Method for producing cell fractions containing pluripotent stem cells

Abstract

To provide a method for efficiently producing a cell fraction containing a pluripotent stem cell.SOLUTION: A method for producing a cell fraction containing a pluripotent stem cell, includes culturing a cell population containing mesenchymal cells, in a medium containing a dissolved platelet component, and separating a cell fraction containing a pluripotent stem cell from the culture.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a method for producing a cell fraction containing pluripotent stem cells. [Background technology]

[0002] Pluripotent stem cells are cells that have the ability to differentiate into various somatic cells, and their practical application as materials for regenerative medicine is progressing. Examples of pluripotent stem cells derived from living organisms include iPS cells obtained by introducing reprogramming factors into somatic cells, ES cells derived from inner cell masses, and Muse cells derived from adult mesenchymal tissue.

[0003] Muse (Multilineage-differentiating Stress Enduring) cells are SSEA-3-positive pluripotent stem cells that are found in mesenchymal tissue and mesenchymal cell fractions and can be obtained from adults without induction treatment such as gene transfer. Unlike iPS cells and ES cells, they do not exhibit neoplastic growth and, when directly transplanted into the body, bring about regenerative and repair effects on damaged areas, leading to advancements in their practical application in regenerative medicine.

[0004] Patent Document 1 discloses a method for obtaining Muse cells from mesenchymal tissue or cultured mesenchymal cells using the positive expression of SSEA-3 as an indicator.

[0005] Patent Document 2 discloses a method for producing Muse cells, which involves applying external stress stimuli to living tissue or cultured mesenchymal cells and then collecting the surviving cells. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Patent No. 5185443 [Patent Document 2] Japanese Patent Publication No. 2016-28614 [Non-patent literature]

[0007] [Non-Patent Document 1] Yasumasa Kuroda, et al. Proc Natl Acad Sci US A. 2010. May 11;107(19):8639-43. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Non-patent document 1 states that the proportion of Muse cells in cultured mesenchymal cells is extremely low, at about 1-2%. Since a large amount of Muse cells is required to implement regenerative medicine using Muse cells, the small amount of Muse cells obtainable from cultured mesenchymal cells is one factor that increases manufacturing costs. However, patent documents 1 and 2 do not show any method for increasing the amount of pluripotent stem cells, such as Muse cells, that can be obtained from cultured mesenchymal cells. The object of the present invention is to provide a method for increasing the amount of pluripotent stem cells that can be obtained from mesenchymal cells. [Means for solving the problem]

[0009] As a result of diligent research to solve the aforementioned problems, the inventors discovered that the yield of pluripotent stem cells can be increased by culturing a cell population including mesenchymal cells in a culture medium containing platelet lysates and separating the cell fraction containing pluripotent stem cells from the culture, thus completing the present invention.

[0010] In other words, the present invention relates to the following [1] to

[13] . [1] A method for producing a cell fraction containing pluripotent stem cells, comprising the steps of culturing a cell population including mesenchymal cells in a culture medium containing platelet lysate, and separating a cell fraction containing pluripotent stem cells from the culture. [2] The method for producing the mesenchymal cells according to [1], wherein the mesenchymal cells are derived from bone marrow. [3] The method for producing the platelet lysate according to [1] or [2], wherein the platelet lysate is of human origin. [4] The production method according to any one of [1] to [3], wherein the concentration of the platelet lysate in the medium is 0.1 to 20% by volume. [5] The production method according to any one of [1] to [3], wherein the concentration of the platelet lysate in the medium is 1 to 3% by volume. [6] The production method according to any one of [1] to [5], wherein the pluripotent stem cells are Muse cells. [7] The production method according to any one of [1] to [6], wherein the method for separating the cell fraction containing the pluripotent stem cells is a method using the positive expression of SSEA-3 as an index. [8] The production method according to any one of [1] to [7], wherein the pluripotent stem cell content rate of the culture is relatively 5% or more higher than that of a culture cultured in a medium containing FBS instead of the platelet lysate. [9] The production method according to any one of [1] to [7], wherein the pluripotent stem cell content rate of the culture is relatively 20% or more higher than that of a culture cultured in a medium containing FBS instead of the platelet lysate.

[10] The production method according to any one of [1] to [9], further comprising a step of further culturing the cell fraction containing the separated pluripotent stem cells.

[11] The production method according to any one of [1] to

[10] , further comprising a step of cryopreserving the cell fraction containing the separated pluripotent stem cells.

[12] The production method according to any one of [1] to

[11] , wherein the ratio of pluripotent stem cells in the culture is 2.2% or more.

[13] The production method according to any one of [1] to [1], wherein the number of pluripotent stem cells in the culture is 18.1% or more with respect to the number of cells seeded in the culturing step. [Effect of the Invention]

[0011] According to the present invention, the ratio of pluripotent stem cells in cultured mesenchymal cells can be increased, and further, the production efficiency of pluripotent stem cells can be increased. [Mode for Carrying Out the Invention]

[0012] One embodiment of the present invention is described below, but the present invention is not limited to this embodiment.

[0013] [1] Explanation of terms (Mesenchymal cells) In this specification, “mesenchymal cells” refers to somatic cells (tissue cells) that can be collected from mesenchymal tissues and organs. The mesenchymal tissues and organs are not particularly limited, but examples include bone, cartilage, fat, blood, bone marrow, dermis, skeletal muscle, ligaments, tendons, heart, dental pulp, gums, umbilical cord, umbilical cord blood, amniotic fluid, amniotic membrane, and placenta. In this specification, “mesenchymal cells” are preferably derived from fat, bone marrow, dental pulp, umbilical cord, umbilical cord blood, amniotic fluid, and amniotic membrane; more preferably derived from fat, bone marrow, dental pulp, umbilical cord, and amniotic membrane; even more preferably derived from fat, bone marrow, and amniotic membrane; and most preferably derived from bone marrow. In this specification, “mesenchymal cells” also include “mesenchymal stromal cells” and “mesenchymal stem cells (MSCs)”.

[0014] (A group of cells including mesenchymal cells) In this specification, "cell population containing mesenchymal cells" refers to an aggregate of one or more cells, including at least one mesenchymal cell. A cell population containing mesenchymal cells may consist solely of mesenchymal cells or may include other cells. Its form is not particularly limited and examples include tissue, tissue fragments, cell pellets, cell aggregates, cell sheets, cell suspensions, cell suspensions, and frozen versions thereof.

[0015] (platelet lysate) In this specification, "platelet lysate" refers to platelet-containing components obtained by lysing platelets, and culture medium additives containing the platelet-containing components. Platelet lysate is one of the body fluid extracts containing multiple types of platelet-containing components, such as growth factors (e.g., platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF2), and epidermal growth factor (EGF)) and cytokines. The platelet lysate of the present invention preferably contains multiple types of platelet-containing components. The method of lysing platelets is not particularly limited and can include, for example, chemical treatment (e.g., treatment with CaCl2), physical treatment (e.g., physical disruption), osmotic pressure (e.g., exposure to a hypotonic solution such as water), and freeze-thaw cycles. As platelet lysate, one prepared in-house from animal body fluids may be used, or commercially available platelet cultures may be used as is or processed. Examples of commercially available platelet lysates include NeoSERA (Kanto Chemical Co., Ltd.), PLTMax Human Platelet Lysate (Merck), Human Platelet Lysate (STEMCELL Technologies), and UltraGRO (AventaCell BioMedical).

[0016] The source of platelet lysates is not particularly limited and may be from the same animal species as the source of the cell population containing mesenchymal cells, or from a different animal species. Examples of sources of platelet lysates include mammals such as humans, monkeys, cattle, horses, dogs, cats, pigs, sheep, mice, rats, and rabbits. In this specification, "platelet lysates" are preferably derived from humans, monkeys, cattle, or horses, and more preferably from humans. Platelet lysates may be derived from a single individual or from multiple individuals. Furthermore, platelet lysates may be derived from the same individual as the individual that provided the cell population containing mesenchymal cells, or from a different individual. Preferably, they are derived from an individual different from the individual that provided the cell population containing mesenchymal cells.

[0017] (pluripotent stem cells) In this specification, "pluripotent stem cells" refers to cells that express pluripotent stem cell markers and / or cells that have the ability to differentiate into three germ layers (endoderm, mesoderm, and ectoderm). Here, pluripotent stem cell markers refer to molecules that are expressed in pluripotent stem cells and whose expression is used as an indicator of pluripotent stem cells. Examples include, but are not limited to, Nanog, Oct3 / 4, Sox2, Klf4, SSEA-1, SSEA-3, SSEA-4, Lin28, and TRA-1-60. Pluripotent stem cell markers may be used alone or in combination of two or more markers.

[0018] Pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), pluripotent stem cells derived from fetal primordial germ cells (EG cells: Proc Natl Acad Sci US A.1998,95:13726-31), pluripotent stem cells derived from testes (GS cells: Nature.2008,456:344-9), induced pluripotent stem cells (iPS cells), Muse (Multilineage-differentiating Stress Enduring) cells, and human somatic stem cells (tissue stem cells). The pluripotent stem cells used in this invention are preferably Muse cells.

[0019] (Cell fraction containing pluripotent stem cells) In this specification, "cell fraction containing pluripotent stem cells" refers to a group of cells containing a certain amount or more of pluripotent stem cells. In the present invention, the cell fraction is preferably a group of mesenchymal cells containing pluripotent stem cells, but may also contain cells other than pluripotent stem cells and mesenchymal cells. The proportion of pluripotent stem cells in the cell fraction containing pluripotent stem cells may be, for example, 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more. Its form is not particularly limited and includes, for example, a group of cells obtained by selecting pluripotent stem cells from a culture, a group of cells obtained by removing non-pluripotent stem cell cells from a culture, and a group of cells obtained by culturing pluripotent stem cells contained in a culture. In this specification, the cell fraction containing pluripotent stem cells is preferably a group of cells obtained by selecting pluripotent stem cells from a culture, and more preferably a group of cells obtained by selecting pluripotent stem cells from a culture using the expression of a pluripotent stem cell marker, such as SSEA-3, as an indicator.

[0020] [2] Method for producing a cell fraction containing pluripotent stem cells The present invention provides a method for producing a cell fraction containing pluripotent stem cells, which involves culturing a cell population including mesenchymal cells in a culture medium containing platelet lysate, and then separating the cell fraction containing pluripotent stem cells from the culture.

[0021] The cell population containing mesenchymal cells in the present invention is not particularly limited as long as it contains at least mesenchymal cells, and may also contain other cells. Although not particularly limited, the proportion of mesenchymal cells in the cell population containing mesenchymal cells is, for example, 0.1% or more, 1% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, and 95% or more.

[0022] The cell population containing mesenchymal cells in the present invention may have undergone any treatment before being subjected to this method. For example, the cell population containing mesenchymal cells in the present invention may or may not have undergone pre-culture treatment or freezing treatment. Specifically, it may be tissue or cells collected from an individual, or frozen products thereof, or a culture of tissue or cells collected from an individual, or frozen products thereof.

[0023] This invention includes a step of culturing a cell population containing mesenchymal cells. In this invention, it is important to use a culture medium containing platelet lysate for culturing the cell population containing mesenchymal cells. By using a culture medium containing platelet lysate for culturing the cell population containing mesenchymal cells, the proportion of pluripotent stem cells in the culture can be increased compared to when using a culture medium that does not contain platelet lysate, for example, a culture medium containing FBS instead of platelet lysate. This leads to improved efficiency in pluripotent stem cell production, reduced production costs, and a reduced risk of non-pluripotent stem cell contamination in the final product. Furthermore, by using a culture medium containing platelet lysate for culturing the cell population containing mesenchymal cells, more cultured cells can be obtained than when using a culture medium containing FBS, and ultimately more pluripotent stem cells can be produced.

[0024] The culture medium is not particularly limited as long as it contains at least platelet lysate, and can be prepared by using any liquid culture medium for animal cells as a base medium and adding platelet lysate. Other components may also be added as needed. Examples of these other components include, but are not limited to, albumin, serum, serum substitute reagents, cytokines, and antibiotics. Furthermore, two or more other components may be added in combination.

[0025] The above-mentioned basal media can be, but are not limited to, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham F10 medium, Ham F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media of these (e.g., DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)). Various commercially available serum-free media can also be used.

[0026] The platelet lysate concentration in a culture medium containing platelet lysates is not particularly limited, but is for example 0.1 to 20 volume percent. Specifically, it can be, for example, 20 volume percent or less, 19 volume percent or less, 16 volume percent or less, 14 volume percent or less, 12 volume percent or less, 11 volume percent or less, 10 volume percent or less, 9 volume percent or less, 7 volume percent or less, 6 volume percent or less, 4 volume percent or less, or 3 volume percent or less. From the viewpoint of increasing the proportion of pluripotent stem cells in the culture, it is preferably 15 volume percent or less, more preferably 10 volume percent or less, even more preferably 8 volume percent or less, even more preferably 5 volume percent or less, and most preferably 3 volume percent or less. The lower limit in this case is also not particularly limited, but is for example 0.1 volume percent or more, 0.2 volume percent or more, 0.3 volume percent or more, 0.5 volume percent or more, 0.7 volume percent or more, 1 volume percent or more, 1.2 volume percent or more, 1.5 volume percent or more, 1.8 volume percent or more, 2 volume percent or more, or 2.5 volume percent or more. From the viewpoint of increasing the amount of pluripotent stem cells ultimately obtained from a cell population containing a certain amount of mesenchymal cells, the concentration is preferably 2% by volume or more, more preferably 5% by volume or more, and most preferably 8% by volume or more. The upper limit in this case is not particularly limited, but for example, it is 15% by volume or less, or 12% by volume or less. The platelet lysate concentration shall be converted to the content of commercially available platelet lysates (e.g., NeoSERA (Kanto Chemical Co., Ltd.), PLTMax Human Platelet Lysate (Merck), Human Platelet Lysate (STEMCELL Technologies), UltraGRO (AventaCell BioMedical), etc.). The conversion method is not particularly limited, but for example, it can be converted based on the proliferation rate of mesenchymal stem cells, based on physical properties such as turbidity, absorbance, dry weight, density or viscosity, based on the concentration of a specific component (e.g., PDGF), or based on the equivalent blood volume.Alternatively, it can be converted according to the protein concentration (e.g., median) per 1 mL. For example, the median protein concentration per 1 mL can be converted to platelet lysates of 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1 mg / mL, 1.25 mg / mL, 1.5 mg / mL, 1.75 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 10 mg / mL.

[0027] The method for culturing a cell population containing mesenchymal cells is not particularly limited. For example, it may be adherent culture or suspension culture. In the culture of a cell population containing mesenchymal cells in this specification, adherent culture is preferably used.

[0028] The seeding density in the step of culturing a cell population containing mesenchymal cells is not particularly limited. For example, it can be seeded at a density of 500 - 100,000 cells / cm 2 . As the lower limit of the seeding density, for example, 500 cells / cm 2 or more, 1,000 cells / cm 2 or more, 2,000 cells / cm 2 or more, 3,000 cells / cm 2 or more, 4,000 cells / cm 2 or more, 5,000 cells / cm 2 or more is preferable. Also, as the upper limit of the seeding density, although not particularly limited, from the perspective of the efficiency of cell culture, it is common to use 100,000 cells / cm 2 or less. In addition, for example, 50,000 cells / cm 2 or less, 30,000 cells / cm 2 or less, 20,000 cells / cm 2 or less, 15,000 cells / cm 2 or less can be used.

[0029] The above culture process may include a subculturing process, or it may include a process of repeating the culture multiple times under different culture conditions.

[0030] The culture conditions, such as temperature, time, and CO2 concentration during cultivation, are not particularly limited. For example, the culture temperature may be 20°C or higher, 35°C or higher, with upper limits of 40°C or lower, 37°C or lower. For example, the CO2 concentration during cultivation may be 2% or higher, 2.5% or higher, 3% or higher, 4% or higher, 4.5% or higher, with upper limits of 7% or lower, 6% or lower, 5.5% or lower, 5% or lower.

[0031] The incubation period for one culture as described above is not particularly limited, but for example, it can be 2 to 14 days, and more specifically, it can be 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0032] The culture period described above may include a step of changing the culture medium. The frequency of changing the culture medium can be, for example, every 1 to 7 days, preferably every 1 day, every 2 days, every 3 days, every 4 days, or every 5 days.

[0033] Cell populations including mesenchymal cells can be cultured using, for example, the following procedure: First, the cell suspension is centrifuged, the supernatant is removed, and the resulting cell pellet is suspended in culture medium. Next, the cells are seeded in a culture vessel and cultured using culture medium at a CO2 concentration of 3% to 5% and a temperature of 37°C. Cells obtained by the above culture method are cells that have been cultured once.

[0034] As described above, even after culturing once, cells can be further subcultured. Further subculture can be carried out, for example, by the following steps. First, cells that have been cultured until the confluence rate reaches 95% or less in the first culture are detached from the culture vessel using a cell detachment means. Next, the obtained cell suspension is centrifuged, the supernatant is removed, and the obtained cell pellet is suspended in culture medium. Finally, the cells are seeded in a culture vessel and cultured using culture medium at a CO2 concentration of 3% or more and 5% or less, and at 37°C. By performing similar subculture and cultivation, cells that have been subcultured n times can be obtained (n is an integer of 1 or more). From the viewpoint of mass production of cells, the lower limit of the number of subcultures n is, for example, 1 or more, preferably 2 or more, more preferably 3 or more, even more preferably 4 or more, and even more preferably 5 or more. Furthermore, from the viewpoint of suppressing cell senescence, the upper limit of the number of subcultures n is preferably, for example, 25 or less, 20 or less, 15 or less, and 10 or less.

[0035] The detachment method used when performing detachment as described above is not particularly limited. For example, a cell detachment agent may be used as the cell detachment method. The cell detachment agent is not particularly limited as long as it has the effect of detaching adherent cells from the culture vessel, and examples include trypsin, collagenase, dispase, and ethylenediaminetetraacetic acid (EDTA). Commercially available cell detachment agents may be used as the cell detachment agent. Examples include, but are not limited to, trypsin-EDTA solution (Thermo Fisher Scientific), TrypLE Select (Thermo Fisher Scientific), Accutase (Stemcell Technologies), and Accumax (Stemcell Technologies). Furthermore, a physical cell detachment method may be used as the cell detachment method, for example, a cell scraper (Corning) may be used, but is not limited to this. The cell detachment methods may be used individually or in combination.

[0036] The form of the culture obtained after culturing a cell population including mesenchymal cells in a medium containing platelet lysate is not particularly limited and can be, for example, a cell pellet, cell aggregates, cell sheets, cell suspension, or cell suspension.

[0037] The pluripotent stem cell content of a culture obtained after culturing a cell population including mesenchymal cells in a medium containing platelet lysate is not particularly limited, but for example, it may be 1% or more, 2% or more, 2.2% or more, 2.25% or more, 2.3% or more, 2.4% or more, 2.5% or more, 2.6% or more, 2.7% or more, 2.8% or more, 2.9% or more, 3.0% or more, 3.5% or more, 4.0% or more, or 4.05% or more.

[0038] The pluripotent stem cell content of a culture obtained by culturing a cell population including mesenchymal cells in a medium containing platelet lysate is preferably 2.5% or more, 3% or more, 4% or more, 5% or more, 7.5% or more, 10% or more, and 15% or more higher than a culture obtained by culturing in a medium without platelet lysate, for example, in a medium containing FBS instead of platelet lysate. It is more preferably 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, and 25% or more higher. It is even more preferably 50% or more, 60% or more, 70% or more, and 80% or more higher. By increasing the pluripotent stem cell content of the culture after culturing, it is possible to improve the efficiency of pluripotent stem cell production, reduce production costs, and reduce the risk of non-pluripotent stem cell contamination in the final product, as described above.

[0039] The efficiency of pluripotent stem cell production, i.e., the number of pluripotent stem cells in the culture (i.e., the number of pluripotent stem cells after culture) relative to the number of cells in the cell population including seeded mesenchymal stem cells (i.e., the total number of cells before culture), is not particularly limited, but is, for example, 18.0% or more, 18.1% or more, 18.5% or more, 19% or more, 20% or more, 25% or more, 27.5% or more, 28% or more, 28.2% or more, 28.5% or more, 29% or more, 30% or more, 30.5% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, or 37% or more.

[0040] The efficiency of producing pluripotent stem cells when a cell population including mesenchymal cells is cultured in a medium containing platelet lysate is not particularly limited, but it should be increased compared to when cultured in a medium without platelet lysate, for example, a medium containing FBS instead of platelet lysate. Specifically, it should be increased by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 66% or more, 67% or more, 68% or more, or 69% or more compared to when cultured in a medium without platelet lysate, for example, a medium containing FBS instead of platelet lysate.

[0041] The present invention includes a step of separating a cell fraction containing pluripotent stem cells from the culture obtained by the above-described culture step. This step is performed simultaneously with or after the above-described culture step.

[0042] The method for separating the cell fraction containing pluripotent stem cells in this process is not particularly limited as long as it is a method that can separate the cell fraction containing pluripotent stem cells from the culture. Examples of such methods include, but are not particularly limited, a method of selecting the cell fraction expressing a pluripotent stem cell marker in the culture using a cell sorter, a method of staining the pluripotent stem cell marker in the culture with magnetic beads and magnetically separating the cell fraction expressing the pluripotent stem cell marker, a method of applying stress stimuli to the culture to induce cell death of cells other than pluripotent stem cells that are not stress-tolerant or have low stress tolerance, and obtaining a cell fraction containing stress-tolerant pluripotent stem cells, and a method of suspension culture to obtain cell aggregates formed by the proliferation of pluripotent stem cells.

[0043] Examples of the above-mentioned pluripotent stem cell markers include Nanog, Oct3 / 4, Sox2, Klf4, SSEA-1, SSEA-3, SSEA-4, Lin28, and TRA-1-60. Alternatively, two or more pluripotent stem cell markers may be used in combination.

[0044] Examples of the stress stimuli mentioned above include protease treatment (trypsin, pepsin, thermolysin, papain, etc.), heat treatment, hypoxic treatment, cultivation under starvation conditions, exposure to reactive oxygen species, pressure treatment, and mechanical stimulation.

[0045] The method for separating cell fractions containing pluripotent stem cells in the present invention is preferably a method of selecting cell fractions expressing pluripotent stem cell markers in a culture using a cell sorter, and more preferably a method of selecting cell fractions expressing SSEA-3 in a culture using a cell sorter.

[0046] The separation process for cell fractions containing pluripotent stem cells can be carried out, for example, by the following steps: First, the culture obtained by culturing a cell population containing mesenchymal cells in the aforementioned culture step is treated with a cell detachment means to detach it from the culture vessel. The pluripotent stem cell marker in the culture is stained, and the cell fraction containing pluripotent stem cells positive for the pluripotent stem cell marker is selected using a cell sorter.

[0047] The confluence rate of a culture of a cell population containing mesenchymal cells is not particularly limited, but from the viewpoint of obtaining more pluripotent stem cells, it is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more. Other examples include 1% or more, 10% or more, 30% or more, and 100%.

[0048] The detachment method used when performing detachment as described above is not particularly limited. For example, a cell detachment agent may be used as the cell detachment method. The cell detachment agent is not particularly limited as long as it has the effect of detaching adherent cells from the culture vessel, and examples include trypsin, collagenase, dispase, and ethylenediaminetetraacetic acid (EDTA). Commercially available cell detachment agents may be used as the cell detachment agent. Examples include, but are not limited to, trypsin-EDTA solution (Thermo Fisher Scientific), TrypLE Select (Thermo Fisher Scientific), Accutase (Stemcell Technologies), and Accumax (Stemcell Technologies). Furthermore, a physical cell detachment method may be used as the cell detachment method, for example, a cell scraper (Corning) may be used, but is not limited to this. The cell detachment methods may be used individually or in combination.

[0049] Staining of pluripotent stem cell markers may be performed by immunocytochemistry using antibodies, by non-immunocytochemistry using antibodies, or by a combination of immunocytochemistry and non-immunocytochemistry. The immunocytochemistry method is not particularly limited and may be monoimmunostaining using one type of antibody or by multiple immunostaining using two or more types of antibodies. It may also be a direct method using a labeled primary antibody or an indirect method using a primary antibody and a labeled secondary antibody. Any antibody known in the art can be used as the labeled antibody, and examples include, but are not limited to, antibodies labeled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), etc.

[0050] In the step of separating the cell fraction containing pluripotent stem cells as described above, the proportion of pluripotent stem cells in the separated cell fraction is not particularly limited, as long as the proportion of pluripotent stem cells is higher than that of the culture before separation. For example, this could be 10% or more, 20% or more, preferably 50% or more, more preferably 70% or more, even more preferably 80% or more, and most preferably 90% or more.

[0051] The present invention may further include a step of culturing the cell fraction containing pluripotent stem cells obtained through the above separation step. This step is carried out simultaneously with or after the above separation step. By further culturing the cell fraction containing the separated pluripotent stem cells, the amount of pluripotent stem cells that can be obtained can be increased. The culture of the cell fraction containing pluripotent stem cells may be adherent culture or suspension culture. The culture medium used is not particularly limited and can be prepared by using any liquid medium for animal cell culture as a base medium and adding other components as appropriate as needed. Examples of the other components include, but are not particularly limited to, albumin, serum, platelet lysate, serum substitute reagents, cytokines, and antibiotics. In addition, two or more other components may be added in combination.

[0052] The above-mentioned basal media can be, but are not limited to, BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium (Iscove's Modified Dulbecco's Medium), Medium 199 medium, Eagle MEM medium, αMEM (Alpha Modification of Minimum Essential Medium Eagle) medium, DMEM medium (Dulbecco's Modified Eagle's Medium), Ham F10 medium, Ham F12 medium, RPMI 1640 medium, Fischer's medium, and mixed media of these (for example, DMEM / F12 medium (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 Ham)). Various commercially available serum-free media can also be used.

[0053] The seeding density in the culture of cell fractions containing pluripotent stem cells is not particularly limited, but for example, 1 to 100,000 cells / cm³. 2 They can be seeded at this density. Furthermore, if the pluripotent stem cells have self-renewal capabilities, they can also be seeded and cultured as single cells.

[0054] Furthermore, the culture conditions for culturing cell fractions including pluripotent stem cells are not particularly limited, but for example, the conditions exemplified in the above culture process can be used.

[0055] The present invention may further include a step of cryopreserving the cell fraction containing pluripotent stem cells obtained through the above separation step. This step is carried out after the culture step, separation step, or cell fraction culture step. Furthermore, for example, when carrying out the separation step or the cell fraction culture step, this step can be carried out before and / or after. The means of cryopreservation are not particularly limited, but examples include a programmable freezer, a deep freezer, or cryopreservation in liquid nitrogen. Not limited to the use of a programmable freezer as the cryopreservation means, the freezing temperature is preferably -30°C or lower, -40°C or lower, -50°C or lower, -80°C or lower, -90°C or lower, -100°C or lower, -150°C or lower, -180°C or lower, or -196°C (liquid nitrogen temperature) or lower. When freezing, and not limited to cases using a programmable freezer, preferred freezing rates are, for example, 15°C / min or less, 11°C / min or less, 10°C / min or less, 9°C / min or less, 5°C / min or less, 2°C / min or less, or 1°C / min or less. When a programmable freezer is used as the above-mentioned freezing and storage method, for example, the temperature can be lowered to a temperature between -50°C and -30°C (e.g., -40°C) at a freezing rate of 1°C / min to 2°C / min or less, and then further lowered to a temperature between -100°C and -80°C (e.g., -90°C) at a freezing rate of 9°C / min to 11°C / min (e.g., 10°C / min). Furthermore, when liquid nitrogen is used as the above-mentioned freezing method, for example, the temperature can be rapidly lowered to -196°C to freeze, and then stored in liquid nitrogen (gas phase). Alternatively, it can be stored in liquid nitrogen (liquid phase).

[0056] When freezing using the freezing method described above, the cell population may be frozen in any storage container. Examples of storage containers include, but are not limited to, cryotubes, cryovials, freezing bags, and infusion bags.

[0057] When freezing by the freezing method described above, the cell population may be frozen in any cryopreservation solution. Commercially available cryopreservation solutions may be used as the cryopreservation solution. Examples include, but are not limited to, BAMBANKER (Lymphotec), STEM-CELLBANKER (Nippon Zenyaku Kogyo Co., Ltd.), CP-1 (Kyokuto Seiyaku Kogyo Co., Ltd.), ReproCryo RM (ReproCELL, Inc.), CryoNovo (Akron Biotechnology Inc.), MSC Freezing Solution (Biological Industries Inc.), and CryoStor (HemaCare Inc.).

[0058] The above cryopreservation solution may contain a predetermined concentration of polysaccharides. Preferred concentrations of polysaccharides are, for example, 1% by mass or more, 2% by mass or more, 4% by mass or more, or 6% by mass or more. Alternatively, preferred concentrations of polysaccharides are, for example, 20% by mass or less, 18% by mass or less, 16% by mass or less, 14% by mass or less, or 13% by mass or less. Examples of polysaccharides include, but are not limited to, hydroxyethyl starch (HES) and dextran (such as Dextran 40).

[0059] The above cryopreservation solution may contain a predetermined concentration of dimethyl sulfoxide (DMSO). Preferred concentrations of DMSO are, for example, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, or 5% by mass or more. Alternatively, preferred concentrations of DMSO are, for example, 20% by mass or less, 18% by mass or less, 16% by mass or less, 14% by mass or less, 12% by mass or less, or 10% by mass or less.

[0060] The above cryopreservation solution may contain albumin at a predetermined concentration greater than 0% by mass. Preferred albumin concentrations are, for example, 1% or more by mass, 2% or more by mass, 3% or more by mass, or 4% or more by mass. Alternatively, preferred albumin concentrations are, for example, 30% or less by mass, 20% or less by mass, 10% or less by mass, or 9% or less by mass. Examples of albumin include, but are not limited to, bovine serum albumin (BSA), mouse albumin, and human albumin. [Examples]

[0061] The present invention will be specifically described in the following examples, but the present invention is not limited to these examples.

[0062] (Comparative Example 1) Bone marrow-derived MSCs (passage 1) were seeded at a density of 5,000 cells / cm². 2 Seeds were seeded in one 10cm dish (Sumitomo Bakelite) and cultured in DMEM containing 10% by volume fetal bovine serum (FBS) and FGF-2 (1 ng / mL) until 100% confluence was reached. It took 7 days to reach 100% confluence. The cultured cells that reached 100% confluence were detached from the 10cm dish using TrypLE Select (Thermo Fisher Scientific) and suspended in PBS containing 0.5% (w / v) BSA and EDTA. 2.29 × 10⁶ cells were obtained from one 10cm dish. 6 A total of 10⁶ cultured cells were collected. The cultured cells were immunostained with rat anti-human SSEA-3 antibody (BioLegend) and FITC-labeled mouse anti-rat antibody (BioLegend), and the SSEA-3 positivity rate was analyzed using a flow cytometer (Guava® easyCyte: Merck). The negative control group was immunostained with rat IgM isotype control (BioLegend) and FITC-labeled mouse anti-rat antibody (BioLegend). As a result, the SSEA-3 positivity rate of the cultured cells was 2.17%. The cultured cells were subjected to a cell sorter (e.g., BD Aria III), and SSEA-3 positive cells were separated, resulting in (2.29 × 10⁶).6 (1) × (2.17%) = 49,693 Muse cells are produced.

[0063] (Example 1) Bone marrow-derived MSCs (passage 1) were seeded at a density of 5,000 cells / cm². 2 Seeds were seeded onto one 10cm dish (Sumitomo Bakelite) and cultured in αMEM containing 2.5% by volume of human platelet lysate (hPL) (protein concentration: approximately 1.25 mg / mL) until 100% confluence was reached. It took 5 days to reach 100% confluence. The cultured cells that reached 100% confluence were detached from the 10cm dish using TrypLE Select (Thermo Fisher Scientific) and suspended in PBS containing 0.5% (w / v) BSA and EDTA. From one 10cm dish, 2.06 × 10⁶ cells were obtained. 6 A total of 100⁶ cultured cells were collected. The cultured cells were immunostained with rat anti-human SSEA-3 antibody (BioLegend) and FITC-labeled mouse anti-rat antibody (BioLegend), and the positivity rate of SSEA-3, a Muse cell marker, was analyzed using a flow cytometer (Guava® easyCyte: Merck). The negative control group was immunostained with rat IgMκ isotype control (BioLegend) and FITC-labeled mouse anti-rat antibody (BioLegend). As a result, the SSEA-3 positivity rate of the cultured cells was 4.08%. The cultured cells were subjected to a cell sorter (e.g., BD Aria III), and SSEA-3 positive cells were separated, resulting in (2.06 × 10⁶) 6 (number of cells) × (4.08%) = 84,048 Muse cells can be produced.

[0064] (Example 2) Using αMEM containing 5% by volume of human platelet lysate (hPL) (protein concentration: approximately 2.5 mg / mL) in the culture medium, bone marrow-derived MSCs (passage 1) were cultured, cultured cells were harvested, and the SSEA-3 positivity rate was analyzed, similar to Example 1. The number of harvested cultured cells was 2.94 × 10⁶. 6The SSEA-3 positivity rate was 2.64%. By subjecting cultured cells to a cell sorter (e.g., BD Aria III) and separating SSEA-3 positive cells, (2.94 × 10⁻⁶) 6 (number of cells) × (2.64%) = 77,616 Muse cells can be produced.

[0065] (Example 3) Using αMEM containing 10% by volume of human platelet lysate (hPL) (protein concentration: approximately 5 mg / mL) in the culture medium, bone marrow-derived MSCs (passage 1) were cultured, cultured cells were harvested, and the SSEA-3 positivity rate was analyzed, similar to Example 1. The number of harvested cultured cells was 4.47 × 10⁶. 6 The SSEA-3 positivity rate was 2.28%. By subjecting cultured cells to a cell sorter (e.g., BD Aria III) and separating SSEA-3 positive cells, (4.47 × 10⁻⁶ cells) were obtained. 6 (number of cells) × (2.28%) = 101,916 Muse cells can be produced.

[0066] The results above can be summarized in Table 1.

[0067] [Table 1]

[0068] As shown in Table 1, culturing bone marrow MSCs using the methods described in Examples 1 to 3 increased the proportion of Muse cells in the cultured cells compared to culturing using the method described in Comparative Example 1. Furthermore, it was found that separating Muse cells from cultured cells cultivated using the methods described in Examples 1 to 3 allowed for the production of more Muse cells than when cultured using the method described in Comparative Example 1.

Claims

1. The process includes the steps of culturing a cell population containing mesenchymal cells in a culture medium containing platelet lysate, and separating a cell fraction containing Muse cells from the culture. The aforementioned mesenchymal cells are derived from bone marrow, dermis, fat, blood, dental pulp, umbilical cord, umbilical cord blood, or amniotic membrane. In the separation step described above, the proportion of Muse cells in the cell fraction containing Muse cells after separation is 10% or more. A method for producing a cell fraction containing Muse cells.

2. The method for producing the product according to claim 1, wherein the mesenchymal cells are derived from bone marrow.

3. The manufacturing method according to claim 1 or 2, wherein the platelet lysate is of human origin.

4. The manufacturing method according to any one of claims 1 to 3, wherein the concentration of the platelet lysate in the culture medium is 0.1 to 20% by volume.

5. The manufacturing method according to any one of claims 1 to 3, wherein the concentration of the platelet lysate in the culture medium is 1 to 3% by volume.

6. The manufacturing method according to any one of claims 1 to 5, wherein the method for separating the cell fraction containing Muse cells is a method that uses the positive expression of SSEA-3 as an indicator.

7. The manufacturing method according to any one of claims 1 to 6, wherein the proportion of Muse cells in the culture is 2.2% or more.

8. The manufacturing method according to any one of claims 1 to 7, wherein the number of Muse cells in the culture is 18.1% or more of the number of cells seeded in the culture step.

9. The method for producing the culture according to any one of claims 1 to 8, wherein the Muse cell content of the culture is 5% or more higher than that of a culture cultured in a medium containing FBS instead of platelet lysate.

10. The manufacturing method according to claim 9, wherein the Muse cell content of the culture is 20% or more higher than that of a culture cultured in a medium containing FBS instead of platelet lysate.

11. The manufacturing method according to any one of claims 1 to 10, further comprising the step of further culturing the cell fraction containing the separated Muse cells.

12. The manufacturing method according to any one of claims 1 to 11, further comprising the step of cryopreserving the cell fraction containing the separated Muse cells.

13. A cell population including bone marrow-derived mesenchymal cells is cultured in a culture medium containing platelet lysate, and a cell fraction including pluripotent stem cells is separated from the culture, The isolation is performed using the positive expression of SSEA-3 as an indicator. A method for producing a cell fraction containing Muse cells.

14. A cell population including bone marrow-derived mesenchymal cells is cultured in a medium containing platelet lysate, and a cell fraction including Muse cells is separated from the culture, The Muse cell content of the culture is 5% or more higher than that of a culture cultured in a medium containing FBS instead of platelet lysate. A method for producing a cell fraction containing Muse cells.

15. A cell population including mesenchymal cells is cultured in a culture medium containing platelet lysate, and a cell fraction including Muse cells is separated from the culture, The aforementioned mesenchymal cells are derived from bone marrow, dermis, fat, blood, dental pulp, umbilical cord, umbilical cord blood, or amniotic membrane. In the aforementioned culturing step, the number of Muse cells in the culture is 20% or more of the number of cells seeded. A method for producing a cell fraction containing Muse cells.

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