Compositions and methods for cryopreserving cells

Abstract

Cryopreserving polymers are provided herein. The cryopreserving polymers comprises polymerized vitamin B5 analogous monomers and a crosslinker such that the crosslinker and the polymerized vitamin B5 analogous monomers are crosslinked. Compositions, cryopreserving colloidal gels, cryopreserving agents comprising the cryopreserving polymers as well as uses thereof are also provided. Methods of cryopreserving cells and methods of preparing the cryopreserving polymer, the compositions, the cryopreserving colloidal gels, and the cryopreserving agents are further provided. The vitamin B5 analogous monomers are defined by formula (I).

Description

Compositions and Methods for Cryopreserving CellsFIELD OF INVENTION

[0001] The present invention relates generally to compositions and methods for cry opreserving cells. More specifically, the present invention relates to cryopreserving compositions comprising cry opreserving polymers and methods thereof for cry opreserving prokaryotic and eukaryotic cells.BACKGROUND

[0002] Maintaining cell viability and recovery during logistics storage, transport, and distribution is a key challenge in biotechnology. Cryopreservation, storing materials at sub-zero temperatures, is essential for fundamental research and the clinical, biomedical and food sciences. Continuous culture to maintain cellular viability is rarely feasible and not practical.1Therefore, lyophilization or cryopreservation is commonly used. Among them, cryopreservation is the most popular among researchers.2

[0003] Bacterial and mammalian cells are the main cell types that are widely used in almost all biological laboratories and which makes up a crucial part of biological materials. Therefore, it is necessary to keep frozen stocks of cells to reduce phenotypic drift from continuous culture as these frozen stocks serve as backups and help preserve valuable or rare cells that are hard to come by, such as specific primary and clinical samples. However, cells are susceptible to temperature changes, and ice crystals formed during cry opreservation damage organelles and membranes at freezing temperatures.1,3Organic solvents such as DMSO and glycerol are the best option to avoid ice crystal formation and maintain cell viability during cry opreservation.1However, these solvents show negative effects since they can damage specific cellular parts and produce concentration-based toxicities.2Minimizing cell death remains a major challenge in the field of cell preservation, particularly with respect to cry opreservation.

[0004] Alternative, additional, and / or improved means of preserving cells are therefore highly desirable.SUMMARY OF INVENTION

[0005] Provided herein are compositions and methods for the cryopreservation of cells. Examples of cells that may be preserved with the compositions described herein include animal cells, plant cells, bacteria cells, as well as cells from other microorganisms. It is an object of the present invention toprovide methods for preserving cells through cry opreservation using the compositions described herein.

[0006] Organic solvents such as DMSO and glycerol are routinely used for cryopreserving cells to avoid the formation of ice crystals and other toxic artefacts, thereby maintaining cell viability during the cry opreservation process and allow subsequent use of the cryopreserved cells.1Indeed, these cryopreserved cells can be stored long term at sub-zero temperatures, until needed. The cells are then typically thawed rapidly and the toxic solvent removed by centrifugation before being cultured for subsequent uses. However, these thawed cells put in culture typically contain a high proportion of dead cells as the yield of cells surviving the cryopreservation process is typically very low due to the toxicity of the selected solvents. Experimental studies described herein indicate that compositions comprising biocompatible vitamin B5 analogous methacrylamide (B5AMA)-based polymers and Di(ethylene glycol) monomethyl ether methacrylate (DEGMEM) can be used for the cry opreservation of a variety of different cell types, including microorganisms and mammalian cells, to increase their viability by reducing toxicity compared to organic solvents such as DMSO and glycerol typically used in the art of cryopreservation of cells.

[0007] According to a first aspect, the present invention is directed to cry opreserving polymer.

[0008] In an embodiment, the cryopreserving polymer comprises polymerized vitamin B5 analogous monomers and a crosslinker, wherein the crosslinker and the polymerized vitamin B5 analogous monomers are crosslinked.

[0009] According to an embodiment, the cryopreserving polymer further comprises a co-polymer.

[0010] The cry opreserving polymer of claim 2, wherein the polymerized vitamin B5 analogous monomers are crosslinked to the co-polymer.

[0011] According to an embodiment, the co-polymer is Di(ethylene glycol) methyl ether methacrylate (DEGMEM).

[0012] According to an embodiment, the crosslinker is a methacrylated ethylene glycol or an OXASPIRO.

[0013] According to an embodiment, the methacrylated ethylene glycol is ethylene glycol dimethylacrylate (EGDMA).

[0014] According to an embodiment, the OXASPIRO is 3,9-Divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane.

[0015] A second aspect of the present invention is directed to a cry opreserving polymer having formula II :Bm-Co,Formula II whereinB is a vitamin B5 analogous monomer, andC is a crosslinker, which is a methacrylated ethylene glycol, wherein m represents the number of monomer repeats present in the cryopreserving polymer, wherein 1 < m < 10.

[0016] A third aspect of the present invention is directed to a cry opreserving polymer having formulaIII :BmDn-Co,Formula III whereinB is a vitamin B5 analogous monomer,D is a co-polymer which is Di(ethylene glycol) methyl ether methacrylate (DEGMEM) or a variant thereof, andC is a crosslinker, which is a methacrylated polyethylene glycol (E) or an OXASPIRO (O), wherein m represents the number of monomer repeats present in the cry opreserving polymer, n represents the number of DEGMEM crosslinked to the cryopreserving polymer and o represents the number of crosslinkers linked to the cry opreserving polymer, wherein 1 < m < 10 and 0 < n < 1.

[0017] According to an embodiment, B is defined by formula I:Formula I

[0018] According to an embodiment, the OXASPIRO is 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

[0019] According to an embodiment, the methacrylated PEG is Di(ethylene glycol) methyl ether methacrylate (EGDMA).

[0020] According to an embodiment, the cryopreserving polymer is Bi-Cs, Bi-Cio, B4-O10, B4-O10 or any combination thereof, wherein C is 3,9-Divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane.

[0021] According to an embodiment, the cry opreserving polymer is B4-O10.

[0022] According to an embodiment, the cryopreserving polymer is B1D1-E4, B1D1-E7, B1D1-O4, BiDi-Oe, BiDi-Os, B1D1-O10, B4D1-O10, B4D1-O8, B4D1-O10, BiDo-Os, B1D0-O10, or any combination thereof, wherein E is EGDMA and O is 3,9-Divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane.

[0023] According to an embodiment, the cryopreserving polymer is B1D1-O10, B4D1-O10, B4D0-O10, or any combination thereof.

[0024] According to an embodiment, the cryopreserving polymer is B4D0-O10.

[0025] A fourth aspect of the present invention is directed to a composition for cryopreserving cells comprising the cry opreserving polymer as described herein and a salt and / or a suitable buffer.

[0026] According to an embodiment, the salt is sodium chloride (NaCl).

[0027] According to an embodiment, the suitable buffer is a phosphate buffer.

[0028] According to an embodiment, the composition further comprises mannitol.

[0029] A fifth aspect of the present invention is directed to a cry opreserving colloidal gel comprising the cry opreserving polymer as described herein.

[0030] According to an embodiment, the cryopreserving colloidal gel further comprises mannitol.

[0031] According to an embodiment, the cry opreserving colloidal gel further comprises a salt.

[0032] According to an embodiment, the salt is sodium chloride (NaCl).

[0033] According to an embodiment, the cryopreserving colloidal gel as described herein is for cry opreserving cells.

[0034] A sixth aspect of the present invention is directed to a method of preparing a cryopreserving polymer.

[0035] According to an embodiment, the method of preparing a cry opreserving polymer comprises polymerizing a plurality of vitamin B5 analogous monomers to obtain a vitamin B5 analogous polymer, and crosslinking the vitamin B5 analogous polymer to obtain the cry opreserving polymer.

[0036] A seventh aspect of the present invention is directed to a method of preparing a cry opreserving polymer.

[0037] According to an embodiment, the method of preparing a cry opreserving polymer comprises polymerizing a plurality of vitamin B5 analogous monomers to obtain a vitamin B5 analogous polymer, and crosslinking the vitamin B5 analogous polymer with Di(ethylene glycol) methyl ether methacrylate (DEGMEM) to obtain the cry opreserving polymer.

[0038] According to an embodiment, the plurality of vitamin B5 analogous monomers are defined by formula (I):Formula I

[0039] According to an embodiment, the polymerizing comprises adding a polymerization initiator to the plurality of vitamin B5 analogous monomers.

[0040] According to an embodiment, the polymerization initiator is potassium persulfate (KPS), VA- 044, VA-051 or 4,4'-Azobis(4-cyanovaleric Acid) (ACVA).

[0041] According to an embodiment, the polymerizing comprises adding a catalyst to the plurality of vitamin B5 analogous monomers.

[0042] According to an embodiment, the catalyst is N,N,N’,N’ -tetramethylethylenediamine (TEMEDA).

[0043] According to an embodiment, the crosslinking comprises adding a crosslinker to the vitamin B5 analogous polymer.

[0044] According to an embodiment, the crosslinker is a methacrylated ethylene glycol or an OXASPIRO.

[0045] According to an embodiment, the methacrylated ethylene glycol is ethylene glycol dimethylacrylate (EGDMA).

[0046] According to an embodiment, the OXASPIRO is 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

[0047] According to an embodiment, the obtained cryopreserving polymer is in a cryopreserving solution.

[0048] According to an embodiment, the method further comprises degassing the cryopreserving solution.

[0049] An eighth aspect of the present invention is directed to a method of cryopreserving cells.

[0050] According to an embodiment, the method of cryopreserving cells comprises mixing the cells with the composition as described herein to obtain a cryopreserving preparation, and freezing the cry opreserving preparation.

[0051] A ninth aspect of the present invention is directed to a method of preparing a cry opreserving colloidal gel.

[0052] According to an embodiment, the method of preparing a cryopreserving colloidal gel comprises pouring an aqueous solution comprising the cryopreserving polymer as described herein into a mold, incubating the poured aqueous solution in the mold for a period of time to obtain a molded colloidal gel, and removing the molded colloidal gel from the mold and washing the molded colloidal gel.

[0053] According to an embodiment, the aqueous solution comprises a salt and / or a suitable buffer.

[0054] According to an embodiment, the suitable buffer is a phosphate buffer.

[0055] According to an embodiment, the salt is sodium chloride (NaCl).

[0056] According to an embodiment, the aqueous solution further comprises mannitol.

[0057] A tenth aspect of the present invention is directed to a method of preparing a cryopreserving agent.

[0058] According to an embodiment, the method of preparing a cry opreserving agent comprises providing the cryopreserving colloidal gel as described herein, solubilizing the cryopreserving colloidal gel in an aqueous solution, wherein the volume of the aqueous solution is determined according to a desired concentration of the cryopreserving polymer, thereby obtaining the cryopreserving agent.

[0059] According to an embodiment, the aqueous solution further comprises mannitol.

[0060] According to an embodiment, the cryopreserving polymer as described herein, the composition as described herein or the cryopreserving colloidal gel as described herein, is for cry opreserving prokaryotic or eukaryotic cells.

[0061] According to an embodiment, the cry opreserving polymer as described herein, the composition as described herein or the cry opreserving colloidal gel as described herein is free of organic solvents such as dimethyl sulfoxide (DMSO) and glycerol.

[0062] An eleventh aspect of the present invention is directed to a use for the cry opreserving polymer as described herein, the composition as described herein or the cry opreserving colloidal gel as described herein for cry opreserving prokaryotic or eukaryotic cells.

[0063] A twelfth aspect of the present invention is directed to a use for the cryopreserving polymer as described herein, the composition as described herein or the cry opreserving colloidal gel as described herein in the manufacture of a cry opreserving agent for cry opreserving prokaryotic or eukaryotic cells.

[0064] A thirteenth aspect of the present invention is directed a cryopreserving agent comprising the cryopreserving colloidal gel as described herein.

[0065] According to an embodiment, the cryopreserving agent further comprises mannitol.BRIEF DESCRIPTION OF DRAWINGS

[0066] These and other features will become better understood with regard to the following description and accompanying drawings, wherein:

[0067] FIGURE 1 shows proton nuclear magnetic resonance (JH NMR spectra) of (A) colloidal gel comprising co-polymers B5AMA and DEGMEM crosslinked with EGDMA (B1D1-E7) in de-DMSO, (B) B5AMA in D2O, (C) DEGMEM in de-DMSO, and (D) EGDMA in de-DMSO, according to an embodiment;

[0068] FIGURE 2 showsJH NMR spectra of (A) colloidal gel comprising co-polymers B5 AMA and DEGMEM crosslinked with OXAS (B1D1-O10) in D2O, (B) B5AMA in D2O, (C) DEGMEM in de- DMSO, and (D) OXAS in de-DMSO, according to an embodiment;

[0069] FIGURE 3 shows results from controlled temperature experiments, showing the effect of the temperature over the effective diameter of the colloidal gel in solution. The shar increase of the particle size allows the calculation of the lower critical solution temperature (LOST), according to an embodiment;

[0070] FIGURE 4 shows rheometric characterization of the colloidal gel prepared with polymer B1D1-E7 reconstituted at 15% w / w in water, according to an embodiment;

[0071] FIGURE 5 shows rheometric characterization of the colloidal gel prepared with polymer B1D1-E4 reconstituted at 15% w / w in water, according to an embodiment;

[0072] FIGURE 6 shows rheometric characterization of the colloidal gel prepared with polymerB4Di-Oio reconstituted at 30% w / w in water, according to an embodiment;

[0073] FIGURE 7 shows rheometric characterization of the colloidal gel prepared with polymer B4D1-O8 reconstituted at 30% w / w in water, according to an embodiment;

[0074] FIGURE 8 shows (A),(B),(C): recovered B. subtilis colonies, after seven freeze (-196°C)-thaw (25°C) cycles, (D),(E),(F): B. subtilis growth profiles after seven freeze (-196°C)-thaw (25°C) cycles, according to an embodiment;

[0075] FIGURE 9 shows (A),(B),(C): recovered E. coli colonies, after seven freeze (-196 C)-thaw (25°C) cycles, and (D),(E),(F): E. coli growth profiles after seven freeze (-196°C)-thaw (25°C) cycles, according to an embodiment;

[0076] FIGURE 10 shows (A),(B),(C): recovered M. luteus colonies, after seven freeze (-196°C)-thaw (25°C) cycles, and (D),(E),(F): M. luteus growth profiles after seven freeze (-196°C)-thaw (25°C) cycles, according to an embodiment;

[0077] FIGURE 11 shows (A),(B),(C): recovered S. cerevisiae colonies, after seven freeze (-196°C)-thaw (25°C) cycles, and (D),(E),(F): S. cerevisiae growth profiles after seven freeze (-196°C)-thaw (25°C) cycles, according to an embodiment;

[0078] FIGURE 12 shows the recovered colonies of E. coli and B. subtilis after 14 days of incubation at -80°C, according to an embodiment;

[0079] FIGURE 13 shows the recovered B. subtilis colonies, after 24 h incubation at -80°C, according to an embodiment;

[0080] FIGURE 14 shows the recovered E. coli colonies, after 24 h incubation at -80°C, according to an embodiment;

[0081] FIGURE 15 shows the recovered M. luteus colonies, after 24 h incubation at -80°C, according to an embodiment;

[0082] FIGURE 16 shows the recovered S. cerevisiae colonies, after 24 h incubation at -80°C, according to an embodiment;

[0083] FIGURE 17 shows the percentage viability of RAW 267.4 cells after cryopreservation at - 80°C for 48 h, according to an embodiment;

[0084] FIGURE 18 shows the percentage viability of RAW 267.4 cells after cry opreservation (A & B): at -80°C for 7 days, (C & D): in liquid nitrogen for 48 h, according to an embodiment;

[0085] FIGURE 19 shows the percentage viability of MDA-MB-231 cells after cry opreservation (A & B): at -80°C for 48 h, and (C & D): in liquid nitrogen for 48 h, according to an embodiment;

[0086] FIGURE 20 shows the percentage viability of 4T1 cells after cryopreservation (A & B): at - 80°C for 48 h, and (C & D): in liquid nitrogen for 48 h, according to an embodiment;

[0087] FIGURE 21 shows the percentage viability of RAW 267.4 cells after 24 hours incubation at 37°C with different concentrations of polymers, according to an embodiment;

[0088] FIGURE 22 shows the percentage viability of B. subtilis after 24 hours incubation at 37°C with different concentrations of polymers, according to an embodiment;

[0089] FIGURE 23 shows the percentage viability of E. coli after 24 hours incubation at 37°C with different concentrations of polymers, according to an embodiment;

[0090] FIGURE 24 shows the percentage viability of RAW 267.4 cells after 48 hours incubation at 37°C with different concentrations of polymers, according to an embodiment;

[0091] FIGURE 25 shows transmission electron microscopes (TEM) images of colloidal gels prepared with polymer B4D1-O10 comprising a w / w ratio of B5AMA:DEGMEM of 4: 1 ratio and crosslinked with 10% OXASPIRO, scale = 500nm;

[0092] FIGURE 26 shows TEM images of colloidal gels prepared with polymer B4D1-O10 comprising a w / w ratio of B5A A:DEGMEM of 4: 1 ratio and crosslinked with 10% OXASPIRO, scale bars = 200nm;

[0093] FIGURE 27 shows TEM images of colloidal gels prepared with polymer B4D1-O10 comprising a w / w ratio of B5AMA:DEGMEM of 4: 1 ratio and crosslinked with 10% OXASPIRO, scale bars = 200nm;

[0094] FIGURE 28 shows TEM images of colloidal gels prepared with polymer B4D1-O10 comprising a w / w ratio of B5AMA:DEGMEM of 4: 1 ratio and crosslinked with 10% OXASPIRO, scale bars = 200nm;

[0095] FIGURE 29 shows TEM images of colloidal gels prepared with polymer B4D1-O10 comprising a w / w ratio of B5AMA:DEGMEM of 4: 1 ratio and crosslinked with 10% OXASPIRO, scale bars = 200nm;

[0096] FIGURE 30 shows TEM images of colloidal gels prepared with polymer B1D0-O10 comprising B5AMA without DEGMEM and crosslinked with 10% OXASPIRO, scale bars = 200nm;

[0097] FIGURE 31 shows TEM images of colloidal gels prepared with polymer B1D0-O10 comprising B5AMA without DEGMEM and crosslinked with 10% OXASPIRO, scale bars = 200nm;

[0098] FIGURE 32 shows the formed ice crystals captured using a 20X magnification, according to an embodiment;

[0099] FIGURE 33 shows the custom-built cryostage, according to an embodiment;

[0100] FIGURE 34 shows the mean longest grain size (MLGS) in the image that was identified and counted, along with the average grain size (AGS), according to an embodiment;

[0101] FIGURE 35 shows metabolic growth rate of Raw 264.7 cells when thawed in the presence of cryoprotectants, according to an embodiment; and

[0102] FIGURE 36 shows metabolic growth rate of Raw 264.7 cells cryopreserved with B4D1-O10 by ultrafast cooling method, according to an embodiment.

[0103] FIGURE 37 shows cryopreservation efficacies of B5AMA nanogels, prepared using Bis as a crosslinker, as was determined by MTS assay 48 hours post-thaw.

[0104] FIGURE 38 shows cryopreservation efficacies of B5AMA nanogels, prepared using DVS as a crosslinker, as was determined by MTS assay 48 and 72 hours post-thaw.DETAILED DESCRIPTION

[0105] Many vitamin B5 derived analogues exhibit desirable biological properties such as antibacterial activity and antifouling capabilities. However, instability of these small molecules under physiological conditions have hindered their widespread applications. To this end, polymers of vitamin B5 derived analogues will be described herein. The cryopreserving ability of polymers of vitamin B5 derived analogues will be described herein. As will be shown, these cryopreserving polymers should be considered as a viable alternative to conventional cryoprotectants in order to minimize cellular damage and cell death during the freezing and thawing process, as these cry opreserving polymers are as efficient and exhibit low toxicity compared to traditional cryopreserving agents such as glycerol and DMSO.

[0106] Pantothenic acid, more commonly referred to as vitamin B5, is a water-soluble vitamin that is essential for the growth and metabolism of prokaryotic and eukaryotic cells. The molecular structure of pantothenic acid is composed of a P-alanine, a non-essential amino acid, moiety and a pantoyl moiety with an amide bond joining the two together. The official IUPAC name for pantothenic acid is 3-[(2R)- 2,4-dihydroxy-2,3,3-trimethylbutanamido]propanoic acid.Molecular Structure of Vitamin B5

[0107] Polymers created from vitamin B5 derived analogues were produced for the first time by the Applicant.4Kabir et al. produced hydrogels from vitamin B5 analogous methacrylamide, B5AMA, and were shown to possess antifouling and hygroscopic properties. In another study by the Applicant, a polymer of B5AMA, poly(B5AMA), was synthesized and displayed salt responsive and bacterial aggregation capabilities.5Described herein are synthesized analogues of polymeric vitamin B5 and their use as cry opreserving polymers.

[0108] Novel compositions for cry opreserving cells and methods for cry opreserving cells using the novel compositions will be described hereinafter. The use of compositions for cryopreserving cells will also be described hereinafter. Although the invention is described in terms of specific illustrative embodiments, it is to be understood that the embodiments described herein are by way of example only and that the scope of the invention is not intended to be limited thereby.

[0109] Provided herein are compositions and methods for the cry opreservation of cells. Examples of cells that may be preserved with the compositions described herein include animal cells, plant cells, bacteria cells, as well as cells from other microorganisms. It is an object of the present invention to provide methods for preserving cells through cry opreservation using the compositions described herein.

[0110] Organic solvents such as DMSO and glycerol are routinely used for cry opreserving cells for avoiding the formation of ice crystals and other toxic artefacts and thereby maintain cell viability during the cry opreservation process to allow subsequent use of the cryopreserved cells.1Indeed, the cryopreserved cells can be stored long term at sub-zero temperatures, until needed. The cells are then typically thawed rapidly and the toxic solvent removed by centrifugation before being cultured for subsequent uses. However, the thawed cells put in culture typically contain a high proportion of dead cells as the yield of cells surviving the cryopreservation process is typically very low due to the toxicity of the selected solvents. Experimental studies described herein indicate that biocompatible vitamin B5 analogous methacrylamide (B5AMA)-based polymer materials can be used for the cryopreservation of a variety of cell types, including microorganisms and mammalian cells, to increase viability by reducing toxicity to the cryopreserved cells compared to organic solvents such as DMSO and glycerol typically used in the art of cryopreservation of cells. Experimental studies described herein indicate that compositions comprising B5AMA and a polyether monomer such as polyethylene glycol can be used as a cry opreservation agent with low toxicity to the cells.

[0111] The terminology used herein is in accordance with definitions set out below.

[0112] By "about", it is meant that the value can vary within a certain range depending on the margin of error of the method or device used to evaluate or measure. A margin of error of 10% is generally accepted.

[0113] The terms “polypeptide” and “protein” refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes of the present invention, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions and substitutions, to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. Further, it is noted that the size of polypeptides referenced herein is not particularly limited.

[0114] The term “peptide” as used herein refers to a fragment of a polypeptide. Thus, a peptide can include a C-terminal deletion, an N-terminal deletion and / or an internal deletion of the native polypeptide, so long as the entire protein sequence is not present. A peptide will generally include at least about 3-10 contiguous amino acid residues of the full-length molecule, and can include at least about 15-25 contiguous amino acid residues of the full-length molecule, or at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 3 amino acids and the number of amino acids in the full-length sequence, provided that the peptide in question retains the ability to elicit the desired biological response.

[0115] The expressions “colloidal gel” and “nanogel” are used interchangeably.

[0116] The term “nanogel” as used herein is defined as nanoscaled hydrogels or crosslinked polymeric structures that can absorb water. Nanogels can stabilize bioactives (e.g. enzymes, proteins), are non-toxic, and are hydrating, making them useful in the preparation of cosmetic formulations.

[0117] The description which follows, and the embodiments described therein are provided by way of illustration of an example of particular embodiments of principles and aspects of the present invention.These examples are provided for the purposes of explanation and not of limitation, of those principles of the invention.Cry opreserving Polymer

[0118] According to one aspect of the present invention, there is provided a cry opreserving polymer.

[0119] According to a preferred embodiment, the cryopreserving polymer comprises vitamin B5 analogous monomers and a crosslinker such that the crosslinker and the polymerized vitamin B5 analogous monomers are crosslinked.

[0120] According to an embodiment, the cryopreserving polymer further comprises a co-polymer such as Di(ethylene glycol) methyl ether methacrylate (DEGMEM).

[0121] According to an embodiment, the polymerized vitamin B5 analogous monomers are crosslinked to the co-polymer.

[0122] According to an embodiment, the crosslinker is a methacrylated ethylene glycol such as ethylene glycol dimethylacrylate (EGDMA) or an OXASPIRO such as 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

[0123] According to an embodiment, there is provided a cryopreserving polymer having formula II:Bm-Co,Formula II

[0124] In the cryopreserving polymer of formula II, B is a B5 analogous monomer, and C is a crosslinker such as a methacrylated ethylene glycol. In formula II, m represents the number of monomer repeats present in the cry opreserving polymer whereas 1 < m < 10.

[0125] According to an embodiment, there is provided a cryopreserving polymer having formula III:BmDn-Co,Formula III

[0126] In the cryopreserving polymer of formula III, B is a vitamin B5 analogous monomer, D is a co-polymer such as Di(ethylene glycol) methyl ether methacrylate (DEGMEM) or a variant thereof, and C is a crosslinker such as a methacrylated polyethylene glycol (E) or an OXASPIRO (O). In formula III, m represents the number of monomer repeats present in the cryopreserving polymer, n represents the number of DEGMEM crosslinked to the cryopreserving polymer and o represents the number of crosslinkers linked to the cry opreserving polymer whereas 1 < m < 10 and 0 < n < 1.

[0127] According to an embodiment, B is defined by formula I:Formula I

[0128] According to an embodiment, the OXASPIRO (O) is 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

[0129] According to an embodiment, the methacrylated PEG (E) is Di(ethylene glycol) methyl ether methacrylate (EGDMA).

[0130] According to an embodiment, the cryopreserving polymer is Bi-Cs, Bi-Cio, B4-O10 or any combination thereof, and C is 3,9-Divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane.

[0131] According to an embodiment, the cry opreserving polymer is B4-O10.

[0132] According to an embodiment, the cryopreserving polymer is B1D1-E4, B1D1-E7, B1D1-O4, BiDi-Oe, BiDi-Os, B1D1-O10, B4D1-O8, B4D1-O10, BiDo-Os, B1D0-O10, or any combination thereof, wherein E is EGDMA and O is 3,9-Divinyl-2,4,8,10-tetraoxaspiro[5.5]undecane.

[0133] According to an embodiment, the cryopreserving polymer is B1D1-O10, B4D1-O10, B4D0-O10, or any combination thereof.

[0134] According to an embodiment, the cry opreserving polymer is B4D0-O10.

[0135] According to an embodiment, the cry opreserving polymer as described herein is for cry opreserving prokaryotic or eukaryotic cells.

[0136] According to an embodiment, the cryopreserving polymer as described herein is free of organic solvents such as dimethyl sulfoxide (DMSO) and glycerol.Cry opreserving Composition

[0137] A second aspect of the present invention is directed to a composition for cry opreserving cells. The composition comprises the cryopreserving polymer as described herein and a salt and / or a suitable buffer.

[0138] According to an embodiment, the salt is sodium chloride (NaCl).

[0139] According to an embodiment, the suitable buffer is a phosphate buffer.

[0140] According to an embodiment, the composition further comprises mannitol.

[0141] According to an embodiment, the composition as described herein is for cryopreserving prokaryotic or eukaryotic cells.

[0142] According to an embodiment, the composition as described herein is free of organic solvents such as dimethyl sulfoxide (DMSO) and glycerol.

[0143] The composition as described herein may comprise between about 0.5% w / v to about 1.0 % w / v of sodium chloride, between about 10% w / v to about 40% w / v of the cry opreserving polymer.Cryopreserving Colloidal Gel

[0144] A third aspect of the present invention is directed to a colloidal gel comprising the cry opreserving polymer as described herein.

[0145] According to an embodiment, the colloidal gel further comprises a salt such as sodium chloride (NaCl). The colloidal gel may comprise between about 0.5% w / v to about 1.0 % w / v of NaCl.

[0146] According to an embodiment, the colloidal gel is for cryopreserving cells.

[0147] According to an embodiment, the colloidal gel as described herein is for cryopreserving prokaryotic or eukaryotic cells.

[0148] According to an embodiment, the colloidal gel as described herein is free of organic solvents such as dimethyl sulfoxide (DMSO) and glycerol.Cry opreserving Agent

[0149] A fourth aspect of the present invention is directed to a cryopreserving agent comprising the cryopreserving colloidal gel as described herein.

[0150] According to an embodiment, the cryopreserving agent further comprising mannitol.

[0151] According to an embodiment, the cry opreserving agent as described herein is for cry opreserving prokaryotic or eukaryotic cells.

[0152] According to an embodiment, the cry opreserving agent as described herein is free of organic solvents such as dimethyl sulfoxide (DMSO) and glycerol.Method of preparing a cry opreserving polymer

[0153] A fifth aspect of the present invention is directed to a method of preparing a cryopreserving polymer.

[0154] According to a preferred embodiment, the method comprises polymerizing a plurality of vitamin B5 analogous monomers to obtain a vitamin B5 analogous polymer, and crosslinking the vitamin B5 analogous polymer to obtain the cry opreserving polymer.

[0155] According to another embodiment, the method comprises polymerizing a plurality of vitamin B5 analogous monomers to obtain a vitamin B5 analogous polymer, and crosslinking the vitamin B5 analogous polymer with Di(ethylene glycol) methyl ether methacrylate (DEGMEM) to obtain the cry opreserving polymer.

[0156] According to an embodiment, the plurality of vitamin B5 analogous monomers are defined by formula (I):Formula I

[0157] According to an embodiment, the polymerizing comprises adding a polymerization initiator to the plurality of vitamin B5 analogous monomers.

[0158] According to an embodiment, the polymerization initiator is potassium persulfate (KPS), VA- 044, VA-051 or 4,4'-Azobis(4-cyanovaleric Acid) (ACVA).

[0159] According to an embodiment, the polymerizing comprises adding a catalyst to the plurality of vitamin B5 analogous monomers.

[0160] According to an embodiment, the catalyst is N,N,N’,N’ -tetramethylethylenediamine (TEMEDA).

[0161] According to an embodiment, the crosslinking comprises adding a crosslinker to the vitamin B5 analogous polymer. The crosslinker may be added in an amount of between about 2 % w / v to about 15% w / v.

[0162] According to an embodiment, the crosslinker is a methacrylated ethylene glycol or an OXASPIRO.

[0163] According to an embodiment, the methacrylated ethylene glycol is ethylene glycol dimethylacrylate (EGDMA).

[0164] According to an embodiment, the OXASPIRO is 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

[0165] According to an embodiment, the obtained cry opreserving polymer is in a cry opreserving solution. The cryopreserving solution may comprise about 50% v / v ethanol in water.

[0166] According to an embodiment, the method further comprises degassing the cry opreserving solution.Method of cry opreserving cells

[0167] A sixth aspect of the present invention is directed to a method of cry opreserving cells.

[0168] According to a preferred embodiment, the method comprises mixing the cells with the composition as described herein to obtain a cry opreserving preparation, and freezing the cry opreserving preparation.

[0169] The cry opreserving preparation may comprise between about 5% w / v and 15 % (w / v) of the cryopreserving polymer. Freezing the cryopreserving preparation may comprise freezing at -20°C for at least 24 hours before freezing at -80°C for long-term storage. The method may further comprise transferring the frozen cells to liquid nitrogen at about -196°C. As commonly known in the art, the frozen cells may be recuperated by thawing the cells rapidly at a selected temperature, for example, at 37°C in a water bath.Method of preparing a cryopreserving colloidal gel

[0170] A seventh aspect of the present invention is directed to a method of preparing a cry opreserving colloidal gel.

[0171] According to a preferred embodiment, the method comprises pouring an aqueous solution comprising the cry opreserving polymer as described herein into a mold, incubating the solution in the mold for a period of time to obtain a molded colloidal gel, and removing the molded colloidal gel from the mold and washing the molded colloidal gel.

[0172] According to an embodiment, the aqueous solution comprises a salt such as sodium chloride (NaCl) and / or a suitable buffer such as a phosphate buffer.

[0173] According to an embodiment, the aqueous solution further comprises mannitol.Method of preparing a cry opreserving agent

[0174] An eighth aspect of the present invention is directed to a method of preparing a cry opreserving agent.

[0175] According to a preferred embodiment, the method comprises providing the cryopreserving colloidal gel as described herein, solubilizing the cryopreserving colloidal gel in an aqueous solution. The volume of the aqueous solution is determined according to a desired concentration of the cryopreserving polymer, thereby obtaining the desired cryopreserving agent.

[0176] According to an embodiment, the aqueous solution further comprises mannitol.Uses

[0177] A ninth aspect of the present invention is directed to the use of the cry opreserving polymer, the composition or the colloidal gel as described herein for cryopreserving prokaryotic or eukaryotic cells.

[0178] A tenth aspect of the present invention is directed to the use of the cry opreserving polymer, the composition or the colloidal gel as described herein in the manufacture of a cry opreserving agent for cry opreserving prokaryotic or eukaryotic cells.

[0179] An eleventh aspect of the present invention is directed to the use of the cryopreserving polymer, the composition or the colloidal gel as described herein as an agent, ingredient or excipient in cosmetics and / or in the preparation of cosmetic formulations.

[0180] A twelfth aspect of the present invention is directed to the cryopreserving polymer, the composition or the colloidal gel as described herein for use as an agent, ingredient or excipient in cosmetics and / or in the preparation of cosmetic formulations.

[0181] The present invention will be further illustrated in the following examples.ExamplesExample 1: Preparation and characterization of cryopreserving polymers and cryopreserving colloidal gels

[0182] A total of eleven cry opreserving polymers were synthesized and assessed for their cryoprotectant ability. The synthesized polymers comprised structural variations and were evaluated for their cryoprotectant ability in order to select the best candidates for cryopreservation. These variations include the number of vitamin B5 analogous monomer repeats, monomer structure, nature of the crosslinker used as well as the proportion of crosslinker used. The synthesis of the polymeric colloidal gels are briefly explained hereinbelow.Materials

[0183] Di(ethylene glycol) methyl ether methacrylate (DEGMEM, 95%, Sigma- Aldrich), ethylene glycol dimethacrylate (EGDMA, 98%, Sigma-Aldrich), 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane (OXAS, 98%, Sigma-Aldrich), potassium persulfate (KPS, 95%, Sigma- Aldrich), N,N,N’,N’ -tetramethylethylenediamine (TEMEDA, 99%, Thermo Scientific), 4,4'-Azobis(4- cyanovaleric acid) (ACVA, 98%, Sigma-Aldrich), and ethanol (absolute, Les Alcools de Commerce), were used as received. Vitamin B5 analogous methacrylamides monomer (B5AMA) was synthesized according to a previously reported procedure and was analyzed usingJH NMR and13C NMR.4MethodsSynthesis of colloidal gels comprising poly(B5AMA- -co- -DEGMEM) crosslinked with EGDMA

[0184] Cry opreserving polymers synthesized from vitamin B5 analogous methacrylamide (B5AMA)-based monomers and the poly ether compound Di(ethylene glycol) monomethyl ether methacrylate (DEGMEM) will now be described.Vitamin B5 analogous methacrylamide (B5AMA)-based monomersDi(ethylene glycol) monomethyl ether methacrylate (DEGMEM)

[0185] Colloidal gels comprising Poly(B5AMA— co-DEGMEM) were prepared by the free-radical polymerization method. B5AMA (0.29M), DEGMEM (0.4M), crosslinker EGDMA (4% and 7% mol), and the polymerization initiator potassium persulfate KPS (3.5% mol) were dissolved in 50% v / v ethanol in water, and the resulting solution was degassed with ultrasonic bath for 5 minutes. Next, the catalyst N,N,N’,N’ -tetramethylethylenediamine (TEMEDA) was added (5%mol). The solution was then transferred to cylindrical molds to form the colloidal gels . The formation of colloidal gels was confirmed by vial inversion test after 4-5 minutes. The colloidal gels were left overnight at room temperature, removed from the mold the following day and washed using an excess amount of deionized water at room temperature for 24 hours to remove the unreacted monomers, ethanol, and by-products. The colloidal gels obtained were freeze-dried and the yield of the polymerization process was calculated to be close to 90%w.Synthesis of colloidal gels comprising poly(B5AMA- -co- -DEGMEM) crosslinked with OXAS

[0186] Colloidal gels comprising poly(B5AMA — co-DEGMEM) were also prepared by the free- radical polymerization method. B5AMA (0.78M), DEGMEM (0.27M), the crosslinker 3,9-Divinyl- 2,4,8,10-tetraoxaspiro[5.5]undecane (OXAS) (10% and 8% mol), and the polymerization initiator 4,4'- Azobis(4-cyanovaleric acid - ACVA)(0.3% mol) were dissolved in 50% v / v ethanol in water. The resulting solution was degassed by three freeze-vacuum-thaw cycles and the sealed reactor was transferred to an oil bath at 70 °C overnight. At the end of the reaction, the solution was dialyzed against deionized water for 48 hours and freeze-dried to obtain the dry colloidal gels. The yield of the polymerization process was calculated to be close to 80%w.Synthesis of colloidal gels comprising poly(B5AMA) crosslinked with OXAS

[0187] To assess the role of the poly ether compound crosslinked to the vitamin B5 analogous polymer, polymers without the crosslinked the polyether compound were synthesized. Colloidal gelscomprising poly(B5AMA) were prepared by the free-radical polymerization method. B5AMA (IM), the crosslinker OXAS (10% and 8% mol), and the polymerization initiator ACVA (0.3% mol) were dissolved in 50% v / v ethanol in water. The resulting solution was degassed by three freeze-vacuum-thaw cycles and the sealed reactor was transferred to an oil bath at 70 °C overnight. At the end of the reaction, the solution was dialyzed against deionized water for 48 hours and freeze-dried to obtain the dry colloidal gels. The yield of the polymerization process was calculated to be close to 80%w.

[0188] The number of vitamin B5 analogous monomer (B5AMA) repeats, the presence or absence of the polyether compound (DEGMEM), the percentage of crosslinkers EGDMA and OXAS (OXASPIRO) as well as the water solubility of the resulting polymer in water are summarized in Table 01.Table 1: Synthesized polymer materials with composition and solubility in waterCode Ratio monomers Ratio monomers Crosslinker (% mol) Solubility in water(» / ») mol / mol g2Q mg / mLB5AMA DEGMEM B5AMA DEGMEM EGDMA OXASPIROB1D1-E4 1 1 1 1.38 4 - Partially solubleB1D1-E7 1 1 1 1.38 7 - Partially solubleB1D1-O4 1 1 1 1.38 - 4 ThermoresponsiveBiDi-Oe 1 1 1 1.38 - 6 ThermoresponsiveBiDi-Os 1 1 1 1.38 - 8 ThermoresponsiveB1D1-O10 1 1 1 1.38 - 10 ThermoresponsiveB4D1-O8 4 1 1 0.35 - 8 Partially solubleB4D1-O10 4 1 1 0.35 - 10 SolubleBiDo-Os 1 0 1 0 - 8 Partially solubleB1D0-O10 1 0 1 0 - 10 SolubleB5AMA 1 - 1 - - - SolubleNuclear Magnetic Resonance (NMR) Spectroscopy

[0189] Nuclear magnetic resonance (NMR) spectroscopy was carried out on a Bruker Advance NMR spectrometer operating at 300 MHz for 'H and 75 MHz for13C using deuterium oxide and DMSO as solvents.

[0190] Figure 1 shows the proton NMR spectra of a representative colloidal gel comprising B5AMA and DEGMEM crosslinked with EGDMA (B1D1-E7), and those of the monomers (B5AMA and DEGMEM) and crosslinker (EGDMA) used in its synthesis. The peaks around 0.95 ppm (methyl group)and 4.20 ppm (methylene group), assigned to B5AMA and DEGMEM, respectively, demonstrate the incorporation of B5AMA and DEGMEM to the colloidal gel. On the other hand, the inclusion of the EGDMA crosslinker is difficult to address due to the characteristic peaks of the crosslinker overlapping with the peaks of the monomers between 3.0 and 4.4 ppm. A similar result was obtained with the colloidal gel comprising B5AMA and DEGMEM crosslinked with OXAS. Figure 2 shows the NMR of one of these colloidal gels (B1D1-O10). Again, the peaks at 0.95 ppm and 4.20 ppm allow the identification of the B5AMA and DEGMEM monomers present in the structure of the colloidal gel, but the inclusion of the OXAS crosslinker cannot be described because of the overlapping of its characteristic peaks.Dynamic Light Scattering (DLS)

[0191] The DLS of the colloidal gel samples were measured using a Nano Brook 90 Plus instrument (Brookhaven, Holtsville, NY, USA). The DLS graph was set to lognormal plot and the data is an average of three runs.Zeta Potential Measurements

[0192] The zeta potential of the solubilized colloidal gels were measured by the light scattering technique using a Nano Brook 90 Plus instrument (Brookhaven, Holtsville, NY, USA). The temperature dependent experiments were performed between 25°C and 61°C, using increments of 3°C.DLS and Zeta Potential Measurements

[0193] The measurements of the particles size and DLS were performed in water solution. For this reason, the results for the colloidal gels crosslinked with OXAS are described because the colloidal gels crosslinked with EGDMA cannot be redispersed in water after the purification process and they remain suspended in the water as swelled particles. Table 2 shows the results obtained for the DLS and Z potential experiments, as well the calculated lower critical solution temperatures (LOST) using the results obtained during the temperature-controlled experiments.Table 2. Obtained results for the DLS and Z potential experiments and the calculated LCST

[0194] As expected, the colloidal gels show a zeta potential close to 0 mv, as expected for hydrophilic neutral polymers.

[0195] The analysis of the effective diameter of the colloidal gels in solution versus the temperature allows the determination of the LCST of the colloidal gels. Figure 3 illustrates the behaviour of the effective diameter when the temperature is increased from 25°C to 61°C. The results obtained show that the monomer ratio of B5AMA and DEGMEM allows the manipulation of the LCST of the colloidal gels.Rheological Characterization

[0196] Rheological characterization of the colloidal gels was carried out on a Kinexus Ultra+ rheometer (Malvern Instruments Ltd, Worcestershire, UK) using parallel plate geometry (diameter = 25.4 mm, gap= 1 mm). All measurements were subjected to a closed environment by using a sample cover to minimize water loss. Amplitude sweep studies (strain 0.01-100% at constant frequency of 1 Hz) were performed at 25°C in order to determine the linear viscoelastic region. Afterward, frequency sweep tests from 100 to 0.1 Hz at a constant strain (y = 0.5%) were performed at 25°C to determine the storage modulus (G') and the loss modulus (G") of colloidal gels. Finally, a continuous ramp experiment fromshear rates of 0.1 s'1to 50 s'1was performed to determine the behaviour of the colloidal gel viscosity upon application of shear.Colloidal Gels Crosslinked With EGDMA

[0197] The results of the amplitude sweep experiment allow the determination of the linear viscoelastic region (LVR), the range where strain and stress are proportional (Figures 4A and 5A). The limit of the LVR region corresponds to the point at which G’ (elastic modulus) becomes strain dependent. In the case of the colloidal gels comprising B1D1-E7 and B1D1-E4 colloidal gels, strains below 0.5% ensure that the experiments are performed in the LVR. For this reason, the frequency sweep was performed at a shear strain of 0.1%. The strain sweep graphs shows that both colloidal gels suffer a drop in the storage modulus at certain strain, indicating that both materials yield. This yield strain is slightly lower for the 4% mol EGDMA colloidal gel, indicating that this colloidal gel may be easier to inject than the 7% mol colloidal gel.

[0198] The absence of a crossover frequency in the frequency sweeps experiments indicates that these colloidal gels are chemically crosslinked permanently, and the relative value indicates that the stiffness of the 7% mol colloidal gel is higher than the stiffness of the 4% mol colloidal gel, making the last more suitable for injection (See Figures 4B and 5B).

[0199] The shear-thinning character of the colloidal gels, an important treat for injectability, is demonstrated by the reduction in viscosity upon application of shear during the shear rate sweep experiment (See Figures 4C and 5C). Again, the observed lower viscosity of the 4 %mol EGDMA colloidal gel could facilitate their injection.Colloidal Gels Crosslinked With OXAS

[0200] The rheometric analyses were performed for the colloidal gels identified with the codes B4D1- O10 and B4D1-O8. These colloidal gels show a different behaviour, where the loss (viscous) modulus, G’, is higher than the storage (elastic) modulus, G”, indicating that the fluid behaviour is more liquid like. Figures 6A and 7A illustrate these results. Additionally, the frequency sweep results allow the classification of the colloidal gels as viscoelastic liquids (Figures 6B and 7B).The reduction of the viscosity with the increase in the shear rate indicates that both colloidal gels display a shear-thinning character, although the colloidal gel B4D1-O8 shows a higher viscosity than the colloidal gel B4D1-O10.Transmission Electron Microscopy (TEM)

[0201] Figures 25 to 31 show higher resolution images using transmission electron microscopy (TEM) of the prepared colloidal gels. Colloidal gels prepared with B4D1-O10 comprising a w / w ratio of B5AMA:DEGMEM of 4: 1 ratio and crosslinked with 10% OXASPIRO, are depicted in Figures 25 to 29 whereas colloidal gels prepared with B1D0-O10 comprising B5AMA without DEGMEM and crosslinked with 10% OXASPIRO are depicted in Figures 30 and 31. As can be clearly seen in Figures 25 to 29, B5AMA nanogels prepared in the presence of DEGMEM (B4D1-O10) showed strikingly different shape than the ones prepared in the absence of DEGMEM (B1D0-O10). The nanogels prepared with B5AMA and DEGMEM monomers, showed homogenous spherical network of -100 nm in diameter, possibly due to the isotropic distribution of both monomers in nanogel structure. In contrast, B5AMA nanogels prepared in absence of DEGMEM exhibited globular and diffused structure with the particle size of 50 nm. The difference in shape of B4D1-O10 and B1D0-O10 was attributed to the difference in comonomer conversions and their distribution across the structure of nanogels.Example 2: Cryopreserving polymers for the cryopreservation of bacteria and fungus

[0202] The main focus of this study was to evaluate the cryoprotectant ability of the synthesized polymers to allow solvent-free cry opreservation of both microorganisms and mammalian cells. A range of synthetic B5AMA polymers were selected for this based on their special characteristics according to the structural differences.

[0203] To evaluate the performance of these polymers versus glycerol, a series of cry opreservation experiments were undertaken. Microorganisms were added to polymers with different concentrations and then exposed to seven freeze-thaw cycles from liquid nitrogen (-196°C) to room temperature (20°C), and the number of viable colonies were counted by growth on agar plates for 24 h was recorded (Figures 8-11 A, B and C). The 7 FT method was chosen to mimic laboratory conditions where stocks are often frozen and thawed during routine use.

[0204] Additional bacterial strains and unicellular fungus were selected for cry opreservation to cover a wide range of genera to ensure that these effects apply to a variety of organisms and are therefore not unique to a specific organism or strain. Bacillus subtilis was chosen as a Gram-positive strain, M. luteus was chosen to represent phylum actinomycetota whereas S. cerevisiae or commercial yeast was selected as a unicellular fungus.

[0205] The cry opreserving polymers were dissolved in 100 pL of 0.9 % NaCl solution to achieve the following final concentration of polymer, 10, 20 and 30 % (w / v). Microorganisms taken from the glycerol stock were cultured in broth media (NB for bacteria and PDB for fungi) and incubated for 24 hours at 37°C. The grown microorganisms were then cultured on agar plates and incubated for 24 hours at 37°C. Microbial suspensions were prepared using 0.9 % NaCl solution and concentrations were adjusted to 0.5 OD-600 absorbance. A volume of 100 pL of bacteria solutions was added to the polymer solution to obtain a final volume of about 200 pL. Therefore the effective final concentrations of polymers were 5, 10 and 15 % (w / v). Glycerol (30 %) solution and 0.9 % NaCl were used as the positive control the negative control respectively.

[0206] The resultant microbial mixtures were then kept in -80 °C freezer for time-based viability assessment. Another set of samples was taken for the freezing-thawing test. The prepared samples were snap-frozen in liquid nitrogen (-196 °C) before thawing at 25 °C in a water bath for 5 min and the freeze-thaw (FT) cycles were repeated seven times. All samples (after 7 FT and -80°C incubation) were diluted to 1 mL with 0.9 % NaCl. Serial dilutions were performed, and the samples were plated on agar plates (NA for bacteria and PDA for fungus). The viable colonies were counted after incubation for 24 hours at 37 °C. After 7 FT cycles, the cells were then grown in NB / PDB (500 pL) media in 24 well plate over 18 h under continuous shaking at 37 °C, and their OD was measured at 600 nm at 20 min intervals and compared to a control sample that underwent no freeze-thaw cycles.

[0207] It was noted that polymers crosslinked with OXASPIRO showed the highest cryoprotection activity with both bacteria and fungus. However, cryoprotection activity increased with decreasing the DEGMEM amount. DEGMEM is a hydrophobic polymer such that polymers with decreased amounts of crosslinked DEGMEM showed more hydrophilic characteristics than polymers with greater amounts of crosslinked DEGMEM. Indeed, hydrophilicity is an important feature to consider for making uniforms (i.e., homogenous?) cryoprotectant compositions to allow cells to be evenly dispersed. This may explainthe better results obtained with polymers with reduced crosslinked DEGMEM. Besides the amount of crosslinked DEGMEM, the concentration of crosslinkers also played a vital role in cryopreservation, as polymers with high crosslinker percentages showed better results since polymers with high crosslinker amounts (10 %) are more homogeneous and make transparent solutions in aqueous media compared to lower crosslinker concentration (8 %). Among all the polymers tested, B1D1-O10, B4D1-O10 and B4D0- O10 is the most promising polymer combination for the cryoprotection of microorganisms.

[0208] Since compositions with higher concentrations (> 10 %) of polymers crosslinked with EDGMA showed reduced solubility, mixing the microorganisms in these compositions was not successfully achieved. Hence, these higher concentration compositions resulted in significantly lower cryoprotectant activity compared to lower concentration compositions. The maximum viability of the tested microorganisms was obtained at a concentration of 10 %. Conclusively, polymers' solubility is directly related to the cryoprotectant ability such that high solubility is indicative of more efficient cryoprotection. Moreover, hydrophilic polymers showed higher levels of cryoprotection, at least with respect to microorganisms. In all cases, glycerol was less cryoprotective due to the intrinsic toxicity of glycerol at cryopreservation concentration which affects cell growth.6A similar pattern of results was obtained with microorganisms incubated at -80°C (Figures 13-16), although the number of recovered colonies was higher than the FT analysis since cells faced a higher degree of temperature fluctuation within a short period of time during the FT analysis, resulting in a harsh environment for cells to survive.7

[0209] To investigate the growth pattern of the frozen microbial sample, the post-thaw growth curves of microbes were also followed by ODeoo (turbidity) measurements, which allowed higher throughput measurements. After seven freeze-thaw cycles in liquid nitrogen (about -196°C), cells cryopreserved with a composition comprising 10 % polymers had essentially identical growth patterns to non-frozen control (Figures 8-11C, D and E). In order to assess the long-term cryoprotectant ability of the cry opreserving polymers, E. coli and B. subtilis were incubated with compositions comprising 10 % cry opreserving polymers at -80°C for 14 days. The recovered colonies are shown in Figure 12. The results summarized in Table 3 below show that the number of recovered colonies was highest when incubated with compositions comprising 10 % crypreserving polymers. This indicates that the cry opreserving polymers also possess long-term cryoprotecting ability at -80°C.Table 3: Recovered colonies and their normalized percentages of polymers tested at 10 % (w / v) concentration against B. subtilis, E. coli, M. luteus, and S. cerevisiae after 7 FT cyclesE. coli B. subtilis M. luteus S. cerevisiae total normalized total normalized total normalized total normalized(%) (%) (%) (%)B1D1-E4 48±8 74 25.3±2.3 52 15±1.3 36 106±12 56B1D1-E7 45±6 69 35±2 73 25±3.3 60 124±16 66B1D1-O4 40±6 62 22±3.3 46 20±4 48 120±14 64BiDi-Oe 42±5 65 15±3 31 28±5 67 128±10 68BiDi-Os 52±4.2 80 27±4.3 56 36±4.7 86 149±12 79B1D1-O10 59±7 91 34±3 71 29±3.3 69 155±18 82B4DI-OS 44±4.4 68 34±2.8 71 22±4 52 132±12 70B4D1-O10 62±3.1 95 48±4.6 100 34±5.2 81 188±5 100BiDo-Os 51±3.7 78 28±5 58 32±2.1 76 129±11 69B1D0-O10 65±2.9 100 42±6.1 88 42±5.6 100 180±9 96B5AMA 0 0 0 0 0 0 12±4 6Gly 30 % 31±8 48 18±6 38 8±3 19 72±7 38Control 5±4 8 l±0.7 2 0 0 35±7 19

[0210] The toxicity of the cry opreserving polymers to bacteria (B. subtilis and E. coli) was tested using OD-600 measurements compared to a negative control (solution with no cry opreserving polymers). Cryopreserving polymer solutions having different concentrations of cryopreserving polymers were prepared in nutrient broth, starting 20 mg / mL to 0.625 mg / mL. Streptomycin / Penicillin (1 mg / mLstreptomycin and 0.6 mg / mL penicillin) was used as the positive control. OD-600 values were measured after 24 hours of incubation at 37°C. The growth of bacteria was calculated against the negative control (100 %).

[0211] Figures 22 and 23 shows that the cry opreserving polymers are not toxic, even at the highest concentration tested (20 mg / mL) except for B5AMA, which showed reduced bacterial growth at higher concentrations.Example 3: Cryopreserving colloidal gels for the cryopreservation of mammalian cellsToxicity evaluation of the cryopreserving polymers

[0212] RAW 267.4, MDA-MB-231 and 4T1 cell lines were selected for toxicity studies. Cells were seeded in 96 well-plate at 10,000 cells per well and incubated overnight at 37°C. The media was then removed and supplemented with polymer-suspended media in serial dilution starting at 20 mg / mL. Cells were incubated for 24 and 48 hours at 37°C and viable cells were measured using MTS assay. The viability was assessed compared to the non-treated control.

[0213] Polymers that produced a transparent solution in an aqueous medium at 37°C were selected to investigate their cryoprotective potential with respect to mammalian cells. Therefore, B4D1-O10 and B4D0-O10 were selected as the ideal polymers that showed the above properties. Though the B4D1-O8 also produced a transparent solution, the resultant aqueous polymer solution showed thermoresponsive behavior and produced micelles-like structures above 35°c, which affected the growth of mammalian cells after recovery.

[0214] B4D1-O10 and B4D0-O10, which produced clear solutions in aqueous media, were selected as cryopreserving polymers for mammalian cells. Cells were grown in respective culture media until 80 % confluent. Firstly, polymers were mixed with 100 pL of fetal bovine serum (FBS) and homogenized at 6000 rpm for 10 min. Then 100 pL of cell suspension (2xl06cells / mL) in FBS were added to each sample and mixed gently. DMSO (10 %) and FBS alone were used as positive and negative control respectively. Then the samples were kept at -80°C and liquid nitrogen for temperature dependent viability assessment. After 48 hours the cells were thawed and diluted with fresh phosphate buffer saline up to 1 mL. The suspension was centrifuged at 1500 rpm for 3 min and the cell pellet was resuspended in 1 mL of freshmedia. The cellular suspension (100 pL) was added to 96 well-plate and incubated at 37°C. After 48 hours, viable cells were counted using MTS cell viability assay.

[0215] The cry opreservation ability of the selected polymers was assessed at both -80°C and in liquid nitrogen (-196°C). DMSO is the widely used cryoprotectant for mammalian cells and was therefore used as the positive control for this study. The viable cells were assessed using MTS assay and the results were normalized to the value of DMSO (100 %). The percentage viability of RAW 267.4 cells at -80°C for 48 hours are summarized in Table 4 and in Figures 17 and 18 whereas the percentage viability of MDA-MB-231 cells and of 4T1 cells at -80°C for 48 hours are summarized in Figures 19 and 20, respectively.Table 4: Percentage viability of RAW 267.4 cells after incubation at -80°C for 48 h with different polymers and polymer concentrations% Viability2 % 5 % 10 % 15 %BiDo-Os 42 55 62 65B4D1-O10 78±6 90±2 97±4 98±4B4D0-O10 38±3 49±5 58±4 62±4B5AMA 14 20 28 25DMSO 10% 100±lControl 23±10

[0216] The results indicate that B4D1-O10 have promising activity as the viable cell count is equal to the DMSO at 10 and 15 % (w / v) concentrations. Although the 15 % polymer concentration showed the highest % viability, the values were not significant compared to the viability results at 10 % (w / v) polymer concentration. Therefore, 10 % concentrated polymer solution could be considered as an effective concentration for cell cryopreservation activity. A similar pattern of results was obtained withthe other two cell lines as the percentage viability of MDA-MB-231 (Fig. 19) and 4Tl (Fig. 20) cell lines at 10 % (w / v) B4D1-O10 were 102 and 105 %, respectively, which were both better than the DMSO control.

[0217] To evaluate the time dependent viability, RAW 267.4 cells were incubated -80°C for seven days with polymer materials and viable cell counts were taken via MTS assay. The same procedure was followed to evaluate the cell viability in liquid nitrogen (-196°C) for 48 hours. Results of both studies indicate that the polymers can protect cell viability after 7-day incubation at -80°C and in liquid nitrogen. The results are shown in Fig. 18. The toxicity study (Fig. 21) showed that the polymers are apparently non-toxic to mammalian cells. Except for three concentrations of two polymers, all polymers showed viability above 80 %. Meanwhile, the highest concentration of polymer tested was 20 mg / mL, which can be considered the upper limit, and most polymers are not toxic even at that concentration. Therefore, the cryopreserving polymers are considered biocompatible polymers. Biocompatible polymers have a wide range of applications in biomedical research and industry; hence these polymers revealed new directions for those applications.8Other than the biocompatibility, the physicochemical properties of these polymers enhance their potential in biomedical application.9Especially, thermoresponsive polymers are extensively used in drug delivery systems to achieve control or modified release of payload.10Effect of Temperature Fluctuation

[0218] The cryopreserving polymer B4D1-O10 was selected to assess the effect of temperature fluctuation on cell viability. B4D1-O10 and cell suspension (RAW 264.7) were prepared in fetal bovine serum to obtain a final polymer concentration of 10 % (w / v). The prepared cell suspensions were frozen at -80°C for 6 hours and transferred to liquid nitrogen (-196°C). The thawing process started from LN2 (liquid nitrogen) and followed the sequence: LN2dry ice (6 hours)thaw at 37°C. The viability of these cells was compared to cells which were thawed directly at 37°C. The viability of cells with temperature fluctuation was measured at each step. Table 5 below summarizes the results.Table 5: Percentage viability of RAW 267.4 cells during thawing process.% ViabilityCryopreserving DMSO polymer B4D1-O10LN2 87 ± 5 100Dry ice 62 ± 3 97±4

[0219] These results indicate that the cells were viable when thawed directly from liquid nitrogen as this approach minimizes the temperature fluctuation during the thawing process. Thus, this demonstrates that avoiding a slow thawing process through incubation at different temperatures will be desirable when cry opreserving cells using the cry opreserving polymers of the present invention.Growth of Mammalian Cells in the Presence of Cryopre serving Polymers

[0220] B4D1-O10 and B4D0-O10 were selected as cryopreserving polymers since they showed the highest cryoprotectant ability and produced a clear solution in an aqueous medium. Cells (RAW 264.7) were mixed with different concentrations of cryopreserving polymers and cultured on 96 well plate for 48 hours at 37°C. The cell viability was calculated using MTS assay and the percentage viability was calculated compared to the non-treated negative control. The maximum concentration of polymers postcryopreservation is 20 mg / mL. Table 6 below summarizes the results obtained, which are also depicted in Figure 24.Table 6: Percentage viability of RAW 267.4 after incubating with different concentrations of cryopreserving polymers (B4D1-O10 and B4Do-Oio)for 48 hoursConcentration % Viability(mg / mL) B4D1-O10 B4D0-O10100 41 ± 2 53 ± 550 122 ± 2* 127 ± 3*25 117 ± 4* 120 ± 1*12.5 116 ± 1* 114 ± 2*6.25 108 ± 2 113 ± 33.13 109 ± 4 108 ± 31.57 107 ± 3 106 ± 3

[0221] These results show that the cells are able to grow in culture when cryopreserved with a concentration of up to 50 mg / mL of cry opreserving polymers. The cry opreserving polymers are therefore compatible with mammalian cells tested for up to 50 mg / mL while the mammalian cells showed reduced viability at 100 mg / mL, possibly due to the osmolality changes of the medium because of the high concentration of polymer as the polymer / cell solutions are highly viscous at 100 mg / mL. The growing cells were carefully observed for morphological changes during the incubation period and no visual changes were observed. Interestingly, there is a trend that the cry opreserving polymer support the growth of the cell tested at higher concentrations (12.5, 25 and 50 mg / mL) and the viability values are significantly higher compared to the negative control.Metabolic Growth Rate

[0222] To further test the applications of B4D1-O10 as next generation cryoprotectants, Raw 264.7 cells stored in the presence of cryoprotectants were studied for their metabolic growth rate when thawed in the presence of cryoprotectants (Figure 35). As shown in Figure 35, Raw 264.7 cells cryopreserved in the presence of nanogels and cultured without the removal of cryoprotectants showed improved metabolic growth rate with time, in contrast DMSO cryoprotectant containing cells (with 4% final concentration of DMSO post-thaw) showing reduced growth rate with time.

[0223] The role of B4D1-O10 as cryoprotectant was evaluated by temperature controlled slow freezing method, an approach widely used in clinical and research laboratories to preserve mammalian cells. The metabolic profile of Raw 264.7 cells was further tested for up to 96 hours post-thaw by both rapid freezing and temperature controlled slow freezing methods to evaluate any potential side effects of cryopreserving agents (CPAs) on cell growth. The metabolic growth rate of cells cryopreserved withB4D1-O10 by ultrafast cooling method was comparable with DMSO and cell growth rate slightly improved as a function of time (Figure 36). Interestingly, Raw 264.7 cells cryopreserved in the presence of B4D1-O10 by temperature controlled slow cooling approach specifically showed higher metabolic rates as a function of time, when compared with DMSO cryoprotectant, post-thaw and removal of CPAs. At 24 hours post-thaw, metabolic rate of cells was comparable to DMSO but significantly improved with time and metabolic growth rate of cells was ~1.5 fold higher than DMSO, at 96 hours post-thaw.Example 4: Cryopreserving colloidal gels for the cryopreservation of mammalian RBC cells

[0224] Sheep red blood cells (RBCs) were subjected to centrifugation at 4000 rpm to separate the RBC pellet. The pellet was then resuspended in phosphate-buffered saline (PBS) to achieve a hematocrit of 30%. Polymers and dimethyl sulfoxide (DMSO) were dissolved in PBS to reach a concentration of 20%. Equal volumes of the RBC suspension and polymer / DMSO solution were mixed and incubated at room temperature for 15 minutes to establish equilibrium and reach a final concentration of cry opreserving agent of 10%. Subsequently, the samples were incubated at temperatures of -20°C, -80°C, and in liquid nitrogen (LN2) for a duration of 4 hours. After the 4-hour incubation period, the cells were thawed and diluted with PBS before measuring their absorbance. The absorbance values were normalized against the absorbance obtained from complete cell lysis using 1% Triton X100™. Finally, the recovery values of RBCs were calculated by comparing the extent of lysis.Table 7: Percentage Recovery of RBC cellsCryopreserving % Recovery agent-20°C -80°C LN2B4D1-O10 (10%) 20 ± 2 23 ± 4 35 ± 2B4D0-O10 (10%) 21 ± 1 19 ± 3 28 ± 3DMSO (10%) 67 ± 4 72 ± 5 67 ± 5PBS (10%) 14 ± 2 12 ± 0 10 ± 1

[0225] As shown in Table 7, dimethyl sulfoxide (DMSO) provided the best cryopreserving ability during the incubation in liquid nitrogen (LN2). The effects of polymers did not yield significant results compared to the efficacy of DMSO. Among the tested polymers, the highest efficacy was demonstrated by polymer B4D1-O10 at a concentration of 10%.Effect of Mannitol

[0226] The polymer that exhibited the highest activity was chosen for further evaluation in combination with mannitol. The experimental procedure was repeated, but this time a hematocrit of 10% was used instead of 30%. The same steps and measurements were performed to assess the activity of the selected polymer in combination with mannitol.Table 8: Percentage Recovery of RBC cellsCryopreserving % Recovery agentLN2DMSO (5%) 64 ± 2B4D1-O10 (5%)Mannitol (5%) 67 ± 2B4D1-O10 (5%) 22 ± 2B4D1-O10 (10%) 29 ± 2Mannitol (5%) 37 ± 4PBS (10%) 10 ± 0

[0227] As shown in Table 8, the addition of mannitol to the cry opreserving agent containing polymer B4D1-O10 provided the best cry opreserving ability during the incubation in liquid nitrogen (LN2). Neither mannitol nor polymer B4D1-O10 alone provided a similar extent of cryopreservation as the combination of B4D1-O 10 with mannitol have superior cryopreserving abilities, which even performed better than DMSO.Example 5: Ice crystallization inhibition capacity of the polymers

[0228] The ice crystallization inhibition capacity of the polymers was assessed using a custom-built cryostage (Figure 33). The tailor-made cryostage attachment was designed to be placed on the stage of an inverted microscope and dry ice were used as cooling agent. The cryo-attachment consists of a plastic depot to contains the dry ice, an aluminum flat bar to transfer the heat from the glass slide to the dry ice, and a central plastic cylinder to encase the glass slide and divert the sublimated CO2 from the visualization zone. The amount of dry ice loaded inside the deposit allows control over the cooling rate and the final temperature of the glass slide, electron

[0229] To achieve the desired temperature range of -5 °C to -10°C, dry ice was utilized. The experimental setup involved placing a 10 pL volume of the sample on a cover slip, followed by carefully placing another cover slip on top. The entire system was then positioned on the prechilled cryostage and allowed to freeze slowly. Once frozen, they formed ice crystals were captured using a 20X magnification (Figure 32). Image analysis was performed using Image J software. The mean longest grain size (MLGS) in the image was identified and counted, along with determining the total number of crystals present (Figure 34). By dividing the area of the field of view by the number of crystals, the average grain size (AGS) was obtained. The results revealed that the nanogel exhibited significantly greater ice recrystallization inhibition (IRI) activity than PBS and results are comparable of DMSO.

[0230] One or more illustrative embodiments have been described by way of example. It will be understood to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.Example 6: Preparation of B5AMA with various crosslinkers

[0231] B5AMA nanogels can be prepared using a variety of crosslinkers, including divinyl sulfone (DVS) and N,N-methylene bis acrylamide (BIS). One example of nanogels prepared using Bisacrylamide is shown below. The following tables contain the obtained results for two batches of B5AMA nanogels crosslinked with BIS synthesized using the following parameters:Table 9. Polymerization reaction results.X % is conversion %.Table 10. DLS results of the nanogels at the end of the reaction.PDI = poly dispersity indexSD = standard deviationEff. Diam. = Effective diameterTable 11. DLS results of the nanogels after purification (dialysis and freeze drying).All the nanogels show cryopreservation efficacies that are comparable to that of DMSO control. Figure 37 shows that cryopreservation efficacies of B5AMA nanogels, prepared using Bis as a crosslinker, neared that of the DMSO control when Bis was used at 7.5% or 10%. Similarly, Figure 38 shows thatcell viability neared the DMSO control when DVS was used as the crosslinker for different cry opreserving polymers.References1. Hasan, M., Fayter, A. E. R. & Gibson, M. I. Ice Recrystallization Inhibiting Polymers Enable Glycerol-Free Cryopreservation of Microorganisms. Biomacromolecules 19, 3371-3376 (2018).2. Engelmann, F. & Dussert, S. Cryopreservation. Conservation of Tropical Plant Species (2012). doi : 10.1007 / 978- 1 -4614-3776-5 _63. Dou, M., Lu, C. & Rao, W. Bioinspired materials and technology for advanced cry opreservation. Trends Biotechnol. 40, 93-106 (2022).4. Kabir, A.; Dunlop, M. J.; Acharya, B.; Bissessur, R.; Ahmed, M. Water Recycling Efficacies of Extremely Hygroscopic, Antifouling Hydrogels. RSC Adv 2018, 8 (66), 38100-38107. https: / / doi.org / 10.1039 / C8RA07915C.5. Nazeer, N.; Ahmed, M. Hydrophilic and salt responsive polymers promote depletion aggregation of bacteria. European Polymer Journal 2019, 119, 148-154.6. Macias Garcia, B. et al. Toxicity of glycerol for the stallion spermatozoa: Effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential. Theriogenology 77 , 1280-1289 (2012).7. Karlsson JO, T. M. Long-term storage of tissues by cryopreservation: critical issues. Biomaterials 1996;17:243-56. 17, 243-256 (1996).8. Green, J. J. & Elisseeff, J. H. applications. 540, 386-394 (2021).9. Nicolas, J., Mura, S., Brambilla, D., Mackiewicz, N. & Couvreur, P. Design, functionalization strategies and biomedical applications of targeted biodegradable / biocompatible polymer-based nanocarriers for drug delivery. Chem. Soc. Rev. 42, 1147-1235 (2013).10. Ward, M. A. & Georgiou, T. K. Thermoresponsive polymers for biomedical applications. Polymers (Basel). 3, 1215-1242 (2011).

Claims

WHAT IS CLAIMED IS:

1. A cryopreserving polymer comprising polymerized vitamin B5 analogous monomers and a crosslinker, wherein the crosslinker and the polymerized vitamin B5 analogous monomers are crosslinked.

2. The cry opreserving polymer of claim 1, further comprising a co-polymer.

3. The cry opreserving polymer of claim 2, wherein the polymerized vitamin B5 analogous monomers are crosslinked to the co-polymer.

4. The cryopreserving polymer of claim 3, wherein the co-polymer is Di(ethylene glycol) methyl ether methacrylate (DEGMEM).

5. The cry opreserving polymer of any one of claims 1 to 4, wherein the crosslinker is a methacrylated ethylene glycol or an OXASPIRO.

6. The cryopreserving polymer of claim 5, wherein the methacrylated ethylene glycol is ethylene glycol dimethylacrylate (EGDMA).

7. The cryopreserving polymer of claim 5, wherein the OXASPIRO is 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

8. A cry opreserving polymer having formula II :Bm-Co,Formula II whereinB is a vitamin B5 analogous monomer, andC is a crosslinker, which is a methacrylated ethylene glycol, wherein m represents the number of monomer repeats present in the cryopreserving polymer, wherein 1 < m < 10.

9. A cryopreserving polymer having formula III :BmDn-Co,Formula IIIwhereinB is a vitamin B5 analogous monomer,D is a co-polymer which is Di(ethylene glycol) methyl ether methacrylate (DEGMEM) or a variant thereof, andC is a crosslinker, which is a methacrylated polyethylene glycol (E) or an OXASPIRO (O), wherein m represents the number of monomer repeats present in the cryopreserving polymer, n represents the number of DEGMEM crosslinked to the cryopreserving polymer and o represents the number of crosslinkers linked to the cryopreserving polymer, wherein 1 < m < 10 and 0 < n < 1.

10. The cry opreserving polymer of claim 8 or 9, wherein B is defined by formula I:Formula I11. The cryopreserving polymer of claim 8 or 9, wherein the OXASPIRO is 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

12. The cryopreserving polymer of claim 8 or 9, wherein the methacrylated PEG is Di(ethylene glycol) methyl ether methacrylate (EGDMA).

13. The cryopreserving polymer of claim 8, wherein the cryopreserving polymer is Bi-Cs, Bi-Cio, B4-O10, B4-O10 or any combination thereof, wherein C is 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

14. The cryopreserving polymer of claim 13, wherein the cryopreserving polymer is B4-O1015. The cryopreserving polymer of claim 9, wherein the cryopreserving polymer is B1D1-E4, B1D1- E7, B1D1-O4, BiDi-Oe, BiDi-Os, B1D1-O10, B4D1-O10, B4D1-O8, B4D1-O10, BiDo-Os, B1D0-O10, or any combination thereof, wherein E is EGDMA and O is 3, 9-Divinyl-2, 4,8,10-tetraoxaspiro[5.5]undecane.

16. The cry opreserving polymer of claim 15, wherein the cry opreserving polymer is B1D1-O10, B4D1-O10, B4D1-O10, B4D0-O10, B4D0-O10, or any combination thereof.

17. The cry opreserving polymer of claim 16, wherein the cry opreserving polymer is B4D0-O10 or B4D0-O10.

18. A composition for cryopreserving cells comprising the cryopreserving polymer of any one of claims 1 to 17 and a salt and / or a suitable buffer.

19. The composition of claim 18, wherein the salt is sodium chloride (NaCl).

20. The composition of claim 18, wherein the suitable buffer is a phosphate buffer.

21. The composition of claim 18 or 19, further comprising mannitol.

22. A cryopreserving colloidal gel comprising the cryopreserving polymer of any one of claims 1 to 17.

23. The cryopreserving colloidal gel of claim 22, further comprising mannitol.

24. The cryopreserving colloidal gel of claim 22 or 23, further comprising a salt.

25. The cry opreserving colloidal gel of claim 24, wherein the salt is sodium chloride (NaCl).

26. The cryopreserving colloidal gel of any one of claims 22 to 25, for cryopreserving cells.

27. A method of preparing a cryopreserving polymer comprising: polymerizing a plurality of vitamin B5 analogous monomers to obtain a vitamin B5 analogous polymer, and crosslinking the vitamin B5 analogous polymer to obtain the cry opreserving polymer.

28. A method of preparing a cryopreserving polymer comprising: polymerizing a plurality of vitamin B5 analogous monomers to obtain a vitamin B5 analogous polymer, and crosslinking the vitamin B5 analogous polymer with Di(ethylene glycol) methyl ether methacrylate (DEGMEM) to obtain the cry opreserving polymer.

29. The method of claim 27 or 28, wherein the plurality of vitamin B5 analogous monomers aredefined by formula (I):Formula I30. The method of any one of claims 27 to 29, wherein the polymerizing comprises adding a polymerization initiator to the plurality of vitamin B5 analogous monomers.

31. The method of claim 30, wherein the polymerization initiator is potassium persulfate (KPS), VA-044, VA-051 or 4,4'-Azobis(4-cyanovaleric Acid) (ACVA).

32. The method of any one of claims 27 to 31, wherein the polymerizing comprises adding a catalyst to the plurality of vitamin B5 analogous monomers.

33. The method of claim 32, wherein the catalyst is N,N,N’,N’ -tetramethylethylenediamine (TEMEDA).

34. The method of any one of claims 27 to 33, wherein the crosslinking comprises adding a crosslinker to the vitamin B5 analogous polymer.

35. The method of claim 34, wherein the crosslinker is a methacrylated ethylene glycol or an OXASPIRO.

36. The method of claim 35, wherein the methacrylated ethylene glycol is ethylene glycol dimethylacrylate (EGDMA).

37. The method of claim 35, wherein the OXASPIRO is 3,9-Divinyl-2,4,8,10- tetraoxaspiro[5.5]undecane.

38. The method of any one of claims 27 to 37, wherein the obtained cryopreserving polymer is in a cryopreserving solution.

39. The method of claim 38, further comprising degassing the cryopreserving solution.

40. A method of cry opreserving cells, comprising:mixing the cells with the composition of any one of claims 18 to 21 to obtain a cryopreserving preparation, and freezing the cry opreserving preparation.

41. A method of preparing a cryopreserving colloidal gel comprising: pouring an aqueous solution comprising the cry opreserving polymer of any one of claims1 to 17 into a mold, incubating the poured aqueous solution in the mold for a period of time to obtain a molded colloidal gel, and removing the molded colloidal gel from the mold and washing the molded colloidal gel.

42. The method of claim 41, wherein the aqueous solution comprises a salt and / or a suitable buffer.

43. The method of claim 42, wherein the suitable buffer is a phosphate buffer.

44. The method of claim 42, wherein the salt is sodium chloride (NaCl).

45. The method of any one of claims 41 to 44, wherein the aqueous solution further comprises mannitol.

46. A method of preparing a cryopreserving agent comprising: providing the cryopreserving colloidal gel of any one of claims 22 to 26, solubilizing the cryopreserving colloidal gel in an aqueous solution, wherein the volume of the aqueous solution is determined according to a desired concentration of the cry opreserving polymer, thereby obtaining the cryopreserving agent.

47. The method of claim 46, wherein the aqueous solution further comprises mannitol.

48. The cry opreserving polymer of any one of claims 1 to 17, the composition of any one of claims 18 to 21 or the cry opreserving colloidal gel of any one of claims 22 to 26, for cry opreserving prokaryotic or eukaryotic cells.

49. The cry opreserving polymer of any one of claims 1 to 17, the composition of any one of claims 18 to 21, or the cry opreserving colloidal gel of any one of claims 22 to 26, which is free of organic solvents such as dimethyl sulfoxide (DMSO) and glycerol.

50. Use of the cry opreserving polymer of any one of claims 1 to 17, the composition of any one of claims 18 to 21, or the cry opreserving colloidal gel of any one of claims 22 to 26, for cry opreserving prokaryotic or eukaryotic cells.

51. Use of the cryopreserving polymer of any one of claims 1 to 17, the composition of any one of claims 18 to 21, or the cry opreserving colloidal gel of any one of claims 22 to 26 in the manufacture of a cry opreserving agent for cry opreserving prokaryotic or eukaryotic cells.

52. A cryopreserving agent comprising the cryopreserving colloidal gel of any one of claims 22 to 26.

53. The cryopreserving agent of claim 52, further comprising mannitol.

54. Use of the cry opreserving polymer of any one of claims 1 to 17, the composition of any one of claims 18 to 21, or the cry opreserving colloidal gel of any one of claims 22 to 26 as an agent in cosmetics and / or in the preparation of cosmetic formulations.

55. The cry opreserving polymer of any one of claims 1 to 17, the composition of any one of claims 18 to 21, or the cry opreserving colloidal gel of any one of claims 22 to 26 for use as an agent in cosmetics and / or in the preparation of cosmetic formulations.

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