that affects its functionality. The importance of the differential contraction between the sealing device and the sealable closed container was previously explained. Therefore, according to the invention, the material the sealing device is made of (i.e., the second shrinkable material) is selected with reference to the material of the sealable closed container (i.e., the first shrinkable material). The second shrinkable material should be selected so as to allow the sealing device to shrink and contract better and stronger, in comparison to the sealable closed container, thereby allowing the sealing device to seal the sealable opening and preventing the sealing device from breaking.

According to some embodiments, sealing device 316 can be made of a second shrinkable material. The selected material has contraction characteristics as explained above, to allow sealing the sealable opening. Alternatively, it is possible to construct a supporting member of the sealing device from a supporting material, such as metal, wherein the supporting member is configured to mechanically clasp an interfacing member, possibly strengthening the interfacing member against breaks while clasping the sealable opening. The interfacing member is configured to interact with the sealable closed container in order to seal the sealable opening and therefore it should be made of a shrinkable material. In FIG. 3D the two members are illustrated as two rings comprising together the sealing device: the supporting member is denoted 320 and the interfacing member is denoted 322. Yet there may exist other embodiments. For example, instead of having an interfacing member alongside a supporting member, all along the O-ring shaped sealing device, it is possible to design a sealing device comprising a supporting member all around, and an interfacing member which is large enough to cover the sealable opening without circling around the sealable opening.

Further to understanding the examples provided via FIGs. 3A-3D, it can be appreciated that other embodiments of sealing devices in accordance with the invention may exist.

FIG. 4A illustrates embodiments of a sealable closed container and a respective sealing device. There is a certain degree of resemblance between the embodiments of FIG. 4A and those of FIGs. 3A-3D, whereupon a sealable container 402 has an adapted cover 404. However, unlike the sealable container 302 of FIGs. 3A-3D, cover 404 is coupleable to sealable container 402 by screwing 408. In addition, while sealable container 302 and cover 304 form together a sealable closed container whose walls are at the same level, in FIG. 4A the cover and the sealable container both have a protruding ring in their perimeter. While closing the container the two rings couple together in order to assure that the two rings are at the same level. FIG. 4A illustrates a sealable closed container 414 and shows, in 416, how sealing device 406 may couple thereto in order to seal the sealable closed container.

It can be appreciated that screwing and having two rings at a same level are not necessarily bound one to the other. In other embodiments the cover may be screwable to the sealable container without same-level rings, while in yet other embodiments same- level rings may exist without screwing. Further to reading the above, a person versed in the art may appreciate that instead of same level rings the sealable container and/or the cover may have another structure ending with a protrusion such as the examples illustrated in FIG. 4B. Moreover, it is non-obligatory that the sealable container and the cover end at the same level. FIG. 4C presents an alternative sealable container 418 with a cover 420 and an alternative sealing device 422. Sealable container 418 and cover 420 couple to yield a sealable closed container 424 with a shoulder 426, unlike same-level coupling as presented, e.g., in FIGs. 3A-3C and 4A-4B. 422 is a cross-section of a sealable device that is coupleable to sealable closed container 424. It can be seen in the cross-section that the sealing device has a structure 428 engaging shoulder 426 of sealable closed container 424. It is noted that in this case cover 420 may be coupleable to sealable container 418 by any applicable mean such as by sliding, by screwing etc. Further to seeing the examples of FIGs. 3A-3D, and those of FIGs.4A-4C, another example is provided with reference to FIG. 5, which presents embodiments of a sealing device alternative to the sealing device of FIG. 4A, according to the invention. The sealable container in the figure resembles the sealable container 302 of FIGs. 3A- 3D, however, the difference is that in FIGs. 3A-3D there in a stand-alone cover 304, that closes sealable container 302 to yield a sealable closed container 314, and the sealing device is also stand alone, utilized to seal slit 312 of the sealable closed container. In the embodiments of FIG. 5 a covering member 502 is coupled to a sealing member 504 in any applicable way that is ultra-cold resistant. For example, covering member 502 may be molded with the sealing member 504 thus forming together a sealing device 506. Alternatively, it can also be welded thereto thus forming together sealing device 506, etc. The sealable opening in this example is opening 306, similar to sealable container 302 of FIGs. 3A-3D. Sealing device 506 therefore comprises two members, namely a sealing member 504 and a covering member 502.

It has been noted and/or illustrated with reference to the embodiments of the previous figures (e.g., FIGs. 3A-3D and FIGs. 4A-4C) that the sealing device can cover the sealable opening, which is the slit formed by the coupling of the cover and the sealable container. This is correct and applicable to the embodiments of FIG 5 as well: The sealing device covers the sealable opening, although it is the covering member that does so, and not the sealing member.

FIG. 6 presents other embodiments of a sealing device 602 configured to seal a sealable container 604. In this example, a sealable opening 606, which has a nearly rectangular form resembling a piggy bank slot, is positioned in the sealable container's walls, in a place having a spherical shape. Unlike the previously presented embodiments, sealing device 602 is not an O-ring. In the figure, 608 is a side-view of sealing device 602, which illustrates that the sealing device grips the sealable container although it doesn't have a shape that closes to a ring. Yet, sealing device 602 applies all the requirements of a sealing device as previously presented: it grips the sealable containers, it covers the sealable opening, and it is shrinkable so as to tighten its grip, thereby sealing the sealable opening.

It is noted that while a sealing device grips a sealable container tight and seals is to prevent passage of nitrogen and contaminants, the container may be referred to also as a "sealed container". Further to understanding what is a sealing deice and seeing several non-limiting embodiments thereof (e.g. 316, 406, 506, 602 and others), it should be remembered that the containers discussed (e.g. 108, 102, 302, 402, and 604) are used for cryopreserving a biological sample. In order to remove a cryopreserved biological sample from a sealed container, it is required to remove the sample via an opening in the container, such as opening 306 in FIG. 3. Therefore, it is required to remove the sealing device that sealed the sealed container in the liquid nitrogen.

Prima facie, in order to remove the seal, it is required to wait until the seal warms up, reliefs the contraction, releases the grip. However, it is important to note that while removing a biological sample from a sealed container, the speed of action is critical to the vitality of the sample. See, for example, S. Yavin and A. Arav's article, mentioned in the background. Waiting for the sealing device to thaw may take too long and harm the biological sample that is in the container. Therefore, it may be preferable to have a mechanism that allows faster release of the sealing device in order to allow faster removal of the biological sample from the previously sealed container.

FIG. 7 illustrates embodiments of a sealing device 702 whose shape is an O- ring. The sealing device comprises, in its perimeter, a releasing member 704. However, it was previously explained, e.g., with reference to FIG. 6, that the sealing device is not necessarily an O-ring. Accordingly, in sealing devices having other shapes the releasing member should be positioned in any place wherein releasing tension therefrom will release the hold of the sealing device and its grip on the sealed container.

FIG. 8A illustrates a releasing member 802 in an O-ring shaped sealing device 804, according to embodiments of the invention. The releasing member in this case is configured to be broken, thus releasing tension from the sealing device's O-ring. The releasing member has a protruding member 806, configured to be held and to be used as a leverage while breaking the sealing device. Supporting member 808 strengthen protruding member 806, so it does not easily and unintentionally break.

FIG. 8B illustrates a releasing handle 810 coupled to releasing member 802. According to embodiments such as those illustrated in the figure, releasing handle 810 comprises a leveraging handle 820 and a coupling body 822. Coupling body 822 comprises two slots: one slot is referred to as a "holding slot" 816 and the other slot is referred to as "supporting slot" 818. Holding slot 816 is configured to engage protruding member 806, while supporting slot 818 is configured for engaging supporting member 808.

In the example of FIG. 8B sealing device 804 seals a sealable container 812. It is noted that only part of sealable container 812 appears in the figure. In order to break the releasing member of the sealing device with the releasing handle used as a lever, the sealing device must be stably positioned. It can be appreciated that when the sealable container, to whom the sealing device is coupled, is submerged in liquid nitrogen or immediately after the removal thereof from the liquid nitrogen, the container may take part is stabilizing the sealing device, thus assisting in breaking the releasing member. The term "immediately" means before the shrinkable material of the sealing device thaws in degree that allows the sealing device to loose grip on the container.

Arrow 824 in the figure represents the forces that should be applied to releasing handle 810, while engaged with releasing member 802, in order to break the releasing member of sealing device 804, thus releasing the seal.

FIG. 8C illustrates a sealing device 804 whose releasing member 802 is broken.

It can be appreciated that the broken sealing device, i.e., the broken O-ring, can no longer tightly grip the sealed container and therefore it (i.e. sealing device 804 ) can be removed from the container, cover 304 can be removed as well, thus exposing the opening and allowing removal of a biological sample stored in the container. Breaking the sealable device is very fast hence the opening is exposed much earlier than it could have been by removing the sealable device after the sealable device thaws and returns to its basic form, before submerging it in the liquid nitrogen.

Further to understanding the embodiments for sealing a sealable container and for releasing seals therefrom, it should be appreciated that further to submerging a sealed container in an ultra-cold environment, the pressure inside the sealed container, constituting "internal pressure", changes.

A person versed in the art would appreciate that upon submerging a sealed container in an ultra-cold environment the internal pressure therein is expected to drop down, thus creating vacuum inside. Such vacuum may turn it difficult to open a sealing device sealing the container, such as any one of the sealing devices presented above. Moreover, if the sealing device is broken and released while the internal pressure is negative, the pressure inside the container will abruptly raise, and the sealing device, parts thereof, or even storage containers (such as containers 112) or other objects in the liquid nitrogen may hit and wound people or damage property around.

In addition, a biological sample, such as sample 102, is normally cryopreserved in order to further process it in the future, e.g., to perform additional processing stages thereon, to implant it in a biological body such as a human body, an animal body or a plant, etc. Hence, upon thawing a cryopreserved biological sample, it should be kept vital. It was mentioned before that upon thawing a biological sample it should be warmed fast to 37°C, and therefore waiting until the sealed container thaws for pressure equalization so as to make it possible to open the sealing device may harm the sample. The releasing member may provide a solution to the problem, where it is possible to snap the releasing member thus breaking the sealing device and allowing immediate pressure equalization. However, other embodiments may suggest other solutions that prevent the pressure from dropping.

FIG. 9 presents a sealable closed container 902 with an airway 904. A cover 906 is coupled to airway 904, wherein the two (cover and airway) are coupled by molding, welding, or any other way that assures that the coupling is sealed. The airway is coupled to the cover while one end of the airway, constituting a "first end" is below the cover, so that when the cover is closing a container the first end is internal to the sealable closed container. An "external part" 908 of the airway is protruding above cover 906. External part 908 has a "second end" 910 with an opening 912. When the cover closes the container the second end is configured to be external to the sealable closed container.

This is non-limiting and other embodiments of airways may exist. For example, FIG. 10 presents alternative embodiments of an airway 1002. Airway 1002 is coupled to a wall 1004 of a container 1006, instead of cover 1008 thereof as is the case in FIG. 9. In FIG. 10, wall 1004 appears as if opened so as to reveal a first end 1010 of airway 1002 that cannot be seen through the walls. Accordingly, the figure illustrates that airway 1002 (like airway 904 of FIG. 9, although the first end cannot be seen there) comprises first end 1010 and a second end 1012. At both ends there are openings, namely opening 1014 at first end 1010 and opening 1016 at second end 1012. Therefore, it should be understood that airway 1010 (and so is airway 904 of FIG. 9) is configured to couple the internal space of the sealable container with an outer environment. Further to describing FIGs 1A and IB it was mentioned that embodiments of a sealing device (e.g., the embodiments presented with reference to FIGs. 3A-3C, in FIG 4, FIG. 5 or FIG 6) can be usable for sealing first containers and/or storage containers. Similarly, airways, such as the airways 904 of FIG. 9 and 1002 of FIG. 10, can be applicable to sealable containers such as first containers and/or storage containers. However, when applied to storage containers that are configured to be stored within a first container, which, in turn, may be submerged in liquid nitrogen, if the first container is sealed as well, it can be appreciated that in order to couple the internal space of the storage container with an outer environment, an airway of the storage container should be coupled to an airway of the first container. Thus, coupling the internal space of a sealable container with an outer environment may be direct, e.g., via an airway, or indirect, e.g., via an airway coupled to another airway.

Remembering that first containers and storage containers are configured to be submerged in an ultra-cold environment, such as liquid nitrogen, and that under such ultra-cold temperatures the pressure inside a sealed container tends to drop, it can be appreciated that by coupling the internal space of such a submerged sealed container with an environment external thereto, whose pressure is higher, the pressure in the internal space would tend to equalize with the external environment.

Accordingly, if the internal space of a sealed container (such as a first container or a storage container) submerged in, e.g., liquid nitrogen is coupled via an airway to, e.g., an environment external to the liquid nitrogen container such as the room's environment, the pressure inside the sealed container would equalize and reach approximately a value similar to the room pressure. Should the airway be very narrow, it is possible that equalization would not be instantaneous though after a while it will reach a value substantially equal to the room pressure, so as to avoid any difficulty arising by the low pressure that could have been in the sealed container without the airway.

Until equilibrium is reached, gas from the environment would flow into the sealed container via the airway. If the airway's second end is open to environmental air, it will be air flowing inside via the airway. If the airway's second end is open to an environment rich with nitrogen, it will be mainly (or solely) nitrogen flowing inside, etc. Whatever the flowing gas is, it should be appreciated that this gas may be contaminated as well. Hence, in cases when such contamination may risk the biological sample in the sealed container, it is possible to couple an applicable filter to the second end, preventing such contamination thereby.

The inward flowing gas would gradually raise the internal pressure in the sealed container, and gradually decrease the pressure difference between the internal pressure in the sealed container and the environment. As the pressure differences decreases the inward flow of gas decreases as well and finally stops. The internal pressure in which inward flow of gas stops is considered as "substantially equal" to the environmental pressure.

It has been shown (e.g., in WO2014/178058) that while the sealed container is submerged in liquid nitrogen, room air (or other gas residing in the environment around the airway's second opening) might flow via the airway into the internal space. Considering that the environmental air might carry a certain degree of humidity therewith, and that at least part of the external airway 908 is submerged in the liquid nitrogen, it can be appreciated that this humidity would freeze in the airway, forming a partial or a complete block thereof. A blocked airway cannot allow pressure equalization (if the block is partial the equalization might be deteriorated). Therefore, it may be beneficial, at least in some embodiments, to prevent humidity from penetrating the airway. This can be achieved, e.g., by coupling a humidity blocking member, such as a humidity filter, to the second end and second opening of the airway, such as to absorb the humidity.

Hence, a contaminants' filter and a humidity filter can prevent contaminants and humidity, respectively, from entering into the airway and to the interior space. Understanding that the inward flow stops when the internal pressure is substantially equal to the environmental pressure, it may be realized that when the flow stops the filters are not required anymore and hence, in some embodiments, the filters can be disconnected from the second end of the airway, possibly, but not necessarily, coupling a plug or a cap, etc. instead.

Returning now to FIGs. 1A and IB, the first container 108 appears therein as if "floating" in the liquid nitrogen. Those versed in the art may appreciate that normally this is not the case and the first container is held in the liquid nitrogen container by a holding element that is, e.g., hanged on the container's edge or on any applicable hanging device. FIG. 11 illustrates hanging a first container 108 submerged within a liquid nitrogen container 104. In the illustrated embodiments an airway 1102 is used as a hanging handle.

It is noted that airway 1102 and first container 108, as illustrated in FIG 11, remind the sealed container and airway of FIG. 9. However, this is non-limiting and other first containers and airways may be used, if applicable.

In the figure, the airway has a bend 1104 that prevents first container 108 from sinking deeper inside liquid air container 104. In addition, a stabilizing member 1106 stabilizes the first container in the liquid nitrogen, preventing it from moving to the sides and possibly even preventing it from floating up inside the liquid nitrogen container. The stabilizing member is non-limiting though and other mechanisms may stabilize the airway and container, while in yet other embodiments the stabilizing member is not required at all.

Further to understanding how it is possible to stabilize a container in liquid nitrogen inside a liquid nitrogen container, and protect the container's internal space from contamination by contaminants coming from the liquid nitrogen, from contaminated samples stored in the same liquid nitrogen container, or from the environmental contaminants, removal of sealed storage containers (and samples) from the liquid nitrogen shall be discussed.

When a sealed storage container is removed from inside a liquid nitrogen container and transferred to a warmer environment, the internal space inside the sealed container would rise due to warm-up. If the sealing device is removed at this stage, the high internal pressure may be abruptly released, the seal and/or the cover and/or elements stored inside the container may erupt, risking both people and property. It can be appreciated that the airway may balance this abrupt pressure increase, by allowing air flow out as necessary, thereby releasing elevated pressure. Accordingly, it may be appreciated that the airway is also a safety mechanism. It is noted that herein "balancing pressure" means decreasing pressure differences though not necessarily achieving equalization of pressures.

It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed systems, methods, and devices. Other embodiments will be apparent to those skilled in the art from consideration of the specification and practice of the disclosed embodiments.

Claims

CLAIMS:

1. A device for cryopreserving a biological sample by liquid nitrogen, the device comprising:

2. The device of claim 1 wherein the first container is sealable with reference to the a cryopreservation space used for storing the liquid nitrogen so as to prevent contaminants carried by the liquid nitrogen from penetrating into the internal space.

3. The device of claim 1 or 2, wherein the airway is coupleable to a humidity blocking member the second end, the humidity blocking member is configured to maintain humidity inside the airway below a threshold so as to prevent blocking the airway by frozen humidity.

4. The device of any one of claims 1-3, wherein the first container comprises a cover;

5. The device of any one of claims 1-4, further comprising:

6. The device of claim 5, wherein the holding element is configured to hold the first container submerged in the liquid nitrogen.

7. The device of claim 6, wherein the holding element is configured to hold the first container fully submerged in the liquid nitrogen.

8. The device of claim 5, wherein the holding element is configured to hold the first container in nitrogen vapors.

9. The device of any one of claims 4-8, wherein the holding element is coupled to the first container.

10. The device of claim 9, wherein the holding element is coupled to the cover.

11. The device of any one of claims 4-8, wherein the holding element is coupled to the airway.

12. The device of any one of claims 4-8, wherein the holding element and the airway are the same.

13. The device of any one of the previous claims, wherein the first container comprises metal elements, characterized by high thermal conductivity.

14. A device for storing a biological sample in a liquid nitrogen container, the device comprising:

a first container comprising an internal space, the first container is configured to seal the internal space from nitrogen while submerged in the liquid nitrogen container; and an airway configured to couple the internal space to an environment external to the liquid nitrogen container;

15. The device of claim 14, wherein the device is configured to allow opening the first container.

16. The device of claim 15, wherein the device is configured to allow re-sealing the first container.

17. A method for storing a biological sample in liquid nitrogen, the device comprising:

Inserting the biological sample into a first container; Verifying that the first container has an open airway coupling the first container and the second end of the airway is susceptible to dry air characterized by a humidity level which is below a threshold.

18. A system for sealing a biological sample in a container while cryopreserving the biological sample by a cryoliquid, the system comprising: a sealable container configured to hold the biological sample in an internal space of the container, the sealable container comprises an opening configured to allow inserting and removing elements to and from the container, respectively; a cover configured to close the opening, wherein the sealable container and the cover are configured together to form a closed sealable container leaving sealable openings; and a sealing device configured to seal a sealable opening which is a gap between the cover and the sealable container in the closed sealable container so as to prevent penetration of molecules comprised in the cryoliquid via the sealable openings; wherein the system is configured to grip the sealable opening from the outside while contracting around the sealable closed container.

19. The system of claim 18 wherein gripping the sealable opening from the outside leaves a gap of 40nm between the sealing device and walls of the sealable closed container.

20. The system of claim 18 wherein gripping the sealable opening from the outside leaves a gap of less than 40nm between the sealing device and walls of the sealable closed container.

21. The system of any one of claims 17-20 wherein the sealable container and the cover are made of a material having weaker contraction characteristics compared to contraction characteristics of sealing device.

22. A system for sealing a biological sample in a container while cryopreserving the biological sample by a cryoliquid, the system comprising: a sealable container configured to hold the biological sample in an internal space of the container, the sealable container comprises walls made of a first shrinkable material configured to shrink while exposed to ultra-cold temperature; and a sealing device made of a second shrinkable material that is characterized by stronger contraction while exposed to the ultra-cold temperature compared to the first shrinkable materials, thereby the sealing device is configured to shrink and grip the sealable container thereby sealing openings in the sealable container.

23. The system of claim 22 wherein the second shrinkable material is Polypropylene (PP).

24. The system of claim 22 wherein the second shrinkable material is silicon.

25. The system of claim 22 wherein the second shrinkable material is Polytetrafluoroethylene (PTFE).

26. The system of claim 22 wherein the second shrinkable material is polyamide (PA).

27. The system of any one of claims 22-26 wherein the sealable container is a closed sealable container.

28. The system of any one of claims 22-27, further comprising: an airway configured to couple the internal space to an external environment, for balancing abrupt pressure changes in the internal space.