WO2013096659A1

WO2013096659A1 - Methods and compositions for storage of animal cells

Info

Publication number: WO2013096659A1
Application number: PCT/US2012/071025
Authority: WO
Inventors: Xu Han; Erik John Woods
Original assignee: Cook General Biotechnology Llc
Priority date: 2011-12-20
Filing date: 2012-12-20
Publication date: 2013-06-27

Abstract

In certain aspects, the present disclosure provides unique compositions and methods for cryopreservation of biological samples. In accordance with some forms of practicing the invention, such compositions and methods are effective at maintaining sample vitrification at temperatures above -100 °C. Accordingly, in one embodiment, the present disclosure provides a cryopreservation media that includes at least a cryoprotectant and a polymer, wherein the cryopreservation media has a devitrification temperature between about -100 °C and about -60 °C.

Description

METHODS AND COMPOSITIONS FOR

STORAGE OF ANIMAL CELLS CROSS REFERENCE TO RELATED APPLICATION

The present application claims the benefit of co-pending provisional application No. 61/577,770, which was filed on December 20, 2011, which is hereby incorporated by reference in its entirety. BACKGROUND

The present disclosure relates generally to the storage of animal cells, and in certain embodiments to polymer-modified cryopreservation media with beneficial vitrification properties, and associated cellular compositions and methods.

Cryopreservation of embryos from animal models enables genetic resource centers to operate in a more cost-effective manner by maintaining and shipping animal lines as cryopreserved germplasm rather than live animals. However, the associated cryopreservation procedures inevitably cause cell damage. Survival of a cell during cryopreservation depends on whether this damage can be minimized. Vitrified samples are generally stored in liquid nitrogen at -196°C or in liquid nitrogen vapor at -120°C. Devitrification is a crystallization process which is detrimental to cells due to the associated mechanical damage and should be avoided during storage or shipping. During storage, shipping or warming, devitrification may occur if the sample is warmed to a temperature higher than its devitrification temperature (T^) typical aqueous solutions designed for cell cryopreservation contain cryoprotectant agents such as dimethyl sulfoxide

(DMSO). Typical cryoprotectant agent T^ values are typically between -120°C and -100°C which are higher than liquid nitrogen and its vapor storage

temperatures so they are stable for storage and shipping of cryopreserved samples in the liquid nitrogen or its vapor. However, storage or shipping in liquid nitrogen requires relatively large space to accommodate heavy liquid nitrogen dewars. These space and weight criteria significantly increase the associated costs. If an aqueous cryoprotectant solution can be developed to be thermodynamically stable at higher temperatures, for example temperatures as high as -80°C, it is then practical to store samples using conventional freezers such as -80°C freezers and ship them safely on dry ice in inexpensive shipping containers, such as polyethylene boxes. The need therefore exists for improved compositions cryopreservation compositions and methods.

SUMMARY

In certain aspects, the present disclosure provides unique compositions and methods for cryopreservation of biological samples. In accordance with some forms of practicing the invention, such compositions and methods are effective at maintaining sample vitrification at temperatures above (warmer than) -100 °C. Accordingly, in one embodiment, the present disclosure provides a

cryopreservation medium that includes at least a cryoprotectant and a polymer, wherein the cryopreservation medium has a devitrification temperature above - 100°C and in particular embodiments between about -100 °C and about -60 °C. In some forms, the cryoprotectant comprises dimethyl sulfoxide, glycerol, and/or lactose-egg yolk extender. In some forms, the polymer comprises Ficoll and/or polyvinylpyrrolidone. In accordance with some forms of practicing the invention, the cryopreservation medium has a devitrification temperature greater than -80 °C. In certain aspects, the cryopreservation medium has a Ficoll concentration between about 1% to about 15%. In certain modes of practicing the invention, the cryopreservation medium is configured for use with mammalian cells, including for example, murine, porcine, and/or human cells. In some forms the

cryopreservation medium of the present disclosure is configured for use with embryonic stem cells, embryos, pluripotent stem cells, and/or spermatozoa.

In another embodiment, the disclosure provides a cellular composition comprised of cells suspended in a cryopreservation medium as described above. In some forms, the suspended cells are mammalian cells including for example, murine, porcine, and/or human cells. In accordance with certain inventive variants, the suspended cells are embryonic stem cells, embryos, pluripotent stem cells, and/or spermatozoa.

In another embodiment, the disclosure provides a method of cryogenically storing cells, the method comprising vitrifying cells in the presence of the cryopreservation medium described above. In some forms, the vitrified cells are mammalian cells including for example, murine, porcine, and/or human cells. In certain embodiments, the vitrified cells may be embryonic stem cells, embryos, pluripotent stem cells, and/or spermatozoa. Additional methods involve devitrifying such cryogenically stored cells, and optionally also administering the cells to a human or other animal for example to treat the animal for a disease state or condition.

Additional embodiments, as well as features and advantages of embodiments of the invention will be apparent from the description herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the exothermic curves of water-Ficoll 70-DMSO solutions with differing R values.

DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS

For the purposes of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the claims is thereby intended, and alterations and modifications in the illustrated device, and further applications of the principles of the disclosure as illustrated therein are herein contemplated as would normally occur to one skilled in the art to which the disclosure relates.

The present disclosure relates to the development of cryopreservation media with preferred devitrification characteristics. In certain embodiments such a cryopreservation medium may comprise a cryoprotectant and a polymer. In some forms, cryopreservation media of the present disclosure will have a devitrification temperature between about -100°C and about -60°C. In accordance with some forms of practicing the invention the cryopreservation medium includes a cryoprotectant. The cryoprotectant of the present disclosure may include for example, dimethyl sulfoxide (DMSO), glycerol and/or lactose-egg yolk extender.

In certain inventive variants, the cryopreservation medium of the present disclosure includes a polymer. In certain embodiments, addition of a polymer may increase the vitrification temperature of the disclosed cryopreservation medium. In some forms, the polymer may thicken or increase the viscosity of the

cryopreservation medium. In certain embodiments, the polymer of the present disclosure may comprise a sucrose polymer. In some forms, the polymer of the present disclosure may comprise for example, Ficoll, and/or polyvinylpyrrolidone. Certain inventive variants may include for example, percoll, hyaluronic acid, albumin and/or glycerol. In some forms, the polymer component may comprise between about 1% and about 20% by weight of the cryopreservation medium. In preferred embodiments the polymer will comprise between about 5% to about 15% by weight of the cryopreservation medium. In certain preferred embodiments, the polymer composition may comprise 10% by weight of the cryopreservation medium. In other preferred embodiments, the polymer may comprise about 5% by weight of the cryopreservation medium. In some forms, the polymer concentration is less than 20% by weight of the cryopreservation medium. It is noted that the concentration should be sufficient to stabilize cryopreservation solutions between about -100°C to about -60°C, preferably greater than about -80°C by preventing devitrification, but polymer concentration should be low enough to attenuate the cell osmotic damage associated with the addition of excessive polymer.

In some forms, the polymer of the present invention comprises Ficoll. As is well known, Ficoll is an uncharged, highly branched polymer formed by the co- polymerization of sucrose and epichlorohydrin. In certain embodiments, the polymer comprises Ficoll or another sucrose polymer having a weight average molecular weight of less than about 500,000 Daltons. In certain embodiments, the polymer of the present invention comprises Ficoll 70, in which the Ficoll molecules of the composition have an average molecular weight of about 70,000 Daltons. In certain embodiments, the polymer of the present invention comprises Ficoll 400, in which the Ficoll molecules of the composition have an average a molecular weight of about 400,000 Daltons.

In certain embodiments, the cryopreservation medium of the present disclosure has a devitrification temperature between about -100°C and about - 60°C. In certain embodiments, the cryopreservation medium of the present disclosure has a devitrification temperature between about -90°C to about -70°C. In preferred embodiments, the cryopreservation medium of the present disclosure has a devitrification temperature greater than about -80°C.

In certain embodiments, the cryopreservation medium of the present disclosure is configured for use with mammalian cells including, but not limited to, murine cells, porcine cells and/or human cells. In some forms, the

cryopreservation medium of the present disclosure is configured for use with cells, including but not limited to, embryonic stem cells, pluipotent stem cells, spermatozoa and/or embryos.

In accordance with some forms of practicing the invention, cells are isolated from patient or donor tissue, for example for autologous, allogenic or xenogenic use in a subject. As described above, a variety of cell types may be preserved according to the present disclosure. Methods of isolating, for example, embryonic stem cells, pluripotent stem cells, spermatozoa and/or embryos are known in the art. The isolated cells may comprise one or more individual cells. In certain embodiments the isolated cells are suspended in the cryopreservation medium of the present disclosure. Suspension may occur, for example, in a cryovial, straw, or other suitable container. In accordance with some forms of practicing the disclosure the composition comprising the cells suspended in the cryopreservation medium may be initially cooled in dry ice. In some forms the composition is stored for a period of time at a temperature between about -100°C and about -60°C. In certain embodiments, the cells may be stored for at least 1 week. In other embodiments the cells may be stored for at least one month, for example for 1-12 months. In yet another embodiment, the cells may be stored for more than 12 months. In accordance with certain modes of practicing the invention, the cells may be thawed after the cryopreservation storage. In some forms, the cells are thawed by immersing the frozen container in a water bath of about 37°C. In certain embodiments the thawed cells are further cultured before administration or other use, and in other embodiments they are used without further culture and/or without expansion in cell number. In accordance with certain inventive variants, the thawed cells are administered to an animal or human patient, e.g. for treatment of a disease state or condition, or in the case of embryos implantation in a uterus of a subject, e.g. for reproductive purposes or in case of sperm for fertilization of an egg, e.g. for reproductive purposes. In some forms, the thawed cells may be utilized for research purposes.

Example 1:

Cryopreservation of Mouse Embryonic Stem Cells Using the Developed Media.

Mouse embryonic stem cells (C57BL/6) were cryopreserved using cryopreservation media of the present disclosure with differing concentrations of Ficoll 70 and stored for one week. Cell culture and preparation followed the same procedure as described in a previous study [Benson, C.J., et al, An improved cryopreservation method for a mouse embryonic stem cell line, 56:210-220 (2008)]. Four different cryopreservation solutions were prepared and their compositions are listed in Table 1. Table 1

Cells cryopreserved in liquid nitrogen using solution A were used the control group. Cells were suspended in 1ml aliquots in five cryovials with cell density as 10⁵/ml. Cryovials were transferred to a commercially available freezing kit, refrigerated at -80°C overnight (a process which cools at a rate of approximately l°C/min), and subsequently transferred to liquid nitrogen. After one week of storage, the cryovials were thawed in a 37°C water bath. The percent post-thaw recoveries of membrane intact cells was determined through Trypan blue stain tests.

Cell cryopreservation at -80°C using solutions A-D followed the similar procedure as described above, except that the cryovials were not plunged into liquid nitrogen, but stored in the same -80°C freezer for one week. For each solution, five cryovials were tested with cell density as 10⁵/ml. The post-thaw membrane intact cell ratios are listed in Table 2.

Table 2

As shown in Table 2, higher concentrations of Ficoll resulted in higher cell survival rate with more stable results (smaller standard deviation across the samples). Use of solutions C and D, Ficoll concentrations of 10% and 15%, respectively, for cryopreservation at -80° C resulted in a higher survival rate than the results from the control group. Example 2

Cryopreservation of Porcine Induced Pluripotent Stem Cells Using the Developed Media

Procine induced pluripotent stem cells were prepared with the procedure described in a previous study [Telugu, B., et al, Porcine Induced Pluripotent Stem Cells Analogous to naive and Primed Embryonic Stem Cells of the Mouse, Int. J. Dev. Biol, 54: 1703-1711 (2010)]. The solutions used for cryopreservation were made with the same compositions as shown in Table 1 of Example 1 except that cultured media for porcine induced pluripotent stem cells was different from that used for murine embryonic stem cells. The cryopreservation procedures and warming procedures were the same as those used for murine embryonic stem cells as described in Example 1. The storage duration was three weeks. The post-thaw cell membrane integrity was also tested by Trypan blue staining and the results for cell survival rates are listed in Table 3. Table 3

As shown in Table 3, addition of sufficient concentrations of Ficoll (10-15%) can significantly increase the cell survival rates from the storage at -80°C for three weeks. More importantly, the use of solutions C and D for storage at -80°C resulted in noticeably higher cell survival rate than that of the control group stored in liquid nitrogen.

Example 3

Cryopreservation of Porcine Spermatozoa using the Developed Media

Porcine spermatozoa were cryopreserved with solutions whose

compositions are listed in Table 4.

Table 4

Approximately 100ml boar semen was purchased from the South Farms at the University of Missouri. Sperm rich fractions were extended and were slowly cooled at 15°C for two hours. After centrifuging the semen pellet, the semen pellet was suspended in lactose egg yolk (LEY) extender. After cooling to 5°C for two hours, the suspensions were extended 2 to 1 with cryoprotectant solutions containing 3x concentration of glycerol and Ficoll as shown in Table 4. The semen was loaded into 0.5ml straws, five straws containing cells in solutions A were used as the control group. They were held in liquid nitrogen vapor 5cm above the liquid for 20 minutes, plunged into the liquid nitrogen and stored for one week. After thawing the straws in a 37°C water bath for 30 seconds, the post-thaw cell survival rates were estimated by calculating the percentage of mobile cells from

microscopic observation. Cell cryopreservation at -80°C using solutions A-D were performed by inserting five straws containing cell suspensions into smashed dry ice for 20 minutes and storing them in a -80°C freezer for one week. With the same warming procedures and post-thaw analysis, the percentages of mobile cells for each treatment are shown in Table 5.

Table 5

Control A B C D

Recovery (%) 41.2 ± 4.2 17.0 ± 9.3 19.1 ± 3.7 39.7 ± 6.8 <10% According to the results shown in Table 5, addition of 5% Ficoll 70 significantly increases the cell survival rate from -80°C storage, comparing with the solutions containing no or 1 % Ficoll 70, and yield similar survival rate as the control group. Example 4

Measurement of the Devitrification Temperatures of Cryopreservation Solutions Containing Polymers using a Differential Scanning Calorimeter (DSC) To demonstrate the influence of the addition of polymers on the values of

Tr_f, cryopreservation media, the values of T^ of the water of a water Ficoll 70 DMSO solution with different solute mass ratios (R) were measured using DSC, where R is defined as the ratio of the mass of polymers over that of DMSO. The total weight percent (W_t) of both solutes (Ficoll 70 and DMSO) in the solutions was fixed as 50%, simulating the composition of the unfrozen fraction of initial cryopreservation solutions (W_t ~ 20% before freezing), after losing approximately 70% of their freezable water from an equilibrium freezing procedure. The R values were chosen as 1.0, 0.5, and 0. A volume of 8μ1 of each solution was sealed in a ΙΟμΙ aluminum crucible. Each sample was cooled to -60°C from 1°C at 100°C/min in a DSC. After being held at -160°C for one minute, the sample was heated to 20°C at a rate of 10°C/min. The exothermic curves of these frozen solutions during warming were recorded and the value of T_rf were calculated using the analytic software provided by the manufacturer. For comparison between different types of polymers and sucrose, the values of T^of other systems with the same values of R (= 1.0) and W, (= 50%), including water-Ficoll 400-DMSO, water-PVP 40-DMSO, water-PVP 360-DMSO and water-sucrose-DMSO, were also measured. The exothermic curves of the water-Ficoll 70-DMSO solutions with differing R values are shown in FIG. 1. The values of T^of all the tested solutions are listed in Table 6. Table 6

From the results shown in FIG. 1 and Table 2, it is clear that Ficoll 70 increases the value of T^of DMSO solutions at about -67°C when R equals 1 and can be used as the polymer to stabilize DMSO solutions stored in a freezer at about -80°C or on dry ice.

Example 5

Storage of Mouse Embryos in a -80°C Freezer

Based on the DSC experimental results reported in Example 4, Ficoll 70 was chosen as the polymer for mouse embryo cryopreservation at about -80°C. Female ICR mice (6-12 weeks old) were induced to super-ovulate and morula- stage embryos were obtained using standard procedures. To determine the optimal Ficoll 70 concentrations, six FHM solutions labeled as A-F containing 1.5M DMSO and different concentrations of Ficoll 70 were prepared and their compositions are listed in Table 7.

Table 7

(The Ficoll 70 concentration was calculated as the mass ratio of Ficoll 70 versus the FHM base solution without DMSO). Approximately 30 embryos were suspended in each solution and given approximately 10 minutes to fully equilibrate with the DMSO. Cell suspensions were drawn into the cryo-straws with a diameter of 2mm and thermally sealed. The straws were first pre-cooled to -4°C and nucleated using a cold metal clamp. They then were cooled to -40°C at approximately 0.5°C/min in a programmable cooling device. After the termination of controlled rate cooling, the straws containing solution A (the control group) were directly plunged into liquid nitrogen for storage. Other straws were plunged into a plastic bucket containing approximately one liter of dry ice and put into a - 80°C freezer for storage. After storage for one week, the straws were thawed by being plunged into water at room temperature. The thawed cell suspensions were mixed with 2x volume of FHM containing 0.2M sucrose for five minutes. When the embryos were recovered and cultured, the number of the early and hatched blastocysts for each group was determined after 48 hours of in vitro culture.

Approximately 150 to 200 embryos were separately tested in five straws for each solution.

For each straw, the percentage of re-warmed embryos that developed to the early and hatched stages was recorded. For each cryopreservation media the results from five straws using the same treatment were analyzed by standard analysis of variants procedures after square root transformation. To examine the effects of the concentration of Ficoll 70 on cryopreservation results, the differences between treatments were analyzed using a student's T-test and considered significant within alpha value of 0.05. The ratios of early and hatched blastocysts developed from the recovered frozen embryos cryopreserved at -80°C using five different cryopreservation media (solutions B-F) are listed in Table 7. The results from the control group which is cryopreserved in liquid nitrogen with solution A is also listed in Table 7 for comparison. A Ficoll concentration of about 10% is the preferred concentration in FHM solutions with 1.5M DMSO for mouse embryo cryopreservation at about -80°C. The resulting cell survival rate (92, ± 7%) was higher than that of the control group (88 ± 7%).

Referring now to FIG. 1, which as stated above illustrates the exothermic curves of water-Ficoll 70-DMSO solutions with differing R values including 1 010, 0.5 020, and 0 030. As illustrated in FIG. 1, a higher concentration of Ficoll 70 results in a higher value of T_A with T_rf being higher than -80°C when R equals 1. Therefore, addition of Ficoll 70 to DMSO aqueous solutions is an efficient approach to increasing the values of T^ and preventing devitrification during storage and shipping of biological samples. From the results shown in Table 7, Ficoll 400 PVP 40, PVP 360 and sucrose can also increase the values of T_rf of DMSO solutions. For example, the solution containing Ficoll 400 has a T^of - 75.7°C which may also satisfy the requirement for cryopreservation media stable at -80°C. From the results listed in Table 1, it can be seen that the use of solution D (FHM solution containing 10% Ficoll 70 and 1.5M DMSO) yields a survival rate not only higher than the solutions containing other Ficoll concentrations, but also higher than the control group stored at -196°C. The reason that lower cell survival rates were obtained when the concentrations of Ficoll 70 was less than 10% can be explained based on results shown in FIG. 1. To prevent devitrification of water- Ficoll 70-DMSO solutions at -80°C the value of R has to be sufficiently high

(approximately 1.0); otherwise, the value of T^ will be lower than -80°C after the equilibrium freezing procedures, resulting in a thermodynamically unstable solidified cell suspension.

When the concentration of Ficoll 70 is higher than 10%, the lower cell survival rates may be caused by the fact that the relatively high concentration of polymers may considerably increase solute osmolalities or reduce the amount of osmotically active water during the freezing procedures. Consequently, the high concentrations of Ficoll may result in severe osmotic damage that cells will undergo during freezing. Therefore, it is likely that an optimal concentration of Ficoll 70 exists for different cell types. The concentration should be high enough to stabilize the cryopreservation media solutions at -80°C by preventing

devitrification, but low enough to attenuate the cell osmotic damage associated with relatively high polymer concentrations.

While the disclosure has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the disclosure are desired to be protected. It will be evident from the specification that aspects or features discussed in one context or embodiment will be applicable in other contexts or embodiments. All publications cited herein are hereby incorporated by reference in their entirety as if fully set forth. In addition, unless expressly indicated otherwise, all embodiments disclosed herein may comprise, or may consist of or consist essentially of, the ingredients or other components specified for those embodiments.

Claims

CLAIMS What is claimed is:

1. A cryopreservation medium composition comprising:

a cryoprotectant; and

a polymer, wherein said cryopreservation medium composition has a devitrification temperature between about -100°C to about -60°C.

2. The cryopreservation medium composition of claim 1, wherein: said cryoprotectant comprises dimethyl sulfoxide.

3. The cryopreservation medium composition of claim 1, wherein: said cryoprotectant comprises glycerol.

4. The cryopreservation medium composition of claim 1, wherein: said cryoprotectant comprises lactose-egg yolk extender.

5. The cryopreservation medium composition of any one of claims 1-4, wherein:

said polymer comprises Ficoll.

6. The cryopreservation medium composition of claim 5, wherein: said polymer comprises Ficoll 70.

7. The cryopreservation medium composition of claim 5, wherein: said polymer comprises Ficoll 400.

8. The cryopreservation medium composition of any one of claims 1-4, wherein:

said polymer comprises polyvinylpyrrolidone.

9. The cryopreservation medium composition of any one of claims 1-8, wherein: said cryopreservation medium composition has a devitrification temperature greater than -80°C.

10. The cryopreservation medium composition of any one of claims 5-7, wherein:

said cryopreservation medium composition has a Ficoll concentration between about 1% and about 15% by weight.

1 1. The cryopreservation medium composition of any one of claims 5-7, wherein:

said cryopreservation medium composition has a Ficoll concentration of about 10% by weight.

12. The cryopreservation medium composition of any one of claims 5-7, wherein:

said cryopreservation medium composition has a Ficoll concentration of about 5% by weight.

13. The cryopreservation medium composition of any one of claims 1-12, wherein:

said cryopreservation medium composition is configured for use with mammalian cells.

14. The cryopreservation medium composition of any one of claims 1-12, wherein:

said cryopreservation medium composition is configured for use with murine cells.

15. The cryopreservation medium composition of any one of claims 1-12, wherein:

said cryopreservation medium composition is configured for use with porcine cells.

16. The cryopreservation medium composition of any one of claims 1-12, wherein:

said cryopreservation medium composition is configured for use with human cells.

17. The cryopreservation medium composition of any one of claims 13-16, wherein:

said cells are embryonic stem cells.

18. The cryopreservation medium composition of any one of claims 13-16, wherein:

said cells comprise an embryo.

19. The cryopreservation medium composition of any one of claims 13-16, wherein:

said cells are pluripotent stem cells.

20. The cryopreservation medium composition of any one of claims 13-16, wherein:

said cells are spermatozoa.

21. A cellular composition, comprising:

the cryopreservation medium composition of any one of claims 1-20; and cells, wherein said cells are suspended within said media.

22 The cellular composition of claim 21, wherein:

said cells are mammalian cells.

23 The cellular composition of claim 22, wherein:

said cells are murine cells.

24. The cellular composition of claim 22, wherein:

said cells are porcine cells.

25. The cellular composition of claim 22, wherein:

said cells are human cells.

26. The cellular composition of any one of claims 22-25, wherein:

said cells are embryonic stem cells.

27. The cellular composition of any one of claims 22-25, wherein:

said cells comprise an embryo.

28. The cellular composition of any one of claims 22-25, wherein:

said cells are pluripotent stem cells.

29. The cellular composition of any one of claims 22-25, wherein:

said cells are spermatozoa.

30. A method of cryogenically storing cells, comprising:

vitrifying cells in the presence of the cryopreservation medium composition of any one of claims 1-20.

31. The method of claim 30, wherein:

said cells are mammalian cells.

32. The method of claim 31 , wherein:

said cells are murine cells.

33. The method of claim 31 , wherein:

said cells are porcine cells.

34. The method of claim 31, wherein: said cells are human cells.

35. The method of any one of claims 31-34, wherein:

said cells are embryonic stem cells.

36. The method of any one of claims 31-34, wherein:

said cells comprise an embryo.

37. The method of any one of claims 31-34, wherein:

said cells are pluripotent stem cells.

38. The method of any one of claims 31-34, wherein:

said cells are spermatozoa.

39. A composition or method according to any preceding claim, wherein the polymer comprises a sucrose polymer.

40. A composition or method according to claim 39, wherein the sucrose polymer is an uncharged, branched polymer formed by the co-polymerization of sucrose and epichlorohydrin and having a weight average molecular weight of less than about 500,000 Daltons.