Cell preservation material, cell preservation solution, and cell preservation method

Abstract

Provided are a cell preservation material, a cell preservation solution, and a cell preservation method with which it is possible to preserve multipotent stem cells at a high survival rate while maintaining undifferentiated characteristics.　A cell preservation material according to one embodiment of the present invention is for use in preserving multipotent stem cells in a liquid culture medium while maintaining undifferentiated characteristics thereof, and contains cello-oligosaccharides. A cell preservation solution according to one embodiment of the present invention contains a liquid culture medium and an aggregate of cello-oligosaccharides dispersed in the liquid culture medium, and is for use in preserving multipotent stem cells while maintaining undifferentiated characteristics thereof. A cell preservation method according to one embodiment of the present invention comprises culturing multipotent stem cells in a state of being dispersed in the cell preservation solution.

Description

Cell preservation materials, cell preservation solutions, and cell preservation methods

[0001] The present invention relates to a cell preservation material, a cell preservation solution, and a cell preservation method.

[0002] Mesenchymal stem cells and other pluripotent stem cells have the ability to differentiate into various tissues and play an important role in the field of regenerative medicine. There is a need for materials that can preserve these pluripotent stem cells while maintaining their undifferentiated state, that is, while keeping their proliferation and differentiation activities stopped.

[0003] In response to such demands, for example, Patent Document 1 proposes a culture material containing cellulose nanofibers derived from crystalline cellulose as a material for maintaining the undifferentiated state of mesenchymal stem cells. Patent Document 2 proposes a cell system in which eukaryotic non-embryonic stem cells, such as mesenchymal stem cells, are contained in an undifferentiated and resting state in a hydrogel containing nanofibril-like cellulose in a cell preservation medium at a temperature in the range of 1 to 25°C.

[0004] On the other hand, Patent Document 3 discloses a liquid culture medium composition containing deacylated gellan gum that can proliferate, differentiate, or maintain cells while maintaining a suspension state. Furthermore, Patent Document 4 discloses a method of suspension culture of adherent cells, such as mesenchymal stem cells, by culturing them while they are attached to chitin or chitosan nanofibers.

[0005] Incidentally, it is known that crystalline cellulose can be synthesized artificially. Cellulose obtained through artificial synthesis is generally an oligomer, and is called cellooligosaccharide. Patent document 5 discloses that cellooligosaccharide can be used as a culture medium additive for suspension culture of cells, and that spheroids (cell aggregates) can be formed by said suspension culture.

[0006] Furthermore, Non-Patent Document 1 discloses that in a liquid culture medium in which cellooligosaccharides are dispersed, cancer cells grow and form spheroids, while normal cells such as human dermal fibroblasts (NHDFs) survive as single cells.

[0007] International Publication No. 2015 / 111734, Japanese Patent Publication No. 2020-103281, Japanese Patent Publication No. 2021-104062, International Publication No. 2018 / 182016, Japanese Patent Publication No. 2021-69339

[0008] Natsuki Hayakawa and 6 others, "Suspension Culture System for Isolating Cancer Spheroids using Enzymatically Synthesized Cellulose Oligomers", ACSAppl. Bio Mater. 2024, 7, 1, 306-314

[0009] As described above, there is a need to preserve pluripotent stem cells in an undifferentiated state. However, according to the inventors' research, cellulose nanofibers cannot necessarily preserve mesenchymal stem cells with a high survival rate, and further improvements are needed. Although Patent Document 5 and Non-Patent Document 1 describe the suspension culture of cells using cellooligosaccharides, they do not describe the ability to preserve pluripotent stem cells while maintaining their undifferentiated state by using cellooligosaccharides.

[0010] In view of the above, embodiments of the present invention aim to provide a cell preservation material, a cell preservation solution, and a cell preservation method that can preserve pluripotent stem cells with a high viability rate while maintaining their undifferentiated state.

[0011] The present invention includes the embodiments shown below: [1] A cell preservation material used for preserving pluripotent stem cells in a liquid culture medium while maintaining their undifferentiated state, comprising a cellooligosaccharide. [2] The cellooligosaccharide is represented by the following general formula (1), [1] The cell preservation material according to the formula, wherein A represents a hydrogen atom or substituent and n represents the average degree of polymerization. [3] The cell preservation material according to [1] or [2], wherein the pluripotent stem cells are somatic stem cells. [4] The cell preservation material according to [1] or [2], wherein the pluripotent stem cells are mesenchymal stem cells. [5] A cell preservation solution comprising a liquid culture medium and an aggregate of cellooligosaccharides dispersed in the liquid culture medium, used to preserve pluripotent stem cells while maintaining their undifferentiated state. [6] The cell preservation solution according to [5], wherein the concentration of the cellooligosaccharides is 0.1 to 2.0% (w / v). [7] A cell preservation method comprising culturing pluripotent stem cells dispersed in the cell preservation solution according to [5] or [6].

[0012] In embodiments of the present invention, pluripotent stem cells can be preserved with a high survival rate while maintaining their undifferentiated state.

[0013] A conceptual diagram showing an aggregate of cellooligosaccharides according to one embodiment. A graph showing the change in cell viability over time in Test Example 1.

[0014] The cell preservation material according to this embodiment is a cell preservation material used to preserve multipotent stem cells in a liquid culture medium while maintaining their undifferentiated state, and contains cellooligosaccharides.

[0015] Cellooligosaccharides are oligosaccharides having a structure in which glucose molecules are linked by β-1,4-glycosidic bonds, and are also called cellulose oligomers. Cellooligosaccharides may be unsubstituted, or they may have substituents such as alkyl groups at the anomeric position of the reducing end.

[0016] The average degree of polymerization (DP) of cellooligosaccharides is not particularly limited, but is preferably 5 to 20. Here, the average degree of polymerization (DP) refers to the average number of glucose units present in one molecule. The average degree of polymerization (DP) is more preferably 6 to 15, more preferably 6 to 12, more preferably 6 to 10, and even more preferably 7 to 9. Cellooligosaccharides are usually mixtures of compounds with different degrees of polymerization, and may include compounds with a degree of polymerization of 4 to 20, 5 to 18, or 5 to 13.

[0017] As the cellooligosaccharide, it is preferable to use a compound represented by the following general formula (1).

[0018] In formula (1), A represents a hydrogen atom or a substituent. The substituent is a group introduced in place of a hydrogen atom, and examples include alkyl groups having 1 to 12 carbon atoms, or alkyl groups having 1 to 5 carbon atoms. In formula (1), the wavy line in the bond between the anomeric position, i.e., the carbon at position 1 of the reducing end (anomeric carbon), and the O-A group indicates that the stereocoordination of the O-A group is α-form, β-form, or a mixture of α-form and β-form.

[0019] In formula (1), n ​​represents the average degree of polymerization (DP) of the cellooligosaccharide, which is preferably 5 to 20, more preferably 6 to 15, more preferably 6 to 12, more preferably 6 to 10, and even more preferably 7 to 9.

[0020] The method for synthesizing cellooligosaccharides is not particularly limited. For example, one method involves reacting α-glucose-1-phosphate (αG1P) with at least one primer selected from the group consisting of glucose, cellobiose, and their derivatives, with cellodextrin phosphorylase (CDP). This reaction is a synthesis method that utilizes the reverse reaction of CDP, in which αG1P is sequentially polymerized as a monomer with respect to the primer. By replacing the hydrogen atom of the hydroxyl group at the anomeric position in the primer with substituents such as alkyl groups, cellooligosaccharides with various substituents represented by formula (1) can be synthesized.

[0021] CDP is known to be produced by microorganisms such as Clostridium thermocellum and Cellulomonas, and can be obtained using known methods with these microorganisms. For example, CDP derived from Clostridium thermocellum YM4 can be prepared using an Escherichia coli expression system according to the method described by M. Krishnareddy et al., J. Appl. Glycosci., 2002, 49, 1-8, but is not limited to this method.

[0022] The concentration of CDP is not particularly limited and may be, for example, 0.1 U / ml or higher, or 0.2 U / ml or higher. Here, the amount of CDP enzyme can be determined, for example, based on enzyme activity. In this case, for example, αG1P, D-(+)-cellobiose, and CDP are incubated, and the amount of phosphate produced by CDP is quantified. The amount of enzyme that releases 1 μmol of phosphate per minute can be defined as 1 U.

[0023] Another method for synthesizing cellooligosaccharides involves reacting the above-mentioned primers and sucrose with sucrose phosphorylase (SP) and CDP in the presence of phosphate. For example, by mixing 10-1000 mM sucrose, 10-200 mM primers (glucose, cellobiose, and their derivatives), 0.1-10 U / mL CDP, and 0.01-10 U / mL or more SP in 1-200 mM phosphate buffer and incubating at 10-80°C for 30 minutes to 30 days, the cellooligosaccharide of formula (1) can be synthesized. Here, "M" is the molar concentration "mol / L", and therefore "mM" is "mol / L".

[0024] The origin of sucrose phosphorylase (SP) is not particularly limited; for example, sucrose phosphorylase derived from microorganisms such as Leuconostoc mesenteroides or Streptococcus mutans may be used. The amount of SP enzyme is determined by defining 1 U as the amount of enzyme that releases 1 μmol of αG1P per minute from 100 mM sucrose and 50 mM phosphate at 37°C and pH 7.

[0025] In cell preservation materials, cellooligosaccharides may be dissolved in an alkaline aqueous solution, for example, but are preferably self-assembled aggregates. Cellooligosaccharide aggregates are formed when cellooligosaccharides aggregate and crystallize. The crystal structure may be cellulose type I (type 1) or cellulose type II (type 2), but cellulose type II is preferred.

[0026] The crystalline structure of cellulose type I is similar to that of naturally occurring cellulose, in which adjacent molecular chains are oriented in the same direction. In contrast, the crystalline structure of cellulose type II is different in which adjacent molecular chains are oriented in opposite directions. Specifically, when cellooligosaccharides are represented by arrows pointing from the reducing end to the non-reducing end of the cellulose chain, as indicated below formula (1) above, in the crystalline structure of cellulose type II, as shown in Figure 1, the cellooligosaccharides are arranged such that the direction of the arrows alternates between adjacent molecular chains.

[0027] In one embodiment, the aggregate of cellooligosaccharides having a cellulose type II crystalline structure may have a sheet-like structure (cellulose nanosheet) consisting of a monolayer, as shown in Figure 1. Here, the sheet-like structure is a concept that encompasses ribbon-like or plate-like structures as shown in Figure 1.

[0028] As a self-assembly process to obtain a cellooligosaccharide aggregate, for example, cellooligosaccharides may be obtained by an enzymatic synthesis reaction and incubated while self-assembly is occurring. Alternatively, the cellooligosaccharides obtained by the enzymatic synthesis reaction may be dissolved in an alkali, then neutralized with an acid to precipitate and self-assemble. Alternatively, the cellooligosaccharides obtained by the enzymatic synthesis reaction may be dissolved in phosphoric acid, then water may be added to precipitate and self-assemble.

[0029] In one embodiment, the cell preservation material may be an aqueous dispersion of cellooligosaccharides. That is, it is preferable that the cell preservation material contains water together with aggregates of cellooligosaccharides, with the aggregates of cellooligosaccharides dispersed in the water. Because the cell preservation material is an aqueous dispersion, the aggregates of cellooligosaccharides can be easily dispersed in the liquid culture medium. In another embodiment, the cell preservation material may contain cellooligosaccharides in powder form.

[0030] When the cell preservation material is an aqueous dispersion, the concentration of cellooligosaccharide is not particularly limited and may be, for example, 0.1-5% (w / v), 0.5-3% (w / v), or 1-2% (w / v).

[0031] In this specification, “%(w / v)” represents mass / volume percentage concentration, which is the mass (g) of the target substance per 100 mL of volume.

[0032] The particle size of cellooligosaccharide in the aqueous dispersion of cellooligosaccharide is not particularly limited. For example, the particle size at the maximum intensity in the measurement by dynamic light scattering (DLS) may be 1 nm or more and 10 μm or less, may be 10 nm or more and 5 μm or less, or may be 100 nm or more and 1.0 μm or less.

[0033] Here, the particle size measurement by DLS is performed on an aqueous dispersion of cellooligosaccharide with a concentration of 0.005% (w / v) using a Zetasizer Nano ZSP (Malvern) at a temperature of 25°C and a scattering angle of 173°. The particle size (average value of three DLS measurements) is calculated as the hydrodynamic diameter by cumulant analysis using Polystyrene Latex as a standard sample.

[0034] In the aqueous dispersion of cellooligosaccharide, the dispersion medium may be water alone, or may be an aqueous solution containing a water-soluble salt such as sodium chloride. In one embodiment, the aqueous dispersion of cellooligosaccharide may be one in which cellooligosaccharide is dispersed in saline, and more specifically, may be one in which cellooligosaccharide is dispersed in physiological saline (PBS).

[0035] In addition to the above components, for example, sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, cytokines, hormones, antibiotics, serum, fatty acids, sugars, etc. may be added to the cell preservation material. Also, for example, fetal bovine serum, human serum, horse serum, chicken serum, insulin, transferrin, lactoferrin, cholesterol, ethanolamine, sodium selenite, monothioglycerol, 2-mercaptoethanol, bovine serum albumin, sodium pyruvate, polyethylene glycol, various vitamins, various amino acids, agar, agarose, collagen, methylcellulose, in addition to various cell function regulators, fibroblast growth factor, epithelial growth factor, vascular endothelial growth factor, platelet-derived growth factor, hepatocyte growth factor, etc. may be included as additives.

[0036] The cell preservation material according to this embodiment is suitably used to preserve pluripotent stem cells in a liquid culture medium while maintaining their undifferentiated state.

[0037] Multipotent stem cells are stem cells that have the property of differentiating into cells that make up specific tissues or organs, and somatic stem cells are a preferred example. Somatic stem cells are stem cells that exist in the body and can differentiate into a limited number of cell types. Examples of somatic stem cells include mesenchymal stem cells, hematopoietic stem cells, neural stem cells, corneal epithelial stem cells, skin stem cells, hair follicle stem cells, intestinal stem cells, liver stem cells, skeletal muscle stem cells, vascular progenitor cells, umbilical cord blood stem cells, and bone marrow stromal cells, and cells obtained by culturing these cells outside the body are also acceptable. Among these, mesenchymal stem cells (MSCs) are preferred as somatic stem cells.

[0038] Mesenchymal stem cells are somatic stem cells naturally present in the body, possessing the ability to differentiate into cells such as bone cells, chondrocytes, adipocytes, nerve cells, glial cells, and hepatocytes. Mesenchymal stem cells can be collected from bone marrow, umbilical cord tissue, umbilical cord blood, adipose tissue, etc. They can be primary human mesenchymal stem cells collected directly from patients for clinical use, mesenchymal stem cells obtained from cell banks suitable for experimental research, or immortalized mesenchymal stem cell lines.

[0039] Maintaining undifferentiated state means suppressing the differentiation of pluripotent stem cells in liquid culture medium and preserving their initial state (culturing them in a dormant state). The initial state refers to the undifferentiated state in which pluripotent stem cells have not yet begun to differentiate into differentiating lines.

[0040] The cell preservation solution according to this embodiment is a liquid used to preserve pluripotent stem cells while maintaining their undifferentiated state, and comprises a liquid culture medium and an aggregate of cellooligosaccharides dispersed in the liquid culture medium. As the aggregate of cellooligosaccharides, the cell preservation material described above can be used. Therefore, the cell preservation solution may also be obtained by adding the cell preservation material to the liquid culture medium and mixing it. The preservation of pluripotent stem cells and their undifferentiated state is as described above.

[0041] Cello-oligosaccharide aggregates exhibit excellent dispersion stability in water, thus effectively maintaining a suspended state of pluripotent stem cells in liquid culture media. In other words, the cello-oligosaccharide aggregates are dispersed in a suspended state within the liquid culture medium, maintaining the pluripotent stem cells in this suspended state and enabling their survival as single cells. Here, a suspended state of pluripotent stem cells means that the pluripotent stem cells are dispersed in the liquid culture medium without adhering to the culture vessel.

[0042] The liquid culture medium included in the cell preservation solution can be appropriately selected depending on the type of pluripotent stem cell, and various liquid culture media can be used. Specifically, examples include Mesenchymal Stem Cell Basal Medium, Dulbecco's modified Eagle medium (DMEM), Eagle's Minimum Essential Medium (EMEM), Minimum Essential Medium Eagle, Alpha Modification (αMEM), Glasgow Minimum Essential Medium (GMEM), Nutrient Mixture F-12 Ham, Dulbecco's modified Eagle Medium / Nutrient Mixture F-12 Ham, Iscove's Modified Dulbecco's Medium (IMDM), RPMI-1640, and McCoy's 5A Medium.

[0043] The components included in the liquid culture medium are not particularly limited, and various components commonly used in culture media, such as proteins, amino acids, inorganic salts, glucose, vitamins, and minerals, can be included.

[0044] The concentration of cellooligosaccharides in the cell preservation solution is not particularly limited, but is preferably 0.1 to 2.0% (w / v), more preferably 0.2 to 1.5% (w / v), and even more preferably 0.3 to 1.0% (w / v). A cellooligosaccharide concentration of 0.1% (w / v) or higher improves the dispersion stability of cellooligosaccharides in the cell preservation solution, thereby stabilizing the suspension state of pluripotent stem cells. A cellooligosaccharide concentration of 2.0% (w / v) or lower allows for easy handling as an aqueous dispersion when dispensing with a pipette or the like.

[0045] The method for producing the cell preservation solution is not particularly limited. For example, when using the above-mentioned aqueous dispersion of cellooligosaccharide, the aqueous dispersion and the liquid culture medium may be sterilized separately beforehand, and then mixed to uniformly disperse the cellooligosaccharide in the liquid culture medium. Alternatively, pluripotent stem cells may be added and dispersed in the liquid culture medium before being mixed with the aqueous dispersion of cellooligosaccharide, in which case the cell preservation solution will also contain pluripotent stem cells when it is obtained. Alternatively, the cell preservation solution may be prepared by mixing the aqueous dispersion of cellooligosaccharide with the liquid culture medium to uniformly disperse the cellooligosaccharide in the liquid culture medium, and then sterilizing it. Note that sterilization of the aqueous dispersion of cellooligosaccharide can also be considered as a sterilization process by preparing the aqueous dispersion via an alkaline aqueous solution, for example.

[0046] The viscosity of the cell preservation solution is not particularly limited; for example, the viscosity at 25°C may be 5.00 mPa·s or less, or 3.00 mPa·s or less. Since a lower viscosity of the cell preservation solution is preferable, the lower limit is not particularly limited and may be, for example, 0.50 mPa·s or more. Here, the viscosity of the cell preservation solution is measured using a tuning fork vibratory viscometer (SV-1A, A&D, 30 Hz) at a measurement temperature of 25°C.

[0047] The cell preservation method according to this embodiment includes culturing pluripotent stem cells dispersed in the cell preservation solution. Specifically, pluripotent stem cells are placed in a culture vessel together with the cell preservation solution to uniformly disperse the pluripotent stem cells in the cell preservation solution, and the pluripotent stem cells are then cultured in that state. The culture vessel is not particularly limited and examples include flasks, centrifuge tubes, microtubes, dishes, Petri dishes, and multiwell plates.

[0048] As a method for dispersing pluripotent stem cells in a cell preservation solution, one may prepare a cell preservation solution containing cellooligosaccharides and then add and mix the pluripotent stem cells to the cell preservation solution, or one may prepare a cell suspension by adding and mixing pluripotent stem cells to a liquid culture medium and then mixing the cell suspension with an aqueous dispersion of cellooligosaccharides.

[0049] Culture may be carried out under static conditions, or rotation, shaking, or stirring may be performed as needed. Preferably, the culture is carried out under static conditions (static culture) in order to reduce damage to the pluripotent stem cells. Here, static conditions mean that the cell preservation solution containing the pluripotent stem cells is kept at rest without external pressure, vibration, or stirring within the cell preservation solution. In a preferred embodiment, the pluripotent stem cells are cultured under static conditions while suspended in the cell preservation solution.

[0050] The cell concentration during culture can be appropriately set according to the pluripotent stem cells being cultured, and is not particularly limited. For example, the number of pluripotent stem cells per 1 mL of cell preservation solution could be 1.0 × 10⁶. ３ pieces / mL ~ 1.0×10 １０ It can also be 1.0 × 10¹ / mL. ４ pieces / mL ~ 1.0×10 ８ It can also be 1.0 × 10¹ / mL. ４ pieces / mL ~ 1.0×10 ６ It may also be expressed as pieces / mL.

[0051] The conditions for culturing, such as temperature, can be appropriately set according to the pluripotent stem cells being cultured. The culture temperature is usually 1 to 37°C, preferably 15 to 30°C. ２The concentration may be, for example, 0 to 10% by volume in the culture atmosphere, and preferably 0 to 6% by volume.

[0052] In culture, the culture medium may or may not be changed. Changing the culture medium means separating the cellooligosaccharides and pluripotent stem cells from the liquid medium, mixing the separated cellooligosaccharides and pluripotent stem cells with new liquid medium, and dispersing the cellooligosaccharides and pluripotent stem cells in the new liquid medium. As a method for separating pluripotent stem cells together with cellooligosaccharides from the liquid medium, for example, the pluripotent stem cells and cellooligosaccharides may be precipitated by centrifugation, and the liquid medium as the supernatant may be removed. By adding new liquid medium to the separated pluripotent stem cells and cellooligosaccharides and mixing, the pluripotent stem cells and cellooligosaccharides can be redispersed in the liquid medium.

[0053] The culture time (i.e., storage period) is not particularly limited. According to this embodiment, pluripotent stem cells can be stored for a long period of time, for example, 10 days or more, more preferably 4 weeks or more, while maintaining their undifferentiated state.

[0054] The cell preservation method according to this embodiment may further include a step of recovering pluripotent stem cells after the culture described above. Recovery is a step of separating the pluripotent stem cells from the cell preservation solution, and the recovery method is not particularly limited. Cellooligosaccharides disperse in the cell preservation solution, thereby maintaining the pluripotent stem cells in a dispersed state within the cell preservation solution, but the cellooligosaccharides and pluripotent stem cells are not adhered to each other. Therefore, separation of cellooligosaccharides and pluripotent stem cells is easy, and thus the recovery rate of pluripotent stem cells can be increased.

[0055] In one embodiment of the recovery method, the cell preservation solution containing pluripotent stem cells after culture is diluted, for example, 10 to 50 times, by adding an excess of liquid culture medium or physiological saline to the cell preservation solution containing pluripotent stem cells after culture, so that the concentration of cellooligosaccharides becomes so low that the pluripotent stem cells cannot be kept suspended. After dilution, the solution is allowed to stand, and the cellooligosaccharides remain dispersed while the pluripotent stem cells settle, allowing the pluripotent stem cells to be separated from the cell preservation solution containing cellooligosaccharides. The pluripotent stem cells that have settled and adhered to the bottom of the container can be detached from the bottom of the container by, for example, trypsin treatment, and the pluripotent stem cells can be recovered.

[0056] The present invention will be described in more detail below based on examples, but is not limited thereto.

[0057] [Synthesis of cellooligosaccharides] Using D-glucose as a primer, cellooligosaccharides with an average degree of polymerization of 8 (i.e., n=8) were synthesized.

[0058] In detail, 200 mM sucrose, 50 mM D-glucose, 0.4 U / mL cellodextrin phosphorylase (CDP), and 0.2 U / mL sucrose phosphorylase (SP) were mixed in 10 mM phosphate buffer and incubated at 40°C for 4 days. The reaction mixture containing the product was centrifuged (12000 g, 10 minutes, 25°C), the supernatant was removed, and the product was redispersed with ultrapure water. This process of redispersing and centrifuging (under the same conditions) was repeated five times to obtain cellooligosaccharides.

[0059] The reaction equation is as follows:

[0060] The average degree of polymerization of cellooligosaccharides was measured by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0061] Standard samples used for mass-to-charge ratio calibration were prepared by mixing 5 μL each of the following in a 1.7 mL tube: 5 μL of an aqueous solution of Bradykin fragment 1-7 (10 nmol / mL Bradykinin fragment 1-7, 0.05% by mass trifluoroacetic acid (TFA), 50% by mass acetonitrile), an aqueous solution of PAMP (10 nmol / mL PAMP, 0.1% by mass TFA), and an aqueous solution of ACTH fragment 18-39 (10 nmol / mL ACTH fragment 18-39, 0.1% by mass TFA), 5 μL of an aqueous solution of 10 mg / mL DHBA, 1 μL of an aqueous solution of 1.0% by mass TFA, and 4 μL of acetonitrile.

[0062] The product measurement sample was prepared by mixing 1 μL of a 0.1% (w / v) aqueous dispersion of the product, 1 μL of a 10 mg / mL aqueous solution of DHBA, and 3 μL of an acetonitrile solution of trifluoroacetic acid (0.2 vol%).

[0063] One μL each of the standard sample and the product sample were mounted on a sample plate and air-dried. After vacuum drying for more than one hour, the samples were measured using MALDI-TOFMS (AXIMA-performance, Shimadzu Corporation). The measurement conditions were: Mode: Liner (positive), Mass Range: 1.0-3000.0, Max Leaser Rap Rate: 10, power: 100, profiles: 100, shots: 2, Ion Gate (Da): Blank 500, Pulsed Extraction optimized at (Da): 1000.0.

[0064] The MS spectra obtained from the measurements were processed under the following conditions: Smoothing method: Gaussian, Smoothing filter width: 19, and Baseline filter width: 1000.

[0065] Since the measurement sample contained sodium ion adducts and potassium ion adducts, the peak areas of these were summed to calculate the peak area for each degree of polymerization. The average degree of polymerization was calculated from the ratio of the calculated peak areas for each degree of polymerization and was defined as the average degree of polymerization.

[0066] [Preparation of aqueous dispersion of cellooligosaccharide] After subjecting cellooligosaccharide to autoclaving treatment (121 °C, 20 minutes), it was diluted with sterilized water in a 15 mL centrifuge tube so that the concentration became 2.0% (w / v), and an aqueous dispersion of cellooligosaccharide was obtained.

[0067] [Test Example 1: Preservation test of mesenchymal stem cells (measurement of survival rate)] 0.5 mL of an aqueous dispersion of cellooligosaccharide with a concentration of 2.0% (w / v) and 1.5 mL of a cell suspension containing human adipose-derived mesenchymal stem cells (hADSCs) dispersed in a liquid medium were mixed in a 15 mL centrifuge tube to prepare 2.0 mL of a cell preservation solution (COs medium(+)) of Example 1 containing pluripotent stem cells. The cell preservation solution of Example 1 contains 0.5% (w / v) of cellooligosaccharide and 5.0×10 ５ cells / mL in the liquid medium.

[0068] The above cell suspension was prepared by the following procedure. hADSCs (ATCC) were cultured in Mesenchymal Stem Cell Basal Medium (ATCC) containing 2 mass% FBS, 5 ng / mL of rhFGFbasic, rhFGFacidic, rhEGF, and 2.4 mM of L-Alanyl-L-Glutamine. After culturing to about 80% confluent in a 10 cm dish under the conditions of 37 °C and 5% CO ２ 2, the medium was removed from the dish, and 5 mL of DPBS was added to the dish. After removing the DPBS, 1 mL of a 0.5 mg / mL trypsin solution was added and spread over the entire surface, and then the excess trypsin solution was removed. Under the conditions of 37 °C and 5% CO ２The cells were incubated for 3 minutes under the specified conditions and detached from the dish. 2 mL of Mesenchymal Stem Cell Basal Medium was added to the suspension, and the cells were collected in a 15 mL centrifuge tube. 2 mL of DPBS (Dulbecc's PBS) was added to the dish, and the remaining cells were similarly collected in a 15 mL centrifuge tube and centrifuged (150 g, 3 minutes, 25°C). The supernatant was removed, and the cells were resuspended in 1 mL of Mesenchymal Stem Cell Basal Medium. Live cells from this suspension and a solution of 0.4% by mass trypan blue solution mixed in a 1:1 (volume ratio) ratio were counted using a cell counter (Invitrogen Countess 3FL, Thermo Fisher Scientific), and the cell concentration was 6.67 × 10⁶. ５ The cells were diluted with Mesenchymal Stem Cell Basal Medium to a concentration of cells / mL to prepare a cell suspension.

[0069] 0.5 mL of a 2.0% (w / v) cellooligosaccharide aqueous dispersion and 1.5 mL of a cell suspension containing human adipose-derived mesenchymal stem cells (hADSCs) dispersed in DPBS were mixed in a 15 mL centrifuge tube to prepare 2.0 mL of the cell preservation solution (COs medium(-)) of Comparative Example 1 containing pluripotent stem cells. The cell preservation solution of Comparative Example 1 contained 0.5% (w / v) cellooligosaccharides and 5.0 × 10⁶ hADSCs in a buffer. ５ The cell suspension used in Comparative Example 1 was prepared by the following procedure: Cells detached in the same manner as above were collected in a 15 mL centrifuge tube and centrifuged (150 g, 3 minutes, 25°C). The supernatant was removed, and 1 mL of DPBS was added to resuspend the cells. Live cells in this suspension and a solution of 0.4% by mass trypan blue solution were mixed in a 1:1 (volume ratio) ratio and counted using a cell counter (Invitrogen Countess 3FL, Thermo Fisher Scientific). The cell concentration was 6.67 × 10⁶. ５ The cells were diluted with DPBS to a concentration of cells / mL to prepare a cell suspension.

[0070] 0.5 mL of a 2.0% (w / v) cellulose nanofiber (CNF) aqueous dispersion (Leocrysta, manufactured by Daiichi Kogyo Seiyaku Co., Ltd.) and 1.5 mL of a cell suspension containing human adipose-derived mesenchymal stem cells (hADSCs) dispersed in a liquid medium were mixed in a 15 mL centrifuge tube to prepare 2.0 mL of the cell preservation solution (CNF medium(+)) of Comparative Example 2, which contains pluripotent stem cells. The cell preservation solution of Comparative Example 2 contained 0.5% (w / v) cellulose nanofibers and 5.0 × 10⁶ hADSCs in a liquid medium. ５ Contains cells / mL. The cell suspension used was prepared in the same manner as in Example 1.

[0071] Centrifuge tubes containing 2.0 mL each of the cell preservation solutions from Example 1 and Comparative Examples 1 and 2 were placed at 25°C and 0% CO2. ２ hADSCs were cultured in a constant temperature bath under specific conditions. Samples were taken 7, 14, and 28 days after the start of culture, and cell viability was calculated. The culture medium was not changed during this period.

[0072] Sampling was performed using a pipettor. After gently pipetting several times to mix the cell preservation solution, which had been left standing in a constant temperature bath, 50 μL was collected in a 1.7 mL microcentrifuge tube.

[0073] Cell viability was calculated by counting live cells using a fluorescence microscope. 50 μL of sampled cell preservation solution and 50 μL of calcein-AM / PI solution (Double staining kit, manufactured by Dojin Chemical Research Institute Co., Ltd.) were mixed in a 1.7 mL microcentrifuge tube, and then incubated at 37°C and 5% CO2. ２ The cells were incubated for 10 minutes under the specified conditions. The entire volume was transferred to a 96-well plate, and green fluorescence (live cells) and red fluorescence (dead cells) were observed using a fluorescence microscope. More than 400 cells were counted per well for three wells, and the average viability was calculated.

[0074]

[0075] The results are shown in Figure 2 and Table 1. In Example 1, where mesenchymal stem cells were preserved in a cell preservation solution containing cellooligosaccharides and liquid culture medium, the survival rate of mesenchymal stem cells remained above 97% for 28 days. In contrast, in Comparative Example 1, where cells were preserved in a cell preservation solution containing cellooligosaccharides but without liquid culture medium, the survival rate decreased significantly after 7 days. Furthermore, in Comparative Example 2, where cellulose nanofibers were used instead of cellooligosaccharides, the survival rate of mesenchymal stem cells was almost halved after 7 days, and no survival was observed after 14 days.

[0076] [Test Example 2: Preservation Test of Mesenchymal Stem Cells (Evaluation of Undifferentiated State)] In the same manner as in Example 1, 0.5 mL of a 2.0% (w / v) cellooligosaccharide aqueous dispersion and 1.5 mL of a cell suspension containing human adipose-derived mesenchymal stem cells (hADSCs) dispersed in liquid culture medium were mixed in a 15 mL centrifuge tube to prepare 2.0 mL of the cell preservation solution for Example 2 containing pluripotent stem cells. The cell preservation solution for Example 2 contained 0.5% (w / v) cellooligosaccharide and 5.0 × 10⁶ hADSCs. ５ Contains cells / mL. A centrifuge tube containing 2.0 mL of the cell preservation solution from Example 2 was placed at 25°C and 0% CO2. ２ hADSCs were cultured in a constant temperature bath under specific conditions. Samples were taken 7, 14, and 28 days after the start of culture, and the maintenance of undifferentiated state was evaluated using a fluorescence microscope.

[0077] Sampling was performed using a pipettor. After gently pipetting several times to mix the cell preservation solution, which had been left standing in a constant temperature bath, 50 μL was collected in a 1.7 mL microcentrifuge tube.

[0078] 50 μL of sampled cell preservation solution was mixed with 200 μL of DPBS, and after centrifugation (150 g, 3 minutes, 25°C), 200 μL of the supernatant was removed. A solution of 5 μL of anti-CD73 antibody solution "CoraLite Plus 488 Anti-Human CD73" (Protein Tech) and 195 μL of DPBS containing 2% by mass of FBS was added, and after several gentle pipettes, the mixture was incubated at room temperature for 1 hour under the protection of light. Subsequently, centrifugation (150 g, 3 minutes, 25°C) was performed, 200 μL of the supernatant was removed, and 200 μL of fresh DPBS was added and redispersed. This procedure was repeated twice. Further centrifugation (150 g, 3 minutes, 25°C) was performed, 200 μL of the supernatant was removed, and the solution was made up to 100 μL with DPBS. The entire volume was transferred to a 96-well plate, and green fluorescence was observed using a fluorescence microscope.

[0079] Microscopic observation revealed fluorescence originating from the anti-CD73 antibody. This indicates that in Example 2, where mesenchymal stem cells were preserved in a cell preservation solution containing cellooligosaccharides and liquid culture medium, the cells maintained their undifferentiated state even after 7, 14, and 28 days. Furthermore, it was confirmed that there was no nonspecific adsorption of the antibody to the cellooligosaccharides. Fluorescence observation was also performed under conditions where no autofluorescence of the cells was observed.

[0080] [Test Example 3: Preservation Test of Mesenchymal Stem Cells (Measurement of Recovery Rate)] In the same manner as in Example 1, 0.5 mL of a 2.0% (w / v) cellooligosaccharide aqueous dispersion and 1.5 mL of a cell suspension containing human adipose-derived mesenchymal stem cells (hADSCs) dispersed in liquid culture medium were mixed in a 15 mL centrifuge tube to prepare 2.0 mL of the cell preservation solution of Example 3 containing pluripotent stem cells. The cell preservation solution of Example 3 contained 0.5% (w / v) cellooligosaccharide and 5.0 × 10⁶ hADSCs. ５ Contains cells / mL. A centrifuge tube containing 2.0 mL of the cell preservation solution from Example 3 was placed at 25°C and 0% CO2. ２ hADSCs were cultured in a constant temperature bath under specific conditions. Samples were taken 7, 14, and 28 days after the start of culture, and the recovery rate and viability were measured.

[0081] Sampling was performed using a pipettor. After gently pipetting several times to mix the cell preservation solution that had been left standing in a constant temperature bath, 30 μL was collected in a 24-well plate. 570 μL of Mesenchymal Stem Cell Basal Medium was added and gently pipetted several times, then the plate was heated at 37°C and 5% CO2. ２ The seeds were incubated for 24 hours under the specified conditions and then resown.

[0082] After engraftment, cells were harvested using trypsin, and the harvest rate and viability were calculated. Specifically, the supernatant of a re-seeded 24-well plate was removed, 500 μL of DPBS was added, and this DPBS was collected in a 15 mL centrifuge tube. 200 μL of 0.5 mg / mL trypsin solution was added and spread over the entire surface, and the excess trypsin solution was collected in a 15 mL centrifuge tube. The 24-well plate was then heated at 37°C in 5% CO2. ２ The cells were incubated for 3 minutes under the specified conditions and detached from the dish. 500 μL of Mesenchymal Stem Cell Basal Medium was added to the suspension, and the cells were collected in a 15 mL centrifuge tube. 500 μL of DPBS was added to the dish, and the remaining cells were similarly collected in a 15 mL centrifuge tube and centrifuged (150 g, 3 minutes, 25°C). The supernatant was removed, and the precipitated cells were made up to 50 μL with Mesenchymal Stem Cell Basal Medium. Live cells from this suspension and a solution of 0.4% trypan blue solution in a 1:1 volume ratio were counted in 3 wells using a cell counter (Countess3FL, Invitrogen), and the average cell concentration and viability were calculated.

[0083] The recovery rate was determined by using the cell concentration obtained when reseeding under the same conditions without cellooligosaccharides as a baseline, with the cell concentration set at 100, and the recovery rate being the ratio to that baseline. Specifically, when the cell concentration was 5.0 × 10⁻⁶ ５ 30 μL of cell suspension, diluted with Mesenchymal Stem Cell Basal Medium to a cell / mL concentration, was added to a 24-well plate. 570 μL of Mesenchymal Stem Cell Basal Medium was added and gently pipetted several times. The plate was then cooled at 37°C and 5% CO2. ２The cells were incubated for 24 hours under the specified conditions and then reseeded. After engraftment, the cells were harvested using trypsin in the same manner as in Example 3 above, and the cell concentration was measured.

[0084] The results are shown in Table 2. Mesenchymal stem cells preserved in a cell preservation solution containing cellooligosaccharides and liquid culture medium were found to engraft on 24-well plates after reseeding. Furthermore, when cells were reseeded 7, 14, and 28 days after the start of culture, both the recovery rate and survival rate were high.

[0085] [Test Example 4] Cell preservation solutions prepared in the same manner as in Example 1 (COs medium(+)), cell preservation solutions prepared in the same manner as in Comparative Example 1 (COs medium(-)), and cell preservation solutions prepared in the same manner as in Comparative Example 2 (CNFmedium(+)) were incubated in a constant temperature bath in the same manner as in Test Example 3 to culture haADSCs. Seven days after the start of culture, 30 μL was sampled and collected in a 24-well plate, as in Test Example 3, and 570 μL of Mesenchymal Stem Cell Basal Medium was added, followed by incubation at 37°C and 5% CO2. ２ The seeds were incubated for 24 hours under the specified conditions and then reseeded. The supernatant from the reseeded 24-well plates was removed and washed with DPBS.

[0086] After removing the supernatant and adding 200 μL of DPBS, phase-contrast images were taken using a fluorescence microscope. As a result, with the cell preservation solution (COs medium(+)), cellooligosaccharides could be removed by removing the supernatant and washing after the cells had settled in the 24-well plate. In contrast, with the cell preservation solution without culture medium (COs medium(-)) and the cell preservation solution using CNF (CNF medium(+)), cells did not settle in the 24-well plate due to the low number of viable cells.

[0087] Furthermore, the various numerical ranges described in this specification can be any combination of their upper and lower limits, and all such combinations are described herein as preferred numerical ranges. Also, the description of a numerical range as "X to Y" means X or greater and Y or less.

[0088] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their omissions, substitutions, and modifications are included in the scope and spirit of the invention, as well as in the claims and their equivalents.

Claims

1. A cell preservation material used to preserve pluripotent stem cells in a liquid culture medium while maintaining their undifferentiated state, the cell preservation material comprising cellooligosaccharides.

2. The cellooligosaccharide is represented by the following general formula (1), The cell preservation material according to claim 1, wherein A represents a hydrogen atom or substituent, and n represents the average degree of polymerization.

3. The cell preservation material according to claim 1, wherein the multipotent stem cells are somatic stem cells.

4. The cell preservation material according to claim 1, wherein the multipotent stem cells are mesenchymal stem cells.

5. A cell preservation solution comprising a liquid culture medium and an aggregate of cellooligosaccharides dispersed in the liquid culture medium, used for preserving pluripotent stem cells while maintaining their undifferentiated state.

6. The cell preservation solution according to claim 5, wherein the concentration of the cellooligosaccharide is 0.1 to 2.0% (w / v).

7. A method for preserving cells, comprising culturing pluripotent stem cells dispersed in the cell preservation solution described in claim 5.

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