JP2020162533A - Cell cryopreservation method, cell cryopreservation composition, and cell cryopreservation culture medium - Google Patents

Abstract

To provide a cell cryopreservation method with excellent cell survival rates after thawing.SOLUTION: A cell cryopreservation method includes the steps of disposing cells in a culture medium containing maltotriose, and freezing the culture medium with the cells disposed therein.SELECTED DRAWING: None

Description

The present invention relates to a cell cryopreservation method, a cell cryopreservation composition, and a cell cryopreservation medium.

As one of the methods for preserving cells, cryopreservation in which cells are frozen and preserved is known and is widely used. In addition, various techniques have been developed regarding the cryopreservation method. For example, when animal cells are cryopreserved, dimethyl sulfoxide (DMSO) or glycerin is conventionally added to the culture medium, but when DMSO or glycerin is used, the cells may be damaged after thawing. Therefore, means for improving the problem are being developed. For example, Patent Document 1 describes a stable vitrification state for an animal cell cryopreservation solution containing an amphoteric polymer compound in which ε-poly-L-lysine is carboxylated for the purpose of increasing the survival rate of cells after thawing. A cryopreservation method using an agent is disclosed. Further, Patent Document 2 discloses a cryopreservation method using a cell cryopreservation composition containing at least sericin, which is a protein produced by silkworms, for the purpose of increasing the survival rate of cells after thawing. ..

JP-A-2016-220672 Japanese Unexamined Patent Publication No. 2006-115837

However, the methods described in Patent Documents 1 and 2 use special compounds. These methods are not always readily available, as preparing the compound is laborious and costly. Therefore, there is still a need for other readily available alternatives.

Therefore, the present invention has been made in view of the above problems, and an object thereof is a cell cryopreservation method and a cell cryopreservation composition which can be easily used and have an excellent cell viability after thawing. The purpose is to provide a substance and a medium for cryopreservation of cells.

The cell cryopreservation method according to the present invention includes a step of arranging cells in a medium containing maltotriose and a step of freezing the medium in which the cells are arranged in order to solve the above-mentioned problems.

Further, in one aspect of the cell cryopreservation method according to the present invention, the medium contains maltose.

Further, in one aspect of the cell cryopreservation method according to the present invention, the content of maltotriose in the above medium is 1% by mass or more and 30% by mass or less.

Further, in one aspect of the cell cryopreservation method according to the present invention, the total content of maltotriose and maltose in the above medium is 1% by mass or more and 40% by mass or less.

Further, in one aspect of the cell cryopreservation method according to the present invention, the cells are mammalian cells.

The cell cryopreservation composition according to the present invention is a cell cryopreservation composition added to a medium for cryopreserving cells in order to solve the above problems, and contains maltotriose.

Further, in one aspect of the composition for cryopreservation of cells in the present invention, maltose is further contained.

The cell cryopreservation medium according to the present invention contains maltotriose in the medium in order to solve the above-mentioned problems.

According to the cell cryopreservation method and the cell cryopreservation composition according to the present invention, the cell viability after thawing is excellent.

[Cell cryopreservation method]
An embodiment of the cell cryopreservation method according to the present invention will be described below.

The cell cryopreservation method of the present embodiment includes at least a step of arranging cells in the medium and a step of freezing the medium in which the cells are arranged.

(Culture medium)
In the cell cryopreservation method of the present embodiment, the medium in which the cells are arranged contains maltotriose. The content of maltotriose in the medium is preferably 1% by mass or more and 30% by mass or less, more preferably 1.5% by mass or more and 20% by mass or less, and further preferably 4% by mass or more and 12.5% by mass or less.

A commercially available maltotriose can be used. Further, it may be prepared by performing an enzymatic reaction using starch as a raw material. The maltotriose added to the medium may or may not be purified.

In addition to maltotriose, the medium may contain monosaccharides, disaccharides, trisaccharides other than maltotriose, and other saccharides selected from polysaccharides of tetrasaccharides and above. Other sugars include, for example, glucose, xylose, arabinose, fructose, galactose, mannose, mannitol, sorbitol, xylitol, myo-inositol, trehalose, sucrose, lactose, maltose, cellobiose, lactitol, maltose, methylcellulose, carboxymethylcellulose, Dextran, glycogen, amylose, amylopectin, inulin, sodium alginate, ethyl cellulose, hydroxyethyl cellulose, raffinose, sucrose, xanthan gum, glucosamine, galactosamine maltose and the like.

When further containing monosaccharides, the content of monosaccharides in the medium can be 0.01% by mass or more and 10% by mass or less, 0.05% by mass or more and 5% by mass or less, and further 0.25% by mass. % Or more and 0.85% by mass or less.

When the disaccharide is further contained, the content of the disaccharide in the medium can be 0.1% by mass or more and 10% by mass or less, 0.5% by mass or more and 7.5% by mass or less, and further 1. It can be 5% by mass or more and 5% by mass or less.

When other trisaccharides other than maltotriose are contained, the content of the other trisaccharides in the medium can be 5% by mass or less, 2% by mass or less, and further 1% by mass or less.

When a polysaccharide of tetrasaccharide or more is further contained, the content of the polysaccharide of tetrasaccharide or more in the medium can be 0.1% by mass or more and 10% by mass or less, and 0.25% by mass or more and 6% by mass or less. It is possible, and it can be 1% by mass or more and 3.5% by mass or less.

When other sugars are contained, maltose is preferable as the other sugars. When maltose is contained as another sugar, the total content of maltotriose and maltose in the medium is preferably 1% by mass or more and 40% by mass or less, more preferably 2% by mass or more and 30% by mass or less, 5 It is more preferably mass% or more and 17.5 mass% or less.

In addition, "arranging the cells in the medium containing maltotriose" means that the cells are present in the medium containing maltotriose before the freezing treatment. Therefore, after transferring the cells to a medium containing no maltotriose, the maltotriose may be added and suspended before freezing, or the maltotriose may be added to the medium in advance. It may be added and cells are added thereto.

In addition to the above-mentioned saccharides, the medium in the cell cryopreservation method of the present embodiment may contain other components conventionally used as protective components in cell cryopreservation. Other conventionally used components include, for example, serum, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, glycerol and the like.

Furthermore, amino acids, vitamins, antioxidants, antibiotics such as penicillin and streptomycin, collagen precursors, trace metal ions or complexes thereof, various salts and the like may be added to the medium.

As the components of the medium, a suitable medium may be selected according to the cells to which the cell cryopreservation method is applied. For example, Dulbecco's Modified Eagle's Medium (D-MEM) is a typical example of a medium used for animal cells. Those skilled in the art of handling the cells can easily understand what kind of medium is suitable.

In the cell cryopreservation method of the present embodiment, as described above, maltotriose is contained in the medium in which the cells are arranged, so that the cell viability after thawing is excellent. In addition, in the present specification, "excellent cell viability after thawing" refers to a case where the proportion of living cells is high based on the determination of life or death immediately after thawing, and also a step of culturing for a predetermined period after thawing. Even when the amount of cells after culturing is larger than that of other scissors, it is assumed that "the cell viability after thawing is excellent".

(cell)
The cells to which the cell cryopreservation method of the present embodiment can be applied are not particularly limited, and may be animal cells, plant cells, microbial cells such as bacteria, fungi, archaea, and protozoa. As used herein, the term "animal" includes vertebrates such as mammals including humans, fish, reptiles, amphibians and birds, and invertebrates such as insects. Among them, the cell cryopreservation method of the present embodiment can be suitably used for animal cells, and more preferably for mammalian cells. Animal cells can be either somatic cells or germ cells. Examples of animal cells include, but are not limited to, CHO cells, HeLa cells, Jurkat cells, HEK293 cells, NIH 3T3 cells, primary culture isolated and cultured from mammalian organs, organs or tissues such as mice, and for reproduction. Examples include livestock sperm cells. Examples of livestock for breeding include horses, cows, pigs and sheep. In addition, examples of microbial cells include, but are not limited to, filamentous fungi that produce various enzymes, amino acids, antibiotics, and the like, fungi such as mushrooms and yeast, and bacteria. Specific examples of the fungi include ascomycetes, zygomycetes, basidiomycetes and imperfecti, and examples of the bacteria include gram-positive bacteria including actinomycetes that produce antibiotics and gram-negative bacteria such as Escherichia coli. And so on.

In addition, the cells are cells that have been established as cultured cells, normal cells that have not been established from biological tissues, transformed cells obtained by genetic engineering techniques, induced pluripotent stem cells (iPS cells), and cells. It may be a cell of any aspect, such as a pluripotent stem cell.

(Crozen storage)
The freezing method in the step of freezing the medium in which the cells are arranged is not particularly limited. Therefore, the cells of interest may be placed in a medium containing maltotriose in a container for cryopreservation such as a tube, and frozen by a conventionally known freezing method. For example, a quick freezing method can be adopted in which cells are placed in a container together with a medium and then the container is placed in a freezer at −80 ° C. to freeze the cells. Alternatively, a slow freezing method may be used in which a container containing cells is placed in a program freezer and the cells are slowly frozen while adjusting the cooling rate or the like.

After the freezing operation, for example, when a freezer at −80 ° C. is used, it may be stored in the freezer as it is, or it may be transferred to liquid nitrogen and stored after the freezing operation is completed. If a program freezer is used, it may be transferred to liquid nitrogen and stored after the freezing operation is completed.

The method of thawing the cells subjected to cryopreservation by the above method for use is also not particularly limited. Therefore, methods and conditions conventionally known to those skilled in the art can be adopted, such as a method of quickly melting in a warm bath at 37 ° C. and a method of gently melting at room temperature.

[Composition for cryopreservation of cells]
Next, one embodiment of the cell cryopreservation composition according to the present invention will be described below.

The composition for cryopreservation of cells of the present embodiment is added to the medium before the cryotreatment for cryopreservation of cells, and contains maltotriose as a component. The content of maltotriose in the cell cryopreservation composition is not particularly limited as long as the above-mentioned maltotriose content in the medium can be achieved by adding the cell cryopreservation composition to the medium. However, as a typical example of the content of maltotriose in the composition for cell cryopreservation, it can be 5% by mass or more and 95% by mass or less, 25% by mass or more and 80% by mass or less, and further. Can be 40% by mass or more and 65% by mass or less.

The cell cryopreservation composition may contain yet another sugar. Examples of other saccharides that can be contained in the cell cryopreservation composition are the same as those described above that can be contained in the medium. When other sugars are contained, maltose is preferable as the other sugars. When maltose is contained as another saccharide, the content of maltose in the composition for cryopreservation of cells can be typically 0.1% by mass or more and 90% by mass or less, and 1% by mass or more and 50% by mass. It can be 10% by mass or less, and further can be 10% by mass or more and 30% by mass or less.

The cell cryopreservation composition can be in the form of powders, liquids and the like.

The composition for cryopreservation of cells may be added to the medium as it is, or may be diluted with a diluent such as water and then added to the medium.

The present invention has been completed by finding that a maltotriose-containing composition used in a completely different application from cell cryopreservation, such as in the food field, has an excellent effect in cell cryopreservation. It is a thing. Therefore, a commercially available maltotriose-containing composition that has been used for other purposes such as in the food field can be used as the cell cryopreservation composition in the present embodiment. Examples of such maltotriose-containing compositions include Gun Ei Chemical Industry Co., Ltd.'s Pure Tooth (registered trademark), Nihon Shokuhin Kako Co., Ltd.'s Fuji Oligo (registered trademark) # 360, Showa Sangyo's Oligo MT500, and Mitsubishi Chemical Foods' Oligo Tooth. And so on. In addition, starch syrup obtained by purifying and concentrating an enzyme-reacted saccharified solution using starch as a raw material can also be used as a maltotriose-containing composition.

Examples are shown below, and embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects can be used for details. Furthermore, the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the claims, and the present invention also relates to an embodiment obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. In addition, all the documents described in the present specification are incorporated by reference.

(Comparative Example 1)
D-MEM medium (containing L-glutamine, penicillin, streptomycin and amphotericin: manufactured by DS-Pharma Biomedical, Catalog No. KBDSM101) supplemented with 10% bovine serum albumin (FBS) (hereinafter, D-MEM ^* medium). (Referred to as) was prepared, and 5 mL of the prepared D-MEM ^* medium was added to a sterilized centrifuge tube having a volume of 50 mL. CHO-K1 cells (Chinese hamster ovary cell K-1 strain, RCB0285) sold from RIKEN BioResource Standard were thawed and transferred to a centrifuge tube containing D-MEM ^* medium. The centrifuge tube to which the cells were transferred was centrifuged at 200 × g for 3 minutes to remove the supernatant, and then 20 mL of D-MEM ^* medium was added. This is transferred to a flask having a bottom surface size of 25 cm ² containing a medium for cell culture, and is confluent while observing under an inverted microscope (phase contrast type) for 7 days at 37 ° C. and a CO ₂ concentration of 5%. The cells were cultured until they were in a state. After becoming confluent, a 0.25% trypsin solution (dissolved in 1 mM EDTA-4 sodium solution, Wako Pure Chemical Industries, Ltd.) was added to the confluent state, and the mixture was incubated at 37 ° C. for 10 minutes with gentle stirring. Cell fluid was obtained. After centrifuging the obtained cell solution at 200 × g for 3 minutes, the supernatant is removed, and D-MEM ^* medium in a volume of 1/2 the volume of the original medium is added to the precipitated cells and gently washed. Then, the cells were centrifuged under the same conditions. This operation was repeated again, and a D-MEM medium ^* having a volume of 1/2 the volume of the original medium was added and suspended, and used as an extracellular fluid for evaluation.

Dispense 200 μL of extracellular fluid for evaluation into two 1.5 mL plastic cryotubes, add 22.2 μL of dimethyl sulfoxide (DMSO) to the final concentration of 10%, and gently stir. did. Each was placed in a paper box and frozen in a freezer at -80 ° C. After storage for 14 days, the extracellular fluid contained in the cryotube was gently thawed at room temperature (23 ° C.). 200 μL of the thawed extracellular fluid was dispensed into a 24-well plate (manufactured by Falcon, Catalog No. 353047), and an equal amount of D-MEM ^* medium was added thereto. The cells were cultured under 37 ° C. and 5% CO ₂ for 7 days (hereinafter, this culture is referred to as "post-culture"), and the cells were confirmed under a 200-fold inverted microscope. Since cells that died due to freezing and thawing can be generally identified from their morphology, cells excluding these cells were counted as viable cells. As a result, no cells were confirmed to be viable under these conditions.

(Comparative Example 2)
The test was carried out in the same manner as in Comparative Example 1 except that 50 μL of a trehalose solution diluted with water to a concentration of 50% by mass was used instead of 22.2 μL of DMSO. The trehalose used in this comparative example contains 10% by mass of water.

The cells were confirmed under a 200-fold inverted microscope, and the number of cells was counted in the same manner as in Comparative Example 1. As a result, about 50% of the cells were alive under this condition. Moreover, the regrowth rear culture period, the number of cells per well became 2.45 × 10 ³ in the average of 2 wells. In each of the following examples, each cell is shown by a relative value with the number of cells as 100.

(Example 1)
The test was carried out in the same manner as in Comparative Example 1 except that 22.2 μL of a Puretos (manufactured by Gun Ei Chemical Industry Co., Ltd.) solution diluted with water to a concentration of 50% by mass was used instead of 22.2 μL of DMSO. The puretoce used in this example is a powdered puretoth containing 3% by mass of water, and has a sugar composition per solid content of glucose: maltose: maltotriose: tetrasaccharide or more = 4: 22 :. It has a composition of 58:16.

The cells were confirmed under a 200-fold inverted microscope, and the number of cells was counted in the same manner as in Comparative Example 1. As a result, under this condition, the cells proliferated during the post-culture period, and the average number of cells per well was 91 compared to Comparative Example 2. That is, it was shown that the same effect as trehalose can be obtained with a smaller amount of maltotriose than trehalose, which is known as a stabilizer during cell cryopreservation.

(Example 2)
The test was carried out in the same manner as in Example 1 except that the amount of the Puretoce solution added in Example 1 was changed to 50 μL.

The cells were confirmed under a 200-fold inverted microscope, and the number of cells excluding dead cells was counted. As a result, under this condition, the cells proliferated during the post-culture period, and the average number of cells per well was 226 compared to Comparative Example 2. That is, it was shown that the amount of maltotriose used, which is smaller than that of trehalose, is superior to that of trehalose in the cell viability after thawing.

(Example 3)
The test was carried out in the same manner as in Example 1 except that the amount of the Puretoce solution added in Example 1 was changed to 130 μL.

The cells were confirmed under a 200-fold inverted microscope, and the number of cells excluding dead cells was counted. As a result, under this condition, the cells proliferated again during the post-culture period, and the average number of cells per well was 229 compared to Comparative Example 2.

The present invention can be utilized in long-term storage of cells and in industries that utilize the preserved cells.

Claims (8)

A cell cryopreservation method comprising a step of arranging cells in a medium containing maltotriose and a step of freezing the medium in which the cells are arranged. The cell cryopreservation method according to claim 1, wherein the medium contains maltose. The cell cryopreservation method according to claim 1 or 2, wherein the content of maltotriose in the medium is 1% by mass or more and 30% by mass or less. The cell cryopreservation method according to claim 2, wherein the total content of maltotriose and maltose in the medium is 1% by mass or more and 40% by mass or less. The cell cryopreservation method according to any one of claims 1 to 4, wherein the cell is a mammalian cell. A composition for cryopreservation of cells added to a medium for cryopreserving cells.
A composition for cryopreservation of cells, which comprises maltotriose. The composition for cell cryopreservation according to claim 6, further comprising maltose. A medium for cryopreservation of cells, which comprises maltotriose in the medium.