CRYOPROTECTIVE AGENT, CRYOPROTECTIVE AND CRYOPRESERVED COMPOSITIONS, USES THEREOF, AND METHODS OF CRYOPRESERVATION

FIELD OF THE INVENTION

The present invention relates to a cryoprotectant, a cryoprotective agent, cryoprotective compositions, cryopreserved compositions, uses thereof, and methods of cryopreservation.

BACKGROUND OF THE INVENTION

Cryopreservation of viable biological samples such as cells, tissues or organs, which have been harvested from a donor source, is of great importance and utility in the scientific and medical communities. Cryopreservation is generally a process where a sample, for example cells or tissue, is preserved by cooling to sub-zero temperatures, typically 77 K (= - 196 °C, the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would cause cell death, is effectively stopped.

Cryopreservation techniques are routinely used for long-time preservation of water-bearing or aqueous materials such as cells and tissues of plants and animals. It is known that upon freezing these materials, ice crystals form, resulting in uneven concentrations of solutes and contaminants excluded by water molecules, called "freeze concentration". In order for the cells or tissues to be preserved, cryoprotectant solutions are typically used to prevent damage due to freezing during the cooling or thawing process. For cryopreservation to be useful, the preserved sample should retain its integrity and viability to a reasonable level at the time of harvest. Thus, the process of preserving the sample should preferably not, in itself, severely damage or destroy for example the cells or tissue architecture.

In conventional cryopreservation techniques, the sample is harvested, placed in a storage solution, and then preserved by freezing. When the sample is to be used, it is thawed, and, as an example, cells taken from human donor sources are brought back to the normal human body temperature (i.e., approximately 37°C) and then placed in a cell culture medium.

Cryopreservation protocols subject the cells to a multitude of stresses and perturbations throughout the process of cell harvesting, freezing, and thawing. These stresses and perturbations can cause irreversible damage to the cell.

Dextran has been used as a cryoprotective agent for human, animal and plant cells (Odavic, R. et al. Experientia 36, 1122 (1980), Ashwood-Smith, M.J. et al. Cryobiology 9, 441 (1972) and Echlin, P. et al . J. Microsc. (Oxford) 110, 239 ( 1977) ) . A mixture of 5% methyl sulphoxide and 9% Dextran 70 was found to afford optimal cryoprotection of human bone marrow committed stem cells (Dextran, Handbook from Amersham Biosciences, 18- 1166- 12, Edition AA, page 35) . Dextran, glycerol and dimethyl sulfoxide ( DMSO), alone or in combination, have been investigated for cryoprotection of human bone marrow cells (Odavic, R. et al . Experientia 36, 1122 ( 1980)) . A significantly better protection against freezing injury was obtained by 9% Dextran 70 in combination with 3 or 5% DMSO, and also with 5 or 10% DMSO alone, than with either 15% glycerol or 9% dextran with 1% DMSO. Dextran 40 is known (Proc. Nati . Acad . Sci . USA, Vol . 92, pp. 10119- 10122, October 1995, Medical Sciences) for cryopreservation of placental/umbilical cord blood in a combination of 50% DMSO in 5% (w/v) Dextran 40.

Shu Guowei et al describes in Advanced Materials Research , Trans Tech Publications Ltd . , Vol. 328 (2012), pp 454-457 the effect of fructo-oligosaccharide, isoma lto-oligosaccharide, inul in and xylo-oligosaccharide on survival of B. bifidium during freeze-drying . Kwan Hwa Park et al . describes in Database CA, XP 002698458 a cryoprotectant containing

fructooligosaccharide, isomaltooligosaccharide or galactooligosaccharide for surimi . In J . Korean Soc. Food Sci . Nutr. , Vol . 30(3) (2001 ), pp 565-568, the effects of a cryoprotectant of fructooligosaccharide, isomaltooligosaccharide and galactooligosaccharide on beef protein is described . Conventiona l cryoprotectants are glycols (alcohols contain ing at least two hydroxyl groups), such as ethylene glycol, propylene glycol, a nd glycerol . Ethylene glycol is commonly used as automobile antifreeze, and propylene glycol has been used to reduce ice formation in ice cream . Dimethyl sulfoxide (DMSO) is also regarded as a conventional cryoprotectant.

Glycerol and DMSO have been used for decades by cryobiologists to reduce ice formation in sperm and embryos that are preserved in liquid nitrogen (- 196° C) . Among these known cryoprotective agents, DMSO is considered the most effective and frequently adopted, but it is physiological ly toxic and known to cause high blood pressure, nausea and vomiting if transfused to a recipient with the cells or for personnel handling it unless precautions are taken . Cox et a l (Cell Tissue Bank (2012) 13 : 203-215) identified by a retrospective review of published literature several hundred adverse reactions (e.g . nausea, chil ls, cardiac arrhythmias, neurological symptoms and respiratory arrest) associated with the

transplantation of stem cells cryopreserved with dimethyl sulfoxide. Further, the toxicity of DMSO tends to debilitate the cells' survival rates a nd/or functions, including genomic alterations after the thawed cells are cultured or transfused into a recipient's body. The toxicity of DMSO thus also affects how long the cells may be exposed to DMSO during handling . Thus, there is still a need for a cryoprotective agent for protecting a sample (such as a biological sample) during freezing as a replacement for other cryoprotectants such as DMSO or as a supplement to such other cryoprotectants to reduce the concentration needed thereof, preferably to non-toxic concentrations, which at the same time have the necessary protective effects and low or no toxicity.

OBJECT OF THE INVENTION

It is an object of embodiments of the invention to provide a cryoprotectant as a replacement for other cryoprotectants such as DMSO or as a supplement to such other cryoprotectants to reduce the concentration thereof, preferably to non-toxic concentrations, or to improve the function thereof, said cryoprotectant having the necessary protective effects with regard to preserving as much functionality such as survival or viability after thawing of the

cryopreserved sample during cryopreserving. It is a further object of embodiments of the invention to provide a cryoprotectant having a low or no toxicity for the personnel handling the cryoprotectant and for the biological samples, whereby the time that the sample can be in contact with the cryoprotectant without being damaged is prolonged, the necessity for washing of samples is reduced, and, if desired, making it possible to return the sample to where it was taken from or to a recipient without having to separate the sample from the cryoprotectant. It is a further object of embodiments of the invention to provide a cryoprotectant, said cryoprotectant being effective as a cryoprotectant for a sample selected from the group consisting of organs, cells and tissues such as selected from the group consisting of mammalian organs, mammalian cells, and mammalian tissues. It is a further object of embodiments of the invention to provide a cryoprotectant, said cryoprotectant being effective as a cryoprotectant for a sample to be transplanted such as an organ, cells or tissues. It is a further object of embodiments of the invention to provide a cryoprotectant, said cryoprotectant being effective as a cryoprotectant of for example cells and result in an acceptable viability of said cells such as survival or viability after thawing. It is a further object of embodiments of the invention to provide a cryoprotectant, said cryoprotectant being effective as a cryoprotectant of for example organs and result in an acceptable physical functionality of said organs. It is a further object of embodiments of the invention to provide a cryoprotectant, said cryoprotectant being effective as a cryoprotectant of for example tissues and result in an acceptable physical functionality of said tissues.

SUMMARY OF THE INVENTION

It has been found by the present inventor(s) that a cryoprotective agent comprising a cryoprotectant selected from the group consisting of carboxyalkyldextran, hydrogenated carboxyalkyldextran, oxidated carboxyalkyldextran, ether of carboxyalkyldextran, ester of carboxyalkyldextran and partly oxidated/hydrogenated carboxyalkyldextran or a combination thereof, is very useful as a cryoprotectant. In one aspect, the cryoprotectant is

carboxyalkyldextran. In another aspect, the cryoprotectant is hydrogenated

carboxya I ky Idextra n .

So, in a first aspect the present invention relates to the use of a cryoprotectant as described herein for cryopreserving a sample, and wherein said sample is selected from the group consisting of organs, cells, and tissues.

In a further aspect, the present invention relates to a cryoprotective agent comprising a cryoprotectant as described herein.

In a further aspect, the present invention relates to the use of a cryoprotectant as described herein for cryopreserving a sample, wherein said sample is selected from the group consisting of organs, cells and tissues.

In a further aspect, the present invention relates to a cryopreservation composition comprising a cryoprotective agent as described herein, which cryopreservation composition further comprises a sample to be cryopreserved, wherein said sample is selected from the group consisting of mammalian organs, mammalian cells, and mammalian tissues.

In a further aspect, the present invention relates to a cryopreserved composition comprising a cryoprotective agent as described herein, which cryopreserved composition further comprises a sample to be cryopreserved. Said sample may be selected from the group consisting of mammalian organs, mammalian cells, and mammalian tissues.

In a further aspect, the present invention relates to a method of cryopreserving a sample, comprising the steps of bringing a sample to be cryopreserved into contact with a cryoprotective agent as described herein to obtain a cryopreservation composition and subsequently reducing the temperature of the cryopreservation composition to a

cryopreservation temperature. Said sample may be selected from the group consisting of organs, cells and tissues. In a further aspect, the present invention relates to a method of cryopreserving a cryopreservation composition as described herein by bringing the composition to a cryopreservation temperature. In a further aspect, the present invention relates to the use of a cryoprotective agent as described herein for cryopreserving a sample, wherein said sample is selected from the group consisting of organs, cells and tissues. In a further aspect, the present invention relates to the use of a cryoprotective agent as described herein for cryopreserving a sample for transplantation.

In a further aspect, the present invention relates to the use of a cryopreservation

composition as described herein for cryopreserving a sample by reducing the temperature of said composition to a cryopreservation temperature, wherein said sample is selected from the group consisting of organs, cells and tissue.

LEGENDS TO THE FIGURE

The Figures show human induced pluripotent stem cells (abbreviated iPSCs or iPS cells) which were cryopreserved and thawed in the cryopreservation compositions described in example 1 and 2. Viability after thawing (Fig. 1, Fig. 3 and Fig. 6) and the proliferative rate of the cells (Fig. 2 and Fig. 4) were evaluated. The figures demonstrate a clear positive effect of including hydrogenated carboxymethyldextran relative to the untreated sample.

Furthermore, it demonstrates that including hydrogenated carboxymethyldextran together with ethylene glycol results in a comparable or better viability than ethylene glycol alone or DMSO alone. It is furthermore demonstrated that after thawing, iPSC lines seeded and as exemplified on Fig. 5 resulted in proliferative and viable iPSC lines capable of be expanded.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

As used herein, cryopreservation means a process where a sample is preserved by cooling to sub-zero temperatures, including vitrification technology in which the cooling rate is faster than a conventional cryopreservation procedure. At such low temperatures activity such as biological activity, including the biochemical reactions that would cause for example cell death, are reduced, and the chemical structure/function of for example proteins/glyco- proteins or lipoproteins are preserved. If cryoprotectant solutions are not used, the samples being preserved are likely to be damaged due to freezing during the cooling or thawing process. In one preferred aspect, cryopreservation means a process where samples are preserved by cooling to sub-zero temperatures, typically 77 K (= - 196 °C, the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would cause cell death, is effectively stopped. As used herein, the term "cryopreservation temperature" designates a temperature of from sub-zero to -196 °C, such as from - 50 °C to -196 °C, such as from - 80 °C to -196 °C, such as a temperature below -55 °C, such as below -60 °C, such as below -65°C, such as below - 70 °C, such as below -75 °C, such as below -80 °C, such as below -85 °C, such as below -90 °C, such as below -95 °C, such as below -100 °C, such as below -105 °C, such as below -110 °C, such as below -115 °C, such as below -120 °C, such as below -125 °C, such as below - 130 °C, such as below -135 °C, such as below -140 °C, such as below -145 °C, such as below -150 °C, such as below -155 °C, such as below -160 °C, such as below -165 °C, such as below -170 °C, such as below -175 °C, such as below -180 °C, such as below -185 °C, such as below -190 °C. As used herein, the term "sample" means any kind of material to be cryopreserved such as organs, cells, or tissue. In one aspect, a sample is selected from the group consisting of organs, cells, tissue and blood. In one aspect, a sample is selected from the group consisting of organs, cells, and tissue, such as selected from the group consisting of mammalian organs, mammalian cells and mammalian tissues. In one aspect, the term "sample" does not comprise the human body at the various stages of its formation and development. In a further aspect, the present invention relates to the use of a cryoprotective agent as described herein for cryopreserving a sample for transplantation. In one aspect, the sample is selected from the group consisting of mammalian organs, mammalian cells and mammalian tissues for transplantation. As used herein, the term "cells" comprises any type of cells such as somatic cells including all kind of cells in tissue or organs, stem cells including all types of totipotent stem cells, pluripotent stem cells, multipotent stem cells and progenitor cells; oocytes; spermatozoa; and germ cells. The cells may be in isolated form or in a not isolated form such as in the form of a cell-containing bodily fluid, a tissue or organ. As used herein, the term "cell-containing bodily fluids" comprises any cell-containing bodily fluid such as for example below defined blood, amniotic fluid, semen, cerebrospinal fluid, bone marrow aspirates and menstrual fluid.

As used herein, the term "blood" comprises any blood containing fluid such as umbilical cord blood, peripheral blood, and mobilized blood. As used herein, the term "tissue" or "tissues" comprises any tissue type comprising any kind of cell type and combinations thereof, including ovarian tissue, testicular tissue, umbilical cord tissue, placental tissue, connective tissue, cardiac tissue, tissues from muscle, cartilage and bone, endocrine tissue, and neural tissue. The term "tissue" or "tissues" also comprise adipose tissue or dental pulp tissue.

As used herein, the term "organ" comprises for example lung, liver, kidney, heart, ovaries and pancreas. The term "organ" also comprise umbilical cord.

As used herein, the term "functional after cryopreservation" in relation to a sample means that the sample such as organs, tissue or cells after cryopreservation retains an acceptable and/or desired function after cryopreservation. In one aspect, the sample after

cryopreservation retains all its function. In another aspect, the sample, such as cells, at least retains 50% of the desired function, such as at least 60% of the desired function, such as at least 70% of the desired function, such as at least 80% of the desired function, such as at least 90% of the desired function, such as at least 95% of the desired function, such as 100% of the desired function, As an example with regards to cells an important function to be preserved is the viability of the cells. As another example with regards to organs an important function to be preserved is the physiological function of such organ, e.g. for a heart the pumping function. As another example with regards to tissue an important function to be preserved is the ability of such tissue to integrate with surrounding tissue (e.g. skin) in the case of transplantation.

The viability of cells after cryopreservation may be measured by using the Nucleocounter system in which dead cells are measured by incubating the cell sample with the DMA binding dye, propidium iodide, which only results in detectable measurement from dead cells as shown in the examples. The viability is given as a percentage of living cells in the population that are being analyzed. The proliferative rate of a cell sample after cryopreservation can be analysed using the colorimetric assay, MTT.

As used herein, the term "banking" means any storage of a sample for future use.

As used herein, the term "clinical banking method" means any storage of a sample relating to clinical treatment of a mammal such as a human being.

As used herein, the term "marrow banking method" means storage of a marrow sample such as bone marrow aspirates and related bodily fluid, or cells isolated from marrow. As used herein, the term "dental pulp tissue banking method" means storage of a dental pulp tissue sample such as cells isolated from dental pulp tissue.

As used herein, the term "fat tissue banking method" means storage of a fat tissue sample such as cells isolated from fat tissue. In the present context the term and in the following, "umbilical cord banking method" means storage of umbilical cord blood, tissues related to umbilical cord or cells isolated from umbilical cord blood or tissue.

In the present context the term and in the following, "mobilized peripheral blood banking method" means storage of peripheral blood after mobilizing with agents that for example releases blood stem cells into the circulation. As used herein the term, "reproduction banking method" means storage of any sample relating to reproduction such as semen, oocytes, spermatozoa, fertilized eggs etc.

The atomic mass unit Dalton (symbol: Da) is the standard unit used for indicating mass on an atomic or molecular scale (atomic mass). It is defined as one twelfth of the remaining mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state. As used herein, the term "weight average molecular weight" (M _w ) is defined as:

wherein g; is the fraction of molecules having the molecular weight M,. The possible values of M make up a set of numbers with discrete values labelled _if defining p.

As used herein, the term "number average molecular weight" is defined as:

wherein N, is the fraction of molecules having the molecular weight M _i( g _{ is the fraction of molecules having the molecular weight M,. The possible values of M make up a set of numbers with discrete values labelled ,, defining p.

As used herein, the term polydispersity (Pd) is calculated by M _w /M _n = Pd. As used herein, the term "Dextran" followed by a number such as "Dextran 1", "Dextran 40" and "Dextran 70" follows the Pharmacopoeial abbreviation for Dextran X meaning that the weight average molecular weight of the dextran is approximately X kDA. Thus, Dextran 1 means a dextran having a weight average molecular weight of 850- 1, 150 Da.

Isomaltooligosaccharide 1, and hydrogenated isomaltooligosaccharide 1 are named similarly. Isomaltooligosaccharide 1 means thus a mixture of isomaltooligosaccharides having a weight average molecular weight of 850- 1, 150 Da conforming to the EP and USP Monographs for Dextran 1. Isomaltooligosaccharide 1 is also named pentaisomaltose in this application . Hydrogenated isomaltooligosaccharide 1 means a mixture of hydrogenated

isomaltooligosaccharides, where the isomaltooligosaccharides conforms to the EP and USP Monographs for Dextran 1. Hydrogenated isomaltooligosaccharide 1 is also named pentaisomaltoside in this application .

As used herein, the term "dextran-based isomaltooligosaccharide" means an

isomaltooligosaccharide having a weight average molecular weight (M _w ) of between 300 and 1,650 Da, such as between 850 and 1,650 Da and obtained from hydrolysed dextran such as by hydrolysis of low molecular weight dextran.

As used herein, the term "cryoprotectant" means a substance which for example In an appropriate solution is used to protect a sample from freezing damage. Examples of known cryoprotectants are for example DMSO, polyols etc.

As used herein, the term "sterile" means free from living germs, microorganisms and other organisms capable of proliferation.

As used herein, the term "substantially free of DMSO" means DMSO in an amount less than O.Olw/w %.

As used herein, "Ci_ ₁₀ alkyl" is a hydrocarbon being a straight-chain or branched Cj. ₁₀ alkyl such as a straight-chain or branched Ci- ₆ alkyi. Examples are methyl, ethyl, 1-propyl, 2- propyl, isopropyl, 1-butyl, 2-methyl- l-propyl, 2-butyl, 1-pentyl, 3-pentyl, 2-methyl-2-butyl, and 3-methyl-2-butyl. As used herein, "carboxyCi- ₁₀ alkyl" means -Ci. ₁₀ alkylCOOH. An example is carboxymethyl

(CM) (-CH ₂ COOH).

As used herein, the term "DEAE" means diethylaminoethyl.

As used herein, the term "hydrogenated carboxyalkyidextran" means hydrogenated dextran substituted with carboxyalkyl. As used herein the term "oxidated carboxyalkyidextran" means oxidated dextran substituted with carboxyalkyl.

Cryoprotective agent

Further described herein is a cryoprotective agent comprising a cryoprotectant selected from the group consisting of carboxyalkyidextran, hydrogenated carboxyalkyidextran, oxidated ca rboxya I ky Idextra n , ether of carboxyalkyidextran, ester of carboxyalkyidextran and partly oxidated/hydrogenated carboxyalkyidextran or a combination thereof, such as

ca rboxya I kyldextra n .

Dextran comprises repeating D-glucose units. Dextrans are a family of neutral branched polysaccharides consisting predominantly of an a-(l→6) linked D-glucose as further described below. Suitably, the carboxyalkyidextran is carboxyCi. ₁₀ alkyidextran.The C _x i ₀ alkyl is suitably methyl. In yet a further aspect, the cryoprotectant is carboxymethyl

isomaltooligosaccharide or carboxyethyl isomaltooligosaccharide.

The molecular weight of dextran is typically determined by means of gel permeation chromatography (GPC) using for example GPC columns of the type polyether hydroxylated gels. The calibration may be performed as described in the European Pharmacopoeia 7 ^th

Edition for dextran and using the iterative mathematical method as described in the European Pharmacopoeia 7 ^th Edition, volume 2, pages 1816-1817, for dextran.

In one aspect, the cryoprotectant described herein is selected from the group consisting of carboxyalkyidextran, hydrogenated carboxyalkyidextran, oxidated carboxyalkyidextran, ether of carboxyalkyidextran, ester of carboxyalkyidextran and partly oxidated/hydrogenated carboxyalkyidextran or a combination thereof such as carboxyalkyidextran and has a weight average molecular weight (Mw) of between 300 and 900,000 Da such as between 300 and 700,000 Da, preferably between 300 and 500,000 Da. In one aspect, the cryoprotectant described herein has a weight average molecular weight (Mw) of above 700,000 Da such as between 700,000 and 900,000 Da. In one aspect, the cryoprotectant described herein has a weight average molecular weight (Mw) of between 500 and 15,000 Da such as between 500 and 6000 Da. In a further aspect, the cryoprotectant described herein has a weight average molecular weight (Mw) of between 500 and 1500 Da or between 3000 and 6000 Da . In one aspect, the cryoprotectant described herein is hydrogenated carboxyalkyldextran and has a weight average molecular weight (Mw) of 300 and 900,000 Da such as between 500 and 15,000 Da, or such as between 500 and 6000 Da . In a further aspect, the hydrogenated ca rboxya I ky Idextra n described herein has a weight average molecular weight (Mw) of between 500 and 1500 Da or between 3000 and 6000 Da. In some aspects, the average molecular weight (Mw) is above 700,000 Da. In one aspect, the hydrogenated cryoprotectant described herein has a weight average molecular weight (Mw) of between 700,000 and 900,000 Da. The cryoprotectant as described herein may be prepared by using hydrogenated dextran or hydrogenated isomaltooligosaccharide or the corresponding polysaccharide having a weight average molecular weight (Mw) of between 300 and 900,000 Da such as between 1000 Da to 700,000 Da as a starting material. In one aspect, the starting material has a weight average molecular weight (Mw) of between 1000 and 7000 Da, such as between 1650 and 6000 Da, such as between 2000 and 5000 Da . The starting material suitably has a polydispersity of > 1 and < 5. Compared to dextran materials of higher average molecular weight such as Dextran 40 (40,000 Da) and Dextran 70 (70,000 Da), the cryoprotective agent having a lower average molecular weight have a lower viscosity. This facilitates preparation of a high concentration, making it possible to add a sample already in a solution and still obtain a composition comprising both cryoprotectant and sample in a concentration suitable for cryopreservation. Furthermore, within the described class of molecules, molecules with lower molecular weight are generally less immunogenic than molecules with high molecular weight. Dextran 1 is for example known as a hapten inhibitor that reduces the risk for anaphylactic reactions when administering dextran and is thus used as pre-injection before injection of d extra ns with higher molecular weight such as Dextran 40 (40,000 Da) and Dextran 70 (70,000 Da). Dextran 1 has also been documented in studies by Richter et al (Int. Arch. Allergy 43 : 252-268 ( 1972) and Int. Arch . Allergy 41 : 826-844 ( 1971)) to have a very low immunological potential in humans.

The cryoprotectant according to the invention suitably has a content of carboxy groups of above 1 % w/w, such as above 2.0 % w/w, such as above 3 % w/w, such as above 4 % w/w, such as for example between 1-7.0 % w/w/, such as between 3-7 % w/w or between 4-7% w/w, as determined by direct titration using for example sodium hydroxide.

In one aspect, the cryoprotectant according to the invention suitably has a content of carboxy groups of between 1- 15 % w/w, such as between 5-12 % w/w, as determined by direct titration using for example sodium hydroxide. The degree of substitution may be measured as described below through titration of the cryoprotectant in its acidic form: x. ^ .· _n/ AxN _NaOH xl00xM

degree of substitution % = '

lOOOxC

A = amount of used NaOl 1 in mL

N _Na0H = Normality of NaOH

M = Molecular weight of Carboxy group = 45 Da

C = Amount af titrateded product Dextran and derivatives thereof

Dextran can be formed by several bacterial strains, mostly gram-positive, facultative anaerobe cocci, e.g. Leuconostoc and Streptococcus strains as for example described in "Advances in polymer science", Volume 205, Polysaccharides II, editor D.KIemm, Springer Verlag. Dextrans for pharmaceutical use have typically been manufactured by specific bacterial strains defined in the US or European Pharmacopoeias such as for example by Leuconostoc Mesenteroides NCTC 10817 or B512 F. The strain NCTC 10817 and B512F is publicly available since 1971 from National Collection Type Cultures (Central Public Health Laboratory) UK,

Dextrans are a family of neutral branched polysaccharides consisting predominantly of an a- (1→6) linked D-glucose having a main chain with varying proportions of linkages and branches depending on the bacteria used in the fermentation. The dextran molecule contains one free terminal aldehyde group which is not shown in Formula I. The o-(l→6) linkages in dextran may vary from 50 to 97 % of the total glucosidic bonds. The remaining glucosidic bonds represent a-(l→2), a-(l→3) and a-(l→4) linkages bound as branches. Formula I illustrates part of the a-( l→6)- linked glucose main chain of dextran with branching points in 2-, 3- and 4- positions. Using the abovementioned strain B512F the ratio of a-(l-6) linkages ist typically 95 % or above.

Formula I

Extremely high values of molecular weight are found for the native dextrans. Values ranging from 10 ⁷ to 4xl0 ⁸ Daltons have been reported. In order to make the dextrans usable for many applications it is therefore necessary to hydrolyse the native dextrans to a lower molecular weight. There are several methods known and available to the skilled person, however, the hydrolysis may be performed at approx. pH 1.5, normally using hydrochloric acid, and at a temperature of approx. 95° C. By the hydrolysis low molecular weight dextrans and glucose are produced. The hydrolysate is typically purified and fractionated by various methods such as sedimentations with alcohol, filtrations and other various chromatographic methods including membrane filtration.

Dextrans for pharmaceutical use have typically been manufactured by specific bacterial strains defined in the US or European Pharmacopoeias such as for example by Leuconostoc Mesenteroides NCTC 10817 or B512 F. The strain B512F and NCTC 10817 is as mentioned above both publicly available from National Collection Type Cultures (Central Public Health Laboratory) UK.

Among dextrans, particularly Dextran 40 and Dextran 70 have been used for human pharmaceutical use. Other molecular sizes such as e.g. Dextran 500 and Dextran 5 and molecular weights in between are used outside the area of cryopreservation as carriers for synthesis, for separation of cells, as excipients in vaccines or in various other applications such as preservation of the human cornea. Furthermore, Dextran 1 has a special use in humans as pre-injection of Dextran 1 exhibits hapten-inhibition and blocks human dextran antibodies, thus preventing potential allergic reactions known to occur occasionally after administration of high molecular weight dextran in humans. Dextran 1, Dextran 40 and Dextran 70 are well described Pharmacopoeia products (European Pharmacopoeia 7 ^th Edition, volume 2, page 1816-1819).

Dextran is also an excellent raw material used for synthesizing water-soluble polymers.

The following are examples of derivatives of dextran, which may be used as starting materials for preparing a cryoprotectant as described herein :

1) Hydrogenated dextran which may be synthesised by reaction of dextran with a reducing agent such as borohydride under alkaline conditions for example at pH 8-12 reducing aldehyde end groups into sorbitol.

2) Ethers of dextran which may be synthesies by methods known to the skilled person. As an example mention can be made of 2-(Diethylamino)ethyl dextran (DEAE dextran) (shown in Reaction scheme 1) which may be synthesised by reaction of dextran with (2- chloroethyl)diethylammonium chloride in alkaline solution.

Reaction scheme 1 : DEAE dextran containing 2-(diethylamino)ethyl (A) and 2- [(2-

(diethylamino)ethyl]diethylammonium]ethyl (B) groups

CarboxyC _1-10 aSkyi dextran such as carboxymethyldextran (CMD) as shown in Reaction Scheme 2 may be synthesised by reaction with monochloric acetic acid (MCA) under strong alkaline conditions. In one aspect, the carboxyalkyldextran has a content of carboxy groups of above 1 % w/w, such as above 2.0 % w/w, or such as above 3 % w/w, such as above 4 % w/w, such as for example 1-7.0 % w/w or such as 4-7% w/w determined by direct titration using for example sodium hydroxide.

Reaction scheme 2

Esters of dextran such as dextran acetate which may be synthesised by reaction of dextran with acetic acid anhydride.

R: H;CH ₃ C0

Reaction scheme 3

4) Oxidated dextran may for example be synthesised by means of a sodium hypochlorite in a basic aqueous solution.

5) Partly oxidated/hydrogenated dextran. A method of preparation of this type of derivatives is for example disclosed in US 6,977,249 which are incorporated herein by reference. As an example mention can be made of a dextran which is prepared by a process in which process the molecular weight of a dextran is reduced by hydrolysis, and functional aldehyde terminal groups thereof converted into alcohol groups by hydrogenation;

characterized in that the hydrogenation is only partial, leaving at the most 15% by weight reducing sugar, calculated on the total amount of carbon hydrates, and said dextran is subsequently subjected to oxidation, said hydrogenation and oxidation being performed to obtain dextran having substantially all aldehyde groups converted into alcohol and carboxylic groups, and said dextran product having no functional aldehyde groups or functional carboxylic acid groups in the intermediate glycosyl groups; wherein the hydrogenation is performed by means of sodium borohydride in aqueous solution; and wherein the oxidation is performed by means of a sodium hypochlorite in basic aqueous solution.

CarboxyCi-jo alkyl-substituted of above hydrogenated and/or oxidated dextran may be prepared by methods known to the skilled person similar to the above described for example as described in the preparation examples herein. Isomaltooligosaccharide and derivatives thereof

Isomaltooligosaccharides are glucose oligomers with an a-D-(l,6)- bound main chain. In one aspect, the isomaltooligosaccharide described herein is dextran-based and is made by hydrolysis of low molecular weight dextran. In a further aspect, the isomaltooligosaccharide described has a weight average molecular weight (M _w ) of between 300 and 1,650 Da such as having a weight average molecular weight (M _w ) of between 850 and 1,650 Da. In one aspect, the isomaltooligosaccharide described herein is hydrolysed dextran having a weight average molecular weight (M _w ) of between 850 and 1,650 Da. Starting from isomaltooligosaccharide derivatives thereof characterised by the changing of the reducing aldehyde end groups into glycitol/sorbitol may be prepared. The conversion from isomaltooligosaccharide to hydrogenated isomaltooligosaccharide may be performed by treating the isomaltooligosaccharide with a reducing agent, such as e.g. borohydride under alkaline conditions as shown in below Reaction scheme 4:

Reaction scheme 4

Reaction scheme 4

With the processes described above for making derivatives of dextran, carboxyalkyl derivatives of isomaltooligosaccharide may also be made by reaction with for example monochloroacetic acid (MCA) for synthesising carboxymethyl isomaltooligosaccharide. To skilled person it will be obvious to make further derivatives of hydrogenated

isomaltooligosaccharide and hydrogenated dextan by reaction with respectively monochloroacetic acid as described above under dextran and isomaltooligosaccharide. Oligo-isomaltose

With d extra ns as starting materials and Dextran 1 (isomaltooligosaccharide) as intermediary, it is possible to synthesise oligo-isomaltose, characterized by a complete lack of branching side-chains a-(l→2), a-( l→3) and a-(l→4) defining the dextran molecules. In the present context oligoisomaltose is considered as a subset of isomaltooligosaccharide. Thus, in one aspect, oligoisomaltose has a weight average molecular weight (Mw) of between 300 and 1,650 Da such as between 850 and 1,650 Da, preferably between 850 and 1,150 Da, and having a complete lack of branching side-chains a-(l→2), a-(l→3) and a-(l→4). Obviously the same synthesis of derivatives described above for isomaltooligosaccharide can be performed using oligo-isomaltose.

Cryopreserva tion

In order to avoid contamination of the sample to be cryopreserved it is preferred that the cryoprotectant is sterile, and that other optional components of the cryopreservation agent/composition also are sterile. In some applications it might be useful to supplement a cryoprotectant as described herein such as carboxyalkyldextran cryoprotectant with an additional cryoprotectant in order to reduce the concentration of such additional cryoprotectant, preferably to non-toxic concentrations. This may be particular useful for specific cell types, like hepatocytes or pluripotent stem cells. In one aspect, the cryoprotective agent according to the invention may comprise at least one additional cryoprotectant selected from the group consisting of acetamide, agarose, alginate, 1-analine, albumin such as human serum albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform, choline, diethylene glycol, dimethyl acetamide, dimethyl formamide, dimethyl sulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, a-glycerophosphate, glycerol monoacetate, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methyl acetamide, methylformamide, methyl ureas, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, pyridine N-oxide, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium sulfate, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, urea, valine and xyiosen. Most suitably, said additional cryoprotectant is ethylene glycol. In one aspect, said additional cryoprotectant is DMSO. In one aspect, an additional cryoprotectant such as ehtylene glycol is present in said agent in an amount from 5 % to 40 % w/w, such as from 5 % to 30 % w/w. In another aspect, the cryoprotective agent may be substantially free of DMSO, e.g. free of DMSO. The cryoprotective agent according to the invention may comprise a cryoprotectant as described herein such as carboxyalkyldextran as the only cryoprotectant. An advantage of adding DMSO in a reduced amount may be that for very fragile cells an additional protection may be obtained. In a preferred aspect, the cryoprotective agent is free of or substantially free of DMSO. Thus, in yet a preferred aspect, said cryoprotectant as described herein such as ca rboxya I ky I dextra n is the only cryoprotectant in the cryoprotective agent. A

cryoprotective agent free or substantially free of DMSO may not require washing after thawing of the sample. The thawed sample may then be directly suspended in a culture medium to immediately start a culturing process without having to wash the sample or may be directly used in a patient without a washing step that potentially leads to substantial cell loss. Another advantage using a cryoprotective agent free of or substantially free of DMSO is that the sample may be exposed to the cryoprotectant for a longer period without damage, enabling a more efficient working process. In another aspect, the cryoprotective agent further comprises at least one anti-freeze protein and/or anti-freeze glycoprotein such as in an amount of from 0.01 to 1 mg/mL of the cryoprotective agent. An example of an anti-freeze glycoprotein is Type I AFP from longhorn sculpin, which is a single, long amphipathic alpha helix.

The cryoproteting agent or composition may comprise further substances for improving the viability of the sample. As examples of such substances mention can be made of IAPs

(inhibitors of apoptosis), inhibitors of the rho-associated protein kinase (ROCK) signaling pathways, growth factors such as EGF, FGF, PDGF, IGF, EPO, BDNF, TGF, TNF, VEGF. In a further aspect, mention can be made of any serum components of human, bovine, equine, canine origin. The cryoproteting agent or composition may also comprise a growth medium. In one aspect, a growth medium comprising -catenin/P300 antagonist and an Activin/TGFp ligand, such as for example ID-8 in conjunction with Activin and TGF3, may be used. This type of medium is especially useful for culturing of pluripotent stem cells, in particular embryonic stem cells as for example described in WO 2013/054112. Another example is the standard knock-out medium comprising KnockOut Serum Replacement, DMEM/F12 with GlutaMAX™ supplement, FGF, EAA and BME. Another example is the mTSER™ system.

Other examples of growth media depending on the sample to be cryopreserved is well known to the skilled person.

The cryoprotective agent as disclosed herein may be in the form of a powder such as a lyophilized or spray dried powder. In a further aspect, said cryoprotective agent is in the form of a solution. The agent may thus further comprise a solvent such as for example sterile water. In one aspect, said agent comprises from 30% to 70% w/w of said cryoprotectant, such as from 40% to 65% w/w or from 50% to 60% w/w of said cryoprotectant. In one aspect, said agent comprises from 5 % to 50 % w/w of said cryoprotectant, such as from 5 % to 40 % w/w, such as from 5 % to 30 % of said cryoprotectant.

The sample such as cells, tissue or organs to be cryopreserved can also be in contact with a freezing compatible pH buffer comprised most typically of at least a basic salt solution, an energy source (for example, glucose) and a buffer capable of maintaining a neutral pH at low temperatures. Well known materials include, for example, Dulbecco's Modified Eagle Medium (DMEM) . This material may also be included as part of the cryopreservation composition and/or agent.

One aspect disclosed herein is a cryopreservation composition comprising a cryoprotective agent as described herein, which cryopreservation composition further comprises a sample to be cryopreserved.

A further aspect disclosed herein is a cryopreserved composition comprising a cryoprotective agent and a sample which has been cryopreserved or is In the process of being

cryopreserved. As described herein the term, "a cryopreserved composition" means either "a cryopreservation composition" which is in the process of being cryopreserved or already has been cryopreserved.

A further aspect disclosed herein is a cryopreserved composition comprising a growth medium or substrate for the sample to be cryopreserved.

Depending on the sample (and growth media) to be cryopreserved, it may be necessary to adjust the pH of the cryopreservation composition for example adjusting the pH of the cryopreservation composition to a pH of between 6.5 and 8.5, such as between 6.5 and 7.8, preferably to a pH 7.0 - 7.4. Depending on the sample to be cryopreserved it may also be preferred to add a freezing compatible pH buffer comprised most typically of at least a basic salt solution, an energy source (for example, glucose) and a buffer capable of maintaining a neutral pH at low temperatures. In one aspect, the sample is selected from the group consisting of organs, cells, and tissues such as mammalian. In a further aspect, the sample is organs, cells, blood or tissues.

Examples of such cells to be cryopreserved are in-vitro-cultured cells including primary cells, cell lines, in vitro-sorted cells including human blood cells, and fertilized eggs of animal and human origin. Further examples are sperm cells, embryonic stem cells, iPS iPSCs, mesenchymal stem cells, haemopoietic stem cells, neuronal stem cells, umbilical cord blood stem cells, hepatocytes, nerve cells, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells and blood cells. In a further aspect, the sample is cells selected from the group consisting of mesencymal stem cells, hematopoietic stem cells, embryonic stem cells, iPS iPSCs, keratinocytes, preferable hematopoietic stem cells such as CD34 positive blood stem cells, mesenchymal stem cells embryonic stem cells and iPSCs. In a further aspect the sample is selected from the group consisting of mesenchymal stem cells and hematopoietic stem cells. In one aspect, the cell is of animal or human origin. Examples of organs are lung, liver, kidney, heart, ovaries and pancreas. Examples of tissues are tissues of bone marrow, skin, ovaries, testis, blood vessels, connective tissue, preferable tissues of ovaries and connective tissue. In a further aspect, the blood is selected from the group consisting of umbilical cord blood, and mobilized peripheral blood, preferable umbilical cord blood. In a further aspect, the sample is a cell-containing body fluid such as blood, menstrual fluid or amniotic fluid.

Depending on the specific sample to be cryoprotected, the cryoprotectant is typically present in the composition to be cryopreserved in an amount from 1 to 50 % w/w such as from 2 to 50 % w/w, such as from 4 to 45 % w/w, or from 6 to 20 % w/w, or from 6 to 12 % w/w, or preferably from 6 to 10% w/w, or more preferably from 7 to 9% w/w. In one aspect, the cryoprotectant is present in the composition to be cryopreserved in an amount of at the most 60% w/w, such as in an amount of at the most 55% w/w, such as in an amount of at the most 50% w/w, such as in an amount of at the most 45% w/w, such as in an amount of at the most 40% w/w, such as in an amount of at the most 35% w/w. In another aspect, the cryoprotectant is typically present in an amount of at the least 2% w/w, such as present in an amount of at the least 4% w/w, such as present in an amount of at the least 6% w/w, such as present in an amount of at the least 6% w/w, such as present in an amount of at the least 7% w/w. In one aspect, the cryoprotectant is typically present in the composition to be cryopreserved in an amount of less than 40 % w/w, less than 30 % w/w such as from 5 % to 50 % w/w, such as 5 % to 40 % w/w, such as from 5 % to 30 % w/w of said cryoprotectant.

In one aspect, an additional cryoprotectant such as ethylene glycol is typically present in the composition to be cryopreserved in an amount less than 40 % w/w, less than 30 % w/w such as from 1-25% w/w. In one aspect, an additional cryoprotectant such as ethylene glycol is typically present in the composition to be cryopreserved in an amount of 5 % to 30 % w/w, such as from 5 % to 25 % w/w of said additional cryoprotectant such as ethylene glycol. If the composition to be cryoprotected comprises an additional cryoprotectant such as DMSO the additional cryoprotectant typically is present in an amount of less than 8 % w/w, such as from 1-8% w/w, such as for example in an amount below 5% w/w, such as below 4 % w/w such as from 1-4% w/w.

In conventional cryopreservation techniques, a sample is harvested, suspended in a storage solution, and then preserved by freezing. When the sample such as cells are to be used, they are thawed, for example, cells taken from human donor sources are brought back to the normal human body temperature (i.e., approximately 37° C), and then placed in a cell culture medium.

In the present method of cryopreservation, the sample is protected during cryopreservation by being brought into contact with a cryoprotective agent as described herein prior to freezing to the cryopreservation temperature. By being brought into contact with the cryoprotective agent is meant that the sample is made to be in contact in some manner with the cryoprotectant so that during the reduction of temperature to the cryopreservation temperature, the sample is protected by the cryoprotectant in the cryopreservation composition. For example, the cells may be brought into contact with the cryoprotective agent by filling the appropriate wells of a plate to which the cells to be protected are attached, by suspending the cells In a solution of the cryoprotective agent or by adding the cryoprotective agent for example in freeze dried form to the cells, blood or organ already in a solution of for example buffer, or by resuspending the cell pellet after centrifugation In the cryoprotective agent bringing the cells into a solution etc. In addition the cells may be brought into contact with the cryoprotective agent by placing a cell-containing scaffold or biomaterial in the cryoprotective agent or by adding the cells in the cryoprotective solution to a scaffold/biomaterial. The organ or tissue may be brought into contact with the

cryoprotective agent by filling the transplant container with the cryoprotective solution.

In one aspect, disclosed herein is a method of cryopreserving a sample, comprising the protection steps of bringing a sample to be cryopreserved into contact with a cryoprotective agent as defined herein to obtain a cryopreservation composition and subsequently reducing the temperature of the cryopreservation composition to a cryopreservation temperature.

In a further aspect, a method of cryopreserving a composition as defined herein by reducing the temperature of said composition to a cryopreservation temperature is disclosed herein.

The rate of change from room temperature to 1-2 °C below the freezing point of the solution may have a major effect on ultimate viability if the cells are sensitive to thermal shock. Between 3.5 °C and -5 °C, the sample Is normally induced to freeze either by the

introduction of an ice crystal, by touching the surface of the media with a cold probe, by mechanical vibration, or by rapidly lowering the temperature until ice nucleation occurs. Since freezing is an exothermic process, heat must be conducted away from the freezing solution. This may be done either by keeping the samples immersed in a liquid with a low freezing point or by providing a substantial heat sink. As ice forms in the extracellular media, more and more free water becomes bound in the ice phase. Cell membranes, being hydrophobic, act as a barrier for the nucleation of intracellular ice and therefore unfrozen cells are exposed to an increasingly hypertonic solution. The extracellular salt concentration increases as a consequence of water sequestration into ice. The unfrozen cells shrink due to the transport of water out of the cell in response to the osmotic imbalance between the intracellular and extracellular fluid phases. The sample is then cooled at a finite rate which must be optimized for each cell type.

The optimal rate of cooling is determined by the permeability of the cell membrane to water, the surface-to-volume ratio of the cell, along the type and concentration of cryoprotective additives in the cryoprotective agent as described herein. For most nucleated mammalian cells frozen in glycerol or DMSO, the optimal cooling rate usually is between about 0.3 to 10 °C per minute. Continuous cooling between about 4 °C and -80 °C is the most commonly used. Once the sample reaches approximately -80 °C, it can be transferred directly into liquid nitrogen (-196 °C) or into the vapor phase of liquid nitrogen for storage. Another method used for cryopreservation is the vitrification technology in which it is possible to obtain very fast cooling rates of 1000 °C - 2000 °C/min. With this technology a specialized vitrification device, containing the cryopreservation composition with the sample, is directly placed into liquid nitrogen. In one aspect, the cryopreservation temperature is reached at a rate of 0.05- 15, such as 0.1-10, such as 0.2-8, such as 0.3-6, such as 0.4-4, such as 0.5-2°C per minute. In another aspect the cryopreservation temperature is reached at a rate of 500-3000, such as 800-2500, such as 1000-2000, such as 1200-1800 °C per minute.

The duration of viable cell storage at liquid nitrogen temperature is dependent predominantly on the rate of generation of free radicals caused by the cosmic ray background.

For example, the half-life for mammalian embryos stored in liquid nitrogen has been estimated to be approximately 30,000 years. It is important not to allow frozen cells to warm above their storage temperature for even brief periods of time. Intermittent warming promotes rapid migratory recrystallization, which can damage cellular structure and decrease overall viability.

In yet a further aspect, the sample are thawed after cryopreservation. The optimal rate of thawing of the sample is dependent on the freezing conditions used and the specific sample to be preserved. In general, for single cells frozen in suspension, and for tissues such as heart valves, a rapid rate of warming is desirable. Such rapid warming limits the growth of ice crystals in the frozen samples and is often an absolute requirement for high survival. With many tissues this warming can be accomplished by agitating the sample in a 37-42 °C water bath. The rationale for rapid warming is that it limits the growth of ice crystals which were formed during cooling.

Some tissues may be sensitive to rapid warming. This is due to transient osmotic shock, because the cells are exposed to an extracellular hypertonic solution as the ice melts and are forced to rehydrate in order to maintain their osmotic equilibrium. For other, more sensitive, samples, metabolic processes can be reactivated or brought up to normal levels by successive dilutions using serum or other high molecular weight polymers in the thawing medium.

Upon completion of the thawing procedure, the cells are still exposed to multimolar concentrations of cryoprotective agents which must be gradually diluted to return the cells to an isotonic media. For mammalian cells, a stepwise dilution protocol is typically used. The dilution of the sample is normally carried out at preferably 37 °C, so as to reduce the effects of both osmotic shock and cryoprotectant toxicity. In a further aspect, the concentration of said cryoprotectant is from 4 to 45 % w/w, such as from 8 to 35 % w/w, such as from 10 to 25 % w/w, or from 12 to 23 % w/w, or preferably from 12 to 22 % w/w, or more preferably from 15 to 21 % w/w cryoprotectant.

In a further aspect, the temperature of the sample in the cryopreservation composition is reduced to a cryopreservation temperature below -50°C, such as between -50°C to - 196°C, such as between -80°C to - 196°C.

In one aspect, the cryoprotective agent is used in a banking method. In one aspect, the cryoprotective agent is used in a clinical banking method. In one aspect, the cryoprotective agent is used in a mobilized peripheral blood banking method. In one aspect, the cryoprotective agent is used in a clinical banking method such as in stem cell transplantation for malignant diseases or in an organ transplantation. In one aspect, the cryoprotective agent is used in a mobilized peripheral blood banking method, marrow banking method or in an umbilical cord banking method.

In one aspect, the cryoprotective agent is used in a marrow banking method or in an umbilical cord banking method. In one aspect, the cryoprotective agent is used in a fat tissue banking method or in a dental pulp tissue banking method. In a further aspect, the cryoprotective agent is used in a reproduction banking method.

FURTHER EMBODIMENTS ACCORDING TO THE INVENTION :

Embodiment 1. Use of a cryoprotectant selected from the group consisting of ca rboxya I ky I dextra n , hydrogenated carboxyalkyldextran, oxidated carboxyalkyldextran, ether of carboxyalkyldextran, ester of carboxyalkyldextran and partly oxidated/hydrogenated ca rboxya I ky Idextran or a combination thereof for cryopreserving a sample, and wherein said sample is selected from the group consisting of organs, cells, and tissues. Embodiment 2. The use according to embodiment 1, wherein the cryoprotectant is hydrogenated carboxyalkyldextran, such as hydrogenated

Embodiment 3. The use according to embodiment 1, wherein the cryoprotectant is carboxyalkyldextran.

Embodiment 4. The use according to any one of embodiments 1-3, wherein the carboxyalkyldextran is carboxyCi_i ₀ alkyldextran.

Embodiment 5. The use according to any one of embodiments 1-4, wherein the a Iky I is C _1-10 alkyl.

Embodiment 6. The use according to any one of embodiments 1-5, wherein the

Q ₀ alky! is methyl. Embodiment 7. Use of a cryoprotectant selected from the group consisting of carboxyalkyldextran, hydrogenated carboxyalkyldextran, oxidated carboxyalkyldextran, ether of carboxyalkyldextran, ester of carboxyalkyldextran and partly oxidated/hydrogenated ca rboxya I ky Idextra n or a combination thereof, wherein the a Iky I is methyl, for cryopreserving a sample, and wherein said sample is selected from the group consisting of organs, cells, and tissues.

Embodiment 8. The use according to any one of embodiments 1-7, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 900,000 Da such as of between 300 and 700,000 Da. Embodiment 9. The use according to any one of embodiments 1-8, wherein the cryoprotectant has a weight average molecular weight (M _w ) of above 700,000 Da such as between 700.000 and 900,000 Da.

Embodiment 10. The use according to any one of embodiments 1-9, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 500,000 Da.

Embodiment 11. The use according to any one of embodiments 1-10, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 15,000 Da.

Embodiment 12. The use according to any one of embodiments 1-11, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 9,500 Da. Embodiment 13. The use according to any one of embodiments 1-12, wherein the carboxyalkyldextran has a weight average molecular weight (M _w ) of between 300 and 700,000 Da such as between 300 and 500,000 Da.

Embodiment 14. The use according to any one of embodiments 1-13, wherein the carboxyalkyldextran has a weight average molecular weight (M _w ) of between 300 and 9,500 Da.

Embodiment 15. The use according to any one of embodiments 1- 14, wherein the carboxyalkyldextran has a weight average molecular weight (M _w ) of between 1000 and 9,500Da.

Embodiment 16. Use of a cryoprotectant selected from the group consisting of carboxyalkyldextran, hydrogenated carboxyalkyldextran, oxidated carboxyalkyldextran, ether of carboxyalkyldextran, ester of ca rboxya Iky Idextra n and partly oxidated/hydrogenated carboxyalkyldextran or a combination thereof, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 9,500 Da, and wherein the alkyl is C,. i ₀ alkyl, such as methyl, for cryopreserving a sample, and wherein said sample is selected from the group consisting of organs, cells, and tissues. Embodiment 17. The use according to any one of embodiments 1-16, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 1000 and 9,500Da. Embodiment 18. The use according to any one of embodiments 1-17, wherein the cryoprotectant is carboxymethyl isomaltooligosaccharide having a weight average molecular weight of 850-1, 150 Da. Embodiment 19. The use according to any one of embodiments 1-18, wherein the cryoprotectant is hydrogenated or oxidated carboxyalkyldextran such as hydrogenated carboxymethyl isomaltooligosaccharide having a weight average molecular weight of 850- 1,150 Da. Embodiment 20. The use according to any one of embodiments 1-19, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 15,000 Da such as approximately 6000 Da.

Embodiment 21. The use according to any one of embodiments 1-20, wherein the cryoprotectant is hydrogenated ca rboxya Iky Idextra n such as hydrogenated

carboxymethyldextran having a weight average molecular weight of 300-15,000 Da.

Embodiment 22. The use according to any one of embodiments 1-21, wherein the cryoprotectant has a content of carboxy groups of above 1 % w/w, such as above 2 % w/w, such as between 1- 15 % w/w, or such as between 5-12 % w/w determined by direct titration using for example sodium hydroxide.

Embodiment 23. The use according to any one of embodiments 1-22, wherein the cryoprotectant has a content of carboxy groups between 1- 7 % w/w determined by direct titration using for example sodium hydroxide.

Embodiment 24. The use according to any one of embodiments 1-23, wherein the cryoprotectant such as carboxyalkyldextran has a content of carboxy groups of above 3 % w/w, such as above 4 % w/w, such as for example 4-7 % w/w determined by direct titration using for example sodium hydroxide.

Embodiment 25. The use according to any one of embodiments 1-24, wherein the cryoprotectant such as carboxyalkyldextran is prepared by using hydrogenated d extra n or hydrogenated isomaltooligosaccharide or the corresponding polysaccharide having a weight average molecular weight (M _w ) of between 300 Da to 900,000 Da such as between 1000 and 900,000 as a starting material. Embodiment 26. The use according to any one of embodiments 1-25, wherein the starting material has a weight average molecular weight (M _w ) of between 1000 and 7000 Da, such as between 1650 and 6000 Da, such as between 2000 and 5000 Da.

Embodiment 27. The use according to any one of embodiments 1-26, wherein said starting material has a polydispersity of > 1 and < 5.

Embodiment 28. The use according to any one of embodiments 1-27, wherein said sample is selected from the group consisting of mammalian organs, mammalian cells, and mammalian tissues.

Embodiment 29. The use according to any one of embodiments 1-28, wherein said sample is cells.

Embodiment 30. The use according to any one of embodiments 1-29, wherein said sample is non-mammalian eukaryotes, bacteria, archea.

Embodiment 31. The use according to any one of embodiments 1-30, wherein said sample is mammalian cells.

Embodiment 32. The use according to any one of embodiments 1-31, wherein said mammalian cells are selected from the group consisting of somatic cells, including all kind of tissue derived cells such as mesenchymal stem cells, tissue-specific progenitor cells, keratinocytes, fibroblasts, chondrocytes, bone cells, or cardiomyocytes; blood derived cells such as hematopoietic stem cells, macrophages, plates, erythrocytes; or stem cells, including all types of pluripotent cells, totipotent cells and unipotent cells, and germ layer cells.

Embodiment 33. The use according to any one of embodiments 1-32, wherein said mammalian cells are selected from the group consisting of mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells.

Embodiment 34. The use according to any one of embodiments 1-33, wherein said mammalian cells are selected from the group consisting of mesenchymal stem cells such as induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC).

Embodiment 35. The use according to any one of embodiments 1-34, wherein said sample is mammalian tissue such as ovarian tissue. Embodiment 36. The use according to any one of embodiments 1-35, wherein said sample is a mammalian organ such as ovaries.

Embodiment 37. The use according to any one of embodiments 1-36, wherein said cryoprotectant such as carboxyalkyldextran is used in combination with another

cryoprotectant such as DMSO or ethylene glycol.

Embodiment 38. The use according to any one of embodiments 1-37, where said cryoprotectant such as carboxyalkyldextran is used for cryopreserving a sample in a cryopreservation composition having a pH of between 6.5 and 8.5, such as between 6.5 and 7.8.

Embodiment 39. The use according to any one of embodiments 1-38, where said use further comprises using any serum component of human, bovine, equine, or canine origin in the cryopreservation composition.

Embodiment 40. The use according to any one of embodiments 1-39, where said use further comprises using albumin.

Embodiment 41. The use according to any one of embodiments 1-40, where said use further comprises using human serum albumin.

Embodiment 42. The use according to any one of embodiments 1-41, wherein said use comprises further using ethylene glycol and human serum albumin .

Embodiment 43. The use according to any one of embodiments 1-42, where said use further comprises using heparin .

Embodiment 44. The use according to any one of embodiments 1-43, where said use further comprises using said cryoprotectant in an aqueous solution .

Embodiment 45. A cryoprotective agent comprising a cryoprotectant selected from the group consisting of carboxyalkyldextran, hydrogenated carboxyalkyldextran, oxidated carboxyalkyldextran, ether of carboxyalkyldextran, ester of carboxyalkyldextran and partly oxidated/hydrogenated carboxyalkyldextran or a combination thereof.

Embodiment 46. The cryoprotective agent according to embodiment 45, wherein the cryoprotectant is carboxyalkyldextran or hydrogenated carboxyalkyldextran. Embodiment 47. The cryoprotective agent according to embodiment 45, wherein the cryoprotectant is hydrogenated carboxyalkyldextran.

Embodiment 48. A cryoprotective agent comprising carboxyalkyldextran.

Embodiment 49. The cryoprotective agent according to any one of embodiment 45-

48, wherein the carboxyalkyldextran is carboxyC _! - ₁₀ a iky Idextra n.

Embodiment 50. The cryoprotective agent according to embodiment 49, wherein the d- _! oalkyl is methyl.

Embodiment 51. The cryoprotective agent according to any one of embodiments

45-49, wherein the carboxyalkyldextran is carboxyCi„ ₁₀ alkyldextran. Embodiment 52. The cryoprotective agent according to any one of embodiments

45-48, wherein the alkyl is Ci-ioalkyl.

Embodiment 53. The cryoprotective agent according to any one of embodiments

45-52, wherein the Q ₁₀ alkyl is methyl.

Embodiment 54. A cryoprotective agent selected from the group consisting of ca rboxya I ky Idextra n , hydrogenated carboxyalkyldextran, oxidated carboxyalkyldextran, ether of carboxyalkyldextran, ester of carboxyalkyldextran and partly oxidated/hydrogenated carboxyalkyldextran or a combination thereof, wherein the alkyl is methyl.

Embodiment 55. The cryoprotective agent according to any one of embodiments

45-54, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 900,000 Da such as of between 300 and 700,000 Da.

Embodiment 56. The cryoprotective agent according to any one of embodiments

45-55, wherein the cryoprotectant has a weight average molecular weight (M _w ) of above 700,000 Da such as between 700.000 and 900,000 Da.

Embodiment 57. The cryoprotective agent according to any one of embodiments

45-56, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 500,000 Da. Embodiment 58. The cryoprotective agent according to any one of embodiments

45-57, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 300 and 9,500 Da. Embodiment 59. The cryoprotective agent according to any one of embodiments

45-58, wherein the cryoprotectant such as carboxyalkyldextran has a weight average molecular weight (M _w ) of between 300 and 900,000 Da such as of between 300 and 700,000 Da. Embodiment 60. The cryoprotective agent according to any one of embodiments

45-59, wherein the cryoprotectant such as carboxyalkyldextran has a weight average molecular weight (M _w ) of between 300 and 500,000 Da.

Embodiment 61. The cryoprotective agent according to any one of embodiments 45-60, wherein the cryoprotectant has a weight average molecular weight (Mw) of between 300 and 15,000 Da.

Embodiment 62. The cryoprotective agent according to any one of embodiments

45-61, wherein the cryoprotectant such as carboxyalkyldextran has a weight average molecular weight (M _w ) of between 1000 and 9,500Da.

Embodiment 63. A cryoprotective agent comprising a cryoprotectant selected from the group consisting of carboxyalkyldextran, hydrogenated carboxyalkyldextran, oxidated carboxyalkyldextran, ether of carboxyalkyldextran, ester of carboxyalkyldextran and partly oxidated/hydrogenated carboxyalkyldextran or a combination thereof, wherein the cryoprotecta t has a weight average molecular weight (M _w ) of between 300 and 9,500 Da, wherein the a Iky I is C _1-10 alkyl, such as methyl.

Embodiment 64. The cryoprotective agent according to any one of embodiments 45-63, wherein the cryoprotectant has a weight average molecular weight (M _w ) of between 1000 and 9,500Da.

Embodiment 65. The cryoprotective agent according to any one of embodiments

45-64, wherein the cryoprotectant is carboxymethyl isomaltooligosaccharide having a weight average molecular weight of 850- 1,150 Da.

Embodiment 66. The cryoprotective agent according to any one of embodiments

45-65, wherein the cryoprotectant is hydrogenated or oxidated carboxyalkyldextran such as hydrogenated carboxymethyl isomaltooligosaccharide having a weight average molecular weight of 850-1,150 Da.

Embodiment 67. The cryoprotective agent according to any one of embodiments 45-66, wherein said cryoprotectant is prepared by using hydrogenated dextran or hydrogenated isomaltooligosaccharide or the corresponding polysaccharide having a weight average molecular weight (M _w ) of between 1000 Da to 900,000 Da such as between 1000 to 700,000 as a starting material. Embodiment 68. The cryoprotective agent according to embodiment 67, wherein the starting material has a weight average molecular weight (M _w ) of between 1000 and 7000 Da, such as between 1650 and 6000 Da, such as between 2000 and 5000 Da.

Embodiment 69. The cryoprotective agent according to any one of embodiments 45-68, wherein said starting material has a polydispersity of > 1 and < 5.

Embodiment 70. The cryoprotective agent according to any one of embodiments

45-69, wherein the cryoprotectant has a content of carboxy groups of above 1 % w/w, such as above 2 % w/w determined by direct titration using for example sodium hydroxide.

Embodiment 71. The cryoprotective agent according to any one of embodiments

45-70, wherein the cryoprotectant has a content of carboxy groups between 1- 7 % w/w determined by direct titration using for example sodium hydroxide. Embodiment 72. The cryoprotective agent according to any one of embodiments

45-71, wherein the cryoprotectant has a content of carboxy groups between 1- 15 % w/w, such as between 5-12 % w/w determined by direct titration.

Embodiment 73. The cryoprotective agent according to any one of embodiments 45-72, wherein the cryoprotectant such as carboxyalkyldextran has a content of carboxy groups of above 3 % w/w, such as above 4 % w/w, such as for example between 4-7% w/w determined by direct titration using for example sodium hydroxide.

Embodiment 74. The cryoprotective agent according to any one of embodiments 45-73 for cryopreserving a sample, and wherein said sample is selected from the group consisting of organs, cells, and tissue such as selected from the group consisting of mammalian organs, mammalian cells, and mammalian tissues. Embodiment 75. The cryoprotective agent according to any one of embodiments

45-74 for cryopreserving a sample, wherein said sample is for transplantation.

Embodiment 76. The cryoprotective agent according to any one of embodiments 45-75 for cryopreserving a sample, wherein said sample is functional after cryopresevation .

Embodiment 77. The cryoprotective agent to any one of embodiments 45-76, wherein said sample is an organ, which organ is functional as measured by physiological function of said organ after cryopreservation, and/or wherein said sample is tissue, which tissue is functional as measured by ability of such tissue to integrate with surrounding tissue and/or wherein said sample is cells, which cells are functional as measured by viability of said cells after cryopreservation.

Embodiment 78. The cryoprotective agent according to any one of embodiments 45-78, comprising at least one additional cryoprotectant selected from the group consisting of acetamide, agarose, alginate, 1-analine, albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform, choline, diethylene glycol, dimethyl acetamide, dimethyl formamide, dimethyl sulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, a-glycerophosphate, glycerol monoacetate, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methyl acetamide, methylformamide, methyl ureas, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, pyridine N-oxide, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium sulfate, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, urea, valine and xylosen.

Embodiment 79. The cryoprotective agent according to embodiment 78, wherein said additional cryoprotectant is ethylene glycol. Embodiment 80. The cryoprotective agent according to embodiment 78, wherein said additional cryoprotectant is DMSO.

Embodiment 81. The cryoprotective agent according to any one of embodiments

45-80, which is substantially free of DMSO.

Embodiment 82. The cryoprotective agent according to embodiment 81, which is free of DMSO. Embodiment 83. The cryoprotective agent according to any one of embodiments

45-82 comprising a cryoprotectant as defined in embodiment 1 as the only cryoprotectant.

Embodiment 84. The cryoprotective agent according to any one of embodiments 45-83, wherein said agent is in the form of a powder.

Embodiment 85. The cryoprotective agent according to any one of embodiments

45-84, wherein said agent is in the form of a lyophilized or spray dried powder. Embodiment 86. The cryoprotective agent according to any one of embodiments

45-85, wherein said agent is in the form of a solution.

Embodiment 87. The cryoprotective agent according to any one of embodiments

45-86, wherein said agent comprises from 30 % to 70 % w/w of said cryoprotectant, such as from 40 % to 65 % w/w or from 50 % to 60 % w/w of said cryoprotectant.

Embodiment 88. The cryoprotective agent according to any one of embodiments

45-87, wherein said agent comprises from 30 % to 70 % w/w of said additional

cryoprotectant, such as from 40 % to 65 % w/w or from 50 % to 60 % w/w of said additional cryoprotectant.

Embodiment 89. The cryoprotective agent according to any one of embodiments

45-88, wherein said agent comprises from 5 % to 50 % w/w, such as 5 % to 40 % w/w, such as from 5 % to 30 % w/w of said cryoprotectant.

Embodiment 90. The cryoprotective agent according to any one of embodiments

45-89, wherein said agent comprises from 5 % to 30 % w/w, such as from 5 % to 25 % w/w of said additional cryoprotectant such as ethylene glycol. Embodiment 91. The cryoprotective agent according to any one of embodiments

45-90 further comprising a growth medium or substrate for a sample to be cryopreserved.

Embodiment 92. The cryoprotective agent according to any one of embodiments

45-91 further comprising any proteins belonging to the IAPs (Inhibitors of apoptosis), inhibitors of the rho-associated protein kinase (ROCK) signaling pathways, and/or any growth factor such as EGF, FGF, PDGF, IGF, EPO, BDNF, TGF, TNF and/or VEGF.

Embodiment 93. The cryoprotective agent according to any one of embodiments

45-92 further comprising any serum component of human, bovine, equine, or canine origin. Embodiment 94. The cryoprotective agent according to any one of embodiments

45-93 comprising albumin in a concentration of less than 40 % (w/w) .

Embodiment 95. The cryoprotective agent according to any one of embodiments

45-94 comprising human serum albumin in a concentration of less than 40 % (w/w).

Embodiment 96. The cryoprotective agent according to any one of embodiments

45-95 comprising heparin in a concentration of less than 2 % (w/w) .

Embodiment 97. The cryoprotective agent according to any one of embodiments

45-96, wherein said cryoprotectant is sterile.

Embodiment 98. The cryoprotective agent according to any one of embodiments

45-97, which is in the form of an aqueous solution .

Embodiment 99. A cryopreservation composition comprising a cryoprotective agent as defined in any one of embodiments 45-98, said cryopreservation composition further comprising a sample to be cryopreserved.

Embodiment 100. The cryopreservation composition according to embodiment 99, wherein the sample is selected from the group consisting of organs, cells such as isolated cells or cell-containing bodily fluids for example blood, and tissues.

Embodiment 101. The cryopreservation composition according to embodiment 100, wherein the sample is selected from the group consisting of mammalian organs, mammalian cells, and mammalian tissues, such as a sample selected from the group consisting of mammalian organs, mammalian cells, and mammalian tissues for transplantation.

Embodiment 102. The cryopreservation composition according to any one of embodiments 99-101, wherein said sample is cells selected from the group consisting of somatic cells, including all kind of tissue derived cells such as mesenchymal stem cells, tissue-specific progenitor cells, keratinocytes, fibroblasts, chondrocytes, bone cells, or cardiomyocytes, blood derived cells such as hematopoietic stem cells, macrophages, plates, erythrocytes, or stem cells, including all types of pluripotent cells, totipotent cells and unipotent cells, and germ layer cells. Embodiment 103. The cryopreservation composition according to any one of embodiments 99-102, wherein said sample is cells selected from the group consisting of keratinocytes, fibroblasts, mesenchymal stem cells, macrophages, and hematopoietic stem cells such as CD34 positive blood stem cells. Embodiment 104. The cryopreservation composition according to any one of embodiments 99-103, wherein said sample is tissue selected from the group consisting of ovarian tissue, testicular tissue, umbilical cord tissue, placental tissue, connective tissue, cardiac tissue, tissue from muscle, bone, and cartilage tissue, endocrine tissue, and neural tissue. Embodiment 105. The cryopreservation composition according to any one of embodiments 99- 104, wherein said sample is a cell-containing bodily fluid selected from the group consisting of blood such as umbilical cord blood, peripheral blood, and mobilized peripheral blood, amniotic fluid, semen, cerebrospinal fluid, menstrual fluid blood, and bone marrow aspirates. Embodiment 106. The cryopreservation composition according to any one of embodiments 99-105, wherein said sample is an organ selected from the group consisting of lung, heart, kidney, liver, umbilical cord and ovaries.

Embodiment 107. The cryopreservation composition according to any one of embodiments 99-106, wherein said mammalian cells are selected from the group consisting of mesenchymal stem cells, hematopoietic stem cells, pluripotent stem cells.

Embodiment 108. The cryopreservation composition according to any one of embodiments 99- 107, wherein said mammalian cells is selected from the group consisting of mesenchymal stem cells such as induced pluripotent stem cells (iPSC) and embryonic stem cells (ESC).

Embodiment 109. The cryopreservation composition according to any one of embodiments 99- 108, wherein said sample is mammalian tissue such as ovarian tissue. Embodiment 110. The cryopreservation composition according to any one of embodiments 99- 109, wherein said sample is a mammalian organ such as ovaries.

Embodiment 111. The cryopreservation composition according to any one of embodiments 99-111 comprising said cryoprotectant in an amount from 1 to 50 % w/w such as from 2 to 50 % w/w, or from 4 to 45 % w/w, or from 6 to 12 % w/w, or preferably from 6 to 10 % w/w, or more preferably from 7 to 9 % w/w.

Embodiment 112. The cryopreservation composition according to any one of embodiments 99-111, comprising said cryoprotectant in an amount of less than 40 % w/w, less than 30 % w/w such as from 5 % to 50 % w/w, such as 5 % to 40 % w/w, such as from 5 % to 30 % w/w of said cryoprotectant.

Embodiment 113. The cryopreservation composition according to any one of embodiments 99-112, wherein said composition comprises an additional cryoprotectant in an amount of less than 40 % w/w, less than 30 % w/w such as from 1-25% w/w, or such as from 5 % to 30 % w/w, or such as from 5 % to 25 % w/w.

Embodiment 114. The cryopreservation composition according to any one of embodiments 99-113, wherein said composition comprises DMSO in an amount of less than 20 % w/w, such as from 1-15 % w/w.

Embodiment 115. The cryopreservation composition according to any one of embodiments 99-114, wherein said composition comprises an additional cryoprotectant such as ethylene glycol in an amount of 40 % w/w, such as less than 40 % w/w, less than 30 % w/w such as from 1-25 % w/w.

Embodiment 116. The cryopreservation composition according to any one of embodiments 99-115, wherein said sample is functional after cryopreservation.

Embodiment 117. The cryopreservation composition according to any one of embodiments 99-116, wherein said composition comprises DMSO in an amount of less than 20 % w/w, less than 15 % w/w such as from 1-12 % w/w. Embodiment 118. The cryopreservation composition according to any one of embodiments 99-117, wherein said composition comprises ethylene glycol in an amount of less than 40 % w/w, less than 30 % w/w such as from 1-25 % w/w.

Embodiment 119. The cryopreservation composition according to any one of embodiments 99-118, wherein said composition comprises albumin in a concentration of less than 40 % (w/w). Embodiment 120. The cryopreservation composition according to any one of embodiments 99-119, wherein said composition comprises human serum albumin in a concentration of less than 40 % (w/w). Embodiment 121. The cryopreservation composition according to any one of embodiments 99-120, wherein said composition comprises heparin in a concentration of less than 2 % (w/w).

Embodiment 122. The cryopreservation composition according to any one of embodiments 99-121, wherein said composition comprises is sterile.

Embodiment 123. The cryopreservation composition according to any one of embodiments 99-122, which is in the form of an aqueous solution. Embodiment 124. The cryopreservation composition according to any one of embodiments 99-123 having a pH of between 6.5 and 8.5, such as between 6.5 and 7.8, preferably at a pH of between 7.0 and 7.4.

Embodiment 125. A method of cryopreserving a sample, comprising bringing a sample to be cryopreserved into contact with a cryoprotective agent as defined in any one of embodiments 45-98 to obtain a cryopreservation composition and subsequently reducing the temperature of the cryopreservation composition to a cryopreservation temperature.

Embodiment 126. A method of cryopreserving a composition as defined in any one of embodiments 99-124 by reducing the temperature of said composition to a

cryopreservation temperature.

Embodiment 127. The method according to any one of embodiments 125-126, wherein the cryopreservation temperature is reached at a rate of 0.05-15, such as 0.1-10, such as 0.2-8, such as 0.3-6, such as 0.4-4, such as 0.5-2 °C per minute.

Embodiment 128. The method according to any one of embodiments 125-127, wherein the concentration of said cryoprotectant is from 4 to 20 % w/w such as from 5 to 15 % w/w, or from 6 to 12 % w/w, or preferably from 6 to 10 % w/w, or more preferably from 7 to 9 % w/w.

Embodiment 129. The method according to any one of embodiments 125-128, wherein the temperature of the sample in the cryopreservation composition is reduced to a temperature below - 50°C, such as between - 50°C to - 196°C, such as -80°C to -196°C. Embodiment 130. The method according to any one of embodiments 125-129, wherein the sample is thawed after cryopreservation. Embodiment 131. The method according to any one of embodiments 125-130, wherein said sample is functional after cryopreservation.

Embodiment 132. The method according to any one of embodiments 125-131, wherein the sample to be cryopreserved is selected from the group consisting of organs, cells, and tissues, such as mammalian organs, mammalian cells, and mammalian tissues, preferably mammalian cells.

Embodiment 133. The method according to any one of embodiments 125-132, which is in a clinical banking method.

Embodiment 134. The method according to any one of embodiments 125-133, which is in a banking method such as a mobilized peripheral blood banking method, marrow banking method, a fat tissue banking method, a dental pulp tissue banking method, a reproduction banking method or in an umbilical cord banking method.

Embodiment 135. The method according to any one of embodiments 125-134, wherein said sample is brought into contact with the cryoprotective agent by placing a cell- containing scaffold or biomaterial in the cryoprotective agent or by adding the cells in the cryoprotective solution to a scaffold/biomaterial.

Embodiment 136. The method according to any one of embodiments 125-135, wherein said sample is brought into contact with the cryoprotective agent by placing a cell- containing scaffold or biomaterial in the cryoprotective agent or by adding the cells in the cryoprotective solution to a scaffold/biomaterial.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions, methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.

EXAMPLES

Preparation Example 1 Production of Isomaltooligosaccharide 1 Hydrolysis of low molecular Dextran

3345kg hydrolysed dextran collected as permeate from a membrane having a cut-off value <5,000 Daltons, is hydrolysed at pH 1.5 at a temperature of 95° C.

The hydrolysis is monitored chromatographically using gel permeation chromatography (GPC), and is terminated by cooling when the molecular weight of the material being hydrolysed is estimated to have achieved the desired value, i.e. a weight average molecular weight of 850-1, 150 Daltons.

By the hydrolysis low molecular weight isomaltooligosaccharide is produced but glucose is also formed. After cooling and neutralization the amount of glucose and very low molecular weight oligomers is reduced by membrane processes having a cut-off value of 340-800

Daltons. After this process, the content of isomaltooligosaccharide is determined by optical rotation (a _D 20 ~200) to be 915 kg, and the amount of reducing sugar is determined by use of Somogyi's reagent to be 22.5 % w/w.

Table 1 : Results from GPC analysis.

3 21.76 0.1282 504 65 0.0432 0.2103 106

4 28.3 0.1667 666 111 0.0425 0.2070 138

5 26.94 0.1587 828 131 0.0325 0.1585 131

6 22.58 0.1330 990 132 0.0228 0.1111 110

7 17.43 0.1027 1152 118 0.0151 0.0737 85

8 13.46 0.0793 1314 104 0.0102 0.0499 66

9 9.73 0.0573 1476 85 0.0066 0.0321 47

10 6.91 0.0407 1638 67 0.0042 0.0206 34

11 4.89 0.0288 1800 52 0.0027 0.0132 24

12 3.66 0.0216 1962 42 0.0019 0.0091 18

13 2.5 0.0147 2124 31 0.0012 0.0057 12

14 5.2 0.0306 2286 70 0.0023 0.0111 25

169.78 1.0000 1020 0.2053 1.0000 827

AUPW: Area under peak (M _w )

AUPN : Area under peak ( _n )

As seen from above Table 1 the Isomaltooligosaccharide has a MW of 1020 Da and Mn equals 827 Da giving a polydispersity Pd = 1.23. The reducing sugar is measured to be 22.5 % w/w. This isomaltooligosacharide is also named pentaisomaltose in this application.

Preparation Example 2

Production of hydrogenated Isomaltooligosaccharide 1 After hydrolysis and fractionation 418 kg isomaltooligosaccharide was left. The reducing sugar was measured to 30.8 % w/w. This amount was treated with 10 kg sodium

borohydride and gave as a result 362 kg hydrogenated isomaltooligosaccharide before final ion exchange. Hereafter the solution was neutralized to pH <7.0, and subsequently de- ionized and finally spray dried. The reducing sugar of the final product was measured to 0.09 % w/w.

Table 2 : Results from GPC analysis.

Number

of

glucose Relative Contribution Relative Contribution units AUPW AUPW Mi to w AUPN AUPN to M _JL ____

1 0.31 0.0033 180 1 0.0017 0.0145 3

2 4.38 0.0472 342 16 0.0128 0.1076 37

3 14.55 0.1568 504 79 0.0289 0.2425 122

4 16.47 0.1775 666 118 0.0247 0.2077 138

5 14.65 0.1579 828 131 0.0177 0.1486 123 6 11.57 0.1247 990 123 0.0117 0.0982 97

7 8.61 0.0928 1152 107 0.0075 0.0628 72

8 6.57 0.0708 1314 93 0.0050 0.0420 55

9 4.77 0.0514 1476 76 0.0032 0.0271 40

10 3.43 0.0370 1638 61 0.0021 0.0176 29

11 2.43 0.0262 1800 47 0.0014 0.0113 20

12 1.76 0.0190 1962 37 0.0009 0.0075 15

13 1.19 0.0128 2124 27 0.0006 0.0047 10

14 2.11 0.0227 2286 52 0.0009 0.0078 18

92.80 1.0000 968 0.1190 1.0000 780

AUPW: Area under peak (M _w )

AUPN : Area under peak (M _n )

As seen from above table 2 the hydrogenated isomaltooligosaccharide has a M _w of 968 Da and M _n equals 780 Da giving a polydispersity Pd = 1.24. This hydrogenated

isomaltooligosahharide is also named pentaisomaltoside in this applicaiton.

Preparation Example 3

32.5 kg hydrogenated dextran 5, containing less than 1.5 % w/w reducing end groups, is mixed with 30.4 kg of sodium hydroxide (27 % w/w) and 32.5 kg of water.

Hereafter 23.4 kg of sodium salt of chloroacetic acid are added while maintaining the temperature below 30 degrees Celsius.

The finished product, hydrogenated carboxymethyl dextran contains 6.6 % carboxylic acid groups and has a specific rotation, measured in a 1 % w/w solution, of 156°.

Preparation Example 4 32.5 kg hydrogenated dextran having the molecular weight as indicated in below table 4 or Hydrogenated Isomoltooligosaccharide 1, containing less than 1.5 % w/w reducing end groups, is mixed with 30.4 kg of sodium hydroxide (27% w/w) and 32.5 kg of water.

Hereafter 23.4 kg of sodium salt of chloroacetic acid are added while maintaining the temperature below 30 degrees Celsius. The finished product, hydrogenated carboxymethyl dextran contains the indicated degree of substitution.

Table 4:

* Degree of substitution calculated as w/w % -COOH after treatment with hydrochloric acid.

EXAMPLE 1

Preparation of cryopreservation agent

The cryopreservation agent used in the following examples were prepared by aseptically solubilizing/diluting the cryoprotectant (such as DMSO, human serum albumin (HSA), hydrogenated carboxymethyldextran, ethylene glycol) in growth medium (mTeSRl + penicillin/streptomycin) to the desired final concentration (e.g. DMSO 10 % w/w, ethylene glycol (EG) 20 % w/w, hydrogenated carboxymethyldextran 20 % w/w). Before use for cryopreservation of a sample, the cryopreservation composition was pH adjusted to pH 7.0 - 7.4 using a laboratory pH-meter.

EXAMPLE 2

Experimental design

The below experiments were performed using hydrogenated carboxymethyldextran (HCMD 5) produced from hydrogenated dextran 5 as described in preparation example 3 having an average weight average molecular weight (M _w ) of 5200 and containing 6.6 % w/w carboxy groups measured by direct titration with sodium hydroxide.

Induced pluripotent stem cells (IPSCs) were grown under standard conditions on matrigel coated surfaces in mTeSRl complete medium from Stemcell Technologies. Passage and harvesting of cells was done with an EDTA (0.2 % w/w) containing solution .

After harvest, cells were collected by spinning and resuspended directly into the

cryopreservation solutions.

1. mTeSR complete

2. mTeSR complete + 10 % w/w DMSO

3. mTeSR complete +20 % w/w ethylene glycol

4. mTeSR complete + 20 % w/w hydrogenated carboxymethyldextran (HCMD 5) (4g in 20 ml)

5. mTeSR complete + 10 % w/w DMSO + 20 % w/w hydrogenated

carboxymethyldextran (HCMD 5) (4g in 20 ml)

6. mTeSR complete + 20 % w/w ethylene glycol + 20 % w/w hydrogenated carboxymethyldextran (HCMD 5) (4g in 20 ml)

Cells were frozen in a Mr Frosty at -80°C, at a rate of 1 degree per minute.

Next day cells were transferred to nitrogen tank

Upon thawing, vials containing cells were incubated in a 37°C water bath until the cells were almost thawed. 5 ml growth medium were added and cells were collected by spinning.

Cells were counted and 200000 cells were plated on matrigel in mTeSRl medium.

On day 7 cells were harvested and counted.

Survival of iPS cells after freezing and thawing and number of cells after 7 days of proliferation is shown in Fig. 1 and Fig. 2. The results show high survival for hydrogenated carboxymethyldextran (HCMD 5) in combination with 10 % w/w DMSO or 20% w/w ethylene glycol, but also with 10 % w/w DMSO alone. 20 % w/w ethylene glycol alone does not perform as well, whereas HCMD 5 alone also has a fairly high degree of survival. Looking at the ability of the cells to adhere and start proliferating, it is clear that freezing with HCMD 5 in combination with either 10 % w/w DMSO or 20 % w/w ethylene glycol largely increases the proliferative ability of the cells. 10 % w/w DMSO alone results in a lower proliferating rate than expected. In conclusion, the results suggest that at least hydrogenated carboxymethyldextran HCMD 5 greatly enhances the ability of iPS cells, grown as colonies on matrigel in mTeS l medium, to survive cryopreservation and, more importantly, enhances their ability to proliferate afterwards.

EXAMPLE 3

The below experiments were performed using hydrogenated carboxymethyldextran as described in preparation example 4 having the indicated average weight average molecular weight (M _w ) of and containing the indicated substitution degree in w/w carboxy groups measured by direct titration with sodium hydroxide.

Human iPS cells were grown under standard conditions on matrigel coated surfaces in mtesrl complete medium from stemcell technologies (growth medium). Passage and harvesting of cells was done with an EDTA (0.2 % w/w) containing solution. After harvest, cells were collected by spinning and resuspended directly into the

cryopreservation solutions.

The following cryopreservation solutions were included in the study: mTeSRl

mTeSRl + 10 % w/w DMSO

mTeSRl + 20 % w/w Ethylene glycol

mTeSRl + 20 % w/w HCMD 5* (without acid treatment), degree of substitution 5.6 % w/w

mTeSRl + 20 % w/w HCMD 5** (with acid treatment), degree of substitution 6.6 % w/w

mTeSRl + 20 % w/w HCMD 1, degree of substitution 9.8 % w/w

mTeSRl + 20 % w/w HCMD 2.5, degree of substitution 9.4 % w/w Cells were freezed In a Mr Frosty™ Freezing Container (Thermo Scientific) at -80°C, with 1 degree per minutes.

Next day cells were transferred to liquid nitrogen tank.

Upon thawing, vials containing cells were incubated in a 37°C water bath until the cells were almost thawed. 5 ml growth medium were added and cells were collected by spinning.

Cells were counted and viability was evaluated using a Nucleocounter system. The results In Fig. 3 demonstrate that human iPS cells survive cryopreservation with HCMD 1, HCMD 2.5, and CMD 5 as the only cryoprotectant present. Although the survival rate is lower than the standard cryopreservation solution containing 10 % w/w DMSO it is significant higher than a cryopreservation solution containing another standard cryoprotectant, ethylene glycol. It is also demonstrated that HCMD 5 is the most efficient HCMD in protecting the cells from damage under the cryopreservation process.

EXAMPLE 4 iPS cells were treated as described in example 2 and the experimental and analytical conditions were also as described in example 1.

The following cryopreservation solutions were included in this study:

• mTeSRl

• mTeSRl + 10 % w/w DMSO

• mTeSRl + 20 % w/w Ethylene glycol (EG) + 20 % w/w HCMD 5* (without acid

treatment), degree of substitution 5.6 % w/w

• mTeSRl + 20 % w/w Ethylene glycol + 20 % w/w HCMD 5** (with acid treatment), degree of substitution 6.6 % w/w

• mTeSRl + 20 % w/w Ethylene glycol + 20 % w/w HCMD 1, degree of substitution 9.8 % w/w

· mTeSRl + 20 % w/w Ethylene glycol + 20 % w/w HCMD 2.5, degree of substitution

9.4 % w/w

The results in Fig. 4 demonstrate that human IPS cells survive cryopreservation with the HCMDs and ethylene glycol as cryoprotectants. Using the HCMD 5 and ethylene glycol results in the same survival rate as the standard DMSO based cryopreservation method. It is also demonstrated that all HCMDs have an additive cryoprotectant effect compared to ethylene glycol used alone.

After thawing, iPSC lines were seeded and as exemplified on Fig . 5 resulted in proliferative and viable iPSC lines capable of being expanded . EXAMPLE 5 iPS cells were treated as described in example 2 and the experimental and analytical conditions were also as described in example 1 added the additive HSA as indicated on

The following cryopreservation solutions were included in this study:

• mTeSRl + 10 % w/w DMSO

• mTeSRl + 4% w/w HSA + 10 % w/w DMSO

• mTeSRl + 4% w/w HSA (human serum albumin)

• mTeSRl + 10 % w/w HCMD 5

• mTeSRl + 10 % w/w HCMD 5 + 4% w/w HSA

• mTeSRl + + 10 % w/w HCMD 5 + 10 % w/w ethylene glycol (EG)

• mTeSRl + + 10 % w/w HCMD 5 + 4% w/w HSA + 10 % w/w ethylene glycol (EG)

• mTeSRl + + 10 % w/w HCMD 5 + 4% w/w HSA + 10 % w/w DSMO

• mTeSRl + 10 % w/w HCMD 750

• mTeSRl + 10 % w/w HCMD 750 + 4% w/w HSA

• mTeSRl + + 10 % w/w HCMD 750 + 10 % w/w ethylene glycol (EG)

• mTeSRl + + 10 % w/w HCMD 750 + 4% w/w HSA + 10 % w/w ethylene glycol (EG)

• mTeSRl + + 10 % w/w HCMD 750 + 4% w/w HSA + 10 % w/w DSMO

The results on Fig. 6 demonstrate that human iPS cells survive cryopreservation with HCMDs, ethylene glycol and HSA as cryoprotectants. HCMD5 combined with HSA and EG results in improved survival compared to DMSO alone.