JP4588598B2 - Cell cryopreservation composition - Google Patents

Description

Background of the Invention

FIELD OF THE INVENTION The present invention relates to a composition for cell cryopreservation that provides good cell viability.

BACKGROUND ART Cryopreservation of cultured cells is performed for the purpose of preventing cell alteration due to passage, prevention of contamination due to germs accompanying passage, and elimination of complexity of passage maintenance. For example, in the case of animal cells, conventionally, the cryopreservation technique for animal cells has been to suspend cells in a culture solution containing 5-15% dimethyl sulfoxide (DMSO) or glycerin, or bovine serum, and place this in a freezing tube or ampoule. Generally, a method of enclosing, cooling at a rate of about −1 ° C./min using a program freezer, and finally storing in liquid nitrogen (−196 ° C.) has been performed.

However, when a culture solution is used during cryopreservation, the viability of frozen cells is lowered, and it often takes time to grow to the desired number of cells when freeze-thawed. On the other hand, if serum is used for cryopreservation, the serum is very expensive, and therefore the storage cost for freezing a large amount of many types of cell lines increases. In addition, since serum contains many components that are not essential for cell preservation such as various cytokines, growth factors, hormones, ions, and the like, there is a risk of altering the properties of the preserved cells. Further, when using serum, even when using albumin obtained by purifying serum or plasma, problems such as the risk of viral infection still exist.
Furthermore, when using a program freezer, the cell freezing procedure itself has to be complicated, and there is also a problem that the survival rate of the frozen cells decreases with time.

  As a cryopreservation solution that does not require the use of a program freezer, for example, Japanese Patent Laid-Open Publication No. Hei 6-46840 discloses that there is no need to use a program freezer, and it can be transferred directly from ice to −80 ° C. Possible cryopreservation solutions have been proposed. However, this cryopreservation solution contained serum components.

  For this reason, a cell cryopreservation solution that can be transferred directly from ice to a −80 ° C. freezer without requiring the use of a program freezer and that can freeze cells for a long period of time, There has been a demand for a culture solution and a serum that does not require serum and serum-derived components.

Conventionally, amino acids, proteins, sugars, alcohols and the like are known as components used in cell cryopreservation solutions. For example, Shinozaki, K. et al .: FEBS Let t., 461 (3), 205, (1999) and Takagi, H. et al .: Appl Environ Microbial., 69 (11), 6527 (2003) It has been reported that one of the amino acids, proline, has a cryoprotective effect. Japanese Patent Laid-Open Publication No. 2002-233356 discloses a cell cryopreservation solution using saccharides (particularly glucose).
However, as far as the present inventors know, the cryoprotective effect of these components alone is not sufficient as compared with the serum components.

  On the other hand, it has been confirmed that sericin, which is a silk protein, has a cell cryoprotective effect in Japanese Patent Publication No. 2002-101869.

Summary of the Invention

  When the present inventors use sericin, which is a silk protein, in combination with amino acids and / or saccharides, it is possible to achieve excellent cell freezing equivalent to the case of using serum-derived components without using serum-derived components. It was found that a protective effect can be shown. This effect became more prominent by selecting and using sericin and an amino acid and / or saccharide with a specific amount and a specific type. The present invention is based on these findings.

Therefore, an object of the present invention is to provide a cell cryopreservation solution capable of freezing cells over a long period of time without using serum and serum-derived components.
The composition for cell cryopreservation according to the present invention comprises at least sericin and one or more components selected from the group consisting of amino acids and saccharides.
The cell cryopreservation method according to the present invention comprises placing the target cell in the cell cryopreservation composition and cryopreserving it.

  The composition for cell cryopreservation according to the present invention exhibits an excellent cryoprotective effect as compared to the case of sericin alone, and is equivalent to or higher than the case of using serum-derived components. It shows the freeze protection effect. Moreover, since the composition for cell cryopreservation of the present invention does not contain serum or serum-derived components, there is little possibility of changing the properties of stored cells and there is almost no risk of contamination with unknown viruses. Since the composition of the present invention is clear, the composition has high safety, is stable in quality, and is advantageous for stable supply. Furthermore, according to the composition of the present invention, the cells can be frozen in a −80 ° C. freezer directly without using a program freezer after filling the cells in a freezing tube or ampoule. Thus, the operation at the time of freezing can be simplified. Moreover, according to the composition of the present invention, it is possible to cryopreserve the target cells for a long period of time, and a high cell survival rate can be achieved even after cryopreservation. Thereby, the time required to grow to the target number of cells can be shortened.

Detailed description of the invention

Cell cryopreservation composition The cell cryopreservation composition according to the present invention comprises, as described above, at least sericin and at least one component selected from the group consisting of amino acids and saccharides. Therefore, as long as the said component is included, the composition by this invention may further contain the other cell cryoprotective component, the buffer component, and other arbitrary components. The composition for cell cryopreservation according to the present invention is usually used as a cryopreservation solution which is in an aqueous solution state by adding water. Therefore, the composition for cell cryopreservation here includes not only the solid state but also the liquid state.

  Here, “cell cryopreservation” refers to freezing and storing cells for the purpose of maintaining the cells for a long time without subculture, and the freezing method and the storage method are not particularly limited. Accordingly, for example, in addition to the case where cells are directly put into the freezer and frozen and stored without using the program freezer, the case where the cells are frozen and stored using the program freezer is also included.

  In the present invention, the “cell” is not particularly limited as long as it may be subjected to cryopreservation, and may be any of microorganisms, bacteria, animal cells, and plant cells. In the present invention, the “cell” is preferably an animal cell. The animals referred to here include mammals including humans, fish, birds, insects and the like. The “animal cell” in the present invention is any type of cell, such as a cell established as a cultured cell, a normal cell not obtained from a biological tissue, or a transformed cell obtained by a genetic engineering technique. It may be.

Sericin In the present invention, sericin may be derived from a natural product, or may be artificially synthesized by conventional chemical and / or genetic engineering techniques. Is included. Therefore, for example, sericin produced by a rabbit or a barb can be used as sericin. Preferably, sericin is purified to a high purity and substantially free of impurities.

  The sericin in the present invention includes sericin and a hydrolyzate thereof. Here, the hydrolyzate of sericin can be obtained by subjecting a non-hydrolyzed product of sericin to hydrolysis using conventional means such as alkali or enzyme.

  In the present invention, sericin can be obtained by extraction from straw or raw silk by a conventional extraction method. Specifically, for example, sericin can be obtained by using, for example, silkworm, raw silk, or silk fabric as a raw material and partially hydrolyzing and extracting it with hot water, acid, alkali, enzyme, or the like. The obtained sericin is purified to a high purity (for example, a total nitrogen amount of 13% or more of sericin solids) by the method described in Japanese Patent No. 3011759 as necessary, and prepared in a solid form such as a powder. The In the present invention, this solid form of sericin can be blended as it is in the cell cryopreservation composition, but if necessary, a solution in which solid form of sericin is dissolved or suspended in water or a buffer solution You may mix | blend with a composition for cell cryopreservation as a state.

  In the present invention, the average molecular weight of sericin is not particularly limited, but is preferably 3,000 to 300,000, more preferably 5,000 to 100,000, and still more preferably 10,000 to 50, 000. When the average molecular weight of sericin is 300,000 or less, the aqueous solution can be prevented from gelation at a concentration of about 0.5% by weight, and the use form can be prevented from being limited. In addition, an average molecular weight of sericin of 3,000 or more is advantageous for exerting a cell cryoprotective effect.

  In the present invention, the content of sericin in the composition for cell cryopreservation is not particularly limited, but preferably contains sericin so that the final concentration is 0.01 to 10.0% by weight, Preferably, sericin is contained so that the final concentration is 0.1 to 1% by weight. A final concentration of sericin of 0.01% by weight or more is advantageous to exhibit a sufficient cryoprotective effect, and 10.0% by weight or less is advantageous for suppressing an increase in storage cost. It is.

  Note that, here, that the composition for cell cryopreservation contains sericin so that the final concentration is 0.01 to 10.0% by weight means that the composition for cell cryopreservation is used for cell freezing treatment. When actually used as a preservation solution, it means that the sericin concentration in the solution is 0.01 to 10.0% by weight. The same applies to amino acids and saccharides.

Amino acids In the present invention, amino acids include both optical isomers, that is, D-form and L-form. The amino acids here include not only 20 kinds of α-amino acids constituting natural proteins, but also other α-amino acids, β-, γ-, δ-amino acids and unnatural amino acids. May be. Thus, for example, the amino acid is a group consisting of alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, glutamine, asparagine, tyrosine, lysine, arginine, aspartic acid, and glutamic acid. It can be one or more selected from more. Preferably, the amino acid is one or more selected from the group consisting of proline, glutamine, serine, threonine, glycine, and alanine, and more preferably, the amino acid is selected from the group consisting of proline and glutamine. One or more types.

  Further, in the present invention, the term “amino acid” is used to include amino acid derivatives in addition to the amino acid itself. Examples of amino acid derivatives include amino acid salts and amino acid solvates. Here, as the amino acid salt, for example, alkali metal or alkaline earth metal salt such as sodium salt, potassium salt or calcium salt, hydrofluoride, hydrochloride, hydrobromide, hydroiodic acid Salts such as hydrohalides, nitrates, perchlorates, sulfates, phosphates and other inorganic acid salts, fumaric acid, succinate, citrate, oxalate, maleate, acetate And organic acid salts such as lactate and ascorbate. Examples of solvates of amino acids include hydrates, alcohol solvates (for example, methanol solvates, ethanol solvates), and ether solvates (for example, diethyl ether solvates).

  In the present invention, the content of the amino acid in the composition for cell cryopreservation is not particularly limited, but it is preferable that the amino acid is contained so that the final concentration is 0.01 to 10.0% by weight, Preferably, amino acids are included so that the final concentration is 0.1 to 1.0% by weight. The final amino acid concentration within the above range is advantageous for exhibiting a sufficient cryoprotective effect.

In the present invention, the saccharide includes any of monosaccharides, oligosaccharides such as disaccharides, polysaccharides and the like. Therefore, as saccharides, for example, glucose, xylose, arabinose, fructose, galactose, mannose, mannitol, sorbitol, xylitol, myo-inositol, trehalose, sucrose, lactose, maltose, cellobiose, lactitol, maltitol, methylcellulose, carboxymethylcellulose, Examples thereof include at least one selected from the group consisting of dextran, glycogen, amylose, amylopectin, inulin, sodium alginate, ethyl cellulose, hydroxyethyl cellulose, raffinose, stachyose, xanthan gum, glucosamine, and galactosamine. Preferably, the saccharide is a maltose type disaccharide. Here, maltose type disaccharide includes, for example, maltose, cellobiose, lactose and the like. More preferably, the saccharide is maltose.

  In the present invention, the content of saccharide in the composition for cell cryopreservation is not particularly limited, but it is preferable that the saccharide is contained so that the final concentration is 0.01 to 10.0% by weight, Saccharides are preferably contained so that the final concentration is 0.1 to 1.0% by weight. The final concentration of the saccharide within the above range is advantageous for exhibiting a sufficient cryoprotective effect.

  According to one preferred embodiment of the present invention, the composition for cell cryopreservation according to the present invention comprises at least sericin, an amino acid, and a saccharide. At this time, the compounding ratio (weight ratio) of these components in the composition for cell cryopreservation is typically 1: 0.01 to 10.0: 0.01 as the weight ratio of sericin: amino acid: saccharide. To 10.0, more preferably 1: 0.1 to 1.0: 0.1 to 1.0, and most preferably 1: 0.4 to 0.8: 0.3 to 0.7. .

Other Cell Cryoprotective Components The composition for cell cryopreservation according to the present invention can further comprise other cell cryoprotective components. Here, the other cell cryoprotective component is, for example, selected from the group consisting of dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol and glycerol. Two or more of these may be used in combination.

  The content of other cell cryoprotective components in the composition for cell cryopreservation according to the present invention can be appropriately changed depending on the type of components used, but preferably the final concentration is 1.0-40. The amount is 0% by weight, more preferably 5.0 to 20.0% by weight. If the content of other cell cryoprotective components is 1.0% by weight or more, it is advantageous for obtaining a sufficient cryoprotective effect, and if it is 40.0% by weight or less, the toxicity to cells is reduced. preferable.

  When the other cell cryoprotective component is dimethyl sulfoxide, the content of dimethyl sulfoxide in the composition for cell cryopreservation is preferably 5.0 to 15.0% by weight. When the other cell cryoprotective component is glycerol, the content of glycerol in the composition for cell cryopreservation is preferably 5.0 to 20.0% by weight. From the viewpoint of cell cryoprotective effect, dimethyl sulfoxide is more preferable than glycerol.

Buffer component The composition for cell cryopreservation according to the present invention may further comprise a buffer component. When the composition for cell cryopreservation is used as a cryopreservation solution in a solution state, the buffer component is 6.0 to 8.5, preferably 7.0 at a room temperature (for example, 25 ° C.). Desirably, it is selected to be ˜7.5. The pH range being within the above range is advantageous in that the influence on cell growth can be reduced.

  In the present invention, the buffer component may be any type as long as the above pH condition is satisfied. Specific examples of the buffer component include phosphate (for example, PBS), BES, TES, acetamide glycine, glycinamide, glycylglycine, TRICINE, trisethanolamine, veronal, and HEPES. Preferably, the buffer component is selected from the group consisting of PBS, TRICINE, and HEPES, more preferably HEPES.

  When the composition for cell cryopreservation is a cryopreservation solution in a solution state, the concentration of the buffer component that can be contained therein is preferably 1 to 1000 mM, more preferably 1 to 200 mM, and still more preferably 5 ~ 200 mM, even more preferably 5-50 mM. When the concentration of the buffer component is within the above range, a buffer effect can be obtained in the cryopreservation solution, and at the same time, the influence on the cells can be suppressed.

Other Optional Components The composition for cell cryopreservation according to the present invention can further comprise other optional components.
Examples of other optional components include additional components such as peptides, other proteins, sugar alcohols, amino sugars, glycoproteins, alcohols, pH adjusters, moisturizers, preservatives, viscosity adjusters, and the like. . Two or more of these may be used in combination. Other optional components can be appropriately selected in terms of the type and amount used in consideration of the type of cells to be cryopreserved, the freezing method, the freezing period, and the like.
The composition for cell cryopreservation according to the present invention preferably contains substantially no conventional culture solution or serum-derived components, but if necessary, denies the use of all or part of them. is not.

According to one preferred embodiment of the invention, the cell cryopreservation composition comprises components (a) to (d) in the following amounts:
(A) Sericin 0.01 to 10.0% by weight;
(B) one or more components selected from the group consisting of amino acids and sugars
0.01 to 10.0% by weight;
(C) an additional cell cryoprotective component selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol
1.0 to 40.0% by weight;
(D) Buffer component 1-1000 mM.

According to one more preferred embodiment of the present invention, the cell cryopreservation composition comprises components (a) to (d) in the following amounts:
(A) Sericin 0.01 to 10.0% by weight;
(B1) Amino acid 0.01 to 10.0% by weight;
(B2) 0.01 to 10.0% by weight of sugars;
(C) an additional cell cryoprotective component selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol
1.0 to 40.0% by weight;
(D) Buffer component 1-1000 mM.

  The composition for cell cryopreservation according to the present invention can be obtained by mixing the above-mentioned components into a solid state as it is, or, if necessary, by dissolving in water to obtain an aqueous solution state.

Cell Cryopreservation The composition for cell cryopreservation according to the present invention is used in a cell cryopreservation treatment. That is, when carrying out the cryopreservation processing of the cells, the target cells are suspended in the composition according to the present invention in a solution state, and the cells are kept under the conditions for cryopreservation and frozen. When the cells are needed, the frozen cells and the composition according to the present invention can be thawed and the cells can be recovered therefrom.

Therefore, according to the present invention, there is provided a method for cryopreserving cells, which comprises placing a target cell in the cell cryopreservation composition according to the present invention and cryopreserving the target cell.
Here, when the target cell is put into the composition for cell cryopreservation, the cell concentration is desirably 10,000 to 10,000,000 cells / ml.

In the present invention, the method for freezing cells is not particularly limited. For example, the suspension of the target cells in the composition for cell cryopreservation in a solution state according to the present invention is placed in a freezing tube and directly put in an ultra-low temperature freezer at −80 ° C. to freeze the cells. be able to. Alternatively, the freezing tube may be placed in a program freezer to slowly freeze the cells. The storage of the frozen cells can be performed, for example, by keeping the cells at a frozen temperature (for example, −80 ° C.).
The method for thawing the cells is not particularly limited. For example, the cells may be thawed by thawing a cryopreserved freezing tube in a 37 ° C. water bath.

  According to another aspect of the present invention, there is provided use of a combination of sericin and one or more components selected from the group consisting of amino acids and saccharides for increasing cell viability in cryopreservation of cells. The Here, it is preferable that the combination of components comprises at least sericin, an amino acid, and a saccharide.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Preparation of sericin After thoroughly washing the silk thread produced by Bombyx mori , 100 g of silk thread was subjected to heat treatment at 96 ° C. for 2 hours in 2 L of 0.2% aqueous sodium hydrogen carbonate solution as an extraction solvent. And sericin hydrolyzate was extracted. The obtained sericin hydrolyzate extract was filtered using a filter having an average pore size of 0.2 μm to remove aggregates, and then the filtrate was desalted with a reverse osmosis membrane to give a colorless transparent solution having a concentration of 0.2%. A sericin aqueous solution was obtained.
Next, this aqueous solution was concentrated using an evaporator until the sericin concentration was about 2%, and then lyophilized to obtain a sericin powder having a purity of 90% or more and an average molecular weight of about 30,000. .
The average molecular weight of the obtained sericin was determined by gel filtration chromatography (LC-9A (manufactured by Shimadzu Corporation) as a measuring instrument and Superdex 75HR 10/30 (manufactured by Pharmacia) as a column). It was measured.

Sericin powder was obtained in the same manner as described above except that the extraction solvent used for extracting sericin from the silk thread was distilled water. In this case, the average molecular weight of the obtained sericin was about 100,000.
A sericin powder was obtained in the same manner as above except that the extraction solvent used for extracting sericin from the silk thread was a 0.5% aqueous sodium bicarbonate solution. In this case, the average molecular weight of the obtained sericin was about 10,000.
The relationship between the type of the extraction solvent and the average molecular weight of the obtained sericin was as shown in Table 1 below.

Example 1: Examination of ingredients a (saccharide)
Preparation of composition for cell cryopreservation According to the composition shown in Table 2 below, a composition for cell cryopreservation in a solution state (hereinafter sometimes referred to as “cryopreservation solution” or simply “preservation solution”) is prepared, It was set as the cryopreservation liquid a1-a9.
Here, sericin having an average molecular weight of about 30,000 obtained above was used.
PBS (phosphate concentration 9.5 mM) was used as a buffer component.
As a serum component, fetal bovine serum (hereinafter FBS) (obtained from GIBCO) was used.

For the cryopreservation test, mouse myeloma P3U1 cells (obtained from Human Science Foundation) were cultured using RPMI1640 medium (Asahi Techno Glass Co., Ltd.) containing 10.0% FBS.
Cells were suspended in each cryopreservation solution so that the cell concentration was about 1.0 × 10 ⁶ cells / ml, and 1 ml each was added to a freezing tube (Asahi Techno Glass Co., Ltd.). Next, the frozen tube was stored frozen for 30 days using a −80 ° C. freezer, and then the tube was immersed in a 37 ° C. water bath and thawed.
The viable cell density in the thawed stock solution was calculated by the trypan blue dye exclusion method, and the survival rate for each frozen stock solution was determined.
The results were as shown in Table 3.
The preservation solution a4 to which maltose was added showed an excellent cryoprotective effect as compared with preservation solutions to which only sericin (preservation solution a2) or other saccharides were added.

Example 2: Examination of component b (amino acid)
Cryopreservation solutions b1 to b9 were prepared in the same manner as in Example 1 except that the composition shown in Table 4 below was followed.

The cryopreservation test was performed in the same manner as in Example 1 except that the preservation solutions b1 to b9 were used as the cryopreservation solutions.
The results were as shown in Table 5.
Stock solution b7 to which proline and glutamine were added at 0.3% each showed an excellent cryoprotective effect compared to a stock solution to which only sericin or amino acid was added alone. In addition, the cryoprotective effect of the preservation solution b7 was comparable to that using serum (preservation solution b1).

Example 3: Examination of ingredients c (other cell cryoprotective ingredients)
Cryopreservation solutions c1 to c6 were prepared in the same manner as in Example 1 except that the composition shown in Table 6 was followed.

The cryopreservation test was performed in the same manner as in Example 1 except that the preservation solutions c1 to c6 were used as the cryopreservation solutions.
The results were as shown in Table 7.
The preservation solutions (preservation solutions c2 to c5) to which dimethyl sulfoxide, ethylene glycol, propylene glycol and glycerol were added showed excellent cryoprotective effects.

Example 4: Examination of ingredients d (buffer ingredient)
Cryopreservation solutions d1 to d6 were prepared in the same manner as in Example 1 except that the composition shown in Table 8 was followed.

The cryopreservation test was performed in the same manner as in Example 1 except that the preservation solutions d1 to d6 were used as the cryopreservation solutions.
The results were as shown in Table 9.
Stock solutions (d2 to d6) supplemented with PBS, HEPES, and TRICINE showed excellent cryoprotective effects. Moreover, the cryoprotective effect which was excellent in any buffer component density | concentration (d4-d6) measured was shown.

Example 5: Examination of ingredients e (average molecular weight of sericin)
Cryopreservation solutions e1 to e6 were prepared in the same manner as in Example 1 except that sericin having different average molecular weights was prepared and the composition shown in Table 10 below was followed.
Here, sericin having an average molecular weight of about 10,000, about 30,000 and about 100,000 obtained in the section “Preparation of sericin” was used, respectively.

The cryopreservation test was also conducted in the same manner as in Example 1 except that the preservation solutions e1 to e6 were used as the cryopreservation solutions.
The results were as shown in Table 11.
The cryopreservation solution (preservation solution e3) to which sericin having an average molecular weight of 30,000 was added showed an excellent cryoprotective effect as compared with the case of adding lower molecular weight sericin and higher molecular weight sericin.

Example 6: Cryopreservation performance of other cell types Similar to the cryopreservation test of Example 1 except that the cells used were changed to other cell types and the cryopreservation solutions were the cryopreservation solutions e1, e3 and e6 in Example 5. The test was conducted.
As other cell types used here, normal human skin fibroblasts (NHDF) (obtained from Bio Whittaker), rat adrenal medullary brown cell line (PC-12) (obtained from Human Science Foundation) Spodopera frugiperda- derived insect cells (Sf-9) (obtained from GIBCO), CHO (Chinese Hamster ovary) cells (obtained from RIKEN), HepG2 cells (obtained from RIKEN), hybridoma cells (Cytotechnology 10, 15 -23, 1992), human epidermal keratinocytes (HEK) (obtained from Cell APPLICATIONS, INC.) And rabbit cornea primary cells (RC4) (obtained from RIKEN). NHDF is used for wound treatment and cytotoxicity research. Hybridoma cells produce monoclonal antibodies, and CHO cells and Sf-9 cells are transformed by genetic engineering techniques to produce physiologically active substances. These cells are used industrially as protein production means. Furthermore, HepG2 cells are useful as models for artificial livers, and PC-12 cells are useful as materials for investigating the process of differentiation into neurons. HEK cells are used for skin irritation tests, and RC4 cells are used for eye toxicity tests such as cosmetics and agricultural chemicals.
Moreover, about the hybridoma cell, after repeating freezing and thawing operation again after normal freezing and thawing operation, the survival rate was measured.
The results were as shown in Table 12.
The cryopreservation solution (preservation solution e3) according to the present invention showed the same survival rate as when serum was used (preservation solution e1) in each cell having a different origin.

Claims (11)

A composition for cell cryopreservation comprising at least sericin, an amino acid, and a saccharide,
The sugar is maltose ,
A composition for cell cryopreservation , wherein the amino acids are proline and glutamine . The composition for cell cryopreservation according to claim 1, wherein the average molecular weight of sericin is 3,000 to 300,000. The composition for cell cryopreservation according to claim 1 or 2 , comprising sericin at a final concentration of 0.01 to 10.0% by weight. The composition for cell cryopreservation according to any one of claims 1 to 3 , comprising an amino acid at a final concentration of 0.01 to 10.0% by weight. The composition for cell cryopreservation according to any one of claims 1 to 4 , comprising a saccharide such that the final concentration is 0.01 to 10.0% by weight. The composition for cell cryopreservation according to any one of claims 1 to 5 , further comprising an additional cell cryoprotective component selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol and glycerol. The composition for cell cryopreservation according to any one of claims 1 to 6 , which is an aqueous solution. The composition for cell cryopreservation according to any one of claims 1 to 7 , further comprising a buffer component so that the pH of the solution is 6.0 to 8.5 at room temperature. . The composition for cell cryopreservation according to any one of claims 1 to 8 , comprising the components (a) to (d) in the following amounts:
(A) Sericin 0.01 to 10.0% by weight;
(B1) Amino acid 0.01 to 10.0% by weight;
(B2) 0.01 to 10.0% by weight of sugars;
(C) an additional cell cryoprotective component selected from the group consisting of dimethyl sulfoxide, ethylene glycol, propylene glycol, and glycerol
1.0 to 40.0% by weight;
(D) Buffer component 1-1000 mM. A method for cryopreserving a cell, comprising placing the target cell in the cell cryopreservation composition according to any one of claims 1 to 9 , and cryopreserving the target cell. Use of a combination of sericin, proline and glutamine , and maltose to increase cell viability in cryopreservation of cells .