Freeze-drying composition, method for producing freeze-dried cells, and pharmaceutical composition

Abstract

The present invention addresses the problem of providing a composition for freeze-drying cells with which it is possible to suppress a decrease in survival rate or a decrease in particle size when the cells are reconstituted after freeze-drying. The composition for freeze-drying cells contains (a) a hydrophobic substance such as (a-1), (a-2), etc., and (b) nanoparticles formed from monolayers or bilayers of amphiphilic molecules, where: (a-1) is one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; and (a-2) is a dipeptide configured from one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine. The composition is characterized by being used added to the cells.

Description

Composition for freeze-drying, method for producing freeze-dried cells, and pharmaceutical composition

[0001] The present invention relates to a composition for freeze-drying, a method for producing freeze-dried cells, and a pharmaceutical composition.

[0002] In recent years, cell-based approaches have been attracting attention in regenerative therapy, cancer treatment, and inflammatory disease treatment. This regenerative therapy is a treatment method that uses cells to treat diseases and injuries, and is adopted as a treatment for various diseases such as myocardial infarction, chronic arterial occlusion, and leukemia, as well as injuries such as spinal cord injury and traumatic cartilage defect.

[0003] Traditionally, cells used in regenerative therapies have been cryopreserved for reasons of transportability, long-term storage, convenience, and other factors. However, the high costs of transporting and storing frozen cells have led to rising prices for regenerative medicine products. Furthermore, cryopreservation of cells is prone to phenomena such as decreased cell viability, decreased cell particle size, cell membrane disruption, cellular dysfunction (e.g., a decrease in the proportion of stem cells in the case of stem cells), and growth inhibition (hereinafter, these phenomena are collectively referred to as "cell damage"). This is thought to be due to the crystallization of intracellular water when cells are frozen, which can damage various organelles such as mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomes, and centrosomes, as well as cell membranes and cell walls. Furthermore, thawing cells can cause a partial loss of intracellular water, resulting in changes in cell shape.

[0004] One known cell cryopreservation method for freezing and preserving cells involves freezing cells in a buffer solution containing a low-molecular-weight compound such as dimethyl sulfoxide (DMSO). However, the use of DMSO has the drawback of reducing cell viability and requiring rapid removal of DMSO after thawing due to its cytotoxicity. Therefore, a technique for freezing cells using a sugar derivative has been disclosed as a method for freezing cells without using DMSO (see Patent Document 1). However, this method of freezing cells still has the drawback of requiring the cells to remain frozen during storage and distribution.

[0005] Therefore, freeze-drying techniques for cells have also been developed. For example, a method for freeze-drying mesenchymal stem cells using one or a mixture of trehalose, sucrose, lactose, glucose, raffinose, dextran, mannitol, sorbitol, or xylitol, or human serum albumin, as a cryoprotectant has been disclosed (see Patent Document 2). Another method for freeze-drying a biological sample placed on a metal surface using a solution containing a combination of one or more of sucrose, sorbitol, glucose, dextran, and trehalose, one or more cryoprotectants selected from DMSO, EG, PG, and glycerol, one or more polymers selected from HSA and FCS, and one or more antioxidants selected from astaxanthin, EGCG, and ascorbic acid has also been disclosed (see Patent Document 3).

[0006] Recently, research has been conducted into freezing and freeze-drying methods using amino acids. For example, a freezing method using an amino acid-based polymer (A) having an amino acid residue-containing structural unit as a cell cryoprotectant has been disclosed (see Patent Document 4). The present inventors have also disclosed a composition for cryopreservation of cells or biological tissues, containing specific nanoparticles (see Patent Document 5). Meanwhile, research into freeze-drying drugs has progressed, and the present inventors have disclosed a freeze-dried composition of a drug, such as a PI3 kinase inhibitor, containing two types of amino acids and a surfactant (see Patent Document 6). Another example is a freeze-dried composition of interferon-γ obtained using a hydrophobic stabilizer, such as a hydrophobic amino acid, a hydrophobic amino acid dipeptide, or a hydrophobic amino acid tripeptide, and a hydrophilic stabilizer, such as a hydrophilic amino acid, a hydrophilic amino acid dipeptide, or a hydrophilic amino acid tripeptide.

[0007] International Publication No. 2021 / 095741 Pamphlet Japanese Patent Application Laid-Open No. 2024-501087 Japanese Patent Application Laid-Open No. 2021-511080 Japanese Patent Application Laid-Open No. 2023-036560 International Publication No. 2024 / 024892 Pamphlet International Publication No. 2014 / 098232 Pamphlet Special Publication No. 2006-509825

[0008] An object of the present invention is to provide a composition for freeze-drying cells that can suppress a decrease in viability or a decrease in particle size when cells are reconstituted after freeze-drying.

[0009] As a result of intensive research to solve the above problems, the inventors discovered that by adding a specific amino acid and a specific nanoparticle and then freeze-drying, it is possible to suppress a decrease in viability or a decrease in particle size even when cells are restored after freeze-drying, and thus completed the present invention.

[0010] That is, the present invention is as follows: [1] (a) a hydrophobic substance selected from the group consisting of (a-1) to (a-4) below: (a-1) one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-2) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-3) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-4) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (b) nanoparticles formed of a monolayer or bilayer of amphiphilic molecules: A composition for freeze-drying cells, comprising (a) and (b) above, wherein the composition is used by adding the composition to cells. [2] The composition for freeze-drying according to [1] above, wherein the hydrophobic substance comprises at least two of phenylalanine and leucine. [3] The composition for freeze-drying according to [1] above, wherein the hydrophobic substance is a combination of phenylalanine and leucine. [4] The composition for freeze-drying according to any of [1] to [3] above, wherein the cells are human-derived cells separated from a living body. [5] The composition for freeze-drying according to any of [1] to [4] above, further comprising trehalose and / or ascorbic acid or a derivative thereof. [6] The composition for freeze-drying according to any of [1] to [5] above, wherein the amphiphilic molecule is a Gemini surfactant. [7] A method for producing freeze-dried cells, comprising the step of adding the composition for freeze-drying according to any of [1] to [6] above to cells.[8] A method for producing freeze-dried cells according to [7] above, characterized in that the composition for freeze-drying according to any one of [1] to [6] above is added to a solution containing the cells so that the concentration of the hydrophobic substance contained in the composition for freeze-drying is 0.3 to 100 mg / mL. [9] A method for producing freeze-dried cells according to [7] or [8] above, comprising the following steps (a) and (b): step (a) of adding the composition for freeze-drying according to any one of [1] to [6] above to cells and freezing them at -196 to -20°C for 8 hours or more; and step (b) of drying the cells frozen in step (a) under vacuum conditions at -50 to 35°C for 10 to 72 hours.

[10] Cells obtained by reconstituting the freeze-dried cells produced by the method according to any one of [7] to [9] above with a solvent.

[11] A pharmaceutical composition comprising the reconstituted cells according to

[10] above and a pharmaceutically acceptable excipient.

[0011] According to the disclosure of the present specification, even if cells are freeze-dried, it is possible to suppress a decrease in viability after restoration or a decrease in particle size, making it easier to store, transport, or process the cells at room temperature.

[0012] Figure 1 shows the results of scanning electron microscopy of human mesenchymal stem cells freeze-dried and restored using Example Product 8 in Example 1. Figure 2 shows the results of examining cell viability in cells freeze-dried and restored without freeze-drying, using the additives in Comparative Product 5 and Example Product 8 in Example 5. Figure 3 shows the results of examining transplantation efficiency in cells freeze-dried and restored without freeze-drying, using the additives in Comparative Product 5 and Example Product 8 in Example 6.

[0013] One embodiment of the composition for freeze-drying cells of the present invention comprises: (a) any one of the following hydrophobic substances (a-1) to (a-4): (a-1) one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-2) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-3) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-4) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (b) nanoparticles formed of a monolayer or bilayer of amphiphilic molecules: The present invention is not particularly limited as long as it is a composition for freeze-drying cells, characterized in that it comprises (a) and (b) above and is used by adding to cells, and is hereinafter also referred to as "the present composition for freeze-drying cells." One embodiment of the method for producing cells of the present invention is not particularly limited as long as it is a method for producing freeze-dried cells, including a step of adding the present composition for freeze-drying to cells, and is hereinafter also referred to as "the present method for producing freeze-dried cells." One embodiment of the present invention is cells obtained by reconstituting freeze-dried cells with a solvent, and is hereinafter also referred to as "the present restored cells." One embodiment of the present invention is a pharmaceutical composition, and is hereinafter also referred to as "the present pharmaceutical composition," and is hereinafter also referred to as "the present pharmaceutical composition."Furthermore, one aspect of the present invention is the use of the following hydrophobic substances in the production of a pharmaceutical composition for the prevention or treatment of a disease that is a target of regenerative medicine or cell therapy: (a) any of the following hydrophobic substances (a-1) to (a-4): (a-1) one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-2) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-3) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; A composition comprising (a) and (b): (a-4) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; and (b) nanoparticles formed of a single layer or two layers of amphiphilic molecules. Another aspect of the present invention is a method for preventing or treating a disease that is a target of regenerative medicine or cell therapy, which comprises administering the pharmaceutical composition to a patient in need thereof.

[0014] 1. Composition for freeze-drying (hydrophobic substance) The composition for freeze-drying contains a hydrophobic substance and nanoparticles formed of a single layer or two layers of amphipathic molecules. The hydrophobic substance is any one of the following (a-1) to (a-4), and it is preferable that the hydrophobic substance contains at least two of phenylalanine and leucine. (a-1) one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-2) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-3) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-4) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine;

[0015] As used herein, a dipeptide refers to a molecule in which two amino acids are linked by one peptide bond, a tripeptide refers to a molecule in which three amino acids are linked by two peptide bonds, and a tetrapeptide refers to a molecule in which four amino acids are linked by three peptide bonds.

[0016] The above-mentioned phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine are all hydrophobic amino acids, and each may independently be in either the D- or L-form. When two or more hydrophobic amino acids are combined, a mixture of D- and L-forms may be used, but it is preferable that all be in the L-form. Furthermore, the above-mentioned phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine may each independently be in the form of a methyl ester. Examples of the methyl ester of phenylalanine include, but are not limited to, N-[N-(3,3-dimethylbutyl)-L-α-aspartyl]-L-phenylalanine 1-methyl ester (Neotame). The use of these hydrophobic amino acids makes it possible to maintain the shape of cells and form a network structure around the cells when freeze-dried.

[0017] In the above (a-1), the one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine may be one hydrophobic amino acid selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, a combination of two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, or a combination of three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine. The hydrophobic amino acids may be a combination of four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, a combination of five hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, a combination of six hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine, or a combination of seven hydrophobic amino acids consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine. Note that the two or more hydrophobic amino acids mentioned above refer to a combination of two or more hydrophobic amino acids.

[0018] When the hydrophobic amino acid (a-1) contains two kinds of hydrophobic amino acids, the combination may include phenylalanine and leucine; phenylalanine and glycine; phenylalanine and valine; phenylalanine and isoleucine; phenylalanine and tryptophan; phenylalanine and alanine; leucine and glycine; leucine and valine; leucine and isoleucine; leucine and tryptophan; leucine and alanine; glycine and valine; glycine and isoleucine; glycine and tryptophan; glycine and alanine; valine and isoleucine; valine and tryptophan; valine and alanine; isoleucine and tryptophan; isoleucine and alanine; or tryptophan and alanine, and preferred examples include phenylalanine or leucine, preferably phenylalanine and leucine.

[0019] When three kinds of hydrophobic amino acids are contained as the hydrophobic amino acids of (a-1) above, the hydrophobic amino acids are selected from the group consisting of phenylalanine, leucine, and glycine; phenylalanine, leucine, and valine; phenylalanine, leucine, and isoleucine; phenylalanine, leucine, and tryptophan; phenylalanine, leucine, and alanine; phenylalanine, glycine, and valine; phenylalanine, glycine, and isoleucine; phenylalanine, glycine, and tryptophan; phenylalanine, glycine, and alanine; phenylalanine, valine, and isoleucine; phenylalanine, valine, and tryptophan; phenylalanine, valine, and alanine; phenylalanine, isoleucine, and tryptophan; phenylalanine, isoleucine, and alanine; phenylalanine, tryptophan, and alanine; leucine, glycine, and valine; leucine, glycine, and isoleucine; leucine, glycine, and It is sufficient to include a combination of tryptophan; leucine, glycine, and alanine; leucine, valine, and isoleucine; leucine, valine, and tryptophan; leucine, valine, and alanine; leucine, isoleucine, and tryptophan; leucine, isoleucine, and alanine; leucine, tryptophan, and alanine; glycine, valine, and isoleucine; glycine, valine, and tryptophan; glycine, valine, and alanine; glycine, isoleucine, and tryptophan; glycine, isoleucine, and alanine; glycine, tryptophan, and alanine; valine, isoleucine, and tryptophan; valine, isoleucine, and alanine; valine, tryptophan, and alanine; or isoleucine, tryptophan, and alanine; and among the three hydrophobic amino acids, a combination of phenylalanine or leucine, preferably two of phenylalanine and leucine, can be preferably included.

[0020] When four types of hydrophobic amino acids are contained as the hydrophobic amino acids of (a-1) above, the hydrophobic amino acids include phenylalanine, leucine, glycine, and valine; phenylalanine, leucine, glycine, and isoleucine; phenylalanine, leucine, glycine, and tryptophan; phenylalanine, leucine, glycine, and alanine; phenylalanine, leucine, valine, and isoleucine; phenylalanine, leucine, valine, and tryptophan; phenylalanine, leucine, valine, and alanine; phenylalanine, leucine, isoleucine Leucine and tryptophan; phenylalanine, leucine, isoleucine, and alanine; phenylalanine, leucine, tryptophan, and alanine; phenylalanine, glycine, valine, and isoleucine; phenylalanine, glycine, valine, and tryptophan; phenylalanine, glycine, valine, and alanine; phenylalanine, glycine, isoleucine, and tryptophan; phenylalanine, glycine, isoleucine, and alanine; phenylalanine, glycine, tryptophan, and alanine; phenyl phenylalanine, valine, isoleucine, and tryptophan; phenylalanine, valine, isoleucine, and alanine; phenylalanine, valine, tryptophan, and alanine; phenylalanine, isoleucine, tryptophan, and alanine; leucine, glycine, valine, and isoleucine; leucine, glycine, valine, and tryptophan; leucine, glycine, valine, and alanine; leucine, glycine, isoleucine, and tryptophan; leucine, glycine, isoleucine, and alanine; leucine, glycine , tryptophan, and alanine; leucine, valine, isoleucine, and tryptophan; leucine, valine, isoleucine, and alanine; leucine, valine, tryptophan, and alanine; leucine, isoleucine, tryptophan and alanine; glycine, valine, isoleucine, and tryptophan, glycine, valine, isoleucine, and alanine, glycine, valine, tryptophan, and alanine, glycine, isoleucine, tryptophan, and alanine, or valine, isoleucine, tryptophan and alanine;Among the four hydrophobic amino acids, it is preferable that the amino acid contains phenylalanine or leucine, preferably two of the four hydrophobic amino acids, phenylalanine and leucine.

[0021] When five kinds of hydrophobic amino acids are contained as the hydrophobic amino acids of (a-1) above, the hydrophobic amino acids are phenylalanine, leucine, glycine, valine, and isoleucine; phenylalanine, leucine, glycine, valine, and tryptophan; phenylalanine, leucine, glycine, valine, and alanine; phenylalanine, leucine, glycine, isoleucine, and tryptophan; phenylalanine, leucine, glycine, isoleucine, and alanine; phenylalanine, leucine, valine, isoleucine, and tryptophan; phenylalanine, leucine, valine, isoleucine, and alanine; phenylalanine, leucine, valine, tryptophan ... isoleucine, tryptophan, and alanine; phenylalanine, glycine, valine, isoleucine, and tryptophan. leucine, glycine, valine, isoleucine, tryptophan, and alanine; phenylalanine, glycine, valine, tryptophan, and alanine; phenylalanine, glycine, isoleucine, tryptophan, and alanine; phenylalanine, valine, isoleucine, tryptophan, and alanine; leucine, glycine, valine, isoleucine, and alanine; leucine, glycine, valine, tryptophan, and alanine; leucine, glycine, valine, isoleucine, tryptophan, and alanine; leucine, glycine, valine, isoleucine, tryptophan, and alanine; or glycine, valine, isoleucine, tryptophan, and alanine; and among the five hydrophobic amino acids, preferred examples include phenylalanine or leucine, preferably two of phenylalanine and leucine.

[0022] When the hydrophobic amino acids (a-1) contain six types of hydrophobic amino acids, they may contain: phenylalanine, leucine, glycine, valine, isoleucine, and tryptophan; phenylalanine, leucine, glycine, valine, isoleucine, and alanine; phenylalanine, leucine, glycine, valine, tryptophan, and alanine; phenylalanine, leucine, glycine, isoleucine, tryptophan, and alanine; phenylalanine, leucine, valine, isoleucine, tryptophan, and alanine; phenylalanine, leucine, valine, isoleucine, tryptophan, and alanine; or leucine, glycine, valine, isoleucine, tryptophan, and alanine; and among the six types of hydrophobic amino acids, preferred examples include phenylalanine or leucine, preferably two of phenylalanine and leucine.

[0023] When the seven hydrophobic amino acids (a-1) are contained, they may include phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine.

[0024] When the dipeptide (a-2) is composed of one kind of hydrophobic amino acid, the dipeptide composed of that one kind of hydrophobic amino acid is, in order from the N-terminus, phenylalanine-phenylalanine; leucine-leucine; glycine-glycine; valine-valine; isoleucine-isoleucine; tryptophan-tryptophan; or alanine-alanine.

[0025] When the dipeptide (a-2) is composed of two hydrophobic amino acids, the combination of the two hydrophobic amino acids is the same as the combination of the two hydrophobic amino acids described in the case where two hydrophobic amino acids are contained as hydrophobic amino acids in the dipeptide (a-1) above. The arrangement of the two hydrophobic amino acids is not particularly limited, and any hydrophobic amino acid may be arranged from the N-terminus to the C-terminus. Preferred examples of the dipeptide (a-2) include leucine-phenylalanine and glycine-phenylalanine, in order from the N-terminus.

[0026] When the tripeptide (a-3) above is composed of one kind of hydrophobic amino acid, the tripeptide composed of that one kind of hydrophobic amino acid is, in order from the N-terminus, phenylalanine-phenylalanine-phenylalanine; leucine-leucine-leucine; glycine-glycine-glycine; valine-valine-valine; isoleucine-isoleucine-isoleucine; tryptophan-tryptophan-tryptophan; or alanine-alanine-alanine.

[0027] When the tripeptide (a-3) is composed of two hydrophobic amino acids, the combination of the two hydrophobic amino acids is the same as the combination of the two hydrophobic amino acids described in (a-2) when two hydrophobic amino acids are contained as hydrophobic amino acids. The arrangement of the two hydrophobic amino acids is not particularly limited, and any hydrophobic amino acid may be arranged from the N-terminus to the C-terminus. Preferably, glycine-glycine-phenylalanine is used in this order from the N-terminus.

[0028] When the tripeptide (a-3) is composed of three hydrophobic amino acids, the combination of the three hydrophobic amino acids may be a combination of two hydrophobic amino acids described in (a-2) above plus one hydrophobic amino acid selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine. The arrangement of the three hydrophobic amino acids is not particularly limited, and any hydrophobic amino acid may be arranged from the N-terminus to the C-terminus.

[0029] When the tetrapeptide (a-4) above is composed of one type of hydrophobic amino acid, the tetrapeptide composed of that one type of hydrophobic amino acid is, in order from the N-terminus, phenylalanine-phenylalanine-phenylalanine; leucine-leucine-leucine-leucine; glycine-glycine-glycine-glycine; valine-valine-valine-valine; isoleucine-isoleucine-isoleucine-isoleucine; tryptophan-tryptophan-tryptophan-tryptophan; or alanine-alanine-alanine-alanine.

[0030] When the tetrapeptide (a-4) is composed of two hydrophobic amino acids, the combination of the two hydrophobic amino acids is the same as the combination of two hydrophobic amino acids described in the case where two hydrophobic amino acids are contained as hydrophobic amino acids in (a-3). The arrangement of the two hydrophobic amino acids is not particularly limited, and any hydrophobic amino acids may be arranged from the N-terminus to the C-terminus.

[0031] When the tetrapeptide (a-4) is composed of three hydrophobic amino acids, the combination of the three hydrophobic amino acids is the same as the combination of two hydrophobic amino acids described in the case where the tetrapeptide (a-3) contains three hydrophobic amino acids as hydrophobic amino acids. The arrangement of the three hydrophobic amino acids is not particularly limited, and any hydrophobic amino acids may be arranged from the N-terminus to the C-terminus.

[0032] When the tetrapeptide (a-4) is composed of four hydrophobic amino acids, the combination of the four hydrophobic amino acids may be a combination of four hydrophobic amino acids obtained by adding one hydrophobic amino acid selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine to the combination of three hydrophobic amino acids described in (a-3) above. The arrangement of the four hydrophobic amino acids is not particularly limited, and any hydrophobic amino acid may be arranged from the N-terminus to the C-terminus.

[0033] Furthermore, the hydrophobic substance may be a combination of the hydrophobic amino acid (a-1), the dipeptide (a-2), the tripeptide (a-3), or the tetrapeptide (a-4). Alternatively, the hydrophobic amino acid, dipeptide, tripeptide, or tetrapeptide (a-1) to (a-4) may be further combined with one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine.

[0034] The dipeptide, tripeptide or tetrapeptide may be straight-chain, cyclic or branched, but is preferably straight-chain.

[0035] The hydrophobic amino acids (monoamino acids) can be produced by known amino acid synthesis methods. The dipeptides, tripeptides, and tetrapeptides can be produced by known peptide synthesis methods such as solid-phase synthesis and liquid-phase synthesis. Examples of solid-phase synthesis methods include the fluorenylmethyloxycarbonyl method (Fmoc method) and the t-butyloxycarbonyl method (tBoc method). Alternatively, commercially available hydrophobic amino acids, dipeptides, tripeptides, and tetrapeptides may be used.

[0036] Furthermore, when two or more hydrophobic amino acids are contained in (a-1) above, the weight ratio of the two or more hydrophobic amino acids is not particularly limited. When two hydrophobic amino acids are contained in (a-1) above, the ratio of one hydrophobic amino acid to the other hydrophobic amino acid may be 0.1 to 10 parts by weight, 0.2 to 5 parts by weight, 0.3 to 3 parts by weight, 0.5 to 3 parts by weight, 0.8 to 2 parts by weight, 0.9 to 1.2 parts by weight, or 1 part by weight, for example.

[0037] (Nanoparticles) In the present composition for freeze-drying, the nanoparticles formed by amphiphilic molecules in a single layer or a double layer may contain only single-layer nanoparticles of amphiphilic molecules, only double-layer nanoparticles of amphiphilic molecules, or both single-layer and double-layer nanoparticles of amphiphilic molecules.

[0038] The monolayer nanoparticles refer to nanoparticles in which amphiphilic molecules are assembled to form a monolayer. A specific example of a monolayer nanoparticle is a micelle. Furthermore, the bilayer nanoparticles refer to nanoparticles containing a bilayer formed by two layers of amphiphilic molecules. Specific examples of the bilayer nanoparticles include (1) liposomes, which are vesicles containing phospholipids as a constituent component and a phospholipid bilayer, and (2) vesicles containing surfactants other than phospholipids as constituent components and a surfactant bilayer. The bilayer-containing vesicles may be small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs), which have one bilayer, or multilamellar vesicles (MVLs), which have two or more bilayers.

[0039] Furthermore, when the nanoparticles contain micelles, the micelles are preferably micelles in which hydrophilic groups are on the outside and hydrophobic groups are on the inside.

[0040] The volume average particle size of the nanoparticles may be 10 nm or more and 300 nm or less, and may be 20 nm or more and 200 nm or less, or 30 nm or more and 150 nm or less.

[0041] The smaller the volume average particle diameter of the nanoparticles, the easier they are to distribute to every corner of the cells, and the easier it is for the shape of the cells to be maintained when the cells are freeze-dried.When the volume average particle diameter of the nanoparticles is 300 nm or less, the nanoparticles are more efficiently introduced into cells.

[0042] The nanoparticles may be cationic, neutral, or anionic nanoparticles, but cationic nanoparticles are more preferred. Typically, the interior of a cell has a negative (negative) potential relative to the exterior of the cell. Therefore, if the nanoparticles are cationic nanoparticles, they can be efficiently introduced into a wide area of ​​the cell through ionic interactions. As a result, cell damage caused by cryopreservation is further reduced.

[0043] Cationic nanoparticles refer to nanoparticles with a zeta potential of more than 10 mV. Neutral nanoparticles refer to nanoparticles with a zeta potential of -10 mV or more and 10 mV or less. Anionic nanoparticles refer to nanoparticles with a zeta potential of less than -10 mV.

[0044] The zeta potential of the nanoparticles is preferably 10 mV or more and 80 mV or less, more preferably 15 mV or more and 70 mV or less, even more preferably 20 mV or more and 60 mV or less, and particularly preferably 25 mV or more and 50 mV or less. When the zeta potential of the nanoparticles is 10 mV or more, the membrane of the nanoparticles is appropriately positively charged. Therefore, the nanoparticles can be more efficiently introduced into cells, and cell damage caused by freeze-drying can be further reduced.

[0045] The nanoparticles may contain cholesterol, and when the nanoparticles are configured to contain cholesterol in the membrane, the fluidity of the membrane of the nanoparticles is improved compared to a membrane that does not contain cholesterol.

[0046] The nanoparticles may further encapsulate other components. The encapsulated components refer to components incorporated within the membrane of the nanoparticles, components incorporated into the surfactants that constitute the nanoparticles, and components encapsulated in the internal aqueous phase. The encapsulated components can be adjusted appropriately depending on the application of the cells to be lyophilized, and include fat-soluble drugs, water-soluble drugs, nucleic acids, proteins, antibodies, crystalline drugs, biologically derived components, water-soluble vitamins, water-soluble vitamin derivatives, fat-soluble vitamins, fat-soluble vitamin derivatives, and labeling substances (e.g., fluorescent substances such as coumarin).

[0047] Specific examples of the encapsulated components include activated vitamin C and activated vitamin A, which have a cell-protecting effect, and ferulic acid and hyaluronic acid, which are moisture-retaining components. In particular, ferulic acid and activated vitamin C have excellent antioxidant properties and moisture-retaining properties, so by encapsulating at least one of these in the nanoparticles, cell damage caused by cell freeze-drying can be further suppressed.

[0048] In the present composition for freeze-drying, the amphiphilic molecule may be a surfactant, which may be a synthetic surfactant or a natural or biologically derived surfactant.

[0049] Examples of synthetic surfactants include Gemini surfactants, which have a hydrophilic group and a hydrophobic group in one molecule, and are linked to each other via a spacer group via the hydrophilic group, and are dimeric surfactants having two types of surface-active units or trimeric surfactants having three types of surface-active units.

[0050] The hydrophobic group constituting the Gemini surfactant may be a linear hydrocarbon chain having 8 to 24 carbon atoms, preferably 12 to 18 carbon atoms, and more preferably 12 or 16 carbon atoms, and a linear saturated hydrocarbon chain is preferred from the viewpoint of nanoparticle stability. Furthermore, the linear hydrocarbon chains constituting the two or three types of hydrophobic groups may be the same or different, but are preferably the same.

[0051] The hydrophilic group constituting the Gemini surfactant may be a polar group or an ionic group. The element that generates the counter ion in the hydrophilic group can be appropriately adjusted depending on the aqueous solution used, and examples thereof include bromine (Br), chlorine (Cl), nitrogen (N), sodium (Na), phosphorus (P), boron (B), iodine (I), fluorine (F), sulfur (S), oxygen (O), carbon (C), beryllium (Be), iron (Fe), calcium (Ca), and magnesium (Mg), and these may also be used in appropriate combination.

[0052] The spacer is usually a hydrocarbon chain, and preferably has a length sufficient to provide a sufficient distance for the hydrophobic groups to act independently of each other. The number of carbon atoms in the spacer is not particularly limited, but may be 2 to 12, preferably 2 to 8, more preferably 2 to 4, and most preferably 2 or 3. A hydroxyl group may be introduced into one of the hydrocarbon chains.

[0053] Natural and biological surfactants include amphipathic lipids. The amphipathic lipids may be non-phosphorus lipids or phospholipids. They may also be cationic lipids, neutral lipids, anionic lipids, or combinations thereof.

[0054] Examples of phosphorus-free lipids include N-(2,3-dioleoyloxy-1-propyl)trimethylammonium methyl sulfate (DOTAP), N-[1-(2,3-oleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), and dioctadecylamide-glycylspermine (DOGS).

[0055] Examples of phospholipids include phosphoethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE); and phosphocholines such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and diphthanoyl-sn-glycero-3-phosphocholine (DPhPC).

[0056] Examples of methods for producing the nanoparticles, such as a method for controlling the volume average particle diameter of the nanoparticles and a method for measuring the zeta potential and volume average particle diameter of the nanoparticles, include the methods described in Patent Document 5.

[0057] The combination of the hydrophobic substance and the nanoparticles is not particularly limited, and examples thereof include a combination of any one of the hydrophobic substances (a-1) to (a-4) and nanoparticles formed in a single or double layer of a Gemini surfactant. For example, examples include a combination of the hydrophobic substance (a-1) and nanoparticles formed in a single or double layer of a Gemini surfactant, a combination of phenylalanine and nanoparticles formed in a single or double layer of a Gemini surfactant, and a combination of phenylalanine and leucine and nanoparticles formed in a single or double layer of a Gemini surfactant. Further examples include a combination of phenylalanine, preferably a combination of phenylalanine and leucine, and nanoparticles formed in a single layer or two layers of Gemini surfactants whose hydrophobic groups are composed of straight-chain hydrocarbon chains with 12 to 18 carbon atoms; a combination of phenylalanine, preferably a combination of phenylalanine and leucine, and nanoparticles formed in a single layer or two layers of Gemini surfactants; and a combination of phenylalanine, preferably a combination of phenylalanine and leucine, and nanoparticles formed in a single layer or two layers of Gemini surfactants whose hydrophobic groups are composed of straight-chain hydrocarbon chains with 12 or 16 carbon atoms.

[0058] (Cells to be added) Cells to which the present composition for freeze-drying of cells can be added include cells derived from mammals, fish, birds, plants, microalgae, and algae, as well as bacteria and unicellular organisms, and preferred examples include cells derived from mammals. Examples of mammals include humans, dogs, cats, monkeys, cows, horses, mice, rats, hamsters, guinea pigs, rabbits, goats, pigs, and sheep. When the cells to be added to the present composition for freeze-drying are human-derived cells, these refer to human-derived cells isolated from a living organism.

[0059] The cells to be added may be differentiated or undifferentiated cells, and specific examples include pluripotent stem cells, organ-specific cells, adipocytes, and germ cells such as embryos, fertilized eggs, sperm, embryonic stem cells, gametocytes, and oocytes. Furthermore, when the cells to be added are human-derived cells, they may be differentiated or undifferentiated cells, and examples thereof include somatic stem cells such as human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) and human bone marrow-derived mesenchymal stem cells (hMSC-BM), neural stem cells, skin stem cells, hematopoietic stem cells, dental pulp stem cells, hepatic stem cells, muscle stem cells, and adipose stem cells; pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells); blood cells such as lymphocytes and neutrophils; organ-specific cells such as epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), smooth muscle cells, melanocytes, hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, and kidney cells; adipocytes; and germ cells such as embryos, fertilized eggs, and sperm. Furthermore, the cells may be single cells or a mixture of multiple cells. The cells may also be in the form of cells that have been cultured in a conventional manner, such as suspension culture, monolayer culture, or 3D culture, or may be in the form of organoids, spheroids, cell sheets, or biological tissues obtained by culturing the above cells.

[0060] (Other Components) The present composition for freeze-drying may contain other components in addition to the hydrophobic substance and the nanoparticles, such as trehalose and / or ascorbic acid or a derivative thereof. Examples of the ascorbic acid derivative include L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, 2-O-α-D-glucopyranosyl-L-ascorbic acid, and glyceryl ascorbate. For example, the present composition for freeze-drying may be a combination of the hydrophobic substance, the nanoparticles, and ascorbic acid 2-phosphate sesquimagnesium salt hydrate, or a combination of the hydrophobic substance, the nanoparticles, ascorbic acid 2-phosphate sesquimagnesium salt hydrate, and trehalose.

[0061] The composition for freeze-drying may also contain a dispersion medium for dispersing the nanoparticles or hydrophobic substance, or amphiphilic molecules or aggregates thereof that did not form a monolayer or bilayer during the process of producing the nanoparticles. Examples of the dispersant include water, buffer solutions, water-soluble solvents, liquid media, isotonic solutions, etc., and they may be used alone or in combination of two or more.

[0062] Examples of the water include distilled water, ion-exchanged water, ultrafiltered water, and pure water, and the water content is preferably 70% by mass or more, more preferably 80% by mass or more and 90% by mass or less, and even more preferably 90% by mass or more and 100% by mass or less, based on the total amount of the dispersion medium. By setting the water content within the above numerical range, the nanoparticles are more stable.

[0063] Examples of the buffer solution include phosphate buffer solution (PBS), trishydroxymethylaminomethane buffer solution (TRIS), hydroxyethylpiperazineethanesulfonic acid buffer solution (HEPES), borate buffer solution, acetate buffer solution, etc. The pH of the buffer solution is not particularly limited, but from the viewpoint of, for example, cell viability, it is preferably pH 5 or more and pH 10 or less, and more preferably pH 6 or more and pH 9 or less.

[0064] As the water-soluble solvent, alcohols such as methanol and ethanol can be used from the viewpoint of the solubility of nanoparticles.

[0065] The liquid medium may be any known medium used for cell culture, and may be adjusted appropriately depending on the type of cells to which the present composition for freeze-drying of cells is added. More specifically, for example, when the cells to which the present composition for freeze-drying of cells is added are human cells, examples of the liquid medium include Dulbecco's Modified Eagle's Medium (DMEM), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM:F-12), Eagle's minimal essential medium (EMEm), and liquid media obtained by adding cell growth factors such as platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and nerve growth factor (NGF), as well as commercially available liquid media for cell growth.

[0066] 2. Method for Producing Freeze-Dried Cells of the Present Invention In the method for producing freeze-dried cells of the present invention, the step of adding the present composition for freeze-drying to cells is not particularly limited, but examples include a method in which the present composition for freeze-drying is added to a predetermined solution containing the cells. The addition of the present composition for freeze-drying to cells may be performed under conditions that allow contact between the composition for freeze-drying and the cells, and the cells may be added to the composition for freeze-drying. Alternatively, the medium containing the cells may be centrifuged to remove the supernatant and then the composition for freeze-drying is added, or the composition for freeze-drying may be introduced into the cells using voltage via electroporation.

[0067] The predetermined solution may be any solution capable of suspending the cells, and examples thereof include isotonic solutions such as phosphate buffered saline (PBS), physiological saline, and simulated body fluids, and liquid culture media.

[0068] In the step of adding the present composition for freeze-drying to cells, the present composition for freeze-drying can be added so that the concentration of the hydrophobic substance contained in the composition for freeze-drying in the predetermined solution containing the cells is 0.2 to 100 mg / mL, or 0.3 to 100 mg / mL. When a hydrophobic amino acid is included as the hydrophobic substance, the concentration of each hydrophobic amino acid can be adjusted to 1 to 60 mg / mL, 3 to 60 mg / mL, 5 to 60 mg / mL, 7 to 30 mg / mL, 8 to 15 mg / mL, 10 to 50 mg / mL, or 20 to 45 mg / mL, although this can be adjusted appropriately depending on the solubility of the hydrophobic amino acid and the type of predetermined solution used. Specifically, when phenylalanine and leucine are used in combination as the hydrophobic substances, the concentration of phenylalanine can be 5 to 60 mg / mL, preferably 10 to 50 mg / mL, and more preferably 20 to 45 mg / mL, and the concentration of leucine can be 5 to 60 mg / mL, preferably 10 to 50 mg / mL, and more preferably 20 to 45 mg / mL. Furthermore, when a dipeptide, tripeptide, or tetrapeptide is used as the hydrophobic amino acid, the concentration of the dipeptide, tripeptide, or tetrapeptide can be 0.3 to 20 mg / mL, preferably 0.4 to 5 mg / mL, and more preferably 0.5 to 1 mg / mL.

[0069] Furthermore, in the step of adding the present composition for freeze-drying to cells, the present composition for freeze-drying can be added so that the solution of nanoparticles formed as a single layer or a double layer of amphiphilic molecules contained in the present composition for freeze-drying, which is prepared by the method described in the Examples below, becomes 0.1 to 1% in a predetermined solution containing cells.

[0070] In the method for producing freeze-dried cells of the present invention, the freeze-drying method is not particularly limited, but preferably comprises the following steps (a) and (b): step (a) of adding the composition for freeze-drying of the present invention to the cells and freezing them at a temperature of −196 to −20° C., preferably −196 to −80° C., for 8 hours or more, preferably 12 hours or more; and step (b) of drying the cells frozen in step (a) in a vacuum, for example, under conditions of −1 MPa or less, at −50 to 35° C. for 10 hours or more, preferably 12 to 72 hours.

[0071] The above step (a) can be carried out at normal pressure. Step (a) enables a predetermined solution containing cells to be rapidly solidified by freezing at a low temperature.

[0072] The drying in step (b) may be performed in one step or multiple steps. For example, when performing the drying in two steps, the drying time can be adjusted appropriately depending on the type, shape, size, and degree of dryness of the cells to be dried. Examples of the primary drying method include slow drying at -40°C and -1 MPa or less for 12 to 48 hours, and rapid drying at -20°C and -1 MPa or less for 4 to 24 hours. Secondary drying methods include drying at -10°C and -1 MPa or less for 4 to 12 hours. Step (b) allows the solvent contained in the predetermined solution containing the cells solidified by freezing to be sublimated and removed.

[0073] After the step (b), a step (c) of further drying at normal pressure and room temperature for 2 hours or more may be carried out.

[0074] The freeze-dried cell production method can also be used for the long-term freeze-drying of cells, such as oocytes and sperm, from rare or endangered animal species for their preservation. The freeze-dried cell production method can further be used for animal husbandry purposes (e.g., animal breeding and husbandry), for example, for the cryopreservation of embryonic stem cells, gametocytes, oocytes, or sperm from animals such as cattle, pigs, and sheep.

[0075] The freeze-dried cells produced by the freeze-dried cell production method of the present invention can be stored for long periods at room temperature or in a refrigerator. The storage period is not particularly limited, and examples thereof include 0.5 days to 50 years, 1 day to 10 years, 1 week to 5 years, and 1 month to 1 year.

[0076] 3. Restored Cells The restored cells may be obtained by restoring freeze-dried cells prepared by the freeze-dried cell preparation method in a solvent, such as distilled water, PBS, or liquid culture medium. The time for restoring in the solvent can be adjusted appropriately depending on the intended use and storage conditions, but may be from 1 minute to 12 hours. The temperature for restoring may be 0 to 40°C, 4 to 37°C, or room temperature. The restored cells can be stored at a temperature appropriate for maintaining the function or morphology of the cells, with the solvent replaced as needed.

[0077] 4. Pharmaceutical Compositions The pharmaceutical compositions of the present invention may contain the restored cells and pharmaceutically acceptable additives, such as saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers, surfactants, buffers, preservatives, tonicity agents, bulking agents, and lubricants.

[0078] The restored cells in the pharmaceutical composition may be autologous cells derived from the patient for whom the pharmaceutical composition is to be used, or may be allogeneic cells derived from another subject of the same species, for example, another patient.

[0079] The dosage of the pharmaceutical composition of the present invention, when in a suspension state, is 1 x 10 as the number of the restored cells contained therein. ２ ~1 x 10 １６ pieces / time, preferably 1 x 10 ４ ~1 x 10 １４ pieces / time, more preferably 1 x 10 ６ ~1 x 10 １２The above dose may be administered multiple times as a single dose, or the above dose may be administered in divided doses. In addition, when administered to an adult, the number of cells per body weight is usually 0.2 × 10 to 5 × 10 １３ pieces / kg, preferably 1 x 10 ２ ~5 x 10 １１ pieces / kg, more preferably 1 x 10 ４ ~5 x 10 ９ The above dose may be administered as a single dose multiple times, or the above dose may be administered in divided doses multiple times.

[0080] The method of administration of the pharmaceutical composition of the present invention is not particularly limited as long as it can provide the desired preventive or therapeutic effect on a disease, and examples thereof include administration by intravenous injection, subcutaneous injection, intramuscular injection, or surgical treatment.

[0081] The pharmaceutical composition can be used for preventing or treating diseases that are targets of regenerative medicine or cell therapy. Examples of the diseases include vascular disorders such as cerebrovascular disorders, peripheral vascular diseases, coronary artery diseases, diabetic vasculopathy, and lymphatic disorders; skin ulcers such as bedsores, arteriosclerosis obliterans, severe limb ischemia, venous insufficiency, diabetic skin ulcers, collagen diseases, and vasculitis; immune diseases such as graft-versus-host disease, graft rejection, autoimmune diseases, chronic inflammatory diseases, inflammatory pain, and neuropathic pain; and cancers such as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, undifferentiated carcinoma, large cell carcinoma, small cell carcinoma, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, pancreatic cancer, spleen cancer, and Examples of cancers that may be affected include lung cancer, tracheal cancer, bronchial cancer, colon cancer, small intestine cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, and ovarian cancer; cancers of tissues such as bone tissue, cartilage tissue, adipose tissue, muscle tissue, vascular tissue, and hematopoietic tissue; sarcomas such as chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma; blastomas such as hepatoblastoma, medulloblastoma, nephroblastoma, neuroblastoma, pancreatoblastoma, pleuropulmonary blastoma, and retinoblastoma; blood cancers such as germ cell tumors, lymphoma, and leukemia; eye diseases such as bullous keratopathy; spinal cord injury, traumatic cartilage defect, endometriosis, and uterine fibroids.

[0082] The present invention will be explained in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0083] The nanoparticles used in the present invention were prepared by organic solvent injection, following the procedure described in Patent Document 5. Nanoparticles containing a bilayer membrane composed of a Gemini surfactant were prepared according to the following procedure: (1) 5.3 mL of PBS (pH 7.4 at 25°C) was placed in a beaker and stirred at 25°C. (2) 1990 μL of ethanol, 38.7 mg of cholesterol, and 11.1 mg of a Gemini surfactant as an amphiphilic molecule were mixed in a centrifuge tube, and the temperature of the mixture was raised to 75°C, above the phase transition temperature of the amphiphilic molecule, to completely dissolve the cholesterol and the Gemini surfactant. (3) The entire mixture was taken with a syringe and rapidly mixed into the PBS. (4) The beaker was shielded from light and stirred at 25°C for 1 hour. (5) Dialysis was carried out in PBS at room temperature for at least 4 hours using a dialysis membrane (SpectraPor (registered trademark) RC Biotech Dialysis Membrane MWCO: 8-10: Repligen). This yielded a nanoparticle solution. (6) The obtained nanoparticle solution was placed in an LF-STB Liposofast Stabilizer (Avestin), and sized by passing through filters with pore sizes of 1000 nm, 800 nm, 400 nm, 200 nm, 100 nm, and 50 nm in this order while applying pressure. The obtained liquid composition containing nanoparticles was used as an additive for Product 1.

[0084] [Example 1] Stem cell culture and freeze-drying Human mesenchymal stem cells (PT-5006: Lonza) were cultured in a 10 cm ２ 12 mL of the special medium was placed in a petri dish and incubated in 5% CO ２ The cells were cultured in an incubator at 37°C and 70-80% of the culture vessel surface area at a density of approximately 5000 cells / cm. ２The cells were maintained by being reseeded at a density of 1000 x g. The above-mentioned human mesenchymal stem cells, which were 80% confluent, were detached using a trypsin / EDTA solution, suspended in a medium (PT-4505: LONZA), and centrifuged to obtain a cell pellet. The obtained human mesenchymal stem cells and the additives in columns 1 to 8 of comparative products and columns 1 to 9 of examples listed in Table 1 or Table 2 below were dispersed in 2 mL of phosphate buffer (pH 7.4: Thermo Fisher Scientific), filled into glass vials, and pre-frozen for 12 to 84 hours in liquid nitrogen or a deep freezer (-80°C). The pre-frozen glass vials were evacuated in a vacuum freeze dryer (VFD-03: AS ONE Corporation), and dried in the following order to prepare freeze-dried products, which were used in the following examples: -20°C, -1 MPa, 24 hours -10°C, -1 MPa, 4 hours

[0085] [Example 2] Scanning electron microscope images of freeze-dried cells The appearance of freeze-dried human mesenchymal stem cells prepared using the additive in Example 1 by the method described in Example 1 was evaluated using a scanning electron microscope (SEM). The sample was removed so as not to disrupt the freeze-dried state, platinum was vapor-deposited, and the sample was observed using a high-resolution scanning electron microscope (FE-SEM) JSM-7500F (JEOL Ltd.). The results are shown in Figure 1.

[0086] As shown in Figure 1, when the additives in Column 2 (i.e., phenylalanine, leucine, and 12-3-12_Br nanoparticles) were added and the cells were freeze-dried using the above method, the cell shape was maintained. In addition, a mesh structure was formed around the cells.

[0087] Example 3: Average Cell Particle Size The particle size of freeze-dried human adipose tissue-derived mesenchymal stem cells (ADSCs) prepared using the method described in Example 1 with the additives listed in columns 1-3 for comparison and columns 1-3 for examples was evaluated using dynamic light scattering (DLS). Freeze-dried human adipose tissue-derived mesenchymal stem cells (ADSCs) prepared using the method described in Example 1 were reconstituted by diluting 50-fold with PBS or PBS containing 10% mannitol. 1 mL of each sample was loaded into a square cell for particle size measurement, and the average particle size of exosomes was measured using a Zetasizer Pro. As a control, the average particle size of human adipose tissue-derived mesenchymal stem cells without freeze-drying was also measured. The results are shown in Table 1. The nanoparticles listed in Table 1 were prepared using the method described in Example 1. In Table 1, the unit of average particle size is nm. Furthermore, the amount of each additive in Table 1 is the amount (mg) of additive per 2 mL of phosphate buffer solution before freeze-drying.

[0088]

[0089] In Table 1 or Table 2 described later, 12-2-12_Br nanoparticles are nanoparticles containing a bilayer composed of Gemini surfactants, each of which has two hydrocarbon chains with 12 carbon atoms, a spacer with 2 carbon atoms, and elements that generate counter ions being bromine and nitrogen; 12-3-12_Br nanoparticles are nanoparticles containing a bilayer composed of Gemini surfactants, each of which has two hydrocarbon chains with 12 carbon atoms, a spacer with 3 carbon atoms, and elements that generate counter ions being bromine and nitrogen; 12-3(OH)-12_Br nanoparticles are nanoparticles containing a bilayer composed of Gemini surfactants, each of which has two hydrocarbon chains with 12 carbon atoms, a spacer with 3 carbon atoms, and a hydroxyl group introduced into one of the three carbon atoms, and elements that generate counter ions being bromine and nitrogen; and 12-3-12_Cl nanoparticles are 12-12-12_Br refers to nanoparticles comprising a bilayer membrane composed of Gemini surfactants each having two types of hydrocarbon chains with 12 carbon atoms, a spacer with 3 carbon atoms, and counter ions of chlorine and nitrogen; 12-12-12_Br refers to nanoparticles comprising a bilayer membrane composed of Gemini surfactants each having two types of hydrocarbon chains with 12 carbon atoms, a spacer with 12 carbon atoms, and counter ions of bromine and nitrogen; 16-3-16_Br refers to nanoparticles comprising a bilayer membrane composed of Gemini surfactants each having two types of hydrocarbon chains with 16 carbon atoms, a spacer with 3 carbon atoms, and counter ions of bromine and nitrogen; and 16-3-12_Cl refers to nanoparticles comprising a bilayer membrane composed of Gemini surfactants each having two types of hydrocarbon chains with 16 carbon atoms, a spacer with 3 carbon atoms, and counter ions of chlorine and nitrogen.

[0090] As is clear from Table 1, when freeze-drying and reconstituting the cells using the additives in columns 1 to 3, i.e., nanoparticles containing a bilayer membrane composed of phenylalanine, leucine, and a Gemini surfactant, the average particle size maintained at 87% or more of the average particle size of the non-freeze-dried cells. On the other hand, when the additives in columns 1 to 3, comparative products, were used, the average particle size was less than one-tenth of the average particle size of the non-freeze-dried cells, and the shape was not maintained. Furthermore, when only the additive 12-3-12_Br nanoparticles in column 2, was used, the shape of the freeze-dried cake could not be maintained even after freeze-drying, to the extent that the particle size distribution of the cells could not be confirmed.

[0091] [Example 4] Proportion of Viable Cells 5 μL of each cell solution, which had been freeze-dried and reconstituted by adding medium using the method described in Example 1, was mixed with 5 μL of trypan blue staining solution, and the number of trypan blue-positive cells was counted using a cell counter. The results are shown in Table 2. In Table 2, the amount of each additive is the amount (mg) of additive per 2 mL of phosphate buffer solution before freeze-drying.

[0092]

[0093] As is clear from Table 2, when nanoparticles containing phenylalanine, leucine, and a Gemini surfactant were used for freeze-drying, the percentage of viable cells was extremely high, ranging from 38.5 to 86.5%. Furthermore, the percentage of viable cells was 1.2 when only the additive phenylalanine (comparison product 6) was used, the percentage of viable cells was 0.7 when only the additive leucine (comparison product 7) was used, and the percentage of viable cells was 8.2 when the additives phenylalanine and leucine (comparison product 9) were used. Considering this, together with the fact that the average particle size was 1 / 36th that of the case without cell freezing when the additive 12-3-12_Br nanoparticles (comparison product 2) in Table 1 were used, it became clear that the combination of a hydrophobic substance and nanoparticles formed of a single or double layer of amphiphilic molecules contributed to an increase in the percentage of viable cells and the maintenance of the average particle size.

[0094] Example 5 Cell Viability Lyophilized human mesenchymal stem cells were prepared without lyophilization, or using the additives in column 5 of the comparative product or column 8 of the experimental product according to the method described in Example 1. The cells were reconstituted with medium and seeded at a density of 2,000 cells / well in a 96-well plate. 10 μl of MTT labeling reagent was added to each well. The cells were incubated in a 37°C incubator for 4 hours. MTT eluate was added, mixed thoroughly, and allowed to stand in a 37°C incubator for 6 hours. To obtain a measurement value, the absorbance of the purple dye was measured at a wavelength of 570 nm using an absorbance meter. The results of the cell proliferation rate are shown in Figure 2.

[0095] As shown in Figure 2, when phenylalanine, leucine, nanoparticles (12-3-12_Br), trehalose, and ascorbyl 2-phosphate sesquimagnesium hydrate were used as additives, the survival rate was maintained at approximately 84% compared to without freeze-drying.

[0096] [Example 6] Transplantation efficiency Human mesenchymal stem cells were prepared without freeze-drying or using the additives in Comparative Product 5 and Example Product 8 by the method described in Example 1, and culture medium was added to restore the human mesenchymal stem cells. Matrigel for spheroid culture and growth factors included in the dedicated culture medium were then added, and the cells were cultured into spheroids one week before transplantation. Human mesenchymal stem cell spheroids were mixed with Matrigel for transplantation at a 1:1 ratio. 10 ７ 300 μL of the transplant mixture containing adipose-derived mesenchymal stem cells from BALB / cAJcl-nu / nu cells was filled into a syringe on ice and transplanted subcutaneously into the dorsal skin of an immunodeficient mouse (strain name: BALB / cAJcl-nu / nu). The weight (mg) of the graft excised three weeks after transplantation was measured using a precision electronic balance. The weight relative to the weight without cell freezing, set at 100, was calculated as the transplant efficiency. The results of the transplant efficiency are shown in Figure 3.

[0097] As shown in Figure 3, when phenylalanine, leucine, nanoparticles (12-3-12_Br), trehalose, and ascorbyl 2-phosphate sesquimagnesium hydrate were used as additives, the transplantation efficiency was maintained at approximately 78% compared to without freeze-drying.

Claims

1. (a) any of the following hydrophobic substances (a-1) to (a-4): (a-1) one or more hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-2) a dipeptide composed of one or two hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-3) a tripeptide composed of one, two, or three hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (a-4) a tetrapeptide composed of one, two, three, or four hydrophobic amino acids selected from the group consisting of phenylalanine, leucine, glycine, valine, isoleucine, tryptophan, and alanine; (b) nanoparticles formed of a monolayer or bilayer of amphiphilic molecules: A composition for freeze-drying cells, comprising (a) and (b) above, which is added to cells when used.

2. The composition for freeze-drying according to claim 1, characterized in that the hydrophobic substance contains at least two of phenylalanine and leucine.

3. The composition for freeze-drying according to claim 1, wherein the hydrophobic substance is a combination of phenylalanine and leucine.

4. A composition for freeze-drying according to any one of claims 1 to 3, wherein the cells are human-derived cells isolated from a living body.

5. A composition for freeze-drying according to any one of claims 1 to 4, further comprising trehalose and / or ascorbic acid or a derivative thereof.

6. A composition for freeze-drying according to any one of claims 1 to 5, characterized in that the amphiphilic molecule is a Gemini surfactant.

7. A method for producing freeze-dried cells, comprising the step of adding the composition for freeze-drying according to any one of claims 1 to 6 to cells.

8. A method for producing freeze-dried cells according to claim 7, characterized in that the composition for freeze-drying according to any one of claims 1 to 6 is added to the solution containing the cells so that the concentration of the hydrophobic substance contained in the composition for freeze-drying is 0.3 to 100 mg / mL.

9. A method for producing freeze-dried cells according to claim 7 or 8, comprising the steps of: (a) adding the composition for freeze-drying according to any one of claims 1 to 6 to cells and freezing them at -196 to -20°C for 8 hours or more; and (b) drying the cells frozen in step (a) under vacuum conditions at -50 to 35°C for 10 to 72 hours.

10. Cells obtained by reconstituting freeze-dried cells prepared by the method of any one of claims 7 to 9 in a solvent.

11. A pharmaceutical composition comprising the restored cells of claim 10 and a pharmaceutically acceptable excipient.

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