Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/02—Preservation of living parts
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/02—Preservation of living parts
  • A01N1/0236—Mechanical aspects
  • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
  • A01N1/0252—Temperature controlling refrigerating apparatus, i.e. devices used to actively control the temperature of a designated internal volume, e.g. refrigerators, freeze-drying apparatus or liquid nitrogen baths
  • A01N1/0257—Stationary or portable vessels generating cryogenic temperatures
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/02—Preservation of living parts
  • A01N1/0236—Mechanical aspects
  • A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
  • A01N1/02—Preservation of living parts
  • A01N1/0236—Mechanical aspects
  • A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
  • A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen

Definitions

• 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.
• US 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.
• US 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.
• US 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.
• US 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.
• US 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.
• US 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.
• US 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.
• PE 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.
• the present invention also disclosed a method for its freezing such that upon thawing, the material has less than 2% free hemoglobin.
• 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.
• biological material is biological material havning a preferred freezing rate of above l°C/min, at times above 5 0 C or even 100°C/min or more.
• cryopreservation means vitrification or freezing
• cryopreserved biological material means vitrified and/or frozen biological material in an essentially solid state.
• 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.
• a system for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis comprising:
• a bag holder for holding said bag, so that the biological material therein has a surface area S, and a volume V; a tank containing a cryogenic fluid, such as liquid nitrogen or the like;
• a guide member extending from said opening into said tank and adapted for engaging walls of said bag holder, when the bag holder is being immersed into the tank via said opening, to reduce widening of the bag during its being frozen by the cryogenic fluid.
• the tank and the immersion mechanism constitute together a submersion freezing device, also referred as SF device or SFD.
• SF device submersion freezing device
• the bag preferably comprises a high surface area-to- volume ratio, and a substantially constant width (e.g. about 4mm to about 27mm, in some embodiments preferably no more than 15mm), 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:
• the guide member may comprise a plurality of guides in the form of tongues depending into said tank from said opening and disposed on two sides of the longitudinal axis of the bag, which define therebetween a narrowing path and may also provide increasing lateral pressure on the bag before freezing thereof.
• the guides may be spaced by a proximal distance at a proximal portion of the guide member and a distal distance at a distal portion of the guide member, said distal distance being equal to or lesser than said proximal distance.
• the tank may comprise a cover, wherein the proximal portion of the guide member is attached to said cover at the opening's periphery.
• the cover may have an inner surface facing the interior of the tank, and the proximal portion of the guide member is attached to the inner surface.
• the tank may comprise level sensors for monitoring a level of the cryogenic fluid within the tank.
• the tank may comprise a floating buoy for preventing overflowing of the cryogenic fluid within the tank.
• the bag holder may be in the form of a cartridge comprising a pair of frame members, which constitute side walls of the cartridge, for placing the bag with the biological material in a space created therebetween.
• the cartridge along or in combination with a holding structure in the tank reduces or prevents forming irregularities or bulges in the shape of the bag.
• the bag holder may further comprise stiffening members to ensure that the bag maintain a generally uniform width when filled with biological material and is held vertically in the cartridge and during freezing.
• the system may comprise means for keeping volume of said cryogenic fluid in said tank within a certain range during the immersion of the bag with the biological material.
• the main purpose is to keep the cryogenic fluid surface at a certain level (within tolerance of 2-3 cm), which may be obtained by automatic control with level sensors or floating sensor and electromagnetic cryogenic valve.
• the system may further comprise a temperature control block (such as a heated block of thermo-conductive metal) for setting an immersion temperature of said bag to a pre-determined value higher than a temperature of said cryogenic fluid, thus providing a sharp temperature gradient in a relatively short distance, which may play a role in providing controlled directional freezing and may help to orient the ice crystals formed during the freezing process in a desired upward and inward direction.
• the temperature control block may be adapted for setting said immersion temperature to between about 0 0 C and about 3O 0 C. hi some embodiments the temperature is maintained at above room temperature, in which case cooling of the control block may be unnecessary.
• the temperature control block provides a starting temperature which is essentially independent of (or above) the ambient temperature.
• this feature provides greater repeatability in terms of the freezing process than may be obtained where the starting conditions are not. controlled and are therefore subject to fluctuation. Similarly, better repeatability may also be provided in terms of the post thaw viability results.
• the system may further comprise a velocity control means allowing to select a pre-determined value of said immersion velocity and to maintain said predetermined value constant during said immersion.
• the immersion velocity can be chosen optimally to maximize and reduce or prevent variations in the heat transfer between the bag and the cold source (while reducing or preventing boiling of the liquid nitrogen, when this is the cold source, which may happen when the immersion velocity is too fast), while reducing the exothermic reaction period associated with the freezing and/or increasing the cooling rate of the biological material. It is noted that a high velocity of immersion may cause boiling of the cryogenic fluid which would significantly reduce the temperature gradient between the fluid and the bag during freezing, thereby reducing the cooling rate at least in some portions of the biological material.
• the immersion velocity can be chosen optimally to maximize and prevent variations in the post thaw viability of the biological material.
• the system may further comprise means for reducing boiling of the" cold temperature source.
• Such means may include, without limitation, suitable cooling means for cooling the cold source to as low temperature as possible without solidification (e.g. liquid nitrogen may be cooled down to about -
• the system may further comprise means for increasing the volume of the cold temperature source and/or adding flow or turbulence thereto.
• the cold temperature source may be chosen to provide a desired cold temperature, being below the freezing temperature of the biological material, and optionally below -80 0 C, below -100 0 C or even lower.
• Examples of cold temperature sources may include liquid nitrogen, liquid helium, liquid hydrogen, and liquid alcohols.
• a system for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis comprising:
• a bag holder for holding said bag, so that the biological material therein has a surface area S, and a volume V;
• a velocity control means allowing to select -a pre-determined value of said immersion velocity from said plurality of values, and to maintain said predetermined value constant during said immersion.
• a system for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis comprising:
• a bag holder for holding said bag, so that the biological material therein has a surface area S, and a volume V;
• a temperature control block for setting an immersion temperature of said bag, prior to said immersion, to a pre-determined value higher than a temperature of said cryogenic fluid.
• a system for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis comprising:
• a bag holder for holding said bag so that the biological material therein has a surface area S, and a volume V;
• 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. , .
• a method for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis comprising:
• a method for cryopreserving a liquid biological material disposed in a bag having a longitudinal axis comprising: placing the bag in a bag holder; and immersing said bag holder into a cryogenic fluid in a direction parallel to said longitudinal axis at an invariant immersion velocity selected so as to ensure that cooling behavior of the biological material in a range of temperatures T within time t at least at three different locations along the longitudinal axis of the bag is defined by a T(t) curve having initial curved portion, central essentially linear portion and final curved portion, the central portions corresponding to more than half of the range of temperatures, and at least the central portions being parallel at said at least three locations.
• a system for warming cryopreserved liquid biological material disposed in a bag, during which the material changes its state from solid into liquid comprising: a heat source;
• a warming device having a space for placing said bag therein, connected to the heat source and adapted to transfer heat from the heat source to the bag; and means for maintaining the heat source in heat transfer contact with a solid portion of said material to allow receiving said heat by said solid portion.
• 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 warming device may comprise a pair of plates with a suitable thermal mass, creating therebetween said space for the bag, at least one of said plates may be used as the heat source.
• the plates may be adapted for sandwiching an amount of cryopreserved (e.g. frozen) biological material therebetween to provide a predetermined desired temperature gradient to generate a high warming (e.g. thawing) rate.
• At least one of the plates may be further adapted to move with respect to the other plate in a direction perpendicular to a longitudinal axis of said bag, thereby applying pressure on the bag.
• the heat source may be adapted to continually supply heat to the warming device until all the cryopreserved material is liquefied and removed.
• the warming device may be adapted to store heat supplied by the heat source.
• the warming device may comprise heat storage means and the heat source is adapted to supply heat to said heat storage means of the warming device only prior to the warming process, during which the heat stored in the warming device is supplied to the bag.
• the bag may have a cryopreserved material section filled with the cryopreserved material before said warming and an empty section constituting said empty space, which is free of the cryopreserved material and is adapted to accommodate said liquefied material.
• the heat source may be an electrical heater.
• the device may be portable. It may be sufficiently light weight and be able to perform adequately using a portable energy source.
• 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.
• a method for warming a cryopreserved liquid biological material disposed in a bag comprising:
• a system for performing the above method may also be referred to as dry thawing device, a "DT device” or simply as "DTD".
• a system for warming a cryopreserved liquid biological material disposed in a bag having a longitudinal axis comprising:
• a warming device having a space for placing said bag therein and fluid accommodating means for accommodating a fluid therein, the device being adapted to transfer heat from the fluid to the bag; and means for mamtaining a liquefied material within the bag evenly distributed along said longitudinal axis thereby improving heat transfer between said fluid and said cryopreserved material.
• a wet thawing device “forced flow thawing device”, “FT device”, “Activator” "FFTD or "WTD”.
• the warming device may comprise a pair of plates creating therebetween said space for the bag.
• the fluid accommodating means may be channels comprised within at least one of said plates.
• the channels such as heat transfer passages, may provide direct thermal contact between the fluid and the bag, and between the bag and those portions of the plates that are between the heat transfer passages.
• At least one of said plates may be adapted to move with, respect to the other plate in a direction perpendicular to said axis, thereby applying pressure on the bag.
• the device may comprise an empty space for accommodating of any liquefied material apart from the cryopreserved material.
• the device may prevent an increase of the surface-to-volume ratio during warming, by ensuring a relatively constant width of the bag, perpendicular to the longitudinal axis of the bag.
• Such increase of the surface-to- volume ratio during warming may occur when, instead of the warming device of the present invention, biological material in a bag is thawed directly in a tank filled with heating fluid, for example due to accumulation of the liquefied portion of the biological sample during warming (typically in the area of the bottom of the bag).
• the system may comprise, in addition to the warming device, an immersion tank filled with said fluid.
• the system may further comprise a pump for pushing said fluid from said tank to said fluid accommodating means.
• the fluid may be warm water.
• 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.
• 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.
• a method for warming a cryopreserved liquid biological material disposed in a bag having a longitudinal axis comprising: providing a heat source;
• a warming device having a space for placing said bag therein and fluid accommodating means for accommodating a fluid therein, for transferring heat from the fluid to the bag; providing means for maintaining a liquefied material within the bag evenly distributed along said longitudinal axis, thereby improving heat transfer between said fluid and said cryopreserved material;
• 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.
• 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.
• the thermal contact between the liquefied portion and the heat source may cause overheating of portions of the biological material.
• 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.
• 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 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.
• 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.
• more than one bag may be placed between the heat transfer plates (e.g. side by side), for example.
• Figs. IA to ID show the effect of cooling rate on homogeneity of freezing provided with a device according to one embodiment of the invention, as obtained in Experiment No. 4.
• Fig. 2 schematically illustrates an embodiment of a freezing system according to one embodiment of the invention.
• Fig. 3 schematically illustrates in isometric view an embodiment of a cartridge for use with the system of Fig. 2.
• Figs. 4 and 4a schematically illustrate, in isometric view and side view respectively, another embodiment of a cartridge for use with the system of Fig. 2.
• Figs 5a and 5b illustrate in plan view and side view, respectively, an embodiment of a freezing and thawing bag for use with the embodiments of Figs. 1 to 4, 7, 9a and 9b.
• Figs. 6a and 6b compare temperature gradient effects obtained with and without a temperature controlled zone above the cold temperature source.
• Fig. 7 schematically illustrates an embodiment of a thawing system according to one aspect of the invention.
• Fig. 8 illustrates in plan view another embodiment of a freezing and thawing bag for use with the embodiments of Figs. 1 to 4, 7, 9a and 9b.
• Figs. 9a and 9b schematically illustrate another embodiment of a thawing system according to one aspect of the invention.
• Fig. 10 illustrates results obtained in Experiment 8.
• Fig. 11 is a schematic representation of a desired cooling behavior according to some embodiments of the invention.
• the chart depicts the temperature (T) taken at different times (t) during immersion of the bag. The temperature is measured at three different locations (channels) along the longitudinal axis of a bag containing biological material.
• Fig. 12 is a chart depicting survival of fresh donkey RBC (closed circles) and freeze-thawed donkey RBC (open circles) during 24 hours after transfusion of FITC labeled cells into donkeys.
• the system comprises an apparatus or device 20 for providing a cold temperature source, including a feed mechanism 50 for immersing and retracting into the device 20 a cassette or cartridge 60 comprising a bag 90, having a longitudinal axis 90a containing the biological materials to be frozen.
• 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 T 2 of about -196°C (or at any other suitable temperature, preferably at or below about -8O 0 C) 5 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 D 1 and a distal distance D 2 therebetween, so that D 2 ⁇ D 1 .
• the distal distance D 2 is equal or lesser than the width dimension W of the opening 28, i.e. D 2 ⁇ 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 fixrtiier comprise suitable maximum and minimum level sensors, LS max and LS m i n for monitoring the level of LN in the tank 21 (although the device may be operated with only one sensor or with no sensors).
• suitable maximum and minimum level sensors LS max and LS m i n for monitoring the level of LN in the tank 21 (although the device may be operated with only one sensor or with no sensors).
• 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 LS max 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.
• 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).
• the cartridge 60 comprises a pair of frame members 61, 62, hinged together at a common pivot axis via hinge 63 at the lower ends thereof, enabling the frames to pivot with respect to one another from a closed position illustrated in Figs. 2 and 3, to an open position.
• the frame members 61, 62 are similar in size and shape, which may be rectangular as illustrated, or any other suitable shape.
• frame member 61 comprises an opening A defined by side elements 61b, 61c, bottom element 61a, and top element 61d
• frame member 62 similarly has an another opening B (not seen in the figures) defined by side elements (only 62c visible in Fig 3), bottom element 62a, and top element 62d.
• Each of the frame members 61, 62 comprises a wall 30 spanning the openings A, B 5 respectively.
• 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.
• 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.
• the frames 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.
• 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).
• 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 inclde 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.
• 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.
• the cartridge 60' comprises all the elements and features of the embodiment of Fig. 3, mutatis mutandis, with some differences, as will be described herein.
• the cartridge 60' comprises a pair of frame elements 61', 62', hinged together at 63', and each frame member 61', 62' defining an opening, A' and B' respectively, comprising a perforated wall 30'.
• cartridge 60' may each comprise one or more webs or rods 68' on the top-inner sides of one or both walls 30, optionally parallel to and below the corresponding top elements 61d' and 62d' of frame members 61' and 62' 5 respectively.
• These webs or rods 68' may provide several functions.
• the rods when cartridge 60 is in the closed position, the rods may serve as spacer elements, to define spacing S between the walls 30 in which the bag 90 may be accommodated.
• the webs/rods 68' may be configured for enabling the top portion of bag 90 to be tightly pressed between them when the cartridge is in the closed position, thus preventing crystal growth above the rods' level. Accordingly, when in use, the bag 90 may be placed in such manner that the portion of the bag immediately above the level of the biological material in the bag is tightly pressed between the rods 68'.
• the frame members 61', 62' may be held together in the closed position via latches 69', which may be provided on one of the handles 64a' and designed to engage with respect to the other handle 64b'.
• cartridge 60 may also comprise rods similar to rods 68' described for cartridge 60', mutatis mutandis.
• the bags 90 each comprise form or shape that is substantially complementary to that of the spacing between the frame members 61, 62 of cartridge 60 (or frame members 61', 62' of cartridge 60'), and allows the bag to be accommodated therein when the cartridge is closed.
• Each bag may be made from a pair of durable flexible sheets 91, 92, having a rectangular or other plan form, and joined together at the peripheral edges 93, to provide a containment volume Q between the sheets, which are spaced by a spacing SQ.
• One or more openings 95 may be provided, for example formed as closable tubes between facing parts of said peripheral edges, to allow the bags to be filled or emptied.
• the bags 90 are also designed to provide a high surface area / volume ratio at a thin width or spacing So-
• a bag 90 may have rectangular dimensions of about 265mm by about 360mm, and/or a holding volume of between about 300ml and about 500ml or 600ml, and/or a spacing So of between about 4mm to about 12mm or to about 27mm or more (including for example between about 8mm to about 10mm).
• 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.
• 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).
• the feed mechanism 50 is located above the tank 21, and comprises a feeding passage 52 having an outlet 53 that is aligned with the slot-shaped opening 28, and adapted for receiving therein a cartridge 60 via inlet 54.
• Suitable drive ⁇ means (not shown) are provided for translating the cartridge 60 along direction Pl linearly along passage 52 and into the tank 21 via opening 58, at a predetermined immersion velocity, v with respect to the tank 21, and particularly the LN therein, as will be further described herein.
• the drive means also enable the cartridge with the bag to be removed from the tank 21 in direction P2 after the freezing process is completed.
• a temperature control block or jacket 55 Surrounding the passage 52 is a temperature control block or jacket 55, which is adapted for setting the datum or start temperature of the bag generally independent of fluctuations in the external ambient temperature.
• the feed mechanism further comprises a heater 57, for example an electrical heater, coupled to the jacket 55, for establishing in the passage 52, and particularly in a bag-containing cartridge 60 therein, a temperature Ti greater than the freezing temperature of the biological material and optionally also above the ambient temperature To arising above the cold temperature source.
• a heater 57 for example an electrical heater
• the jacket 55 may have coupled to it also cooling elements and closed loop control using thermocouples, and other suitable features and components.
• 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 binge 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 bag 90 With its contents.
• 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 Pl is as shown in Fig. 11 for three different channels Cl, C2 and C3.
• Fig. 11 shows that in a range of temperatures T within time t the cooling behavior is defined by a T(t) curve having initial curved portion HA 3 central essentially linear portion HB and final curved portion HC, wherein the central portions correspond to more than half of the range of temperatures, and at least the central portions for all the three channels are parallel.
• 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.
• TMs arrangement ensures that the bags have an essentially constant width So throughout, minimizing the bulging that otherwise accompanies material frozen in bags.
• 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.
• 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.
• 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 LS max and LS m j n .
• the rninimal 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 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.
• 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.
• an immersion velocity v of between about lmm per second and about 3mm 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.
• the preferred cooling rate is high (above 5°C) and the biological sample is bulky
• 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.
• a device and method for warming e.g. thawing
• biological materials for example blood and blood components according to some embodiments.
• the device generally designated herein with the numeral 200 and illustrated in Fig. 7, comprises a housing 210 having an upper part 214 and a lower part 212, respectively accommodating an upper heating plate 234 and a lower heating plate 232.
• the upper part 214 is displaceable with respect to the lower part 212, for example by means of a hinge arrangement, between an open position, in which a frozen bag 90 may be placed over the lower heating plate 232, and a closed position in which the plates 232, 234 are in generally overlying relationship sandwiching the bag 90 therebetween.
• Each plate 232, 234 comprises a pad 222, 224, respectively having a high thermal mass, and a heating element, 252, 254, respectively, for example electrical heaters, for heating the respective pads 222, 224.
• Suitable springs 270 are provided between each plate 232, 234 and the respective casing part 212, 214, that provide a mechanical pressure force onto the bags when this is in the device 200 when in the closed position.
• a pneumatic, hydraulic or other arrangement may be provided to generate this pressure force.
• a ramp 260 (in this example, a downwardly sloping ramp) is provided on the bottom part 212 for supporting an empty (vacuumed) part 99 of the bag 90, i.e. a part thereof in which no biological material was cryopreserved.
• a conduit leading from the bag interior may be supported by the ramp, the conduit being connected to a vessel or other bag to collect the liquefied contents directly.
• the heaters 252, 254 heat the pads 222, 224, to a suitable temperature T w , which by way of non-limiting example may be between about 5O 0 C and about 9O 0 C, preferably about 70 0 C, and sufficient to thaw a particular volume (or width) in a desired time period.
• T w a suitable temperature
• 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.
• 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.
• thermal (heat transfer) contact between the frozen contents of me bag and the heated pads 222, 224 is maintained (or even maximized) and thermal contact between the thawed contents and the pads is reduced.
• the pads 222, 224 may be continually heated by the heaters 252, 254 during the thawing operation.
• 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.
• a bag filled 500ml 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 0 C.
• 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 0 C.
• a variation of a freezing bag, designated 90' is illustrated, having a freezing compartment 192 and a collection compartment 194, joined via a communicating passage 195, but otherwise similar to bag 90 as described above, mutatis mutandis, having at least one inlet tube 197 to the freezing compartment.
• Other mechanisms for removal of the thawed portion of the biological material are mentioned above.
• the collection compartment is sealed off via a valve 196 or by clamping shut the passage 195, for example using an external clamp or via the internal clamping action of rods such as rods 68' when the cartridge is fitted with such rods, and the biological material is introduced into the freezing compartment 192 only.
• the bag 190 and contents are then frozen.
• the whole bag, or just the freezing compartment 192 is placed between the plates 232, 234, and the passage 195 opened, thereby enabling the thawed contents to be received in the collection compartment 194, while enabling the thermal contact between the pads and the frozen contents, via the bag walls, to be maximized.
• a filter or mesh may be provided in device 200 for preventing small frozen fragments from leaving the bag volume that is being heated by the pads.
• 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.
• 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;
• the warming device generally designated herein with the numeral 300 and illustrated in Figs. 9a and 9b, comprises a housing 310 defining an immersion tank 312 and optionally a cover 314, and an integrated unit 350 comprising a heat exchanger 320 in fluid communication with pump unit 340.
• the heat exchanger 320 comprises opposed vertically arranged heat exchanger plates 322, 324, defining a holding space M therebetween.
• the tank 312 comprises a duty fluid such as water, capable of heat transfer at desired rates.
• 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.
• 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.
• cryopreserved material e.g. frozen biological material
• 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 0 C and about 60°C, and preferably between about 37°C and about 45 0 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.
• the two chambered bag 90' of Fig. 8 may be used with the embodiment of Figs 9a and 9b instead of bag 90, in a manner similar to that described with respect to the embodiment of Fig 7, mutatis mutandis.
• EGCG - Epigallocatechin gallate a green tea catechin. Purchased from Zhejiang Yixin Pharmaceutical Co., Ltd.
• IMT-I - A freezing solution composed of 20% (w/v) Dextran 40 (Pharmacosmos, Denmark) and 0.945mg/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 unfrozen fraction of the frozen solution, to buffer osmolality and/or to stabilize the membrane and prevent mechanical damage caused by ice crystals.
• CPAs examples include 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.
• 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
• 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 -8O 0 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 0 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:
• Hct Hematocrit
• Samples were placed into 310X200mm aluminum/PE bags. Sample volume in each bag was 300ml. 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 -8O 0 C freezer (Forma, USA).
• 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 -8O 0 C freezer had a sample thickness of 7.66 ⁇ 1.lmm.
• 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 depicts the MCV, cell number and hemoglobin assayed in thawed cells.
• 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).
• cells were frozen in a -8O 0 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.).
• sample thickness after freezing was highest in bags that were inserted manually..
• Blood solution (packed RBC with IMT-I freezing solution) was divided to four 300ml portions each placed in a 310mmX200mm 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.
• Short dated RBC i.e. blood cells that were stored for some time; "sdRBC"
• sdRBC Short dated RBC
• RBC units collected into CPDA-I and stored for 28 days at 4 0 C.
• Sometimes there is need to preserve sdRBC e.g. when rare blood type RBC is near expiry.
• sdRBC were treated as described above for fresh "new" packed RBC. 3 units, with a final volume of 300ml 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 0 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.
• 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).
• 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).
• 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-I (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, hi Israel most of the donated blood is collected into bags containing CPDA-I (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-I can be stored up to 35 days in refrigeration.
• CPDA-I Anticoagulant Citrate Phosphate Dextrose Adenine Solution
• USP Dextrose (monohydrate) 31.9 g/L Sodium Citrate (dihydrate) 26.3 g/L
• Monobasic Sodium Phosphate (monohydrate) 2.22 g/L and Adenine 0.275
• a pool of 4 Packed RBCs units was used to produce 500ml units in four nylon/PE bags (produced from PerfecFlex 30652 W, 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 5Kg 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 0 C).
• Nylon/PE and Aluminum/PE bags are preferred. Aluminum/PE is also preferred for better conductivity and nylon/PE also preferred for transparence. Both alumiriurn/PE and nylon/PE bags provided acceptable RBC recovery (data not shown).
• Samples comprising RBC and IMT-I were prepared essentially as described above in Experiment 1 and placed into 310X200mm aluminum/PE bags.
• Sample volume in each bag was 300ml.
• a sample from each bag was taken before freezing (but after addition of IMT-I 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 0 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-I solution (RBC+IMT-1 freezing solution).
• Hb free hemoglobin
• Donkey blood was obtained from "Segera Farm", Ilania, Israel. The blood was collected in a regular collection bag, containing CPDA-I (MacoPharma, France). All donkeys transfusions were autologous.
• FITC Fluorescein isothiocyanate
• the saline-glucose buffer is comprised of 118.5 niM glucose, 3.68 inM Na barbiturate, 1.25 mM barbituratic acid, 0.55 mM Mg sulafate-7H2O, 0.15 mM CaCl-2H2O, and 58.2 mMNaCl.
• the FITC stained RBCs were transferred to a sterile bag containing CPDAl, and kept at 4 0 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.
• the donkey's RBC were stained and then mixed in a 1:1 ratio with IMT-I freezing solution, frozen essentially as described above, and stored overnight at -80 0 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.
• RBC recovery and survival was determined by measuring the percent of stained cells out of the total RBC population.
• blood samples were taken from the animals in Vacutainer- K2EDTA tubes (BD, Franklin Lakes, NJ, USA). 20 ⁇ l of this blood were then suspended in 980 ⁇ l of Dulbecco's Modified Eagle's Medium (DMEM, 4500mg/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.
• FACS Fluorescence-Activated Cell Sorter

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