HK1078438A - Method of preparing biological materials for cryopreservation using pre-chilled protectant - Google Patents

Description

Method for producing biological substances for cryopreservation using pre-chilled protectant

Technical Field

The present invention relates generally to cryopreservation, and more particularly to cryopreservation processes using protectants.

Background

It has been known from the eighteenth century to use cryopreserved cells, when experiments with canine sperm have found that cells can be frozen and subsequently thawed, after which a small percentage of sperm cells can restore normal physiological function. In the early twentieth century, it was discovered that the recovery rate of cells could be improved if the cells were chemically treated to undergo cycles of freezing and thawing using compounds collectively known as cryoprotectants. In the late twentieth century, a great deal of research was conducted to develop cytoprotective agents while optimizing freezing temperatures and freezing rates for a variety of different cells. However, despite the advances in technology today, cell recovery rates after cryopreservation are still often 50% or less.

Typically, cryoprotectants consist of water, various salts, various sugars, protein sources, and chemical compounds known as cryoprotectants or chemoprotectants. Various salts act as buffers to maintain the pH within the limits tolerated by cells or molecules when frozen, while various sugars act as energy sources and osmotic pressure factors. Various proteins chemically stabilize the cell membrane structure prior to freezing to prevent reactions of shock proteins.

Various cryoprotectants are used today, for example, Dimethylsulfoxide (DMSO), propylene glycol (PPO), and egg yolk/glycerol solutions. The standard cryoprotectant widely accepted in the industry for cryopreservation of most cell types is DMSO. This is due to the widespread experience and knowledge of DMSO-based cryopreservation solutions, which is generally believed to remove water from cells such that ice crystal formation during freezing is reduced, providing superior protection and cell survival at best.

To date, research efforts to improve the restoration rate of cryopreservation have generally focused on new cryoprotectants and freezing techniques. Both efforts have been made to reduce cell damage caused by the expansion of intracellular water due to ice crystal formation during freezing. In theory, a very slow or fast freezing rate will reduce or eliminate the formation of ice crystals in the cells. Mechanisms for very slow rate freezing include controlled descent, by moving the sample from vapor nitrogen to liquid nitrogen or in a super-freezing compound, followed by immersion in liquid nitrogen. The rapid freezing technique immerses the cells directly in liquid nitrogen to rapidly freeze water in the cells, thereby inhibiting ice crystal formation. This extreme temperature drop over a short period of time often results in stress-induced rupture of the cell membrane, thus adversely affecting the recovery rate.

During freezing, the molecules that make up the chemical components of the cryoprotectant medium are forced into alignment during freezing. This forced alignment causes the chemical constituents of the media to undergo an endothermic reaction, which releases energy in the latent heat phase. This released heat causes a brief increase in the temperature of the cryoprotectant when the frozen material is subjected to the latent heat phase (with an endothermic reaction). This latent heat, also known as heat of conversion, is simply a change in enthalpy if measured in a pressure-invariant phase transition (e.g., thawing, boiling, sublimation). The change in enthalpy during isobaric is equal to the transfer of heat during a system undergoing a minimum from initial equilibrium state to final equilibrium state.

Two important cryopreservation parameters that must be optimized at most for cell survival are freezing time and freezing rate. The observed change in freezing rate and increase in freezing temperature in the latent heat phase are obstacles to optimizing cell survival during cryopreservation or during conventional freezing.

Summary of The Invention

Thus, there is a need for improved methods that can protect single cells, tissues, organs, nucleic acids, or other bioactive molecules from survival during cryogenic processes, while avoiding certain problems inherent in prior methods. Therefore, at least one embodiment of the present invention provides a method for improving the recovery rate of cryopreservation by reducing the amount of heat of sublimation of the protectant by pre-cooling the protectant to cause an irreversible phase change before the thawed protectant is applied to the biologically active substance.

In one embodiment, the protectant is frozen to induce an endothermic reaction. After the endothermic reaction has occurred, the protectant is thawed and used to prepare the biologically active substance for freezing. Thawed protectants in biologically active cells have no endothermic reaction upon subsequent freezing, so the methods disclosed herein can substantially increase the number of cells that survive in biological material undergoing cryopreservation processes.

Another embodiment of the present invention provides a method for reducing heat release during cryopreservation by a cryoprotectant. The method comprises pre-cooling the protectant to produce an irreversible phase change in the protectant, treating the biologically active substance with the protectant, and subsequently freezing the treated biologically active substance.

Another embodiment of the present invention provides a biological material undergoing a cryopreservation process comprising pre-chilling a protectant until it freezes to cause the protectant to irreversibly release energy, thawing the protectant to a temperature suitable for application to a biologically active material, applying the thawed protectant to the biological material, and freezing the treated biological material.

It is an object of at least one embodiment of the present invention to improve the survival rate of biologically active substances during cryopreservation.

An advantage of at least one embodiment of the present invention is a reduction in the rate of cell viability loss because the rate of freezing is not adversely affected by the release of heat from the preservative during cryopreservation.

Brief Description of Drawings

Other objects, advantages, features, and characteristics of the present invention, as well as the methods, operations, and functions of the same, related to the structure, the method of operation, and the combination of parts and economies of manufacture, will become apparent upon consideration of the following description and the appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals designate corresponding parts in the various figures, and wherein:

FIG. 1 is a graph of temperature measurements for three cryoprotectants subjected to a pre-treatment of rapid freezing with short time intervals in accordance with at least one embodiment of the present invention;

FIG. 2 is a flow chart illustrating a method according to at least one embodiment of the present invention;

FIG. 3 is a bar graph of comparative experimental results of a cryopreservation method with liquid nitrogen and a control group and a cryopreservation method of the invention and a control group, according to at least one embodiment of the invention;

FIG. 4 is a bar graph illustrating the percentage of pig sperm cells remaining viable after undergoing freeze-thaw cycles in accordance with at least one embodiment of the present invention;

fig. 5 is a side sectional view of a refrigeration apparatus suitable for use in at least one embodiment of the present invention.

Detailed description of the drawings

Fig. 1-5 depict methods of using pre-chilled protectants in cryopreservation of biologically active substances that result in increased cell survival through the freezing process, according to various embodiments disclosed herein. In various embodiments, the bioactive agent includes viable single cells, viable tissues, viable organs, viable nucleic acids, viable ribonucleic acids, viable amino acid-based compounds, and viable fat-based compounds.

In theory, most chemical reactions are bi-directional (reversible). In practice, however, many chemical reactions are found to be unidirectional (irreversible) depending on the energy requirements of the particular reaction. In the case of the protective agent contained in the present invention, the heat release in the latent heat phase is a one-way chemical reaction. Thus, once frozen, the protective agents encompassed by the present invention exhibit long-lasting phase change capabilities (irreversible phase change) upon subsequent thawing and refreezing.

Latent heat release during freezing is seen in fig. 1, which is a graph of temperature measurements for three cryoprotectants subjected to a pre-treatment with rapid freezing for short time intervals, according to various embodiments of the present invention. Cryoprotectants measured in figure 1 include dimethyl sulfoxide, as indicated by DMSO 110; egg yolk/glycerol solution, as shown by Gly 115; propylene glycol, as shown by PPO 120. The effect of heat release in the energy conversion during freezing is clearly visible in the measurements of time interval 5 (at time 75 seconds) and interval 6 (at time 90 seconds), with all three substances being visible with a distinct increase in temperature, or a peak 125. After the peak 125, measurements in later time intervals show a drop in temperature until the end of the measurement time.

From a series of measurements on solute freezing, it was possible to determine that heat release in the latent heat phase is a brief increase in temperature in the protectant medium, as shown by the peak 125 in fig. 1. However, as disclosed herein, the temperature increase noted above is not observed when the protectant is first rapidly cooled (over-cooled) in the preconditioning step, as the preconditioned protectant undergoes a change in chemical properties, exhibiting long-lasting phase change capability. The effect of the long duration phase change capability of such pre-treated solutes has been measured and it has been found that the pre-treated protective agents disclosed herein do not have significant heat release during freezing as described herein.

In one embodiment, there is no particular temperature storage requirement for the protectant after undergoing freeze/thaw cycles. After the pretreatment described herein, the protectant exhibits a phase change capability of long duration, which can be reused if desired, without undesirable temperature spikes again occurring during freezing. According to various embodiments of the invention, a reduction in temperature spikes should increase the viability and viability of cells and molecules after cryopreservation.

Turning now to fig. 2, fig. 2 illustrates a method according to one embodiment of the present invention. The illustrated method begins at step 1010, where the protectant is rapidly frozen as discussed above to form an irreversible release of energy (irreversible phase change). Protective agents used in various embodiments may include, but are not limited to, the following: glycerol, DMSO, or propylene glycol. In step 1015, the protectant is returned to a pre-cooled consistency by thawing the protectant to a temperature above 0 degrees celsius. Once thawed, the rapid cryoprotectant to-18 degrees celsius or less has no separation of the liquid layer. It is advantageous that there is no separation of the liquid layers, as the dissolution of the protective agent in subsequent freezing cycles increases after the first freezing and thawing cycle. After the protectant is dissolved to a sufficient consistency, the biological material to be frozen is dipped into the thawed protectant in preparation for freezing the biological material, as shown in step 1020. In step 1025, the biological material impregnated with the protectant is flash frozen. In one embodiment, biological substances to which the method can be applied include biologically active substances such as viable single cells, viable tissues, viable organs, viable nucleic acids, viable ribonucleic acids, viable amino acid-based compounds, and viable fat-based compounds.

Alternatively, some biological substances require additional chemical preparation prior to freezing. For example, chemical preparation of substances involves pre-treatment of agents (stabilizers) that increase cell viability by removing harmful substances secreted by cells during growth or death. Useful stabilizers include chemicals and chemical compounds, many of which are known to those skilled in the art, which sequester highly reactive and damaging molecules such as oxidizing groups.

The steps shown in fig. 2 are shown and discussed in sequential order. However, the illustrated method is characterized in that some or all of the steps are performed continuously, and may be performed in a different order. For example, if an existing batch of protectant has been subjected to freeze/thaw cycles, the protectant need not be re-frozen before application to the biological material.

Studies using the techniques disclosed herein have shown that improved cell survival rates of 40% or greater can be obtained. The results of the experiments with porcine muscle cells are shown in fig. 3, which is a bar graph showing the results of a method for cryopreservation of Liquid Nitrogen (LN) and a control group and an embodiment of the invention (SC) and a control group, as referred to in bar graph 400. Bar graph 400 compares the number of viable porcine muscle cells that have undergone cryopreservation with a pre-chilled protectant to a control group. According to various embodiments disclosed herein, the cryoprotectant applied to the control group was not pre-chilled.

Control group without pre-chilled protectant 405 was chilled to approximately minus-25 degrees celsius, while control group without pre-chilled protectant 410 was chilled to approximately minus 196 degrees celsius in liquid nitrogen, according to the high temperature freezing method shown herein. Two control groups 405 and 410, and pre-chilled protectant groups 420 and 425 were taken from common tissue sources, and these tissues were divided into a number of groups to be used for different treatment and freezing techniques (LN and SC). Porcine muscle group 425 is used for cryopreservation with Liquid Nitrogen (LN) after pre-chilled protectant treatment as shown herein. Porcine muscle group 420 was cryopreserved following the same high temperature freezing procedure used for frozen control group 405 following pre-chilled protectant treatment as described herein.

As shown in bar graph 400, pre-chilled group 425 showed a percent survival after thawing of about 60-70% using Liquid Nitrogen (LN) technology, while control group 410 without pre-chilled protectant, using the same LN freezing technology, showed a percent survival after thawing of about 40-50%. Using the freezing technique of the high temperature freezing method shown herein, pre-chilled group 420 showed a percent survival of about 80-90%, while control group 405 without pre-chilled protectant showed a percent survival of about 80-90%. LN cryopreservation techniques have considerably less cell viability than high temperature freezing techniques, depending on overall cell viability. In groups 410 and 425 using liquid nitrogen, cells using pre-chilled protectants as shown herein had a significant increase in viability compared to cells using non-pre-chilled protectants. However, as shown by comparison of groups 420 and 425 treated with pre-chilled protectant, the high temperature freezing method produced an increase in cell viability that was nearly doubled over the LN technique. Although freezing of the same cells using a pre-chilled protectant with a high temperature freezing method did not significantly change survival rates, e.g., groups 405 and 420, other cell types (porcine sperm as shown in fig. 4) from other experiments showed significant improvement in both freezing techniques (LN and SC).

Turning now to fig. 4, fig. 4 illustrates the percent of motility measured after cryopreservation (freezing) and thawing of a porcine sperm sample treated with a conventional protectant and a porcine sperm sample treated with a pre-chilled protectant according to an embodiment of the invention. The protectant used in this study was a mixture of glycerol and water, with each concentration of glycerol having a respective weight percentage. Thus, the numbers 1%, 2%, etc. indicate on the ordinate the final glycerol concentration of 1%, 2%, 3%, 4%, or 5%. Control 505 shows sperm samples treated with different concentrations of glycerol (1% -5% by weight) of a protectant that was not pre-chilled according to the embodiments shown herein. Pre-cooling group 510 shows sperm samples treated with protectants having different concentrations of glycerol, which have been pre-cooled as described herein. The high temperature freezing methods contained herein are used to freeze various porcine sperm samples.

As shown in fig. 4, pre-chilled group 510 showed a higher percentage of exercise performance at all glycerol concentrations than control group 505, except for the 3% glycerol data point where the two were nearly equal. These data show that cells more sensitive to freezing exhibit higher survival rates if frozen with pre-treated media, as shown herein, show long duration phase change capability (irreversible phase change). The results of the study further demonstrate that the techniques disclosed herein can be used to cryopreserve biological species that were previously thought to be unavailable using these techniques, as are all other mammalian species. In addition to sperm, there are many other areas such as skin, cell lines, proteins and other biologically active substances that may benefit from the methods described herein.

In one embodiment, the application of the methods described herein can be extended to humans, providing low cost treatment for infertility in the areas of artificial insemination and in vitro fertilization. Because some of the cryoprotectants described herein, such as propylene glycol, do not exhibit the toxic effects of other cryoprotectants, such as DMSO, long duration phase change protectants have no adverse effects on patients who have introduced sperm using the protectant.

Turning to fig. 5, a refrigeration apparatus suitable for use in the present method is illustrated in accordance with at least one embodiment of the present invention and is generally referred to as a refrigeration unit 800. Freezer unit 800 preferably includes a tank 810 containing a freezing fluid 840. Immersed in cooling fluid 840 are circulation mechanism 834, such as a motor and impeller combination, and heat exchanging coil 820. Substances that can be frozen include, but are not limited to, viable single cells, tissues, organs, nucleic acids, ribonucleic acids, amino acid-based compounds and fat-based compounds, and other bioactive molecules. Outside of tank 810, coupled to heat exchanging coil 820 is refrigeration unit 890.

Tank 810 must be sufficient to submerge the material to be frozen in the volume of cooling fluid 840 and may be any size, which may be a multiple of 12 inches by 24 inches by 48 inches. Other sizes of water tanks may be used consistent with the requirements herein. For example, in one embodiment (not shown), tank 810 is sized to contain a cooling fluid 840, so that a container for rapidly freezing a suspension including biological material and a cryoprotectant may be placed in tank 810. In another embodiment, the water tank 810 is large enough to completely submerge the entire organism for rapid freezing. The tank 810 may be larger or smaller, and prepared as needed to accommodate various sizes and quantities of substances to be frozen, which is advantageous.

Tank 810 contains a cooling fluid 840. In one embodiment, the freezing fluid is a food grade solution. Good examples of food grade quality liquids are liquids based on propylene glycol, sodium chloride solution, glycerol, etc. In a preferred embodiment, the freezing fluid is propylene glycol, a protectant. While various containers for holding biological materials have been used, embodiments of the present invention utilize containers that allow rapid and efficient freezing of biological materials by direct immersion in a freezing fluid.

In order to freeze material without the formation of ice crystals, one embodiment of the present invention circulates cooling fluid 840 through the material being frozen at a relatively constant rate of 35 liters per minute for each portion of cooling fluid contained in a range exceeding 24 inches wide by 48 inches deep. The necessary circulation is provided by one or more circulation mechanisms 834, such as a combination of an engine and propeller. In at least one embodiment of the present invention, a circulation mechanism 834, immersed in a circulating fluid, circulates cooling fluid 840 through the material being cooled. Other circulation mechanisms 834, including various pumps (not shown), may be used consistent with the articles of the present invention. At least one embodiment of the present invention uses at least one circulation mechanism 834 to circulate the cooling fluid, increasing the area and volume of the circulating cooling fluid. In embodiments using multiple circulation mechanisms 834, the increase in area and volume of cooling fluid circulation is directly proportional to each circulation mechanism increasingly used. For example, in a preferred embodiment, an additional circulation mechanism is used to circulate each portion of the circulating fluid in a range no more than 24 inches wide by 28 inches deep.

Preferably, the motor in the circulation mechanism 834 can maintain a fixed predetermined speed to control the flow of the cooling fluid through the material to be cooled while maintaining a uniform distribution of cooling fluid temperature within +/-0.5 degrees Celsius at each point in the tank 810. Circulation of the cooling fluid through the substance or product at a fixed and predetermined rate provides continuous, measurable heat transfer so that cooling and freezing of the substance can occur. In one embodiment, the characteristics of the cooling fluid, such as viscosity, temperature, etc., are measured and processed, and control signals are sent to the circulation mechanism 834 so that the motor in the circulation mechanism 834 can increase or decrease the rotational speed and torque of the propeller as desired. In other embodiments, the engine is configured to maintain a particular speed of rotation without generating additional heat over a range of fluid conditions. In this case, the torque or rotational speed of the engine to the propeller is not externally controlled. It is important that there be no external pumps, shafts or pulleys required for the refrigeration equipment. The combination of motor and impeller, or other circulation mechanism 834, is immersed directly in cooling fluid 840. As a result, cooling fluid 840 not only freezes the substance placed in tank 810, but cooling fluid 840 also freezes components (i.e., the motor and impeller) within circulation mechanism 834.

Heat exchanging coil 820 is preferably a "multi-path coil" that allows refrigerant to be diverted through multiple paths (i.e., three or more paths), as compared to conventional refrigeration coils where refrigerant is typically confined to one or two continuous paths. Moreover, coil size is directly related to the cross-sectional area containing a measurable amount of cooling fluid 840. For example, in a preferred embodiment, the tank 810 is one foot long, two feet wide and four feet deep, using one foot by two feet of heat exchange coils. If the length of the water tank 810 is increased to twenty feet, the length of the heat exchanging coil 820 is also increased to twenty feet. As a result, heat exchanging coil 820 only needs about fifty percent of the size of a conventional coil to handle the same heat load. Circulation mechanism 834 circulates chilled cooling fluid 840 to the material being chilled and then delivers heated cooling fluid to heat exchanging coil 820, which is immersed in cooling fluid 840. In at least one embodiment, heat exchanging coil 820 is designed to remove no less heat from cooling fluid 840 than the heat removed from the substance being frozen, thus maintaining the temperature of the cooling fluid within a predetermined range. The heat exchanging coil 820 is connected to a refrigeration unit 890 that removes heat from the heat exchanging coil 820 and the system.

In a preferred embodiment, refrigeration unit 890 is designed to match the load requirements of heat exchanging coil 820 so that heat can be removed from the system in a balanced and efficient manner, resulting in controlled, rapid freezing of the substance. The efficiency of refrigeration unit 890 is directly related to the method used to control the suction pressure created by the efficient delivery of heat exchange coil 820 and the efficient output of the compressor used by refrigeration unit 890.

This method requires that a small deviation in the temperature of the refrigerant and the cooling fluid 840, between the condensation temperature and the ambient temperature, be maintained. These temperature standards, together with the heat exchange coil 820, make the delivery of the heat exchange coil 820 more efficient, which allows the compressor to be delivered in a balanced and tightly controlled manner, such that the compressor achieves performance in excess of twenty-five percent of the compressor manufacturer's rating.

Directing attention to the embodiment depicted in fig. 5, refrigeration unit 890 is an external, remotely located refrigeration system. However, in another embodiment (not shown), refrigeration unit 890 is incorporated into another portion of tank 810. It is advantageous that the various shapes of refrigeration unit 890 are more or less adapted to certain shapes of refrigeration unit 800. For example, if tank 810 is particularly large, a separate refrigeration unit 890 may be desirable, while a portable embodiment would benefit from an integrated refrigeration unit 890. Such integration is only possible by achieving efficiency by implementing the principles set forth herein, particularly using heat exchange coils of reduced size.

By virtue of the refrigeration unit 890 and the heat exchanging coil 820, in a preferred embodiment, the chilled liquid is chilled to a temperature between-20 degrees Celsius and-30 degrees Celsius, with a temperature differential within the chilled liquid of less than about +/-0.5 degrees Celsius. In another embodiment, the freezing fluid is frozen to a temperature in excess of-20 degrees Celsius and-30 degrees Celsius to control the rate at which the substance is frozen. Other embodiments control the circulation rate of the freezing fluid to achieve the desired freezing rate. Alternatively, the volume of the freezing fluid may be varied to facilitate a particular freezing rate. Various combinations of chilled liquid circulation rates, chilled liquid volumes, and chilled liquid temperatures that can be used to achieve a desired freezing rate are advantageous.

In the foregoing detailed description, reference has been made to the accompanying drawings that form a part hereof, and in which is shown by way of illustration specific embodiments in which the invention may be practiced. These embodiments have been described in sufficient detail to enable those skilled in the art to practice the invention, and it is to be understood that other embodiments may be utilized and that logical, mechanical, chemical and electrical changes may be made without departing from the spirit and scope of the present invention. To avoid detail not necessary to enable those skilled in the art to practice the invention, the description may omit certain information known to those skilled in the art. The preceding detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims.

Claims (40)

1. One method comprises the steps of:

cryoprotectants provide irreversible release of energy from the protectant;

treating the biologically active substance with the protectant; and

freezing the treated bioactive substance.

2. The method of claim 1, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 6.5 ℃ per minute.

3. The method of claim 1, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled from room temperature to a temperature of less than about-23 ℃.

4. The method of claim 1, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled from room temperature to between about-23 ℃ and-26 ℃.

5. The method of claim 1, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 6.5 ℃ to 8.5 ℃ per minute.

6. The method of claim 1, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 17 ℃ per minute for at least a portion of the time.

7. The system of claim 1, wherein the heat absorption rate of the pre-treated solute is about 135BTU between about-23 ℃ and-26 ℃.

8. The method of claim 1, further comprising the step of heating the protectant prior to the step of treating the biologically active material.

9. The method of claim 8, wherein the step of heating the protectant comprises heating the protectant to a temperature in excess of 0 degrees celsius.

10. The method of claim 1, wherein the protectant comprises propylene glycol.

11. The method of claim 1, wherein the protectant comprises glycerol.

12. The method of claim 1, wherein the protectant comprises DMSO.

13. The method of claim 1, wherein the bioactive agent comprises viable single cells, viable tissue, viable organs, viable nucleic acids, viable ribonucleic acids, viable amino acid-based compounds, and viable fat-based compounds.

14. One method comprises the steps of:

freezing the protectant to below about-23 degrees celsius to allow irreversible release of energy from the protectant;

heating the protectant to a temperature in excess of 0 ℃;

treating the biologically active substance with the protectant; and

freezing the treated bioactive substance.

15. The method of claim 14, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 6.5 ℃ per minute.

16. The method of claim 14, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled from room temperature to a temperature of less than about-23 ℃.

17. The method of claim 14, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled from room temperature to between about-23 ℃ and-26 ℃.

18. The method of claim 14, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 6.5 ℃ to 8.5 ℃ per minute.

19. The method of claim 14, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 17 ℃ per minute for at least a portion of the time.

20. The method of claim 14, wherein the heat absorption rate of the pretreated solute is about 135BTU between about-23 ℃ and-26 ℃.

21. The method of claim 14, wherein the protectant comprises propylene glycol.

22. The method of claim 14, wherein the protectant comprises glycerol.

23. The method of claim 14, wherein the protectant comprises DMSO.

24. The method of claim 14, wherein the bioactive agent comprises viable single cells, viable tissue, viable organs, viable nucleic acids, viable ribonucleic acids, viable amino acid-based compounds, and viable fat-based compounds.

25. A cryopreserved treated biological material, the cryopreservation treatment comprising:

cryoprotectants provide irreversible release of energy from the protectant;

treating the biologically active substance with the protectant; and

freezing the treated bioactive substance.

26. The biological material as in claim 25, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 6.5 ℃ per minute.

27. The biological material as in claim 25, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled from room temperature to a temperature of less than about-23 ℃.

28. The biological material as in claim 25, wherein said pre-conditioned solute is a solute having been conditioned by being super-cooled from room temperature to between about-23 ℃ and-26 ℃.

29. The biological material as in claim 25, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 6.5 ℃ to 8.5 ℃ per minute.

30. The biological material as in claim 25, wherein the pre-conditioned solute is a solute having been conditioned by being super-cooled at an average rate of at least about 17 ℃ per minute for at least a portion of the time.

31. The biological material as in claim 25, wherein the heat absorption rate of the pretreated solute is about 135BTU between about-23 ℃ and-26 ℃.

32. The biological material as in claim 25, wherein the cryopreservation process comprises heating the protectant prior to the step of treating the biologically active material.

33. The biological material as in claim 32, wherein the cryopreservation process comprises heating the protectant to a temperature in excess of 0 degrees celsius.

34. The biological material as in claim 25, wherein said biological material comprises viable single cells.

35. The biological material as in claim 25, wherein said biological material comprises viable tissue.

36. The biological material as in claim 25, wherein said biological material comprises a living organ.

37. The biological material as in claim 25, wherein the biological material comprises viable nucleic acids.

38. The biological material as in claim 25, wherein said biological material comprises viable ribonucleic acids.

39. The biological material as in claim 25, wherein the biological material comprises viable amino acid-based compounds.

40. The biological material as in claim 25, wherein the biological material comprises viable fat-based compounds.