The present invention relates to apparatuses and methods for freezing and thawing liquid biological materials; e.g. blood and blood components.
Cryopreservation of biological material is intended to extend the preservation period of biological material while maintaining their functionality and viability after warming. This may be done by freezing or vitrification. In vitrification, a vitrification solution is added to the biological material, such that the freezing point of the sample is reduced, which in turn leads to vitrification rather than freezing (i.e. ice is essentially not formed). Freezing is a transient non-equilibrium process, during which ice crystallization occurs with release of latent heat as liquid or fluid cools below freezing temperature due to ambient cooling conditions.
Different biological materials require different cooling rates in order to survive cryopreservation. At times the best cooling rates are low (e.g. 0.5-5° C./min) while for other biological materials the best survival is observed at relatively high cooling rates (>5° C.; e.g. ca. 50° C./min for semen and 500-5,000° C./min for red blood cells).
The cooling rate during cryopreservation is affected by factors that include the volume of the biological material, its geometrical shape, and the cooling method (i.e. liquid nitrogen, nitrogen vapor, freezers, dry ice, etc.). When the samples are bulky, the rate of removal of heat through the bulk of the sample may limit the cooling rate.
In directional cooling (such as disclosed in U.S. Pat. No. 5,873,254, entitled “Device and method for multigradient directional cooling and warming of biological samples”) biological material is frozen by being moved along a temperature gradient. In such cases, the cooling rate is also influenced by the velocity of movement along the gradient.
The following references are presented by way of general background interest.
U.S. Pat. No. 5,863,715, entitled “Methods for bulk cryopreservation encapsulated islets” discloses method for cryopreservation of encapsulated islets. The method includes the steps of providing a flexible container, such as a freezer bag, containing the islets; placing the bag in a holder that maintains the cross-sectional area of the bag essentially constant and small; treating it with a cryoprotectant and freezing it.
U.S. Pat. No. 4,018,911, entitled “Method for large volume freezing and thawing of packed erythrocytes” discloses a rigid metal holder having perforated side plates for containing plastic bags of human red blood cells for both freezing and thawing such cells is provided. Hydroxyethylstarch, HES, is used as the cryoprotective agent.
U.S. Pat. No. 5,309,723, entitled “Method of freezing red blood cells”, discloses a method of freezing a standard donor unit of red blood cells and involves centrifuging a blood unit to remove plasma and platelets and to provide a Packed Cell Volume of the red blood cells of not less than 90%, adding the red blood cells to a freezing bag, containing HES solution such that the ratio of HES/red blood cell freezing unit is not more than 7% (preferably 6%) w/v, positioning the freezing bag in a freezing frame adapted to maintain the thickness of the contents of the bag constant, and placing the frame without shaking, into liquid nitrogen.
U.S. Pat. No. 5,935,848, entitled “Deep-freezing container” discloses a container having two plane-parallel thin-walled metallic plates secured in swivellingly interconnected frame halves. When the frame halves are superimposed, the plates secured therein are arranged with parallel faces at a slight defined distance from each other and form an intermediate cavity in which the bag is placed. When the container is closed, the plates press the bag arranged inside the container and flatten it until it has a small defined thickness. A microporous layer is secured to the outer side of plates by an adhesive layer.
U.S. Pat. No. 6,022,344, entitled “Cryopreservation bag”, relates to a bag for the cryopreservation of blood cells, comprising a joining piece and a shrink tube to connect the bag with a non-PVC tubing.
U.S. Pat. No. 4,327,799, entitled “Process and apparatus for freezing living cells” discloses a process for freezing cell suspensions by locating the suspension in a freezing chamber and simultaneously monitoring the temperature of the suspended cells and of the chamber. The cooling of the chamber is regulated at predetermined rates in response to give temperature levels of the sample. The cooling chamber includes a fan, a heater, and a source of refrigerant. The process includes the steps of selectively decreasing and increasing the temperature of the freezing chamber responsive to predetermined temperature points on the freezing curve of the cell sample.
U.S. Pat. No. 5,250,044, entitled “Blood cryopreservation container”, relates to bags for the cryopreservation of mammalian cells and particularly for the long-term freezing of red blood cell, and to methods of manufacturing such bags.
US 2005/129887, entitled “Medical freezer bag”, discloses a medical freezer bag made from a three-layer film where both sides of an ultra-high molecular weight polyethylene (PE) film are welded respectively with a thermoplastic resin film having a lower melting point than that of the ultra-high molecular weight polyethylene and having compatibility with the ultra-high molecular weight polyethylene.
WO 2004/003444, entitled “Changing the temperature of a liquid sample and a receptacle useful therefor”, discloses a method of changing the temperature of a liquid sample, comprising: providing a receptacle having inner and outer walls defining an annular portion therebetween for receiving therein a liquid sample, inserting said liquid sample, at a first temperature, into said annular portion, and exposing said receptacle to a second temperature different from said first temperature. Further disclosed are a receptacle useful in the method and a chamber useful for performing said method.
WO 2005/072523, entitled “Biological material and methods and solutions for preservation thereof”, discloses a preservation solution for preserving biological material at low temperature comprising one or more polyphenols and a method for preservation of biological material, said method comprising adding the preservation solution to biological material, cooling the biological material and storing it at appropriate storing conditions. The method may be used for hypothermic preservation or for cryopreservation, including freezing and lyophilization, and may be used with any biological material, including cells selected from RBC, WBC, MNC, UCB MNC and bacteria. In case of RBC the present invention also disclosed a method for its freezing such that upon thawing, the material has less than 2% free hemoglobin.
Herein, “biological material” includes any material which comprises biological material (e.g., cells, tissue, or portion thereof), and may include, inter alia, any liquid suspension comprising biological cells, blood, blood components, blood fractions, blood preparations, blood plasma, semen, stem cells, bone marrow cells, platelets, oocytes, embryos, lymphocytes, white blood cells and/or red blood cells. At times, the biological material is biological material having a preferred freezing rate of above 1° C./min, at times above 5° C. or even 100° C./min or more.
The term “cryopreservation” means vitrification or freezing, and “cryopreserved biological material” means vitrified and/or frozen biological material in an essentially solid state. The term “warming” of a cryopreserved biological material means the warming of such material, which may be performed until the major portion or whole of the biological material becomes liquid. In case of frozen biological material the warming may mean thawing, while in the case of vitrified biological material, warming may mean liquefying.
According to one aspect of the present invention, there is provided a system for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis, the system comprising:
The tank and the immersion mechanism constitute together a submersion freezing device, also referred as SF device or SFD.
The bag preferably comprises a high surface area-to-volume ratio, and a substantially constant width (e.g. about 4 mm to about 27 mm, in some embodiments preferably no more than 15 mm), which helps to provide uniformity of thermal history through the biological material (or a major portion thereof) where appropriate, and/or to reduce the likelihood of the bag being damaged during freezing, thawing, handling, storage, transportation, and so on, and/or to potentially increase the post-thaw viability of at least some types of biological materials, for example blood cells or other cells.
Any one or more of the following features may be included in the system:
According to another aspect of the present invention, there is provided a system for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis, the system comprising:
According to another aspect of the present invention, there is provided a system for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis, the system comprising:
According to another aspect of the present invention, there is provided a system for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis, the system comprising:
The bag when held by the bag holder has two large side surfaces and a thickness therebetween which is significantly smaller than the dimensions of the large side surfaces, wherein said surface area is the total area of the side surfaces. The prevention of, the surface area-to-volume ratio of the biological material from increasing may be obtained by reducing, or even eliminating, the widening, i.e. increasing the thickness, of the bag during the immersion. This may be achieved, for example, by using the guides, selecting the rate of the immersion or using the stiffening members, or by a combination of any of them.
According to another aspect of the present invention, there is provided a method for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis, the method comprising:
According to another aspect of the present invention, there is provided a method for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis, the method comprising:
According to another aspect of the present invention, there is provided a system for warming cryopreserved liquid biological material disposed in a bag, during which the material changes its state from solid into liquid, the system comprising:
Disclosed below is an embodiment of such system also referred to as dry thawing device, a “DT device” or simply as “DTD”. One of the benefits of using a dry device (in this and other aspects of the invention) is that there is no need to manage a heating fluid (e.g. water).
The system may comprise an empty space for accommodating any liquefied material apart from the cryopreserved material, which space may be a part of the bag spaced from the part where the solid portion of the cryopreserved material is located in heat transfer contact with the heat source. The liquefied portion of the biological material may be forced away from the area of said contact by suitable means, including for example a vacuum (applied within the bag), gravity, centrifugation, osmotic pressure, spring pressure and/or pneumatic or hydraulic pressure and any combination thereof.
Any one or more of the following features may be included in this aspect of the invention:
The system is adapted for applying a suitable mechanical pressure onto the bag, which can urge any liquefied material to a volume outside of or away from the frozen contents of the bag, to maintain substantially constant the thermal contact between the heated plates and the cryopreserved material via the bag walls only, without any substantial interference from liquefied thawed material and without overheating the biological material.
This aspect takes advantage, among other things, of the relatively better heat conductivity of solids, when compared with liquids.
According to another aspect of the present invention, there is provided a method for warming a cryopreserved liquid biological material disposed in a bag, the method comprising:
A system for performing the above method may also be referred to as dry thawing device, a “DT device” or simply as “DTD”.
According to another aspect of the present invention, there is provided a system for warming a cryopreserved liquid biological material disposed in a bag having a longitudinal axis, the system comprising:
Such system may also referred to as a wet thawing device, “forced flow thawing device”, “FT device”, “Activator” “FFTD or “WTD”.
Any one or more of the following features may be included in this aspect of the invention:
The warming device may be adapted for applying a suitable mechanical pressure onto the bag, which at times removes liquified material to a volume outside of or away from the cryopreserved contents of the bag. For example, the plates may be mounted on springs which urge the plates towards the bag; as the material thaws, the bag presents less resistance to the plates, which effectively move closer the cryopreserved bulk within the bag, and a portion of the liquified contents thereof may be squeezed out to the upper extremity of the bag away from the cryopreserved core, and/or the bag walls may deform and bulge into the heat transfer passages, with liquefied material occupying these bulges rather than accumulating at the bottom of the bag and minimizing thermal transfer to any cryopreserved material thereat. This serves to maintain substantially constant the thermal contact between the heat transfer plates and the cryopreserved material still in the bag via the bag walls only, without any substantial interference from bulk liquified material.
According to another aspect of the present invention, there is provided a method for warming a cryopreserved liquid biological material disposed in a bag having a longitudinal axis, the method comprising:
The method may be performed using a wet thawing device, “forced flow thawing device”, “FT device”, “Activator” “FFTD or “WTD” according to one or more embodiments of the invention.
In warming biological material of significant bulk, the liquefied portions of the material may insulate the portions of the material that are still cryopreserved (solid) and thus reduce the warming rate of the solid portion. Also, the thermal contact between the liquefied portion and the heat source may cause overheating of portions of the biological material. Thus, according to some aspects of the invention, thawing is carried out in a relatively rapid rate and in a manner to minimize recrystallization, which can otherwise occur due to the endothermic effect of thawing, whereby a thawing crystal may cause surrounding water to recrystallize. At the same time, thermal contact between the heat source used for warming and the cryopreserved part of the biological material is maximized during warming, thereby minimizing negative effects that may occur due to slow warming (or thawing). Additionally overheating of the biological material may be avoided or eliminated, for example, by removal of liquefied material from the thermal contact areas, and/or preventing accumulation of liquefied material at locations in the bag.
A system for cryopreserving and a system for warming biological materials, according to any of the embodiments of the present invention, may be provided as a kit.
It is to be noted that warming and/or cryopreserving according to aspects of the present invention of more than one sample of biological material may be performed in sequence or simultaneously. For cryopreserving for example, more than one bag may be placed in a cartridge (e.g. side by side) or multiple cartridges may be inserted simultaneously (e.g. one next to the other) or in sequence (e.g. one above the other) with respect to a single cold temperature source. For warming, more than one bag may be placed between the heat transfer plates (e.g. side by side), for example.
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:
a schematically illustrate, in isometric view and side view respectively, another embodiment of a cartridge for use with the system of
a and 5b illustrate in plan view and side view, respectively, an embodiment of a freezing and thawing bag for use with the embodiments of
a and 6b compare temperature gradient effects obtained with and without a temperature controlled zone above the cold temperature source.
a and 9b schematically illustrate another embodiment of a thawing system according to one aspect of the invention.
According to one aspect of the invention, a system and method for cryopreserving (freezing or vitrifying) biological materials, for example blood and blood components according to some embodiments is provided. According to one embodiment, the system, generally designated herein with the numeral 100 and illustrated in
The device 20 is in the form of liquid nitrogen (LN) dewar (or any other suitable cold temperature source), and comprises a vessel or tank 21, having side walls and a base and defining a volume V containing LN at a temperature T2 of about −196° C. (or at any other suitable temperature, preferably at or below about −80° C.), and a cover 25 having an inner surface 25a which faces the interior of the tank 21. The cover comprises a slot-like access opening 28 having a width dimension W smaller than a length dimension. Downwardly depending into volume V from the periphery of the opening 28 is a plurality of tongues 29. The tongues 29 may be arranged in two sets, one along each of the opposite long sides (i.e., along the length direction) of the opening 28. The tongues 29 have a proximal distance D1 and a distal distance D2 therebetween, so that D2≦D1. In addition, the distal distance D2 is equal or lesser than the width dimension W of the opening 28, i.e. D2≦W. One reason for the above relations is that the frozen bag should be removed; therefore it can not be wider than the opening width W. The lower ends of the tongues may be joined to a lower frame member 27 to prevent or minimize the ends separating when the system is in operation.
The tank 21 may further comprise suitable maximum and minimum level sensors, LSmax and LSmin for monitoring the level of LN in the tank 21 (although the device may be operated with only one sensor or with no sensors). When the level of LN drops below the minimum measured by LSmin, an alarm may be generated, advising the user to top up the tank 21 with more LN, and/or suitable means may be provided for automatically feeding the tank 21 from a suitable LN source. The other sensor LSmax advises when the maximum level has been reached, and to discontinue filing. Additionally or alternatively, filling may be discontinued at a predetermined time or after a predetermined volume has been added, without the use of an additional sensor. If the level LSmax is exceeded, it may be required to remove some LN, for example via natural evaporation (e.g. opening the cover 25) and/or via a tank drain (not shown). Above the level of LN in the tank 21 a headspace may be provided. LN level may be measured using any one of known methods, for example including use of thermocouples, by weighing, and by measuring conductivity. One commercially available sensor that may be used in the present invention is LS2 Liquid Nitrogen Sensor (Teragon, USA).
Referring to
Each of the frame members 61, 62 comprises a wall 30 spanning the openings A, B, respectively. Preferably, these walls are formed of thermally conductive material, and are perforated, to increase heat transfer from the biological material to the cold temperature source. Suitable holes of other perforations (schematically shown at X) in the walls 30, allow fluid communication between the outside of the cartridge, through the walls 30, and into the space between the walls 30 of the two frame members 61, 62. The perforated walls 30 are each designed to provide a structure of relatively high rigidity, and also to provide good fluid communication therethrough. The open area provided by the perforations can be maximized while maintaining sufficient rigidity in the walls 30 to contain the shape of a bag 90 placed between the walls 30 when the bag is frozen. By way of non limiting example, the open area of the perforations may comprise between 60% and about 70%, say 63%, of the plan area of the corresponding wall.
Frame 62 further optionally comprises a handle element 64 extending from top element 62d in a direction away from the bottom element 62a; alternatively or additionally, frame member 61 may comprise a similar handle. In the closed position, the frames are spaced from one another by a spacing, which is adapted for receiving and holding therein a bag 90, and the frames can be locked in this position by means of one or more closure members 65, 66. A pair of closure members 65 may be provided, one each hinged at 69 on a different side of the cartridge 60 to one of side elements of the frame members 61 (or alternatively of frame member 62), and each comprising a U-shaped cross-section and extended sides that retain frame members 61, 62 along the length thereof in the closed position. When the closure members 65 are swung about their corresponding hinges 69, the extended sides thereof come out of engagement with the frames 61, 62, which may then open to the open position. Closure member 66 is similar to closure members 65, and is hingedly mounted to a bottom element of frame member 61 (or alternatively to a bottom element of frame member 62). Alternatively, any other suitable closure members, including clamps or belts for example, or other arrangement may be used for enabling the frame members to reversibly close, to define the aforesaid spacing, and open to allow a bag to be inserted therebetween or removed therefrom.
The cartridge 60 optionally or alternatively further comprises a plurality of stiffening members 67, joined at their ends to the top element 61d and bottom element 61a of frame member 61. Each stiffening member 67 may comprise a U cross section, open at the longitudinal ends 38 thereof, and comprises a plurality of through holes 68. The stiffening members are intended to keep a width of the bag uniform.
The cartridge 60 optionally or alternatively further comprises one or more separators, allowing an insertion of more than one bag, with each of the two outmost bags abutting one of the perforated walls. The separators may in a form of a flexible sheet, made of a having a little thermal mass, and optionally also being permeable to LN and/or having high heat transfer capability. Examples for such material include polyurethane foam, thin aluminum sheets (perforated or not). The inventors found that use of such one or more separators according to an embodiment of the invention may reduce the post freezing thickness of the bag and improve cell post thaw viability. In addition, the post thaw viability may also be improved by decreasing the thickness of the cartridge and by inserting more than one bag thereinto, thereby decreasing the thickness of each bag.
Referring to
Optionally, cartridge 60 may also comprise rods similar to rods 68′ described for cartridge 60′, mutatis mutandis.
Referring to
The bags 90 may be made from any suitable biologically compatible material, and which preferably facilitates heat transfer between the inside and the outside of the bag 90. To reduce or avoid damage to the bag during cryopreservation due to the expansion of the biological material, it is preferred that the material would have a glass transition temperature that is below that of the biological material. By way of non-limiting example, such bags 90 may be made from sheets of aluminium foil laminated with polyethylene, for example commercially available metalized polyethylene sheets (for example as purchased from Aran Packaging, Israel), or from sheets made from nylon and polyethylene, for example PerfecFlex 30652W, provided by PerfecSeal (UK).
Referring to
The system 100 may be operated as follows. A bag 90 is filled with a biological material, for example blood (optionally also comprising one or more added cryoprotectant agents), and closed in any suitable manner, for example vacuum sealed. The cartridge 60 is opened by releasing the closure members 65 and 66, and pivoting frame members 61 and 62 away from one another about hinge 63. The bag 90 is placed in the cartridge 60, and the frame members pivoted back to the closed position, wherein the closure members are clamped to the closed position, trapping the bag 90 in the space between the frame members in a tight fitting manner and optionally having the top portion of the bag 90 tightly pressed between or under the frames' rods immediately above the level of the biological material when the cartridge 60 is thus configured. Most or a majority of the outer surface of each side 91, 92 of the bag 90 are exposed via the perforations of the walls 30 covering openings A and B of the frames. The thus loaded cartridge 60 is then placed in the passage 52 via opening 54, and driven towards the tank 21 along direction P1, the heater having heated at least the lower part of jacket 55 to a temperature T1 above ambient T0 and thus also the cartridge 60 (being in thermal contact with the jacket), the bag 90 with its contents. By way of non-limiting example, the bag is thus heated and maintained at a temperature of between 0° and about 30°, preferably between about room temperature (e.g. 20°) to about 25° prior to being immersed in the tank 21.
The cartridge 60 is then lowered at a predetermined controlled velocity v into the volume V via the opening 28. The drive means allow to select a predetermined value of the immersion velocity from plurality of values, and to maintain this value constant during the immersion of the cartridge 60. The velocity is selected so as to ensure that cooling behavior of the biological material at least at three different locations along the direction P1 is as shown in
The tongues 29 act as guides, for example rail guides, for the cartridge 60, as this is being lowered into the LN-containing volume V. The tongues 29 engage the corresponding walls 30 of the cartridge and/or the stiffening members 67, so as to abut and press against them and prevent the bag from increasing in volume and/or bulging at any point.
This arrangement ensures that the bags have an essentially constant width S0 throughout, minimizing the bulging that otherwise accompanies material frozen in bags.
Optionally, the tongues may be replaced with a cage or other arrangement that presses the bags to keep and maintain a substantially uniform thickness when held vertically, and while maximising the direct fluid communication between the LN and the bag. Thus, in other embodiments of the invention, the tongues may be replaced with perforated double wall structures, while the frame members are provided without walls 30, so that the bag 90 in the cartridge has substantially fully exposed sides via opening A, B. When immersed in the tank 21, the sides of the bag are in thermal contact with these perforated walls and thus the LN (in relative terms, in a similar manner to the embodiment described above having the perforated walls on the cartridge).
The construction of the cartridge 60 and the tongues 29 is such as to maximize direct thermal contact between the LN in volume V and the bag 90. The tank 21 contains a sufficiently large volume of LN to enable it to absorb substantially enough the heat from the biological sample to enable it to freeze, without reducing the level of LN below a predetermined limit. Any LN that evaporates away is replaced, either manually or automatically, and the level is maintained almost constant by monitoring with level sensors LSmax and LSmin. Optionally, the minimal level of cooling fluid is the tank is no more than 2 cm (or 1 inch) below the maximal level of the cooling fluid.
The structure and operation of the system with respect to cartridge 60′ is similar to that described herein for cartridge 60, mutatis mutandis.
Without being restricted to theory, inventors consider that as the cartridge is immersed into the LN, the sharp temperature gradient between the LN and the temperature controlled jacket 55, which is kept in close proximity to the cover 25, results in reproducible freezing conditions and together with the controlled velocity for immersion results in ice crystals growing quickly from the outer walls towards the inner part of the bag in a direction having significant lateral and upwards components (
The immersion velocity v is such as to provide as uniform as possible a cooling rate for different parts of the bag 90, while minimizing exothermic reaction effects. The faster the immersion velocity v, the faster LN is evaporated around the bag, which may cause nitrogen gas to form around the bag in bubbles, temporarily reducing the heat transfer from the bag, and introducing chaotic cooling rates in the bag. On the other hand, slowing the rate R results in a longer the exothermic period (since the cooling rate is limited by the velocity of immersion); and the longer the exothermic period, the more damage than can occur to cells, blood cells for example, when these are comprised in the biological material. By way of non limiting example, an immersion velocity v of between about 1 mm per second and about 3 mm per second may be optimal. The immersion velocity v may vary with the thickness of the bag 90, and thus also indirectly with respect to the bag surface and volume.
At times, when the preferred cooling rate is high (above 5° C.) and the biological sample is bulky, it is desired to maintain a reduced width of the bag (perpendicular to the longitudinal axis) and to set the velocity of immersion such that it will be proportional to the rate of heat removal from the bag (e.g. less than 3 mm/sec or even less than 1 mm/sec). This may provide essentially identical cooling rates for different portions of the biological material. A faster immersion velocity would lead to higher boiling of LN. This would mean that the leading end of the bag would experience a higher temperature difference and hence have a higher cooling rate than the middle and tailing portions of the bag.
According to another aspect of the invention, a device and method for warming (e.g. thawing) biological materials, for example blood and blood components according to some embodiments, is provided.
According to one embodiment, the device, generally designated herein with the numeral 200 and illustrated in
In operation, the heaters 252, 254 heat the pads 222, 224, to a suitable temperature Tw, which by way of non-limiting example may be between about 50° C. and about 90° C., preferably about 70° C., and sufficient to thaw a particular volume (or width) in a desired time period. A bag 90 is sandwiched between the pads 222, 224, with the empty (vacuumed) part projecting from the device 200 and resting on ramp 260. As the pads 222, 224 heat the bag, the weight of the upper plate 234 and part 214, together with the force exerted by springs 270 force the thawed material away from the space between the plates 232, 234, and via the ramp towards the empty part 99 of the bag or to a waiting vessel, for example. By taking thawed material away from the bag volume continuously, thermal (heat transfer) contact between the frozen contents of the bag and the heated pads 222, 224 is maintained (or even maximized) and thermal contact between the thawed contents and the pads is reduced.
Optionally, the pads 222, 224 may be continually heated by the heaters 252, 254 during the thawing operation. Alternatively, the pads 222, 224 may have a higher thermal mass and are heated only prior to the thawing operation, and thawing progresses due to the stored heat in the pads 222, 224.
By way of non-limiting example, a bag filled 500 ml of cryopresereved biological material may be thawed in about 1 to about 3 minutes using a device 200 having preheated pads at a temperature of about 70° C. Typically, a sample of biological material having a width of 10-15 mm after it has been frozen, will be thawed in about 1 to 3 minutes at a temperature of about 70° C.
Referring to
Optionally, a filter or mesh (not shown) may be provided in device 200 for preventing small frozen fragments from leaving the bag volume that is being heated by the pads.
Optionally, the device 200 may comprise one or more safety features, such as for example: a lock to prevent premature opening of the device, before operation (to assure a desired temperature before warming) and/or during operation (until a preset time sufficient to warm a bag); a detector for detecting that a bag is actually in place at the warming location between the pads; displays showing the temperature of the pads and/or of the bag; alarms for indicating that the thawing cycle has finished, and/or for alerting that the pad temperature has not reached, or is exceeding the desired temperature; and so on.
According to another embodiment, the warming device, generally designated herein with the numeral 300 and illustrated in
The plates 322, 324 each have a plurality of facing open channels 332, 334, respectively, having open inlet openings and outlet openings allowing free passage of a fluid medium therethrough. In the illustrated embodiment, the passages 332, 334 are shown as vertical, but in other embodiments the passages may have any suitable orientation. The plates 322, 324 are accommodated in a housing 330, plate 322 statically, while plate 324 is horizontally movable towards and away from plate 322, but is urged towards the static plate 322 by means of springs 335 or any other suitable arrangement that can provide this effect. A suitable pump 360, which may be located inside the housing or outside thereof, comprises an outlet 362 in fluid communication with an inlet end 332 of the housing 310, aligned with inlet openings of the channels 332, 334, via conduit 364.
Holding space M is configured for vertically accommodating therein a bag 90 containing cryopreserved material, e.g. frozen biological material, such that the channels 332, 334 are facing the walls 91, 92, respectively of the bag.
In operation, the water in tank 312 is suitably heated, for example by means of an electric heater (not shown) to attain a generally uniform temperature of, say between about 22° C. and about 60° C., and preferably between about 37° C. and about 45° C., by way of non-limiting example. A bag 90 with cryopreserved (e.g. frozen) contents is vertically inserted between the plates 322, 324, the movable plate 324 having first been separated against the springs 335. As water is pumped through the channels 332, 334 by means of pump 360, which has a pump inlet (not shown) in fluid communication with the water in tank 312, heat in the water is transferred directly to the bag and its contents, via the surface of the bag facing the channels, and indirectly, through the contact with the blocks between the channels, enabling the frozen contents to begin to thaw. As the material in the bag thaws, there is less resistance by the bag to the force provided by the springs 335, which force the plate 324 towards the static plate 322. Thawed material is pushed upwards to the empty space 99, and the channel form of the plates cause a bulging of the side walls of the bag into the channel, the bulging portions of the bag filling with thawed material. By pressing the two sides of the bag closer together and preventing buildup of thawed material at the bottom of the bag by means of the pads 322, 324, thermal contact between the frozen contents of the bag and the heat exchange channels 332, 334 is maximized.
Alternatively, the two chambered bag 90′ of
Terms and Abbreviations:
EGCG—Epigallocatechin gallate, a green tea catechin. Purchased from Zhejiang Yixin Pharmaceutical Co., Ltd.
LN—liquid nitrogen
IMT-1—A freezing solution composed of 20% (w/v) Dextran 40 (Pharmacosmos, Denmark) and 0.945 mg/ml EGCG dissolved in saline (0.9% (w/v) sodium chloride in double distilled water (DDW))
MCV—Mean corpuscular volume, this is a value measured by the automatic cell counter as part of the complete blood count (CBC) and gives an insight to the RBC volume.
Aluminum/PE bag—A bag made of aluminum foil (metallized polyethylene (PE). The aluminum/PE raw material is manufactured by Kolon Industries Inc., South Korea and laminated by Aran Packaging, Israel.
Cryoprotectant agent—denotes any agent that is added to a solution it improves the post cryopreservation viability (i.e. after thawing or liquefying) of a biological material cryopreserved in that solution. Intracellular CPAs are thought to replace water inside the cells, thus preventing crystallization therein, to enlarge the un-frozen fraction of the frozen solution, to buffer osmolarity and/or to stabilize the membrane and prevent mechanical damage caused by ice crystals. Examples of CPAs are DMSO, glycerol, ethylene glycol, poly ethylene glycol, propylene glycol, sugars, such as sucrose, dextrose, trehalose, and proteins, carbohydrates such as hydroxy ethyl starch (HES), dextran, etc.
Material and Methods:
Except where indicated otherwise, in the following experiments the following materials and methods were used:
Packed RBCs from several donors (received from the Israeli Blood Bank, Magen David Adorn) were pooled, and mixed with IMT-I freezing solution in a 1:1 (v/v) ratio. It is noted that the freezing solution may be added to the RBC or the RBC added to the freezing solution, with comparable results (not shown). Before freezing, but after adding a freezing solution, a sample of blood was taken to be used as a control (“fresh”).
For freezing, blood mixed with IMT-1 freezing solution was placed in freezing bags, and each bag was frozen in one of the following methods: (1) Submersion Freezing Device (SFD)—the bag was placed in a frame and the frame was lowered by the device into LN at a controlled velocity of 1 mm/sec, (2) manual insertion into LN of a “naked bag” (i.e. without a freezing frame) and (c) slow freezing by placing the samples in a freezing frame, in a −80° C. conventional freezer (Forma, USA) until thoroughly frozen. For LN freezing, the bag was held in LN for one minute after insertion, to ensure complete freezing. After freezing was complete, all samples were stored for 24 hours in the −80° C. mechanical freezer (Forma, USA).
Thawing was done in as follows: The bags were removed from the freezing frame (if a frame was used) and their thickness was measured and then immediately thawed by one of the following: (1) placing the sample in an Activator (wet thawing device) or (2) by dipping samples in a still water bath, or (3) placing the sample in a DT device. Thawing was done, in each device, at a temperature of 22°, 37° or 45° C., as detailed in the experiment. The time needed for complete thawing was measured.
The blood was evaluated before freezing (“fresh”) and after thawing for one or more of the following parameters:
Supernatant free Hemoglobin (Hb) measurement were done by using the cyanmethemoglobin method using Drabkin's reagent (Sigma, USA) and reading absorbance at 540 nm using an ELISA reader (EL-800, Bio-Tek Industries, USA).
Free Hb was Calculated as Follows:
% Free Hb=100×(Absorbance of the supernatant) (Absorbance of supernatant+Absorbance of the pellet)
Specific Details:
Samples were placed into 310×200 mm aluminum/PE bags. Sample volume in each bag was 300 ml. Each sample was a pool of blood from at least 3 different donors (all belong to the same blood group). Hb readings were performed in triplicates. Results are shown as the mean and standard deviation. The samples were frozen using the SFD or by manual insertion or by placing the sample in a mechanical −80° C. freezer (Forma, USA).
In the following experiment frozen blood bags having a width of 6.5-9 mm were thawed either in the Activator or water bath, at 22° C., 37° C. or 45° C.
Results:
Freezing in a Submersion freezing Device (6 bags), resulted in 8.38±0.38 mm thickness of the bag. Freezing by manual insertion to LN, without a freezing frame, resulted in 27±0 mm thickness (3 bags). Samples frozen in the mechanical −80° C. freezer had a sample thickness of 7.66±1.1 mm.
The thawing time results are depicted in Table 1. As seen, at any given temperature, the Activator thaws the sample much faster than the water bath. Additionally, for each thawing method the thawing time generally increases as the thawing temperature is lower.
Table 2 below depicts the MCV, cell number and hemoglobin assayed in thawed cells. As seen in Table 2, the MCV and cell number for cells frozen with SFD were about 100% (within the error margin of the Pentra device used to assay these parameters). Contrarily, when cells were frozen in a −80° C. freezer, cells had 100% free hemoglobin regardless of the thawing conditions (water bath of Activator, at different temperatures) and appeared almost always as “all ghosts” (not shown) as can be seen in the samples that were manually frozen. These samples were inserted into LN gradually, similarly to the insertion by the SFD device (but without a metal frame, or controlled pre-freeze environment, or controlled velocity, etc.). As also seen in Table 2, sample thickness after freezing was highest in bags that were inserted manually.
Generally, as the thawing temperature increased the survival was higher, probably due to the fact that this allows faster thawing and as a consequence less damage from recrystallization.
Specific Details
Blood solution (packed RBC with IMT-1 freezing solution) was divided to four 300 ml portions each placed in a 310 mm×200 mm aluminum/PE bag. The bags were frozen in SFD by submersion in LN at 1 mm/sec in one of two directions: two bags were positioned vertically in respect to the LN (standing tall) and 2 were positioned horizontally (i.e. lying flat). Thawing was in the Activator device. Free Hb readings were performed, each repeated twice. Results are shown in Table 3 as the mean and standard deviation, compared to fresh blood.
Results:
Material and Methods:
In this experiment RBC were frozen in 500 ml volume of RBC/IMT-1 mixture in aluminum/PE bags (210×300 mm) using SFD. Each bag contained RBC from a different donor. After freezing the bags (12) were stored for 24 hours in a −80° C. freezer (Forma, USA). Each thawing condition was repeated in triplicates. Samples were thawed either in a regular water bath or in the activator at 37 and 45° C., each condition repeated thrice. The results are depicted in Table 4.
As seen in Table 4, cell numbers and MCV were about the same for all samples, being within the margin of error for the assay. In terms of free Hb the survival of RBC after being thawed in the activator were slightly improved using the Activator. However, the standard deviation of the free Hb levels, when thawed in a regular water bath, was higher, indicating a higher variability between samples.
Additionally, when looking at the thawing time (Table 1 above), we can see it was much longer in a regular water bath compared to the activator at any given temperature. As expected, the decrease in temperature to 37° C. from 45° C. increased thawing time in both cases. However, this increase in thawing time was significantly larger for the water bath than for the Activator. In the present experiment this difference was even higher, with the thawing time measuring about 2 minutes for the water bath, compared to only 30 seconds for the Activator (data not shown), although the samples thawed in the Activator were slightly thicker. It is therefore concluded that thawing in the Activator is less prone to be affected by slight experimental variation (e.g. of the thawing temperature or other process steps, solution composition, etc.) than simple thawing in a water bath. This provides a thawing method with improved dependability.
Specific Details:
Aluminum/PE bags were filled with 300 ml of IMT-1 solution (no RBC). 5 thermocouples (Pico, UK) (connected to a PC for data recording) were place along the longitudinal axis of the freezing bag, and the bags were frozen in SFD, each in one of the following velocities: 0.5, 1, 3 and 6 mm/sec. Each freezing velocity was repeated 3 times, and the temperature was measured by the thermocouples continuously. All samples were frozen to a thickness of 9 mm. The results are shown in
Comparing
At a cooling rate of 0.5 mm/sec (
Background
Short dated RBC (i.e. blood cells that were stored for some time; “sdRBC”) are packed RBC units collected into CPDA-1 and stored for 28 days at 4° C. Sometimes there is need to preserve sdRBC (e.g. when rare blood type RBC is near expiry).
After such storage the sdRBC were treated as described above for fresh “new” packed RBC. 3 units, with a final volume of 300 ml each, were prepared. One unit was pooled from two donors and two others were from a single donor each. Freezing was done using the SFD at 1 mm/sec. All bags were stored in −80° C. overnight before thawing in the Activator at 45° C. Thawing time measured 75 seconds. Hct, cell count, MCV and free hemoglobin were measured and the results are shown in Table 5.
It is known in the art that sdRBC have approximately the same amount of free Hb as freshly harvested RBC (with a slight immeasurable increase). As seen in Table 5, sdRBC survived freezing and thawing, resulting with only about 2-3% hemolysis (see free Hb values).
Specific Details:
The present experiment was conducted similarly to Experiment No. 5, with the following differences: Packed RBC (300 ml) were provided by the Red Cross and stored in 4° C., for a total time of 34 days from the donation. The Red Cross in the USA usually collects blood into CPD containing bags. Later, the blood is centrifuged and ADSOL (aka AS-1 and SAG-M) is added to the packed cells (pellet). These units of packed RBC (with ADSOL) can be stored in refrigeration (2-8° C.) for up to 42 days. Each blood unit was mixed with 250 ml IMT-1 freezing solution and transferred into a nylon/PE bag (total volume 550 ml; produced from PerfecFlex 30652W, from PerfecSeal). The results are depicted in Table 6. Het and MCV are presented as compared to the fresh control.
As seen above, 34 days old sdRBC survived the prolonged storage followed by freezing (SFD, 1 mm/see), storage at 80° C. and thawing (Activator 45° C.). The cell count, MCV and Hct were normal. In addition, free Hb levels of thawed units were very low, indicating about 97% survival of RBC.
Background:
The Red Cross normally collects blood into bags containing CPD (Anticoagulant Citrate Phosphate Dextrose USP containing Sodium Citrate (dihydrate) 26.3 g/L Dextrose (monohydrate) 25.5 g/L Citric Acid (anhydrous) 3.27 g/L and Monobasic Sodium Phosphate (monohydrate) 2.22 g/L). The blood is centrifuged and to the packed RBC AS-1 (ADSOL, also termed SAGM) is added (containing Sodium Citrate (dihydrate) 26.3 g/L, Dextrose (monohydrate) 25.5 g/L, Citric Acid (anhydrous) 3.27 g/L, Monobasic Sodium Phosphate (monohydrate) 2.22 g/L, Sodium Chloride, Mannitol and Adenine). Packed units that are with ADSOL can be stored up to 42 days in refrigeration. In Israel most of the donated blood is collected into bags containing CPDA-1 (Anticoagulant Citrate Phosphate Dextrose Adenine Solution USP Dextrose (monohydrate) 31.9 g/L Sodium Citrate (dihydrate) 26.3 g/L Citric Acid (anhydrous) 3.27 g/L Monobasic Sodium Phosphate (monohydrate) 2.22 g/L and Adenine 0.275 g/L). packed RBC units in CPDA-1 can be stored up to 35 days in refrigeration.
Packed RBCs, received from the Red Cross (RC) and Magen David Adorn (MDA) were each mixed in a ratio of 1:1 (v/v) with IMT-1 freezing solution to a final volume of 440 ml. The mixture was transferred to a nylon/PE bag (produced from PerfecFlex 30652W, from PerfecSeal) and then frozen by the SFD at 1 mm/sec. The bags were stored for 3 hours in a −80° C. freezer before thawing. Thawing lasted about 120 sec in an Activator. The results are depicted in Table 7, where Hct is depicted as percentage from fresh sample.
The results in Table 7 show that there was no significant difference between the fresh and the post thaw cell count and MCV. In addition, when comparing the free Hb levels all samples showed very high survival rates (about 97-98% survival) with apparently no significant difference between the different storage conditions/solutions.
Specific Conditions:
A pool of 4 Packed RBCs units was used to produce 500 ml units in four nylon/PE bags (produced from PerfecFlex 30652W, from PerfecSeal). Freezing was in SFD (1 mm/sec). After freezing, the bag was immediately transferred to storage in dry ice for two hours and was then thawed. The three other bags were stored first in LN for two hours and then were transferred into dry ice for 19, 25 and 48 hr. Storage in dry ice was done in an expanded polystyrene box. The box contained 8 Kg dry ice placed below the blood bags and 5 Kg dry ice was placed on top of the frozen RBC bags. No ice was added during storage. Thawing of all the bags was done in the water bath activator (45° C.).
Since during freezing ice was not added to the ice box, the temperature in the ice box was followed during storage by measuring the temperature of frozen dextran solution kept in dry ice in five different locations in the box. The results are shown in
As seen in
Upon removal from the ice box, the bags were thawed, and then assayed for MCV, Het and free Hb. The results are shown in Table 8.
As seen in Table 8, storage in dry ice has resulted in very high survival rates up to 25 hours with more then 98% RBCs survival (manifested by free hemoglobin values). During this time, the box temperature was between −72 and −77° C. (below −70° C). An increase in hemolysis was detected at storage for 48 hours which resulted in about 94% RBC survival. This may be explained by the rise in temperature during the period between 25 and 47 hours of storage. The final temperature, in the vicinity of −50-−55° C. may be higher then the glass transition temperature of the sample causing recrystallization due to which there was increase in cells lysis.
To date blood bags are normally manufactured of PVC. Such bags are not chilled below −80° C., and would not endure exposure to LN. Stem cells are frozen in EVA bags. These bags remain intact after freezing of up to 100 ml preparations in LN.
The present inventors found that EVA bags holding more than 100 ml blood preparation tended to crack during freezing, thawing and/or storage. Therefore it was desirable to find a new material to prepare freezing bags. Three types of bags were used in various experiments. The bags were made to order from the materials as detailed in Table 9. 300 ml blood preparation (RBC/IMT-1 mixtures) were frozen in each bag.
It was found that 35785 bags tended to crack significantly more often thank the other two. Therefore Nylon/PE and Aluminum/PE bags are preferred. Aluminum/PE is also preferred for better conductivity and nylon/PE also preferred for transparence. Both aluminum/PE and nylon/PE bags provided acceptable RBC recovery (data not shown).
Samples comprising RBC and IMT-1 were prepared essentially as described above in Experiment 1 and placed into 310×200 mm aluminum/PE bags. The average blood unit age before freezing was 5.67 days (SD=1.32). Sample volume in each bag was 300 ml. A sample from each bag was taken before freezing (but after addition of IMT-1 freezing solution) to serve as a “fresh” control. The samples were frozen using the SFD essentially as described above. After freezing was completed, the bags were transferred to a mechanical freezer for storage at −80° C. (Forma, USA) for overnight storage.
Thawing was performed in a DTD, wherein the plates were heated to a temperature of 70° C. before thawing, and heating was stopped as soon as the frozen bag was inserted to the device.
Table 10 compares RBCs parameters (Mean±SD) of 9 units of RBCs before and after freeze-thawing. The control value for each measurement was obtained from samples taken from the same blood unit prior to freezing after the addition of IMT-1 solution (RBC+IMT-1 freezing solution).
Packed RBCs from 2 donors were mixed in a 1:1 ratio (v/v) with IMT-1 freezing solution. A nylon bag with 500 ml was frozen using SFD. The post freeze thickness of the bags was 9 mm. After freezing the bags were stored in a −80° C. mechanical freezer (Forma, USA) for two hours. Thawing was done in the DTD set to 70° C. before thawing, and thawing lasted 135 sec,
Cell count, mean corpuscular volume (MCV) and hematocrit were measured as described above. The level of free hemoglobin (Hb) was determined as well before and after freeze thawing using HemoCue plasma/low Hb photometer device (HemoCue AB, USA). The results are shown in Table 11.
As seen in Table 11, in both donors cell count, hematocrit and MCV did not change significantly before and after thawing. In addition, free hemoglobin levels were very similar for the same freezing and thawing conditions resulting with about 3.5% free hemoglobin after freeze thawing.
Experiment No. 12. Freezing and Thawing a RBC Sample at 3 mm Thickness with Low Hematocrit
Packed RBC were mixed in a 1:2 ratio (v/v) with IMT-1 freezing solution. 250 ml samples were placed in each nylon bag, and were frozen using the SFD, with the following modification.
Two bags were placed side by side in a single cartridge, each abutting an opposing perforated wall of the cartridge. The bags were separated by a 3 mm flexible foam sheet of polyurethane. The post freeze thickness of each bag was 3 mm. After freezing the bags were stored in a −80° C. mechanical freezer (Forma, USA) for two hours. Thawing was done in the DTD set to 70° C. before thawing.
Cell count, mean corpuscular volume (MCV), hematocrit and free hemoglobin (Hb) were determined as described in Experiment No. 11. The results are shown in Table 12. As seen in Table 12, the low hematocrit (20% or less or even 15% or less, or about 12-13%) improved post thaw viability. Similarly it is concluded that reducing the width of the bag to below 9 mm (or even below 5 mm, or to about 3 mm) improved post thaw viability.
Donkey blood was obtained from “Segera Farm”, Ilania, Israel. The blood was collected in a regular collection bag, containing CPDA-1 (MacoPharma, France). All donkeys transfusions were autologous.
A day before the transfusion, approximately 500 mL of whole donkey blood was drawn out into CPDA-1 bags and sent to CD laboratories. The blood was centrifuged (4000 g, 10 min) and the supernatant plasma removed. The packed RBC were washed three times with saline-glucose buffer (1000 g, 10 min), and then mixed in a 1:1 ratio with the same buffer.
Donkey blood was labeled with Fluorescein isothiocyanate (FITC) (Sigma, USA), a common fluorescent dye, according to the protocol of Horie [Horie Y, Kato S, Ohki E, Hamamatsu H, Fukumura D, Kurose I, Suzuki H, Suematsu M, Miura S, Ishii H.: Effect of lipopolysaccharides on erythrocyte flow velocity in rat liver, J Gastroenterol., 783-90;32(6): December 1997]. After adding the FITC solution; the cells were gently shaken for 1 hour at room temperature and then washed four times with the saline- glucose buffer. The saline-glucose buffer is comprised of 118.5 mM glucose, 3.68 mM Na barbiturate, 1.25 mM barbituratic acid, 0.55 mM Mg sulafate.7H2O, 0.15 mM CaCl.2H2O, and 58.2 mM NaCl.
When fresh (unfrozen) RBCs were to be transfused, the FITC stained RBCs were transferred to a sterile bag containing CPDA1, and kept at 4° C. overnight. The next day the unit was transported on ice (approx. 5° C.) to the farm and then transfused to the donkey over the course of an hour.
When frozen-thawed cells were to be transfused, the donkeys RBC were stained and then mixed in a 1:1 ratio with IMT-1 freezing solution, frozen essentially as described above, and stored overnight at −80° C. The following day the frozen sample was thawed using DTD as described in experiment 12, and its contents were transferred into a transfusion bag for administration to the donkey.
After completion of the transfusion, blood samples were drawn out from each donkey at the following time points: ten minutes and 2, 4 and 24 hours. The time point of 10 minutes after transfusion was used as the base line measurement (i.e. the concentration of labeled cells at that time was defined 100%).
RBC recovery and survival was determined by measuring the percent of stained cells out of the total RBC population. To that end, blood samples were taken from the animals in Vacutainer-K2EDTA tubes (BD, Franklin Lakes, N.J., USA). 20 μl of this blood were then suspended in 980 μl of Dulbecco's Modified Eagle's Medium (DMEM, 4500 mg/l D-Glucose) (Biological Industries Inc., Beit-Haemek, Israel). Stained RBC were counted using a Fluorescence-Activated Cell Sorter (FACS), the number of stained and un-stained cells counted, and the ratio between them determined.
The results are depicted in
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/IL07/01120 | 9/11/2007 | WO | 00 | 10/29/2009 |
Number | Date | Country | |
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60843394 | Sep 2006 | US |