Cell population containing mesenchymal stem cells, cell sheet, and method for producing the cell sheet

Abstract

The present invention obtains a cell population containing mesenchymal stem cells that have excellent proliferative ability and osteoblast differentiation ability and that are useful for regeneration of periodontal tissues. Further, provided are: a cell sheet in which the material thereof is a cell population containing mesenchymal stem cells that have excellent proliferative ability and osteoblast differentiating ability and that are useful for regeneration of periodontal tissues; and a production method for the cell sheet.　The present invention involves acquiring, through selection, a cell population having an alkaline phosphatase activity of 1 U or more before undergoing calcification induction, from a cell population containing mesenchymal stem cells derived from a tooth or a periodontal tissue. The cell sheet is obtained by: (a) a step for acquiring a cell population that contains mesenchymal stem cells having an alkaline phosphatase activity; (b) a step for culturing the cell population in a calcification induction medium and growing same on a plastic substrate; and (c) a step for recovering and rinsing a sheet-like cell population formed on the plastic substrate.

Description

[Technical Field]

[0001] This invention relates to a cell population containing mesenchymal stem cells, a cell sheet, and a method for producing a cell sheet. [Background technology]

[0002] Mesenchymal stem cells (MSCs), which are included in stromal cells, are stem cells that have the ability to differentiate into tissues derived from the mesoderm, such as bone, cartilage, blood vessels, and cardiomyocytes. The International Society for Cell Therapy has proposed that the following are the minimum requirements for defining human MSCs: (1) adherence to plastic under standard culture conditions, (2) positivity for the cell surface markers CD73, CD90, and CD105, and negativity for CD79α, CD34, CD45, and HLA-DR, and (3) the ability to differentiate into osteoblasts, chondrocytes, or adipocytes (Non-Patent Literature 1).

[0003] MSCs are known to have anti-inflammatory, growth factor-inducing, and angiogenesis-promoting effects, playing an important role in tissue repair. In recent years, the application of MSCs to regenerative medicine has been progressing, and the practical application of new treatments that promote the regeneration and functional improvement of tissues and organs is rapidly advancing. For example, Patent Document 1 discloses a sheet of periodontal ligament-derived cells containing MSCs that can be used for the regeneration of periodontal tissue damaged by periodontal disease, etc. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Public Gazette 2005 / 103233 [Non-patent literature]

[0005] [Non-Patent Document 1] M. Dominici, MD et al., Cytotherapy, : 8, 315-317 (2006) [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Several useful markers have been proposed for evaluating MSCs. For example, as mentioned above, Non-Patent Document 1 discloses CD105, CD73, and CD90 as essential markers for MSCs. However, cells expressing these markers do not always show good proliferation, so more useful markers are desired.

[0007] Since the proliferative and differentiation capabilities of mesenchymal stem cells (MSCs) vary greatly depending on their originating tissue, useful markers for evaluating MSCs may also differ depending on the originating tissue and intended use. Patent Document 1 discloses a method for producing cell sheets of periodontal ligament-derived cells, but it does not disclose markers for selecting the cells to be used as material.

[0008] The present invention aims to obtain a cell population containing mesenchymal stem cells that have excellent proliferative and osteoblast differentiation capabilities and are useful for the regeneration of periodontal tissue. Furthermore, the present invention aims to provide a cell sheet and a method for producing a cell sheet using a cell population containing mesenchymal stem cells that have excellent proliferative and osteoblast differentiation capabilities and are useful for the regeneration of periodontal tissue as a material. [Means for solving the problem]

[0009] The inventors of this invention searched for a marker that can identify MSCs with superior proliferative capacity and osteoblast differentiation capacity before differentiation induction, for use in periodontal tissue regeneration, and have found such a marker. Furthermore, they found that by using this marker as a selection indicator, it is possible to obtain MSCs with high periodontal tissue regeneration capacity, and that cell sheets useful for periodontal tissue regeneration can be obtained using these as a material, thus completing the present invention.

[0010] In other words, the following inventions are provided according to this specification. (1) A cell population comprising mesenchymal stem cells derived from teeth or periodontal tissue, wherein the alkaline phosphatase activity before calcification induction is 1 U or more. (2) The cell population described in (1), having alkaline phosphatase activity of 5 U or more after calcification induction. (3) The cell population according to (1) or (2), wherein the mesenchymal stem cells are mesenchymal stem cells derived from the periodontal ligament. (4) A cell sheet comprising a cell population including mesenchymal stem cells derived from teeth or periodontal tissue, wherein the alkaline phosphatase activity of the cell population after calcification induction is 5 U or more. (5) The cell sheet according to (4), wherein the alkaline phosphatase activity of the cell population before calcification induction is 1 U or more. (6) A method for producing a cell sheet, comprising the steps (a) to (c) below: (a) A step of obtaining a cell population that has alkaline phosphatase activity and includes mesenchymal stem cells derived from teeth or periodontal tissue; (b) A step of culturing the cell population in a calcification-inducing medium and growing it on a plastic substrate; and (c) A step of collecting and washing the sheet-like cell population formed on the plastic substrate. (7) The method according to (6), wherein the alkaline phosphatase activity of the cell population in step (a) is 1 U or more. (8) The method according to (6) or (7), wherein the alkaline phosphatase activity of the cell population after induction of calcification is 5 U or more. (9) A method for preserving and / or transporting a cell sheet comprising a cell population comprising mesenchymal stem cells derived from teeth or periodontal tissue, wherein the cell population has high alkaline phosphatase activity, and the cell sheet is maintained in α-modified Eagle minimal essential medium at room temperature. (10) The method according to (9), wherein the cell sheet can be maintained in a usable state for 7 days or more in α-modified Eagle minimum essential medium at room temperature. (11) The method according to (9) or (10), wherein the cell sheet is the cell sheet according to (4) or (5), or the cell sheet produced by the method according to any one of (6) to (8). This specification incorporates the disclosure of Japanese Patent Application No. 2021-188216, which is the basis of the priority of this application.

Advantages of the Invention

[0011] According to the present invention, it is possible to obtain mesenchymal stem cells that are excellent in proliferation ability and osteoblast differentiation ability and are useful for periodontal tissue regeneration. Further, it is possible to provide a cell sheet using mesenchymal stem cells that are excellent in proliferation ability and osteoblast differentiation ability and are useful for periodontal tissue regeneration as a material, and a method for producing the cell sheet.

Brief Description of the Drawings

[0012] [Figure 1] It is a graph showing the results of measuring the alkaline phosphatase activity of a cell population containing each MSC before and after induction of calcification. [Figure 2] It is a graph showing the results of measuring the cell proliferation ability of a cell population containing each MSC before induction of calcification. [Figure 3] It is a graph showing the results of measuring the colony formation ability of a cell population containing each MSC before induction of calcification.

Embodiments for Carrying Out the Invention

[0013] [1] Cell population containing mesenchymal stem cells The cell population containing mesenchymal stem cells (MSCs) of the present invention (hereinafter also referred to as "the cell population of the present invention") is a cell population containing MSCs derived from teeth or periodontal tissue, characterized in that its alkaline phosphatase activity (hereinafter, alkaline phosphatase is also referred to as "ALP," and alkaline phosphatase activity as "ALP activity") before calcification induction is 1 U or more. The cell population of the present invention has the advantage of having excellent proliferative capacity and osteoblast differentiation capacity, and is useful for the regeneration of periodontal tissue. Furthermore, the cell population of the present invention has the advantage of being usable in the production of cell sheets, particularly cell sheets useful for the regeneration of periodontal tissue.

[0014] In this invention, "mesenchymal stem cells (MSCs)" refers to somatic cells (tissue cells) that can be collected from various tissues and organs and that satisfy the following definition. (1) Adhesion to plastic under standard culture conditions; (2) The cell surface markers CD73, CD90, and CD105 are positive, and CD79α, CD34, CD45, and HLA-DR are negative; (3) Having the ability to differentiate into osteoblasts, chondrocytes, or adipocytes.

[0015] In the present invention, "cell population containing MSCs" refers to a cell population that mainly contains MSCs. Here, "mainly containing MSCs" means a state in which 90% or more, particularly 95% or more, of cells satisfy the condition of "positive for CD73, CD90, and CD105, and negative for CD79α, CD34, CD45, and HLA-DR" in flow cytometry analysis. The origin of the cell population containing MSCs is a cell population containing MSCs derived from teeth or periodontal tissue. Examples of MSCs derived from teeth or periodontal tissue include MSCs derived from dental pulp, periodontal ligament, dental follicle, dental papilla, and deciduous teeth, but MSCs derived from the periodontal ligament are particularly preferred. The following describes examples of embodiments using periodontal ligament-derived MSCs, but this is not intended to limit the scope of the present invention.

[0016] In this invention, the "ALP activity" of the cell population before calcification induction refers to the enzyme activity measured by the following method: Place the cell population before calcification induction into a 96-well microplate in a 1 × 10⁶ well. 4 Seeds are seeded to a cell / well (100 μL). Cells are cultured for 2 days in basal medium (α-modified Eagle's minimal essential medium (α-MEM) with 10% FBS) at 37°C in a 5% CO2 environment. The medium is changed and the cells are cultured for a further 5 days. After removing the medium and washing, substrate (e.g., p-nitrophenyl phosphate (pNPP)) buffer is added and the mixture is reacted under conditions of 37°C, and absorbance is measured after adding stop solution as needed. The ALP of the above cell population is calculated using a calibration curve created from measurements of p-nitrophenol (pNP) solution of known concentration. That is, the ALP activity (U) shown herein is for an initial cell number of 1 × 10⁶ cells. 4 This refers to the activity level in a cell population after culturing cells under specified conditions for 7 days.

[0017] The specific method for measuring ALP activity (U) is not particularly limited, but for example, it can be calculated by the following method: Dispense pNP standard solutions of multiple concentrations into a microwell plate, measure the absorbance at 405 nm, and derive the calibration curve shown below. Y=aX+b (X is pNP concentration (mmol / L), Y is absorbance at 405 nm) The ALP activity of a sample is calculated from the absorbance at 405 nm measured after reacting 100 μL of the sample with pNPP solution for 5 minutes. The concentration X (mmol / L) of pNP produced by the ALP in the sample is calculated by substituting the absorbance into Y on the calibration curve above. Next, the ALP activity per 1 μL (unit / μL) is calculated. In this specification, 1 unit of ALP activity refers to the enzyme activity that produces 1 nmol of pNP per minute at pH 9.8 and 37°C. ALP activity (unit / μL)=X(mmol / L) / 5(min) The total activity (U) of 100 μL of sample is calculated using the following formula. ALP activity (U) = ALP activity (unit / μL) × 100

[0018] In the present invention, the ALP activity of the cell population before calcification induction is 1 U or more, preferably 1.2 U or more, more preferably 1.4 U or more, and even more preferably 2 U or more.

[0019] Furthermore, in the present invention, the ALP activity of the cell population after calcification induction is preferably 5 U or more, more preferably 7 U or more, even more preferably 8.5 U or more, and even more preferably 15 U or more. "ALP activity after calcification induction" refers to the ALP activity of the cell population obtained by culturing the cell population of the present invention in a calcification induction medium.

[0020] As a "calcification-inducing medium," any commercially available medium for calcification induction can be used. For example, a medium prepared by adding L-ascorbic acid magnesium phosphate n-hydrate, dexamethasone, β-glycerophosphate, etc., to a basal medium (e.g., 10% FBS-added α-MEM) can be used.

[0021] The conditions for culturing a cell population containing MSCs using calcification-inducing medium are not particularly limited as long as the conditions allow for sufficient induction and proliferation of MSCs, but for example, the following conditions can be used: The cell suspension obtained using ALP activity before calcification induction as an indicator is placed in a 96-well microplate in a 1 × 10⁶ layer. 4 Seeds are seeded to a cell / well (100 μL). Cells are cultured for 2 days in basal medium (α-modified Eagle's minimal essential medium (α-MEM) supplemented with 10% FBS) at 37°C and 5% CO2. Subsequently, the medium is replaced with calcification-inducing medium (e.g., α-MEM containing magnesium L-ascorbic acid phosphate n-hydrate, dexamethasone, β-glycerophosphate, etc.), and the cells are cultured for another 5 days at 37°C and 5% CO2. The ALP activity of the cell population after calcification induction is measured using the same method as for the cell population before calcification induction.

[0022] Typically, in MSCs derived from the periodontal ligament, ALP activity before calcification induction is lower than that after calcification induction, and inter-individual differences are relatively small. However, it was confirmed that cell populations containing MSCs exhibiting higher ALP activity before calcification induction possess high proliferative capacity. Furthermore, a significant increase in ALP activity was observed in these cells after calcification induction, confirming their high osteoblast differentiation potential. Therefore, by using ALP activity as an indicator, it is possible to select and obtain cell populations containing MSCs with high proliferative capacity and osteoblast differentiation potential.

[0023] The cell population containing the obtained MSCs exhibits high cell proliferation and colony formation capabilities. "Cell proliferation capacity" can be measured, for example, by the following method: 1 × 10⁻⁶ 3 Cells were seeded in 96-well plates and cultured in basal medium for 2 and 4 days at 37°C under 5% CO2 conditions. The medium was changed with 100 μL of fresh basal medium, and measurements were performed using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega).

[0024] "Colony-forming ability" can be measured, for example, by the following method: 100 cells in a 60cm² cell. 2 Seeds are seeded in a Petri dish and cultured in basal medium for 14 days at 37°C under 5% CO2 conditions. During this time, the medium is changed twice a week. Afterwards, the cells are stained with crystal violet according to the standard method. The number of cells that form colonies out of 100 seeded cells (i.e., the number of colonies) is counted, and the percentage (%) of these colonies is used as an indicator of colony-forming ability.

[0025] Cell proliferation ability and colony formation ability can be measured, for example, by the method described in Iwata et al., J. Clin. Periodontol., Vol. 37, pp. 1088-1099 (2010).

[0026] [2] Cell sheet The cell sheet of the present invention is a cell sheet comprising a cell population including MSCs, characterized in that the ALP activity of the cell population after calcification induction is 5 U or more. The "cell population including MSCs" referred to here may be the cell population described in the section "[1] Cell population including mesenchymal stem cells". The cell sheet of the present invention has the advantage of having high ALP activity, which gives it high engraftment and regenerative ability in periodontal tissue, and is therefore particularly useful as a cell sheet for the regeneration of periodontal tissue.

[0027] In this invention, "cell sheet" refers to cells that have been confluently attached to a plastic substrate and then peeled off in a sheet shape, or to a stack of such sheet-shaped cells. In particular, by growing cells on a temperature-responsive cell culture substrate (for example, UpCell® (manufactured by CellSeed Co., Ltd.)), cells can be easily peeled off in a sheet shape by temperature changes alone, without the need for enzymatic treatment or other procedures. Since such cells have not undergone enzymatic treatment, their original functions are maintained. Furthermore, because the adhesion proteins between cells are retained, they have the advantage of engrafting more quickly when applied to living organisms.

[0028] In this specification, "plastic" refers to a synthetic resin having plasticity. The material is not particularly limited as long as it is a synthetic resin material that can be formed into a flat plate shape, and for example, polyethylene, polystyrene, polypropylene, polycarbonate, poly(meth)acrylamide, etc. can all be used. Specifically, examples include materials obtained by the homopolymerization or copolymerization of monomers such as (meth)acrylamide compounds and N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives described in Japanese Patent Application Publication No. 2-211865, and in particular, a temperature-responsive cell culture substrate (e.g., UpCell®) that can detach adhered cells by temperature changes can be preferably used.

[0029] The ALP activity after calcification induction in a cell population containing MSCs constituting the cell sheet of the present invention is 5 U or more, preferably 7 U or more, more preferably 8.5 U or more, and even more preferably 15 U or more. Furthermore, the ALP activity of the cell population before calcification induction is preferably 1 U or more, more preferably 1.2 U or more, and even more preferably 1.4 U or more. The definition of the ALP activity value used herein is as described in the section "[1] Cell population containing mesenchymal stem cells," and the induction of calcification and the selection and acquisition can also be carried out by the method described in the section "[1] Cell population containing mesenchymal stem cells."

[0030] The cell sheet of the present invention can be manufactured by the method described in the section "[3] Method for manufacturing a cell sheet" described later. More specifically, the cell sheet of the present invention is a cell sheet manufactured by the method described in the section "[3] Method for manufacturing a cell sheet," and the detailed conditions such as the properties, materials, and manufacturing method of the cell sheet are as described in the sections "[1] Cell population containing mesenchymal stem cells" and "[3] Method for manufacturing a cell sheet," unless otherwise specified.

[0031] [3] Method for producing cell sheets The method for producing a cell sheet of the present invention (hereinafter also simply referred to as "the method of the present invention") is characterized by comprising the following steps (a) to (c). (a) A step of obtaining a cell population containing MSCs that have ALP activity and are derived from teeth or periodontal tissue; (b) A step of culturing the cell population in a calcification-inducing medium and growing it on a plastic substrate; and (c) A step of collecting and washing the formed sheet-like cell population. The method of the present invention, having the above-mentioned features, makes it possible to produce cell sheets that are particularly useful for the regeneration of periodontal tissue, using MSCs, which have excellent proliferative and osteoblast differentiation capabilities, as a material.

[0032] [3-1] Step to obtain a cell population containing MSCs (Step (a)) The method of the present invention includes a step of obtaining a cell population containing MSCs having ALP activity (hereinafter also referred to as "step (a)").

[0033] The "cell population containing MSCs with ALP activity" obtained in this process is a cell population selected and obtained from among the cell populations containing MSCs derived from teeth or periodontal tissue (preferably MSCs derived from the periodontal ligament) as described in the section "[1] Cell population containing mesenchymal stem cells," based on the indicator that the ALP activity before calcification induction, measured by the method described in the section "[1] Cell population containing mesenchymal stem cells," is above a certain value.

[0034] In step (a), the ALP activity used as an indicator for selecting and obtaining cell populations, i.e., the ALP activity of the cell population before calcification induction, is preferably 1 U or more, more preferably 1.2 U or more, and even more preferably 1.4 U or more.

[0035] [3-2] A step of culturing a cell population including MSCs in calcification-inducing medium (step (b)) The method of the present invention includes a step of culturing the cell population containing MSCs obtained in step (a) in a calcification-inducing medium and growing it on a plastic substrate (hereinafter also referred to as "step (b)").

[0036] In step (b), the cell population is cultured in a container having a plastic substrate. The bottom and / or walls of the container may be made of flat plastic, and the inner surface of the bottom and / or walls may serve as the plastic substrate. Alternatively, a flat plastic material, formed separately from the material forming the container, may be placed inside the container and function as the plastic substrate. The plastic substrate is preferably formed on the inner surface of the bottom and / or walls of the container, and more preferably on the inner surface of the bottom of the container.

[0037] As the material of the plastic substrate used in step (b), in particular, a temperature-responsive cell culture substrate (for example, UpCell (registered trademark)) can be preferably used. Examples of the container used for culturing in step (b) include a microwell plate, a Petri dish, a tissue culture flask, etc. In particular, a microwell plate, a Petri dish, etc. using a temperature-responsive cell culture substrate on the bottom or wall can be preferably used. Hereinafter, a mode of using a Petri dish provided with a temperature-responsive cell culture substrate will be exemplarily described, but it is not intended to limit the scope of the present invention.

[0038] As the calcification induction medium used in step (b), any medium generally commercially available as a calcification induction medium can be used. For example, a medium obtained by adding magnesium phosphate L-ascorbate n-hydrate, dexamethasone, and β-glycerophosphate to a basal medium (for example, α-MEM supplemented with 10% FBS) can be used.

[0039] The culture conditions for culturing a cell population containing MSCs using a calcification induction medium are not particularly limited as long as the conditions allow sufficient induction and proliferation of MSCs. For example, the following conditions can be adopted.

[0040] (1) Seeding The cell suspension obtained using the ALP activity before calcification induction as an index was thawed, pre-cultured as necessary, and then added to the calcification induction medium (for example, α-MEM containing magnesium phosphate L-ascorbate n-hydrate, dexamethasone, and β-glycerophosphate) in a Petri dish at a density of 1×10 4 ~1×10 6 cells / cm 2 , preferably 1×10 4 ~1×10 5 cells / cm 2 and seeded.

[0041] (2) Culture and calcification induction Culturing can be carried out in calcification-inducing medium under conditions such as 37°C and 5% CO2. Culturing is preferably carried out for about 7 to 10 days, with the culture medium being changed every 3 to 4 days. Due to their nature, the cell population, including MSCs, usually grows attached to the plastic substrate of the culture system and takes on a sheet shape.

[0042] The cell population containing MSCs whose calcification was induced in step (b) preferably has higher ALP activity than before calcification. The ALP activity after calcification is particularly preferably at least twice, and more preferably at least three times, the activity before induction. When the ALP activity of the cell population containing MSCs after calcification is measured by the method described above, it is preferably 5 U or more, more preferably 7 U or more, even more preferably 8.5 U or more, and even more preferably 15 U or more.

[0043] [3-3] Steps to collect and wash the sheet-like cell population. The method of the present invention includes a step (b) in which a sheet-like cell population formed on a plastic substrate is collected and washed (hereinafter also referred to as "step (c)").

[0044] The cell population calcified in step (b) is formed into a sheet on the plastic substrate while adhered to it. The adhered sheet of cell population can be detached by any known method, such as trypsin treatment or scraping, provided that the sheet shape can be maintained. If a temperature-responsive cell culture substrate is used as the plastic substrate, it is possible to detach the cell without degrading the cell adhesion factors by applying a temperature change to the culture medium.

[0045] The sheet-like cell population that has undergone the detachment process is washed with a washing solution. The washing solution used in step (c) can be physiological saline, PBS, serum-free basal culture medium, etc. The washed sheet is placed in a storage container containing a preservation solution. Further washing of sheets of other cell populations is also possible, and these sheets may be laminated on top of the previously placed sheet. The lamination of sheets is preferably 2 to 5 layers, for example, 3 layers. As the storage container, for example, a container as described in Japanese Patent Publication No. 2018-121568 can be used. The preservation solution placed in the storage container is preferably α-MEM.

[0046] [4] Methods for storing and / or transporting cell sheets The present invention provides a method for storing and / or transporting a cell sheet containing a cell population including MSCs derived from teeth or periodontal tissue (hereinafter also referred to as the "storage / transportation method"). The cell population constituting the cell sheet stored and / or transported by the storage / transportation method of the present invention has high ALP activity. Furthermore, in the storage / transportation method of the present invention, the cell sheet is maintained in α-MEM and stored and / or transported at room temperature.

[0047] Cell sheets are sometimes stored and transported in dedicated containers. Conventionally, cell sheets have been stored and transported under low-temperature conditions of 2-10°C, particularly 2-5°C, which required costs and effort for preparing refrigeration equipment. To simplify the handling of cell sheets, storage and transport at room temperature is desirable. In this specification, "room temperature" refers to the general temperature under normal living conditions without any special heating or cooling, but for the storage and transport conditions of cell sheets, 15-25°C is preferred.

[0048] The inventors have found that immersing cell sheets in α-MEM during storage and / or transport allows them to be maintained in a usable state at room temperature. In the storage / transportation method of the present invention, cell sheets can be maintained in a usable state even after being stored in α-MEM at room temperature for 7 days or more, preferably 10 days or more. However, the actual storage period is preferably 7 days or less, particularly 5 days or less.

[0049] In this invention, a cell sheet in a "ready-to-use state" specifically refers to a cell sheet in which the number of cells, cell viability, ALP activity, and ALP purity are all above the predetermined standard values. The respective standard values ​​are as follows: Cell number: 1×10 6 Cells / product (top view: 21mm diameter circle) Cell viability: 80% ALP activity: 10U or more ALP positive cell rate: 50%

[0050] The "ALP activity" of a cell sheet refers to the ALP activity measured from cell sheet fragments recovered from the cell sheet product using a biopsy trephine (φ8.0 mm).

[0051] The "cell count" and "cell viability" of a cell sheet can be measured, for example, by the following method: The remaining cell sheet recovered with a biopsy trephine is treated with collagenase, followed by trypsin treatment to prepare a cell suspension. After washing the cells, trypan blue staining is performed, and the cell count and viability are confirmed under a microscope.

[0052] The "ALP-positive cell rate" can be measured using FACS after reacting an anti-ALP antibody with a cell suspension recovered from a cell sheet.

[0053] The "cell count," "cell viability," and "ALP-positive cell rate" of a cell sheet can be measured, for example, by the method described in Washio, et al., Int. J. Oral Maxillofac. Implants, vol. 29, No. 1, e117-121 (2014).

[0054] In the storage / transportation method of the present invention, the cell sheet to be stored and / or transported is preferably a cell sheet described in section "[2] Cell Sheet" or a cell sheet manufactured by the method described in section "[3] Method for Manufacturing a Cell Sheet". Unless otherwise specified, the properties, materials, and manufacturing method of the cell sheet to be stored are as described in sections "[1] Cell Population Including Mesenchymal Stem Cells", "[2] Cell Sheet", and "[3] Method for Manufacturing a Cell Sheet". [Examples]

[0055] The present invention will be described in more detail below with reference to examples, but it is not intended to limit the present invention to the scope of these examples.

[0056] [Example 1] Collection, isolation, and culture of periodontal ligament tissue cells With the approval of the Ethics Committee, samples were collected from seven healthy donors (P303-P309) who had given informed consent. One third molar or equivalent tooth was extracted from each healthy donor under local anesthesia, and periodontal ligament tissue was collected from the central one-third of the root. The obtained periodontal ligament tissue was treated with enzymes to isolate cells. The actual procedure followed the one described in Iwata et al., Regen. Ther., Vol. 9, pp. 38-44 (2018).

[0057] The collected cells were cultured for about a week at 37°C in a 5% CO2 environment using α-MEM (containing 10% FBS, L-glutamine, gentacin, and fungizone), and then subcultured. At passage 3, 1 × 10⁶ cells were subcultured using STEM-CELLBANKER®. 5 The cells were prepared into tubes and cryopreserved in liquid nitrogen.

[0058] [Example 2] Measurement of alkaline phosphatase activity ALP activity was measured in cell populations derived from each sample using the following method: 1 × 10⁻⁶ 5 Cells were seeded in a 96-well plate at a concentration of cells / mL (100 μL: 1 × 10⁶). 4Cells were cultured for 2 days at 37°C in a 5% CO2 environment in basal medium (α-MEM with 10% FBS), then the medium was changed and the cells were cultured for another 5 days. The medium was removed and the cells were washed twice with 200 μL of physiological saline. 100 μL of LabAssay ALP (Fujifilm Wako Pure Chemical Industries, Ltd.) substrate buffer was added and incubated at 37°C for 5 minutes. Then 80 μL of stop solution was added, and the absorbance at 405 nm was measured using a microplate reader. Using a calibration curve created based on the absorbance of LabAssay ALP standard solutions, the ALP activity (U) of each cell population was calculated from each absorbance.

[0059] ALP activity was measured in each cell population after calcification induction. 1 × 10 5 Cells were seeded in a 96-well plate at a concentration of cells / mL (100 μL: 1 × 10⁶). 4 Cells were cultured in basal medium at 37°C and 5% CO2 for 2 days. Then, the medium was changed to calcification induction medium (α-MEM supplemented with 10% FBS, magnesium L-ascorbic acid phosphate n-hydrate, dexamethasone, and β-glycerophosphate), and cultured for a further 5 days at 37°C and 5% CO2. The medium was removed and the cells were washed twice with 200 μL of physiological saline. 100 μL of LabAssay ALP (Fujifilm Wako Pure Chemical Industries, Ltd.) substrate buffer was added and incubated at 37°C for 5 minutes. Then, 80 μL of stop solution was added, and the absorbance at 405 nm was measured using a microplate reader. Using a calibration curve created based on the absorbance of LabAssay ALP standard solutions, the ALP activity (U) of each cell population was calculated from each absorbance.

[0060] Figure 1 shows the results of ALP activity measurements for each cell population before and after calcification induction. Cell populations derived from tissues with high ALP activity before calcification induction (hereinafter simply referred to as "P304," "P305," etc.) showed significantly higher ALP activity after induction. On the other hand, P307 and P309, which had low ALP activity before induction, did not show much increase in ALP activity after induction. This indicates that a significant increase in ALP activity occurs after induction in cell populations with high ALP activity before calcification induction.

[0061] [Example 3] Measurement of cell proliferation ability For each cell population before induction, 1 × 10 3 Cells were seeded in 96-well plates and cultured in basal medium for 2 and 4 days at 37°C under 5% CO2 conditions. Then, the basal medium was replaced with 100 μL of fresh medium, 20 μL of CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) was added, and the cells were incubated at 37°C under 5% CO2 conditions for 1 hour. Afterward, absorbance was measured at 490 nm using a microplate reader (Model 450; Bio-Rad). The detailed conditions for this measurement were those described in Iwata et al., J. Clin. Periodontol., Vol. 37, pp. 1088-1099 (2010).

[0062] Figure 2 shows the cell proliferation capacity of each cell population. Sufficiently high cell proliferation capacity was observed in P304 and P305. This indicates that cell populations with high ALP activity before calcification induction possess sufficiently high cell proliferation capacity.

[0063] [Example 4] Measurement of colony-forming ability For each cell population, 100 cells in a 60cm space. 2 Seeds were seeded in Petri dishes and cultured in basal medium at 37°C under 5% CO2 conditions for 14 days. During this period, the medium was changed twice a week. Afterwards, the cells were stained with 0.5% crystal violet and washed twice with distilled water. After drying the Petri dishes, the stained areas were counted. The number of cells that formed colonies out of 100 seeded cells, i.e., the colony count, was defined as the colony-forming ability (%). The detailed conditions for this measurement were those described in Iwata et al., J. Clin. Periodontol., Vol. 37, pp. 1088-1099 (2010).

[0064] Figure 3 shows the colony-forming ability of each cell population. Sufficiently high colony-forming ability was observed in P304 and P305. This indicates that ALP activity prior to calcification induction contributes to colony-forming ability.

[0065] [Example 5] Preparation of cell sheets A cell population of P304 was used as the material for the cell sheet. The cell population was divided into 3-4 × 10⁶ cells. 5 Cells were seeded in UpCell (CellSeed Co., Ltd.) and cultured in calcification-inducing medium at 37°C under 5% CO2 conditions for 9-10 days. During this time, the cell condition was checked and the medium was changed every 3-4 days. The medium was removed and the cells were washed twice with 1 mL of physiological saline warmed to 37°C. The cell sheets were then collected using a ring-shaped support. The collected cell sheets were placed on top of other washed cell sheets and collected again. Finally, a three-layer cell sheet product was prepared.

[0066] [Example 6] Storage / transport stability test of cell sheets The stability of the cell sheets prepared in Example 5 under storage / transportation conditions was investigated. The cell sheets prepared in Example 5 were placed in a dedicated transport container containing α-MEM. The transport containers containing the cell sheets were left standing at room temperature for 0 days and 7 days. After the transport containers were left standing for 7 days, they were transported for 1 hour under room temperature conditions (on foot and by train). As a control, cell sheets were similarly placed in transport containers containing PBS instead of α-MEM, left standing for 7 days, and then transported for 1 hour under room temperature conditions.

[0067] For each cell sheet in the transport container, cell number, cell viability, ALP activity, and ALP-positive cell percentage were measured. ALP activity was measured using the following method: A portion of the cell sheet was collected from the cell sheet product using a biopsy trephine (φ8.0 mm) and placed in a 96-well plate (n=3). Subsequently, ALP activity was measured using LabAssay ALP under the same conditions as in Example 2.

[0068] Cell count and cell viability were measured using the following method. 200 μL of α-MEM was added to the remaining cell sheet recovered with a biopsy trephine, followed by 20 μL of collagenase, and the mixture was incubated at 37°C under 5% CO2 conditions. After 10 minutes, the cell sheet was removed, loosened by pipetting, and incubated for another 5 minutes. Then, 200 μL of 0.25% trypsin-EDTA was added, the cell sheet was loosened again by pipetting, and incubated for another 5 minutes. After confirming that the cell sheet was sufficiently loosened and a cell suspension had been formed, 600 μL of cell washing solution (PBS + 10% FBS) was added, and the mixture was transferred to a 1.5 mL tube and centrifuged at 500 g for 5 minutes at room temperature. After removing the supernatant, 1 mL of cell washing solution was added again to prepare a cell suspension, and 15 μL was transferred to a new 1.5 mL tube. 15 μL of trypan blue was added, and the cells were dispersed by pipetting to confirm the cell count and viability.

[0069] The ALP-positive cell rate was measured using the following method. Cells collected from cell sheets were suspended in cell washing solution as described above. The cell suspension was divided into ALP reagent and control reagent. Phycoerythrin-labeled anti-ALP antibody was added to the ALP reagent and reacted, while phycoerythrin-labeled mouse nonspecific γG1 immunoglobulin was added to the control reagent and reacted. After incubation at 4°C for 30 minutes, the cells from each reagent were washed with cell washing solution and fixed with 1% paraformaldehyde to prepare cell samples. Flow cytometry was used to measure each cell sample and calculate the ALP-positive cell rate (%).

[0070] Detailed measurement conditions for ALP activity, cell number, cell viability, and ALP-positive cell percentage were based on those described in Washio, et al., Int. J. Oral Maxillofac. Implants, vol. 29, No. 1, e117-121 (2014).

[0071] Table 1 shows the cell count, cell viability, ALP activity, and ALP-positive cell percentage for cell sheets in each transport container. Cell sheets stored in α-MEM maintained sufficient product performance even after 7 days of storage at room temperature followed by 1 hour of transport. On the other hand, cell sheets immersed in PBS instead of α-MEM showed a 0% cell viability after 7 days of storage at room temperature followed by 1 hour of transport.

[0072] [Table 1] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A cell population containing mesenchymal stem cells derived from teeth or periodontal tissue, wherein the alkaline phosphatase activity before calcification induction is 1 U or more. The alkaline phosphatase activity (ALP activity (U)) is calculated by seeding cells into a microwell plate at approximately 1 × 10⁴ cells / well (100 μL), culturing them for 2 days in α-modified Eagle's Minimum Essential Medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ environment, then changing the medium and culturing for another 5 days. After removing the medium from the microwell plate and washing, 100 μL of p-nitrophenyl phosphate (pNPP) solution at pH 9.8 is added and reacted at 37°C for 5 minutes. The resulting concentration X (millimol / L) of p-nitrophenol (pNP) is then calculated using the following formulas (I) and (II). ALP activity (unit / μL) = X (mmol / L) / 5 (min) ... (I) ALP activity (U) = ALP activity (unit / μL) × 100 (II), A group of cells.

2. The cell population according to claim 1, wherein the alkaline phosphatase activity after calcification induction is 5 U or more.

3. The cell population according to claim 1, wherein the mesenchymal stem cells are mesenchymal stem cells derived from the periodontal ligament.

4. A cell sheet comprising a cell population including mesenchymal stem cells derived from teeth or periodontal tissue, wherein the alkaline phosphatase activity of the cell population after calcification induction is 5 U or more. The alkaline phosphatase activity (ALP activity (U)) is calculated by seeding cells into a microwell plate at approximately 1 × 10⁴ cells / well (100 μL), culturing them for 2 days in α-modified Eagle's Minimum Essential Medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ environment, then changing the medium and culturing for another 5 days. After removing the medium from the microwell plate and washing, 100 μL of p-nitrophenyl phosphate (pNPP) solution at pH 9.8 is added and reacted at 37°C for 5 minutes. The resulting concentration X (millimol / L) of p-nitrophenol (pNP) is then calculated using the following formulas (I) and (II). ALP activity (unit / μL) = X (mmol / L) / 5 (min) ... (I) ALP activity (U) = ALP activity (unit / μL) × 100 (II), Cell sheet.

5. The cell sheet according to claim 4, wherein the alkaline phosphatase activity of the cell population before calcification induction is 1 U or more.

6. A method for producing a cell sheet, comprising the following steps (a) to (c): (a) A step of obtaining a cell population that has alkaline phosphatase activity and includes mesenchymal stem cells derived from teeth or periodontal tissue; (b) A step of culturing the cell population in a calcification-inducing medium and growing it on a plastic substrate; and (c) A step of collecting and washing the sheet-like cell population formed on the plastic substrate. The alkaline phosphatase activity of the cell population in step (a) is 1 U or more. The alkaline phosphatase activity (ALP activity (U)) is calculated by seeding cells into a microwell plate at approximately 1 × 10⁴ cells / well (100 μL), culturing them for 2 days in α-modified Eagle's Minimum Essential Medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ environment, then changing the medium and culturing for another 5 days. After removing the medium from the microwell plate and washing, 100 μL of p-nitrophenyl phosphate (pNPP) solution at pH 9.8 is added and reacted at 37°C for 5 minutes. The resulting concentration X (millimol / L) of p-nitrophenol (pNP) is then calculated using the following formulas (I) and (II). ALP activity (unit / μL) = X (mmol / L) / 5 (min) ... (I) ALP activity (U) = ALP activity (unit / μL) × 100 (II), method.

7. The method according to claim 6, wherein the alkaline phosphatase activity after induction of calcification of the cell population is 5 U or more.

8. A method for storing and / or transporting a cell sheet according to claim 4 or 5, or a cell sheet manufactured by the method of claim 6 or 7, wherein the cell population has high alkaline phosphatase activity, and the cell sheet is maintained in α-MEM at room temperature.

9. The method according to claim 8, wherein the cell sheet can be maintained in a usable state for 7 days or more in α-MEM at room temperature.

Images