WO1991013091A1

WO1991013091A1 - Cryoperservation of red blood cells

Info

Publication number: WO1991013091A1
Application number: PCT/US1991/001412
Authority: WO
Inventors: John F. Carpenter
Original assignee: Agouron Pharmaceuticals, Inc.
Priority date: 1990-03-01
Filing date: 1991-03-01
Publication date: 1991-09-05

Abstract

A composition is provided for enhancing survival of red blood cells during freezing and thawing. The composition includes at least one cryoprotectant and a thermal hysteresis peptide in an amount effective for enhancing the viability of the red blood cells. A method of enhancing survival of red blood cells during freezing and thawing is also provided.

Description

Cryopreservation of RED BLOOD CELLS

This application is a continuation-in-part of U.S. Serial 07/486,799, filed March 1, 1990, still pending, which is hereby expressly incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a method for increasing t percentage of red blood cells remaining viable after cryopreser tion and storage at ultracold temperatures and thawing. That i the process described herein results in an enhancement of the preservation capacity provided by classical cryoprotectants.

According to certain advantageous features, the present invention allows one to use a lower concentration of classical cryoprotectant (e.g., dimethylsulfoxide) and still achieve the same level of cell viability after thawing. In addition, with certain cryoprotectants (e.g., polyvinylpyrrolidone and hydroxyethyl starch), the use of the process described herein results in higher viability than is achievable when the cryoprotectant is used alone. Furthermore, the present inventi

minimizes the damage arising due to suboptimal warming rates a hence allows one more leeway in thawing samples.

Current medical technology allows the use of red blood ce for transfusion to correct congenital, disease- or trauma-indu or degenerative failure of a recipient's cells.

Cryopreservation and ultracold storage of cells and tissu became possible after the discovery in 1949, by Polge, Smith a Parks, that glycerol could be used to protect cells from injur due to freezing. Workers in the medical and biological fields have been seeking better ways to cryopreserve cells and to circumvent some of the drawbacks of classical cryopreservation regimes (e.g., toxicity of cryoprotectants at physiological temperatures and need to thaw samples very rapidly) .

Several methods for freezing cells and tissues have been reported. For example, U.S., Patent No. 3,303,662 refers to a process for cell preservation that utilizes a cryoprotectant i the freezing process. One drawback of many cryopreservation protocols is that at high concentrations the cryoprotective ag can be toxic to cells. For example, more than 80% of fetal pancreas cells remain viable after freeze-thawing in the prese of 2M dimethylsulfoxide, but less than 50% recovery is noted w 3M dimethylsulfoxide (Mazur, In "Organ Preservation for Transplantation," 2nd edition, pp. 143-175, Mariel Deker, New York, 1981). (see Fahy, Cryobiology, 23:1-13, 1986). Therefo it would be advantageous to develop methods that allowed for a equal level of cell preservation with a reduced amount of cryoprotectant.

In addition, an inherent component of most cryopreservat procedures is the formation of ice crystals. Intracellular i formation and crystal growth is usually thought to be lethal cells. Even extracellular ice can cause damage to frozen cel and tissue, especially if they are thawed at a slow rate, whi allows migratory recrystallization (the increase in ice cryst size as the sample is warmed from ultracold temperatures ) . T growing crystals can cause direct mechanical disruption of th tissue or cellular integrity. Finally, even when cells or ti are frozen in such a manner that ice crystals do not form (i. vitrification), devitrification and ice crystal growth during thawing can be damaging. The nature and extent of ice formation during cooling pl an important role in the survival of red blood cells during freeze-thawing (See Mazur, Organ Preservation for Transplanta (Karow et al., editors), pp. 143-175 (1981); Pegg et al., Cryobiology 21:491 (1984)). In addition, it has been shown t the propagation of large ice crystals at the expense of small crystals during warming also contributes to cellular damage ( et al., 1984). For example, Pegg, the Biophysics of Organ Preservation (Pegg et al., editors), pp. 117-136 (1987) demonstrated that slowly warming (0.3°C/min) solutions of red blood cells, which had previously been cooled to -100°C (in t presence of 2 M glycerol), resulted in a massive increase in t crystal size of the extracellular ice and greater than 50% hemolysis. In contrast, hemolysis was less than 10% for prepa tions warmed at 100°C/min, in which ice simply melted instead recrystallizing. The prevalence of such recrystallization, ei alone or following devitrification, during slow warming proba accounts for the fact that red blood cells frozen in hydroxyet starch must be warmed very rapidly to realize preservation of high percentage of cells (Weatherbee, DOD Technical Report AD- 387 (1975); Lionetti, et al. DOD Technical Report AD-A020 513

(1975)).

The present inventor has surprisingly discovered that cer naturally-occurring molecules and derivatives thereof provide enhancement of red blood cell survival during cryopreservation thawing. These molecules are called thermal hysteresis peptid

(THPs). THPs are found in both polar fish and insects (DeVrie Phil. Trans. R. Soc. Lond. B 304:575, 1984; Knight and Du an, Cryobiology 23:256, 1986).

These peptides have also been characterized as "antifreez peptides (AFP)" or "antifreeze glycopeptides (AFGP)." It has known for many years that fish have been able to inhabit the i laden waters of the polar oceans. However, only recently has been learned that survival of these fish in such freezing envi ments is largely due to the presence in the serum of macromole ular ice crystal control peptides and glycopeptides, which dep the freezing temperatures of the body fluids. These ice cry control materials reduce or prevent ice crystal growth, and also been characterized as antifreezes. Four classes of thes fish macromolecular antifreezes have been identified to date. The first class of macromolecular antifreezes, found in

Nototheniids and Gadoids, are antifreeze glycopeptides. The range in molecular weight from 2,600 to 37,000 and contain h proportions of alanine and threonine with a disaccharide moi covalently linked to the threonine residues. The second, th and fourth classes of antifreezes are peptides, and are refe to in Hew et al.. Journal of Chromatoσraphv. 296.213-219 (198 The second class of antifreezes are small peptides, molecular weight 3,300 to 4,000, which also show a high alanine content have no carbohydrate attached. These are found in winter flo and shorthorn sculpin. The third class is a 9,900 molecular weight peptide found in sea raven. It also contains no carbohydrate but unlike all other known antifreezes it contai cysteine (7.6%). The fourth class is isolated from ocean pou has a molecular weight of approximately 6,000 and is neither alanine-rich nor cysteine-rich.

The macromolecular peptides and glycopeptides show freez point depression in a non-colligative manner. Thus, the pept and glycopeptides lower the freezing temperature of water but little to no effect on its melting temperature. Hence, the t "hysteresis" of the term "THP" means that there is the inhibit of freezing with little to no effect on melting.

The capacity to depress freezing point per se is not beneficial during cryopreservation procedures, since at concen tions around 40 mg/ml (usually the highest level tested in published reports) the THPs depress freezing point only to abo 1.2°C. For most applications, cryopreservβd samples are coole and stored at the temperature of liquid nitrogen (-196°C) or t of liquid nitrogen vapor (about -130°C) . It has also been fo that antifreeze glycopeptides at concentrations of about 10^"

10

10~ M effect the morphology of growing ice crystals. Knight et al.. Nature, 308:295-296, 1984. Peptide antifreeze isolate from winter flounder also produces freezing hysteresis and similarly effects ice growth. Kniσht et al. , at page 296. These peptides also have been shown to inhibit the recrystallization of ice under isothermal conditions and at a nealing temperatures no lower than -8°C. Knight et al., Cryobiology 23: 256-262, 1986; Knight et al., Cryobiology 25: 60, 1988. Those experiments do not suggest the use of such protein for recrystallization inhibition during cryopreservat to ultracold temperatures. First, the temperature range used ing cryopreservation is much lower than that used by Knight e to characterize recrystallization inhibition by THPs. Second, during freezing and thawing, cryopreserved samples are subjec to changing temperatures and are not held isothermally for lo time periods at high subzero temperatures (temperatures above

-130°C).

In certain initial studies, the present inventors also n that the presence of THP induced damage to cells during freez thawing, and negated the protective effects of cryoprotective compounds such as glycerol and dimethylsulfoxide. However, i subsequent studies, the present inventors surprisingly discov methods by which THPs could be used to enhance the level of cryopreservation provided by cryoprotectants . SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a method of enhancing the survival of cryopreserved r blood cells upon thawing.

It is yet another object of the present invention to uti thermal hysteresis proteins to effect this increase in red bl cell survival by enhancing the degree of cryopreservation pro by penetrating and extracellular cryoprotectants .

It is yet another object of the present invention to use thermal hysteresis proteins to reduce the concentration of a cryoprotectant that is needed to achieve a given degree of pr tion, and thus, reduce the possibility of cryoprotectant toxi

It is yet another object of this invention to use thermal hysteresis proteins to increase the degree of preservation to higher level than can be achieved by even the most effective concentration of a given cryoprotectant, when used without THP It is yet another object of this invention to use thermal hysteresis peptide, to minimize the damage to red blood cells arising during thawing, thus allowing one to thaw samples at suboptimal warming rates and still achieve a high degree of ce survival.

It is yet another object of this invention to use thermal hysteresis peptide to minimize the damage to red blood cells arising during freeze-drying.

Additional objects and advantages of the invention will b set forth in part in the description which follows, and in par will be obvious from the description or may be learned from practice of the invention. The objects and advantages may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims. These and other objects, features, and advantages of the present invention are achieved by providing a method for enhan survival of red blood cells during cryopreservation and thawin which is comprised of freezing and thawing red blood cells in presence of a penetrating or nonpenetrating (extracellular) cryoprotectant and a thermal hysteresis peptide.

It is to be understood that both the foregoing general description and the following detailed description are exempla and explanatory only and are not restrictive of the invention, claimed. The accompanying drawings, which are incorporated in constitute a part of the specification, illustrate various embodiments of the invention and, together with the descript serve to explain the principles of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 compares the influence of thermal hysteresis pep on the level of hemolysis of human red blood cells frozen at ferent rates in the presence of 1.0 M glycerol . In the experiments with red blood cells, a decrease in the percenta hemolysis indicates an increase in cell survival. Samples w cooled at 20°C/min ( .) or 100°C/min (O ) or by plunging dire in liquid nitrogen (#) and stored in liquid nitrogen vapor.

Another set of samples was frozen in a -20°C freezer and sto that temperature overnight ( O ) . Fig. 1A shows the results f samples thawed by immersion in a 45°C water bath. Fig. IB s the results for samples thawed in air at room temperature . point represents the mean ± SE for triplicate samples.

Fig. 2 compares the influence of thermal hysteresis pept on the level of hemolysis of human red blood cells frozen at ferent rates in the presence of 1.5 M glycerol. Samples were cooled at 20°C/min ( ) ^or 100°C/min (□ ) or by plunging dire in liquid nitrogen (# ) and stored in liquid nitrogen vapor.

Another set of samples was frozen in a -20°C freezer and stor that temperature overnight ( O ) • Fig. 2A shows the results f samples thawed by immersion in a 45°C water bath. Fig. 2B sh the results for samples thawed in air at room temperature. E point represents the mean ± SE for triplicate samples. Fig. 3 compares the influence of thermal hysteresis pepti on the level of hemolysis of human red blood cells frozen at d ferent rates in the presence of 15% (wt/vol) hydroxyethyl star (450/0.7). Samples were cooled at 20°C/min (^) or 100°C/min or by plunging directly in liquid nitrogen ( ) and stored in liquid nitrogen vapor. Another set of samples was frozen in a 20°C freezer and stored at that temperature overnight ( O ) • Fig. 3A shows the results for samples thawed by immersion in a 45°C water bath. Fig. 3B shows the results for samples thawed air at room temperature. Each point represents the mean SE triplicate samples.

Fig. 4 shows the influence of thermal hysteresis peptide the level of hemolysis of human red blood cells frozen by plun directly into liquid nitrogen in the presence of 15% (wt/vol ^< hydroxyethyl starch (450/0.7). Fig. 4A shows the results for samples thawed by immersion in 45°C water bath. The open circ represent values for individual samples and the closed triangl with error bars represent the mean ± SD (Standard Deviation, f the triplicate samples. Fig. 4B shows the results for samples thawed in air at room temperature. Each point represents the

± SD for triplicate samples .

Figure 5 shows the influence of thermal hysteresis peptid the stability of cryopreserved human red blood cells stored at -80°C in the presence of 15% hydroxyethyl starch (450/0.7) . Fi 5A shows the results for samples diluted in physiological sali Figure 5B shows the results for samples diluted in human plas Each point represents the mean +. (SD) for triplicate samples.

Fig. 6A compares the influence of THP on the level of hemolysis (after a post-thaw saline dilution) of human red blo cells frozen in 15% HES 450/0.7 and thawed in water baths set different temperatures. The warmer the water bath, the more rapidly the samples thaw. Fig. 6B shows results for samples treated in an identical manner, except that they were thawed i air at room temperatures. This treatment results in a much sl warming rate than that seen in any of the water bath thawing treatments. Each point represents the mean +, SD for triplicat samples.

Fig. 7 shows the effect of varying the concentration of T across a broad range, on the level of hemolysis of human red b cells that were frozen in 15% HES 450/0.7 and thawed in air at room temperature. Each point represents the mean +. SD for triplicate samples.

Fig. 8 compares the level of hemolysis (after the post-th saline dilution) of human red blood cells frozen and thawed in presence of THP and different preparations of HES 200/0.5.^" Ea point represents the mean ± SD triplicate samples.

Fig. 9 shows the effect of the THP on the level of hemoly of full-units of human red blood cells frozen and thawed in HE Values are given for the initial percentage of hemolysis after thawing and for the degree of hemolysis arising after a post-t dilution in saline. Each data set represents the mean ^ SD fo three separate full-units of red blood cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Reference will now be made in detail to the presently preferred embodiments of the invention, which, together with t following examples, serve to explain the principles of the in tion. All references cited herein are hereby incorporated by reference.

The following amino acids are abbreviated herein as follo Ala is Alanine,

Arg is Arginine, Asn is Asparagine, Asp is Aspartic acid, Gin is Glutamine, Glu is Glutamic acid,

His is Histidine, lie is Isoleucine, Ser is Serine, Thr is Threonine, and Val is Valine.

The present invention encompasses a method for enhancing blood cell survival during cryopreservation and thawing, whic comprised of freezing and thawing red blood cells in the pres of a penetrating or nonpenetrating cryoprotectant(s) and a th hysteresis peptide. In the field of cryopreservation of red cells, the goal is to maximize the viability and functionalit that is present upon thawing.

To achieve this goal, often it is desirable to reduce th level of cryoprotectant used to preserve red blood cells, sin many of the classical penetrating cryoprotectants are toxic t blood cells at physiological temperatures as discussed above. (Fahy, Cryobiology 23:1-13, 1986). In addition, some cryopro tectants are more preferable because they are less toxic and/ pose less of an osmotic stress to red blood cells during free thawing regimes, for example, nonpenetrating cryoprotectants as polyvinyl-pyrrolidone and hydroxyethyl starch. However, t compounds when used alone often do not provide a sufficient l of cryoprotection to red blood cells. Moreover, often extrem rapid thawing is needed to achieve red blood cell survival du freeze-thawing with these compounds. By the use of combinati of thermal hysteresis peptide and cryoprotectants, the presen invention provides a novel and surprising solution to these l standing problems in cryobiology.

As used herein, the term "thermal hysteresis peptide (TH refers to a class of macromolecular ice crystal control pepti and glycopeptides. They are referred to as thermal hysteresis molecules or antifreezes because they lower the freezing point water without altering the melting point significantly. These crystal control materials also reduce or prevent migratory ice crystal growth, a process known as recrystallization. THPs ar found in nature in certain polar fish, winter flounder and in certain freeze-tolerant insects.

The discoveries described by the present invention were b on research with THP from the winter flounder, which has a molecular weight of 3300, is comprised of 37 amino acids and the following formula: (NH₂) ASP-THR-ALA-SER-ASP-ALA-ALA-ALA- A___-AI_A-LEU-THR-AI_A-AI_A-ASN-AI_A-LYS--_I_A-AI_A-AIJ_-GLU-LEU-THR-AI AI_A-ASN-AI_A-AI_A-AI_A---LA-AIA-AI_A-ALA-THR-ALA-ARG (COOH) . The protein was produced by use of a synthetic gene and was expres in the bacterium _E. coli, as described in patent application s rial no. 07/394,881. However, the methods and compositions described herein are applicable to any of the THP molecules derived by purification from natural sources, chemical synthe using solid phase techniques or synthesis by use of recombina gene technology, which molecules demonstrate the properties o controlling ice crystal growth.

The term ultracold temperatures is defined as temperatur lower than -100°C Room temperature is a term known in the a and according to certain preferred embodiments encompasses temperatures of 20°C to 24°C.

The term "cryoprotectant", as used herein, refers to bro groups of organic molecules that have been shown to protect c lular viability and function, the morphological and functiona integrity of tissues and their component cells, and the struc and function of biomaterials, for example, liposomes and isol enzymes, during freezing and storage at ultracold temperatures and thawing. Penetrating cryoprotectants are those molecules can permeate across the plasma membrane into the interior of c and examples include dimethylsulfoxide, glycerol, ethylene gly propane diols, butane diols, amide compounds (e.g., formamide) and others . Extracellular or nonpenetrating cryoprotectants a those molecules that do not permeate across the plasma membran and include, for example, relatively large organic polymers su as polyvinylpyrrolidone, hydroxyethyl starch, dextrans, FICOLL PHARMACIA, polyethylene glycol, and others. Other classes of compounds that may serve as extracellular cryoprotectants incl sugars (e.g., sucrose, maltose and trehalose), amino^' acids (e. proline, glutamate, and glycine), methylamines (e.g., betaine sarcosine), and polyhydroxy alcohols (e.g., sorbitol and mannitol). The foregoing examples of suitable cryoprotectants exemplary only, and should not be considered exhaustive. Enha cryoprotection can be realized using the present invention by combining THP with individual cryoprotectants or mixtures of cryoprotectants, and mixtures can comprise a combination of completely different cryoprotectant materials and/or variation the same materials, e.g., different commercial preparations of same material.

The present inventor has also discovered that certain pretreatments of cryoprotectants before combining with THP far minimizes cellular damage. According to certain preferred embodiments, such pretreatments can be described as fractionat processes. Fractionation processes can be defined as process that separate out lower molecular weight constituents from th cryoprotectant preparation. Certain examples of fractionatio processes include dialysis, ultrafiltration, and washing with solvent such as alcohol, e.g., isopropanol. These examples a not to limit the present invention, because those of skill in art will be able to determine processes that achieve the fractionation as described herein. According to certain preferred embodiments, the pretreat includes processes that remove contaminants from cryoprotecta preparations with or without actual fractionation. Such proc may include washing with alcohol or other solvents. Again, t of skill in the art, given the present disclosure, would be a to determine other processes that are encompassed by the pres described pretreatment processes, and thus, the example provi should in no way limit the present invention.

According to certain preferred embodiments, starch cryoprotectants, e.g., hydroxyethyl starch (HES), are pretrea However, any cryoprotectant can be pretreated as described he to enhance the protection afforded when the pretreated cryoprotectant is combined in a treatment with THP. For exam it is contemplated by the present inventor that any of the cryoprotectant polymers could be pretreated to enhance cryopreservation when such pretreated polymers are combined w

THP according to the presently claimed processes.

The methods and formulations described herein apply to r blood cells that are sensitive to freeze-thawing, but which c cryopreserved with one or a combination of the compounds list above. The red blood cells are placed in a suitable tissue culture medium, physiological saline or buffer, which contain least one cryoprotectant in combination with at least one the hysteresis peptide. The choice of aqueous medium, as well as cryoprotectant, will depend on various factors such as the material to be preserved, any constraints on freezing rate (e. sample volume and geometry), and sensitivity to cryoprotectan toxicity. The present inventors discovered that the cooling warming protocols and the concentrations of cryoprotectant an thermal hysteresis peptide must be chosen such that the therma hysteresis peptide enhances cryoprotection. Without proper matching of these parameters, the thermal hysteresis peptide c actually negate the protective effect of the cryoprotectant an induce damage to the frozen and thawed sample. The frozen sample can then be stored at ultracold temperatures, for example, in liquid nitrogen (-196°C) or in liquid nitrogen vapor (about -130°C). Alternatively, a mechan freezer that maintains temperatures below -100°C can be used. additional advantage of the present invention is that samples be stored at relatively high subzero temperatures, or temperat 1 greater than -100°C, for example, -80° or -20°C, and the presen of the thermal hysteresis peptide will minimize damage that can induced by migratory ice recrystallization during long term storage.

5 One further advantage of the present invention is that, regardless of the type of sample to be frozen, the presence of thermal hysteresis peptide can enhance the degree of preservati provided by the cryoprotectant, when the proper combination of parameters is used.

.0 It is possible to use known dilution assay techniques to determine the level of cell protection after thawing. Those o skill in this art are familiar with such assay techniques, whi are thought to be a predictor of how cells will survive when infused into a living body. Such techniques are not necessary 5 showing the effects of cryoprotection, but are merely another technique that can be used to determine such effects. Example which follows, describes such a technique, but because such techniques are known by those skilled in the art a more detail discussion is not deemed necessary.

20 The following specific examples will illustrate certain embodiments of the invention applied to the cryopreservation o human red blood cells. The THP used in these examples was the type described in the sentence bridging pages 11 and 12 of the present application. Various alternatives will be apparent to 25

30 those of ordinary skill in the art from the teachings herein, the invention is not limited to the specific illustrative examples.

EXAMPLE 1 Human blood was collected into hepinarized tubes and centrifuged to pack the red cells. The red cells were washed phosphate buffered saline (PBS) and centrif ged. The superna was removed and the wash was repeated. After centrifugation, supernatant was removed and the red cells were brought to a hematocrit of approximately 70% in PBS. To an aliquot of the cell suspension was added an equal volume of 2.0 M glycerol ( PBS) with 2-times the final desired concentration of THP. Th samples were held on ice for 0.5 hour, after which 0.4 ml ali were placed into 1.5 ml Eppendorf test tubes for freezing. Fo different freezing protocols were compared: 1) Samples were c at 20°C/min to -80°C, transferred to liquid nitrogen vapor and stored overnight ( ) } 2 ) Samples were cooled at 100°C/min to -80°C, transferred to liquid nitrogen vapor and stored overnig (□); 3) Samples were placed into a -20°C freezer and stored overnight (O ) ; and 4) Samples were frozen by plunging the tes tubes directly into liquid nitrogen, and then stored overnight liquid nitrogen vapor (#). For each freezing regime, two dif ent thawing protocols were tested: 1) samples test tubes were immersed into a 45°C water bath; and 2) samples were thawed in at room temperature. Cell damage arising during freeze-thawing was quantified b measuring the percentage of hemolysis. An aliquot of the supernatant of the centrifuged red cell samples was mixed with

Drabkin's reagent to convert free hemoglobin into cyanomethemoglobin. Unfrozen cell solutions were used as the control (blank) and saponin lysed cells as the 100% hemolysis standard. Optical density of samples was measured at 540 nm wi a microtiter plate spectrophotometer. The percentage hemolysis was calculated according to Pegg et al. , Cryobiology 21:491 usi the following equation:

ODsample - ODblank x 100 ODsaponin std. - ODblank

The results for cells that were thawed in the 45°C water b are shown in Fig. 1A. The presence of THP had minimal influenc on the degree of hemolysis of red cells frozen by any of the protocols, except for cells frozen in a -20°C freezer. In this case, the THP induced increased hemolysis. For cells thawed i air at room temperature, THP also caused increased hemolysis i samples frozen in a^' -20°C freezer (Fig. IB). In contrast, the presence of THP led to reduced hemolysis of red cells frozen b plunging into liquid nitrogen. These results demonstrate that method used to freeze and thaw red blood cells can greatly inf ence the effect of THP on red cell survival, and that under th proper conditions, THP can enhance cryopreservation of red blo cells by glycerol. EXAMPLE 2

Red blood cells were prepared, frozen and thawed as descr in Example 1, except that 1.5 M glycerol was used as the cryoprotectant. The results for samples that were thawed by i mersion into a 45°C water bath are shown in Fig. 2A. The effe of the THP were more dramatic when 1.5 M glycerol was used as cryoprotectant. For all freezing protocols, except plunging i liquid nitrogen, the presence of THP led to increased hemolysi With this latter method, however, the presence of THP led to a relatively large decrease in hemolysis. Similar results were for samples that were thawed in air at room temperature (Fig.

These findings demonstrate that under the proper conditio THP can be used to reduce substantially damage to red blood ce during freeze-thawing in the presence of glycerol. EXAMPLE 3

Red blood cells were prepared, frozen and thawed as descr in Example 1, except that 15% (wt/vol) hydroxyethyl starch (450/0.7) was used as the cryoprotectant. The results for sam that were thawed by immersion into a 45°C water bath are shown Fig. 3A. For all freezing protocols, except plunging into liq nitrogen, the presence of THP led to no change or a slight increase in hemolysis. With this latter method, however, the presence of THP led to a small, in absolute terms, but signifi decrease in hemolysis. That is, without the THP, hemolysis wa 3.3%, but with 10 μg/ml THP hemolysis was reduced to 1.3%. Th effects of THP were much more dramatic for samples that were thawed in air at room temperature (Fig. 3B) . For samples that were frozen by plunging into liquid nitrogen, the degree of hemolysis was reduced from 42.8%, without the THP, to about 18 the presence of 10-40 μg/ml THP.

These findings demonstrate that under the proper conditio THP can be used to reduce substantially damage to red blood ce during freeze-thawing in the presence of the extracellular cryoprotectant, hydroxyethyl starch.

EXAMPLE 4

Human blood was collected into heparinized tubes and centrifuged to pack the red cells. The buffy coat was removed the remaining red cells were centrifuged. Sufficient plasma w removed from the supernatant to give a final hematocrit of ap¬ proximately 70%. To an aliquot of the red cell suspension was added an equal volume of 30% (wt/vol) hydroxyethyl starch (450/0.7) with 2-times the final desired concentration of THP. The samples were held on ice for 0.5 hour, after which 0. ml aliquots were placed into 1.5 ml EPPENDORF (microcentrifuge) t tubes for freezing. Samples were frozen by plunging the test tubes directly into liquid nitrogen, and then stored overnight liquid nitrogen vapor. Two different thawing protocols were tested: 1) sample test tubes were immersed in a 45°C water bat and 2) samples were thawed in air at room temperature. Hemolysis was assessed as described in Example 1. The results for red cells that were thawed in the 45°C water bath shown in Fig. 4A. The average percentage of hemolysis (close triangles) was reduced from 3.6 %, without THP, to as low as in the presence of 10 μg/ml THP. In addition, examination of values for individual samples (open circles) reveals that wit THP, two out of three samples had hemolysis levels exceeding and only a single sample had hemolysis less than 2%. In cont in the presence of 10 _/_g/ml THP, the values for all samples w tightly grouped at less than 1.4% hemolysis.

These results demonstrate that not only does THP foster enhanced cryopreservation, but it also minimizes the variabil in the degree of hemolysis between individual samples. That the reproducibility of red cell recovery during freeze-thawin much greater when THP is present. Therefore, the addition of would serve to assure that all samples processed would meet stringent guidelines for cell survival (e.g., less than 3% hemolysis) .

The degree of THP-induced enhanced cryopreservation was greater when samples were thawed in air at room temperature (

4B) . The degree of hemolysis was reduced from 45.5 %, without THP, to as low as 11% in the presence of 20 _/_g/ml THP.

Taken together, the results of this experiment show that, without the addition of THP, slow thawing in air results in a greater degree of hemolysis compared to that seen after rapid thawing in a 45°C water bath (Fig. 4A, B). In contrast, in the presence of the THP, slow thawing is much less detrimental. Th the addition of THP to the cryoprotectant solution serves to minimize the damage that arises due to thawing samples at suboptimal warming rates. Moreover, THP allows one to achieve very high levels of cell preservation consistently, even if the warming rates vary.

EXAMPLE 5 The influence of THP on the stability of cryopreserved red blood cells stored at -80°C was tested. Human red blood cells were washed with PBS (as described in Example 1) and combined HES 450/0.7 to give a final hematocrit of 35% and a final HES concentration of 15% (wt/vol). Another mixture was prepared i the identical manner except that 10 μg/ml THP was added. 0.4 aliquots were placed into 1.5 ml EPPENDORF (microcentrifuge) t tubes and the samples were frozen by plunging the tubes direct into liquid nitrogen. The frozen samples were then transferre into a -80°C mechanical freezer. Samples were removed at intervals during a four week period and thawed by immersion in 45°C water bath.

The durability of the frozen-thawed red blood cells was tested by diluting the cell suspension in physiological saline human plasma. The degree of hemolysis induced by these treatm was assessed by the following method. After thawing, the red cell suspension was mixed well an

25 μl aliquot was placed into 975 μl of PBS and into 975 μl o human plasma. From the resulting suspensions, 400 μl was rem and mixed with 20 μl of 0.5% saponin (in PBS). This mixture freeze-thawed twice to induce complete lysis of the red cells

(saponin standard). The remaining 600 μl of the red cell suspension was centrifuged to pack the red cells. 400 μl of supernatant was removed and treated with saponin as above. Hemoglobin content of the samples was determined by mixing 300 of the sample with 500 μl of Drabkin's Reagent and reading absorbance at 540 nm. The percentage of red cells undergoing hemolysis after dilution was calculated with the following equation:

Abs54nsupernatant - AbS54pblank x 100 Abs54Qsaponin standard - Abs54Q blank

The results are given in Figures 5A and 5B. The presence

THP greatly enhances the percentage of cells surviving after dilution into either saline or plasma. These results indicate that THP protects red cells from damage arising during storage relatively high temperatures (> - 100°C), which are normally considered suboptimal temperatures.

EXAMPLE 6

Comparison of the influence of THP on survival of cryopreserve red cells thawed at different rates. The red cells were prepared and frozen as described in Example 4. The frozen samples were thawed in a water bath set different temperatures (or in air at room temperature). After thawing, the red cell suspension was mixed well and a 25 μl aliquot was placed into 975 μl of phosphate buffered saline (PBS). From the resulting suspension, 400 μl were removed and mixed with 20 μl of 1.0% saponin to induce complete cell lysis (saponin standard). To another 400 μl aliquot was added 20 μl of PBS; simulating the degree of dilution arising upon addition saponin. These samples were then centrifuged to pack the red cells. Hemoglobin contents of the lysed samples and the supernatant of the other samples were determined by mixing 300 of the appropriate sample with 500 μl of Drabkin's reagent. T percentage of red cells undergoing hemolysis after dilution in saline was calculated as described in Example 5.

The results in Fig. 6A demonstrate that improved cell recovery is realized in the presence of THP, at all thawing ra the warmer the water bath, the more rapidly the samples will t In addition, these data document that greater cell recovery is noted with THP, even at the least optimal thawing rate (23°C w bath), than is seen without THP at the optimal warming rate (4 water bath). The data in Fig. 6B show that even at the slowes warming rate (in air at room temperature) the THP greatly impr cell survival. 1 EXAMPLE 7

Concentration dependency of THP's effects on cell survival du cryopreservation.

5 Red cells were prepared and frozen as described in Examp

The samples were thawed in air at room temperature and hemoly was measured as described in Example 1. The results in Figur demonstrate that at concentrations _^■ 100 μg/ml, THP greatly enhances cell survival. This effect is lost when the

0 concentration is increased to 500 μg/ml, and at 1000 μg/ml th presence of THP actually leads to increased cell damage.

EXAMPLE 8

Effect of fractionating HES on cryoprotective capacity in the presence of THP. 5

Human blood was collected and prepared as described in

Example 4. To an aliquot of the red cell suspension was added equal volume of 30% (wt/vol) HES 200/0.5 containing 2-times t final desired concentration of THP. Three different preparati

20 of HES 200/0.5 were tested. 1) HES 200/0.5 was used as receiv from the manufacturer, with no further processing. Since this preparation already contains NaCl, the solution was prepared i distilled water. 2) HES 200/0.5 was washed with isopropanol a follows. Approximately 20 g of HES was mixed with 200 ml of i

25 cold isopropanol in a 250 ml centrifuge bottle. The bottles w centrifuged (16,000 x g) at 4°C for 30 minutes. The resulting supernatant and suspended particles were decanted off, and the wash procedure was repeated two-times. After the final wash,

^•30 HES was dried in air, followed by vacuum drying on a lyophiliz

This washing procedure does not remove the NaCl from the HES preparations, therefore the isopropanol-washed HES was dissolv in distilled water. 3) HES was dialyzed (Spectrapor tubing wi molecular weight cutoff of 3500) for several days (at room temperature) against distilled water to remove NaCl and low molecular weight organic constituents. The water outside the dialysis bags was changed several times during this process. resulting, dialyzed HES solution was then freeze-dried. Since osmotic contribution of the HES molecules themselves is negligible, the dialyzed HES was dissolved in phosphate buffer saline for the cryopreservation experiments.

Samples were frozen as described in Example 4 and thawed 48°C water bath. The degree of cell damage was quantified usi the saline dilution assay described in Example 6. The results presented in Figure 8 demonstrate that cell recovery is much greater when either isopropanol-washed or dialyzed HES is used combination with THP, than when the unprocessed HES is used wi

THP. EXAMPLE 9

Cryopreservation of full units of red cells with isopropanol- washed HES

For this experiment a blend of HES's was employed. 330 g (dry weight) of isopropanol-washed HES 200/0.5 was mixed with

(dry weight) of isopropanol-washed HES 450/0.7. The HES sampl were washed as described in Example 8. This mixture was disso in distilled water and brought to a final volume of 1400 ml., t resulting in a 30% (wt/vol) solution of HES. 700 ml of this solution was mixed with THP to give a final THP concentration 20 μg/ml. Full units of packed red blood cells were washed 3-tϊmes with phosphate buffered saline and centrifuged. A ful unit of red blood cells is defined as the amount of red blood cells obtained from about 450 ml of whole blood, the normal vo of one donation. Whole blood comprises about 40 % red blood cells.

The cells were brought to a hematocrit of approximately 8 To 220 ml of the red cell mixture was added an equal volume of HES, either with or without 20 μg/ml THP. After thorough mixi and chilling to 4°C, the 440 ml samples were frozen by direct plunging into liquid nitrogen and stored in liquid nitrogen va overnight. Triplicate samples were prepared for each treatmen i.e., with and without THP. The samples were thawed by exposu to air at room temperature for 20 minutes, followed by complet of the thawing process in a 22°C water bath. This process resulted in relatively slow, suboptimal rate of warming.

The initial hemolysis was compared to that for aliquots subjected to a 1/40 dilution in phosphate buffered saline. Th results presented in Figure 9 indicate that THP, used in combination with the blend of isopropanol-washed HES's, greatl enhanced the survival of the red blood cells. It will be apparent to those skilled in the art that variou modifications and variations can be made in the processes and products of the present invention. Thus, it is intended that th present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method for enhancing survival of red blood cells during cryopreservation comprising freezing and subsequently t ing red blood cells in the presence of at least one cryoprotec and a thermal hysteresis peptide in an amount effective for enhancing viability of the red blood cells.

2. A method as in claim 1, wherein said freezing and tha ing include using at least one cryoprotectant capable of penetrating into an intracellular area of the red blood cells.

3. A method as in claim 1, wherein said freezing and tha ing include using at least one cryoprotectant which does not penetrate into an intracellular area of the red' blood cells.

4. A method as in claim 1, wherein said freezing and tha ing include using a thermal hysteresis peptide having the follo ing amino acid sequence: (NH₂) ASP-THR-ALA-SER-ASP-ALA-ALA-ALA AI_A-ALA-A]_A-LEU-THR-AI_A-AI_A-ASN-ALA-LYS-AI_A-AI_A-ALA-GLU-LEU-THR AI_A-A__A-ASN-A1_A-AI_A-A__A-ALA-ALA-AIA-AI_A-THR-__LA-ARG (COOH) .

5. A method as in claim 1, wherein said freezing and thawing include using a thermal hysteresis peptide selected fro the group consisting of a glycopeptide having a molecular weigh of about 2,600 to 37,000 having high proportions of alanine and threonine, a peptide having a molecular weight of about 3,300 t 4,000 having a high alanine content, a peptide having a molecul weight of about 9,900 containing cysteine and a peptide having molecular weight of about 6,000 being neither alanine rich nor cysteine rich.

6. A method as in claim 1, wherein prior to said freezin and thawing said cryoprotectant is subjected to at least one of fractionation and extraction of certain impurities.

7. A method as in claim 6, wherein prior to said freezin and thawing said cryoprotectant is subjected to at least one of dialysis, ultrafiltration, gel filtration chromatography, and washing with an alcohol.

8. A method as in claim 6, wherein prior to said freezin and thawing said cryoprotectant is subjected to washing with isopropanol.

9. A method as in claim 6, wherein said cryoprotectant i hydroxyethyl starch and prior to said freezing and thawing said hydroxyethyl starch is subjected to washing with isopropanol.

10. A method as in claim 6, wherein said cryoprotectant i hydroxyethyl starch and prior to said freezing and thawing said hydroxyethyl starch is subjected to dialysis.

11. A method as in claim 1, wherein said freezing and thawing includes using at least one cryoprotectant selected fro the group consisting of dimethylsulfoxide, glycerol, ethylene glycol, propane diols, butane diols, amides, hydroxyethyl starc dextrans, polyethylene glycol, FICOLL, polyvinyl-pyrrolidone, trehalose, maltose, glucose, sorbitol, proline, glutamate, mannitol and betaine.

12. A method as in claim 1, wherein said freezing and thawing includes using at least one cryoprotectant selected .fr the group consisting of dimethylsulfoxide, glycerol, ethyleneglycol, hydroxyethyl starch, dextrans, polyethylene glycol, polyvinyl-pyrrolidone, trehalose, sorbitol, proline a glutamate.

13. A method as in claim 1, wherein said freezing and thawing includes using thermal hysteresis peptide in an amoun greater than 200 μg/ml.

14. A method as in claim 1, wherein said freezing and thawing includes using thermal hysteresis peptide in an amount about 1-100 μg/ml.

15. A method as in claim 1, wherein said freezing includ plunging into liquid nitrogen.

16. A method as in claim 1, wherein said freezing and thawing further includes using glycerol or hydroxyethyl starch the cryoprotectant.

17. A method as in claim 1, wherein said freezing and thawing of red blood cells in the presence of at least one cryoprotectant and a thermal hysteresis peptide enhances the viability of the red blood cells at warming rates that can dama the red blood cells, in the absence of thermal hysteresis pepti

18. A method as in claim 17, wherein said thawing include thawing in air at approximately room temperature.

19. A method as in claim 1, wherein said freezing includ long-term storage at temperatures greater than -100°C.

20. A method as in claim 1, wherein said freezing and th ing include using a full unit volume of red blood cells.

21. A composition for enhancing survival of red blood ce during cryopreservation and thawing comprising at least one cryoprotectant and a thermal hysteresis peptide in an amount effective for enhancing viability of the red blood cells.

22. A composition as in claim 21, wherein said at least cryoprotectant is capable of penetrating into an intracellular area of the red blood cells.

23. A composition as in claim 21, wherein said at least cryoprotectant does not penetrate into an intracellular area o the red blood cells.

24. A composition as in claim 21, wherein said thermal hysteresis peptide has the following amino acid sequence: (NH ASP-THR-ALA-SER-ASP-ALA-AI_A-AI_A-ALA-ALA-ALA-LEU-THR-ALA-ALA-AS A__A-LYS-ALA-ALA-__LA-GLU-LEU-THR-AI_A-ALA-ASN-A__A-ALA^ ALA-ALA-THR-ALA-ARG (COOH) .

25. A composition as in claim 21, wherein said cryoprotectant has been subjected to at least one of fractiona and extraction of certain impurities.

26. A composition as in claim 25, wherein said cryoprotectant has been subjected to at least one of dialysis, ultrafiltration, gel filtration chromatography, and washing wi an alcohol.

27. A composition as in claim 25, wherein said cryoprotectant has been subjected to washing with isopropanol.

28. A composition as in claim 25, wherein said cryoprotectant is hydroxyethyl starch and said hydroxyethyl st has been subjected to washing with isopropanol.

29. A composition as in claim 25, wherein said cryoprotectant is hydroxyethyl starch and said hydroxyethyl st has been subjected to dialysis.

30. A composition as in claim 21, wherein said cryoprotectant is selected from at least one of the group consisting of dimethylsulfoxide, glycerol, ethylene glycol, propane diols, butane diols, amides, hydroxyethyl starch, dextrans, polyethylene glycol, FICOLL, polyvinyl-pyrrolidone, trehalose, maltose, glucose, sorbitol, proline, glutamate, mannitol and betaine.

31. A composition as in claim 21, wherein said thermal hysteresis peptide is in an amount no greater than 200 μg/ml.

32. A composition as in claim 21, wherein said thermal hysteresis peptide is in an amount of about 1-100 μg/ml.

33. A composition as in claim 21, wherein said at least cryoprotectant and a thermal hysteresis peptide are in an amou effective for enhancing viability of the red blood cells in a f unit volume of red blood cells.