KR102298738B1 - Anti-freezing composition comprising oligopeptide self-assebly nanostructure - Google Patents

Abstract

The present invention relates to a material and method in which an oligopeptide composed only of amino acids, which plays an important role in antifreeze protein, is formed in nano and micro sizes through a self-assembly process in an aqueous solution, and the self-assembled material composed of the oligopeptide is non-toxic, In addition, it is a cryopreservative that has excellent cell and tissue protection and can be produced at low cost, and has a high potential for use in various related industries such as medicine and food.

Description

Anti-freezing composition comprising oligopeptide self-assebly nanostructure

The present invention relates to an oligopeptide self-assembled nanostructure exhibiting freezing control properties.

For long-term preservation of water-containing substances such as cells, tissues, or food of plants or animals, cryopreservation at a temperature of 0° C. or less is routinely used. However, when these substances containing water are frozen, the water molecules crystallize while excluding the medium of the solute or mixed substance during freezing to form ice crystals composed of only water molecules. It is known that freeze-concentration occurs. In order to prevent such freeze-concentration, the method of adding various low molecular weight compounds is implemented. For example, when cryopreserving cells, a method of adding dimethyl sulfoxide (DMSO) or glycerol as a cryoprotectant is widely used to minimize cell damage caused by intracellular water crystallization that occurs during cryopreservation. .

That is, the cells are suspended in a physiological solution such as a culture solution containing a cryopreservative such as 5 to 20% dimethyl sulfoxide, glycerin, ethylene glycol, and propylene glycol, filled in a cryo tube and cooled, and finally -80 ° C or - It is common to cryopreserve at a cryogenic temperature of 196 °C.

Among them, dimethyl sulfoxide is the most effective and frequently used. However, it is also known that dimethylsulfoxide is physiologically toxic, particularly at high concentrations, and causes hypertension, nausea, and vomiting in patients receiving cryopreserved cells. There is also a problem in that the viability and function of cells that have been thawed due to the toxicity of dimethyl sulfoxide are reduced during culture or after injection into a living body. In addition, since other cryopreservatives such as glycerin have a low effect, it is necessary to freeze the cells after suspending the cells and leaving them at room temperature or refrigeration for some time, and strict temperature control by a program freezer or the like is required. In addition, there are problems such as a low cryoprotective effect and a decrease in function after thawing.

On the other hand, in hepatocytes such as ES cells and iPS cells, sperm, egg, fertilized egg, etc., for example, a rapid freezing method (freezing method) is known. The vitrification method is a method in which water contained in cells is made into a glassy form by rapidly lowering the temperature, and an obstacle due to formation of ice crystals is avoided. However, since the high-concentration anti-freeze agent used at this time is highly toxic, it is highly likely to damage cells or tissues, and thus has only been used limitedly for cryoprotection of tissues. On the other hand, when manufacturing pharmaceuticals, foods, and ice for display, adding salts, such as sodium chloride, and sugars, such as glucose, trehalose, is also implemented.

Further, addition of immobilized proteins and immobilized glycoproteins produced by plants, fish, insects, and the like is known. Polar organisms are living in extreme environments by expressing anti-freezing proteins in vivo in order to maintain life in extreme environments. Antifreeze protein (AFP) is a protein produced and secreted by fish, plants, fungi, and bacteria. Amico et al., 2006). This protein inhibits the growth of ice crystals, which are fatal to cells, and prevents recrystallization, allowing living things to live without freezing even at sub-zero temperatures.

Anti-freeze proteins found in polar fish generally have a TH activity of about 1 degree, whereas anti-freeze proteins found in insects have a relatively higher (about 4-7 degrees) TH activity. Anti-freeze proteins found in insects have a beta-helical structure, a flat ice-binding site, and repetitive ice-binding functional groups (T-X-T motif). Antifreeze proteins recently discovered in two species of longhorn beetles insects (Rhagium inquisitor and Rhagium mordax) are known to have the highest TH activity among antifreeze proteins known so far. They were found to have a longer and more repetitive ice-binding functional group (TxTxTxT) than previously known anti-freeze proteins.

The activity of the freezing control material can be determined by measuring both thermal hysteresis (TH) and recrystallization inhibition (RI). Thermal hysteresis activity is an indicator of anti-freezing activity and is expressed as the difference between the melting point and the freezing point, and can be measured using a nanoliter osmometer. In the thermal hysteresis (TH) temperature range, ice crystals no longer grow. Ice recrystallization inhibitory activity (RI) is the action of preventing small ice crystals formed by freezing from growing into larger ice crystals after thawing. The control substance binds and prevents the growth of ice crystals during the thawing process.

Japanese Patent Publication No. 10-511402 Japanese Patent No. 3694730

Lovelock JE and Bishop MWH, Nature 183:1394-1395, 1959 Polge C, Smith AU, Parkes AS, Nature 164:666-666, 1949 Miszta-Lane H, Gill P, Mirbolooki M, Lakey JRT. Cell Preserv Technol 5, 16-24,

In the current cryopreservation method including the rapid freezing method, it is difficult to preserve the cells and tissues in their complete form after freezing and thawing, and new cryopreservation substances with low toxicity are desired. In addition, since N,N-dimethylsulfoxide (DMSO) is potentially toxic as a chemical, it may induce the differentiation of specific cells, and some cells are not suitable for cryopreservation. On the other hand, the antifreeze protein or the antifreeze glycoprotein has a low level of cell cryopreservation effect than DMSO, but has the advantage of not being toxic. However, since the manufacturing cost is high, there is a limit to its application to various industrial fields (food, pharmaceutical, etc.).

Therefore, the present invention relates to a material formed through the self-assembly process of an oligopeptide composed of amino acids that play an important role in the antifreeze protein. An object of the present invention is to provide a cryopreservative that can be produced at low cost.

1. (X) a composition for cryopreservation of cells and tissues comprising an oligopeptide represented by _n:

wherein X is at least one amino acid selected from the group consisting of Ser, Asp, Glu, Cys, Ala, Val, Tyr, and Thr;

Said n is an integer of 1 to 15.

2. The composition according to item 1, wherein X is any one selected from the group consisting of Ala, Val, Tyr, and Thr.

3. The composition according to item 1, wherein n is an integer from 5 to 10.

4. The cell according to item 1, wherein the cell is a prokaryotic cell, a eukaryotic cell, a microorganism, an animal cell, a cancer cell, a sperm, an egg, an adult stem cell, an embryonic stem cell, a retrodifferentiated stem cell, an umbilical cord blood, a leukocyte, a red blood cell, a platelet, a kidney cells, liver cells, or muscle cells.

5. (X) A composition for controlling freezing, comprising the oligopeptide represented by _n:

wherein X is at least one amino acid selected from the group consisting of Ser, Asp, Glu, Cys, Ala, Val, Tyr, and Thr;

Said n is an integer of 1 to 15.

6. The composition according to item 5, wherein X is any one selected from the group consisting of Ala, Val, Tyr, and Thr.

7. The composition according to item 5, wherein n is an integer from 5 to 10.

8. The composition according to item 5, which inhibits recrystallization of ice, lowers the freezing point of water, or inhibits recrystallization of ice and lowers the freezing point of water.

9. (X) A composition for cryopreservation of food comprising an oligopeptide represented by _n:

wherein X is at least one amino acid selected from the group consisting of Ser, Asp, Glu, Cys, Ala, Val, Tyr, and Thr;

Said n is an integer of 1 to 15.

10. The composition according to item 9, wherein X is any one selected from the group consisting of Ala, Val, Tyr, and Thr.

11. The composition according to item 9, wherein n is an integer from 5 to 10.

The oligopeptide according to the present invention is a short peptide composed of amino acids that interact with ice in the freezing control protein. It has the advantage of having higher thermal history activity and ice recrystallization inhibitory activity than the known freezing control protein, but also very low manufacturing cost. Since the cell cryopreservation effect is at the same level as that of DMSO, it has a very high possibility of being used as a material for cryopreservation of cells and tissues.

1 is an electron micrograph of a material formed through self-assembly and the structure of an oligopeptide linked to five alanines showing a cryopreservation effect according to an embodiment of the present invention.
2 is an electron micrograph of the structure of an oligopeptide with five balnins linked to it and a material formed through self-assembly, showing a cryopreservation effect according to an embodiment of the present invention.
3 is an electron micrograph of a material formed through self-assembly and the structure of an oligopeptide linked to five tyrosines showing a cryopreservation effect according to an embodiment of the present invention.
4 is an electron micrograph of a material formed through self-assembly and the structure of an oligopeptide with five threonines linked thereto showing a cryopreservation effect according to an embodiment of the present invention.
5 is an experimental result related to the ice crystal growth inhibitory activity of four types of self-assembled oligopeptides according to an embodiment of the present invention.
6 is a result of comparing the cell cryopreservation effect of four types of oligopeptides made by connecting five consecutive amino acids according to an embodiment of the present invention with DMSO.
7 is a result of a comparison experiment with DMSO on the cell cryopreservation effect of four types of oligopeptides made by connecting 10 consecutive amino acids according to an embodiment of the present invention.

The present invention will now be more fully described below with reference to the accompanying drawings. However, only some, but not all, embodiments of the present invention are illustrated. Indeed, these inventions may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. As used in this specification and the appended claims, the singular forms include plural referents unless clearly indicated otherwise.

For long-term preservation of water-containing substances such as cells, tissues, or food of plants or animals, cryopreservation at a temperature of 0° C. or less is routinely used. However, when these substances containing water are frozen, the water molecules crystallize while excluding the medium of the solute or mixed substance during freezing to form ice crystals composed of only water molecules. It is known that freeze-concentration occurs. In order to prevent such freeze-concentration, the method of adding various low molecular weight compounds is implemented. For example, when cryopreserving cells, a method of adding dimethyl sulfoxide (DMSO) or glycerol as a cryoprotectant is implemented to minimize the adverse effect on cells due to intracellular crystallization that occurs during cryopreservation. is becoming That is, the cells are suspended in a physiological solution such as a culture solution containing a cryopreservative such as 5 to 20% dimethyl sulfoxide, glycerin, ethylene glycol, and propylene glycol, filled in a cryo tube and cooled, and finally -80 ° C or - It is common to cryopreserve at a cryogenic temperature of 196 °C.

An object of the present invention is to provide an ice-crystallization-inhibiting substance that has excellent ice-crystallization inhibitory activity suitable for practical use and can be produced simply, efficiently and inexpensively in a safe process that can be used for food production. In addition, the present invention provides a method for producing the ice crystallization inhibitor, a polypeptide that is an active part of the ice crystallization inhibitor, and an ice crystallization inhibitory composition containing the ice crystallization inhibitor or polypeptide, a food, a biological sample protection agent and cosmetics. It is also intended to provide.

During cryopreservation, ice recrystallization during thawing damages cell membranes and causes cell and tissue damage due to cell dehydration. Because organisms living in cold environments are more susceptible to damage from ice recrystallization, AFP or AFGP was developed. Since AF(G)P adheres to the ice surface and inhibits ice growth, it has been used as an additive in fields where ice recrystallization is a problem. In the present invention, a material for controlling freezing using a peptide derived from AF(G)P was developed.

In the present invention, “anti-freeze protein”, “anti-freeze protein” or “AFP” may be used interchangeably, and “anti-freeze glycoprotein”, “anti-freeze glycoprotein” or “AFGP” are interchangeable It can be used as “AF(G)P” by combining AFP and AFGP. AF(G)P is found in a variety of animals, plants, fungi and bacteria, and these proteins are known to inhibit ice growth and recrystallization by binding to ice crystals. This property of AF(G)P has been used to preserve biological samples at low temperatures. According to a recent study, AF(G)P has a special type of tertiary structure for binding to ice according to its type. .

Accordingly, the present inventors _{synthesized (Thr) 5} , and (Thr) ₁₀ oligopeptides, and surprisingly confirmed that these oligopeptides form nanostructures through self-assembly. From this, the present invention was completed by confirming that a nanostructure prepared from an oligopeptide having a residue capable of hydrogen bonding or hydrophobic interaction with water or ice crystals can control freezing.

In an embodiment of the present invention, (X) _{a composition comprising an oligopeptide represented by n} , wherein X is at least one amino acid selected from the group consisting of Ser, Asp, Glu, Cys, Ala, Val, Tyr, and Thr; , wherein n is an integer of 1 to 15, and provides a composition. wherein X is a negatively charged amino acid residue having a carboxyl group capable of forming a hydrogen bond with a water molecule, Asp, and Glu; Cys having a thiol group capable of forming a hydrogen bond with a water molecule; Ser in which the -CH ₃ group of Thr is substituted with -H; Val in which the -OH group of Thr is substituted with -CH _{3 ;} Ala in which the -OH group of Thr is substituted with H; Alternatively, Tyr in which -OH of Thr is substituted with H and CH ₃ is substituted with a phenol group may be used. In another embodiment of the present invention, X is Ala, Val, Tyr, or Thr.

In one embodiment of the present invention, n is an integer from 1 to 15, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In another embodiment of the present invention, n is an integer from 2 to 14, an integer from 3 to 13, an integer from 4 to 12, or an integer from 5 to 10. In another embodiment of the present invention, n is 5 or 10.

In addition, it was confirmed that when the nanostructures are administered to water, thermal hysteresis occurs and/or ice recrystallization is inhibited, and when cells are frozen and then thawed, the survival rate of cells can be increased. In particular this phenomenon occurs _{not only in the oligopeptides of (Tyr) 5} , and (Tyr) ₁₀ , but in particular (Val) ₅ , (Val) ₁₀ , (Ala) ₅ , (Ala) ₁₀ , (Thr) ₅ , and (Thr) ) was prominent in the oligopeptides of _10.

As used herein, the terms “anti-freeze”, “anti-freeze”, “freeze control”, “freeze control”, “freeze suppression” and “freeze suppression” can be used interchangeably, and lower the freezing point, It refers to the action of preventing ice from forming or slowing the rate of ice formation, preventing ice from recrystallizing, slowing down the rate of ice recrystallization, or keeping the size of ice crystals small.

"Thermal hysteresis" means a phenomenon in which the freezing temperature and the melting temperature are different. When the nucleating agent is present in the water, the freezing and melting temperatures are substantially the same. However, if the nucleating agent is not present or the nucleating agent is an antifreeze protein, the freezing temperature is lower than the melting temperature.

“Ice recrystallization” refers to the process of growing from small ice crystals to larger ice crystals, and Ostwald ripening refers to this recrystallization that occurs at room temperature and pressure due to the difference in surface energy between the crystals. -diffusion-refreezing or sublimation-diffusion-condensation mechanism.

As used herein, the terms “oligopeptide”, “polypeptide”, “peptide” and “protein” are used interchangeably and refer to a polymeric compound composed of amino acid residues covalently linked through peptide bonds. In the present invention, a peptide synthesized through a known technique may be used. The method for synthesizing the peptide may be a chemical method or a biological method, and the chemical method includes, for example, a solution phase method; Solid-phase methods including the tert-butyloxycarbonyl (Boc)/benzyl (Bzl) strategy and the 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (t-Bu) strategy (Kent SBH, Mitchell AR, Engelhard M, Merrifield RB (1979) Mechanisms) and prevention of trifluoroacetylation in solid-phase peptide synthesis (Proc Natl Acad Sci USA 76(5):2180-2184); a method of immobilizing the first amino acid on a resin and extending the peptide chain in sequence order; Alternatively, the method may be a method using a microwave, and the biological method may be a method using a microorganism, but is not limited thereto.

The nanostructure according to the present invention has a property of inhibiting freezing, and this property can be used for cryopreservation of cells and/or food. When cells or foods are frozen and thawed, cells are destroyed due to recrystallization of ice, and thus, cell viability is lowered or food texture is significantly deteriorated. However, when the nanostructure of the present invention is used as a cryopreservative for cells and/or food, it is possible to inhibit cell destruction by inhibiting ice recrystallization or inducing thermal hysteresis. As a result, in the case of thawing cryopreserved cells, the cell viability is higher than in the conventional method, and in the case of cryopreserved foods, the texture before freezing can be maintained.

In one embodiment of the present invention, cells that can be cryopreserved using the composition for cryopreservation of cells according to the present invention include prokaryotic cells; eukaryotic cells; microbe; zooblast; cancer cells, sperm; egg cell; stem cells, including adult stem cells, embryonic stem cells, and retrodifferentiated stem cells; blood cells, including cord blood, white blood cells, red blood cells, and platelets; tissue cells including, but not limited to, kidney cells, liver cells, and muscle cells.

In one embodiment of the present invention, a method for controlling freezing by adding the nanostructure according to the present invention to a solvent, a method for cryopreserving cells, including adding the nanostructure according to the present invention to a solution containing cells It provides a method for freeze-preserving food, including the step of treating the food with the nanostructure according to the present invention.

When numerical values are described as ranges in the present specification, all numerical values falling within that range are considered to be disclosed herein.

A person skilled in the art will understand the case where "about" must be used inevitably, and in this specification, the term "about" will be understood to include a numerical value within a range of error.

The term “substantially the same” used in the present invention means the same or an unrecognizable difference within the error range. As used herein, the terms “substantially absent”, “substantially free” or similar expressions refer to cases where 0 or 0 is included within an error range, or a case that is negligible for a person skilled in the art to recognize do.

Hereinafter, the present invention will be described with reference to specific examples. The following examples are intended for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

Example 1. Nanostructure of an oligopeptide linked to 5 alanines

For the oligopeptide with 5 or 10 alanine linked, a peptide synthesized with a purity of 98% or higher by Anygen Co., Ltd. was used, and 2 mg of the peptide was added to 100 μL of distilled water, and the result was transparent after undergoing a water bath at an elevated temperature of 50 degrees and sonic for 2-3 minutes. After confirming that the solution was in the solution phase, it was observed that the solution was suspended when left at room temperature for more than 30 minutes. For electron microscopic analysis, about 5 μL of this solution was taken and placed on a grid for electron microscopy analysis, and then dried at room temperature for 2 hours or more. Uranyl acetate solution (5 μL) was added to the dye, and after further drying, the resulting structure was confirmed through electron microscopic analysis (FIG. 1).

Example 2. Nanostructure of an oligopeptide linked to 5 valines

For the oligopeptide with 5 or 10 valines, a peptide synthesized with a purity of 98% or higher by Anygen Co., Ltd. was used, and 2 mg of the peptide was put in 100 μL of distilled water, and it was subjected to a water bath at an elevated temperature of 50 degrees and sonic for 2-3 minutes. After confirming that the solution was in the solution phase, it was observed that the solution was suspended when left at room temperature for more than 30 minutes. For electron microscopic analysis, about 5 μL of this solution was taken and placed on a grid for electron microscopy analysis, and then dried at room temperature for 2 hours or more. Uranyl acetate solution (5 μL) was added to the dye, and after further drying, the resulting structure was confirmed through electron microscopic analysis (FIG. 2).

Example 3. Nanostructure of oligopeptide with 5 tyrosine linked

For the oligopeptide with 5 or 10 tyrosine chains, a peptide synthesized with a purity of 98% or higher by Anygen Co., Ltd. was used, and 2 mg of the peptide was put in 100 μL of distilled water, and it was subjected to a water bath at an elevated temperature of 50 degrees and sonic for 2-3 minutes. After confirming that the solution was in the solution phase, it was observed that the solution was suspended when left at room temperature for more than 30 minutes. For electron microscopic analysis, about 5 μL of this solution was taken and placed on a grid for electron microscopy analysis, and then dried at room temperature for 2 hours or more. Uranyl acetate solution (5 μL) was added to the dye, followed by further drying, and then the resulting structure was confirmed through electron microscopic analysis (FIG. 3).

Example 4. Nanostructure of an oligopeptide with 5 threonines linked

For the oligopeptide with 5 or 10 threonine linked, a peptide synthesized with a purity of 98% or higher by Anygen Co., Ltd. was used, and 2 mg of the peptide was added to 100 μL of distilled water, followed by a water bath at an elevated temperature of 50 degrees and sonic for 2-3 minutes. After confirming that it became a transparent solution phase, it was observed that the solution was suspended when left at room temperature for more than 30 minutes. For electron microscopic analysis, about 5 μL of this solution was taken and placed on a grid for electron microscopy analysis, and then dried at room temperature for 2 hours or more. Uranyl acetate solution (5 μL) was added to the dye, and after further drying, the resulting structure was confirmed through electron microscopic analysis (Fig. 4).

Example 5 Thermal hysteresis ( TH) activity

A nanoliter osmometer (Otago osmometers, New Zealand) was used to observe the change of the ice crystal morphology by the anti-freezing protein. After filling the sample chamber with immersion oil, the measurement sample was placed on the oil. The sample chamber was placed on the stage and flash frozen to -20°C and the temperatures were raised slowly. Most of the ice crystals melted, leaving only the crystals to be observed. The formation of ice crystals was observed while slowly lowering the temperature of the stage again. The temperature at which ice crystals grow rapidly while the ice crystals do not grow even when the temperature is lowered (freezing temperature) was measured (Kuiper et al., 2003).

As a result, as shown in Table 1, it was confirmed that the nanostructure formed of the oligopeptide according to the present invention lowered the freezing point of water.

Example 6. RI activity

For RI activity (Recrystallization inhibition activity), the Smallwood method using sucrose was performed using a Linkam TMHS600 cold stage (Linkam scientific instruments, Surrey, UK) connected to an Olympus BX51 uplight microscope. After putting 2 μl of the sample in 30% sucrose, it was placed between two glass covers. The temperature was lowered to -80°C at a rate of 90°C and maintained for 1 minute. Then, the temperature was adjusted to -6°C and maintained for 60 minutes.

As a result, as shown in FIG. 5 , the growth of single ice crystals in distilled water and the growth of ice crystals in an aqueous solution containing an oligopeptide self-assembled material are completely different, and the growth of ice crystals is clearly different depending on the type of amino acid. The difference was confirmed (Fig. 5). This indicates that the nanostructures according to the present invention have an activity of inhibiting recrystallization of ice.

Example 7. Cell cryopreservation experiment

By subculture of HUVEC cells, ^{more than 5x10 4} cells were secured (n=1 : 1x 10 ⁴ cells), and a freezing solution [FBS (9 ml) + DMSO (1 ml)] was prepared. After taking 1 ml, 1x10 ⁴ pellets were added to the cells (in falcon tube). 1ml of the freezing solution containing cells was transferred to a vial and placed in a freezing container ([1x10^5ea/Freezing solution 1ml] total 5ea per vial). Through the container, the temperature of the vial was cooled to -80°C at a rate of -1°C/min in a deepfreezer, and after 1 day, it was transferred to a liquid nitrogen container and stored for a certain period (1 day, 3 days, 10 days). After thawing, the freezing solution was dissolved in a hot water bath at 37°C for 2 mins, and the cells were collected by centrifugation at 1000 rpm for 5 mins. thawed for 2 minutes. 2) The freezing solution containing the cells was transferred to a 15 ml tube and mixed with 9 ml media. 3) After centrifugation at 1000 rpm for 5 minutes, the freezing solution was removed by removing the supernatant. 4) Fresh media was put into the cell pellet, and after cell counting, the dish was seeded.

Cell viability was measured through MTS, and cell viability was measured by replacing it with one solution MTS solution, and the results are shown in FIGS. 6 and 7 .

The nanostructures of the oligopeptide composed of 5 alanine, valine, tyrosine, or threonine and the nanostructure of the oligopeptide composed of 10 alanine, valine, tyrosine, or threonine were all negative controls (without antifreeze administration). group), it was confirmed that a significantly higher anti-freeze effect was observed. In addition, it was confirmed that the cell viability was significantly higher in all groups except for the nanostructures made of tyrosine, even when compared to the DMSO treatment group, which has a well-known anti-freeze efficacy. In the case of the nanostructure composed of tyrosine, although it exhibited a low cell viability compared to DMSO, the nanostructure of the present invention has substantially no cytotoxicity, considering that it is significantly lower than that of DMSO, cells, tissues, or organs This suggests that it can be usefully used during freeze-drying.

Claims (11)

(X) A composition for cryopreservation of cells and tissues comprising a self-assembled construct of a plurality of oligopeptides represented by _n:
wherein X is at least one amino acid selected from the group consisting of Ser, Asp, Glu, Cys, Ala, Val, Tyr, and Thr;
Said n is an integer of 2 to 15. The composition of claim 1, wherein X is any one selected from the group consisting of Ala, Val, Tyr, and Thr. The composition of claim 1, wherein n is an integer from 5 to 10. The method according to claim 1, wherein the cell is a prokaryotic cell, a eukaryotic cell, a microorganism, an animal cell, a cancer cell, a sperm, an egg, an adult stem cell, an embryonic stem cell, a retrodifferentiated stem cell, umbilical cord blood, a leukocyte, a red blood cell, a platelet, a kidney cell, liver cells, or muscle cells. (X) A composition for controlling freezing, comprising a self-assembled structure of a plurality of oligopeptides represented by _n:
wherein X is at least one amino acid selected from the group consisting of Ser, Asp, Glu, Cys, Ala, Val, Tyr, and Thr;
Said n is an integer of 2 to 15. The composition of claim 5, wherein X is any one selected from the group consisting of Ala, Val, Tyr, and Thr. The composition of claim 5, wherein n is an integer from 5 to 10. The composition of claim 5 , wherein the composition inhibits recrystallization of ice, lowers the freezing point of water, or inhibits recrystallization of ice and lowers the freezing point of water. (X) A composition for cryopreservation of food, comprising a self-assembled construct of a plurality of oligopeptides represented by _n:
wherein X is at least one amino acid selected from the group consisting of Ser, Asp, Glu, Cys, Ala, Val, Tyr, and Thr;
Said n is an integer of 2 to 15. The composition of claim 9, wherein X is any one selected from the group consisting of Ala, Val, Tyr, and Thr. The composition of claim 9, wherein n is an integer from 5 to 10.

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