Thawing methods and apparatus

Abstract

The invention provides a method of thawing a sample comprised in a container, the method comprising the steps of: a) calculating an agitation program as a function of either or both of the sample volume and the type of the container, and the ice fraction in the sample, and optionally the thermal conductivity of the sample container; b) agitating said sample according to the program to agitate at least one region of the sample; and c) changing the agitation program applied to at least one region of the sample in response to changes in the sample volume and/or sample ice fraction. The invention further provides a method of reducing shearing to cells during a method of agitation, and methods for thawing a sample wherein a sample container is differentially heated. An apparatus for use in the methods is also provided, as is an apparatus for thawing and/or cooling a sample which comprises a resilient vessel wall.

Claims

1. A method of thawing a sample wherein said sample is within a container comprising an outer wall and an inner wall, said method comprising a) heating at least one region of the outer wall of said container to a temperature equal to or above that at which cellular damage occurs and wherein the inner wall of the container is at a temperature lower than that at which cellular damage occurs, or b) heating the container such that an outer wall region of said container corresponding to an inner wall region which contacts the sample, is heated to a temperature higher than another outer wall region corresponding to another inner wall region which does not contact the sample; and c) i) calculating an agitation program as a function of 1) either or both of a sample volume and a type of container, 2) a sample ice fraction in the sample, and 3) the thermal conductivity of the sample container; ii) agitating said sample according to the agitation program to agitate at least one region of the sample; iii) changing the agitation program applied to the at least one region of the sample in response to changes in the sample volume and the sample ice fraction; and iv) further agitating said sample according to the changed agitation program to agitate the at least one region of the sample.

2. The method according to claim 1, wherein in step b) the outer wall region of the container corresponding to the inner wall region which contacts the sample is heated to a temperature at which cellular damage would occur, and wherein the outer wall region of the container corresponding to the inner wall region which does not contact the sample is at a temperature less than that at which cellular damage would occur.

3. The method according to claim 1, wherein step b) further comprises a step of monitoring the temperature of the inner wall region contacting the sample and the inner wall region not contacting the sample and adjusting the heat or energy applied to one or both of the corresponding outer wall regions of the sample container in response to changes in the temperature of the two regions and/or a step of monitoring the sample temperature and/or the ice fraction in the sample and adjusting the heat or energy applied to the outer wall region, the corresponding inner wall region to which contacts the sample material.

4. The method according to claim 1, wherein in b) the inner wall of the container is at a temperature less than that at which cellular damage would occur.

5. The method according to claim 1, wherein in step a) or b) said temperature at which cellular damage would occur is a temperature above 37° C. and/or said temperature less than that at which cellular damage would occur is a temperature at or less than 37° C.

6. The method as claimed in claim 1, wherein in step a) or b) said sample is agitated.

7. The method according to claim 1, wherein in step c) steps ii) and iii) are repeated at least once, or wherein steps ii) and iii) are carried out continuously.

8. The method according to claim 1, wherein in step c) said method comprises a step of monitoring sample volume and/or sample ice fraction carried out within or sequentially to step ii) and/or a step of determining the container type and/or the volume of the sample material and optionally comprises determining the thermal conductivity of the sample container, before step i).

9. The method according to claim 1, wherein in step c) (1) agitation of the sample comprises agitation of a single region of the sample, of multiple regions of the sample, or the whole sample, (2) the change in agitation applied to the at least one region of the sample in step iii) comprises changing the frequency, amplitude or both frequency and amplitude of the agitation, (3) the sample is heated and/or (4) a heating and agitation program as a function of the volume of the sample, and the ice fraction is calculated, wherein the sample is heated according to the heating program to heat at least one region of the sample and wherein the heating program is changed to at least one region of the sample in response to changes in the sample volume and/or ice fraction in the sample.

10. The method of claim 1, wherein in step c) the method reduces shearing during the agitation of the sample comprised in the container.

11. The method as claimed in claim 1, wherein said sample is a biological sample comprising a biopharmaceutical, a cellular material, a biological tissue, a biological organ or a part thereof, nucleic acids, proteins, polypeptides and/or amino acids.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) In order that the invention may be more clearly understood embodiments thereof will now be described by way of example only with reference to the accompanying drawings of which:

(2) FIG. 1 illustrates a sample thawing and/or cooling apparatus of the third and fifth aspects of the invention with a biological sample container there above;

(3) FIGS. 2a and 2b illustrate cross-sectional views of the biological sample thawing and/or cooling apparatus of FIG. 1 with an inserted sample container;

(4) FIG. 3 illustrates a second embodiment of a sample thawing apparatus of the invention with an inserted frozen biological sample container;

(5) FIGS. 4a and 4b illustrate cross-sectional views taken from the boxed area of FIG. 2a, showing thermal gradients across the sample container wall during different heating/thawing regimes;

(6) FIG. 5 is a graph illustrating thawing speed versus outside wall temperature in relation to thawing of a cryopreserved sample;

(7) FIG. 6 is a graph illustrating three distinct thawing stages of a cryopreserved sample under a constant temperature;

(8) FIG. 7 illustrates the equilibrium ice fraction curve (light line) and enthalphy curve (dark line) for a solution of glycerol (10% w/w) in 0.15 m NaCl; and

(9) FIG. 8 illustrates the internal temperature distribution of a liquid under a) an internal temperature gradient (left-hand image) and b) a uniform temperature distribution within the liquid (right-hand image).

(10) An embodiment of a sample thawing and/or cooling apparatus of the third and fifth aspects of the invention is shown in FIGS. 1, 2a and 2b. The apparatus 10 comprises an outer vessel 12 surrounding an inner heating or cooling vessel 14 separated by an airspace 16. The outer vessel 12 and inner heating or cooling vessel 14 are substantially cylindrical in cross-section and form nested containers with the airspace 16 separating them. The inner heating or cooling vessel comprises a vessel connection rim 22 which connects to the outer vessel 12 and seals the airspace 16. The outer vessel 12 is constructed from an inflexible plastics material such as polypropylene, polystyrene or the like. The inner heating or cooling vessel 14 comprises an inner vessel wall 18 in the form of a flexible membrane which is constructed from a resilient material such as a styrene-butadiene copolymer. The inner vessel wall 18 comprises four heating or cooling devices in the form of four heating or cooling bands 20a-20d which are embedded within the vessel wall 18 and extend there-around in an undulating or serpentine manner. Each heating or cooling band is spaced apart from adjacent heating or cooling bands 20a-20d. The heating bands 20a-20d comprise resistive heating elements which apply heat energy to the vessel wall 18 when activated.

(11) The resilient inner vessel wall 18 is flexible and moveable by way of negative or positive pressure applied through airspace 16, which is connected to an air and vacuum pump (not shown).

(12) Use of the application 10 will now be described with reference to FIGS. 1, 2a and 2b. A sample container in the form of a vial 2, as shown in FIG. 1, is inserted into the inner heating vessel 14. The vial 2 comprises a vial wall 4 which holds a frozen sample material 8, consisting of frozen cellular or biological tissue matter and frozen cryopreservant comprising glycerol containing 0.15% wt NaCl. The sample material 8 partially fills the vial 2 and the vial wall 4 is sealed with a cap 6.

(13) In order to place the vial 2 in the inner heating vessel 14 the inner vessel wall 18 is biased from a first resting position in which the vessel wall 18 has a diameter substantially smaller than that of the vial 2, to a second, open position to which the vessel wall 18 has a diameter substantially larger than that of the vial 2, such that the vial 2 can be inserted within the vessel wall 18. Movement from the first position to the second position is achieved via applying a partial vacuum in the airspace 16, in order to bellow the inner wall 18 outwardly. When the vial 2 has been inserted within the vessel wall 18 the partial vacuum is released and the vessel wall 18 moves back to the first, resting position and conforms to, and abuts, the vial 2, as shown in FIGS. 2a and 2b. The membranous inner wall 18 adheres to and conforms with a large region of the vial wall 4. In the embodiment shown in FIGS. 2a and 2b, the sample 8 is of a size such that only the inner wall region which includes the heating bands 20c and 20d contacts the vial wall 4 which contacts the sample 8. The heating bands 20a and 20b of the inner wall 18 are therefore above the level of the sample 8 and abuts regions of the vial wall 4 above the level of the sample 8.

(14) When the vial 2 is in the correct position within the inner wall 18, the sample 8 is thawed in the following manner, which is an embodiment of the methods of the first, second and fourth aspects of the invention.

(15) Firstly, the temperature of the sample 8 is measured, as is the volume of the sample 8 and the volume of the vial 4, and each of the heating elements 20a-20d are activated according to an initial heating program. The initial heating program sets the heating elements 20a and 20b, which are above the level of the sample 8, to 37° C., whilst the heating elements 20c and 20d are initially set to 70° C. In this way, the heating bands 20c and 20d, which are present in the inner wall region 18 that contacts the vial wall 4 which is in turn in contact with the sample 8, ensure that a higher heat/energy transfer is effected through the region of the inner wall 18 of the inner heating vessel 14, through the wall 4 of the vial 2, into the sample 8, than the region above the level of the sample 8.

(16) After the initial program is activated, the apparatus 10 is also agitated, in order to agitate the sample 8, and ensure movement of the sample within the vial 2 so that no portion of the sample 8 contacts the vial wall 4 for a prolonged length of time.

(17) As initial heating progresses, and the sample 8 begins to thaw, the temperature and the ice fraction are measured in the sample 8 and the heating program adjusted accordingly.

(18) FIG. 4b shows the temperature isotherm of a zoomed in region bounded by the dotted box of FIG. 2a at the end of the initial heating program as can be seen in FIG. 4b, due to the differential power and heat transfer to the sample 8, through the vessel wall 18 and vial wall 4 in the areas of the vial wall 4 contacting the sample, compared to the heat and power heat transfer through the vessel wall 18 and vial wall 4 in the regions above the level of the sample 8, a temperature gradient is created by the heat and energy flow through the walls to maintain an inner vial wall temperature of 37° C. in both regions. This is the optimal condition possible for rapid thawing by conduction.

(19) FIG. 4a shows the situation where each of the heating bands 20b, 20c and 20d apply a constant and uniform heat transfer across the vial wall 4 in both the region contacting the sample 8 and above the sample 8, in which the heating bands, 20b, 20c and 20d are set at 37° C. It can be seen that having each of the heating bands 20b-20d set to 37° C., this results in a lower than optimal inner wall 18 and vial wall 4 temperature at the same time stage as shown in FIG. 4b for thawing the sample 8 quickly, as the temperature gradients across sections of the vial 2 in contact with the sample 8 lead to a much lower internal vial wall temperature (10° C. in the set-up shown in FIG. 2a).

(20) In addition, if the inner wall 18 is heated to a single fixed temperature, the vial wall 4 will reach the inner wall 18 temperature above the sample 8 (which acts as a heat sink). If the sample is agitated (which is generally required in all cryothawing processes), the sample 8 would then be in contact with areas of the vial wall 4 that are at the external temperature. If this external temperature was above 37° C., this would damage any cellular material.

(21) As thawing progresses, it is imperative to decrease the overall temperature of the internal wall 18 to a maximum of 37° C., so as not to damage the sample 8. The heating program, under closed loop control, may achieve this by employing one of the following methods: a) inferring or knowing the height of the ice within the container before thawing; b) monitoring the applied power of the heating bands 20a-20d as a function of the temperature of the vial wall 4 during the thawing process; and c) determining that the ice-fraction within the sample 8 has fully thawed (phase change enthalpy is overcome), which normally occurs when the bulk temperature of the sample 8 rises rapidly.

(22) In some embodiments, the sample 8 will thaw such that there are separated regions of frozen material within liquid aqueous components (or thawed solid components) at various points in time during the thawing process. In such cases, it would also be advantageous to enable targeted heating of areas of the vial wall 4 which abut and contact the frozen material using more heat/energy than applied to areas of the vial wall 4 not contacting the frozen material. In such instances, the heating program can monitor the presence of regions of frozen material, such as by measuring the ice fraction, and selectively activate any or all of heating bands 20a-20d to increase or decrease heat energy applied across the inner wall 18 and vial wall 4 at regions where the vial wall 4 contacts the frozen material of the sample 8.

(23) Although the embodiments shown in FIGS. 1, 2a and 2b utilises resistive heating bands 20a-20d as the heating element to physically contact and transfer heat to the vial wall 4, other heating elements may be used, for example radiative heating (e.g. infra-red heating) of the vial 4 via infra-red heating elements replacing heating bands 20a-20d, or heating the vial 4 using heated fluid conduits replacing the heating bands 20a-20d. In such embodiments, spatial control of heating (e.g. heating of specific regions of the sample 8 may be achieved by the use of shuttering, windowing or focusing/imaging of the radiative heat source and/or spectral control of the radiative heat source, or through shuttering of different fluid temperature zones or flows around the inner wall 18 in the case of heated fluid embodiments.

(24) Although not shown in FIGS. 1, 2a and 2b, the apparatus 10 also includes a number of components to monitor the temperature of the sample 8, vial wall 4 and inner wall 18. These components may be heat or temperature sensors embedded within the inner wall 18 or infra-red sensors embedded within the inner wall 18 and arranged to detect infra-red emissions from the surface of the vial 4, or from the sample 8 itself. Spatial resolution of any of the aforesaid measurements is achieved by using multiple sensors physically separated within the inner wall 18 and these may also be combined as a single component with the heating bands 20a-20d or equivalent heating elements.

(25) When the region of the inner wall 18 and the region of the vial wall 4 adjacent to the sample 8 reach the same temperature as the region of the inner wall 18 and vial wall 4 above the level of the sample 8, i.e. 37° C., the heating bands 20a-20d may be set to maintain the temperature. Alternatively, the heating bands 20a-20d may be set to 37° C. once the sample 8 has fully thawed.

(26) Once the sample 8 has fully thawed, the vial 2 may be removed from the apparatus 10 as required.

(27) FIG. 3 illustrates a second embodiment of a thawing apparatus of the invention, for use in any one of the methods of the first, or third aspects of the invention. The apparatus is similar to that of the embodiments as shown in FIGS. 1, 2a and 2b, but includes agitation means 122a, 122b. The embodiments shown in FIG. 3 comprises an apparatus 110 including an inner vessel 113 of generally circular cross-section arranged to hold a sample container in the form of a vial 114. The inner vessel 113 includes four spatially separated heating elements in the form of heating bands 120a, 120b, 120c and 120d, which extend around the wall 112 of the inner vessel 113, in a similar manner to the embodiments shown in FIGS. 1, 2a and 2b. The heating bands 120a-120d are as described for the embodiment shown in FIGS. 1, 2a and 2b. The vial 114 includes a vial wall 116 and a sealing cap 118. The vial contains a sample of frozen material 119 and in the embodiments as shown in FIG. 3, the heating bands 120a and 120b are located above the level of the sample 119, while the heating bands 120c and 120d are located in the inner wall 112 contacting the vial wall 116 adjacent to the sample 119. As stated previously, the inner vessel 113 includes two agitation means in the form of agitators 122a and 122b, located within the vessel 112. The agitator 122a is located in the bottom of the inner vessel 113, while the agitator 122b is located around the sides of the inner wall 112.

(28) Use of the apparatus 110 will now be described with reference to FIG. 3.

(29) To optimise thermal power transfer into the sample 119, the sample 119 is thawed in a manner by which the applied wall temperature of the inner wall 112 and vial wall 116 and applied agitation are varied as a function of the ice-fraction (ratio of frozen material to non-frozen material of the sample 119). The ice-fraction can be obtained as a function of direct temperature measurement, by calorimetry based deduction, by light transmission, or by measurement of changing conditions of agitation, for example.

(30) At the beginning of thawing, the vial 114 is inserted into the inner vessel 113 and overall agitation of the sample 119 is activated by activating the agitators 122a and 122b. The frequency and amplitude of agitation may be controlled as a function of any of the above parameters such as ice-fraction, temperature of the sample 119, volume of the vessel 113, volume of the sample 119 etc. In addition spatially differentiated agitation of different regions of the sample 119 may be effected by activating either of the agitators 122a or 122b separately. For example if the lower region of the sample 119 needs to be agitated, whilst the upper region does not, the agitator 122b can be shut off, while the agitator 122a at the bottom of the vessel 113 continues to agitate the lower region. Likewise, the agitators 122a and 122b can be differentially controlled to agitate at different frequencies and/or oscillations at the same or different times, to fine tune the agitation of different regions of the sample 119.

(31) In the embodiments shown in FIG. 3, the following parameters of the sample 119 and vessel 113 are determined prior to initial agitation: the type and thermal conductivity of the vial 114, the volume of the sample 119, the initial temperature of the sample 119 and the composition of the sample 119. Once these parameters have been determined, an initial agitation program is calculated as a function of the volume of the vial 114, volume of the sample, the ice fraction in the sample 119 (which initially will be 100% frozen material) and the thermal conductivity of the vial 114. The sample volume and sample ice fraction is monitored throughout thawing and the agitation program changed to apply variable agitation (spatially variable agitation or varying frequency and oscillation) to the sample 119 or regions thereof over time, in response to any changes of sample volume and sample ice fraction.

(32) In addition, thawing of the sample 119 is achieved by heat/energy across the inner wall 112 and vial wall 116 using the heating bands 120a-120d. Initial heating is effected by heating the sample 119 to a temperature above its glass transition temperature (Tg) but below the melting temperature of any aqueous components of the sample for a period of approximately two minutes. In this embodiment the frozen sample 119 comprising biological material and a glycerol/NaCl cryoprotectant, is warmed from its cryogenic storage temperature which may be below −100° C. (−196° C. when stored in liquid nitrogen), to a temperature of around −30° C., at which point approximately 20% of the ice-fraction has melted. This is achieved by rapidly heating the sample 119 at a temperature of approximately 40° C. utilising all the heating bands 120a-120d set at 40° C. At the end of this heating period, the heating of the frozen sample 119 is slowed, and heating is maintained over a second time period of approximately fifteen minutes until the sample 119 reaches a temperature of approximately −2° C. to −3° C., in which the majority of the aqueous components have changed phase from solid to liquid. Once the sample 119 is fully melted it is important not to overheat, so the heating bands 120a-120d may be switched off, agitation is stopped and the vial 114 can be removed from the apparatus 110 if desired. However, in this embodiment heating is then maintained for a third time period until the sample 119 reaches a temperature of around 4° C., at which point both the frozen biological material and the aqueous components have fully thawed. This third time period is approximately five to ten minutes. During each of the three thawing phases and time periods, agitation of the sample 119 is maintained according to the agitation program described hereinbefore.

(33) In the embodiments described for FIGS. 1-4, the sample 119 is frozen cellular material in cryoprotectant comprising glycerol containing 0.15% wt sodium chloride. Other cryoprotectants may be used such as dimethyl sulphoxide (DMSO) or propylene glycol for example. In the embodiment utilising glycerol/NaCl, approximately 80% of the ice-fraction melts in the temperature zone −12.5° C. to −2.5° C., whilst the remaining 20% of the ice melts in the temperature zone −64° C. (the Tg of this cryoprotectant solution) to −12.5° C. The most damaging temperature zone during thawing is associated with the thawing of the final 80% of the ice-fraction, which occurs between −12.5° C. and −2.5° C. The melting of the first 20% of the ice-fraction can therefore be carried out to a relatively slow rate of heating without any detrimental effects on cell viability.

(34) The apparatus 110 could be adapted to include the facility to actively control the post-thaw sample temperature, by utilising a Peltier element in place of the heating bands 120a-120d, to both heat the sample during thawing, and to cool the sample post-thaw, to maintain its temperature at around 4° C. until it is desired to remove the sample 119.

(35) The above embodiments are described by way of example only. Many variations are possible without departing from the scope of the invention, as defined in the appended claims.