WO2011098996A2

WO2011098996A2 - Improved method for changing the temperature of a biological specimen

Info

Publication number: WO2011098996A2
Application number: PCT/IL2011/000113
Authority: WO
Inventors: Ginadi Shaham; Victor Rzepakovsky
Original assignee: Core Dynamics Ltd.
Priority date: 2010-02-09
Filing date: 2011-02-01
Publication date: 2011-08-18
Also published as: WO2011098996A3

Abstract

A method for changing the temperature of one or more biological specimens from an initial temperature to a final temperature, comprising: providing at least two temperature control plates defining a space therebetween, a first temperature control plate having a first inner surface and the second temperature control plate having a second inner surface; providing one or more heat conducting structures, each having a first outer surface and a second outer surface spaced from said first outer surface by a body of the heat conducting structure, said body having at least one channel extending along at least a portion thereof; placing said biological specimens received within a container within at least one of said channels; placing said heat conducting structures within said space such that said first outer surface is in contact with said first inner surface of the first temperature control plate and second outer surface is in contact with said second inner surface of the second temperature control plate; and changing a temperature of at least one temperature control plate from an initial temperature to a final temperature changing thereby a temperature of said heat conducting structures and biological material specimens received therein.

Description

IMPROVED METHOD FOR CHANGING THE TEMPERATURE OF A BIOLOGICAL SPECIMEN

FIELD OF THE INVENTION

This present disclosure generally relates to cryopreservation. Specifically, the invention relates to a method and a system for freezing and freeze-drying of biological specimens.

LIST OF REFERENCES

The following references are considered to be pertinent for the purpose of understanding the background of the present invention:

1. US 5,873,254;

2. WO 03/056919;

3. WO 03/020874;

4. US 6,337,205;

5. US 5,863,715;

6. WO2006/016372;

BACKGROUND OF THE INVENTION

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 (l°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 exosmosis 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 of a large volume is disclosed in US 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 US Patent 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 biopharmaceutical is disclosed in US 6,337,205. The sample to be frozen is inserted into special oblong vials that have special appendages, termed " ce 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 US 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).

A method for changing the temperature of a biological material from a first temperature to a second temperature within a time period is described in WO2006/016372. This publication discloses, inter alia, changing the temperature of a biological material from a first temperature to a second temperature where one of the first or second temperature is above freezing temperature and the other being below freezing temperature of the material. The temperature change 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 freezing stage may constitute only a part of a process, such as, for example, a lyophilization process which takes place in a Lyophilizer and consists of three identifiable stages:

(1) A freezing stage, at which the temperature of the substance being lyophilized is reduced below the triple point of the substance (at the operational pressure of the next stage);

(2) A primary drying stage, at which the pressure is lowered, and enough heat is supplied to the material for the water to sublimate. The amount of heat necessary can be calculated using the sublimating molecules' latent heat of sublimation. In this initial drying phase, about 95% of the water in the material is sublimated. This phase may be relatively slow, due to the fact that adding too much heat could alter the material's structure; and

(3) A secondary drying stage, at which unfrozen water molecules are removed, since the ice was removed in the primary drying phase. This part of the freeze- drying process is governed by the material's adsorption isotherms. In this phase, the temperature is raised higher than in the primary drying phase, and can even be above 0°C, to break any physico-chemical interactions that have formed between the water molecules and the frozen material. SUMMARY OF THE INVENTION

The present invention thus provides a method for changing the temperature of one or more biological specimens from an initial temperature to a final temperature, comprising:

providing at least two temperature control plates defining a space therebetween, a first temperature control plate having a first inner surface and the second temperature control plate having a second inner surface;

providing one or more heat conducting structures, each having a first outer surface and a second outer surface spaced from said first outer surface by a body of the heat conducting structure, said body having at least one channel extending along at least a portion thereof;

placing said biological material specimens within at least one of said channels;

placing heat conducting structures within said space such that said first outer surface in contact with said first inner surface of the first temperature control plate and second outer surface in contact with said second inner surface of the second temperature control plate; and

changing a temperature of at least one temperature control plate from an initial temperature to a final temperature changing thereby a temperature of said heat conducting structures and biological material specimens received therein.

The initial or the final temperatures may be above freezing temperature of the biological specimen and said final or said initial temperatures, respectively, being below freezing temperature of the biological specimen.

The change of temperature of the at least one specimens may be freezing. The temperature of said first temperature control plate may be changed independently of said second temperature control plate.

The temperature gradient between the two temperature control plates may be maintained substantially constant at least along certain period of time. The initial temperature and the final temperature may create at least one linear temperature gradient within said heat conducting structure, which may be other than 0°C/cm.

The initial temperature and the final temperature may create two or more linear temperature gradients within said heat conducting structure.

There is also provided a system for changing the temperature of one or more biological material specimens from an initial temperature to a final temperature, comprising:

at least two temperature control plates defining a space therebetween, a first temperature control plate having a first inner surface and the second temperature control plate having a second inner surface;

a heat conducting structures configured to be received between said first and second plates, each having a first outer surface contacting said first inner surface of the first temperature control plate and a second outer surface spaced from said first outer surface by a body of the heat conducting structure, contacting said second inner surface of the second temperature control plate, said body having at least one channel extending along at least a portion thereof and configured for receiving therein said biological material specimens; and;

a control unit for controlling temperature of at least one of the temperature control plates, imposing thereby a temperature gradient to said one ore more heat conducting structures and said biological material received therein.

At least one channel of said heat conducting structure may extend along the body of the conduction structure between the said first inner surface and said second inner surface.

The channel has a cross-section in a direction perpendicular to said plates, which may be uniform or non-uniform.

The first outer surface, the second outer surface or both first and second surfaces of at least one conducting structure may be in a form of a wall separating between said channel and said first or second inner surfaces. The heat conducting structure may comprise one or more heat conducting blocks, each configured to be received between said first and second inner surfaces of the temperature control plates.

The blocks may be detachably attachable one to the other or fixed one to the other.

The control unit may control the temperature of said first temperature control plate independently of said second temperature control plate.

The device may comprise a plurality of temperature control plates, each two plates defining said space therebetween.

The temperature control plates may constitute shelves of a device, such as a lyophilizer. During the freezing stage, the control over the freezing of the substance is very limited to only controlling the temperature of the lyophilizer shelf, as a result, the size of the ice crystal is not controlled and there are large differences between various containers containing the substance to be lyophilized on the same shelf. A way to address some of the challenges of the lyophilization process is to try to improve dramatically the freezing phase and improve considerably the primary drying phase by using control directional freezing. When using the method and system according to the present invention, the advantages are: (1) the control over the freezing process is very precise; (2) due to the tight contact between the heat conducting structures and the shelves and the heat conducting structures and the containers, almost all the heat is supplied by means of direct conduction. (3) In general, this method of lyophilization might be much shorter and with much more repeatable results.

The method and system in an embodiment of the invention enable imposing a controlled, essentially linear, temperature gradient in the heat conducting structure, thereby imposing a temperature gradient within the biological specimen, and to cause directional freezing of the specimen. Specifically, it has been found that the method and system disclosed herein provides improved post-reconstitution viability of the biological specimen. In this invention, a temperature gradient means a temperature profile of an object (e.g. block or specimen) at a given moment, measured as temperature/length (e.g. °C/cm).

A temperature regime, in this invention, is the temperature change in time (e.g. °C/minute). In a single operation, a plurality of temperature changes may be imposed on a single object resulting in a complex temperature regime.

Without being bound by theory, it is assumed that due to the creation of temperature gradient within the heat conducting structure the temperature gradient within the biological specimen is better controlled than in the absence of a heat conducting structure. The interface between a frozen portion and a non frozen portion in the biological specimen creates a "ice-front propagation", which may then gradually propagate in said specimen as other portions of the specimen gradually freeze. The rate of propagation may be controlled by the temperature differential between temperature controlled plates and also by a change in temperature in one or both of the temperature controlled plates. This allows promoting the temperature gradient along the heat conducting structure at a predetermined rate. This allows highly controlled directional freezing of the biological specimen, similar to the directional freezing disclosed in US 5,873,254, but without moving parts.

As may be appreciated, at times, the at least two temperature controlled plates are not independent bodies but rather may be part of one temperature controlled device. For example, in one embodiment the at least two temperature controlled plates may be integrally formed as a single unit that holds the temperature control arrangement.

The heat conducting structure and the one or more channels therein may have various shapes and forms. Without being limited thereto, the heat conducting structure and the channels may independently have the form of a tubular body, which may have a cylindrical, elliptical, oval, polygonal, square, rectangular or any other suitable cross-sectional shape. Notwithstanding the versatility in shape, the temperature controlled plates will be constructed to encase the heat conducting structure.

In accordance with one embodiment of the invention more than one, and preferably all of the temperature controlled plates comprise or are associated with a cooling arrangement for cooling the internal, biological specimen-facing surface thereof.

In accordance with another embodiment, at least one, and preferably all of the temperature controlled plates comprise or are associated with a heating arrangement for assisting in the temperature control and, at times, for defrosting the temperature controlled surfaces (e.g. after use or before a new use).

In accordance with one embodiment of the invention, the cooling arrangement includes conduits for cooling fluids, which conduits are associated with or are formed within the temperature controlled plates. Such conduits are in flow communication with a cooling fluid reservoir (e.g. liquid nitrogen, liquid hydrogen, liquid oxygen, or cooled alcohol), typically through flow control valving means. The cooling rate of the temperature controlled plates may be controlled by controlling the flow rate of the cooling fluid within the conduits.

The heating arrangement may also involve such conduits, which may be the same or different than the conduits used for the cooling. An example of a heating fluid is water or alcohol. In accordance with another embodiment, the heating arrangement includes electric heating modules.

The heat exchange system is typically insulated from the environment, so as to minimize heat loss and improve heat transfer to or from the biological material. The insulating material may comprise Styrofoam, glass wool, cellulose wool, ceramic foams, polyethylene, vacuum, and generally in any type of insulation known per se.

In accordance with some embodiments of the invention, the heat conducting structure may have channels having an irregular internal shape, for example, so as to fit the external contours of the biological specimen, e.g. the external contours of a heart, a kidney, or a specially shaped container holding the biological specimen or to impose a predetermined shape on a deformable container (e.g. a bag) etc. The channels and block should be constructed in such a manner that the biological material or its container will have direct or indirect heat transfer contact with the surface of the channels. Preferably, the biological material and its container may be introduced into the heat conducting structure for freezing and be removable therefore after freezing is complete. Nonetheless, the heat conducting structure may also be an integral part of the container such that the frozen biological specimen would be stored and/or transferred therein.

The system disclosed herein may comprise, in additional to the elements described above, at least one environment control utilities for monitoring and controlling at least the temperature of one or more of the temperature controlled surfaces, the heat conducting block, and said biological specimen. The environment control utility may comprise, in one embodiment, one or more temperature sensors for sensing the temperature of the temperature controlled surfaces and/or the heat conducting structure and/or that of the biological specimen.

The control unit 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 temperature regime.

In addition, in some cases it may be desirable to provide additional information to the control unit (information such as freezing temperature 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 specimen. The sensor reading may also allow the control unit and/or user to adapt the temperature regime to the actual changes within the biological specimen. Finally, the collected data may be stored in any form (such as digital data or printed documentation) for any use, including research and development.

Thus, in addition to temperature sensors the system might include additional sensors such as: 1. One or more CCD cameras that may be used for observation of the biological specimen and crystals formed therein;

2. One or more temperature sensors (e.g. a thermocouple or infrared camera or detector) at one or more locations within the biological specimen, that may be used to record the temperature pattern at any time and the changes in temperature during operation;

3. One or more electrical resistance (impedance) measuring units that allow detection of changes within the biological specimen during operation.

Finally, ultrasound may also be used to follow the freezing temperature front propagation inside the biological specimen. In such case an ultrasound transmitter may be used, for example within the chamber, and the propagation of the interface (cold front) may be monitored by ultrasound readings as known in the art.

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. 1 is a schematic cross sectional view of a system according to an embodiment of the present disclosure.

Fig. 2 is a schematic cross sectional view of a device forming part of a system according to one embodiment of the present disclosure.

Fig. 3 is a schematic perspective views of heat conducting structure in accordance with another embodiment disclosed herein.

Fig. 4A is a front view of a device according to the present invention having temperature control plates constituting its shelves.

Figs. 4B and 4C are schematic perspective and cross-sectional views, respectively, of a heat conducting structure used with the device shown in Fig. 4A. Fig. 5 is a graph illustrating temperature profiles at different locations in a device according to one embodiment of the present disclosure.

Figs. 6A and 6B are graphs illustrating temperature profiles at different time points during operation of the system according to two alternative embodiments of the present disclosure.

Fig. 7 is a schematic view of a system according to another embodiment of the present disclosure.

DETAILED DESCRIPTION OF NON-LIMITING EMBODIMENTS

The apparatus and method of the present invention are for changing the temperature of a biological specimen past a freezing point for freezing a biological material e.g. for subsequent transplantation of a preserved organ in an organ recipient. The controlled freezing in accordance with the invention allows a controled, directional ice crystal generation in the organ/tissue/biological material.

Reference is first being made to Fig. 1 which is a schematic representation of a system in accordance with the invention generally designated 100. The main components of the system include a heat exchange system generally designated 102, a control unit 104 and a cooling liquid supply system generally designated 106.

The heat exchange system 102 includes, in this specific embodiment, two temperature controlled plates 110 each having a first inner surface 110a, a second inner surface 110b defining a space S between them and a longitudinal axis The system further comprises a heat conduction 116 structure configured for receiving therein biological material, to be received within the space S between the surfaces 110a and 110b of the plates 110. The structure may comprise one or more blocks of various shapes and sizes each having one ore more channels adapted for receiving and holding biological specimens. The channels may extend along a portion of the structure or may be through channels extending between the surfaces 110a and 110b. The channels may be perpendicular to the axis Ai of plates 110, however, do not have to be. The channels have any regular or irregular shapes, and may have a cross-section in a direction perpendicular to the axis A_\ of a uniform or non-uniform shape.

In particular, the heat conducting structure 116 comprises a first outer surface 113a contacting the inner surface 110a and a second outer surface 113b contacting the inner surface 110b, and channels 116a, 116b, 116c, 116d and 116e extending along a portion of the structure 116, the channels adapted for receiving and holding biological specimens.

Reference is now made to Fig. 2 which is a schematic cross sectional illustration of another arrangement 200 in accordance with the present disclosure. In this embodiment, a heat conducting structure 216 comprising three channels 216a, 216b and 216c is fitted within two temperature controlled plates 210, such that a first inner surface 210a of the first plate 210 is in tight contact with a first outer surface 218a of the heat conducting structure 216 and a second inner surface 210b of the second plate 210 is in tight contact with a second outer surface 218b of the heat conducting structure 216. Channels 216a, 216b and 216c extend between the outer surfaces 218a and 218b of the heat conducting structure 216.

Further illustrated in Fig. 2 are two biological specimens 240a and 240b, optionally placed within a suitable container or a pouch being positioned within the channels. The biological specimen is typically harvested osteochondral tissue (e.g. a plug or portion of a joint) or liquid biological sample (such as blood or semen or stem cells or any other cell suspension) intended for cryopreservation for subsequent use for in vitro purposes or in transplanting in an organ recipient.

Referring back to Fig. 1, the temperature controlled plates 110 and the heat conducting structure 116 are typically made of material with high thermal conductivity, e.g. a metal such as brass, gold, aluminum, gold plated brass, and others. The temperature controlled plates 110 and the heat conducting structure 113 may be made of the same or different material. For cooling, temperature controlled plates 110 are provided with conduits 114 that are linked to cooling system 106 through tubings 120. Cooling system 106 comprises flow control valves 122, disposed within tubings 120 and a cooling fluid reservoir 124, with the cooling fluid being typically liquid nitrogen, although other cryogenic fluids may also of course be used.

Illustrated herein is a single conduit 114 in each of the temperature controlled bodies 110 although as may be appreciated, the heat conducting system may be equipped with more than one conduit.

In accordance with one embodiment, spent cooling fluid is discharged to the atmosphere through an exhaust 126. Alternatively, it is also possible to have a recycling arrangement whereby spent fluid is cooled again and returned back to reservoir 124.

The heat exchange system 102 further includes electrical heating modules 130 which are in tight association with the heat conducting temperature controlled plates 110. Thus, the temperature of temperature controlled plates 110 and accordingly, heat conducting structure 113 may be tightly controlled through a combination of cooling and heating, and a fine temperature control may be achieved. The rate of cooling of the surfaces 110a and 110b of the temperature controlled plates 110 by controlling the flow rate of cryogenic fluid may be very fast whilst still reaching a predetermined final temperature that is above that of the cooling fluid. The rate of cooling may be as fast as 10°C/minutes, 20°C/minute, 30°C/minute, 40°C/minute or even faster, and the desired final temperature may be obtained with an accuracy of about 0.5°C.

The heat exchange system is typically insulated by means of insulator 150.

The system also comprises environment control utilities 132, such as temperature sensors, connected to control unit 104. Two environment control utilities are shown in this schematic illustration, although it may be appreciated that more temperature sensors may be included, e.g. different sensors at different zones of the temperature controlled surfaces, sensors for sensing the temperature of the temperature controlled surfaces, as well as, at times, sensors for sensing the temperature of the biological specimen, etc. Control unit 104 is also connected to electric heating elements 130, valves 122 and cooling reservoir 124.

With reference to Fig. 3, there is illustrated a heat conducting structure 700 according to another embodiment of the present invention. The structure 700 comprises two heat conducting blocks 710 and 720 received between the plates 110 so that their outer surfaces 710a, 710b, 720a and 720b, respectively, contact the inner surfaces 110a and 110b of the plates 110. The block 710 comprises a though channel 712 extending between the surfaces 710a and 710b. The block 720 comprises a wall 721 constituting its outer surface 720a and a channel 722 extending between the wall 721 and the surface 720b. The blocks 710 and 720 are separated one from the other, however, may be detachably or fixedly attached one to the other.

The configuration of the block 710 allows an easy extraction of the biological material received within the block.

The temperature control plates of the heat conducting structure may constitute shelves of a device configured for receiving therein biological specimens. One example of such a device is a lyophilization device.

With reference to Figs. 4A to 4C, a lyophilization device 800 comprises a lyophilization chamber 810 having a plurality of shelves 820_A to 820_N, each separately controlled by a cooling mechanism (not shown), configured for applying different temperatures to different shelves. The device 800 further comprises one ore more heat conducting structures in a form of a metal (for example aluminum) block 830 (shown also in Figs. 4B and 4C) configured for receiving therein containers 840, such as a Vaile, cup or a tray with biological specimen therein. In particular, the block 830 comprises a plurality of passages 835 (shown also in Fig. 4C) extending between a lower surface 831 and an upper surface 833 of the block 830, each passage configured for receiving a single container 840 therein. The containers 840 may be formed with closures 841 When placed within the lyophilization chamber 810, the block 830 is in contact with the surfaces of the shelves (as will be further explain in detail), i.e. the lower surface 831 of the block 830 contacts an upper surface 821 of the shelf 820 and the upper surface 833 of the block 830 contacts the lower surface 823 of the shelf 820.

The shelves 820 are movable along Y axis (Fig. 4A), which is perpendicular to the surfaces of the shelves.

Operation of an Apparatus

In operation the temperature controlled plates are set to an initial temperature. A biological specimen is placed within channels of heat conducting structure such that the biological specimen (or the container holding the biological material) is held in heat transfer contact with the channel's surface. Preferably the biological specimen is placed in the device at a predetermined initial temperature, which may be different from that of the temperature controlled surfaces of the system. It should be noted, that before operation, the specimen and the heat conducting structure may have the same temperature, or the heat conducting structure may have the temperature gradient as imposed by temperature controlled surfaces before the specimen is inserted therein.

After the biological specimen is in place, the temperature of at least one of the temperature controlled plates of the system is changed for a period of time. As detailed below, the pattern and rate of temperature change may be preset or may be modified during operation in response to processes in the biological specimen.

In case that the temperature control plates constitute shelves of a lyophilization device, as described with reference to Figs. 4A to 4C, the method of operation is the following:

(a) An adequate vacuum level is set within the lyophilization device, which is about 5 mTorr ÷150 mTorr.

(b) The lowest shelf 820_A is cooled to an initial temperature of about -10°C ÷ 10 °C. (c) The shelf above the lowest 820_B is cooled to an initial temperature of about 0°C ÷ 20 °C.

(d) The block 830 containing the substance to be lyophilized are placed on the shelf 820A. For relatively small quantities, the containers 840 may undergo external seeding process prior placing them within the lyophilization device. For very large quantities, the external seeding process may take place using automation (robot).

(e) The shelf 820_B is moved downwardly along the axis Y so that the block 830 is in tight contact with the shelf 820_B, i.e. the upper surface 833 of the block 830 contacts the lower surface 823 of the shelf 820_B. At this stage the temperature of both shelves 820_A and 820_B will start dropping according the predetermined cooling rate, while keeping the temperature difference between the shelves. This process will ensure the freezing is controlled and directional. After the shelf 820_B reaches the initial temperature of the shelf 820_A, next shelf 820_c will move downwardly along the axis Y and a process similar to that described above will take place between shelves 820B and 820c- The cooling process will continue until the uppermost shelf 820_N reaches the temperature associated with the end of freezing process, and a primary drying phase will begin.

It is well established that different biological specimen require different freezing protocols in order to survive freezing and thawing and remain biologically active. Many such protocols are known in the art. Examples for such protocols are given in US 5,873,254. To achieve a desired protocol, the apparatus may be set such that a sensor within the block or one or more of the temperature controlled surfaces or the biological specimen would dictate the changing of rate of temperature change, or a CCD camera or ultrasound transmitter would be used to detect the time when a change of rate is desired (e.g. after lipid phase transition or crystallization are complete) and then change the temperature controlled surfaces temperature regime, either automatically or by a user. The rate of temperature change in the biological specimen would be proportional to the temperature difference between the specimen and the heat conducting structure, the rate of change in the temperature controlled surfaces' temperature and the thermal properties of the biological specimen.

The time upon which a change in the temperature regime of any heat conducting material in the system (or portion of a temperature controlled surface or block) would be affected may be set according to any calculable or observable parameter. Accordingly the control unit may be configured to change the temperatures of the temperature controlled surfaces upon reaching of certain time or temperature thresholds. Some non-limiting examples are:

(a) A time-dependent change, namely a change that occurs within a given time after operation began;

(b) A temperature-dependent change that begins at a time when the temperature controlled surfaces, the heat conducting structure or the biological specimens' measured temperature reaches a specific temperature or is within a pre-defined temperature range; or

Alternatively, the change of temperature regime may be in accordance with a process observed within the biological specimen, such as upon the beginning or termination of any one of the following processes: seeding, lipid phase transition, nucleation, crystallization, glass transition. The change of a regime may be effected automatically by the controller through feedback from one or more sensors of any kind. Alternatively this change of regime may be manipulated manually by the user in real time in accordance with said sensor readings.

Finally, as noted above, the temperature regime of each of the temperature controlled surfaces may be different from that of the other, and the temperature difference between them may change during operation. This difference may also be preset, but may also be changed during operation, according to the sensor feedback from the temperature controlled surfaces, the heat conducting structure or the biological specimen or according to any of the abovementioned processes that are observed within the biological specimen. Fig. 5 shows three temperature profiles at three different time points (t=0, 0<tl<t2) of two temperature controlled surfaces, TCSl and TCS2 and of a heat conducting structure positioned therebetween, as a function of point of measurement along a cross sectional axis extending from TCSl to TCS2. In the temperature profiles the width of the block that comprises TCSl along said axis is marked as "location I", the width of the block that comprises TCS2 along said axis is marked as "location II", and the width of the heat conducting structure along the axis is marked as "location III". In this particular example, the temperature of TCSl is above that of TCS2 (at the three shown times). . Also illustrated in a schematic fashion is a freezing front temperature (dashed line) of a biological specimen held within said heat conducting structure. As seen in the drawing, at t=0, the freezing front is adjacent TCS2 (which is colder than TCSl), and it moves closer to TCSl, as the temperature reduces.

Fig. 6 A shows another example for a temperature regime which may be utilized during operation of the system according to an embodiment of the present invention. The abscissa shows the time of measurement and the ordinate shows the temperature. According to Fig. 7 A the system has two temperature controlled surfaces, TCSl, the temperature profile of which being illustrated by a continuous line, and TCS2, the temperature profile of which being illustrated by a dashed line.

At time equal to tl, operation of the system is initiated, where TCSl has a temperature Tl, TCS2 has a temperature T2 being lower than Tl. After a period of time, namely, at t2, the temperatures of both TCSl and TCS2 are lowered at the same controlled rate (C 1), thus maintaining a fixed temperature difference Dl between them. After a further time period, namely at t3, when the temperatures of TCSl and TCS2 are T3A and T3B, respectively, the temperatures of each of the temperature controlled surfaces are lowered at a controlled rate (CR2) which is greater than CR1, until a final temperature T4 is reached in each of the two heat conducting structures. The cooling rate between t2 and t3 (CR1), is selected to match the biological specimen (i.e. the cooling rate that is required to provide best post thaw viability)

Once the temperatures of the temperature controlled surfaces (and consequently also the heat conducting structure) are such that provide a temperature of the biological specimen below its freezing point or below its glass transition point, the specimen may be removed from the system.

Fig. 6B provides a temperature regime alternative to that present in Fig. 6A (the temperature regime of TCS1 being illustrated by a continuous line, and the temperature regime of TCS2 being illustrated by a dashed line). Specifically, at tl, TCS1 and TCS2 have the same temperature Tl. The temperature of TCS2 is lowered at a desired cooling rate (CR1) until t2, when a desired temperature difference (Dl) is formed between TCS1 and TCS2. At t2, the temperature of TCS1 is lowered at a cooling rate CR1 while the temperature of TCS2 is lowered as the same rate, thus maintaining the temperature difference Dl between the two temperature controlled surfaces, At t3, TCS2 reached a desired final temperature T3 and its temperature is thus maintained (cooling rate being null), while the temperature of TCS1 continues to be decreased until it reaches the desired final temperature T3 as well.

The system according to the present invention may comprise an internal seeding system, i.e. a system movable with respect to the temperature control plates and comprising cooling elements having an access to the containers with biological material for a predetermined period of time.

In particular, with reference to Fig. 7, there in shown a system 300 according to the present invention, similar to the systems described above, comprising a first temperature control plate 301, a second temperature control plate 303, a heat conducting structure 305 with a containers 307 with a biological material, received within through channels 302 conforming with the shape of the containers 307. The second plate 303 comprises through apertures 309 of a width W_A providing an access to the containers 307 through the channels 302. The system 300 further comprises a seeding device 310 having cooling elements 313 of a width W_E smaller then the width W_A of the apertures 309, allowing the elements 313 to pass through the apertures when the seeding device moves towards the plate, as detailed below, and a driving mechanism 315, such as a piston, for movement of the seeding device 310 along the direction D, shown by an arrow.

When in use, the seeding device moves towards the plate 303 so that the cooling elements 313, which are cooled to a temperature range between -80°C and - 160°C are brought into contact with lower surfaces 308 of the containers 307 for a predetermined period of time allowing seeding to occur (usually between 10 sec and 10 min). When the seeding process is over, the device 310 is then moves apart from the plate 303.

It is appreciated that the above discussion regarding operation of a system according to a specific, non-limiting embodiment of the present invention, applies, mutatis mutandis, also to the method of the invention, even when a system according to the invention is not used, and vice versa.

Those skilled in the art will readily appreciate that various modifications and changes can be applied to the embodiments of the invention as hereinbefore exemplified without departing from its scope defined in and by the appended claims.

Claims

CLAIMS:

1. A method for changing the temperature of one or more biological specimens from an initial temperature to a final temperature, comprising:

placing said biological specimens received within a container within at least one of said channels;

placing said heat conducting structures within said space such that said first outer surface is in contact with said first inner surface of the first temperature control plate and second outer surface is in contact with said second inner surface of the second temperature control plate; and

2. The method according to Claim 1, wherein said initial or said final temperatures being above freezing temperature of the biological specimen and said final or said initial temperatures, respectively, being below freezing temperature of the biological specimen.

3. A method according to Claims 1 or 2, comprising changing the temperature of said first temperature control plate independently of said second temperature control plate.

4. A method according to Claim 3, wherein a temperature gradient between the two temperature control plates is maintained substantially constant at least along certain period of time.

5. The method according to Claim 4, wherein said temperature gradient is linear.

6. The method according to Claim 5, wherein said linear temperature gradient is other than 0°C/cm.

7. The method according to Claims 1 to 6, further comprising seeding of said biological material specimens by means of cooling elements configured to be in a direct contact with said containers for at least a predetermined period of time.

8. The method according to Claim 7, wherein at least one of said temperature control plate comprises apertures providing an access of said cooling elements to said containers.

9. A system for changing the temperature of one or more biological material specimens from an initial temperature to a final temperature, comprising:

- at least two temperature control plates defining a space therebetween, a first temperature control plate having a first inner surface and the second temperature control plate having a second inner surface;

- one ore more heat conducting structures configured to be received between said first and second plates, each having a first outer surface contacting said first inner surface of the first temperature control plate and a second outer surface spaced from said first outer surface by a body of the heat conducting structure, contacting said second inner surface of the second temperature control plate, said body having at least one channel extending along at least a portion thereof and configured for receiving therein said biological material specimens received within containers; and

- a control unit for controlling temperature of at least one of the temperature control plates, changing thereby a temperature of said one ore more heat conducting structures and said biological material received therein.

10. The system according to Claim 9, wherein said first and second temperature control plates have a longitudinal axis and at least one of said channels extends substantially perpendicular to said axis.

11. The system according to Claims 9 or 10, wherein at least one channel of said heat conducting structure is a through channel extending along the body of the conduction structure between the said first inner surface and said second inner surface.

12. The system according to Claims 10 or 11, wherein said channel has a cross-section in a direction perpendicular to said axis of the plates, said cross- section is uniform or non-uniform.

13. The system according to any one of Claims 9 to 12, wherein said heat conducting structure comprises one or more heat conducting blocks, each configured to be received between said first and second inner surfaces of the temperature control plates.

14. The system according to Claim 13, wherein said blocks are detachably attachable one to the other.

15. The system according to Claim 13, wherein said blocks are fixed one to the other.

16. The system according to any one of Claims 9 to 15, wherein said control unit controls the temperature of said first temperature control plate independently of said second temperature control plate.

17. The system according to any one of Claims 9 to 16, wherein the device comprises a plurality of temperature control plates, each two plates defining said space therebetween.

18. The system according to any one of Claims 9 to 17, wherein the temperature control plates constitute shelves of a device.

19. The system according to Claim 18, wherein such a device is a lyophilizer.

20. The system according to any one of Claims 9 to 19, comprising at least one environment control utility for monitoring and controlling at least the temperature of one or more of the temperature control plates, heat conducting structures, and said biological specimens.

21. The system according to any one of Claims 9 to 20, further comprising cooling elements configured to be in a direct contact with said containers for at least a predetermined period of time for seeding of said biological material specimens.

22. The system according to Claim 21, wherein at least one of said temperature control plate comprises apertures providing an access of said cooling elements to said containers.