This invention relates to a method and an apparatus for freezing of a biological material.
The following references are considered to be pertinent for the purpose of understanding the background of the present invention:
1. WO06/016372;
2. U.S. Pat. No. 5,873,254;
3. WO 03/056919;
4. WO 03/020874;
5. U.S. Pat. No. 6,337,205 (Wisniewski);
6. U.S. Pat. No. 5,863,715 (Rajotte).
In freezing of biological material, two freezing stages are recognized: nucleation and crystallization. In the first stage ice nucleation occurs in the solution outside the cells. In order to minimize cellular damage, it is critical to control during this stage (nucleation) both the interface velocity of the cold front and the direction of thermal gradient within the object. Normally, in some biological materials (e.g. blood, cell suspensions, plasma, semen and other liquid samples) the best survival is obtained when the freezing rate at this stage is relatively rapid (10° C./min or more). In other cases (e.g. organs or organ fragments), it is accepted that a slow freezing rate at this stage (0.5° C./min or less) would improve freezing.
The next stage is that of crystallization, an exothermic process that produces latent heat within the frozen material, causing a period of time when the biological material remains isothermal, or even experiences an increase in temperature: latent heat exudes from the biological material and thus, although the material is being cooled no temperature change is observed or the temperature may even rise. This, in turn, causes spontaneous freezing and thawing cycles which are hazardous to the biological material.
Permitting osmosis of water out of the cells at this stage would reduce damage to the cells, and the increase of intracellular concentration would cause the cells to vitrify rather than freeze. This is affected by the rate of freezing, and thus, in order to optimize the biological material's survival of this stage control of the rate of freezing is important. The optimal rate depends on the type and composition of the biological material being frozen.
In addition to the above, cryopreservation of material having a large volume (e.g. tissues, organs or portions thereof) is associated with heat transfer and mass problems that are not associated to the same extent with cryopreservation of isolated cells. For example, in conventional freezing methods, ice grows at an uncontrolled velocity and morphology and may disrupt and kill cells by mechanical destruction of the tissue architecture. Due to the large size of macroscopic material, large uncontrolled thermal gradients may develop from the surface of the sample to its interior.
One method that was devised to allow freezing biological material is disclosed in WO2006/016372. In this publication, the temperature of a biological material to be frozen is changed from a first temperature to a second temperature within a time period, one of the said first or second temperature being above freezing temperature and the other being below freezing temperature. Freezing is achieved by placing the biological material in tight contact with at least one, preferably between two heat exchangers, and controlling the temperature in at least one of said heat exchangers such that a freezing temperature front propagates in said material away from at least one of the two heat exchangers.
A method of freezing biological material of a large volume is disclosed in U.S. Pat. No. 5,863,715. In this patent, the biological material is placed in a flexible container, such as a bag. The bag is then flattened in a holder that maintains an essentially constant cross-sectional area of the bag in order to minimize thermogradients. The holder is then cooled along with the bag contained therein.
It is well established that directional freezing, a process in which a cold front propagates in a controlled manner through the frozen object, improves the chances of biological material to survive freezing and thawing. In this process a temperature pattern (or gradient) is established in the object being frozen to form a propagation cold front within the object, resulting in improved chances of survival.
A successful method of directional freezing is disclosed in U.S. Pat. No. 5,873,254. In this patent, a freezing apparatus is used to establish a laterally varying thermal gradient and the biological sample is moved along the thermal gradient at a controlled velocity. Additional methods were developed in order to improve the freezing of large volume objects. For example, WO 03/056919 discloses freezing biological samples via an isothermal stage, wherein the temperature is changed until temperature of the sample in an outer zone equals intermediate temperature and changing temperature until the majority of the sample is in a final temperature. This method may be used in conjunction with directional freezing but is not limited thereto. Another process is disclosed in WO 03/020874 in which the biological sample is agitated during its migration along a thermal gradient.
A method for cryopreservation of a biological is disclosed in U.S. Pat. No. 6,337,205. The sample to be frozen is inserted into special oblong vials that have special appendages, termed “ice crystal-nucleating structures”, situated at the opposite ends of the vial's oblong cross-section. The vials are placed within a compartment of a cryopreservation apparatus, said compartment containing a cryopreservation fluid. A freezing front is then generated at one of the walls of the apparatus that is adjacent to one of the appendages, and propagates through the cryopreservation fluid. Due to the special shape of the appendage, nucleation begins at the appendage, and thus the cold front propagates within the sample in a direction that is away from the cooling wall and along the oblong cross section of the vial. In an alternative disclosed in U.S. Pat. No. 6,337,205, two cold fronts may be generated in the compartment, in opposing directions, by opposing walls of the apparatus. In this method, the freezing of the sample is achieved indirectly, in the sense that the cooling wall of the apparatus cools the cryopreservation fluid, which in turn cools the vial (and the sample within it).
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:
In accordance with one aspect of the present invention there is provided a device for freezing biological material, comprising:
The slot within the device is adapted for receiving therein a container having a first container portion for accommodating said biological material and a second container portion free of said material, such that when the container is mounted within said slot, the first container portion is disposed in the first slot portion and a portion of the second container portion is within the second slot portion.
The term “free” when used in connection with biological material, means that essentially all the biological material needed to be viable after thawing is located away from the second container portion. “Essentially all” means that at least 80%, or at least 90% or even at least 95% and at times even all the biological material to be frozen is located away from the second container portion. In case the biological material contains many small members (small particulate matter such as cells, clusters of cells, microorganisms, tissue fragments for example bone chips, etc.) some of the small members may be found in the second container portion. Small members are members measuring no more than 5mm or even no more than 2 mm in any direction. In case the biological material comprises larger (e.g. measuring at least 2 mm, or even at least 5 mm in at least one direction) solid or semi-solid body of biological material (namely, non-fluid material, for example one or several slices of tissue or an osteochondral cylinder)) the term “free” may be taken to mean that the body of the biological material is absent (located away) from the second container portion, while free cells or other biological debris material may still be present in the second container portion.
The device may comprise one of more of the following features:
In accordance with another aspect of the present invention, there is provided a container for accommodating therein of a biological material, said container having a longitudinal container axis and comprising a first container portion having a first dimension and a second container portion having a second dimension smaller than the first dimension, both measured perpendicularly to said container axis; the container portions being in fluid communication; said container being adapted for carrying said biological material in said first container portion, and a solution different from said material, fully filling said second container portion, which is free of said biological material, and at least partially filling said first container portion so as to be in contact with said biological material; said container further comprising retention means for preventing passage of said biological material from said first container portion to said second container portion.
The retention means may be attached to a stopper adapted to be received within an opening of said first container portion. The retention means may also be in a form of a mesh located within said first container portion or in a from of a membrane. The retention means may also be part of the construction of the container, such that the first container portion may have an obstruction or narrowing in it that would prevent a solid biological material from passing therethrough.
The biological material carried by the container may comprise cells, tissues or organs (portion or whole organs). Examples of cells may include, without being limited thereto, blood cells (whole blood, blood fraction, blood constituents, etc.), semen, oocytes (ova), germ cells, zygotes, bone chips and embryos. Examples of tissues include, without being limited thereto, cartilage-containing tissue, osteochondral cartilage (such as osteochondral plugs or cylinders) excised cartilage-containing tissue and viable tissue. Examples of organs include, without being limited thereto, whole organs (optionally, including vasculature, i.e. blood vessels responsible for local supply of blood to the removed organ), e.g. whole gonadal organ; as well as fragments of organs.
According to another aspect of the present invention, there is provided a kit for freezing biological material, comprising:
said container further comprising retention means for preventing passage of said biological material from said first container portion to said second container portion.
The kit may further comprise a protective pouch for placing said container therein.
According to yet another aspect of the present invention, there is provided method for freezing biological material comprising:
Without being bound by theory, as such knowledge may not be necessary to practice the invention, it appears that ice-seeding (ice-crystal formation) inside the sample container propagates from the second section of the sample container (being near the heat sink) towards the first section of the sample container and thus, inherently, towards the biological material located (at least mainly) in the upper part of the sample container, until the biological material is frozen.
The rate of propagation may be controlled by changing (during operation or a priori) one or more of the device's and/or the container's parameters such as the temperature differential between the cooling unit and the biological material, the distance between the front section (i.e. the tip) of the sample container and the biological material, the dimension and geometrical shape of the upper and lower parts of the sample container (and/or the corresponding dimension and geometrical shape of portions of the device that are adapted for holding the sample container or a portion thereof), the shape of the front section of the sample container etc.
Without being bound by theory, a greater the distance between the level of the cooling fluid or cooling block (in which the tip of the container is embedded) and the biological material will result in a lower cooling rate of the biological material; a greater cross sectional surface area at least at the second section (i.e. tip) of the sample container, will result in a greater cooling rate of the biological material. One or a combination of two or more of the above parameters may be taken into consideration when considering freezing of a biological sample and those versed in the art will know how to select the desired parameters based, for example, on the specific biological material to be frozen (e.g. known preferred cooling rates) and after conducting simple experimentations (e.g. measuring the temperature change during freezing and/or testing post thaw viability).
The construction of the device disclosed herein permits a temperature change front that propagates from the front section of the sample container towards the biological material carried within the upper section of the sample container. Thus, temperature change is one direction throughout the entire biological material, i.e. unidirectional temperature change.
The device and process in accordance with the invention are suitable for example for providing viable biological material following freezing with the device. The term “viable biological material” in the context of this disclosure means biological material comprising cells that are capable of surviving and maintaining their original function, provided that they are given the necessary conditions (e.g. nutrients, temperature and the like) after being thawed.
Reference is made to
In the example of
The chamber 14 is made of a thermally insulating material, for example of the type of a vacuum flask (“thermos”). The flask may be made of glass, metal or plastic with double, spaced apart walls, the region between the inner and outer walls being evacuated of air or filled with appropriate, heat-insulating plastic substance, as will be appreciated by those versed in the art.
The heat insulating unit 16 may either be fixed in place with respect to chamber 14 (e.g. by hooks or flaps that allow the heat insulating unit to hang on top of the cooling fluid medium M contained within chamber 14) or is made of material that has a specific gravity lower than that of the cooling medium M, thereby resulting in the floatation of the insulating unit 16 on top of the said cooling fluid medium M. The heat insulating unit 16 may comprise any type of insulation known per se, such as, without being limited thereto Styrofoam, cellulose wool, ceramic foams, polyethylene, vacuum.
The cooling medium may be any fluid including liquid or gas. For the purpose of freezing a sample, the medium, being a cooling medium may comprise, without being limited thereto, liquid nitrogen (LN), liquid helium or other fluid, the temperature of which is or may be controlled to be below the freezing temperature of the material to be frozen by the device.
According to some embodiments of the invention, the cooling medium M is a liquid, and the insulating unit 16 is constructed such that it would float on the cooling medium. This allows for continued contact between a tip 72 of a sample container 70 (as will be further explained) and the cooling medium, even if a portion of the cooling medium evaporates. As the level of the liquid reduces, the insulating unit 16 would move lower, such that the vertical location of the insulating unit may be used as an indicator for the need to add cooling medium M to chamber 14. The insulating unit 16 may further comprise one or more longitudinal channels and/or tubes that would allow adding cooling medium M to chamber 14 through the insulating unit and/or around it, without removing it from the chamber.
In alternative embodiments of the invention, the cooling medium M is a gas (e.g. LN vapor above liquid LN or the gaseous atmosphere above dry ice). In such cases the insulating unit 16 is fixed in place with respect to chamber 14.
The chamber 14 is typically insulated from the environment, so as to minimize temperature fluctuations (e.g. heat loss) and improve heat transfer to or from the medium carrying the biological material. The insulating material of the chamber 14 may be the same or different from that constituting the heat insulating unit 16 and may comprise glass wool, cellulose wool, ceramic foams, polyethylene, vacuum, and generally in any type of insulation known per se. The insulating material depends on the cooling medium M within the chamber. For example, if the cooling medium M is LN, a simple vacuum chamber is suitable for containing thereof. However, other insulation materials may be used, especially if circulation of the cooling medium M is required. At times, the chamber may also include a cover or be otherwise sealed, to prevent evaporation and/or contamination of the cooling medium.
The device 10 further comprises a sample slot 20 having a central axis A. While not illustrated, it is noted that the device 10 may comprise more than one sample slot 20 each having a central axis A. The sample slot 20 comprises two portions 20A and 20B. The lower portion 20A of the slot 20 has a height H1 and diameter D1, and the upper portion 20B of slot 20 has a height H2 and diameter D2, where D1<D2.
As shown, an interface I, defined between the upper portion 20B and lower portion 20A of slot 20, is located within the heat insulating unit 16 at a distance H1 from the lower surface 16A of the insulating unit 16. The slot 20 has, at its top, a first opening 25 for allowing insertion of an elongated sample container into slot 20. Further, at its bottom, slot 20 defines a second opening 27, allowing a portion of the sample carrier to be immersed in the cooling medium M within container 14 and thus the direct contact between the sample carrier 70 and the cooling medium M. Alternatively, while not illustrated, insulating unit 16 may comprise several portions that may be opened (e.g. along a hinge) to allow insertion of one or more containers to respective slots, after which the insulating unit 16 is closed and at times fastened before introduction into cooling chamber 12.
In the example of
The device 50 is different from the device 10 of
When using a solid cooling unit 18, the cooling unit may be constructed from a single unit or a plurality of sub-units together encasing the sample container. It may be also constructed from cooling units or from fluid cooling conduits. When using conduits, the conduits may be in fluid communication with a cooling fluid reservoir, typically through flow control valving means. By controlling the rate of flow and/or the temperature of the fluid that enters the conduits, the temperature of the cooling unit may be controlled. The cooling fluid transferred through the conduit will be typically liquid nitrogen.
In some embodiments, the lowest portion 20A′ of slot 20 that is encased by the cooling unit 18, as shown in
The device disclosed herein may comprise one or more temperature sensors for sensing the temperature of one or more of the cooling unit, such as cooling units 12, 18, the cooling medium M, the solution surrounding or in close proximity with the biological material, or that of the biological material per se.
The device may further comprise a control utility for controlling, inter alia, the operational steps of the device. The control utility may comprise a dedicated computer or external desktop or laptop computer or PLC (Programmable Logic Controller). It may also comprise a user interface allowing a user to control or override the pre-set operational steps of the device. In addition, in some cases, it may be desirable to provide additional information to the control utility (information such as the temperature change front propagation feedback). This information may be used for example as feedback for control of operation and also for quality assurance of the resultant temperature change of the biological material.
Furthermore, the data so collected may be stored in any form, such as digital data or printed documentation, for any use, including clinical use (e.g. for process control and verification such as to confirm that the process took place correctly, thereby indicating viability of frozen biological material for medical applications), as well as for research and development.
In addition to temperature sensors the device may include additional sensors such as:
Ultrasound may be used to follow the freezing temperature front propagation inside the biological material. In such case an ultrasound transmitter may be used, for example within the sample container, and the propagation of the temperature front may be monitored by ultrasound readings as known in the art.
Reference is now made to
The term “sample” as disclosed herein typically comprises biological material within a physiologically acceptable solution S. In the following description, when referring to “biological material” it includes also the physiological solution in which it is carried, unless otherwise stated.
The sample container 70 is constructed to provide adequate conditions for holding therein the biological material. It is thus made of material that withstands sterilization (using steam, gamma irradiation or other conventional methods) and during use can remain sealed (aseptic/sterile conditions can be maintained). The sample container is made of material that is compatible with the biological material and has compatibility with the cooling medium. This material may be rigid (e.g. polymers used for manufacturing test tubes) or flexible (e.g. polymers used for preparing blood bags), and should be capable of withstanding the freezing temperatures as needed for use (e.g. exposure to liquid nitrogen). Various polymers can be used for forming the sample container 70, as known in the art, and include, without being limited thereto, polytetrafluoroethylene, polystyrene, polyethylene, polypropylene or glass. A flexible container may, for example, be made of one or more layers of materials such as nylon, polyethylene, Teflon, Compton, or of materials suitable for creating a peelable and/or vacuum sealed cover, in which case a portion of the container may be sealable (e.g. welded) after filling.
The sample container 70 is further constructed so as to fit within slot 20 of the device. In the examples of
As illustrated in
Referring to
Referring to
The sample of biological material may be of any size which fits and may be encased within container 70. Thus, when referring to large sample it is meant any sample of biological material having a minimal dimension of a cross-section that is perpendicular to the longitudinal axis of the upper portion 70B of the elongated sample container 70, exceeds 0.5 cm. This minimal dimension may also exceed 1.6 cm and even 2.5 cm. The sample of biological material may be of large volume, meaning, any volume exceeding 5 mL. The volume may also exceed 12 mL and even 50 mL or more.
The biological material is maintained within a physiologically acceptable solution S contained within the internal space of the sample container 70 (filling the lower portion 70A and at least partially filling the upper portion 70B. The physiologically acceptable solution S is selected such to be compatible with the biological material and to improve the post thaw viability and/or functionality of the biological material, as determined by parameters known to those versed in the art. Examples for components included in physiologically acceptable solution S comprise cryoprotectant agents as known in the art, including, without being limited thereto, one or more of DMSO, glycerol, ethylene glycol, poly ethylene glycol, propylene glycol; sugars, such as, without being limited thereto, sucrose, dextrose, trehalose; and proteins, such as, without being limited thereto, serum proteins, albumin, fetal calf serum, of human or other source, etc; carbohydrates such as, without being limited thereto, hydroxy ethyl starch (HES), dextran, and polyphenols, such as, without being limited thereto, epigallocatechin gallate (EGCG).
As shown in
Alternatively or additionally, as shown in
It is noted that slot 20 in insulation unit 16 should be modified to accommodate the shape and dimension of the container or a cover or any suitable adaptor added to prevent the container's displacement in the cooling chamber.
A variety of different geometries for the sample container may be applicable in accordance with the invention. These may include different cross sectional shapes of the container, being, without being limited thereto, circular, oval, elliptic, polygonal, irregular etc. Further, since ice-seeding occurs at the front section of the sample container, various geometries are applicable for the lower part of the sample container, and in particular for the front section, which may facilitate the seeding process.
Reference is now made to
The pouch 30 may comprise two layers, so that an outer layer insures the sterility of an inner layer (not shown).
Referring to
The pouch 30 may be made of materials such as nylon, polyethylene, Teflon, Compton, or other materials suitable for creating a peelable and/or vacuum sealed cover.
Reference is now made to
The sample container 70 is inserted within the slot 20, the dimensions of which correspond to the dimensions of the container 70, through the first opening 25. As shown, the sample container 70 is fitted within slot 20 such that at least a part the lower portion 70A of the sample container 70 is in thermal contact with the cooling medium M (
With respect to
In the device 10 (
The upper opening 74 of the sample container 70 ends at the upper surface 16B of the insulating unit 16, so as to ensure complete thermal isolation of the biological material within the sample container 70. In these particular embodiments, the biological material 84 is shown to be suspended over, albeit, distant from the spacer element 86.
It is essential that the temperature of the cooling unit 12, or 18 is below the freezing temperature of the biological material to be frozen. Once the sample container 70 is inserted within the respective device 10, 50, the temperature of the physiologically acceptable solution carrying the biological sample is gradually reduced. More specifically, a temperature gradient is formed within the solution. Consequently, a portion of the solution proximal to the cooling unit 12, or 18 is cooled first, and eventually freezes. The interface between a frozen portion and non-frozen portions of the solution creates a “freezing front”, which then gradually propagates in the solution as other portions of the solution gradually freeze.
The location of the biological material within the upper portion 70B is preferably adjustable so as to permit better control of its cooling. Once a location of a desired cooling rate along the sample container 70 is determined, the location of the biological sample is adjusted accordingly, i.e. within the range of the desired cooling rate. Adjustability may be achieved, inter alia, by displacing the spacer element 86 within the sample container 70, buy selecting a supporting member of a predefined length, and/or by selecting or constructing the device and sample container 70 with the appropriate proportions etc.
The cooling of the biological material is achieved by heat transferred through the solution M, from the portion of the container 70 that is remote from the cooling unit 12, or 18 (in which the biological material is accommodated, as described above) to the portion of the container 70 that is in direct contact therewith.
The temperature of the cooling unit may be essentially constant throughout the temperature change operational steps. Alternatively, the device may be constructed such that the temperature of the cooling unit is changed during the temperature change process (i.e. until the biological material is frozen). When using a fluid cooling substance for the cooling unit the temperature change may be achieved by changing the temperature of the fluid within chamber 14 in a controlled manner, using dedicated refrigerators and/or heaters.
As appreciated, the device of the invention thus provides means for changing a temperature of a biological sample, by convection, i.e. the bulk movement of thermal energy in fluids; as well as by conduction.
Once the temperature of the biological material reaches a desired temperature the sample container 70 may be removed from the slot typically for storage. This may, for example, be determined by the use of a dictated sensor or after a predetermined minimal period of time elapsed.
It is appreciated that the above discussion regarding operation of devices according to specific, non-limiting embodiments applies, mutatis mutandis, also to the method of the invention. Those skilled in the art will readily appreciate that various modifications and changes can be applied to the embodiments as hereinbefore exemplified, without departing from its scope defined in and by the appended claims.