JP7198524B2 - Method for producing spheroids and method for expressing pluripotent stem cell markers - Google Patents

Description

The present invention relates to a method for producing spheroids and a method for expressing pluripotent stem cell markers .

In recent years, in regenerative medicine, clinical applications of techniques for repairing tissues and organs by transplantation of stem cells have been investigated in various organs. For example, there is a method for preparing pluripotent stem cells that are highly capable of differentiating into cardiomyocytes (Patent Document 1). Heart tissue-derived myocardial stem cells, bone marrow-derived hematopoietic stem cells, and mesenchymal stem cells are known as stem cells, but if the degree of differentiation into cardiomyocytes is low, they cannot be used for clinical applications such as cardiac tissue regeneration. Patent document 1 prepares a cell suspension derived from heart tissue and separates a cell group containing stem cells by a density gradient method. The isolated cell group was subjected to suspension culture in a medium containing fibroblast growth factor and epidermal growth factor at a cell concentration of 1×10 ⁴ to 2×10 ⁴ cells/ml at the start of culture, and a suspension sphere was obtained. are selected and separated to obtain pluripotent stem cells. These pluripotent stem cells are said to have the ability to differentiate into cardiomyocytes and smooth muscle cells. The floating spheres are formed by repeated cell division of pluripotent stem cells (Patent Document 1, paragraph “0034”). Since stem cells proliferated from a single cell are selectively separated, the stem cells themselves are highly uniform and clinically useful (Patent Document 1, paragraph "0015").

There is also a method for efficiently inducing the differentiation of stem cells into cerebral cortex progenitor cells (Patent Document 2). A stem cell differentiation culture method including a step of forming uniform stem cell aggregates in a serum-free medium, and growth of Nodal signal promoter, Wnt signal promoter, FGF signal promoter, BMP signal promoter, retinoic acid, etc. Differentiation is induced by culturing pluripotent stem cells as floating aggregates in a serum-free medium that does not substantially contain factors and insulins, and hypothalamic neuron progenitor cells can be isolated from the culture (Patent Document 2, paragraph “0020”). Since there are many types of nerve cells in the brain, it is possible to accurately and efficiently differentiate in vitro disease-related nerve cells for the purpose of cure. In Examples, mouse ES cells were used for differentiation culture, and it was observed that about 70% of the cells in aggregates 10 days after the start of culture expressed the cerebrum-specific marker Bf1.

Furthermore, there is also a method of selecting or growing pluripotent stem cells derived from human skin fibroblasts (Patent Document 3). Characteristic cell masses are formed from the untreated human bone marrow mesenchymal cell fraction at an extremely low frequency, and stress stimulation activates tissue stem cells in a sleepy state (Patent Document 3, paragraph " 0024"), mesenchymal cells such as bone marrow mesenchymal cell fractions and skin fibroblast fractions, or mesenchymal cells are cultured and subjected to stress stimulation by various methods, and surviving cells is to collect. By isolating stem cells using SSEA-3 expression as an index and culturing them in suspension culture in a methylcellulose-containing medium, embryoid body-like cell clusters with a maximum diameter of 150 μm can be obtained. This stem cell is a pluripotent stem cell characterized by SSEA-3 positive and CD105 positive, self-renewal ability, etc., and is named Muse cell. Muse cells are present in mesodermal tissue, mesenchymal tissue, or the like in vivo. Cells or cell fractions present in these tissues are separated (Patent Document 3, paragraph “0040”), non-stem cells are removed by stress stimulation, and remaining stem cells are enriched (Patent Document 3, paragraph "0167"). Muse cells differ from iPS cells and ES cells in that they can be obtained directly from living organisms or tissues. It also has the feature of being obtained without requiring reprogramming or induction of dedifferentiation (Patent Document 3, paragraph “0036”). In the examples, human dermal fibroblast fractions were used as mesenchymal cells, incubated for 16 hours in trypsin (0.25% trypsin-HBSS) as a stress stimulus, and viable cells were collected to contain methylcellulose. It is cultured in a medium at a density of 8,000 cells/ml for 7 days (Patent Document 3, paragraph "0138"). The cell population formed from the human fibroblast fraction that has been subjected to the above-mentioned incubation treatment with trypsin for 16 hours is referred to as a "rich Muse cell fraction", and single cells obtained from the population are suspended and cultured to form a rich Muse cell fraction. Approximately 9 to 10% of Muse cells are contained in the Muse cell-rich fraction (Patent Document 3, paragraph "0139").

While the above Patent Documents 1 to 3 are techniques for selecting and proliferating stem cells contained in biological tissues, there is also a technique for generating pluripotent cells by reprogramming differentiated cells (Patent Document 4). . Subjecting somatic cells to a stress such as low pH exposure is said to form pluripotent cells without the introduction of exogenous genes, transcripts, proteins, nuclear components or cytoplasm, exogenous reprogramming factors, or without cell fusion. In the examples, CD45-positive lymphocytes derived from Oct4-GFP mice were used, and when exposed to a low pH solution, spherical colonies were observed within 3 days of exposure, and markers of embryonic stem cells were expressed (Patent Document 4, paragraph "0149"). In addition, when mature cells evaluated effective stress for changing to Oct4-expressing cells, exposure to an acidic solution of pH 5.4 to 5.6 was found to be efficient (Patent Document 4, paragraph "0155"). ). Collected from the spleen of Oct4-GFP (GOF) mice, exposing CD45-positive cells to low pH, sorting GFP-expressing cells and culturing them in B27-LIF medium, GFP-expressing spherical colonies were observed within 5 days, It is said that it grew to a diameter of 70 μm over 7 days. These cultured cells were dissociated and cultured, and phenotypic changes in CD45-positive cells were observed at the single-cell level. CD45-negative cells or CD45-negative/Oct4-positive cells were shown, and CD45 expression disappeared on day 7, and Oct4-expressing cells were observed (Patent Document 4, paragraph "0158"). To confirm reprogramming, day 7 cells were assessed for the appearance of early embryogenesis markers, demonstrating that pluripotent cell markers such as Oct4, Nanog, and Sox2 were expressed and reprogrammed by external stimuli. (Patent Document 4, paragraph “0159”).

In addition, there are introduced proteins that render cells into a pluripotent stem cell-like state (Non-Patent Document 1). It is called reprotin, and fibroblasts undergo epithelial-mesenchymal transition in a short period of time to form spheroids, and reprogramming and reprogramming are said to be markedly enhanced. When fibroblasts are reprogrammed using Reprotin, pluripotent stem cell colony-like spheroids can be produced in a process of about 1 to 3 days with an efficiency of 10% or more. The spheroids were strongly positive in alkaline phosphatase staining, and the pluripotent markers SSEA-3 and TRA-1-81 were also positive at the same time. However, the structure of Reprotin is unknown. When colonies of the prepared pluripotent-like stem cells are cultured on gelatin, the neural stem cell marker Nestin becomes positive, making it possible to easily prepare pluripotent stem cells and neural stem cells from fibroblasts.

Japanese Patent No. 4783909 JP 2016-5465 A Japanese Patent No. 5185443 Japanese translation of PCT publication No. 2015-516812

Reprotech (gene-free pluripotent stem cell production technology) and neural stem cell markers, Nakanobu Hayashi, GeneTry, Inc, Reprotech homepage, http://reprotech.jimdo.com/reprotech%E3%81%A8%E3%81%AF /, retrieved on August 29, 2016

However, Patent Document 1 described above is a method for selectively separating pluripotent stem cells having excellent differentiation potential into cardiomyocytes and the like from cardiac tissue. The obtained cells are limited to pluripotent stem cells that have excellent cardiomyocyte differentiation potential. In addition, a cell suspension derived from cardiac tissue is used as a raw material, and a cell population containing the target stem cells is separated by a density gradient method. A step of culturing and selectively isolating floating pluripotent stem cell spheres from the cell group contained in the culture is required, and the operation is complicated. On the other hand, in the method described in Patent Document 2, by aggregating stem cells in a serum-free medium, a qualitatively uniform stem cell group is formed, the aggregated stem cells are cultured in suspension in a serum-free medium, and cerebral cortical nerves are formed. It forms a network in vitro. This method is not a technique for creating stem cells, but a method for tuning existing stem cell populations to the same properties. Both Patent Document 1 and Patent Document 2 are for culturing stem cells that already exist in small amounts, and are not techniques for preparing pluripotent stem cells from differentiated mature cells.

The Muse cells described in Patent Document 3 are also obtained by culturing pre-existing stem cells in the same manner as in Patent Document 1 and the like. Extremely rare pluripotent stem cells contained in human dermal fibroblasts are sorted and proliferated using the SSEA-3 marker as an indicator. It is excellent in that easily available human dermal fibroblasts can be used as raw materials, but most of the cells are killed by stress overload, and Muse cells are selected from the surviving cells. is extremely low. In addition, expensive equipment must be used to separate the target cells, and the operation is complicated due to the use of special techniques. Furthermore, Patent Document 3 describes that Muse cells are proliferated by a series of cycles of Muse cell-derived embryoid body cell clusters and clonal proliferation, whereby a large amount of Muse cells can be obtained from a mesenchymal cell population. (Patent Document 3, paragraph “0142”). However, the size of the cell aggregates is about 150 μm in maximum diameter (Patent Document 3, paragraph “0140”). According to Patent Document 3, Muse cells have multiple security systems to prevent explosive proliferation and can avoid abnormal proliferation (Patent Document 3, paragraph “0167”). If a large amount of spheroids can be prepared, they can be used for stem cell research, regenerative medicine, treatment, etc. without performing a proliferation operation, and have excellent versatility.

On the other hand, Patent Document 4 generates pluripotent cells from differentiated cells by exposure to a low pH solution. However, when CD45-positive cells collected from the spleens of Oct4-GFP (GOF) mice were exposed to an acidic solution of pH 5.4-5.6, and GFP-expressing cells were sorted and cultured in B27-LIF medium, the first It is said that the number of dead cells increases after 7 days of culture. Patent Document 4 states that the characteristics of cells were gradually changed by stress treatment and specific culture, and successfully changed cells expressing Oct4 were selected (Patent Document 4, paragraph “0158”), but the cells were allowed to remain for 7 days. Only cell clumps with a diameter of 70 μm are obtained by growth over a period of time. Therefore, there is a demand for the development of a method for producing spheroids composed of large-sized pluripotent stem cells and a production technique for converting all the somatic cells used into stem cells.

In addition, Non-Patent Document 1 prepares pluripotent-like stem cells by culturing using an introduced protein called Reprotin. Although gene transfer operation is not required, the structure of Reprotin is not clear, and safety and the like are insufficient for use in regenerative medicine, stem cell therapy, and the like. Therefore, it is desired to develop a method for preparing pluripotent stem cells without adding extracellular compounds.

In view of the above situation, an object of the present invention is to provide a simple method for producing pluripotent stem cell-like spheroids expressing pluripotent stem cell markers from differentiated somatic cells.

Another object of the present invention is to provide polyclonal pluripotent stem cell-like spheroids with a diameter of 0.5 to 3 mm.

The present inventors prepared a cell suspension containing spherical fibroblasts that were individually separated and suspended by enzymatically treating differentiated fibroblasts, and placed the spherical cells in a non-adhesive cell culture vessel. When the cells are statically cultured at high density or in close contact with each other, the cells begin to aggregate, and the aggregates increase in size with the passage of time to form spheroids, and the cells that make up the spheroids are Oct3 /4, SSEA-3 and other proteins characteristic of pluripotent stem cells were found to be expressed, thereby completing the present invention. These pluripotent stem cells do not require introduction of artificial compounds, vectors/genes, exposure to strong stress environments (low pH, culture temperature lower or higher than 37 degrees, hypoxic conditions, etc.), hormones, proliferation Spheroids exhibiting properties of pluripotent stem cells can be produced from mature somatic cells without the use of stimulating factors such as factors, pluripotency-inducing proteins, or other pluripotent cell-inducing factors. The inventor named this pluripotent stem cell as natural stem cell.

That is, the present invention treats differentiated fibroblasts with serine protease to individually separate the fibroblasts from 1×10 ⁶ /ml to 2×10 ⁷ /ml, or 1×10 ⁵ /cm ² to 7×10 ⁶ cells/cm ² , stimulation with one or more selected from the group consisting of hormones, growth factors, pluripotency-inducing proteins, and pluripotent cell-inducing factors Static culture at 30 to 40°C without using factors, in D-MEM medium containing glucose and fetal bovine serum or equivalent medium containing glucose and fetal bovine serum in a cell non-adherent cell culture vessel, while supplying _CO2 . forming spheroids,
The spheroids are characterized by expressing pluripotent stem cell markers of OCT3/4, SOX2, NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and ALP. The present invention provides a method for producing spheroids .

The present invention also provides a method for producing the spheroids , wherein the spheroids have a diameter of 0.5 to 3 mm.

In addition, the present invention provides a method for treating differentiated fibroblasts with serine protease and separately separating the fibroblasts from 1×10 ⁶ /ml to 2×10 ⁷ /ml, or 1×10 ⁵ . /cm ² to 7×10 ⁶ cells/cm ² , stimulation with one or more selected from the group consisting of hormones, growth factors, pluripotency-inducing proteins, and pluripotent cell-inducing factors Static culture at 30 to 40°C without using factors, in D-MEM medium containing glucose and fetal bovine serum or equivalent medium containing glucose and fetal bovine serum in a cell non-adherent cell culture vessel, while supplying _CO2 . characterized by forming spheroids,
A method is provided for expressing the pluripotent stem cell markers of OCT3/4, SOX2, NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and ALP in said spheroids . It is.

According to the present invention, cells that express pluripotent stem cell markers are obtained by statically culturing living cells that have been individually separated and spheroidized in a cell non-adhesive cell culture vessel in a high-density state. spheroids can be formed.

FIG. 1 shows the results of Example 1, and is differential interference contrast microscope images after 0 hour of culture, 5 hours of culture, 14 hours of culture, and 26 hours of culture. FIG. 2 is a diagram showing the results of Example 2, showing the results of expression of ALP, which is a pluripotent stem cell marker. FIG. 4 is a diagram showing the results of Example 2, showing the results of the expression of NANOG, which is a pluripotent stem cell marker. FIG. 4 is a diagram showing the results of Example 2, showing the results of expression of OCT3/4, which are pluripotent stem cell markers. FIG. 4 is a diagram showing the results of Example 2, showing the results of expression of PAR4, which is a pluripotent stem cell marker. FIG. 2 shows the results of Example 2, showing the results of expression of SSEA-3, which is a pluripotent stem cell marker. FIG. 2 shows the results of Example 2, and shows the results of expression of SSEA-4, which is a pluripotent stem cell marker. FIG. 2 shows the results of Example 2, showing the results of expression of SOX2, which is a pluripotent stem cell marker. FIG. 4 is a diagram showing the results of Example 2, showing the results of the expression of TRA-1-60, which is a pluripotent stem cell marker. FIG. 2 shows the results of Example 2, and shows the results of expression of TRA-1-81, a pluripotent stem cell marker. FIG. 10 is a diagram showing the results of (4) of Example 3; The spheroids obtained in step (1) of Example 3 were dropped and adhered to human skin fibroblasts of normal morphology adherently surviving on an adherent cell culture vessel and co-cultured. The spheroids transferred (transformed) into normal morphology of human dermal fibroblasts. The figure shows differential interference contrast microscope images at the start of co-culture, 2 hours after co-culture, 4 hours after co-culture, and 26 hours after co-culture. FIG. 10 is a diagram showing the results of (5) of Example 3; When the spheroids obtained by static culture for 6 hours in step (1) of Example 3 were cultured using a cell culture vessel for adherent cells without adding normal adherent human dermal fibroblasts. is a differential interference microscope image. Fig. 4 shows the results of Example 4, in which a cell suspension solution (30 µl, 60 µl, 125 µl) of enzyme-treated spheroidized biological cells was transferred to a centrifuge tube, and the biological cells accumulated at the bottom of the centrifuge tube by centrifugation. It is a stereomicroscope. The left shows countless spheroidized biological cell cultures that have sunk to the bottom of the centrifuge tube at the start of culture, and the right shows spheroids formed by the culture. Fig. 4 is a diagram showing the results of Example 4, which is a microscopic image of the bottom of a centrifuge tube when cells separated by enzymatic treatment and sphered were transferred to a centrifuge tube in different amounts (250 µl, 500 µl) and cultured. . The left image shows a countless number of spheroidized biological cell cultures that have sunk to the bottom of the centrifuge tube before the start of culture, and the right image shows spheroids formed by the culture. Fig. 10 shows the results of Example 5, showing stereomicroscopic images of a plurality of cell droplets and one droplet at the start of culture of sphered biological cells in droplets of 8 µl and 10 µl, at 15:00, and at 24:00. be. The image of one droplet is the result of observing the same droplet over time. FIG. 10 shows the results of Example 5, and is a stereoscopic microscope image of a plurality of cell droplets and one droplet at the start, 15:00, and 24:00 of culturing of the spheroidized biological cells in each droplet. The image of one droplet is the result of observing the same droplet over time. It should be noted that the drawings of 1 to 5 μl droplets are diagrams of static culture of droplets of 1 μl, 2 μl, 3 μl, and 5 μl from bottom to top, and the image of one droplet is the result of a 5 μl droplet. is. FIG. 10 is a diagram showing the results of Example 6; FIG. 10 shows the results of Example 7, and is a stereoscopic microscope image of spheroids with a diameter of about 0.1 to 0.3 mm. FIG. 7 shows the results of Example 7, showing the DNA-stained images of the obtained spheroids and the results of the expression of pluripotent stem cell markers TRA-1-81, OCT3/4 and NANOG. It is a fluorescence microscope image. FIG. 3 shows the results of Comparative Example 1, and is an optical microscope image of human dermal fibroblasts normally cultured in a low cell density state using a cell culture vessel for adhesive cells. FIG. 10 shows the results of Comparative Example 2, and is a differential interference microscope image of human dermal fibroblasts cultured in a low cell density state using a cell culture vessel for adhesive cells and 19 hours after the start of culture.

In the first aspect of the present invention, differentiated living cells are treated with an enzyme to separate the living cells individually, and the separated living cells are 3×10 ⁵ cells/ml to 7×10 ⁸ cells/ml. A method for producing pluripotent stem cell-like spheroids, characterized by statically culturing in a cell non-adhesive cell culture vessel at a cell density of . The present invention will be described in detail below.

The term “living cell” as used herein can broadly refer to cells that constitute mammalian tissues and organs. Examples of such mammals include primates such as humans and monkeys, rodents such as mice and rats, lagomorphs such as rabbits, artiodactyls such as bovines, equines such as horses, cetacean artiodactyls such as pigs, Meat such as dogs and cats can be exemplified.

Biological cells as used herein are limited to those that have differentiated. "Differentiated biological cell" means a cell that has become specialized since the organism's development, and is intended to include both terminally differentiated cells and cells that have become more specialized than development but before terminal differentiation. . Tissue stem cells are included among differentiated biological cells because they can differentiate into final tissue cells. On the other hand, embryonic stem cells (ES cells) and embryonic germ stem cells (EG cells) are not included in "differentiated living cells". As used herein, "differentiated biological cells" include muscle tissue, connective tissue, circulatory tissue, excretory tissue, reproductive tissue, bone, cartilage, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon. , heart, etc., preferably skin fibroblasts and fibroblasts present in various organs. The differentiated biological cells may be commercially available products, and the biological tissue is separated into individual cells that constitute the body tissue by physical or chemical treatment, and the cells containing the target cells are fractionated by a density gradient method or the like, and placed in a medium. may have been saved in

Differentiated biological cells are usually cultured in media or culture vessels most suitable for the cells. Such media include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, D-MEM medium, Ham medium, RPMI 1640 medium, Fisher medium, and the like. be. Culture vessels for adherent cells include plastic or glass culture vessels coated with extracellular matrices such as collagen, fibronectin, laminin, gelatin, elastin, proteoglycan, vitronectin, and various types of poly-lysine. Non-adhesive cell culture vessels include plastic or glass culture vessels that are not coated with a cell-adhesive extracellular matrix. It can be appropriately selected according to the living cells to be used.

In this method, the differentiated biological cells are individually separated by enzymatic treatment. The separated living cells spheroidize and float in the culture medium. For example, fibroblasts are adherent cells, and adhere to culture vessels during culture, or adhere to each other. In order to avoid such adhesion between culture vessels and cells, individual cells are separated by enzymatic treatment. Therefore, "enzymatic treatment" as used herein is an operation for obtaining a cell suspension in which living cells are individually separated. Examples of enzymes to be used include serine proteases such as trypsin and chymotrypsin, aspartic proteases such as pepsin, cysteine proteases such as papain and chymopapain, metalloproteases such as thermolysin, glutamic proteases, N-terminal threonine proteases, collagenase, dispase and other proteases. There is Trypsin is preferred for this method.

Conditions for enzyme treatment are concentrations used for peeling off living cells adhered to a culture vessel when treating adherent cells. .3 mass %. The temperature is 30-40°C, more preferably 33-38°C. The treatment time is 1 to 60 minutes, preferably 1 to 30 minutes, more preferably 2 to 5 minutes. When using collagenase, it is 0.1 to 0.6% by mass, preferably 0.2 to 0.4% by mass. The temperature is 25-40°C, more preferably 30-40°C. Moreover, the processing time is 30 minutes to 24 hours. When any enzyme is used, it is sufficient that the enzymatic treatment produces a cell suspension in which living cells are individually separated. It should be noted that stress may be applied to living cells depending on the type of enzyme used and the treatment method. The purpose of the enzymatic treatment in this method is to separate individual living cells and not to apply stress. As an example of stress load, Patent Document 3 describes that incubation for 16 hours in trypsin (0.25% trypsin-HBSS) is a stress stimulus.

The culture medium, preservation medium, etc. for the "differentiated biological cells" may contain inhibitors of the enzymes used in the enzyme treatment. In that case, prior to the enzyme treatment, the inhibitor is removed by washing the cells with a culture solution containing no enzyme inhibitor, PBS, or another buffer solution, and then the enzyme treatment is performed.

Individually separated biological cells contained in the cell suspension are spherical. Centrifugation is suitable for recovering the spherical living cells. Usually, the cells are centrifuged at 500-1,500 rpm for 2-5 minutes, the supernatant is removed, and the precipitated cells are collected.

Then, the collected spheroidized biological cells have a cell density of 3×10 ⁵ cells/ml to 7×10 ⁸ cells/ml, preferably 4×10 ⁵ cells/ml to 7×10 ⁷ cells/ml, Static culture is more preferably carried out at 5×10 ⁵ cells/ml to 3×10 ⁷ cells/ml, particularly preferably 1×10 ⁶ cells/ml to 2×10 ⁷ cells/ml. In general, cells are broadly classified into adherent culture cells and suspension culture cells. In conventional cell culture, adherent culture cells are seeded in a culture container coated with an adhesive substance such as poly-L-lysine, and cultured and grown while adhering to the container. On the other hand, suspension culture cells are cultured in a state of being suspended in a cell non-adhesive cell culture vessel. In this method, static culture is performed regardless of whether the differentiated living cells are adherent or suspension culture. In addition, "static culture" means culturing without stirring or swirling the culture medium.

Individually separated biological cells, even adhesive cells such as fibroblasts, become spherical and float in solution. In general, floating cells do not grow or proliferate by adhering to a culture vessel, so non-adhesive cell culture vessels that are not coated with adhesive substances such as poly-L-lysine are used. In this method, when the individually separated biological cells are statically cultured, even if the biological cells are adhesive cells, in order to prevent the spherical biological cells from adhering to the container, the cell non-adhesive Use sex cell culture vessels. For example, poly-D-lysine, poly-L-lysine, 2-methacryloyloxyethyl Phosphoryl Choline and poly (2-hydroxyethyl methacrylate) (Poly(2-hydroxyethyl methacrylate)), A so-called non-coated container, which is not coated with collagen or the like, is suitable. Therefore, no feeder cells are required.

There are no restrictions on the shape of the vessel for static culture, and examples include flasks, tissue culture flasks, dishes, Petri dishes, cell culture dishes, multidishes, microplates, microwell plates, multiplates, multiwell plates, Chamber slides, petri dishes, test tubes, centrifuge tubes, trays, culture bags, roller bottles, plastic plates, plastic microtubes, and the like can be used. For example, when statically culturing individually isolated fibroblasts, a flat-bottomed floating cell culture flask may be used, or a deep vessel with a curved bottom such as a test tube or centrifuge tube may be used. Static culture may be used. A preferable cell density for culturing on a flat bottom is about 1×10 ⁵ cells/cm ² to 7×10 ⁶ cells/cm ² per medium area. In the case of a deep container, the cell density per medium area is higher than this, but if the cell density is 3×10 ⁵ cells/ml to 7×10 ⁸ cells/ml and static culture is performed, the medium There is no limit to the number of cells per area.

When the individually separated cells are statically cultured at the above cell density, the cells naturally fall into the culture solution over time, and other cells pile up on top of the cells that have fallen to the bottom of the vessel to form a stratified layer. Under the cell density conditions described above, many cell layers are formed on top of the bottom layer of cells that have sunk to the bottom of the vessel, and the cells may contact and adhere to each other in the culture solution.

The feature of this method is that when the individually separated living cells are statically cultured, the culture is started after adjusting the cell density to 3×10 ⁵ cells/ml to 7×10 ⁸ cells/ml. As a basic property of mammalian cells, when the cell culture vessel is full, that is, in a confluent state, adherent cells spontaneously detach and die, and floating cells also die by apoptosis. When performing cell culture, even if a culture environment such as a culture vessel and nutrient sources is in place, the cells cannot survive for a long period of time if the high-density state in which the cells adhere to each other continues. A general cell density state including normal cells, cell lines, and cancer cells is 8×10 ³ to 2×10 ⁴ cells/cm ² with respect to the area of the culture vessel. Since normal cell culture is performed at a lower cell density than this, the cells hardly come into contact with each other, avoiding a decrease in cell proliferation ability and the risk of cell death. It is possible to retain the cell morphology and properties peculiar to the cell type. In this method, stationary culture is performed at high density, and the cell density is 1×10 ⁵ cells/cm ² to 7×10 ⁶ cells/cm ² in the case of a flat-bottomed vessel, or the cell density per solution is is between 3×10 ⁵ cells/ml and 7×10 ⁸ cells/ml. Usually, at the cell density described above, the number of seeded cells is too large and the distance between the cells is short or the cells are in close contact, resulting in a confluent state in a short period of time. This may result in decreased proliferative capacity, failure of cell-specific morphology, and risk of cell death. However, when the individually separated spheroidized biological cells are statically cultured at the above cell density using a cell non-adhesive cell culture vessel, the cells floating in the culture medium naturally fall over time and fall to the bottom of the vessel. It was found that the cells came into contact with each other and formed spheroids in 5 to 8 hours. Surprisingly, the cells that make up these spheroids express markers of embryonic stem cells and pluripotent stem cells. As shown in the examples below, when the cell density is less than 3×10 ⁵ cells/ml, spheroids can be produced by stationary culture in a non-adherent cell culture vessel or an adherent cell culture vessel. do not form Therefore, this method can be said to be a method of forming spheroids from differentiated living cells and a method of expressing pluripotent stem cell markers in spheroids derived from differentiated living cells.

Pluripotent stem cell markers include NANOG, SOX2, Oct-3/4, Klf4, Lin28, TRA-1-60, SSEA-1, SSEA-4, c-Myc, PAR4, TRA-1-81, SSEA- 3, and ALP. NANOG, SOX2, Oct-3/4 are essential markers for pluripotent stem cells. The spheroids formed by the above include at least one pluripotent stem cell marker selected from the group consisting of OCT3/4, SOX2 and NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3 , SSEA-4 and at least one pluripotent stem cell marker selected from the group consisting of ALP. In the examples below, spheroids formed by the above manipulations expressed NANOG, SOX2 and Oct-4, and PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4 and ALP was expressed. Expression of a pluripotent stem cell marker indicates that the cell has changed to a pluripotent state. As used herein, the term "pluripotent stem cell-like spheroids" refers to aggregates of cells, and the cells constituting the aggregates are at least one type of multipotent cell selected from the group consisting of OCT3/4, SOX2 and NANOG. expressing a potent stem cell marker and at least one pluripotent stem cell marker selected from the group consisting of PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4 and ALP and

Pluripotent stem cells express markers of different cell types as they differentiate, for example albumin is expressed when they differentiate into hepatocytes. Terminally differentiated hepatocytes express albumin and lose expression of pluripotent stem cell markers. Although the degree of expression of markers for different cell types and the degree of loss of pluripotent stem cell markers differ depending on the degree of differentiation, the expression of both embryonic stem cells and pluripotent stem cell markers is different. It means that it is in a non-initialized state. The spheroids prepared as described above are pluripotent stem cell spheroids in a reprogrammed state because they express embryonic stem cells and pluripotent stem cell markers. Therefore, this method can be said to be a method for reprogramming differentiated living cells.

The method for producing pluripotent stem cell-like spheroids of this method does not require stress load due to long-term enzyme treatment, and artificial nucleic acids such as OCT3/4, SOX2, KLF4, and c-Myc and Reprotin (non-patent literature) 1) does not need to be used. Using fibroblasts as an example, individually separated spherical fibroblasts were adjusted to 3×10 ⁵ cells/ml to 7×10 ⁸ cells/ml by enzymatic treatment and treated with D containing 10% fetal bovine serum. - using MEM medium at a temperature of 30-40°C, more preferably 33-38°C, supplying CO ₂ for 3 hours to 30 days, more preferably 5 hours to 10 days, more preferably 5 hours to 7 Static culture may be performed for days in a cell-non-adhesive cell culture vessel. When the individually separated and spherical living cells are cultured under the above conditions, adjacent cells aggregate with each other, the aggregate increases over time, and almost all living cells aggregate to form spheroids.

Among the cells constituting the pluripotent stem cell-like spheroids of this method, the percentage of cells expressing the pluripotent stem cell marker is 95-100%. In addition, almost no dead cells are observed because there is no stress load in the operation process. The number of dead cells during static culture is extremely low at 0-5%. This means that differentiated living cells could be converted into cells expressing pluripotent stem cell markers with a conversion rate of 90-100%, more preferably 95-98%.

Since the conversion rate from differentiated living cells to cells expressing pluripotent stem cell markers is high, it is possible to adjust the spheroid diameter according to the cell number by adjusting the cell number during static culture. can. In particular, when static culture is performed using a test tube, a centrifuge tube, or the like as a culture vessel, the cells aggregate together over time. For fibroblasts, 30 μl of a cell suspension with a cell density of 1.25×10 ⁷ cells can be cultured to form spheroids with a diameter of about 1 mm, and 500 μl can be cultured to form spheroids with a diameter of about 4 mm. can do. By statically culturing different numbers of cells in this way, pluripotent stem cell-like spheroids with a diameter of 0.1 to 4 mm can be produced depending on the number of cultured cells. For this reason, there is no need for a concentration operation using an antibody against pluripotent stem cells, or a step such as separation into single cells followed by suspension culture to increase or enlarge spheroids. In this respect, it is different from Muse cells in which the maximum diameter of the cell aggregates is about 150 μm.

The spheroids formed by this method are formed by agglutination of neighboring cells during static culture, and are not formed by proliferation of a single cell. Therefore, it can be said that it is a "polyclone" in which different cells, that is, different clones are aggregated. Moreover, the cell aggregation occurs over time and without exception, and the size of the spheroids increases along with the progress of aggregation. In Examples described later, pluripotent stem cell-like spheroids are prepared in a short time of 6 hours. That is, by preparing spherical fibroblasts, it is possible to prepare a large amount of pluripotent stem cell-like spheroids in a short period of time in one step of static culture. On the other hand, the Muse cells described in Patent Document 3 are cultured and selected by giving a stress stimulus of incubation treatment with trypsin for 16 hours to human skin fibroblasts, and the resulting single cells are enriched by suspension culture. It is a cell cluster formed by the Muse cell fraction. Since it proliferates in culture, it can be said to be a "single clone." Moreover, the preparation of the Muse cell-rich fraction is not easy, and the number of Muse cells is increased by subjecting the single cells obtained from this fraction to suspension culture and adhesion culture multiple times. It requires a multi-step cell proliferation procedure and takes a long time to obtain the desired number of cells.

According to this method, a large amount of pluripotent stem cell-like spheroids can be prepared in a short time. Moreover, spheroids with a diameter of 0.5 mm, for example, can be visually observed and can be recovered without the need for an expensive centrifugal device because they settle under their own weight. It can be easily prepared in the required amount when required for research or treatment, and can save time and money. Since the operation is simple, there is little stress on the cells, and unnecessary operational errors can be avoided.

Pluripotent stem cell-like spheroids prepared by this method can be applied to regenerative medicine. In recent years, a technique has been developed in which subject-derived iPS cells are injected into pig blastocysts, which are unable to generate specific organs, to regenerate pigs with organs composed of the iPS cell-derived cells. By using pluripotent stem cell-like spheroids prepared from differentiated living cells of a subject by this method instead of iPS cells, problems such as canceration can be avoided and organs can be regenerated. Stem cells derived from subjects include stem cells obtained from the subject's own bone marrow, placenta, umbilical cord blood, umbilical cord, amniotic membrane, etc., and there is also a "stem cell bank" in which these cells are ultra-cold preserved in liquid nitrogen or the like. However, according to this method, since pluripotent stem cell-like spheroids can be prepared from living cells such as fibroblasts of a subject, there is no need for cryopreservation, and there are problems such as a decrease in the number of viable cells during lysis. No need to.

The second of the present invention is at least one pluripotent stem cell marker selected from the group consisting of OCT3/4, SOX2 and NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3, 0.1-4 mm in diameter, polyclonal pluripotent stem cell-like spheroids composed of cells expressing at least one pluripotent stem cell marker selected from the group consisting of SSEA-4 and ALP. . These pluripotent stem cell-like spheroids can be produced according to the first aspect of the present invention using differentiated living cells as raw materials, but are not limited to this. Monoclonal spheroids formed by proliferating stem cells have conventionally existed. For example, Muse cells had a diameter of about 150 μm. Muse cells have multiple security systems to prevent explosive proliferation. Therefore, it is not easy to prepare large spheroids. In contrast, the pluripotent stem cell-like spheroids of the present invention are approximately 0.1-4 mm in diameter, more preferably 0.5-3 mm. Since they are larger than conventional spheroids, they do not need to be enlarged by cell proliferation.

The pluripotent stem cell-like spheroids of the present invention can express pluripotent stem cell markers, function as pluripotent stem cells, and differentiate into any tissue/cell type. It can be used for regenerative medicine and the like. The pluripotent stem cell-like spheroids of the present invention can also be used for transplantation of spheroids, etc., by preparing a cell suspension of pluripotent stem cell-like spheroids, attaching them to tissues and proliferating them, and applying them to various organ cell types. can. For example, transplanted tissue includes skin, cerebrospinal cord, liver, muscle, and the like. By administering the pluripotent stem cell-like spheroids of the present invention directly to or in the vicinity of a damaged or damaged tissue, organ, etc., cells expressing pluripotent stem cell markers enter the tissue or organ, They can differentiate into tissue-specific cells and regenerate tissues and organs.

Administration can be carried out by subcutaneous injection, injection into veins or arteries, intramuscular injection, intraperitoneal injection, parenteral administration such as direct injection into damaged or missing organ tissues, intrauterine injection into embryos, and the like. The dosage can be appropriately determined according to the type of organ or tissue to be regenerated, the desired size, and the degree of regeneration.

Alternatively, the pluripotent stem cell-like spheroids of the present invention can be induced to differentiate in advance, and the differentiated cells can be used in regenerative medicine. As shown in Example 6 below, when the size after spheroid culture was evaluated over time, it reached a maximum at 67 hours after culture, and then maintained the size at the time of transformation until 94 hours after culture. The pluripotent stem cell-like spheroids of the present invention are not prepared by introducing an oncogenic gene or the like, and tumorigenesis, which can be inferred from an increase in size after culturing, is not observed. The possibility of carcinogenesis is extremely low even if it is used for

The pluripotent stem cell-like spheroids of the present invention can be used for basic research such as elucidation of disease mechanisms, development of therapeutic agents, screening for efficacy and toxicity of drugs, evaluation of drugs, and the like. For example, drug evaluation and drug screening of embryoid-like cell clusters made from pluripotent stem cell-like spheroids, pluripotent stem cell-like spheroids, and cells, tissues and organs obtained by differentiating from the embryoid-like cell clusters It can be used as a material for For example, the pluripotent stem cell-like spheroids of the present invention contain basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), dimethylsulfoxide (DMSO) and isoproterenol, or fibroblasts as differentiation inducers. By adding cell growth factor 4 (FGF4), hepatocyte growth factor (HGF) or the like to the medium and culturing, differentiation can be induced to obtain desired cells. On the other hand, various factors can be added to the pluripotent stem cell-like spheroids of the present invention to evaluate the presence or absence of differentiation, and used to identify differentiation-inducing factors. By using the pluripotent stem cell-like spheroids of the present invention, it is possible to promote differentiation induction into each cell type and research on fate-determining factors.

According to the present invention, spheroids expressing pluripotent stem cell markers can be obtained from differentiated biological cells without the introduction of exogenous genes, transcripts, proteins, nuclear components, cytoplasm, or other exogenous reprogramming factors. can. However, the reason for expressing pluripotent stem cell markers is unknown. Assuming that this phenomenon is reprogramming, the only difference from conventional cell culture is that the cells are cultured at a high density or in a contact state. High-density contact culture can be considered as a kind of stress load, but it is difficult to think of it as a stress because cells in vivo survive in contact in the first place. Even if the above phenomenon is interpreted as reprogramming, the mechanism of reprogramming expression in the pluripotent stem cell-like spheroids of the present invention is currently unknown.

EXAMPLES Next, the present invention will be specifically described with reference to examples, but these examples are not intended to limit the present invention in any way.

(Example 1)
(1) Using a cell culture petri dish for adhesive cells (Sumitomo Bakelite Co., Ltd., trade name “cell culture petri dish 90φ (deep type)”), 1.5 × ¹⁰ human skin fibroblasts (HDF, 10 ml of D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., containing 10% fetal bovine serum, 1% penicillin/streptomycin), 37° C., 5% CO ₂ to 80% confluency. It was cultured for 48 hours until .
(2) Remove the same D-MEM medium in the petri dish with an aspirator, add 15 ml of 0.01 M phosphate buffer (PBS) and stir to remove the remaining D-MEM medium containing fetal bovine serum components. , washing of adhering human dermal fibroblasts.
(3) 500 μl of trypsin was added to the same PBS-washed cells, and the cells were treated at 37° C. for 3 minutes to peel off adherent cells and separate them individually. Next, 5 ml of the same D-MEM medium was added to stop the trypsin reaction, and the mixture was transferred to a 15 ml centrifuge tube and centrifuged at 1,000 rpm for 5 minutes to precipitate 3.9×10 ⁶ individually separated human dermal fibers. blast cells were obtained.
(4) 3 ml of the same D-MED medium was added to the individually separated human skin fibroblasts obtained by centrifugation, and 1.5 ml (2×10 ⁶ cells) of the cell suspension was placed in a glass bottom dish. (manufactured by FPI Co., Ltd., non-coated, hole diameter 1.4 mm, culture area 9.6 cm ² ), and statically cultured under conditions of 37° C. and 5% CO ₂ . The cell density per culture area was 2.1×10 ⁵ /cm ² . Twenty-four hours after the start of culture, almost all of the human skin fibroblasts formed countless spheroids with a diameter of about 0.1 mm. The results are shown in FIG. FIG. 1 shows differential interference contrast microscope images at the start of culture, after 5 hours of culture, after 14 hours of culture, and after 26 hours of culture. Few dead cells were observed.

(Example 2)
(1) Two cell culture dishes for adherent cells (Sumitomo Bakelite Co., Ltd., trade name "cell culture petri dish 90φ (deep type)") and two cell culture dishes for adherent cells (Sumitomo Bakelite Co., Ltd., trade name "cells") 2×10 ⁶ and 0.6×10 ⁶ human dermal fibroblasts (HDF, purchased from Kurabo Industries, Ltd.) were added to D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., containing 10% fetal bovine serum and 1% penicillin/streptomycin) were used and cultured for 48 hours at 37° C. and 5% CO ₂ . Cells were washed and trypsinized according to steps (2) and (3) of Example 1, and combined to obtain 1.87×10 ⁷ individually isolated human dermal fibroblasts. Obtained.
(2) 5 ml of the same D-MEM medium was added to the human skin fibroblast sediment obtained by centrifugation (1,000 rpm, 5 minutes) to prepare a cell suspension, and 1.5 ml of the same was added to a 96-well plate. The same cell suspension was placed on a lid (manufactured by greiner bio-one, trade name “LID FOR MICROPLATE, non-coating”, lid area 4.84 cm ² ). This was statically cultured for 24 hours under conditions of 37° C. and 5% CO ₂ . The cell density was 3.7×10 ⁶ /ml, approximately 1.2×10 ⁶ /cm ² . Numerous spheroids were formed from almost all of the human skin fibroblasts in the upper lid.
(3) Dispense 150 µl of the suspension containing spheroids into nine 0.5 ml microtubes, and perform immunocytochemical staining using an antibody specific to a marker protein of pluripotent stem cells to determine OCT3/4. , PAR4, TRA-1-60, TRA-1-81, SSEA3, SSEA4, ALP, SOX2 and NANOG. As a result, all cells were found to be positive for OCT3/4, PAR4, TRA-1-60, TRA-1-81, SSEA3, SSEA4, ALP, SOX2 and NANOG. The results are shown in FIGS. 2-10. In the figure where each stem cell marker was detected, the upper left is a differential interference microscope image (DIC image), the lower left is an image stained with 4',6-diamidino-2-phenylindole and observed with a fluorescence microscope (DAPI image), and the right The lower image is an image (fluorochrome image) observed under a fluorescence microscope after the marker protein of each pluripotent stem cell was stained with a fluorescent dye (Alexa488), and the upper right image is an image in which the DAPI image and the fluorescent dye image are superimposed.

In addition, OCT3/4, PAR4, TRA-1-60, TRA were analyzed by immunocytochemical staining method in the same manner as above for human skin fibroblasts (HDF, purchased from Kurabo Industries) that do not form spheroids. -1-81, SSEA3, SSEA4, ALP, SOX2, NANOG and other proteins were evaluated. However, none of the pluripotent stem cell markers were expressed.

(Example 3)
(1) Using four cell culture dishes for adhesive cells (manufactured by Sumitomo Bakelite Co., Ltd., trade name "cell culture dish 90φ (deep type)"), (1) to (3) of Example 1 A similar procedure yielded 1.1×10 ⁷ individually isolated human dermal fibroblasts. 10 ml of D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., containing 10% fetal bovine serum, 1% penicillin/streptomycin) was added to the obtained human skin fibroblasts to prepare a cell suspension. 500 μl (5.5×10 ⁵ cells) was transferred to a multiplate for suspension culture (manufactured by Sumitomo Bakelite Co., Ltd., trade name “plate for suspension culture 12F (independent well) with lid”, non-coated, flat bottom, 1 well bottom area: 3). 0.6 cm ² ) and incubated statically at 37° C., 5% CO ₂ for 9 hours. Cell densities are 1.1×10 ⁶ /ml, 1.5×10 ⁵ /cm ² . Spheroids were formed from human dermal fibroblasts by culturing.
(2) Transfer 200 μl of the cell suspension prepared in step (1) to a chamber slide (manufactured by IWAKI, AGC Techno Glass Co., trade name “Chamber Slide II”, collagen coating, bottom area of 1 well: 9 cm ² ). 1 ml of D-MEM medium was added and static culture was carried out at 37° C. and 5% CO ₂ for 9 hours. Cell densities are 1.8×10 ⁵ /ml, 2.4×10 ⁴ /cm ² . The cultured human skin fibroblasts had the morphology of normal human skin fibroblasts adhered to glass slides.
(3) The spheroids obtained in step (1) of Example 3 were collected in a 1.5 ml microtube and centrifuged at 6,200 rpm for 10 seconds to obtain spheroid pellets.
(4) The spheroid pellet collected in step (3) of Example 3 was added to normal human skin fibroblasts obtained in step (2) of Example 3, and co-cultured for 26 hours. Through co-culture, spheroids in contact with normal-morph human skin fibroblasts changed into normal human skin fibroblasts over time. The results are shown in FIG. FIG. 11 shows differential interference contrast microscope images at the start of coculture, 2 hours, 4 hours, and 26 hours later.

(5) In step (1) of Example 3, static culture was performed for 6 hours to prepare spheroids. The spheroids were collected and placed on a chamber slide (manufactured by IWAKI, AGC Techno Glass Co., trade name “Chamber Slide II”, collagen coating, 1 well) in D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., 10% bovine Fetal serum, containing 1% penicillin/streptomycin) was used and cultured in an environment free of normal morphology of human dermal fibroblasts. As a result, the spheroids maintained the shape of cell aggregates. The results are shown in FIG. FIG. 12 shows differential interference contrast microscope images at the start of culture in the adherent cell incubator, 2 hours after the start of culture, 4 hours after the start of culture, and 23 hours after the start of culture. Even after culturing for 23 hours using a cell culture vessel for adherent cells, the spheroids did not return to fibroblasts.

(Example 4)
(1) By operating in the same manner as in Example 2, 1.25×10 ⁷ individually separated human skin fibroblasts were obtained. This was suspended in 1 ml of D-MEM medium.
(2) Put the suspension in a 1.5 ml centrifuge tube, 30 μl (3.8×10 ⁵ ), 60 μl (7.5×10 ⁵ ), 125 μl (1.6×10 ⁶ ), 250 μl ( 3.1×10 ⁶ cells), 500 μl (6.3×10 ⁶ cells) were transferred, centrifuged at 1,000 rpm for 5 minutes, and the supernatant was removed. D-MEM medium was added to each test tube so that the final volume was 90 μl, and static culture was carried out at 37° C. and 5% CO ₂ for 15 hours. One spheroid was formed in each test tube, and its diameter was 1 mm, 1.4 mm, 1.8 mm, 2.8 mm and 4 mm, respectively. The results are shown in FIGS. 13 and 14. FIG.

(Example 5)
(1) By operating in the same manner as in Example 1, 3×10 ⁶ individual human skin fibroblasts were obtained. This was suspended in 1 ml of D-MEM medium to prepare a cell suspension.
(2) 1 μl, 2 μl, 3 μl, 5 μl (1.5× 10 ⁴ pieces), 8 μl (2.4 × 10 ⁴ pieces), 10 μl (3 × 10 ⁴ pieces) were added dropwise, and static culture was carried out in droplet form under the conditions of 37 ° C and 5% CO _2. The results were obtained. Fig. 15 and Fig. 16. Fig. 15 and Fig. 16 show each droplet of cells at the start of culturing of each droplet, 15 hours after culturing, and 24 hours after culturing, and a stereomicroscopic image of one droplet. The image of one droplet is the result of observing the same droplet over time.In addition, the drawing of each well plate of 1 to 5 μl in FIG. It is a diagram of stationary culture of 3 μl and 5 μl droplets, and the image of one droplet is the result of a 5 μl droplet.

(Example 6)
By operating in the same manner as in Example 1, a cell suspension of 7.5×10 ⁶ individually separated human dermal fibroblasts was obtained. 15 ml of D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., containing 10% fetal bovine serum, 1% penicillin/streptomycin) was added to obtain a cell suspension of 5×10 ⁵ cells/ml. . 25 μl of it was seeded in one well of a 96-well plate for suspension cell culture (manufactured by Sumitomo Bakelite Co., Ltd., trade name “Multiplate 96U with lid”, well volume 0.3 ml), and placed at 37° C. in 5% CO ₂ atmosphere. Stationary culture was carried out under these conditions for 17 hours, 36 hours, 67 hours and 94 hours. Results of the same well images are shown in FIG. A plurality of spheres were formed over time from the start of the culture, and large fused spheroids were formed after 67 hours. However, the size of the spheroids after 94 hours was similar to that at 67 hours.

(Example 7)
The procedure was carried out in the same manner as in Example 1, except that normal human skin fibroblasts (CCD-1079SK) were used instead of human skin fibroblasts (HDF, purchased from Kurabo Industries). Formation of spheroids was confirmed after culturing for 9 hours. The results are shown in FIG. FIG. 18 is a stereomicroscopic image of spheroids with a maximum diameter of approximately 0.1 to 0.3 mm.

In addition, in the same manner as in step (3) of Example 2, OCT3/4, TRA-1-81 and OCT3/4, TRA-1-81 and NANOG protein expression was confirmed. The results are shown in FIG. Spheroids obtained from human normal skin fibroblasts (CCD-1079SK) also expressed OCT3/4, TRA-1-81 and NANOG and were considered pluripotent stem cell-like spheroids.

(Comparative example 1)
D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., ¹⁰ % fetal bovine serum, 1 % penicillin/streptomycin) was added to adjust the cell density to 2.5×10 ⁵ /ml, and 1.1 ml of this was added to a slide chamber (manufactured by AGC Techno Glass Co., type I collagen coating, 1 well area 8.36 cm ² ). ) and statically cultured under conditions of 37° C. and 5% CO ₂ . The cell density per culture area is 3.3×10 ⁴ /cm ² . No spheroids were formed even after 15 hours of culture. The results are shown in FIG. FIG. 20 is an optical microscope image.

(Comparative example 2)
D-MEM medium (High Glucose, Wako Pure Chemical Industries, Ltd., ¹⁰ % fetal bovine serum, 1 % penicillin/streptomycin) was added to make the cell density 1.2×10 ⁶ /ml, and 20 μl of the resulting D-MED medium and 1 ml of the D-MED medium were added to a glass bottom dish (manufactured by FPI Co., Ltd., non-coated, hole diameter 10 ml). 0.4 mm and a culture area of 9.6 cm ² ), and statically cultured under conditions of 37° C. and 5% CO ₂ . The cell density per culture area is 2.5×10 ³ /cm ² . After culturing for 19 hours, the cells adhered and survived, albeit weakly, but did not form spheroids. FIG. 21 shows differential interference contrast microscope images after 19 hours from the start of culture.

(result)
(1) As shown in Example 1, FIG. 1, when individually isolated human skin fibroblasts are statically cultured in a high-density, floating state, cells in the vicinity aggregate over time to form spheroids. formed. The formed spheroids were not formed by proliferation of one cell, but were derived by aggregation of proximal cells. Therefore, it is a polyclonal spheroid formed by aggregation of a plurality of cells. In addition, as shown in Example 7, spheroids were similarly formed in normal human skin fibroblasts of the same cell type and other cell lines.
(2) As shown in Example 2 and FIGS. 2 to 10, the cells forming the spheroids expressed pluripotent stem cell markers. Moreover, about 100% of the cells forming the spheroids expressed pluripotent stem cell markers, and the living cells were transformed into pluripotent-like cells at an extremely high rate.
(3) As shown in Example 3, when pluripotent stem cell-like spheroids are co-cultured with normal human skin fibroblasts, they change to normal human skin fibroblasts. Even when cultured using an adherent cell incubator, the spheroid morphology was maintained without transforming into normal fibroblasts. It was suggested that stimulation by secretory components of human dermal fibroblasts and intercellular interactions may induce pluripotent stem cells to differentiated cells.
(4) Example 6 shows that the pluripotent stem cell-like spheroids of the present invention have no cell proliferation. It can be said that this indicates that the pluripotent stem cell-like spheroids of the present invention have no carcinogenic action. Cancerous cells have a doubling time of about 24 hours, so they double after being cultured for 24 hours and become 16 times after culturing for 4 days. No proliferative or cancerous cells seen in cancerous cells were detected even after 4 days of culture.
(5) As shown in Example 5, when using a cell suspension with the same cell density and changing the amount of droplets and static culture in a floating state, droplets as shown in FIGS. 15 and 16 The amount of spheroids increased corresponding to the amount of cells, ie, the amount of cells. It was shown that a large number of spheroids can be prepared by increasing the number of cells. In addition, the reproducibility was confirmed by the fact that spheroids were formed in all droplets 24 hours after the start of culture.
(6) As shown in Example 4, it was found that spheroids with a large diameter were formed by using a culture vessel with a shape that facilitates aggregation of cells, such as a centrifuge tube, during static culture in a floating state. did. Combined with Example 5, the number and size of spheroids can be adjusted by selecting the number of cultured cells and the shape of the culture vessel.
(7) As shown in Comparative Examples 1 and 2, spheroids are not formed when the cell density is less than 2.5×10 ⁵ /ml. Since the cell density per medium area in this case is 3.3×10 ⁴ cells/cm ² , it can be said that spheroids are not formed when the cell density is less than 3.3×10 ⁴ cells/cm ² . In addition, when compared with Example 1 in which culture is performed at a cell density of 2×10 ⁶ cells/ml and a cell density of 2.1×10 ⁵ cells/cm ² per medium area, the present invention is 3×10 ⁵ cells/cm 2 . Cells/ml to 7×10 ⁸ cells/ml or cell densities of 1×10 ⁵ cells/cm ² to 7×10 ⁶ cells/cm ² were cultured statically in suspension by a simple method. It can be said that this is a method capable of forming potential stem cell-like spheroids.

The present invention is based on Japanese Patent Application No. 2016-042126 filed on March 4, 2016. The entire specification, claims, and drawings of Japanese Patent Application No. 2016-042126 are incorporated herein by reference.

[Appendix]
[Appendix 1]
enzymatically treating the differentiated biological cells to separate the individual biological cells;
A pluripotent cell characterized by statically culturing the individually separated biological cells at a cell density of 3×10 ⁵ cells/ml to 7×10 ⁸ cells/ml in a cell non-adhesive cell culture vessel. A method for producing sex stem cell-like spheroids.

[Appendix 2]
The method for producing pluripotent stem cell-like spheroids according to Appendix 1, wherein the enzymatic treatment is to treat the differentiated biological cells with a solution having a trypsin concentration of 0.1 to 1% for 1 to 60 minutes.

[Appendix 3]
3. The method for producing pluripotent stem cell-like spheroids according to any one of Appendix 1 or 2, wherein the differentiated biological cells are fibroblasts.

[Appendix 4]
The pluripotent stem cell-like spheroids contain at least one pluripotent stem cell marker selected from the group consisting of OCT3/4, SOX2 and NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA- 3, SSEA-4, and at least one pluripotent stem cell marker selected from the group consisting of ALP. manufacturing method.

[Appendix 5]
The separated biological cells were statically cultured in a cell non-adhesive cell culture vessel at a cell density of 3×10 ⁵ to 7×10 ⁸ /ml, and the diameter corresponding to the cell number was 0.1. 5. A method for producing pluripotent stem cell-like spheroids according to any one of Appendices 1 to 4, characterized in that spheroids of ~4 mm are formed.

[Appendix 6]
at least one pluripotent stem cell marker selected from the group consisting of OCT3/4, SOX2 and NANOG, and PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4 and ALP 0.1-4 mm in diameter, polyclonal pluripotent stem cell-like spheroids composed of cells expressing at least one pluripotent stem cell marker selected from the group.

[Appendix 7]
enzymatically treating the differentiated biological cells to separate the individual biological cells;
statically culturing the individually separated living cells in a flat-bottom non-adherent cell culture vessel at a cell density of 1×10 ⁵ cells/cm ² to 7×10 ⁶ cells/cm ² per medium area; A method for producing pluripotent stem cell-like spheroids, characterized by

[Appendix 8]
enzymatically treating the differentiated biological cells to separate the individual biological cells;
The individually separated living cells are statically cultured in a cell non-adhesive cell culture vessel at a cell density of 3×10 ⁵ cells/ml to 7×10 ⁸ cells/ml to form spheroids. A method for expressing a pluripotent stem cell marker.

Claims (3)

The differentiated fibroblasts were treated with serine protease and the individualized fibroblasts were treated with 1×10 ⁶ cells/ml to 2×10 ⁷ cells/ml, or 1×10 ⁵ cells/cm ² to 7×. at a high cell density of 10 ⁶ cells/cm ² without using any one or more stimulating factors selected from the group consisting of hormones, growth factors, pluripotency-inducing proteins, and pluripotent cell-inducing factors, In a cell non-adhesive cell culture vessel, in D-MEM medium containing glucose and fetal bovine serum or its equivalent medium, while supplying CO ₂ , stationary culture at 30 to 40 ° C. to form spheroids,
The spheroids are characterized by expressing pluripotent stem cell markers of OCT3/4, SOX2, NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and ALP. , a method for producing spheroids . The method for producing spheroids according to claim 1, wherein the spheroids have a diameter of 0.5 to 3 mm. The differentiated fibroblasts were treated with serine protease and the individualized fibroblasts were treated with 1×10 ⁶ cells/ml to 2×10 ⁷ cells/ml, or 1×10 ⁵ cells/cm ² to 7×. at a high cell density of 10 ⁶ cells/cm ² without using any one or more stimulating factors selected from the group consisting of hormones, growth factors, pluripotency-inducing proteins, and pluripotent cell-inducing factors, In a cell non-adhesive cell culture vessel, in D-MEM medium containing glucose and fetal bovine serum or its equivalent medium, while supplying CO ₂ , static culture at 30 to 40 ° C. to form spheroids. characterized by
A method of expressing the pluripotent stem cell markers of OCT3/4, SOX2, NANOG, PAR4, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and ALP in said spheroids .

Images