JP2021093996A - Cell cryoprotectant - Google Patents

Abstract

To provide a cell cryoprotectant having an excellent cell survival rate.SOLUTION: A cell cryoprotectant has a polymer having a constitutional unit represented by the following formula (1) (where, R is a monovalent hydrocarbon group, and * is a binding site).SELECTED DRAWING: None

Description

The present invention relates to a cell cryoprotectant.

So far, cell cryoprotectants include low molecular weight compounds such as dimethylsulfoxide (DMSO), ethylene glycol, glycerol, and saccharides, natural high molecular weight compounds such as dextran, albumin, and sericin, polyvinyl alcohol (PVA), and polyvinylpyrrolidone. (PVP), polyethylene glycol (PEG), carboxylated polylysine, synthetic polymer compounds such as solid phase synthetic peptides and the like are used. Of these, DMSO, for example, is a low-molecular-weight compound that is permeable to cell membranes, and is known to have a function of preventing damage such as destruction of cell membranes and organelle membranes due to crystallization of intracellular ice during cell cryopreservation. ing. However, on the other hand, it is known that when the concentration is high, cytotoxicity is exhibited and pluripotent stem cells can be induced to differentiate into the mesoendoderm lineage.

Therefore, for example, Patent Document 1 below discloses a cryoprotectant containing at least one selected from dextrin, dextran, isomaltooligosaccharide and its derivatives, and these compounds are DMSO's toxicity to cells and the like. It is stated that it can be an alternative.

JP-A-2019-4904

Although the conventional cell cryoprotectant as described above can reduce the amount of DMSO showing toxicity to some extent, the performance as a cell cryoprotectant such that the survival rate of cells decreases when DMSO is completely removed. There is still room for improvement.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a cell cryoprotectant having an excellent cell viability.

The present invention provides a cell cryoprotectant containing a polymer having a structural unit represented by the following formula (1).

（式（１）中、Ｒは１価の炭化水素基を示し、＊は結合部位を示す。）

(In formula (1), R indicates a monovalent hydrocarbon group, and * indicates a binding site.)

The cell cryoprotectant may further contain a cell membrane penetrating low molecular weight compound.

According to the present invention, it is possible to provide a cell cryoprotectant having an excellent cell viability.

Hereinafter, one embodiment of the present invention will be described in detail, but the present invention is not limited thereto.

The cell cryoprotectant according to the present embodiment contains a polymer having a structural unit represented by the following formula (1).

（式（１）中、Ｒは１価の炭化水素基を示し、＊は結合部位を示す。）

(In formula (1), R indicates a monovalent hydrocarbon group, and * indicates a binding site.)

The monovalent hydrocarbon group in the substituent R is preferably a linear, branched or cyclic alkyl group. The alkyl group preferably has 1 to 20 carbon atoms, more preferably 1 to 3 carbon atoms. Examples of such an alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a cyclopropyl group and the like.

The polymer having the structural unit represented by the above formula (1) may be, for example, a polymer having a structural unit derived from the compound represented by the following formula (1'). That is, the polymer having the structural unit represented by the formula (1) can also be said to be a polymer obtained by polymerizing the compound represented by the following formula (1'). As the compound represented by the formula (1'), one kind may be used alone, or two or more kinds may be used in combination.

（式（１’）中、Ｒは式（１）と同義である。）

(In equation (1'), R is synonymous with equation (1).)

The polymer is preferably a polymer consisting of only structural units represented by the above formula (1), but further contains other structural units as long as the effects of the present invention are not significantly impaired. May be good. In this case, the content of the structural unit represented by the formula (1) in the polymer may be, for example, 1% by mass or more, 20% by mass or more, or 50% by mass or more. Further, the upper limit of the content of the structural unit represented by the formula (1) in the polymer is not particularly limited, and may be, for example, less than 100% by mass.

The other structural unit is not particularly limited as long as it is a structural unit derived from a monomer copolymerizable with the compound represented by the above formula (1'), and is, for example, styrene, methyl methacrylate, 2-. Examples thereof include structural units derived from monomers such as hydroxyethyl methacrylate and ε-caprolactone. Further, as another structural unit, a structural unit obtained by reacting a homopolymer of a compound represented by the above formula (1'), for example, a homopolymer of a compound represented by the above formula (1') can be used. Examples thereof include structural units of linear ethyleneimine obtained by hydrolysis.

The polymer according to this embodiment is produced by carrying out a polymerization reaction (ring-opening polymerization) by appropriately selecting the compound represented by the above formula (1') and, if necessary, the other monomer. be able to. The preferable amount of the monomer used in the polymerization reaction is the same as the preferable content of the structural unit derived from these monomers in the polymer.

The polymerization reaction is preferably carried out in the presence of a polymerization initiator. As the polymerization initiator (cationic ring-opening polymerization initiator) for producing a polymer, for example, a strong acid such as hydrochloric acid or methyl tosylate, a Lewis acid such as aluminum trichloride; a sulfonium salt compound composed of a sulfur atom, or the like is preferable. is there.

The amount of the polymerization initiator used is preferably 0.1 g or more and 10 g or less, more preferably 0.2 g or more and 8 g or less, and 0.3 g or less, based on 100 g of the monomer used. As mentioned above, it is more preferably 7 g or less, and most preferably 0.5 g or more and 5 g or less.

The above-mentioned polymerization reaction may be carried out without using a solvent, but it is preferable to use a solvent. Examples of the solvent include water, acetonitrile, tetrahydrofuran, dioxane, acetone, methyl ethyl ketone, methyl isobutyl ketone, methanol, ethanol, isopropyl alcohol and the like. These solvents may be used alone or in combination of two or more. Of these solvents, water is most preferred. The amount of the solvent used is preferably 40 to 250 parts by mass with respect to 100 parts by mass of the monomer used in the polymerization reaction.

The polymerization reaction is usually preferably carried out at 0 ° C. or higher, and is preferably carried out at 150 ° C. or lower. More preferably, it is 40 ° C. or higher, further preferably 60 ° C. or higher, and particularly preferably 80 ° C. or higher. Further, it is more preferably 120 ° C. or lower, and further preferably 110 ° C. or lower. The polymerization temperature does not have to be kept substantially constant in the polymerization reaction, and may fluctuate (heat or cool) by 1 ° C. or 2 ° C. or higher. The polymerization reaction may be carried out under any conditions of normal pressure, pressurization and reduced pressure.

In the above polymerization reaction, the monomer, the polymerization initiator and the like may be added to the reactor all at once or sequentially.

The method for producing a polymer may include steps other than the above-mentioned polymerization reaction step. Examples of other steps include a aging step, a neutralizing step, a deactivating step of a polymerization initiator and the like, a diluting step, a drying step, a concentration step, and a purification step. When a polymer is obtained by polymerization in a solvent other than water, the solvent is removed by a drying step or a reprecipitation step by dropping the reaction solution into a poor solvent such as methanol, and then the solution is added to water. It is preferable to obtain an aqueous solution of the polymer by the step of dissolving.

The cell cryoprotectant according to the present embodiment may contain only a polymer having a structural unit represented by the above formula (1), or may be used in combination with other components. ..

The other components are not particularly limited as long as they are components used in a cell antifreeze protectant, and are, for example, dimethylsulfoxide (DMSO), glycerol, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol and the like. Cell membrane permeable low molecular weight compounds, cell membrane impermeable low molecular weight compounds such as sorbitol, trehalose, acetamide, lactoamide, biological high molecular weight compounds such as dextrin, dextran, sericin, albumin, antifreeze protein, polyvinyl alcohol (PVA) ), Polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), carboxylated polylysine, synthetic polymer compounds such as solid phase synthetic peptides and the like. From the viewpoint of further improving the cell viability, it is preferable to further contain a cell membrane-permeable low molecular weight compound such as dimethyl sulfoxide (DMSO). The content of these other components may be 0.1% by mass or more, 1% by mass or more, or 10% by mass or less, based on the total amount of the cell cryoprotectant. It may be mass% or less. Regarding the content ratio of these other components to the polymer, when the content of the polymer is C ₁ part by mass and the content of the other component is C ₂ parts by mass, C ₂ / C ₁ is 0. It may be 003 or more, and may be 0.1 or more. Further, the upper limit of the content ratio may be 10 or less for _{C 2} / C _{1 and 0.3 or less.}

Further, the cell cryoprotectant according to the present embodiment is preferably used as a solution by dissolving the above-mentioned polymer and, in some cases, other components in a solvent from the viewpoint of improving handleability. The solvent can be appropriately selected depending on the intended use, and for example, various media for cell culture, water, phosphate buffered saline, Tris hydrochloric acid buffer, HEPES hydrochloric acid buffer and the like are preferably used. .. In this case, the content of the polymer having the structural unit represented by the above formula (1) in the cell cryoprotectant may be 1% by mass or more based on the total amount of the cell cryoprotectant, and may be 5% by mass. It may be the above, it may be 30% by mass or less, and it may be 20% by mass or less.

The cell cryoprotectant according to the present embodiment has an excellent cell viability after freezing and thawing. The cells to be cryopreserved are not particularly limited, and may be all types of cells in tissues or organs, such as somatic cells, stem cells, germ cells, and cultured cells used experimentally. The cell may be in an isolated form or in a non-isolated form such as a cell-containing body fluid, tissue or organ morphology. Examples of cells to be preserved include sperm cells, ES cells, iPS cells, mesenchymal stem cells, hematopoietic stem cells, nerve stem cells, stem cells such as umbilical cord blood cells, hepatocytes, nerve cells, myocardial cells, vascular endothelial cells, and blood vessels. Animal cells including humans such as smooth muscle cells and blood cell cells, plant cells and the like are particularly suitable.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[Preparation of cell cryoprotectant]
(Examples 1 to 3 and Comparative Examples 1 to 4)
Using each component shown below, a cell cryoprotectant having the composition shown in Table 1 (mass% based on the total amount of the cell cryoprotectant) (Examples 1 to 3 and Comparative Examples 1 to 3) was prepared. Specifically, the polymer A or the polymer B is dissolved in ultrapure water, purified by ultrafiltration using Amicon Ultra-15 (NMWL = 3 kDa) (manufactured by Merck, product number: UFC900324), and then freeze-dried. Was dissolved in PBS (−) to adjust the pH to 7.2. As Comparative Example 4, a commercially available cell cryoprotectant (trade name: BAMBANKER, containing 10% DMSO) was prepared.
PBS (-): Phosphate buffered saline (without Ca and Mg)
Polymer A: A homopolymer having a structural unit in which R in the formula (1) is an ethyl group (manufactured by SIGMA-ALDRICH, product number: 741906).
Polymer B: A homopolymer having a structural unit in which R in the formula (1) is a methyl group (manufactured by Tokyo Chemical Industry Co., Ltd., product number: P2506).
DMSO: Dimethyl sulfoxide

[Measurement of cell viability after freezing and thawing]
After preparing L929 cells to 1 × 10 ⁶ cells / ml, 1 mL was dispensed into a 1.5 mL tube and centrifuged at 230 × g for 3 minutes. After removing the supernatant, 1 mL of the cell cryoprotectant prepared above was added to suspend the precipitated cells, the suspension was transferred to a cryotube, and the cryotube was placed in a bicelle manufactured by Nippon Freezer Co., Ltd. at -80 ° C. , Stored frozen for 7 days. After freezing and thawing in a 37 ° C. water bath, the cell cryoprotectant is removed by centrifugation, and the cell culture medium (manufactured by Nakaraitesk Co., Ltd., trade name: Dalveco modified Eagle's medium (1.0 g / L glucose)) (L- Glutamine, pyruvic acid contained) (liquid)) was suspended. The viability of each cell was measured by trypan blue staining. The results are shown in Table 1. Regarding the cell cryoprotectants of Example 1 and Comparative Example 1, the same operation as above was performed using normal human skin fibroblasts (NHDF cells) in addition to L929 cells (for NHDF cells, manufactured by Nacalai Tesque, Inc.). Product name: MEM medium (using Earl's salt, containing L-glutamine (liquid)) was used, and the cell viability was measured. The results are shown in Table 1.

Each example using the cell cryoprotectant containing the polymer according to the present invention does not contain DMSO, or even if the content of DMSO is low, is it comparable to the commercially available cell cryoprotectant (Comparative Example 4)? It showed better cell viability. On the other hand, in Comparative Examples 1 and 2 containing no polymer according to the present invention, the cell viability was lower than that of each Example and Comparative Example 4, and in order to increase the cell viability, as in Comparative Example 3. It was necessary to increase the content of DMSO.

Claims (2)

A cell cryoprotectant containing a polymer having a structural unit represented by the following formula (1).

(In formula (1), R indicates a monovalent hydrocarbon group, and * indicates a binding site.) The cell cryoprotectant according to claim 1, further comprising a cell membrane penetrating low molecular weight compound.