Composition for cryopreservation, cryopreservation method, and frozen cell or biological tissue

Abstract

The present invention addresses the problem of providing a composition for cryopreservation that reduces cell damage caused by cryopreservation, and a cryopreservation method that reduces cell damage caused by cryopreservation. In the present invention, a composition for cryopreservation of a cell or a biological tissue is produced, said composition containing nanoparticles having an amphiphilic molecule as a constituent component, and the amphiphilic molecule is in a single layer or a double layer.

Description

Technical Field

[0001] The present invention relates to a composition for cryopreservation, a cryopreservation method, and frozen cells or biological tissues. Background Art

[0002] Regenerative medicine is a method of treating diseases, injuries, etc. using cells, etc. In recent years, regenerative medicine has been used as a method of treating various diseases such as myocardial infarction, chronic arterial occlusion, leukemia, spinal cord injury, traumatic cartilage defects, etc. From the perspective of transportability, long-term preservation, convenience, etc., cells used for regenerative medicine have always been cryopreserved cells. However, when cells are cryopreserved, it is easy to cause a decrease in cell survival rate, impaired cell function (for example, in the case of stem cells, a decrease in the proportion of stem cells), proliferation inhibition, and other phenomena (hereinafter, these phenomena are also collectively referred to as "cell damage"). As the main reason, it can be considered that the water contained in the cell crystallizes when the cell is frozen, and the crystallization causes damage to the cell wall, organelles, etc., or the water in the cell is partially lost when the cell is thawed, thereby changing the shape of the cell.

[0003] As one of the cell cryopreservation methods for freezing and preserving cells, as described in Patent Document 1, a method of freezing cells in a buffer solution containing a low molecular weight compound such as dimethyl sulfoxide (DMSO) is known.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: Japanese Patent No. 5773347 Summary of the invention

[0007] Problems to be solved by the invention

[0008] However, the method using DMSO has a low cell survival rate after cell thawing, and there is room for improvement. The present invention was completed under such circumstances.

[0009] An object of one embodiment of the present invention is to provide a composition for cryopreservation of cells or biological tissues that reduces cell damage caused by cryopreservation.

[0010] Another embodiment of the present invention aims to provide a method for cryopreservation of cells or biological tissues, which reduces cell damage caused by cryopreservation.

[0011] Means for solving problems

[0012] Specific means for solving the above-mentioned problems include the following.

[0013] <1> A composition for cryopreservation of cells or biological tissues, comprising nanoparticles, wherein the nanoparticles have amphiphilic molecules as constituent components, and the amphiphilic molecules are monolayer or bilayer.

[0014] <2> The composition for cryopreservation according to <1> above, wherein the nanoparticles are cationic nanoparticles.

[0015] <3> The composition for cryopreservation according to <1> or <2> above, wherein the amphiphilic molecule is a Gemini surfactant.

[0016] <4> The composition for cryopreservation according to <1> or <2> above, wherein the nanoparticles have a zeta potential of 10 mV to 50 mV.

[0017] <5> The composition for cryopreservation according to <1> or <2> above, wherein the volume average particle size of the nanoparticles is from 30 nm to 300 nm.

[0018] <6> The composition for cryopreservation according to <1> or <2> above, wherein the nanoparticles contain ferulic acid or active vitamin C.

[0019] <7> A method for cryopreservation of cells or biological tissues, comprising:

[0020] A step of mixing cells or biological tissues with the cryopreservation composition described in any one of <1> to <6> above and introducing nanoparticles into the cells or biological tissues; and

[0021] A step of freezing the cells or biological tissues into which the aforementioned nanoparticles have been introduced.

[0022] <8> Frozen cells or biological tissues, comprising nanoparticles, wherein the nanoparticles are composed of amphiphilic molecules, and the amphiphilic molecules are monolayer or bilayer.

[0023] Effects of the Invention

[0024] According to one embodiment of the present invention, a composition for cryopreservation of cells or biological tissues can be provided, which reduces damage to cells or biological tissues caused by cryopreservation.

[0025] According to another embodiment of the present invention, a method for cryopreservation of cells or biological tissues can be provided, which reduces damage to the cells or biological tissues caused by cryopreservation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] [ Figure 1 ] is a graph showing the distribution of particle sizes of nanoparticles contained in the composition of Example 3 in Test Example 1.

[0027] [ Figure 2 ] is a diagram showing the results of observing the nanoparticles introduced into cells using a transmission electron microscope in Experimental Example 3. DETAILED DESCRIPTION

[0028] Hereinafter, embodiments of the present invention will be described. These descriptions and examples are examples of the present invention and do not limit the scope of the present invention.

[0029] In the present specification, a numerical range expressed using “to” means a range including the numerical values ​​described before and after “to” as the minimum value and the maximum value, respectively.

[0030] <Composition for cell cryopreservation>

[0031] The composition for cryopreservation of cells or biological tissues involved in this embodiment (hereinafter also referred to as "the composition for cryopreservation of the present application") contains nanoparticles (hereinafter also referred to as "nanoparticles of the present application") having amphiphilic molecules as constituent components and the amphiphilic molecules being a single layer or a double layer.

[0032] If the cryopreservation composition of the present application is mixed with cells or biological tissues, the nanoparticles of the present application can be introduced into the cells or cells constituting the biological tissues. That is, the nanoparticles of the present application are introduced into the cells.

[0033] With regard to the cell or organism tissue after freezing under the nanoparticle of the present application that has been imported into the intracellular state, the nanoparticle of the present application shows a relaxation function (buffering function) for the external force, the temperature variation that is applied to the cell, thereby suppresses the crystallization and the crystal growth of the water contained in the cell, and cell wall and organelle etc. become difficult to be damaged. In addition, with regard to the cell after freezing under the nanoparticle of the present application that has been imported into the intracellular state, even when thawing, the shape of the cell also becomes easy to be maintained because of the nanoparticle that has the present application. It can be believed that the result is the impairment that reduces the cause of cryopreservation.

[0034] It should be noted that nanoparticles such as liposomes and micelles have been used as drug delivery systems and other technologies for introducing pharmacological components into target sites. In contrast, the invention to which the present disclosure relates focuses on the buffering function of the nanoparticles of the present application themselves against cells, based on the following insight: the physical properties possessed by the nanoparticles of the present application effectively act on the reduction of cell damage caused by the cryopreservation of cells.

[0035] [Nanoparticles]

[0036] The cryopreservation composition of the present application comprises nanoparticles, wherein the nanoparticles are composed of amphiphilic molecules, and the amphiphilic molecules are monolayer or bilayer. The cryopreservation composition of the present application may comprise only monolayer nanoparticles, may comprise only bilayer nanoparticles, or may comprise both monolayer nanoparticles and bilayer nanoparticles.

[0037] The above-mentioned single-layer nanoparticles refer to nanoparticles obtained by aggregating amphiphilic molecules in a single-layer manner. As single-layer nanoparticles, micelles can be specifically mentioned. In addition, the above-mentioned double-layer nanoparticles refer to nanoparticles containing a double-layer membrane formed by amphiphilic molecules in a double layer. As the above-mentioned double-layer nanoparticles, specifically, (1) vesicles, i.e., liposomes, which have lipids as constituent components and contain a double-layer membrane of the lipids; (2) vesicles, which have surfactants other than lipids as constituent components and contain a double-layer membrane of the surfactant. The above-mentioned vesicles containing a double-layer membrane can be small unilamellar vesicles (SUVs) or large unilamellar vesicles (LUVs) having one double-layer membrane, or can be multivesicular liposomes (MVLs) having two or more double-layer membranes.

[0038] Furthermore, when the nanoparticles of the present application contain micelles, the micelles are preferably micelles in which hydrophilic groups are aggregated on the outside and hydrophobic groups are aggregated on the inside.

[0039] The volume average particle size of the nanoparticles of the present application may be 10 nm to 300 nm, may be 20 nm to 200 nm, or may be 30 nm to 150 nm.

[0040] The smaller the volume average particle size of the nanoparticles of the present application, the easier it is to distribute throughout the cells, and when the cells are frozen, the shape of the cells is more easily maintained.

[0041] When the volume average particle size of the nanoparticles of the present application is 300 nm or less, the nanoparticles of the present application can be more easily and efficiently introduced into cells.

[0042] The method for controlling the volume average particle size of the nanoparticles of the present application is not particularly limited, and for example, methods such as sizing by adjusting the pore size of the filter used when manufacturing the nanoparticles of the present application can be cited. Specifically, the nanoparticles of the present application can be cited as being pressurized to pass through a filter having fine pores, and an extrusion process using physical shearing force to homogenize the particle size can be cited.

[0043] Nanoparticle of the present application can be cationic nanoparticle, can be neutral nanoparticle, also can be anionic nanoparticle, more preferably cationic nanoparticle.Usually, in the cell, relative to the extracellular, be in negative (negative) potential.Therefore, when nanoparticle of the present application is cationic nanoparticle, by ionic interaction, nanoparticle of the present application is easily imported into the wide area in the cell efficiently.As a result, further reduce the cell damage caused by cryopreservation.

[0044] Cationic nanoparticles refer to nanoparticles with a zeta potential greater than 10 mV.

[0045] Neutral nanoparticles refer to nanoparticles having a zeta potential of not less than -10 mV and not more than 10 mV.

[0046] Anionic nanoparticles refer to nanoparticles with a zeta potential of less than -10 mV.

[0047] The zeta potential of the nanoparticles of the present application is preferably 10 mV to 80 mV, more preferably 15 mV to 70 mV, further preferably 20 mV to 60 mV, particularly preferably 25 mV to 50 mV.

[0048] When the zeta potential of the nanoparticles of the present application is above 10 mV, the membrane of the nanoparticles of the present application is appropriately positively charged. Therefore, the nanoparticles of the present application are more easily and efficiently introduced into cells, further reducing cell damage caused by cryopreservation.

[0049] The zeta potential and volume average particle size of the nanoparticles of the present application can be measured, for example, in the following manner.

[0050] The cryopreservation composition of the present application was diluted 100 times with phosphate buffered saline (PBS, pH 7.4), 1 mL of the resulting solution was collected and filled into a 1 mL sample cell for measurement. Then, the particle size and zeta potential measuring device (ZETASIZER PRO, manufactured by Malvern) was used to measure it at room temperature (25°C).

[0051] The surface charge of the nanoparticle of the present application, i.e. the value of Zeta potential is subject to the influence of the electric charge of the amphiphilic molecule constituting the nanoparticle of the present application. Therefore, when preparing the nanoparticle of desired Zeta potential, the method for using amphiphilic molecule, preparing the nanoparticle of the present application can be enumerated according to its Zeta potential suitable selection or change.

[0052] Examples of the amphiphilic molecule include surfactants, which may be synthetic surfactants or surfactants derived from natural or biological organisms.

[0053] As synthetic surfactants, Gemini surfactants can be cited. Gemini surfactants have a hydrophilic group and a hydrophobic group in one molecule, which are connected to each other via a spacer group through the hydrophilic group, and are dimer surfactants having two surface active units or trimer surfactants having three surface active units.

[0054] As the hydrophobic group constituting the Gemini type surfactant, there can be mentioned a linear hydrocarbon chain having 8 to 24 carbon atoms, preferably 12 to 18 carbon atoms, and more preferably 12 or 16 carbon atoms. From the viewpoint of the stability of the nanoparticles, a linear saturated hydrocarbon chain is preferred. In addition, the linear hydrocarbon chains constituting two or three hydrophobic groups may be the same or different, but are preferably the same.

[0055] As the hydrophilic group constituting the Gemini surfactant, polar groups and ionic groups can be cited. As the element that generates the counter ion in the hydrophilic group, it can be appropriately adjusted according to the aqueous solution used, and 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) can be cited, and they can also be used in appropriate combinations.

[0056] The spacer is usually a hydrocarbon chain, preferably having a length to provide sufficient distance so that the hydrophobic groups can function independently of each other. The number of carbon atoms in the spacer is not particularly limited, and may be 2 to 12, preferably 2 to 8, more preferably 2 to 4, and most preferably 2 or 3.

[0057] As the surfactant of natural and organism origin, amphipathic lipids can be enumerated. As amphipathic lipids, it can be phosphorus-free lipids, or phospholipids. In addition, it can be cationic lipids, neutral lipids, anionic lipids, or a combination thereof.

[0058] Examples of the phosphorus-free lipids include N-(2,3-dioleoyloxy-1-propyl)trimethylammoniummethyl sulfate (DOTAP), N-[1-(2,3-oleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), and dioctadecylamidoglycylspermine (DOGS). In addition, examples of phospholipids include 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhPE) and other phosphoethanolamines, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dibutyryl-sn-glycero-3-phosphocholine (DPhPC) and other phosphocholines.

[0059] The amphiphilic molecule can be composed of a surfactant having a labeling substance (for example, a fluorescent molecule such as coumarin, FITC (fluorescein isothiocyanate), a maleimide group, a PEG group, etc.) introduced into the molecule.

[0060] The nanoparticles of the present application preferably contain cholesterol, and more preferably the nanoparticles of the present application contain cholesterol in their membranes. When the nanoparticles of the present application are constructed in such a way that cholesterol is contained in the membrane, the fluidity of the membrane in the nanoparticles of the present application is improved compared to a membrane without cholesterol.

[0061] [Other ingredients]

[0062] The composition for cryopreservation according to the present embodiment may further contain other components (hereinafter, also simply referred to as “other components”) in addition to the nanoparticles of the present application.

[0063] Examples of other components include internal components contained in the nanoparticles of the present application, dispersion media, amphiphilic molecules that do not form a monolayer or bilayer in the process of producing the nanoparticles of the present application, or aggregates thereof.

[0064] ·Ingredients included

[0065] The internally contained components refer to components incorporated into the membrane of the nanoparticles of the present application, components in the surfactant constituting the nanoparticles of the present application, and components contained in the internal aqueous phase.

[0066] Examples of the internal components include labeling substances (e.g., fluorescent substances such as coumarin), 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 the like.

[0067] Specific examples of the internal components include active vitamin C and active vitamin A having a cell-protecting effect, and ferulic acid and hyaluronic acid as moisture-retaining components.

[0068] The nanoparticles of the present application preferably contain at least one selected from the group consisting of water-soluble vitamins, water-soluble vitamin derivatives, and ferulic acid, and more preferably contain at least one of ferulic acid and active vitamin C.

[0069] In particular, ferulic acid and active vitamin C are excellent in antioxidant capacity and water retention. Therefore, by enclosing at least one of them in the nanoparticles of the present application, cell damage caused by cell cryopreservation is further suppressed.

[0070] Dispersion medium

[0071] The nanoparticles of the present application are preferably dispersed in a dispersion medium.

[0072] Examples of the dispersion medium include water, a buffer solution, a water-soluble solvent, a liquid culture medium, etc. The dispersion medium may be used alone or in combination of two or more.

[0073] Examples of water include distilled water, ion-exchanged water, ultrafiltered water, and pure water.

[0074] The water content is preferably 70% by mass or more, more preferably 80% by mass or more and 90% by mass or less, and further preferably 90% by mass or more and 100% by mass or less relative to the total amount of the dispersion medium. By setting the water content within the above numerical range, the nanoparticles of the present application exist more stably.

[0075] Examples of the buffer include phosphate buffer (PBS), tris(hydroxymethylaminomethane) buffer (TRIS), hydroxyethylpiperazineethanesulfonic acid buffer (HEPES), borate buffer, and acetate buffer.

[0076] The concentration of the buffer solution is preferably based on the osmotic pressure value and is used within a range approximately equivalent to the human plasma osmotic pressure of 285±5m0sm / L.

[0077] The pH of the buffer solution is not particularly limited, but is preferably pH 5 or higher and pH 10 or lower, and more preferably pH 6 or higher and pH 9 or lower, from the viewpoint of cell viability and the like.

[0078] The pH is a value measured at 25° C. using a pH meter (the same applies hereinafter).

[0079] The water-soluble solvent refers to a solvent that can be mixed with water.

[0080] Examples of the water-soluble solvent include alcohols such as methanol and ethanol from the viewpoint of the solubility of lipids.

[0081] As the water-soluble solvent, for example, the water-soluble solvent used to dissolve the prepared amphiphilic molecules such as lipids in the production process of the nanoparticles of the present application can be used as it is.

[0082] As the liquid culture medium, for example, a known culture medium used in the culture of stem cells can be cited. More specifically, as the liquid culture medium, for example, DMEM (Dulbecco's Modified Eagle's Medium), DMEM: F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12), EMEM (Eagle's minimal essential medium), and a liquid culture medium to which 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) are added to these culture media, etc.

[0083] The liquid culture medium may be a commercially available product. Examples of commercially available products include ADSC-BulletKit (registered trademark) PT-4505 manufactured by Lonza Corporation.

[0084] The pH of the liquid culture medium (at 25°C) may be, for example, pH 7.0 to 8.0 or pH 7.3 to 7.4.

[0085] The pH (at 25° C.) of the composition for cell cryopreservation is not particularly limited, but is preferably pH 4 or higher and pH 12 or lower, and more preferably pH 5 or higher and pH 11 or lower, from the viewpoint of cell viability and the like.

[0086] ·Method for producing a composition for cryopreservation

[0087] The method for producing the composition for cryopreservation of the present application is not particularly limited, and a known method can be applied.

[0088] The method for producing a composition for cryopreservation may be, for example, a method comprising the following steps: a first step of mixing a solution obtained by dissolving components constituting the membrane of the nanoparticles of the present application (materials such as surfactants (including the internal inclusion components when there are internal inclusion components that enclose the nanoparticles of the present application)) in a water-soluble solvent with water or a buffer solution to obtain a dispersion containing the nanoparticles of the present application; and a second step of subjecting the aforementioned dispersion to a filter treatment.

[0089] In particular, when the second step is included, the particle size of the nanoparticles of the present application can be adjusted.

[0090] (1st step)

[0091] In the first step, a dispersion liquid containing at least one of the nanoparticles of the present application is obtained. As for the dispersion liquid, a solution obtained by dissolving the components of the film constituting the nanoparticles of the present application in a water-soluble solvent (e.g., alcohol, etc.) and mixing with water or a buffer solution, and controlling conditions (e.g., pH, temperature) are prepared. It should be noted that, as described above, the amphiphilic molecules constituting the nanoparticles of the present application are omitted from the description herein.

[0092] For example, a dispersion liquid containing liposomes can be prepared by mixing ethanol and lipids to dissolve the lipids, and then mixing with water while adjusting the pH and stirring.

[0093] In the first step, if there are excess cholesterol, amphiphilic molecules such as phospholipids, etc. that do not contribute to the formation of the nanoparticles of the present application, or components that further encapsulate the nanoparticles, the unencapsulated components can be removed using a dialysis membrane or the like.

[0094] In the second step, the dispersion obtained in the first step is subjected to extrusion treatment using a filter.

[0095] The size of the nanoparticles of the present application can be adjusted by extrusion (size adjustment). The filter can be selected to have a desired pore size according to the purpose, etc., and can be appropriately selected from commercially available products.

[0096] <How to store in a freezer>

[0097] The cryopreservation method of the present application includes the following steps: mixing cells or biological tissues with the cryopreservation composition of the present application, and introducing the nanoparticles of the present application into the cells (hereinafter, also referred to as the "introduction step"); and freezing the cells or biological tissues into which the nanoparticles of the present application have been introduced (hereinafter, also referred to as the "freezing step"). It should be noted that, in the case of freezing biological tissues, the meaning of "introducing into cells" is "introducing into cells constituting biological tissues".

[0098] According to the cryopreservation method of the present application, cell damage caused by cryopreservation can be reduced.

[0099] The cells to be frozen as the object of the cryopreservation method of the present application are not particularly limited, and known cells for cryopreservation can be used. For example, as cells, mesenchymal stem cells such as mesenchymal stem cells (hAD-MSC) derived from human adipose tissue and mesenchymal stem cells (hMSC-BM) derived from human bone marrow, neural stem cells, skin stem cells, hematopoietic stem cells, dental pulp stem cells, liver stem cells, muscle stem cells, adipose stem cells, etc., adult stem cells; induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells) and other pluripotent stem 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 (pancreatic exocrine cells, etc.); differentiated cells such as brain cells, lung cells, kidney cells and fat cells; germ cells such as embryos, fertilized eggs, and sperms. In addition, examples of the freezing target of the cryopreservation method of the present application include organoids, cell spheres, cell sheets, and biological tissues composed of the above cells.

[0100] [Introduction process]

[0101] In the introduction step, cells or biological tissues are mixed with the cryopreservation composition of the present application, and the nanoparticles of the present application are introduced into cells (in the case of frozen biological tissues, cells constituting the biological tissues, and the same applies hereinafter).

[0102] The culture medium is not particularly limited, and examples thereof include known culture media used for culturing stem cells. For example, examples of the culture medium include the liquid culture medium described above.

[0103] The means for introducing the nanoparticles of the present application into cells is not particularly limited, and for example, the following methods (I) to (IV) can be cited:

[0104] (I) Method of adding the cryopreservation composition of the present application to a culture medium containing cells

[0105] (II) A method of centrifuging a culture medium containing cells, removing the supernatant, and then adding the cryopreservation composition of the present application

[0106] (III) A method of adding cells detached from a culture vessel to the cryopreservation composition of the present application.

[0107] (IV) A method of introducing voltage into cells by electroporation.

[0108] [Freezing process]

[0109] In the freezing step, the cells or biological tissues into which the nanoparticles of the present application have been introduced are frozen.

[0110] The means for freezing cells or biological tissues is not particularly limited, and a known freezing method can be used.

[0111] The temperature for freezing cells or biological tissues is preferably within the range of -30°C to -196°C (preferably -80°C to -196°C). As for the means for freezing cells or biological tissues, for example, from the viewpoint of further reducing cell damage caused by cryopreservation, it is preferred to use an ultra-low temperature refrigerator or liquid nitrogen to freeze cells or biological tissues. The freezing time is not particularly limited, and examples include 0.5 days to 50 years, 1 day to 10 years, 1 week to 5 years, and 1 month to 1 year.

[0112] [Other processes]

[0113] The cryopreservation method of the present application may further include other steps in addition to the above-mentioned introduction step and freezing step. Examples of the above-mentioned other steps include a storage step of storing the frozen cells at an appropriate temperature.

[0114] [Freezing cells]

[0115] The frozen cells or biological tissues involved in this embodiment are frozen cells or biological tissues containing the nanoparticles of the present application. The frozen cells or biological tissues can be prepared by the above-mentioned cryopreservation method of the present application.

[0116] [Example]

[0117] The present invention is further specifically described by enumerating examples below. For the material, usage amount, ratio, processing steps etc. shown in the following examples, as long as it does not exceed the gist of the present invention, then appropriate changes can be made. Therefore, the scope of the present invention should not be interpreted as restrictive according to the specific examples shown below.

[0118] <Example 1>

[0119] Preparation of composition for cryopreservation

[0120] As Example 1, a double-layer membrane vesicle containing DOTAP as a constituent component was prepared by an organic solvent injection method according to the following procedure.

[0121] (1) Add 5.3 ml of PBS (pH = 7.4 at 25°C) to a beaker and stir at 25°C.

[0122] (2) 1990 μL of ethanol, 38.7 mg of cholesterol, and 11.1 mg of DOTAP, a lipid that is an amphiphilic molecule, were mixed in a centrifuge tube, and the temperature of the mixed solution was set to 75°C, which is above the phase transition temperature of the lipid, to completely dissolve the cholesterol and the lipid. It should be noted that when evaluating the cell introduction property described later, fluorescently labeled coumarin was further added, and adjustments and tests were performed under light-shielding conditions to prevent attenuation. It should be noted that coumarin is hydrophobic and is therefore mainly taken into the membrane of the formed nanoparticles.

[0123] (3) Use a syringe to take the total amount of the above mixed solution and quickly mix it into the above PBS.

[0124] (4) Shield the beaker from light and stir at 25°C for 1 hour.

[0125] (5) Using a dialysis membrane (Spectra / Por RC Biotech Dialysis Membrane MWCO: 8-10), dialyze in PBS at room temperature for more than 4 hours to obtain a nanoparticle solution.

[0126] (6) The obtained nanoparticle solution was placed in a LF-STB LiposoFast stabilizer manufactured by Avestin, and the size was adjusted by passing the solution through the filter pore size in the order of 1000 nm, 800 nm, 400 nm, 200 nm, 100 nm and 50 nm. The obtained liquid composition containing nanoparticles was used as the cryopreservation composition of Example 1. The pH (under 25°C) of the cell cryopreservation composition was 7.2.

[0127] <Examples 2 to 5>

[0128] The type of amphiphilic molecule was changed from DOTAP to DOPE, Gemini type, DPPC, and DSPE, and the same specifications as in Example 1 were used to obtain the compositions for cryopreservation of Examples 2 to 5. "Gemini type" means "Gemini type surfactant". It should be noted that the molecular structure of the Gemini type surfactant used in Example 3 and Examples 8 and 13 described later is as follows. It is a dimer surfactant having two surface active units, the hydrophobic group is composed of a hydrocarbon chain with 12 carbon atoms, the spacer group has 3 carbon atoms, and the elements that generate the counter ions are composed of bromine and nitrogen.

[0129] [Chemical formula 1]

[0130]

[0131] <Examples 6 to 15>

[0132] The cryopreservation compositions of Examples 6 to 15 were obtained by the same method as Examples 1 to 5 except that the operation (1) for preparing the cryopreservation composition of Example 1 was set to the operation (1') shown below. Examples 6 to 10 are cryopreservation compositions containing nanoparticles containing ferulic acid, which have DOTAP, DOPE, Gemini type, DPPC, and DSPE as constituent components, respectively. Examples 11 to 15 are cryopreservation compositions containing nanoparticles containing active vitamin C, which have DOTAP, DOPE, Gemini type, DPPC, and DSPE as constituent components, respectively.

[0133] (1') 5.3 ml of PBS (pH = 7.4 at 25°C) was added to a beaker, and 10 μM ferulic acid or active vitamin C was dissolved as an internal component, followed by stirring at 25°C.

[0134] The active vitamin C used in Examples 11 to 15 is a compound having the following molecular structure.

[0135] [Chemical formula 2]

[0136]

[0137] <Comparative Example 1>

[0138] In Comparative Example 1, 1 mL of a mixed solution of 10% by volume of dimethyl sulfoxide (DMSO), 40% by volume of fetal bovine serum (FBS), and 50% by volume of a stem cell culture medium was prepared into a composition for cryopreservation.

[0139] <Comparative Example 2>

[0140] In Comparative Example 2, CELLBANKER (registered trademark) 1 (1 mL) manufactured by ZNQ Corporation which does not contain nanoparticles was used as a composition for cryopreservation.

[0141] [Test Example 1]

[0142] <Volume average particle size and zeta potential>

[0143] For the cryopreservation composition of each example, the volume average particle size and Zeta potential were measured. Specifically, the cryopreservation composition of Examples 1 to 5 was diluted 100 times with phosphate buffered saline (PBS, pH 7.4), 1 mL of the obtained solution was collected, and filled into a 1 mL sample cell for measurement. Then, it was measured at room temperature (25°C) using a particle size and Zeta potential measuring device (ZETASIZER PRO, manufactured by Malvern). The results are shown in Table 1. In addition, the distribution of the particle size of the nanoparticles contained in the cryopreservation composition of Example 1 is shown in Figure 1 .

[0144] [Table 1]

[0145]

[0146] [Test Example 2]

[0147] <Evaluation of cell transduction ability>

[0148] Regarding the cryopreservation compositions of Examples 1 to 5, the cell-introducing properties of the nanoparticles contained in the cryopreservation compositions were evaluated according to the following procedure.

[0149] (1) Human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) were seeded at 2000 cells / well on an 8-well chamber slide coated with collagen.

[0150] (2) The cryopreservation compositions of Examples 1 to 5 labeled with the fluorescent pigment coumarin were added to the wells and incubated at 37° C. for 30 minutes.

[0151] (3) DAPI solution (4',6-diamidino-2-phenylindole dihydrochloride solution) was used for staining of cell nuclei.

[0152] (4) Using a fluorescence microscope (manufactured by KEYENCE Japan, BZ-9000) at a magnification of 20 times, the fluorescence intensity of the cytoplasm and the cell nucleus observed was measured at an exposure time of 1 / 2 second and a black balance of 42. The value of the ratio of the fluorescence intensity IN of the coumarin contained in the nanoparticles present in the cell nucleus to the fluorescence intensity OUT of the coumarin contained in the nanoparticles present in the cytoplasm and outside the cell nucleus (IN / OUT) is shown in the evaluation item of cell transposability [Fluorescence intensity ratio (cell nucleus / cytoplasm)] in Table 2.

[0153] [Table 2]

[0154]

[0155] As shown in Table 2, the value of the fluorescence intensity ratio (nucleus / cytoplasm) was positive, which showed that part of the nanoparticles contained in the cryopreservation compositions of Examples 1 to 5 were introduced not only into the cytoplasm but also into the nucleus.

[0156] [Test Example 3]

[0157] <Transmission electron microscope>

[0158] The nanoparticles introduced into the cells by the method of Experimental Example 2 were observed using a transmission electron microscope. Mesenchymal stem cells (hAD-MSC) derived from adipose tissue of healthy individuals were cultured using 5 mL of a dedicated culture medium containing growth factors at a density of approximately 5,000 cells / cm 2 The density of inoculation was 3.5 cm 2 Culture dish. The cryopreservation composition of Example 3 (Gemini type) was labeled with gold nanoparticles (Cytodiagnostics). The labeled cryopreservation composition was exposed to ADSCs for 30 minutes. In order to prepare samples for transmission electron microscopy, ADSCs were fixed overnight at 4°C using a phosphate buffer containing 2% glutaraldehyde as a pre-fix. After washing the fixed sample with phosphate buffer, it was post-fixed with a 2% osmium aqueous solution at 4°C for 2 hours. For dehydration, the concentration of ethanol was increased stepwise from 30% to 100%, and each treatment was performed for 15 minutes. The sample was embedded in epoxy resin at 60°C for 48 hours, and ultrathin sections of 80-90nm were made using an ultrathin microtome and mounted on a Cu 200 mesh. Observation was performed using a transmission electron microscope H-7600 at 100kV. The results are shown in Figure 2 .

[0159] like Figure 2As shown, the nanoparticles contained in the cryopreservation composition of Example 3 were taken up into the cytoplasm. In addition, a part of the nanoparticles with a small particle size were taken up into the nucleus.

[0160] [Test Example 4]

[0161] <Cryopreservation and thawing of cells using the cryopreservation composition>

[0162] Cryopreservation of cells

[0163] (1) Human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) at 70% to 90% confluence were treated with trypsin, detached from the culture vessel, and centrifuged to obtain a cell pellet.

[0164] (2) Regarding the above-mentioned approximately 5×10 6 For each cell, 10 μL of the cryopreservation composition of each example was added to the cells, and 990 μL of the cell culture medium was added to obtain a cell suspension. For the comparative example, 1 mL of the cryopreservation composition of each example was added to obtain a cell suspension.

[0165] (3) The cell suspension was dispensed into cryotubes and stored in a -80°C ultra-low temperature freezer.

[0166] Thawing of cryopreserved cells

[0167] (1) The cryopreserved cells (within 1 month) are rapidly thawed while being shaken in a 37°C warm bath.

[0168] (2) The thawed cell suspension was mixed with 10 mL of a stem cell-specific culture medium to obtain a mixed solution.

[0169] (3) The mixed solution was centrifuged (1,200 rpm, 5 minutes, 4°C), the supernatant was removed by aspirator, and then the mixture was suspended in an appropriate amount of the medium.

[0170] <Evaluation of cell survival rate>

[0171] The cell viability before and after cell cryopreservation was evaluated using a cell proliferation assay kit (MTT Cell Viability Assay Kit, AR1156, BTI: manufactured by COSMOBIO CO., LTD.) according to the following procedure.

[0172] (1) Cells that were cryopreserved using the cryopreservation compositions of Examples 3 and 6 to 15 and then thawed were seeded in a 96-well plate at 2,000 cells / well.

[0173] (2) Add 10 μL of the MTT standard reagent in the above kit to each well and incubate at 37°C for 4 hours.

[0174] (3) Add 100 μL of formazan solution to each well and incubate at 37°C for 18 hours.

[0175] (4) Using a microplate absorbance detector, measure the absorbance A of the pigment at an absorption wavelength of 570 nm.

[0176] (5) The above-mentioned operations (1) to (3) were performed using hMSC-AT before cell freezing, and then the absorbance B of the pigment at an absorption wavelength of 570 nm was measured using a microplate absorbance detector.

[0177] (6) The ratio of the absorbance A after thawing obtained in (4) above to the absorbance B before cryopreservation (=A / B×100%) was taken as the cell survival rate. The results are shown in Tables 3 and 4.

[0178] [Table 3]

[0179]

[0180] [Table 4]

[0181]

[0182] As shown in Tables 3 and 4, it can be seen that when the cryopreservation composition of Example 3 and the cryopreservation compositions of Examples 6 to 15 containing ferulic acid or active vitamin C are used for freezing, the cell survival rate is higher than when the cryopreservation compositions of the comparative examples are used for freezing, that is, cell damage caused by cryopreservation can be reduced.

[0183] [Test Example 5]

[0184] <Evaluation of stem cell ratio>

[0185] Cells were cryopreserved using the cryopreservation compositions of Examples 3 and 6 to 15, and the stem cell rate was evaluated using the Human Mesenchymal Stem Cell Multi-Color Flow Kit (FMC020, manufactured by R&D systems) based on the following procedure. This kit uses CD90 as a positive marker and CD45, CD34, CD11b, CD79A, and HLA-DR as negative markers.

[0186] (1) 5×10 6Each cell was mixed with 100 μL of the flow cytometry staining buffer attached to the above kit, and the mixture was transferred to a flow cytometer tube to prepare a cell suspension.

[0187] (2) 10 μL of each of two fluorescent dye-labeled antibody solutions (one positive marker antibody solution and one negative marker mixed antibody solution) provided in the kit was added to the cell suspension and incubated in the dark at room temperature (25°C) for 45 minutes.

[0188] (3) Wash the incubated cell suspension with 2 mL of the above-mentioned flow cytometry staining buffer.

[0189] (4) Flow cytometry analysis was performed using a flow cytometer to quantify cells showing the characteristics of CD90(+), CD45(-), CD34(-), CD11b(-), CD79A(-) and HLA-DR(-) after the cells were cryopreserved. The proportion of these cells when cryopreserved using the cryopreservation compositions of Examples 3, 6 to 15 is shown in Tables 5 and 6 as "the proportion of stem cells".

[0190] According to Tables 5 and 6, when cryopreservation is performed using a cryopreservation composition containing the nanoparticles of Examples 3 and 6 to 15, the proportion of stem cells is higher than when the cryopreservation composition of the comparative example is used, that is, it is useful for maintaining the stem cell function based on cryopreservation.

[0191] [Table 5]

[0192]

[0193] [Table 6]

[0194]

[0195] [Test Example 6]

[0196] <Comparison of Gemini-type surfactants>

[0197] In the above test, the cryopreservation compositions of Examples 3, 8, and 13 were used, that is, Gemini-type surfactants were used in which the carbon number of the hydrocarbon chain of the hydrophobic group was 12, the carbon number of the spacer group was 3, and the elements forming the counter ion were bromine and nitrogen. Therefore, as Gemini-type surfactants, the carbon number of the two alkyl chains, the carbon number of the spacer group, and the element forming the counter ion were changed, and the cryopreservation compositions of Examples 16 to 19 were prepared by the same method as in Example 1, and the survival rate after freezing was investigated together with the cryopreservation composition of Example 3 by the same method as in Test Example 4. The results are shown in Table 7. In Table 7, 12-3-12_Br means that the carbon number of each of the two hydrocarbon chains is 12, the carbon number of the spacer group is 3, and the elements that generate the counter ion are bromine and nitrogen, and 16-3-16_Cl means that the carbon number of each of the two hydrocarbon chains is 16, the carbon number of the spacer group is 3, and the counter ion is chlorine and nitrogen.

[0198] [Table 7]

[0199]

[0200] According to Table 7, not only the Gemini surfactants with 12 carbon atoms in the hydrocarbon chain, but also the freezing of the composition for cryopreservation of nanoparticles composed of Gemini surfactants with 16 carbon atoms in the hydrocarbon chain maintained a high survival rate. In addition, the use of chlorine instead of bromine as the element for generating the counter ion in the Gemini surfactant also maintained a high survival rate. In addition, the high survival rate was maintained when the carbon atoms of the spacer in the Gemini surfactant were 2 and 3.

[0201] [Test Example 7]

[0202] <Long-term cryopreservation>

[0203] The cryopreservation composition of Example 3 was introduced into cells and cryopreserved in the same manner as in Test Example 4. The cells were cryopreserved for 3, 6, and 12 months, and the survival rate after thawing was investigated in the same manner as in Test Example 4. The results are shown in Table 8.

[0204] [Table 8]

[0205]

[0206] According to Table 8, even when the cryopreservation composition of Example 3 was introduced into cells and cryopreserved for 12 months, the survival rate after freeze-thawing was still maintained as high as 87.8%.

[0207] [Test Example 8]

[0208] <Excretion of the composition to the outside of cells>

[0209] The cryopreservation composition of Example 3 was introduced into cells and frozen in the same manner as in Test Example 2, and the fluorescence intensity including the cytoplasm and nucleus was quantified 0, 1, 7, and 14 days after thawing to investigate the excretion of the introduced cryopreservation composition to the outside of the cells. The results are shown in Table 9.

[0210] [Table 9]

[0211]

[0212] According to Table 9, the fluorescence intensity was less than 1 / 10 7 days after thawing, and decreased to a level that was almost undetectable 14 days after thawing. Therefore, it was confirmed that when freezing with the cryopreservation composition of Example 3, the cryopreservation composition was excreted outside the cells after thawing.

[0213] [Test Example 9]

[0214] <Doubling time of cells after thawing>

[0215] The cryopreservation composition of Example 3 was introduced into cells and frozen in the same manner as in Test Example 4. Cell proliferation was measured 0 and 7 days after thawing, and the time it took for the total number of cells to double (cell doubling time: PDT (Population Doubling Time)). The results are shown in Table 10.

[0216] The doubling time was calculated based on the following formula.

[0217] PDT (Population Doubling Time) calculation method:

[0218] 1) The cells were measured at two points in the logarithmic growth phase.

[0219] 2) The measured value of the cell density at the first point is set as N0, and the measured value at the second point is set as N.

[0220] In addition, the time from the start of culture to the first point is t0, and the time to the second point is t.

[0221] PDT=(t-t0)log2 / (logN-logN0)

[0222] [Table 10]

[0223]

[0224] According to Table 10, the doubling time of cells was maintained even after 7 days from thawing. Therefore, it was confirmed that even when the cells were frozen using the cryopreservation composition of Example 3, the proliferation activity of the cells was not affected by the cryopreservation.

[0225] [Test Example 10]

[0226] <Survival rates of embryos, sperm, blood cells, and 3D organoids after freezing>

[0227] The composition for cryopreservation of Example 3 was introduced into cells by the same method as in Test Example 4. As cell types, embryos, sperm, blood cells, and 3D organoids were used together with mesenchymal stem cells derived from human adipose tissue. In addition, the survival rate after freezing was investigated by the same method as in Test Example 4, and the results are shown in Table 11.

[0228] The cells used were described below.

[0229] Embryos (species: mouse, strain: C57BL / 6J Jcl, stage: 2cell, number of embryos: 30, purchasing institution: National Institute of Pharmaceutical Research, Health and Nutrition)

[0230] · Sperm (species: mouse, strain: C57BL / 6NCr Slc, number: 1 straw, purchased by: National Research and Development Corporation Pharmaceutical Foundation, Health and Nutrition Research Institute)

[0231] Blood cells (species: human, source: peripheral blood of patients with acute monocytic leukemia, type: monocytes, purchased from ATCC)

[0232] 3D organoids (species: human, source: healthy adipose tissue, type: using mesenchymal stem cells, Corning (registered trademark) Matrigel basement membrane matrix for organoid culture, purchased by Lonza)

[0233] [Table 11]

[0234]

[0235] From Table 11, it was confirmed that when the cryopreservation composition of Example 3 was used, not only mesenchymal stem cells but also cells such as embryos, sperm, blood cells, and 3D organoids were frozen at a high survival rate.

[0236] [Test Example 11]

[0237] <Evaluation of transplantation efficiency in immunodeficient mice>

[0238] The composition for cryopreservation of Example 8 was introduced into cells by the same method as in Test Example 4, and the transplantation efficiency in immunodeficient mice was evaluated using the frozen and thawed mesenchymal stem cells.

[0239] Mesenchymal stem cells (ADSCs) derived from healthy human adipose tissue were cultured into spheroids one week before transplantation by adding matrigel for spheroid culture and growth factors included in the dedicated culture medium. Stem cell spheroids were mixed with matrigel for transplantation at a ratio of 1:1. 7 The transplantation mixture of ADSCs containing 100 cells was filled into a syringe on ice and transplanted subcutaneously into the back of immunodeficient mice. The vertical and horizontal heights of the transplanted site 2 weeks after transplantation were measured with a vernier caliper and the volume (mm 3 ). In addition, the weight (mg) of the transplanted piece removed 3 weeks after the ADSC transplantation was weighed using a precision electronic balance. As a control, ADSCs frozen without the composition of Example 8 were used. The results are shown in Table 12.

[0240] [Table 12]

[0241] Comparison Gemini cationic nanoparticles Example 8 <![CDATA[Volume of the graft (mm 3 )]]> 1250 2020 Graft weight (mg) 1060 2420

[0242] According to Table 12, when the cells treated with the cryopreservation composition of Example 8 were transplanted into mice, the physiological activity was maintained and the enlargement of the transplant was confirmed.

[0243] [Test Example 12]

[0244] <In vitro evaluation of carcinogenicity>

[0245] Using the composition for cryopreservation of Example 3, a soft-agar colony formation assay, which is an in vitro evaluation of carcinogenicity as a safety index, was performed.

[0246] A 1.8% agar solution was sterilized and dissolved in an autoclave and kept warm in a water bath at 45°C.

[0247] A bottom mix (2×DMEM 37.5 ml, serum 9 ml, 2.8% NaHCO 3 5.25 ml, 10× penicillin / streptomycin 0.9 ml) was prepared and kept warm in a 45° C. water bath.

[0248] The well-insulated lower layer mixture is mixed with 1.8% agar solution in a sterilized reagent bottle or tube at a ratio of 1.4 vol:1 vol according to 2.5 ml×necessary number of slices+α, and incubated in a 45°C water bath (final 0.75% agar).

[0249] Use a disposable pipette to inject 2.5 ml of the bottom agar into a 35 mm dish or a 6-well plate, let it stand at 4°C for 8 minutes, and keep it warm in an incubator after solidification.

[0250] The fully insulated upper layer mixture (upper layer: 2×DMEM 37.5ml, serum 9ml, 2.8% NaHCO3 5.25ml, 10× penicillin / streptomycin 0.9ml, sterile water 19.5ml) is mixed with 1.8% agar solution in a sterilized reagent bottle or tube at a ratio of 4vol:1vol at 1.5ml×the required number of slices+α, and incubated in a 45°C water bath (final 0.36% agar).

[0251] Mesenchymal stem cells derived from human adipose tissue were separated according to the usual procedure and passed through a cell sieve to separate into single cells, and the number of cells was adjusted to 2×10 5 cells / ml.

[0252] 100 μl of the cell suspension was transferred to a 15 ml tube, and 3 ml / tube was added to the top agar using a disposable pipette. The mixture was pipetted without bubbling and 1.5 ml was spread on the bottom agar in portions.

[0253] Let stand at 4°C for 8 minutes and keep warm in an incubator after solidification.

[0254] After 2 weeks, the colonies were stained and counted. The results are shown in Table 13.

[0255] [Table 13]

[0256]

[0257] According to Table 13, no colonies were generated in the cases where the storage time was 0, 3, and 6 months, and carcinogenicity due to the introduction of the cryopreservation composition of Example 3 into cells was not confirmed.

Claims

1. A composition for cryopreservation of cells or biological tissues, comprising nanoparticles, wherein the nanoparticles have amphiphilic molecules as constituent components, and the amphiphilic molecules are monolayer or bilayer.

2. The composition for cryopreservation according to claim 1, wherein The nanoparticles are cationic nanoparticles.

3. The composition for cryopreservation according to claim 1 or 2, characterized in that The amphiphilic molecule is a Gemini surfactant.

4. The composition for cryopreservation according to claim 1 or 2, wherein The zeta potential of the nanoparticles is greater than or equal to 10 mV and less than or equal to 50 mV.

5. The composition for cryopreservation according to claim 1 or 2, wherein The volume average particle size of the nanoparticles is greater than or equal to 30 nm and less than or equal to 300 nm.

6. The composition for cryopreservation according to claim 1 or 2, wherein The nanoparticles contain ferulic acid or active vitamin C.

7. A method for cryopreservation of cells or biological tissues, comprising: A step of mixing cells or biological tissues with the cryopreservation composition according to any one of claims 1 to 6 and introducing nanoparticles into the cells or biological tissues; and A process of freezing the cells or biological tissues into which the nanoparticles have been introduced.

8. Frozen cells or biological tissues, comprising nanoparticles, wherein the nanoparticles have amphiphilic molecules as constituent components, and the amphiphilic molecules are in the form of a single layer or a double layer.

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