Apparatus for freezing of biological products

Abstract

To provide a method of determining an amount of cryoprotectant to be added to a biological product prior to cryopreservation.SOLUTION: A method comprises: a. determining the total surface area of an approximated geometry of the biological product, including an initial amount of cryoprotectant, to be cryopreserved; b. estimating thermal properties of the sample; c. performing computational fluid dynamics analysis on the sample within the apparatus; d. determining an average temperature reduction rate of the core of the sample at a predetermined sample surface temperature and corresponding heat exchange fluid flow rate to obtain the average temperature reduction rate; and e. if the fluid flow rate calculated at step (d) corresponds to a pump duty of the apparatus that is below a predetermined pump duty, selecting an amount of cryoprotectant that is a predetermined amount less than the initial amount to define a new initial amount.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for freezing biological products for storage. [Background technology]

[0002] The ability to store red blood cells (RBCs) outside the body has been relevant to the performance of life-saving procedures for many years. More recently, the use of cryopreserved RBCs in transfusion medicine has been widely evaluated. During cryopreservation of RBCs, they gradually deteriorate, and infusion of long-term stored RBCs has been associated with poor medical outcomes in terms of postoperative infections, prolonged hospital stays, and survival rates.

[0003] Problems with transfusion of stored RBCs remain, and restrictive transfusion strategies are still preferred. This has led to a resurgence of interest in cryopreservation. Storage of RBCs at ultra-low temperatures halts cellular metabolism and prevents the progressive cellular deterioration that is subsequently associated with adverse medical outcomes.

[0004] Cryopreservation initially emerged as a viable approach to maintain RBCs in a viable state for extended periods of time, but the medical application of cryopreserved RBCs (commonly known as frozen RBCs) was hindered by the high cost, time-consuming, and inefficient storage methods.

[0005] Requirements for cryopreserved RBCs Currently, RBCs are often stored at 2 to 6 °C for up to 5 to 6 weeks, depending on the preservation solution used. Cryopreservation, on the other hand, allows the storage of RBCs for several years. Cryopreservation is currently a useful approach for long-term storage of RBCs from donors with rare blood types and for military deployment. However, stockpiles of frozen RBCs are also beneficial in emergency or medical situations where the demand for RBCs exceeds the supply. The shelf life of frozen RBCs using current methods is up to 10 years.

[0006] International guidelines require that hemolysis in RBC cryopreservation units must be kept below acceptable levels (i.e., 0.8% in Europe and 1% in the United States) and that at least 75% of infused RBCs must circulate for an additional 24 hours after infusion.

[0007] However, the guidelines do not specifically reflect the ability of RBCs to function after infusion.

[0008] Quality of stored RBCs Storage at 4°C slows down biological processes in RBCs, but cellular metabolism is not completely inhibited at these temperatures. During cryopreservation, various changes have been observed that compromise the ability of RBCs to function after infusion. These changes include reduced concentrations of 2,3-diphosphoglycerate (DPG), adenosine triphosphate (ATP), and membrane sialic acid content within RBC storage units. Other changes include translocation of phosphatidylserine (PS) to the cell surface, oxidative damage to membrane lipids and proteins, shape change to globular echinocytes, membrane blebbing, and accumulation of potassium, free hemoglobin (Hb), cytokines, bioactive lipids, and (procoagulant) microvesicles.

[0009] The rheological properties of RBCs are also impaired during cryopreservation. Frozen RBCs show an increased tendency to aggregate and adhere to endothelial cells (ECs) and a decreased deformability from the second week of storage. These changes may impede the proper functioning of RBCs in the microcirculation.

[0010] Storage of RBCs at ultra-low temperatures stops the biological activity of RBCs, thereby allowing them to be stored for long periods of time. Generally, either high concentrations of cryopreservation additives or rapid freezing are required to prevent cell damage. At slow cooling rates, extracellular ice formation occurs. As ice forms, the non-frozen fraction becomes richer in solute content. The resulting osmotic imbalance causes fluid to move out of the RBCs, resulting in intercellular dehydration. On the other hand, at rapid cooling, RBC cell lipids become supercooled, resulting in intercellular ice formation, which subsequently leads to mechanical damage.

[0011] Cryopreservation additives are important to minimize freezing damage. Over the years, different non-permeable and osmotic additives for cryopreservation of RBCs have been investigated. Non-permeable additives such as hydroxyethyl starch and polyvinylpyrrolidone, as well as various glycols and sugars, seemed promising because they were proposed not to require removal from thawed RBCs prior to transfusion.

[0012] Conversely, glycerin, an osmotic additive, is known to have the ability to protect RBCs at cryogenic temperatures. The concentration of glycerin required to protect RBCs depends on the cooling rate and storage temperature. Glycerin protects RBCs by slowing the rate and extent of ice formation while minimizing cellular dehydration and solute effects during freezing.

[0013] Requirements for cryopreserved RBCs Protection of RBCs at ultra-low subzero temperatures allows for storage for many years, but once thawed, RBCs have a limited shelf life. RBCs removed from glycerol are primarily stored in SAGM (saline-adenine-glucose-mannitol) preservation solution for up to 48 hours or in AS-3 preservation solution for up to 14 days. Frozen stored RBCs must be deglycerolized to reduce the residual glycerol content to 1% or less. RBCs are also subject to the aforementioned international guidelines, which require that hemolysis within the RBC unit must remain below acceptable levels (i.e., 0.8% in Europe and 1% in the United States) and that RBC thaw recovery after deglycerolization (i.e., freeze-thaw-wash recovery) must exceed 80%. At least 75% of frozen RBCs must also circulate for 24 hours after transfusion.

[0014] Freezing method using glycerol Currently, there are two accepted freezing methods for preserving RBCs with glycerol. 1. RBCs are rapidly frozen in liquid nitrogen using the low glycerol method (LGM) at a temperature below 140°C with a final concentration of glycerol of approximately 20% (wt / vol). 2. RBCs are slowly frozen using the high glycerol method (HGM), which allows storage of RBCs units at temperatures between -65°C and -80°C with a final concentration of approximately 40% (wt / vol) glycerol.

[0015] Frozen RBCs are less efficient due to cell loss that occurs during the processing procedure. This cell loss is more pronounced in HGM cryopreserved RBCs (approximately 10-20%) because RBCs require more extensive washing. However, while LGM RBCs have a higher yield, HGM cryopreserved RBCs are generally considered to be able to tolerate a wide range of temperature fluctuations during freezing and are stable during the post-thaw stage. Also, HGM cryopreserved RBCs do not require liquid nitrogen, which facilitates storage and transport conditions. As a result, HGM is currently the most applicable RBC cryopreservation method in Europe and the United States. Summary of the Invention [Problem to be solved by the invention]

[0016] Storage methods associated with cryopreservation of HGM result in intercellular dehydration due to the high glycerol content and storage temperature range. Preferred embodiments of the present invention seek to use a lower glycerol content, thereby minimizing intercellular dehydration and solute effects while extending the shelf life of cryopreserved RBCs. [Means for solving the problem]

[0017] As used herein, "biological product" (or "biological material") includes the following non-exhaustive list of materials: blood, plasma, platelets, white blood cells or other blood products, germs, bacteria, fungi, or other microorganisms, organs, semen, eggs, colostrum, skin, lymph, vaccines, stem cells (e.g., from bone marrow, umbilical cord blood, amniotic fluid, etc.), umbilical cord, bone marrow, bacterial cells, tumor cells, semen, vaccines, and plant cells.

[0018] According to a first aspect of the present invention, there is provided an apparatus for storing a biological product comprising an inner housing disposed within an outer insulated housing, the apparatus being characterized in that the walls of the inner housing define a compartment for receiving the biological product, the walls having an inlet wall for flowing heat exchange fluid into the compartment and an opposite outlet wall for flowing heat exchange fluid out of the compartment, a side wall and a base, the side wall and the base joining the inlet wall to the outlet wall, the inlet wall and the outlet wall each including a series of apertures for accommodating a continuous flow of heat exchange fluid through the apparatus such that, during operation, a biological product received in the compartment of the inner housing is immersed in the heat exchange fluid for exchanging heat with the heat exchange fluid.

[0019] According to a second aspect of the present invention, there is provided a method for determining the amount of cryoprotectant to add to a biological product prior to storage, the method comprising: a. determining the total surface area of ​​the approximate geometric shape of the biological product to be preserved, including an initial amount of cryoprotectant, where the biological product, cryoprotectant, and any packaging define the sample; b. estimating thermal properties of the sample; c. performing a computational fluid dynamics analysis on the sample within the apparatus of the first aspect based on flow constraints including one or more of the approximate geometry of the sample, the thermal properties of the sample, the geometry of the apparatus, a predetermined location of the sample within the apparatus, a predetermined inlet temperature of the heat exchange fluid, and a predetermined increase in temperature of the heat exchange fluid from the inlet to the outlet; d. determining an average temperature drop rate of the core of the sample at a predetermined temperature of the sample surface and correlating the heat exchange fluid flow rate to obtain the average temperature drop rate; e. if the fluid flow rate calculated in step (d) corresponds to a pump duty of the device that is lower than the predetermined pump duty, selecting an amount of cryoprotectant that is a predetermined amount less than the initial amount to define a new initial amount, and if the fluid flow rate calculated in step (d) corresponds to a pump duty equal to the predetermined pump duty, selecting the initial amount of cryoprotectant as the amount of cryoprotectant to be added to the biological product prior to storage; and f. if the fluid flow rate calculated in step (d) corresponds to a pump duty lower than the predetermined pump duty, repeating steps (a) through (e) until the fluid flow rate calculated in step (d) corresponds to a pump duty equal to the predetermined pump duty.

[0020] According to a third aspect of the present invention there is provided a method for determining an amount of cryoprotectant to add to a biological product prior to storage, the method comprising: a. determining the total surface area of ​​the approximate geometric shape of the biological product to be preserved, including an initial amount of cryoprotectant, where the biological product, cryoprotectant, and any packaging define the sample; b. estimating thermal properties of the sample; c. performing a computational fluid dynamics analysis on the sample within the apparatus of the first aspect based on flow constraints including any one or more of the approximate geometry of the sample, the thermal properties of the sample, the geometry of the apparatus, a predetermined position of the sample within the apparatus, and a predetermined temperature increase of the heat exchange fluid from the inlet to the outlet; d. determining an average temperature drop rate of the core of the sample at a predetermined temperature of the sample surface and correlating the inlet temperature of the heat exchange fluid to obtain the average temperature drop rate; e. if the inlet temperature of the thermal fluid determined in step (d) corresponds to an evaporator duty of the apparatus that is less than the predetermined evaporator duty, selecting an amount of cryoprotectant that is a predetermined amount less than the initial amount to define a new initial amount, and if the fluid flow rate calculated in step (d) corresponds to a pump duty equal to the predetermined pump duty, selecting the initial amount of cryoprotectant as the amount of cryoprotectant to be added to the biological product prior to storage; and f. if the fluid flow rate calculated in step (d) corresponds to an evaporator duty lower than the predetermined evaporator duty, repeating steps (a) through (e) until the fluid flow rate calculated in step (d) corresponds to a pump duty equal to the predetermined pump duty.

[0021] According to a fourth aspect of the present invention there is provided a use of the device of the first aspect for storing a biological product.

[0022] The device may have trays, racks, or baskets designed to hold the relevant biological products to be stored.

[0023] Embodiments of the present invention will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a bottom perspective view of a tank for storage of biological products. [Diagram 2] FIG. 2 is a top perspective view of a tank for storage of biological products. [Diagram 3] FIG. 3 is a graph showing the specific enthalpy of blood at various temperatures. [Figure 4] FIG. 4 is a graph showing the thermal conductivity of blood at various temperatures. [Diagram 5] FIG. 5 depicts a cold storage vial of blood. [Figure 6] FIG. 6 shows the temperature plot of blood at various time intervals. [Figure 7] FIG. 7 shows the temperature / time profile of geometric increments of blood in a polypropylene walled cryogenic storage vial exposed to cryogenic storage with a heat exchange fluid inlet temperature of 25° C. [Figure 8] FIG. 8 shows the geometrically incremental temperature / time profile of blood exposed to cryopreservation with a heat exchange fluid inlet temperature of −50° C. [Figure 9] FIG. 9 shows the geometrically incremental temperature / time profile of blood exposed to cryopreservation with a heat exchange fluid inlet temperature of −70° C. [Figure 10] FIG. 10 shows the geometric increment temperature / time profile of blood exposed to cryopreservation with a heat exchange fluid inlet temperature of −50° C. [Figure 11] FIG. 11 shows the geometrically incremental temperature / time profile of blood exposed to cryopreservation with a heat exchange fluid inlet temperature of −50° C. and the relative movement between the cryopreservation vial and the heat exchange fluid at 0.2 m / s. [Figure 12] FIG. 12 shows the temperature / time profile of geometric increments of blood exposed to cryopreservation with a heat exchange fluid inlet temperature of −50° C. and the horizontal orientation of the cryopreservation vial. [Figure 13] FIG. 13 is a graph showing hemolysis for different storage scenarios. [Figure 14] FIG. 14 is a diagram of the piping instrumentation for the cooling system. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] 1 and 2 show an immersion tank 1 for storing biological products (or biological materials). Tank 1 is made of steel conforming to ASTM A240. Tank 1 has two heat exchange fluid inlets 2 and two heat exchange fluid outlets 3, the inlets 2 being disposed on an inlet wall 4 and the outlets 3 being disposed on an outlet wall 5.

[0026] 2 shows an inner housing 10 disposed inside the outer wall of the vessel 1. The inner housing 10 has an inlet wall 14, an outlet wall 15, and a base 16, each including apertures 11 to allow the flow of heat exchange fluid into and out of the inner housing 10. The apertures 11 are provided in four rows of ten on the inlet wall 14 and outlet wall 15, and in ten rows of ten on the base 16. The apertures 11 on the inlet wall 14 and base 16 are 10 mm in diameter, and the apertures 11 on the outlet 15 are 20 mm in diameter. The inlet wall 14 is spaced 100 mm from the inner surface 12 of the inlet wall 2, thereby providing a void 13. A similar void is provided between the outlet wall 15 and the inner surface of the straight wall 5.

[0027] A void spacing of 100 mm is further provided at the base. The interior surface 12 is defined by a steel sheet frame located 50 mm from the inlet wall 2 and secured by brackets to provide a cavity into which polyurethane foam insulation is pumped during manufacture of the vessel 1. Insulation is similarly provided on all four walls of the vessel from the vessel ceiling to approximately 595 mm below the vessel walls.

[0028] A row of 30 mm diameter holes 22 are provided along the bottom of each wall along strips 23 disposed at an angle of approximately 45° between the base 7 and the walls of the vessel. The strips 23 are provided to reinforce the vessel structure and may be used to guide trays or brackets to prevent them from touching the walls or base of the inner housing 10. It will be appreciated that other structures are possible that reinforce the vessel structure and perform the guide function. The holes 22 help reduce stagnation of heat exchange fluid that accumulates in areas of the vessel due to the presence of the strips 23.

[0029] Below the heat exchange fluid outlet 3 there is a drain 6 formed as an elbow pipe oriented from the base 7 of the vessel 1 to extend beyond the outlet wall 5 of the vessel 1. The heat exchange fluid inlet 2 and the heat exchange fluid outlet 3 have a diameter of 80 mm.

[0030] The tank 1 further has a lid formed from sheet steel (not shown). The base 7 of the tank 1 has four central legs 8 which support the central weight of the tank 1, as well as feet 16 located at the corners of the tank 1 and formed into the edges of the tank walls. Cutouts 9 are provided in the lower edges of the tank walls to provide access for maintenance of the tank base 7. The tank 1 is approximately 1.105m high and is square with a side length of 1.705m.

[0031] In use, the vessel 1 is filled with a heat exchange fluid that will not freeze at temperatures above -70°C. The heat exchange fluid is pumped into the cavity 13 of the vessel 1 through the heat exchange fluid inlet 2 at a volumetric flow rate of 17 cubic meters per hour. As the heat exchange fluid is forced through the restricted area of ​​the aperture 11, the pressure in the cavity 13 increases, thereby decreasing the volumetric flow rate but increasing the velocity of the fluid entering the inner housing 10. Some fluid flows under the inner housing 10 and is forced to the opposing cavity in the outlet wall 5, and some fluid flows through the aperture 11 in the base 7 of the inner housing 10. The aperture 11 improves the distribution of refrigerant to all parts of the vessel and minimizes the occurrence of hot spots that tend to occur away from the inlet area. As the heat exchange fluid continues to flow through the vessel 1, heat is removed from the biological product and the heated heat exchange fluid leaving the vessel 1 is then exchanged with a cooling system that continuously cools the heat exchange fluid. The heat exchange fluid itself is in heat exchange with a refrigerant in a cooling system.

[0032] Preferably, a low range heat exchange fluid is used for the immersion fluid bath which advantageously has a relatively low viscosity even at very low temperatures, thereby reducing the pump power requirements of the system. The following table (Table 1) provides some illustrative examples of thermal properties of heat exchange fluids.

[0033] [Table 1]

[0034] At each of the above temperatures, the heat exchange fluid is very thin, having a density less than that of water. Advantageously, if any breakage or leakage occurs during operation of the vessel, the broken or leaking material will tend to sink to the lower portion of the vessel, facilitating drainage of the material without substantial loss of heat exchange fluid. It will be appreciated that any suitable heat exchange fluid may be used, provided it has a sufficiently low viscosity without requiring excessive pumping power at the low temperatures required for storage. Preferably, the heat exchange fluid is safe for biological products.

[0035] [Table 2]

[0036] Table 2 gives the temperature between the vessel inlet and outlet for various flow rates of the heat exchange fluid, assuming that 20 kW of heat is extracted from the fluid in the vessel. From Table 2, it can be seen that a temperature difference of 3°C between the inlet and outlet is achievable using a mass flow rate of approximately 4 kg / s. This temperature difference was deemed to be an acceptable temperature rise for the required evaporator duty and the required cooling of the product. The acceptable temperature rise must be balanced against the cost for the maximum number of products that can be processed at one time to make the system economically viable. It can be seen that a higher flow rate is desirable in increasing the heat transfer between the heat exchange fluid and the consumables. However, a higher flow rate also creates a higher thermal resistance and therefore a higher pumping power is required.

[0037] The inventors have discovered that by using increased flow rates of heat exchange fluid to rapidly reduce the temperature of a biological product, the biological product can be preserved with reduced levels of cryoprotectant while minimizing damage from ice crystal formation that normally occurs during the freezing process.

[0038] The methodology for determining the correct temperature and rate to achieve vitrification of RBCs and other biological materials is based on calculations that increase thermal properties, surface area and product load volume. The calculated heat transfer coefficients inform the final device operating conditions that will enable the production of cryopreservation.

[0039] In one example, freezing of 0.5 ml of blood in a cryovial was investigated.

[0040] FIG. 5 shows a CAD model of a cryovial made from polypropylene with an internal volume of 0.5 mL, an outer diameter of 6 mm, a length of 27.5 mm, and a wall thickness of 0.5 mm.

[0041] Computational fluid dynamics (CFD) was used to calculate the freezing time for a cryovial filled with blood. For the CFD analysis, the cryovial was assumed to have a uniform 2°C temperature at the start of the simulation. It was immersed in heat exchange fluids at temperatures of minus 25°C, minus 50°C, and minus 70°C, respectively. Transient CFD then automatically calculates the heat exchange between the heat exchange fluid and the external surface of the cryovial. CFD also calculates the conduction of heat through the cryovial (blood and polypropylene).

[0042] Movement of blood during freezing has been ignored; i.e., blood is assumed to be a "solid". The thermal properties of blood are based on the assumption that it is composed of 85% water and 15% protein. Thermal properties of biological products can be obtained through methods known to those of skill in the art or can be found in thermal property tables known to those of skill in the art.

[0043] Figures 3 and 4 show the specific enthalpy and thermal conductivity, respectively, as calculated for blood. Thermal conductivity has been calculated by the Kopelman method.

[0044] The table below gives the core cooling times for heat transfer fluid temperatures of minus 25°C, minus 50°C, and minus 70°C.

[0045] [Table 3]

[0046] The temperature / time plots corresponding to each of the scenarios in Table 3 are shown in Figures 7 through 9. Figure 6 shows the temperature plots for minus 50°C for the heat transfer fluid at 10 second intervals between 30 and 100 seconds.

[0047] 7, the core temperature starts at about 2° C. at time 0 s and cools to a temperature of about −24° C. every 220 s, resulting in an overall average temperature drop rate of about 7° C. per minute. However, when observing the time window between about 125 s and 150 s, a rapid decrease in core temperature of about 14° C. occurs, resulting in an average temperature drop rate of about 34° C. per minute over that time interval.

[0048] 8, the core temperature starts at about 2° C. at time 0 s and cools by about −49° C. every 180 s, resulting in an overall average temperature drop rate of about 17° C. per minute. However, observing the time window between about 69 s and 75 s, a rapid decrease in core temperature of about 14° C. occurs, resulting in an average temperature drop rate of about 140° C. per minute over that time interval.

[0049] 9, the core temperature starts at about 2° C. at time 0 s and cools to a temperature of about −50° C. every 77.5 s, resulting in an overall average temperature drop rate of about 40° C. per minute. However, observing the time window between about 49 s and 52.5 s, a rapid decrease in core temperature of about 14° C. occurs, resulting in an average temperature drop rate of about 240° C. per minute over that time interval.

[0050] Advantageously, the rapid temperature drop may occur between about -2°C and -15°C, which is believed to be the range of interest for supercooling many biological products. In the example given above, the window for rapid temperature drop corresponds approximately to this temperature range.

[0051] In the above example, the steepest slopes of the graph are visually evaluated to determine the approximate maximum rate of temperature change. Another option is to model the data and plot the derivative, thereby determining the maximum instantaneous rate of change of the product. The required rate of change may be defined as being at least equal to a given rate of fall over a given time interval. For example, the rate of temperature fall may be required to be greater than or equal to 100° C. per minute for a 20 s interval.

[0052] The following additional scenarios were performed to explore the effects of alternative scenarios. All analyses below were performed with the heat transfer fluid at minus 50°C. These include the effects of: a. Effect of polypropylene walls: Polypropylene has a low thermal conductivity and therefore a high resistance to heat flowing from the blood to the heat transfer fluid. In this case, polypropylene was replaced with a steel wall. This shows the effect of using a higher thermal conductivity material and / or a thinner wall thickness. b. Effect of moving a cryovial horizontally through a heat transfer fluid at 0.2 m / s: The movement of the cryovial (or heat transfer fluid) enhances the heat transfer between the cryovial surface and the heat transfer fluid. c. Effect of orienting the cryovial horizontally: During the freezing process, the cryovial returns heat to the heat transfer fluid. The hotter fluid increases, and therefore the warmer heat transfer fluid adjacent to the cryovial, creating a movement of heat transfer fluid that increases the heat transfer from the cryovial surface. Orienting the cryovial horizontally may have a surface heat transfer effect.

[0053] The following table shows the freezing times versus the freezing scenarios for the alternative scenarios mentioned above. The results for Scenario 2 as described in Table 3 are repeated for ease of reference. Figures 10 to 12 show the corresponding temperature / time profiles, respectively.

[0054] [Table 4]

[0055] These results indicate that using a higher thermal conductivity / smaller thickness cryovial material has a significant effect. Thus, the movement of the heat transfer fluid / cryovial increases the surface heat transfer and has an effect on the freezing time. Orienting the cryovial horizontally also has a positive effect.

[0056] Tests were performed on the RBCs after exposure to this cryopreservation method.

[0057] Approximately 30 ml (6 x 5 mls) of blood was obtained from healthy volunteers by venipuncture. Whole blood was centrifuged at 1100 x g for 12 minutes at 4°C. The buffy coat and most of the plasma were removed and discarded. The enriched RBCs were then washed twice with phosphate buffered saline (PBS; pH 7.4) and resuspended in PBS to a final hematocrit (Hct) value of 50 ± 10%. The RBC enrichment was then transferred to a 50 ml plastic Falcon tube for glycerolization.

[0058] Glycerolation RBCs were glycerolized at room temperature to obtain a final concentration of 20% or 40% glycerol.

[0059] Six combinations were measured (three of each).

[0060] [Table 5]

[0061] An equal amount of standard 57% (wt / vol) glycerol was added to the RBC concentrate to achieve a final concentration of approximately 40% (wt / vol) glycerol. A 25% (wt / vol) glycerol mixture was added to the RBCs in a 5:1 ratio to achieve a final concentration of glycerol of approximately 20% (wt / vol). The final solution was subjected to cryopreservation, thawed, and kept at 4°C overnight. A control RBC concentration (not subjected to cryopreservation treatment) was also kept at 4°C overnight.

[0062] Deglycerolization The RBC concentrate was equilibrated at room temperature for 30 min by gentle inversion. The suspension was then centrifuged at 1100 x g for 12 min and the supernatant was discarded. The RBC suspension (0.5 mls) was deglycerolized by repeated washing with hypoosmolar NaCl solutions as follows: 0.125 ml of 8% (12% for 40% glycerol samples) NaCl, incubated at room temperature for 3 minutes 0.625 ml of 0.9% NaCl, incubated at room temperature for 3 minutes 0.75 ml of 0.9% NaCl, incubated at room temperature for 3 minutes 4 ml of 0.9% NaCl, incubated at room temperature for 3 minutes

[0063] The suspension was centrifuged at 1100×g for 12 min and the supernatant was discarded.

[0064] Assessment of hemolysis Deglycerolized cells (described above) were stored in PBS at 4°C for 2 min, after which hemolysis was measured. The cells were centrifuged at 2860xg for 1 min to separate the RBCs from the supernatant. The supernatant was transferred to a plastic cuvette (1 cm) and the free hemoglobin fraction was determined by measuring the absorbance at 540 nm in a spectrophotometer.

[0065] result The raw data for each treatment is given in the table below. The amount of free hemoglobin correlates to the amount of lysed RBCs. Both methods produced low levels of hemolysis.

[0066] [Table 6]

[0067] FIG. 13 shows the amount of hemolysis in each of the above scenarios compared to the control.

[0068] The results show that the cryopreservation method described here protected RBCs from deterioration when 40% glycerol was used as the cryoprotectant. At both temperatures tested (-25°C and -50°C), the treatment was superior to the control. With system and process optimization, it is believed that the 20% glycerol case will perform at least as well as the control.

[0069] Determining cryopreservation requirements The analysis was performed by investigating the effect of changing the input parameters of the storage system. This may include identifying the product geometry, the starting temperature of the product, the packaging characteristics, and the racking system to be utilized. The method involves dividing the biological product into geometric increments (e.g. cylindrical shells for bottles or test tubes). For each of these increments, an energy conservation equation is solved; that is, during a given time step, an amount of energy is removed from a shell, resulting in a drop in the temperature of that shell. The amount of energy removed is a fraction of the temperature of the adjacent shells, and the resistance to heat flow between the shells. This is relevant to consider the thermal properties of the biological product as a function of temperature.

[0070] The analysis was performed assuming that blood can be treated as a solid mass with a starting temperature of 2° C. and thermal properties that can be identified, estimated or calculated using methods known to those skilled in the art.

[0071] Based on the total surface area of ​​the product, the volumetric loading of the product in the vessel, a preselected inlet temperature of the heat exchange fluid, a preselected allowable outlet temperature of the heat exchange fluid (e.g., 3°C above the inlet temperature), the thermal properties of the product (including cryoprotectant) and packaging, and a preselected rate of fluid flow through the vessel, the rate of temperature drop of the product can be simulated as described above.

[0072] The temperature drop rate within a given snapshot in time may be, for example, 90° C. per minute or greater. If the production rate and temperature drop are acceptable from a practical standpoint (e.g., if the pump duty is acceptable based on the viscosity of the heat exchange fluid at the selected temperature or if the evaporator duty is acceptable based on the amount of heat removal required), then a higher temperature drop rate can be selected along with a correspondingly lower amount of cryoprotectant in the product (so that preservation can occur without damage to the product). The newly selected temperature drop rate, and thus the higher fluid rate, can then be simulated to determine whether it is selectable from a practical standpoint as described above.

[0073] Once the practical maximum temperature drop rate has been determined (and the corresponding minimum level of cryoprotectant determined), the product is mixed with cryoprotectant to the determined level and subjected to storage based on the determined temperature drop rate. In practice, a safety factor of, for example, 10% may be used for each cryoprotectant level and temperature drop rate.

[0074] Cooling System 14 is a piping and instrumentation diagram of a cooling system that continuously cools a heat exchange fluid. The cooling system has a heat exchanger for exchanging heat between a heat transfer fluid and a refrigerant, which may be, for example, R404A.

Claims

1. 1. An apparatus for use in the cryopreservation of a biological product comprising an inner housing disposed within an insulated outer housing, the walls of the inner housing defining a compartment for receiving the biological product, the walls including an inlet wall for flowing heat exchange fluid into the compartment, an opposed outlet wall for flowing heat exchange fluid out of the compartment, side walls and a base, the side walls and base joining the inlet wall to the outlet wall; The outer housing includes: an inlet side corresponding to the inlet wall of the inner housing, the inlet side defining an inlet space between the inlet side and the inlet wall; an outlet side corresponding to the outlet wall of the inner housing, the outlet side defining an outlet space between the outlet side and the outlet wall; having the inlet wall and the outlet wall each include a series of apertures for accommodating a continuous flow of heat exchange fluid through the device such that, during operation, a biological product received within the compartment of the inner housing is immersed in the heat exchange fluid for exchanging heat with the heat exchange fluid; The inlet side includes at least one inlet communicating from the outside of the outer housing into the inlet space, the outlet side includes at least one outlet communicating from the outlet space to the outside of the outer housing, the apparatus further comprising a pump for pumping a heat exchange fluid through the at least one inlet and into the inlet space; During operation, the heat exchange fluid is introduced into the apparatus through the at least one inlet and removed from the apparatus through the at least one outlet. An apparatus comprising:

2. The apparatus of claim 1 , wherein the base has a series of apertures.

3. 10. The apparatus of claim 1, wherein said apparatus comprises a structure receivable within said compartment for holding said biological product, said structure being one or more of a tray, a rack and a basket.

4. 4. The apparatus of claim 3, wherein the structure is suspended from a lid of the apparatus.

5. The apparatus of claim 1 , wherein the inlet volume and the outlet volume are fluidly connected.

6. 2. The apparatus of claim 1, wherein said at least one inlet and said at least one outlet are 80 mm in diameter.

7. 10. The apparatus of claim 1, wherein, in use, the apparatus is coupled to an external cooling system whereby the heat exchange fluid exchanges heat with a refrigerant.

8. 13. Use of the device according to claim 1 for cryopreservation of biological products.

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