HK1156970B - Cryopreservative composition for cell and tissue - Google Patents

Description

Composition for cryopreservation of cells and tissues

[ technical field ] A method for producing a semiconductor device

The present invention relates to a reagent for cryopreservation of human and animal cells and tissues, which can reduce damage or injury to the cells and tissues when the cells and tissues are frozen and thawed. This cryopreservation technique is expected to be highly demanded in transplantation medicine requiring cryopreservation of living tissues (such as skin, cornea, pancreatic islets and heart valves) and in regenerative medicine requiring cryopreservation of cells (such as hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, iPS cells (induced pluripotent stem cells) and the like).

[ background of the invention ]

Cryopreservation techniques at temperatures of 0 ℃ or below are routinely used for long-term storage of water-borne or aqueous materials (e.g., plant and animal cells and tissues and food products). It is known that when these substances are frozen, ice crystals form, resulting in a non-uniform concentration of solutes and contaminants that are removed by water molecules, referred to as 'freeze concentration'.

To prevent freeze concentration, each low molecular weight compound may be added to the cryopreservation medium. For example, dimethyl sulfoxide (DMSO), glycerol, and the like are added as a cryoprotectant to minimize damage to cells and tissues caused by water crystallized in cells during cryopreservation.

Therefore, cells are usually suspended in a physiological solution containing 5-20% cryopreservative (e.g., DMSO, glycerol, ethylene glycol and propylene glycol) in a cryovial, in a culture medium, and stored at low temperatures, either-80 ℃ or-196 ℃.

Of these agents, DMSO is the most effective and commonly used, but is physiologically toxic and is known to cause hypertension, nausea and vomiting when cells are infused into a recipient. Moreover, DMSO toxicity tends to attenuate the viability and/or function of cells following culture or infusion of thawed cells into a recipient's body.

Of these reagents, glycerol has a lower cryopreservation effect, and requires freezing after keeping the cell suspension only at room temperature or a non-frozen low temperature, or precise control of the reduced temperature by using a program freezer or the like. Moreover, because of their low protective effect on cell survival and function, the cryopreservatives are harmful to the thawed cells.

Cryopreservation of stem cells (e.g., embryonic stem cells or iPS cells) or germ cells (e.g., sperm, unfertilized or fertilized eggs) is performed using high concentrations of cryoprotectants (e.g., DMSO, acetamide, propylene glycol and polyethylene glycol) for rapid freezing or vitrification. Vitrification rapidly brings intracellular water into a vitrified state to avoid damage or injury to cells due to ice crystal formation. Also, highly toxic damage of cells or tissues by concentrated cryopreservatives is highly likely; therefore, this technique is employed only in some limited cases.

In the preparation of pharmaceutical products, food products and ice sculptures for display purposes, additives such as sodium chloride or sugar, glucose and trehalose are used. Other additives such as anti-freeze proteins or anti-freeze glycoproteins made by organisms such as plants, fish and insects have also been used (Japanese patent laid-open Nos. 2005-126533 and 2003-250506).

In a fuel cell, water is generated at either electrode by an electrochemical reaction. For example, in proton-exchange membrane fuel cells, water is produced at the cathode electrode; and a part of the generated water moves toward the anode side through the electrolyte membrane. Water will also be generated from the condensation of steam in the gas entering the cell. This water potentially impedes gas flow, depletes the supply of gas itself and ultimately reduces cell performance. These complications can be prevented by treating the gas barrier surface with a hydrophilic coating material (e.g. protein), thereby limiting water condensation, but liquid water occasionally freezes even at low temperatures under the conditions described leads to other complications. To prevent this problem, a polymer electrolyte having a freeze-resistant protein is added to a resin layer, which then coats the surface of the polymer electrolyte membrane (Adler et al, below), but this method has a problem of high cost.

[ Prior art documents ]

[ patent document ]

[ patent document 1 ] Japanese Kohyo publication Hei 10-511402

[ patent document 2 ] patent No. 3694730

[ patent document 3 ] Japanese patent application laid-open No. 2005-126533

[ patent document 4 ] Japanese patent application laid-open No. 2003-250506

[ patent document 5 ] Japanese patent application laid-open No. 2008 & 041596

[ non-patent literature ]

[ non-patent document 1 ] Lovelock JE and Bishop MWH, Nature 183: 1394-1395, 1959

[ non-patent document 2 ] Polge C, Smith AU, Parkes AS, Nature 164: 666-666, 1949

[ non-patent document 3 ] Miszta-Lane H, Gill P, Mirbooki M, LakeyJRT. cell Preserv Technol 5, 16-24, 2007

[ Nonpatent document 4 ] Ha SY, Jee BC, Suh CS, Kim HS, Oh SK, Kim SH, Moon SY. human Reproduction 20, 1779-

[ Nonpatent document 5 ] Yu HN, Lee YR, Nob EM, et al. INT JHEMATOL ≡ 87: 189-; 2008

[ non-patent document 6 ] Adler S, Pellozzer C, Paparella M, Hartung T, Bremer S.Toxicol in Vitro 20: 265-271; 2006

[ summary of the invention ]

[ technical problem to be solved by the invention ]

Conventional cryopreservation methods (including rapid freezing techniques) do not preserve the complete structural integrity of the cells or tissues after freezing and thawing; therefore, there is a strong need for new cryopreserved materials with low toxicity. Moreover, DMSO is known to induce cells (e.g., HL-60 cells) to differentiate; therefore, it is not suitable for certain types of cells. Anti-freeze proteins and glycoproteins have excellent preservation capacity but are too costly for use in food (JPY1, 300,000YEN/g), and are even more commonly used in cells and tissues.

The present invention aims to provide a cryopreservative having an excellent protective effect and low toxicity to cells or tissues, thereby substituting DMSO. The present invention also aims to provide an inexpensive and safe cryopreservation agent having properties similar to those of anti-freeze proteins and glycoproteins which prevent condensation, thereby allowing the cryopreservation and freeze-drying of materials (e.g. food and pharmaceutical products).

[ technical means for solving problems ]

The cryopreservation liquid of the present invention comprises: substantially 1-50% of a polyamine having pendant amino groups; and physiological solutions (e.g., saline or culture media).

[ Effect of the invention ]

By immersing each of the animal cells (including human cells) and the plant cells in a cryopreservation solution and then cryopreserving at-80 ℃ or cooling with liquid or vapor nitrogen, they can be preserved with their viability and biological activity maintained without using highly toxic DMSO or other conventional cryopreservative agents. Since conventional cryopreservatives (e.g., DMSO, glycerol, ethylene glycol) and the like are not used, toxicity to cells is kept low, and cells can be cryopreserved for a long time without reducing biological activity of the cells. Furthermore, the cryopreservation solution is free from protein components (such as fetal bovine serum and albumin), and therefore, there is no fear of infection with diseases, and is not affected by great changes that are occasionally seen in pharmaceutical products made of biological materials.

Polyamines having a side chain amino group (e.g.,. epsilon. -poly-L-lysine and polyallylamine) are considered to have a cell protective effect because they have affinity for cell membranes due to the side chain amino group. The polymer compound having abundant carboxyl groups also has high affinity with water, and therefore, will assist in the removal of intracellular water to the surrounding medium during freezing, and thus is expected to have a cryopreservation effect. The polymer compound having both amino and carboxyl groups in a sufficient ratio is expected to have a further improved cryopreservation effect on cells when frozen. Therefore, the present invention has been made in an effort to provide a cryopreservation solution having high efficacy and high safety, and the conditions and requirements of a polymer compound having a cationic group (e.g., a side chain amino group) (e.g., a polyamino acid), a polymer compound having an anionic group (e.g., a carboxylic acid group), and a polymer compound having both a cationic group and an anionic group have been studied.

The cryopreservative of the invention is less toxic than DMSO and does not require post-thaw washing of cells or tissues. The thawed cells or tissue can then be suspended directly in culture medium to immediately begin the culture process.

The cryopreservation of cultured cells for experiments can be stably performed according to the present invention; and is expected to be preserved by maintaining the cell function of functional cells such as pancreatic islets and stem cells (e.g., ES cells, mesenchymal stem cells, and iPS cells). Therefore, it is expected to improve the efficiency of transplantation of these cells.

By using the non-frozen poly-amino acid of the present invention, inactivation of a physiological substance during freezing of a water-bearing substance having the physiological substance can be prevented. Furthermore, by using non-frozen poly-amino acids, a uniform diffusion of components other than water molecules can be achieved in the process of obtaining a frozen product or a freeze-dried product by freezing or freeze-drying of a water-bearing or water-containing substance. The frozen product can be ice cream, fruit juice milk jelly, other frozen dessert, ice for exhibition, quick-frozen soup, etc.; and the freeze-dried product may be a food or pharmaceutical product in the form of a freeze-dried powder.

The non-frozen reagents of the invention can also be applied to industrial fuel cells to prevent their deterioration due to the initial behaviour of the liquid freezing.

[ description of the drawings ]

FIG. 1 is a graph showing the relationship between the percentage of blocked amino groups in epsilon-poly-L-lysine and the percentage of viable L929 cells cryopreserved by epsilon-poly-L-lysine partially blocked by succinic anhydride using amino groups;

FIG. 2 is a graph showing the relationship between the concentration of partially blocked poly-L-lysine and the percentage of viable L929 cells cryopreserved by using epsilon-poly-L-lysine (PLL succinic anhydride 63%) to which succinic anhydride has been added in a molar amount corresponding to 63% of the amino groups of epsilon-poly-L-lysine;

FIG. 3-1 is a microscope image showing a culture of L929 cells that have been frozen in 10% DMSO/fetal bovine serum, then thawed and immediately cultured on plates for 24 hours without washing or dilution;

FIG. 3-2 is a microscope image showing a culture of L929 cells that have been frozen in a 7.5% PLL solution containing 63% succinic anhydride, then thawed and immediately transferred to a plate for 24 hours without washing or dilution;

FIG. 4 is a set of graphs showing Rat Mesenchymal Stem Cells (RMSCs) frozen in 7.5% PLL solution containing 63% succinic anhydride and 10% DMSO/fetal bovine serum, and their pluripotency in differentiation into bone, adipose body, and cartilage was evaluated. Unfrozen and undifferentiated cells were included for comparison.

FIG. 5 is a series of microscope images showing that ice recrystallization was prevented by adding 0.1-15% PLL (. epsilon. -poly-L-lysine) (PLL succinic anhydride 63%) to a 30% sucrose aqueous solution.

Fig. 6 is a microscope image showing crystal structures in a frozen 5% PLL solution without succinic anhydride (e-poly-L-lysine) and a 5% PLL solution with 63% succinic anhydride (PLL succinic anhydride 63%).

FIG. 7 is a photograph of a freeze-dried agar gel (1). The gel on the left is additive free and the gel on the right has 5% PLL with 63% succinic anhydride.

FIG. 8 is a photograph of a frozen-thawed agar gel (2). PLL concentrations with 63% succinic anhydride were 0% (left), 1% (middle) and 3% (right).

[ embodiment ] A method for producing a semiconductor device

Dissolving 1-50 w/w% in physiological solution; preferably 2 to 20 w/w%, particularly preferably 3 to 15w/w%, and more preferably 5 to 10w/w% of a polymer (e.g., poly-lysine) to obtain the cryopreservation liquid of the present invention. The physiological solution to be used is physiological saline and a medium for culturing each cell and tissue. For example, Dulbecco-modified eagle MEM medium (DMEM) may be one of the preferred media. Polyallylamine may be used instead of, or in addition to, poly-lysine. Instead of these, or in addition to at least one of these, the compounds to be used are selected from: other polyamines (e.g., amino group-introduced polysaccharides) and poly-amino acids (e.g., poly-arginine, poly-glutamic acid, and poly-aspartic acid); also selected from the group consisting of dextrans, dextrins, pullulans and chitosan, and polycarboxylic acids (e.g., polyacrylic acid). Among these polymers, preferred are polymers having a structure obtainable by polymerization of a monomer compound having both cationic and anionic substituents in the same monomer molecule; and particularly preferred are poly-amino acids. More particularly, a polymer having a repeating unit having both an amino group and a carboxyl group is particularly preferable.

The poly-lysine to be used may be epsilon-poly-L-lysine or epsilon-poly-D-lysine or alpha-poly-L-lysine. The cryoprotectant polymer has a molecular weight between 100 and 100,000. The most preferred polymers fall into a group of epsilon-poly-L-lysine that is conventionally used as a food additive. These are either enzymatically synthesized or produced by Streptomyces fungi and have an average molecular weight of 1000 to 20,000, and in particular an average molecular weight of 1000 to 10,000 (http:// www.chisso.co.jp/fine/jp/polylisin/index. html), with attempts to produce a degree of polymerization varying between 15 and 35, and a degree of polymerization of 20 or less (for example, Japanese patent application laid-open Nos. 2003-171463 and 2005-318815). The average molecular weight or the average polymerization degree can be easily measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by, for example, using an electrophoresis apparatus and a densitometer (type AE-6920V) supplied by Atto Co. Standard protein markers were used for the measurements. Poly-lysine can be heat treated to increase its molecular weight above 30,000 and used as a polymer compound. However, the above-mentioned molecular weight range is preferred due to the viscosity with increasing molecular weight. Since poly-lysines with free terminal carboxyl groups have pendant primary amino groups, their partial amidation by dicarboxylic anhydrides as described below largely gives excellent miscibility and solubilization performance. Other particularly advantageous polymer compounds which can also be employed according to the invention are polyallylamines having an average molecular weight of 1000 to 1,000,000, preferably 1000 to 20,000. For example, the polymers that can be used are: adding acetic anhydride or acetic acid to an aqueous solution of an allylamine polymer (PAA-03 of Nitto textile Co., Ltd.); and partially-methoxy-carbonylated allylamine polymers (PAA-U5000 by Nidong textile Co., Ltd.). Allylamine polymers, like poly-lysine, have only primary amino groups as side groups, but the density of primary amino groups per unit molecular weight is greater in allylamine polymers than in poly-lysine. Furthermore, when allylamine is partially carboxylated, the resulting polymer compound is thought to function as if it were partially-carboxylated poly-lysine as described later.

Preferably, the amino groups of the polyamine are partially blocked by carboxylation or acetylation with a carboxylic acid anhydride. The blocking is carried out by carboxylation or acetylation of the amino group to an extent of preferably 50 to 99 mol%, particularly 50 to 93mol%, more preferably 50 to 90 mol%, still more preferably 55 to 80 mol%, and most preferably 58 to 76 mol%. About 50% of the amino groups will be blocked by reaction with 52-53 mol% of an anhydrous carboxylic acid (relative to the molar amount of amino groups in the polyamine). Under normal reaction conditions, when the compound reacts with 100 mol% of anhydrous carboxylic acid, 90-95% of amino groups are blocked. Blocking rates above or below the above-mentioned range will reduce the cryopreservation effect. Carboxylic anhydrides which may be employed herein include: acetic anhydride, citric anhydride, succinic anhydride, glutaric anhydride, malic anhydride, fumaric anhydride, and malic anhydride. Among these, succinic anhydride and acetic anhydride are particularly preferable.

However, polyamines in which the amino group is not blocked and is in a free state may also be used; therefore, the carboxylation and acetylation degree can be in the range of 0 to 100 mol/mol%. In the present invention, partially carboxyl aminated polycarboxylic acids can be used. More particularly, polycarboxylic acids can be partially aminated by reacting their carboxyl groups with compounds such as diamines, triamines and polyamines. Diamines which may be used are ethylenediamine and hydrazides, for example adipic acid dihydrazide. The reaction of these amino compounds with carboxylic acids proceeds in an addition reaction with carbodiimides. In this case, the amination degree may be in the range of 0 to 100 mol/mol%. As with the amino group blocking, the percentage of residual carboxyl groups is preferably in the range of 50 to 99 mol%, more preferably in the range of 60 to 97 mol%, wherein the residual percentage is the residual percentage of aminated carboxylic acid groups. For example, polyacrylic acid having an average molecular weight of 1000 to 3,000,000, or particularly 1000 to 10,000 is used; and 1to 50 mol%, preferably 3 to 40 mol%, of the carboxyl groups of the polyacrylic acid are blocked with an amine or a carbodiimide (e.g., ethylenediamine dihydrazide). The cryopreservation solution of the present invention may also contain 0.3 to 15w/w%, or particularly 0.1 to 50w/w%, of a conventional cryopreservation substance such as DMSO, glycerol, ethylene glycol, trehalose or sucrose. The addition of antioxidants to cryoprotectants is expected to improve their preservation effect as cells are subjected to damage caused by oxidative stress during freezing and thawing. For example, antioxidants such as catalase, peroxidase, superoxide dismutase, vitamin E, vitamin C, polyphenols (e.g., epigallocatechin gallate) or glutathione may be used.

The osmotic pressure of the cryopreservation agent is 200-1000 mOsm/kg, more preferably 300-700 mOsm/kg, and still more preferably 400-600 mOsm/kg. The cryopreservation agent of the present invention can be applied to the preservation of not only cells but also tissues. Examples of the cells and tissues to be cryopreserved with the cryopreservative are cultured cell lines, fertilized eggs of animal and human origin. Further examples are: sperm cells, embryonic stem cells, iPS cells, mesenchymal stem cells, hematopoietic stem cells, neuronal stem cells, umbilical cord blood stem cells, hepatocytes, nerve cells, cardiac muscle cells, vascular endothelial cells, vascular smooth muscle cells, and blood cells. Not only animal or human cells but also plant cells may be included. Tissues and organs which can be preserved by the cryopreservative according to the invention are skin, nerves, blood vessels, cartilage, cornea, liver, kidney, heart and pancreatic islets.

Furthermore, the above-mentioned polymer compounds can also be applied to produce frozen or freeze-dried foods or medicines by adding the polymer compounds to aqueous or water-carrying substances for foods or medicines to avoid freeze concentration and thereby obtain frozen or freeze-dried products in which the ingredients are uniformly dispersed. In particular, compounds selected from the following group for blocking the amino group by carboxylation or acetylation by reaction with succinic anhydride, acetic anhydride or other carboxylic anhydrides can be used: epsilon-poly-L-lysine, alpha-poly-L-lysine, polyarginine, other polyamino acids, aminated polysaccharides and polyallylamine. It is not necessary to dissolve the polymer compound using a physiological solution. For example, poly-lysine having a partially blocked amino group, other poly-amino acid or aminated poly-sugar is added to the aforementioned water-borne or water-containing substance for ice cream or freeze-dried food, so that the concentration of the polymer compound is 1to 15%. In this way, freeze concentration is prevented. When succinic anhydride is used for blocking a polymer group, an excellent effect of preventing freeze concentration is obtained when succinic anhydride in a molar amount corresponding to 50 to 85 mol% of an amino group reacts with a polymer, wherein an actual amino-blocking ratio is in a range of about 48 to 80 mol%.

The above-described polymer compounds may be used in industrial fuel cells, whereby the polymer compounds are added to the fuel cells to prevent their deterioration in initial performance (which may otherwise be caused by liquid freezing at the time of initiation). More particularly, it is possible to use polymer compounds formed of units having amino groups, selected from: epsilon-poly-L-lysine, alpha-poly-L-lysine, polyarginine, other polyamino acids, aminated polysaccharides and polyallylamine; wherein the amino group of the polymer compound is blocked by carboxylation or acetylation by reaction with succinic anhydride, acetic anhydride or other carboxylic anhydrides; and the polymer compound may be added to the surface layer material exposed to the inside of the fuel cell. For example, the polymer compound may be incorporated at 1to 15w/w% into a coating material forming the surface of a separator or a solid electrolyte membrane, or particularly a UV curable resin liquid.

[ examples ] A method for producing a compound

Examples of the present invention and comparative examples are shown below, but the present invention is not limited to the following examples.

Example 1: preparation of cryopreservation solutions

A25% aqueous solution of epsilon-poly-L-lysine (manufactured by Chisso Corp.; molecular weight: 4000) was used; and polyallylamine (Nittobo, molecular weight 5000[ PAA-05L ], 15000[ PAA-L ], 60000[ PAA-H ]) in 20% aqueous solution was used. 0 to 100 mol% of succinic anhydride (manufactured by Wako pure chemical industries, Ltd.) is added to each solution with respect to the amino group of the polyamine polymer to obtain poly-amines having blocked amino groups with different amino-blocking ratios. Each poly-amine solution was added to Dulbecco's modified Eagle Medium (DMEM, Sigma Aldrich) to 0-10 w/w%. At this time, the pH of the medium is adjusted to 7.0 to 8.0 with 1N hydrochloric acid or sodium hydroxide solution. Furthermore, the medium osmolality was measured with a vapor pressure osmometer (type 5 520, Wescor) and adjusted with 10% aqueous sodium chloride solution.

Example 2: cryopreservation of cultured cells ]

In a freezer bottle (Simport Plastics), 1X 10⁶Cell types of L929, MG63, Caco-2 (manufactured by Sumitomo, Japan), Colon26, HT1080, B16F1 and KB cell (ATCC) were suspended in 1ml of each freezing solution; then frozen in a-80 ℃ freezer. After one week, cells were quickly thawed in a 37 ℃ water bath, washed in DMEM and tested for cell death rate with trypan blue dye. Then theThawing the cells at 1X 10⁵Cells/well were seeded in 6-well culture plates and cell viability was assessed with trypan blue dye after 6 and 24 hours of culture. 10% DMSO in Fetal Bovine Serum (FBS) as a commonly used cryopreservative was used as a cryopreservative for the comparative example.

As shown in fig. 1, when each 7.5% solution of poly-lysine (PLL) which had been modified by adding 50% or more molar amount of succinic anhydride with respect to amino group was used as a cryopreservation solution in cryopreservation of L929 cells, cell viability almost the same or higher than that of the comparative example using DMSO solution was achieved. Carboxylated poly-lysine (PLL), which has been modified by the addition of 100% molar amount of succinic anhydride, shows a 93% amino-blocking percentage as a result of quantitative measurement of residual amino groups by the ninhydrin and TNBS method. Poly-lysine (PLL) which had been modified by adding succinic anhydride in a molar amount of 10 mol%, 27 mol%, 45 mol%, 52 mol%, 63 mol% and 79 mol% with respect to the amino group of poly-lysine showed amino-blocking percentages of 10%, 25%, 43%, 50%, 60% and 76%, respectively. As shown in FIG. 1, the poly-lysine solution having an amino-blocking percentage in the range of 50 to 93% shows a cryopreservation effect; and a particularly high cryopreservation effect is achieved by a poly-lysine solution having a 60% blocking percentage (63 mol% succinic anhydride has been added) and a 76% blocking percentage (79 mol% succinic anhydride has been added). When an aqueous solution of polyallylamine (which is an allylamine polymer having a molecular weight of 5000 that has been reacted with succinic anhydride at 45 to 90 mol% relative to the molar amount of amino groups in the allylamine polymer) having partially blocked amino groups is used, as also shown above, the cell survival rate improves as the amino-blocking percentage increases.

FIG. 2 shows the relationship between cell viability of L929 cells and the concentration of partially blocked ε -poly-L-lysine modified by the addition of a 63% molar amount of succinic anhydride relative to amino groups, which is indicated as "PLL (0.63)" in the figure and referred to as "PLL succinic anhydride 63%" throughout the specification below, during cryopreservation. As shown in fig. 2, when the partially blocked poly-L-lysine or PLL succinic anhydride concentration of 63% is 7.0% or more, the cell survival rate is almost the same as or higher than that obtained using the DMSO solution. The tendency as described above is also exhibited when an aqueous solution of polyallylamine having a partially blocked amino group, which is an allylamine polymer having a molecular weight of 5000 that has been reacted with succinic anhydride in an amount of 63 to 85 mol% based on the molar amount of the amino group, is used.

In FIGS. 1to 2, the range showing the best results, as shown when the osmotic pressure of the preservation solution was obtained, corresponds to an osmotic pressure in the range of 400 to 600 mOsm/kg. More particularly, the best preservation effect is obtained when the osmotic pressure is in the range of 400 to 600 mOsm/kg.

Table 1 shows the cryopreservation effect of other cell species when the cells were cryopreserved in a PLL succinic anhydride 63% 7.5% solution. As shown in Table 1, all cell species achieved cell viability nearly identical to or higher than that obtained from DMSO solutions (10% DMSO/fetal bovine serum). Although data is not shown, polyallylamine with partially blocked amino groups gave similar results.

[ TABLE 1 ] cryopreservation Effect of 7.5% PLL (0.63) on individual cells

Cryopreserved cells	Survival rate 24 hours after thawing
		MG63	93.1±2.3
HT1080	90.2±4.3
		Colon26	92.3±2.3
B16F1	94.2±0.6
		KB	91.8±0.9
Caco2	93.7±1.9

Example 3: toxicity test

Toxicity testing was performed on L929 cells. Cells suspended in DMEM medium containing 10% fetal bovine serum were seeded in 96-well plates (1.0X 10)³Cells/well) and cultured at 37 ℃ for 72 hours. Then, the epsilon-poly-L-lysine and the modified poly-lysine, each of which had been added with succinic anhydride at different concentrations, were added to the medium to reach a final concentration of 0-10%. Then, after 48 hours of culture, the concentration value at 50% inhibition of cell growth was measured as IC by MTT assay relative to the cell growth in the medium without added polymer₅₀. Table 2 shows the results; and the preservation solution of the comparative example was a DMSO solution (10% DMSO/fetal bovine serum). IC of PLL succinic anhydride 58%, 63% and 79% as shown in Table 2₅₀The value is 2-3 times of that of the DMSO solution; this indicates that the toxicity of poly-lysine is 1/2-1/3 in the widely used cryopreservation solution. In particular, IC of 63% PLL succinic anhydride and 58% PLL succinic anhydride₅₀The largest values, which are the best polymer compounds for high cell viability in the data shown in figure 1.

Meanwhile, the cryopreservation solution containing L929 cells was frozen, thawed, and directly seeded on a 12-well plate, and cultured at 37 ℃ for 24 hours. More specifically, cells were cryopreserved in a 63% solution of PLL succinic anhydride in 7.5% and thawed as in example 2, except that neither the liquid nor the cells were diluted, nor washed, and the liquid containing the cells was transferred directly to the culture plate. Observation of the cells showed that: as shown in FIG. 3-1, cells that had been cryopreserved in DMSO solution (10% DMSO/fetal calf serum) were apparently round and dead; and as shown in FIG. 3-2, the cells cryopreserved in the cryopreservation solution of the present invention attached to the plate and survived well. Polyallylamine with blocked amino moieties (this is a 5000 molecular weight polymer of allylamine that has been reacted with 63-85 mol% succinic anhydride, equivalent to the molar amount of amino groups) gave test results with similar low toxicity.

[ TABLE 2 ] 50% cell growth-inhibitory concentration of L929 by cryopreservative

	IC50/％
		DMSO	2.035±0.017
PLL(0)	1.194±0.006
		PLL(0.44)	2.025±0.013
PLL(0.58)	＞7.500
		PLL(0.63)	6.777±0.005
PLL(0.68)	3.412±0.097
		PLL(0.79)	4.801±0.017

Example 4: preservation of mesenchymal Stem cells

Rat Mesenchymal Stem Cells (RMSC) were cryopreserved. The preservation solution of the comparative example was 10% DMSO fetal bovine serum; and the preservation solution used in the examples was a 63% solution of PLL succinic anhydride in 7.5%, which is represented in fig. 4 as 7.5% PLL (0.63).

Table 3 shows that the survival rate of Rat Mesenchymal Stem Cells (RMSC) after thawing was almost the same for the cryopreservation solution of the present invention and the DMSO solution. DMEM supplemented with polyallylamine (allylamine polymer with a molecular weight of 5000, which reacts with succinic anhydride at 63-85 mol% equivalent to the amino content) with 7.5% amino moiety blockade exhibited similarly high cell viability.

[ TABLE 3 ] cryopreservation Effect on rat mesenchymal stem cells

	Immediate use	After 6 hours	After 24 hours
				10％DMSO	92.3±2.3	88.3±1.1	92.8±3.5
7.5％PLL(0.63)	95.4±3.8	92.9±2.0	95.7±1.3

The cells are now cryopreserved and thawed as described in example 2; and inducing them to differentiate into osteocytes, adipocytes and chondrocytes to evaluate their differentiation potential. Figure 4 shows that the pluripotency of the cells is maintained almost the same as that of unfrozen cells and as that of cells cryopreserved and thawed in DMSO solution. Carrying out color separation on the image data of the color microscopic image to 3 primary colors of red, green and blue; and only the red portion is shown in figure 4. Thus, the red color in the color image is turned to white; and converting the blue color in the color image to black. Differentiation potential into osteocytes was assessed by assessing calcium deposition in a manner stained with alizarin red S; as a result, each sample was colored red. As shown in the upper image in fig. 4, the image of the fully differentiated cells appears similarly as a diluted or low-grayscale monochrome pattern compared to the undifferentiated cells. The diluted monochromatic patterns indicated that the color microscopic images had red shades throughout their area. Meanwhile, the cells cryopreserved in each cryopreservation solution showed higher alkaline phosphatase activity than the cells that were not frozen. Differentiation potential into adipocytes was assessed by staining fat droplets with oil red O. Red-colored fat droplets were observed in microscopic images of the cells cryopreserved in each of the cryopreservation solutions. The fat droplets appear in the middle row image of fig. 4 as a circular or elliptical pattern with low grayscale and a diameter of tens of μm. Differentiation potential into chondrocytes was assessed by staining proteoglycans in cell aggregates with Alcyan blue. Blue-stained proteoglycans were observed in the microscopic images of the cells cryopreserved in each of the cryopreserved solutions, as in the unfrozen cells. Protein aggregates appear as dark black parts in the down-line image of fig. 4. When DMEM added with polyallylamine (allylamine polymer of molecular weight 5000, which reacts with 63-85 mol% of succinic anhydride corresponding to the amino group content) partially blocked with 7.5% of amino groups was used as a preservation solution; the differentiation potential of the cells is maintained in a similar manner even after freezing.

Example 5: preservation of umbilical cord blood ]

Cord blood was collected from human umbilical cord using a 7mL plastic evacuated blood collection tube (Venoject II, Terumo corporation) containing 10.5mg of anticoagulant (EDTA2 Na). Subsequently, the cord blood to which PLL succinic anhydride 63% was added to a concentration of 7.5% (as expressed as 7.5% PLL (0.63)) was cryopreserved at-80 ℃ for 3 months in a freezer. The cord blood was then flash thawed in a 37 ℃ water bath and the undiluted cord blood sample was analyzed by flow cytometry for expression of the surface marker CD 34. The number of hematopoietic cells expressing CD34 was measured according to standard methods described in the literature (modified methods of a. higuchiet al., j.biomed.mater.res., 68A, 34-42 (2004)). Therefore, the number of hematopoietic cells expressing CD34 was estimated using a Stem-kit (Beckman-Coulter) according to the protocol in the manual (International society for blood therapy and transplantation, ISHAGE guidelines). When 63% PLL succinic anhydride added at 7.5% was added to umbilical cord blood, the counted cell number of hematopoietic cells expressing CD34 was estimated to be about 70% of that of the first day even after 3 months of cryopreservation; however, when cord blood in a 10% DMSO solution state was cryopreserved, the number of hematopoietic cells expressing CD34 was estimated to be about 20% of the first day. Thus, it was revealed that when umbilical cord blood was stored in a storage solution to which epsilon-poly-L-lysine was added, hematopoietic cells expressing CD34 could be stored in an undifferentiated state for a long period of time.

These results show that the cryopreservation liquid according to the embodiment of the present invention is remarkably excellent in the effect of preserving umbilical cord blood.

Example 6: anti-cryoprotein Activity

PLL (epsilon-poly-L-lysine) and succinic anhydride modified PLL were investigated for their ability to resist freeze protein activity or prevent ice recrystallization. Anti-freeze proteins are known to have specific activities and are known to cause thermal hysteresis, prevent ice recrystallization growth, and change ice crystal morphology to hexagonal or bipyramidal. Please see JP2005-126533a, JP2003-250506A and JP 2008-041596A.

A30% sucrose solution in water was added to 20% of unmodified PLL and PLL succinic anhydride, 46% of PLL succinic anhydride, 50% of PLL succinic anhydride, 65% of PLL succinic anhydride, 76% of PLL succinic anhydride and 84% to 1-15% of PLL succinic anhydride. The actual amino-blocking ratios of these succinic anhydride-modified PLLs were measured by the methods described previously and showed about 0.20, 0.43, 0.48, 0.62, 0.73 and 0.80, respectively. 4 microliters (4 μ L) of each of the unmodified PLL and the modified PLL solution was placed on a glass plate and covered with another glass plate; then placing the sample on a temperature control microscope stage of Linkam company or a fast cooling stage 10002L; and rapidly cooling to-30 ℃ to induce ice crystal formation. Subsequently, the table temperature was gradually raised and then kept at-9 ℃ for 30 minutes; and during this process, ice crystal growth was observed by microscopy. As shown in a series of photomicrographs of fig. 5, the results show that the effect of preventing ice recrystallization can be imparted to the PLL by introducing carboxyl groups up to 50% or more of the amino groups. Fig. 5 shows the results of adding unmodified PLL and modified PLL to 5 weight percent in solution, however PLL succinic anhydride 50% (PLL (0.50)) -PLL succinic anhydride 84% (PLL (0.84)) at concentrations of 1% to 15% showed efficacy in preventing ice recrystallization.

Subsequently, on the rapid cooling stage, the ice crystal morphology of the 5% solution of unmodified PLL and the 5% solution of modified PLL (PLL succinic anhydride 65%) was studied. More particularly, first, the solution is rapidly cooled to-30 ℃ to induce the formation of abundant ice crystals; the solution temperature was then raised at a rate of 0.02 deg.c/min to a temperature at which one ice crystal with a diameter of about 10 μm was present in the field of view of the microscope. As shown in the microscope image of fig. 6, the ice crystals in the succinic anhydride-modified PLL solution appeared to have a hexagonal crystalline shape. It is noted that the hexagonal crystal shows that the concentration of any PLL succinic anhydride from 50% (PLL (0.50)) to 84% (PLL (0.84)) is in the range of 1% to 15%. Succinic-anhydride modified PLLs give a thermal hysteresis (which is one of the differences between the melting temperature and the crystallization-growth-initiation temperature, and the property of anti-freeze proteins) of up to 0.1 ℃. This shows that anti-freeze protein activity can be obtained by introducing 50 mol% or more of carboxylic acid groups of amino groups of PLL.

Example 7: food preservation

Agar gel to prevent freeze concentration-freeze-thaw: PLL succinic anhydride 63% was added to agar powder (Nakaraitesque Co.; grade 1 reagent); a 5% solution was then prepared. Adding red ink into the solution, placing into a plastic bottle, and freezing at-20 deg.C; and then thawed at room temperature. The results obtained are shown in FIG. 7; add 5% PLL succinic anhydride to get the right gel; and left gel without addition. The gel on the left side of the view shows a clear distinction between the red opaque part on the upper half of the view and the translucent part on the lower half of the view, thereby presenting the web pattern of the tissue, but it induces shadows adjacent to the upper right of the view. Meanwhile, the right-hand gel obtained by adding PLL succinic anhydride showed a red color uniformly throughout the gel; thereby indicating that freeze concentration has been prevented.

Freeze-dried agar gel: to agar powder (Nakaraitesque; grade 1 reagent) were added 0%, 1% and 3% PLL succinic anhydride 63%, the actual amino blocking ratio was 0.6. Putting the solution into a plastic bottle, and then freezing at-20 ℃; and then freeze-dried by vacuuming at 1Torr for 2 to 3 days to obtain a freeze-dried agar gel. A photographic image of the resulting freeze-dried product is shown in fig. 8. The freeze-dried agar gels with 0% PLL succinic anhydride added to the left side of the view showed a volume shrinkage of about one third, whereas the freeze-dried agar gels with 1% and 3% PLL succinic anhydride added (center and right side of the view) showed only a small degree of volume shrinkage. This result shows that freeze-drying of a solution containing a non-frozen polyamino acid of the invention results in drying that is efficient and maintains product quality.

Claims (5)

1. A composition for cryopreservation of cells and tissues,

it includes:

● a combination of a polymer and a metal,

■ which includes a polymer having units of amino groups, and

■ selected from: epsilon-poly-L-lysine and alpha-poly-L-lysine; and

● a physiological solution; and

dissolving the polymer compound in the physiological solution to 1-50 w/w%,

wherein the content of the first and second substances,

50 to 93mol% of the amino groups of the polymer compound are blocked by a carboxylic acid group through reaction with succinic anhydride;

the polymer compound has a number average molecular weight in the range of 1000 to 20,000; and is

When the polymer compound is dissolved in the physiological solution to 7.5 to 10w/w%, the osmotic pressure of the composition for cryopreservation is in the range of 400 to 600 mOsm/kg.

2. The composition of claim 1, wherein the polymer compound has a number average molecular weight in the range of 1000 to 10,000.

3. The composition of claim 1, wherein the physiological solution is saline, Dulbecco-modified eagle MEM medium (DMEM), or a medium for cells or tissues.

4. The composition according to any one of claims 1to 3, wherein the polymer compound is dissolved in the physiological solution to 3 to 15 w/w%.

5. An additive composition for cryopreservation or freeze-drying of foods or medicines,

which comprises a polymer compound which is a mixture of a polymer compound,

● which includes a polymer having units of amino groups, and

● selected from: epsilon-poly-L-lysine and alpha-poly-L-lysine; and is

Wherein the content of the first and second substances,

50 to 93mol% of the amino groups of the polymer compound are blocked by a carboxylic acid group through reaction with succinic anhydride;

the polymer compound has a number average molecular weight in the range of 1000 to 20,000; and is

When the polymer compound is dissolved in a physiological solution to 7.5 to 10w/w%, the osmotic pressure of the solution thus obtained is in the range of 400 to 600 mOsm/kg.