Method for culturing or inducing cells

Abstract

A method of culturing or deriving cells is provided, which comprises culturing or deriving cells in a closed system.

Description

[Technical Field]

[0001] The present invention relates to cell technology and to methods for culturing or deriving cells. [Background technology]

[0002] Embryonic stem cells (ES cells) are stem cells established from early human or mouse embryos. ES cells have the pluripotency to differentiate into all cell types present in the body. Currently, human ES cells are available for cell transplantation therapy for many diseases, including Parkinson's disease, juvenile diabetes, and leukemia. However, ES cell transplantation has its drawbacks. In particular, ES cell transplantation can trigger immune rejection similar to the rejection that occurs following unsuccessful organ transplants. Furthermore, the use of ES cells, which are established by destroying human embryos, has been criticized and opposed on ethical grounds.

[0003] Against this background, Professor Shinya Yamanaka of Kyoto University succeeded in establishing induced pluripotent stem cells (iPS cells) by introducing four genes: OCT3 / 4, KLF4, c-MYC, and SOX2 into somatic cells. This led to Professor Yamanaka receiving the Nobel Prize in Physiology or Medicine in 2012 (see, for example, Patent Documents 1 and 2). iPS cells are ideal pluripotent cells that are free from rejection and ethical issues. Therefore, iPS cells are expected to be used in cell transplantation therapy. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4183742 [Patent Document 2] Japanese Patent Application Laid-Open No. 2014-114997 Summary of the Invention [Problem to be solved by the invention]

[0005] There is a demand for a method that can efficiently and simply culture or induce various cells, not just iPS cells. Therefore, one of the objects of the present invention is to provide a method that can efficiently and simply culture or induce cells. [Means for solving the problem]

[0006] According to an aspect of the present invention, there is provided a method of culturing or deriving cells, comprising culturing or deriving cells in a closed system.

[0007] In the above-described method, the induction may include at least one of reprogramming, reprogramming, transdifferentiation, differentiation induction, and cell fate change.

[0008] In the above method, gas exchange between the inside and outside of the closed system may not occur.

[0009] The method may further include controlling the temperature within the closed system.

[0010] In the culturing of the above method, the closed system may be sealed.

[0011] In the above method, the closed system may be sealed and outside air may not enter the closed system.

[0012] In the above method, the closed system is sealed so that cells, microorganisms, viruses, and dust particles outside the closed system do not enter the closed system.

[0013] In the above method, the closed system may be sealed and the substance in the closed system may not flow out of the closed system.

[0014] In the above method, at least one of carbon dioxide gas, nitrogen gas, and oxygen gas may not be supplied into the closed system.

[0015] In the above method, the pH of the medium in the closed system may be maintained within a predetermined range.

[0016] In the above method, at least a part of the closed system may be formed by being embedded in a gas-impermeable material.

[0017] In the above method, at least a portion of the closed system may be made of a gas-impermeable material.

[0018] In the above method, the cells may be cultured or induced in a closed system while the medium is replenished or exchanged.

[0019] In the above method, the cells may be cultured or induced in a closed system while circulating the medium.

[0020] In the above method, the closed system may include a culture tank for culturing cells, and the culture tank may be provided with a supply port for supplying a fluid into the culture tank and a discharge port for discharging the fluid in the closed system, and the supply port and the discharge port may be sealable.

[0021] In the above method, the supply device for supplying the fluid to the supply port may be detachable, and the discharge device for discharging the fluid to the discharge port may be detachable. When the fluid is supplied from the supply device into the culture tank, the fluid in the culture tank may move into the discharge device.

[0022] In the above method, when the culture medium is supplied from the supply device into the culture tank, air in the culture tank may move into the discharge device.

[0023] In the above method, when the medium is supplied from the supply device into the culture tank, the medium in the culture tank may move into the discharge device.

[0024] In the above method, the medium may contain cells.

[0025] In the above method, when a fluid is supplied from the supply device into the culture tank, outside air does not need to enter the culture tank.

[0026] In the above method, the carbon dioxide concentration in the closed system does not need to be controlled during the culturing.

[0027] In the culturing method described above, the carbon dioxide concentration outside the closed system does not need to be controlled.

[0028] During the culturing in the above method, substances within the closed system may move through a semipermeable membrane within the closed system.

[0029] In the above method, the closed system may include a culture vessel for culturing cells and a flow path connected to the culture vessel, and the medium may circulate between the culture vessel and the flow path.

[0030] In the above method, gas exchange with the outside does not need to occur in the flow channel.

[0031] In the above method, the pH of the medium in the culture tank may be maintained within a predetermined range by circulating the medium.

[0032] In the above method, the culture may be a suspension culture.

[0033] In the above method, the culture may be an adherent culture.

[0034] In the above method, the cells may be cultured in a gel medium in a closed system.

[0035] In the above method, the cells may be cultured in a liquid medium in a closed system.

[0036] In the above method, the medium in the closed system may be stirred.

[0037] In the above method, the medium in the closed system does not need to be stirred.

[0038] The above method may further comprise passaging the cells.

[0039] In the above method, it is not necessary to add or replace the medium between seeding and passaging.

[0040] In the above method, the medium may be added or replaced between seeding and subculture.

[0041] In the above method, it is not necessary to add or replace the medium between passages.

[0042] In the above method, the medium may be added or replaced between passages.

[0043] In the above method, the cells may be stem cells.

[0044] In the above method, the stem cells may be iPS cells, ES cells, or somatic stem cells.

[0045] During the culturing in the above method, the stem cells may be maintained in an undifferentiated state.

[0046] In the above method, the stem cells may maintain pluripotency during culturing.

[0047] In the above method, the cells may be somatic cells.

[0048] In the above method, the cells may be at least one selected from blood cells, nervous system cells, cardiac muscle cells, epithelial cells, mesenchymal cells, hepatic cells, insulin-producing cells, retinal pigment epithelial cells, and corneal cells.

[0049] In the above method, the cells may be cells into which an inducer has been introduced.

[0050] In the above method, an inducer may be added to the medium in the closed system to introduce the inducer into the cells cultured in the closed system.

[0051] In the above method, the cells may be induced to become stem cells.

[0052] In the above method, the stem cells may be iPS cells.

[0053] In the above method, the cells may be blood cells.

[0054] In the above method, the cells may be induced to become cells of another type.

[0055] In the above method, the cells may be blood lineage cells, and an inducer may be added to the culture medium in the closed system to introduce the inducer into the blood lineage cells cultured in the closed system, thereby inducing the blood lineage cells to iPS cells.

[0056] In the above methods, the inducer may be contained in a plasmid.

[0057] In the above method, the inducer may be RNA.

[0058] In the above method, the inducer may be contained in the Sendai virus.

[0059] Furthermore, according to an aspect of the present invention, there is provided a method for culturing or inducing cells, comprising: preparing a cell culture vessel including a culture component permeable member that is permeable to culture components, a culture vessel that holds a cell-containing culture medium and covers one side of the culture component permeable member, and for culturing cells; and a culture medium holding vessel that covers the other side of the culture component permeable member, and for holding the culture medium; and culturing or inducing cells in the culture vessel.

[0060] In the above-described method, the induction may include at least one of reprogramming, reprogramming, transdifferentiation, differentiation induction, and cell fate change.

[0061] In the above method, the inside of the cell culture vessel may be closed from the outside.

[0062] In the above method, the pH of the medium in the cell culture vessel may be maintained within a predetermined range.

[0063] In the above method, the culture may be a suspension culture.

[0064] In the above method, the cells may be stem cells.

[0065] In the above method, the cells may be somatic cells.

[0066] In the above method, the cells may be at least one selected from blood cells, nervous system cells, cardiac muscle cells, epithelial cells, mesenchymal cells, hepatic cells, insulin-producing cells, retinal pigment epithelial cells, and corneal cells.

[0067] In the above method, the cells may be cells into which an inducer has been introduced.

[0068] In the above method, an inducer may be added to the medium in the culture vessel to introduce the inducer into the cells cultured in the culture vessel.

[0069] In the above method, the cells may be induced to become cells of another type.

[0070] In the above-mentioned method, the cell culture vessel may further include a culture-side plate having an opening and placed on the surface of the culture component permeable member facing the culture vessel.

[0071] In the above-described method, the cell culture vessel may further include a culture medium side plate having an opening and placed on the surface of the culture component permeable member facing the culture medium holding tank.

[0072] In the above method, the culture plate may be dark in color.

[0073] The above method may further comprise observing the cells or cell clusters made of cells against the background of a portion of the culture-side plate where no openings are provided.

[0074] The above method may further include photographing the cells or cell clusters made of cells with a portion of the culture-side plate where no openings are provided as a background.

[0075] The above method may further comprise replenishing or replacing the medium in the medium holding tank. [Effects of the Invention]

[0076] According to the present invention, it is possible to provide a method by which cells can be cultured or induced efficiently and simply. [Brief explanation of the drawings]

[0077] [Figure 1] FIG. 1 is an exploded perspective view of a cell culture vessel according to an embodiment. [Figure 2] FIG. 1 is a perspective view of a cell culture vessel according to an embodiment. [Figure 3] FIG. 2 is a front view of a portion of the cell culture vessel according to the embodiment. [Figure 4] FIG. 2 is a perspective view of a portion of the cell culture vessel according to the embodiment. [Figure 5] FIG. 2 is a front view of a portion of the cell culture vessel according to the embodiment. [Figure 6] FIG. 2 is a rear view of the cell culture vessel according to the embodiment. [Figure 7] FIG. 1 is an exploded perspective view of a cell culture vessel according to an embodiment. [Figure 8] 1 is an optical microscope photograph of cells cultured by the culture method according to Example 1. [Figure 9] 1 is a histogram showing the results of flow cytometry analysis of cells cultured by the culture method according to Example 1. [Figure 10] Fig. 10(a) is a photograph of a tube used in the culture method according to Example 2. Fig. 10(b) is an optical microscope photograph of cells cultured by the culture method according to Example 2. [Figure 11] 10 is a histogram showing the results of flow cytometry analysis of cells cultured by the culture method according to Example 2. [Figure 12] 10 is an optical microscope photograph of cells cultured by the culture method according to Example 3. [Figure 13] 10 is a histogram showing the results of flow cytometry analysis of cells cultured by the culture method of Example 3. [Figure 14] 10 is a photograph of a flask used in the method for producing induced pluripotent stem cells according to Example 4. [Figure 15] 10 is an optical microscope photograph of cells produced by the method for producing induced pluripotent stem cells according to Example 4. [Figure 16] 10 is a graph showing the number of cell colonies for each colony type produced by the method for producing induced pluripotent stem cells according to Example 4. [Figure 17] 10 is a histogram showing the results of flow cytometry analysis of cells produced by the method for producing induced pluripotent stem cells according to Example 4. [Figure 18] 10 is an optical microscope photograph of cells produced by the method for producing induced pluripotent stem cells according to Example 5. [Figure 19] 10 is a histogram showing the results of flow cytometry analysis of cells produced by the method for producing induced pluripotent stem cells according to Example 5. [Figure 20] FIG. 10 is an exploded perspective view of the cell culture vessel according to Example 6. [Figure 21] FIG. 10 is a perspective view of a cell culture vessel according to Example 6. [Figure 22] 10 is a micrograph of a cell aggregate according to Example 6. [Figure 23] 10 is a histogram showing the results of flow cytometry of iPS cells according to Example 6. [Figure 24] 10 is a micrograph of a cell aggregate according to Example 7. [Figure 25] 10 is a histogram showing the results of flow cytometry of iPS cells according to Example 7. [Figure 26] 10 is a micrograph of a cell aggregate according to Example 8. [Figure 27] 10 is a histogram showing the results of flow cytometry of iPS cells according to Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0078] A cell culture method according to an embodiment includes culturing cells in a closed system. A closed system is, for example, a completely closed system in which gas exchange does not occur between the inside of the closed system and the outside. A closed system is, for example, sealed, in which outside air does not enter the closed system. For example, cells, microorganisms, viruses, and dust from outside the closed system do not enter the closed system. For example, substances in the closed system do not flow out of the closed system.

[0079] Cells may be cultured in a liquid medium or a gel medium in a closed system. Cells may also be cultured in an adherent or suspension culture in a closed system. While cells are cultured in a closed system, the medium may or may not be stirred. When cells are cultured in an adherent culture, feeder cells may or may not be used. When cells are cultured in a suspension culture, feeder cells may or may not be used.

[0080] The cells cultured in the closed system may be animal cells, including human cells, insect cells, or plant cells.

[0081] The cells cultured in the closed system may be, for example, somatic cells, differentiated cells, undifferentiated cells, or stem cells. The cells cultured in the closed system are not particularly limited, and may be, for example, blood cells, nervous system cells, cardiac muscle cells, epithelial cells, vascular endothelial cells, mesenchymal cells, fibroblasts, hepatocytes, insulin-producing cells, retinal pigment epithelial cells, and corneal cells.

[0082] The blood cells may be blood cells such as T cells, B cells, NK cells, NKT cells, megakaryocytes, macrophages, granulocytes, neutrophils, eosinophils, hematopoietic stem cells, blood stem / progenitor cells, red blood cells, white blood cells, and platelets. The nervous cells may be neurons and glial cells, oligodendrocytes, and neural stem cells. The cardiomyocyte cells may be cardiac stem cells, cardiomyocytes, and pacemaker cells. The epithelial cells may be keratinocytes, intestinal epithelial cells, oral epithelial cells, and corneal epithelial cells. The mesenchymal cells may be dermal cells, osteoblasts, adipocytes, muscle cells, chondrocytes, and the like.

[0083] The stem cells include, for example, induced pluripotent stem (iPS) cells, embryonic stem cells (ES cells), and somatic stem cells. The somatic stem cells may be mesenchymal stem cells. When the cells are stem cells, they proliferate in a culture medium while maintaining an undifferentiated state and pluripotency.

[0084] For example, before being cultured in suspension, stem cells are disaggregated into single cells or cell clusters, and the disaggregated stem cells are placed in a medium. The single cells or cell clusters grow while maintaining clonality, and form colonies in the medium.

[0085] Stem cells are cultured, for example, in a stem cell medium, such as a human ES / iPS medium such as mTeSR1 (registered trademark, STEMCELL TECHNOLOGIES).

[0086] However, the stem cell medium is not limited to this, and various stem cell media can be used. For example, a medium containing 20% ​​KnockOut SR (registered trademark, ThermoFisher Scientific), GlutaMAX (registered trademark, ThermoFisher Scientific), and non-essential amino acids (NEAA) may be used as the stem cell medium. Alternatively, stem cell culture media such as Primate ES Cell Medium, Reprostem, ReproFF, ReproFF2, ReproXF (Reprocell), TeSR2, TeSRE8, ReproTeSR (STEMCELL Technologies), PluriSTEM (registered trademark) Human ES / iPS Medium (Merck), NutriStem (registered trademark) XF / FF Culture Medium for Human iPS and ES Cells, Pluriton reprogramming medium (Stemgent), PluriSTEM (registered trademark), Stemfit AK02N, Stemfit AK03 (Ajinomoto), ESC-Sure (registered trademark) serum and feeder-free medium for hESC / iPS (Applied StemCell), and L7 (registered trademark) hPSC Culture System (LONZA) may be used.

[0087] The medium in the closed system is appropriately selected depending on the type of cells to be cultured in the closed system. For example, if the cells are blood cells, a medium suitable for blood cells is placed in the closed system. For example, if the cells are mesenchymal cells, a medium suitable for mesenchymal cells is placed in the closed system.

[0088] The medium may not contain a growth factor such as basic fibroblast growth factor (bFGF), or may contain a growth factor such as bFGF at a low concentration of 400 μg / L or less, 40 μg / L or less, or 10 μg / L or less.

[0089] The medium may not contain TGF-β, or may contain TGF-β at a low concentration of 2 μg / L (2 ng / mL) or less, 600 ng / L or less, 300 ng / L or less, or 100 ng / L or less.

[0090] The medium may contain at least one substance selected from the group consisting of cadherin, laminin, fibronectin, and vitronectin.

[0091] When the medium is a gel medium, the gel medium is prepared, for example, by adding deacylated gellan gum to the medium to a final concentration of 0.001% by weight to 0.5% by weight, 0.005% by weight to 0.1% by weight, or 0.01% by weight to 0.05% by weight.

[0092] The gel medium may contain at least one polymer compound selected from the group consisting of gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, keratosulfate, chondroitin sulfate, deltamannan sulfate, rhamnan sulfate, and salts thereof. The gel medium may also contain methylcellulose. By including methylcellulose, cell aggregation is further suppressed.

[0093] Alternatively, the gel medium may contain at least one temperature-sensitive gel selected from poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypropyl methacrylate) (PHPMA), Poly(N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide (NHS) ester terminated, triethoxysilane terminated, Poly(N-isopropylacrylamide-co-acrylamide), Poly(N-isopropylacrylamide-co-acrylic acid), Poly(N-isopropylacrylamide-co-butylacrylate), Poly(N-isopropylacrylamide-co-methacrylic acid), Poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-isopropylacrylamide.

[0094] In the present disclosure, gel-like medium or gel medium includes polymer medium.

[0095] While cells are being cultured in a closed system, the temperature of the medium in the closed system is maintained at, for example, 0°C or higher, 4°C or higher, 15°C or higher, 20°C or higher, or 34°C or higher. Also, while cells are being cultured in a closed system, the temperature of the medium in the closed system is maintained at, for example, 45°C or lower, 39°C or lower, or 20°C or lower. While cells are being cultured in a closed system, the temperature of the medium in the closed system may be controlled using a temperature control device such as a heater or cooler.

[0096] The pH of the medium placed in the closed system is, for example, 4.0 or higher, 5.0 or higher, 6.0 or higher, 7.0 or higher, or 8.0 or higher. The pH of the medium placed in the closed system is, for example, 10.0 or lower, 9.0 or lower, 8.8 or lower, or 8.0 or lower. The pH of the medium placed in the closed system tends to be maintained within the above range while cells are being cultured due to its presence in the closed system. A pH of 7.0 or higher or 8.0 or higher is preferred because it neutralizes lactic acid during cell culture in the closed system and prevents a decrease in pH.

[0097] While cells are being cultured in a closed system, at least one or all of carbon dioxide gas, nitrogen gas, and oxygen gas may not be supplied to the closed system. Furthermore, while cells are being cultured in a closed system, the carbon dioxide concentration within the closed system may not be controlled. While cells are being cultured in a closed system, the carbon dioxide concentration outside the closed system may not be controlled. For example, the closed system may not be placed in a carbon dioxide (CO2) incubator. However, the closed system may be placed in a carbon dioxide (CO2) incubator.

[0098] When culturing cells in a closed system, it is preferable that there is no or little gas layer such as an air layer in the closed system. Therefore, it is preferable that the closed system is filled with a medium so that there is no or little gas layer remaining in the closed system.

[0099] During cell culture in the closed system, the medium may be circulated within the closed system to prevent contact with the outside air. In the closed system, a semipermeable membrane may be placed between the cell suspension and the circulating medium, and the active ingredients of the medium may be permeated into the cell suspension through the semipermeable membrane.

[0100] In the closed system, cells are cultured for, for example, 3 hours or more, 1 day or more, 14 days or more, or 30 days or more, although the closed system may be opened for passaging, medium change, and medium addition.

[0101] It is not necessary to replace or add the medium between the seeding and the passaging of the cells. In this case, the closed system is not opened at all between the seeding and the passaging, and the cells are cultured in the closed system. The closed system is not opened at all between the seeding and the passaging, for example, for one day or more, five days or more, or ten days or more.

[0102] The medium does not need to be replaced or added between successive passages of cells. In this case, the cells are cultured in a closed system without being opened at all between successive passages. The cells are cultured in a closed system without being opened at all between successive passages, for example, for one day or more, five days or more, or ten days or more.

[0103] Alternatively, the closed system may be opened between seeding and subculturing the cells to add or replace the medium. Also, the closed system may be opened between subculturing the cells to add or replace the medium. The addition or replacement of the medium may be performed every one day or more, every two days or more, or every five days or more. The cells in the closed system are cultured without being opened at all except when adding or replacing the medium or when subculturing the cells.

[0104] Furthermore, the cell induction method according to the embodiment includes inducing cells in a closed system. The closed system is as described above. Induction refers to reprogramming, initialization, transdifferentiation or lineage reprogramming, differentiation induction, cell fate reprogramming, and the like.

[0105] The cells induced in the closed system may be cells into which an inducer has been introduced outside the closed system in advance, or the cells may be induced in the closed system by adding the inducer to the medium in the closed system and introducing the inducer into cells that have not been introduced with the inducer and are being cultured in the closed system.

[0106] The cells induced in the closed system may be animal cells or plant cells.

[0107] The cells may be induced in a liquid medium in a closed system, or in a gel medium in a closed system. The cells may also be induced while being cultured in adhesion or suspension in a closed system. During cell induction in a closed system, the medium may or may not be stirred. When cells are induced while being cultured in adhesion, feeder cells may or may not be used. When cells are induced while being cultured in suspension, feeder cells may or may not be used.

[0108] In a closed system, cells may be induced to become stem cells such as iPS cells. In a closed system, cells may be induced to become cells of a different type other than stem cells. In a closed system, stem cells such as iPS cells and ES cells may be induced to become cells of a different type.

[0109] Cells induced to become iPS cells in a closed system may be blood cells, such as blood cells. Alternatively, cells induced to become iPS cells in a closed system may be fibroblasts, marrow stem cells, keratinocytes, hair papilla cells, oral epithelial cells, somatic stem progenitor cells, etc. Cells induced to become iPS cells in a closed system may be induced to become, for example, blood cells, nervous system cells, cardiac muscle cells, epithelial cells, mesenchymal cells, hepatocytes, insulin-producing cells, retinal pigment epithelial cells, and corneal cells.

[0110] Blood cells are separated from blood, such as, but not limited to, peripheral blood and umbilical cord blood. Blood may be collected from adults or minors. Anticoagulants, such as ethylenediaminetetraacetic acid (EDTA), heparin, and biologic standard blood preservation solution A (ACD-A), are used during collection.

[0111] Blood cells are nucleated cells such as mononuclear cells, neutrophils, eosinophils, lymphocytes, macrophages, blood stem / progenitor cells, and vascular endothelial cells, but do not include red blood cells or platelets. Blood cells may be, for example, endothelial progenitor cells, blood stem / progenitor cells, T cells, or B cells. T cells are, for example, αβT cells.

[0112] Mononuclear cells are separated from blood using a blood cell separation medium and a centrifuge, etc. When Ficoll (GE Healthcare) is used as the blood cell separation medium, the method for separating mononuclear cells is as follows.

[0113] Because low temperatures tend to reduce the accuracy of mononuclear cell separation, set the centrifuge to 4°C to 42°C, preferably 18°C. 10 μL to 50 mL of blood is collected from an adult or minor. A chelating agent containing EDTA is added to the blood and gently mixed to prevent blood clotting. 5 mL of human lymphocyte separation medium (Ficoll-Paque PREMIUM, GE Healthcare Japan) is dispensed into two 15 mL tubes. 5 mL of blood is diluted with 5 mL of PBS, and 5 mL of the medium is layered on top of the human lymphocyte separation medium in each tube. The diluted blood is then slowly added to the medium by sliding it down the tube wall to avoid disturbing the interface.

[0114] The solution in the tube is centrifuged at 10 x g to 1000 x g, preferably 400 x g, at 4°C to 42°C, preferably 18°C, for 5 minutes to 2 hours, preferably 30 minutes. After centrifugation, a cloudy white intermediate layer will appear in the tube. This cloudy white intermediate layer contains mononuclear cells. Slowly collect the cloudy white intermediate layer in the tube with a pipette and transfer it to a new 15 mL tube. Be careful not to aspirate the bottom layer. Approximately 1 mL of the cloudy white intermediate layer can be collected from one tube. Transfer the intermediate layers from two tubes together into one tube.

[0115] To the collected mononuclear cells, 1 mL to 48 mL, preferably 12 mL, of PBS is added, and the solution is further centrifuged at 10 × g to 1000 × g, preferably 200 × g, at 4°C to 42°C, preferably 18°C, for 1 minute to 60 minutes, preferably 10 minutes. The supernatant is then aspirated and removed using an aspirator, and 1 mL to 12 mL, preferably 3 mL, of chemically defined serum-free hematopoietic cell medium (X-VIVO® 10, Lonza) is added and suspended to obtain a mononuclear cell suspension. 10 μL of the mononuclear cell suspension is stained with trypan blue and counted using a hemocytometer.

[0116] When Vacutainer (registered trademark, BD) is used as a blood collection tube, the method for isolating mononuclear cells is as follows.

[0117] Because low temperatures tend to reduce the accuracy of mononuclear cell separation, the centrifuge is set to 4°C to 42°C, preferably 18°C. Eight milliliters of blood is collected from an adult or minor using a blood collection tube (Vacutainer®, BD) and mixed with an anticoagulant by inversion. The balance is then adjusted, and the solution is centrifuged in a swinging bucket rotor at 4°C to 42°C, preferably 18°C, at 100 x g to 3000 x g, preferably 1500 x g to 1800 x g, for 1 minute to 60 minutes, preferably 20 minutes. After centrifugation, the upper layer (plasma) is removed, and the mononuclear cell layer and blood cells adhering to the gel are suspended by pipetting to obtain a suspension. The resulting suspension is transferred to another 15 mL tube.

[0118] Add 1 mL to 14 mL, preferably 12 mL, of PBS to the suspension in the 15 mL tube, and centrifuge the suspension at 4°C to 42°C, preferably 18°C, at 100 x g to 3000 x g, preferably 200 x g, for 1 to 60 minutes, preferably 5 minutes. After centrifugation, remove the supernatant using an aspirator. Dilute the hemolysis agent (PharmLyse®, 10x concentration, BD) to 1x concentration with sterile water. Loosen the pellet in the 15 mL tube by tapping, and add 1 mL to 14 mL, preferably 1 mL, of hemolysis agent. Then, leave the solution at room temperature, protected from light, for 1 to 60 minutes, preferably 1 minute.

[0119] Next, 1 mL to 14 mL, preferably 12 mL, of PBS is added to a 15 mL tube and centrifuged at 100 × g to 3000 × g, preferably 200 × g, for 1 to 60 minutes, for 5 minutes, at 4°C to 42°C, preferably at room temperature. After centrifugation, the supernatant is removed using an aspirator, and 1 mL to 15 mL, preferably 3 mL, of chemically defined serum-free hematopoietic cell medium (X-VIVO® 10, Lonza) is added and suspended to obtain a mononuclear cell suspension. 10 μL of the mononuclear cell suspension is stained with trypan blue and counted using a hemocytometer.

[0120] The method for separating mononuclear cells from blood is not limited to the above method, and mononuclear cells may be separated from blood using, for example, a dialysis membrane. Filters such as the PureCell Select System (registered trademark, PALL) for whole blood mononuclear cell concentration, a purifier for blood cell removal (CellSorber E, registered trademark, Asahi Kasei), and a leukocyte removal filter for platelet preparations (SepaCell PL, registered trademark, PLX-5B-SCD, Asahi Kasei) can also be used.

[0121] Mononuclear cells may be separated using an erythrocyte sedimentation agent capable of separating nucleated cells by gravity sedimentation or centrifugation of red blood cells. Examples of erythrocyte sedimentation agents include HetaSep (registered trademark, STEMCELL Technologies) and HES40 (NIPRO).

[0122] Furthermore, mononuclear cells such as CTL-UP1 available from Cellular Technology Limited and PBMC-001 available from Sanguine Biosciences may also be used.

[0123] Alternatively, blood cells may be thawed and used after being cryopreserved using a cell cryopreservation solution such as Cellbanker 1, Stem Cellbanker GMP Grade, or Stem Cellbanker DMSO-Free GMP Grade (Zenoac).

[0124] To thaw mononuclear cells, first place 1 mL to 15 mL, preferably 8 mL, of chemically defined serum-free hematopoietic cell medium (X-VIVO® 10, Lonza) in a 15 mL tube. Then, place the tube containing the frozen mononuclear cells in a warm water bath at 4°C to 42°C, preferably 37°C, to begin thawing the mononuclear cells. Then, with some ice remaining, remove the tube containing the mononuclear cells from the warm water bath and transfer the mononuclear cells to a tube containing chemically defined serum-free hematopoietic cell medium. Then, 10 μL of the mononuclear cell suspension is stained with trypan blue and counted using a hemocytometer.

[0125] Blood cells may be separated based on cell surface markers. Blood stem and progenitor cells are CD34 positive. T cells are CD3, CD4, or CD8 positive. B cells are CD10, CD19, or CD20 positive. Blood stem and progenitor cells, T cells, or B cells are separated from blood cells using, for example, an automated magnetic cell separator and immunomagnetic beads. Alternatively, pre-separated mononuclear cells may be prepared. However, blood cells that have not been separated based on cell surface markers may also be used.

[0126] CD34+ cells are stem / progenitor cells that tend to be easily reprogrammed. Furthermore, when iPS cells are generated from CD3+ T cells, the T cell-derived iPS cells retain the TCR recombination pattern and tend to be efficiently induced to differentiate into T cells.

[0127] An inducer is introduced into cells in adherent culture. Alternatively, an inducer is introduced into cells in suspension culture in a gel medium. The inducer may be RNA. The inducer may be contained in a Sendai virus. Alternatively, the inducer may be introduced into cells by transfection. The inducer may be DNA. The inducer may be contained in a plasmid.

[0128] Inducers may be contained in, for example, adenoviruses, lentiviruses, and retroviruses.

[0129] The inducer may be a protein.

[0130] CytoTune (registered trademark, Invitrogen) can be used as the Sendai virus. The multiplicity of infection (MOI) can be used as an indicator of the titer of the Sendai virus. The MOI of the Sendai virus is, for example, 0.1 to 100.0, or 1.0 to 50.0.

[0131] When inducing cells into stem cells, for example, inducers introduced into the cells include OCT3 / 4 mRNA, SOX2 mRNA, KLF4 mRNA, and c-MYC mRNA. M3O, an improved version of OCT4, may also be used as the inducer. The inducer may further comprise mRNA of at least one factor selected from the group consisting of LIN28A, FOXH1, LIN28B, GLIS1, p53-dominant negative, p53-P275S, L-MYC, NANOG, DPPA2, DPPA4, DPPA5, ZIC3, BCL-2, E-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, KLF2, KLF5, ESRRB, miR-291-3p, miR-294, miR-295, NR5A1, NR5A2, TBX3, MBD3sh, TH2A, TH2B, and P53DD. These mRNAs are available from TriLink.

[0132] The mRNA contained in the inducer contains pseudouridine (Ψ), 5-methylcytosine (m 5 C), 5-methyluridine (5meU or m 5 U), N1-methylpseudouridine (me1Ψ), 5-methoxyuridine (5moU), 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), 5-carboxymethylesteruridine (5camU), thienoguanosine (thG), N4-methylcytidine (me 4 C), 5-methylcytidine (m 5C), 5-methylcytidine (5moC), 5-hydroxymethylcytidine (5hmC), 5-hydroxycytidine (5hoC), 5-formacytidine (5fC), 5-carboxycytidine (5caC), N 6 -methyl-2-aminoadenosine (m 6 DAP), diaminopurine (DAP), 2'-O-methyluridine (Um or m 2’-O U), 2-thiouridine (s 2 U), and N 6 -methyladenosine (m 6 A) may be modified with at least one selected from the group consisting of:

[0133] Cytosine is 5-methylcytosine (m 5 C) Uracil may be substituted with pseudouracil.

[0134] The mRNA contained in the inducer may be polyadenylated.

[0135] The mRNA contained in the inducer may be prepared by polyadenylation of in vitro transcribed (IVT) RNA. The mRNA may be polyadenylated during IVT by using a DNA template encoding a poly(A) tail. The mRNA may be capped. To maximize the efficiency of expression in cells, it is preferable that the majority of mRNA molecules contain a cap. The mRNA may have a 5' cap [m7G(5')ppp(5')G] structure. This sequence stabilizes the mRNA and promotes transcription. The 5' triphosphate may be removed from mRNA with a 5' triphosphate by dephosphorylation treatment. The mRNA may have an anti-reverse cap analog (ARCA) [3'O-Me-m7G(5')ppp(5')G]. ARCA is a sequence inserted before the transcription start site, doubling the efficiency of transcribed mRNA. The mRNA may have a polyA tail.

[0136] The mRNA contained in the inducer may be treated with ribonuclease III (RNase III).

[0137] Furthermore, the mRNA contained in the inducer may be replicative RNA, which has the ability to self-replicate. Replicative RNA is RNA with the ability to self-replicate, and unlike normal RNA, it also has the ability to express proteins necessary for RNA replication. Replicative RNA is derived from the Venezuelan equine encephalitis (VEE) virus, a type of alphavirus. Transfecting replicative RNA into cells allows the cells to express RNA that continues to produce reprogramming factors, making it possible to avoid introducing inducer RNA into cells multiple times.

[0138] The replicative RNA sequence may comprise a sequence obtained from an alphavirus selected from the group consisting of alphavirus replicon RNA, Eastern equine encephalitis virus (EEE), Venezuelan equine encephalitis virus (VEE), Everglades virus, Mucambo virus, Pixuna virus, and Western equine encephalitis virus (WEE).

[0139] The replicative RNA may also comprise sequences from an alphavirus selected from the group consisting of Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, O'nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus.

[0140] For example, the replicative RNA contains, from 5' to 3', (VEE RNA replicase)-(promoter)-(RF1)-(self-cleaving peptide)-(RF2)-(self-cleaving peptide)-(RF3)-(IRES or core promoter)-(RF4)-(IRES or any promoter)-(optional selectable marker)-(VEE 3'UTR and polyA tail)-(optional selectable marker)-promoter. RF1-4 above are factors that induce dedifferentiation of cells into pluripotent cells. RF2-3, RF3-4, and RF4 above are optional. The RF1-4 may be selected from the group consisting of OCT-4, KLF4, SOX-2, c-MYC, LIN28A, LIN28B, GLIS1, FOXH1, p53-dominant negative, p53-P275S, L-MYC, NANOG, DPPA2, DPPA4, DPPA5, ZIC3, BCL-2, E-RAS, TPT1, SALL2, NAC1, DAX1, TERT, ZNF206, FOXD3, REX1, UTF1, KLF2, KLF5, ESRRB, miR-291-3p, miR-294, miR-295, NR5A1, NR5A2, TBX3, MBD3sh, TH2A, and TH2B.

[0141] The medium in which the cells into which the inducer is introduced are cultured is appropriately selected depending on the type of cells into which the inducer is introduced. Also, the medium in which the cells into which the inducer is introduced are cultured is appropriately selected depending on the type of cells into which the inducer is introduced.

[0142] As described above, the medium may contain no growth factors, such as bFGF, or may contain a low concentration of a growth factor. Alternatively, the medium may contain no TGF-β or a low concentration of TGF-β. The medium may contain at least one substance selected from the group consisting of cadherin, laminin, fibronectin, and vitronectin.

[0143] When the medium is a gel medium, the medium may contain at least one polymer compound as described above. The gel medium may also contain methylcellulose. Alternatively, the gel medium may contain a temperature-sensitive gel as described above.

[0144] During the induction of cells in the closed system, the temperature of the medium in the closed system is, for example, similar to the temperature during the culturing of cells in the closed system described above.

[0145] The pH of the medium placed in the closed system in which the cells are derived is similar to the pH of the medium placed in the closed system in which the cells are cultured, for example, as described above.

[0146] While cells are being induced in the closed system, at least one or all of carbon dioxide gas, nitrogen gas, and oxygen gas may not be supplied to the closed system. Furthermore, while cells are being induced in the closed system, the carbon dioxide concentration in the closed system may not be controlled. While cells are being induced in the closed system, the carbon dioxide concentration outside the closed system may not be controlled. For example, the closed system may not be placed in a carbon dioxide (CO2) incubator. However, the closed system may be placed in a carbon dioxide (CO2) incubator.

[0147] When inducing cells in a closed system, it is preferable that there is no or little gas layer such as an air layer in the closed system. Therefore, it is preferable that the closed system is filled with a medium so that there is no or little gas layer remaining in the closed system.

[0148] During the induction of cells in the closed system, the medium may be circulated within the closed system so as not to come into contact with the outside air. In the closed system, a semipermeable membrane may be placed between the cell suspension and the circulating medium, and the active ingredients of the medium may be permeated into the cell suspension through the semipermeable membrane.

[0149] The cells are induced in the closed system while being cultured for, for example, 1 day or more, 14 days or more, or 30 days or more, although the closed system may be opened for passaging, medium change, and medium addition.

[0150] It is not necessary to change or add the medium between the seeding and the subculture of the cells. In this case, the closed system is not opened at all between the seeding and the subculture, and the cells in the closed system are cultured and induced. For example, for one day or more, five days or more, or ten days or more, the closed system is not opened at all between the seeding and the subculture, and the cells in the closed system are cultured and induced.

[0151] The medium does not need to be replaced or added between successive passages of cells. In this case, the closed system is not opened at all between successive passages, and the cells in the closed system are cultured while undergoing induction. The closed system is not opened at all between successive passages, for example, for one day or more, five days or more, or ten days or more, and the cells in the closed system are cultured while undergoing induction.

[0152] Alternatively, the closed system may be opened between seeding and subculturing of cells to add or replace the medium. Furthermore, the closed system may be opened between subculturing of cells to add or replace the medium. Addition or replacement of the medium may be performed every one or more days, every two or more days, or every five or more days. Except for the addition or replacement of the medium and subculturing, the closed system is not opened at all, and the cells in the closed system are induced while being cultured.

[0153] Whether cells transfected with an inducer have been induced (reprogrammed) into iPS cells can be confirmed, for example, by cell morphology. Alternatively, whether cells have been induced into iPS cells can be determined by analyzing using a cytometer whether they are positive for at least one surface marker selected from TRA-1-60, TRA-1-81, SSEA-1, and SSEA5, which are cell surface markers indicating undifferentiation. TRA-1-60 is an antigen specific to iPS / ES cells and is not detected in differentiated cells. Since iPS cells can be generated only from the TRA-1-60-positive fraction, TRA-1-60-positive cells are considered to be the seed of iPS cells.

[0154] A closed system for culturing or inducing cells inside according to an embodiment may include, for example, a cell culture vessel as shown in Fig. 1. The cell culture vessel includes a culture component permeable member 10 that is permeable to culture components, a culture vessel 30 that covers one side of the culture component permeable member 10 and holds a cell-containing culture medium for culturing cells, and a culture medium retention tank 40 that covers the other side of the culture component permeable member 10 and holds the culture medium. The cell-containing culture medium in the culture vessel 30 can come into contact with the culture component permeable member 10. Furthermore, the culture medium in the culture medium retention tank 40 can come into contact with the culture component permeable member 10. The culture medium in the culture medium retention tank 40 does not contain cells.

[0155] The culture component permeable member 10 allows active ingredients of the culture medium in the culture medium holding tank 40 to permeate into the cell-containing culture medium in the culture tank 30. The culture component permeable member 10 may also allow waste products in the cell-containing culture medium in the culture tank 30 to permeate into the culture medium in the culture medium holding tank 40. For example, a semipermeable membrane or a mesh can be used as the culture component permeable member 10. Semipermeable membranes include dialysis membranes.

[0156] When the culture component permeable member 10 is a semipermeable membrane, the molecular weight cutoff of the semipermeable membrane is, for example, 0.1 KDa or more, 10 KDa or more, or 50 KDa or more. Examples of semipermeable membranes include cellulose ester, ethyl cellulose, cellulose esters, regenerated cellulose, polysulfone, polyacrylonitrile, polymethyl methacrylate, ethylene-vinyl alcohol copolymer, polyester polymer alloy, polycarbonate, polyamide, cellulose acetate, cellulose diacetate, cellulose triacetate, cupric ammonium rayon, saponified cellulose, hemophane membrane, phosphatidylcholine membrane, and vitamin E-coated membrane.

[0157] When the culture component permeable member 10 is a mesh, the mesh has pores smaller than the cells or cell clusters cultured in the culture vessel 30. This prevents the cells or cell clusters in the culture vessel 30 from migrating into the culture medium holding vessel 40. The material of the mesh is, for example, resin or metal, but is not particularly limited. The surface of the culture component permeable member 10 may be non-cell adhesive.

[0158] The cell culture vessel according to the embodiment may further include a culture-side plate 21 and a culture medium-side plate 22, each having an opening, that sandwich the culture component permeable member 10. The culture-side plate 21 and the culture medium-side plate 22 hold the culture component permeable member 10 by sandwiching it between them to prevent the culture component permeable member 10 from being displaced by the pressure of the cell-containing medium in the culture vessel 30 and the medium in the culture medium retention tank 40. This prevents the culture component permeable member 10 from contacting the inner wall of the culture vessel 30 or the culture medium retention tank 40 due to pressure fluctuations. The culture-side plate 21 and the culture medium-side plate 22 have a hardness that prevents them from being displaced by the pressure applied by the cell-containing medium in the culture vessel 30 and the medium in the culture medium retention tank 40. The material of the culture-side plate 21 and the culture medium-side plate 22 is, for example, resin or metal, but is not particularly limited. The surface of the culture-side plate 21 may be non-cell-adhesive. If the culture component permeable member 10 does not fluctuate due to the pressure exerted by the cell-containing medium in the culture vessel 30 and the medium in the culture medium holding vessel 40, the culture-side plate 21 and the medium-side plate 22 may be omitted.

[0159] The culture-side plate 21 has an opening so that the cell-containing medium in the culture vessel 30 can come into contact with the culture component permeable member 10. The culture medium-side plate 22 has an opening so that the medium in the culture medium holding tank 40 can come into contact with the culture component permeable member 10. Components of the cell-containing medium in the culture vessel 30 and components of the medium in the culture medium holding tank 40 can permeate the culture component permeable member 10 through the opening in the culture-side plate 21. The shape of the openings provided in each of the culture-side plate 21 and the culture medium-side plate 22 is, for example, circular, but is not particularly limited. The openings provided in each of the culture-side plate 21 and the culture medium-side plate 22 have a size within a range that can suppress movement of the culture component permeable member 10. The openings are provided in each of the culture-side plate 21 and the culture medium-side plate 22, for example, in a grid pattern or randomly.

[0160] The culture-side plate 21 may have a dark color, such as black. When the culture-side plate 21 has a dark color, the cells in the cell-containing medium can be visualized or imaged with high contrast against the background of the culture-side plate 21. When the area, etc., of the portion of the culture-side plate 21 without openings is larger than the size of the cells or cell clusters, the cells or cell clusters can be visualized or imaged with high contrast against the background of the portion of the culture-side plate 21 without openings. However, by adjusting the light irradiated onto the cells or cell clusters, it is also possible to visualize or image the cells or cell clusters even if the culture component permeable member 10 or the culture-side plate 21 is transparent.

[0161] The culture vessel 30 and the culture medium holding vessel 40 may be fixed with screws, pins, electromagnets, or the like. The contact portion of the culture vessel 30 and at least a portion of one side of the culture-side plate 21 are in close contact. At least a portion of the other side of the culture-side plate 21 are in close contact with at least a portion of one side of the culture component permeable member 10. At least a portion of the other side of the culture component permeable member 10 are in close contact with at least a portion of one side of the culture medium-side plate 22. At least a portion of the other side of the culture medium-side plate 22 are in close contact with the contact portion of the culture medium holding vessel 40. When achieving close contact, a packing or the like may be used as appropriate. The packing may be disposed, for example, between the culture component permeable member 10 and the culture vessel 30. The packing may be disposed between the outer periphery of the culture component permeable member 10 and the culture vessel 30. The outer diameter of the packing disposed between the culture component permeable member 10 and the culture vessel 30 may be larger than the outer diameter of the culture component permeable member 10. Furthermore, the packing may be disposed, for example, between the culture component permeable member 10 and the culture medium holding tank 40. The packing may be disposed between the outer periphery of the culture component permeable member 10 and the culture medium holding tank 40. The outer diameter of the packing disposed between the culture component permeable member 10 and the culture medium holding tank 40 may be larger than the outer diameter of the culture component permeable member 10.

[0162] The culture vessel 30 includes, for example, a housing 31 and a cover 32 that covers the housing 31. The housing 31 and the cover 32 may be integrated. The inner wall of the culture vessel 30 may be coated with a cell-non-adhesive substance such as poly-HEMA (poly 2-hydroxyethyl methacrylate) to prevent cells from adhering, thereby making the inner wall of the culture vessel 30 non-adhesive. The housing 31 is provided with an opening 131 for exposing the culture component permeable member 10 through the opening in the culture-side plate 21. As shown in FIG. 2, the cover 32 of the culture vessel 30 is provided with a window 132 that allows observation of the cell-containing medium in the culture vessel 30. The window 132 can be made of, for example, glass or resin.

[0163] The cell incubator according to the embodiment may include a temperature control unit for heating and cooling the window 132. The temperature control unit may be a transparent heater, such as a transparent conductive film, disposed on the window 132 and heating the window. Alternatively, the cell incubator according to the embodiment may include a temperature control unit for heating and cooling the housing 31 or cover 32 of the culture vessel 30. The temperature of the cell-containing medium in the culture vessel 30 can be controlled by controlling the temperature of any of the housing 31, the cover 32, and the window 132 with the temperature control unit. The cell incubator according to the embodiment may further include a thermometer for measuring the temperature of the cell-containing medium in the culture vessel 30. The thermometer may measure the temperature of the cell-containing medium based on the temperature of the culture vessel 30 without contacting the cell-containing medium, or may directly measure the temperature of the cell-containing medium by contacting the cell-containing medium. In this case, the temperature control unit may be feedback-controlled so that the temperature of the cell-containing medium is maintained at a predetermined temperature.

[0164] As shown in FIG. 1, the culture tank 30 is provided with a supply port 231 for supplying a fluid into the culture tank 30 and a discharge port 331 for discharging the fluid from the culture tank 30. For example, a plug 33 shown in FIG. 2 is inserted into the supply port 231 to which a supply device such as a bag, bellows, or syringe for supplying a fluid can be connected. The supply device may be a fluid machine such as a pump. However, an injection device may be directly connected to the supply port 231 shown in FIG. 1. The supply device is detachable from the supply port 231, and when the supply device is not connected to the supply port 231, the supply port 231 can be sealed, and no fluid is exchanged between the inside and outside of the culture tank 30 via the supply port 231.

[0165] The plug 33 may be a needleless connector. The needleless connector may be a split septum type or a mechanical valve type. When the plug 33 is a split septum type needleless connector, the plug 33 has a disk valve with a slit. When supplying a fluid into the culture vessel 30, a supply device or a flow path connected to the supply device is inserted into the slit of the disk valve. When the supply device or a flow path connected to the supply device is not inserted into the slit, the slit is sealed. When the supply device or a flow path connected to the supply device is inserted into the slit, the disk valve tightly fits against the outer periphery of the supply device or the flow path connected to the supply device. Therefore, even when the supply device or a flow path connected to the supply device is inserted into the plug 33, outside air does not enter the culture vessel 30 through the plug 33. However, the plug 33 may be a connector into which a needle is inserted.

[0166] Furthermore, for example, a plug 34 shown in FIG. 2 is inserted into the outlet 331. The plug 34 can be connected to an ejector, such as a bag, bellows, or syringe, for discharging fluid from the culture tank 30. The ejector may be a fluid machine such as a pump. However, the ejector may be directly connected to the outlet 331 shown in FIG. 1. The ejector may actively suck fluid from the culture tank 30. Alternatively, the ejector may passively increase its internal volume in response to the pressure inside the culture tank 30 to receive fluid pushed out from the culture tank 30. The ejector is detachable from the outlet 331. When the ejector is not connected to the outlet 331, the outlet 331 is sealable, and no fluid is exchanged between the inside and outside of the culture tank 30 via the outlet 331. The plug 34 may be a needleless connector. The needleless connector may be a split septum type or a mechanical valve type. Even when an ejector or a flow path connected to an ejector is inserted into the plug 34, outside air does not enter the culture tank 30 through the plug 34. However, the plug 34 may be a connector into which a needle is inserted.

[0167] For example, when the culture vessel 30 is in close contact with the culture medium holding vessel 40 with the culture-side plate 21, the culture component permeable member 10, and the culture medium-side plate 22 sandwiched therebetween, and air is present in the culture vessel 30, the cell-containing medium can be introduced into the culture vessel 30 shown in FIG. 2 by injecting the cell-containing medium into the culture vessel 30 through the supply port 231 while discharging the air from the culture vessel 30 through the outlet 331. It is also possible to completely eliminate the air layer in the culture vessel 30. However, an air layer may remain in the culture vessel 30. When the cell-containing medium is already contained in the culture vessel 30, at least a portion of the cell-containing medium in the culture vessel 30 shown in FIG. 2 can be replaced by injecting another cell-containing medium into the culture vessel 30 through the supply port 231 while discharging the cell-containing medium from the culture vessel 30 through the outlet 331 shown in FIG. 1.

[0168] The cell culture vessel according to the embodiment may further include a culture vessel holding member that can hold the culture vessel 30 and adjust the inclination of the culture vessel 30. Adjusting the inclination of the culture vessel 30 makes it easier to discharge gases such as air from within the culture vessel 30.

[0169] The supply port 231 and the discharge port 331 of the culture tank 30 can be closed with a stopper or the like. Alternatively, the plugs 33 and 34 connected to the supply port 231 and the discharge port 331 of the culture tank 30, respectively, can be closed. Alternatively, the supply port 231 of the culture tank 30 can be shielded from the outside by being connected to a supply device, and the discharge port 331 of the culture tank 30 can be shielded from the outside by being connected to a discharge device. When the supply port 231 and the discharge port 331 are closed and the culture tank 30 is tightly attached to the culture medium holding tank 40 as shown in FIG. 2, the inside of the culture tank 30 is sealed from the air outside the culture tank 30. This prevents outside air from entering the culture tank 30, suppressing changes in the pH of the cell-containing culture medium in the culture tank 30 and maintaining it within a predetermined range. Based on the findings of the present inventors, since cells can be cultured in a completely closed, sealed space, it is not necessary to actively supply carbon dioxide gas, nitrogen gas, oxygen gas, or the like to the culture tank 30. Therefore, the culture tank 30 does not need to be placed in a CO2 incubator. Furthermore, since cells, microorganisms, viruses, dust, and the like present outside the culture tank 30 do not enter the sealed culture tank 30, the cleanliness of the inside of the culture tank 30 is maintained. Therefore, the culture tank 30 does not need to be placed in a clean room. The culture tank 30 may be embedded in a gas-impermeable material. In other words, the culture tank 30 may be embedded in a gas-impermeable material.

[0170] The culture medium holding tank 40 shown in Fig. 1 is provided with an opening 140 shown in Fig. 3 for exposing the culture component permeable member 10 through the opening in the culture medium-side plate 22. The opening 140 is covered with the culture component permeable member 10 shown in Fig. 1. The culture medium holding tank 40 shown in Fig. 3 is also provided with an inlet 240 for introducing a fluid into the culture medium holding tank 40 and an outlet 340 for discharging the fluid inside the culture medium holding tank 40. Furthermore, a plurality of rectifying plates 41 may be arranged inside the culture medium holding tank 40. The plurality of rectifying plates 41 are arranged, for example, so as to alternately protrude from opposing inner walls of the culture medium holding tank 40.

[0171] For example, when the culture medium holding tank 40 is in close contact with the culture tank 30 with the culture medium side plate 22, the culture component permeable member 10, and the culture side plate 21 shown in Fig. 1 sandwiched therebetween, and air is present in the culture medium holding tank 40, the cell culture medium can be introduced into the culture medium holding tank 40 by injecting the cell culture medium into the culture medium holding tank 40 from the inlet 240 while discharging the air from the culture medium holding tank 40 from the outlet 340 shown in Fig. 3. Furthermore, when the culture medium is already contained in the culture medium holding tank 40, the cell culture medium can be introduced into the culture medium holding tank 40 by injecting the cell culture medium into the culture medium holding tank 40 from the inlet 240 while discharging the cell culture medium from the culture medium holding tank 40 from the outlet 340, thereby causing the cell culture medium to flow into the culture medium holding tank 40.

[0172] When multiple rectifying plates 41 are arranged in the culture medium holding tank 40, the culture medium flows along the multiple rectifying plates 41 from the inlet 240 toward the outlet 340 in the culture medium holding tank 40. This ensures that the components of the culture medium have an opportunity to come into contact with the culture component permeable member 10.

[0173] Alternatively, as shown in FIG. 4, one or more outlets 241 communicating with the inlet 240 shown in FIG. 3 may be provided on the inner wall of the culture medium holding tank 40. The multiple outlets 241 shown in FIG. 4 are provided, for example, in a horizontal row. The number and arrangement of the multiple outlets 241 may be uniformly or randomly arranged. The number and arrangement of the multiple outlets 241 are set according to the viscosity and other characteristics of the culture medium. As shown in FIG. 5, by discharging the culture medium from the multiple outlets 241, it is possible to improve the uniformity of the culture medium that comes into contact with the culture component permeable member 10 within the culture medium holding tank 40.

[0174] A discharge block 145 having one or more discharge ports 241 may be insertable into the inner wall of the culture medium holding tank 40. For example, discharge blocks 145 having different patterns, such as the number or arrangement of the multiple discharge ports 241, may be prepared and used depending on the characteristics of the culture medium and the cells to be cultured. The upper side of the inner wall of the culture medium holding tank 40 with respect to gravity may be bent or curved upward or downward. The lower side of the inner wall of the culture medium holding tank 40 with respect to gravity may be bent or curved upward or downward.

[0175] 4 and 5, openings 242 may be provided near the multiple discharge ports 241 on the inner wall of the culture medium holding tank 40. As the culture medium discharged from the multiple discharge ports 241 accumulates in the culture medium holding tank 40, air inside the culture medium holding tank 40 flows out through the openings 242. After the culture medium is placed in the culture medium holding tank 40, the openings 242 may be sealed.

[0176] As shown in FIG. 3 , the inlet 240 and outlet 340 of the culture medium holding tank 40 may be connected by a culture medium flow path 200, allowing the culture medium to circulate between the culture medium holding tank 40 and the culture medium flow path 200. The culture medium flow path 200 may include a resin tube, a silicone tube, or the like. The culture medium flow path 200 may be embedded in a gas-impermeable material. In other words, the culture medium flow path 200 may be embedded in a gas-impermeable material. For example, the culture medium flow path 200 may be a hole formed in a member made of resin, glass, metal, or the like. In this case, the culture medium flow path 200 may be formed, for example, by bonding together members having recesses. The culture medium flow path 200 may be provided with a fluid machine for introducing the culture medium into the culture medium holding tank 40 and discharging the culture medium from the culture medium holding tank 40. The fluid machine may include, for example, an introduction fluid machine 51 for introducing the culture medium into the culture medium holding tank 40 and a discharge fluid machine 52 for discharging the culture medium from the culture medium holding tank 40.

[0177] Positive displacement pumps can be used as the introduction fluid machine 51 and the discharge fluid machine 52 shown in FIG. 1. Examples of positive displacement pumps include reciprocating pumps, including piston pumps, plunger pumps, and diaphragm pumps, and rotary pumps, including gear pumps, vane pumps, and screw pumps. Examples of diaphragm pumps include tubing pumps and piezoelectric (piezo) pumps. Tubing pumps are sometimes called peristaltic pumps. Alternatively, a microfluidic chip module combining various types of pumps may be used.

[0178] When a hermetic pump such as a peristaltic pump (registered trademark), a tubing pump, or a diaphragm pump is used, it is possible to send the liquid without the pump coming into direct contact with the culture medium inside the culture medium flow path 200 shown in Fig. 3. Alternatively, a syringe pump may be used as the introduction fluid machine 51 and the discharge fluid machine 52. Pumps other than hermetic pumps can also be reused by subjecting them to heat sterilization or the like.

[0179] When the introduction fluid machine 51 is a hermetic pump, as shown in FIG. 1 , the introduction fluid machine 51 includes a pump head 151 and a drive unit 251 such as a motor. The pump head 151 and the drive unit 251 are detachable. The pump head 151 includes rollers that squeeze a culture medium flow path such as a tube from the outside. The drive unit 251 rotates the rollers of the pump head 151. When the discharge fluid machine 52 is a hermetic pump, the discharge fluid machine 52 includes a pump head 152 and a drive unit 252 such as a motor. The pump head 152 and the drive unit 252 are detachable. The pump head 152 includes rollers that squeeze a culture medium flow path such as a tube from the outside. The drive unit 252 rotates the rollers of the pump head 152.

[0180] 3, a culture medium tank 60 into which the culture medium can be placed may be provided in the culture medium flow path 200. The culture medium that has entered the culture medium tank 60 from the culture medium flow path 200 flows out again into the culture medium flow path 200. By providing the culture medium tank 60, it is possible to increase the volume of the culture medium circulating between the culture medium flow path 200 and the culture medium holding tank 40.

[0181] The culture medium tank 60 may be provided with a supply port for supplying a fluid into the culture medium tank 60 and a discharge port for discharging the fluid from the culture medium tank 60. For example, a plug 61 shown in FIG. 6 is inserted into the supply port of the culture medium tank 60. The plug 61 can be connected to a supply device, such as a bag, bellows, or syringe, for supplying a fluid. The supply device may be a fluid machine, such as a pump. However, the supply device may be directly connected to the supply port of the culture medium tank 60. The supply device is detachable from the supply port. When the supply device is not connected to the supply port, the supply port can be sealed, and no fluid is exchanged between the inside and outside of the culture medium flow path 200 through the supply port. Alternatively, the supply port is sealed from the outside by being connected to the supply device. The plug 61 may be a needleless connector. The needleless connector may be a split septum type or a mechanical valve type. Even when a supply device or a flow path connected to the supply device is inserted into the plug 61, outside air does not enter the culture medium tank 60 through the plug 61. However, the plug 61 may also be a connector into which a needle is inserted.

[0182] Furthermore, for example, a plug 62 to which an ejector such as a bag, bellows, or syringe can be connected for discharging fluid from the culture medium tank 60 is inserted into the outlet of the culture medium tank 60. The ejector may be a fluid machine such as a pump. However, the ejector may also be directly connected to the outlet of the culture medium tank 60. The ejector may actively suck fluid from the culture medium flow path. Alternatively, the ejector may passively increase its internal volume in response to the pressure in the culture medium flow path to receive fluid pushed out from the culture medium flow path. The ejector is detachable from the outlet. When the ejector is not connected to the outlet, the outlet is sealable, and no fluid is exchanged between the inside and outside of the culture medium flow path 200 through the outlet. Alternatively, the outlet is sealed from the outside by being connected to the ejector. The plug 62 may be a needleless connector. The needleless connector may be a split septum type or a mechanical valve type. Even when an ejector or a flow path connected to an ejector is inserted into the plug 62, outside air does not enter the culture medium tank 60 through the plug 62. However, the plug 62 may be a connector into which a needle is inserted.

[0183] 1 is in close contact with the culture tank 30 with the culture medium side plate 22, the culture component permeable member 10, and the culture side plate 21 sandwiched therebetween, and when air is present in the culture medium holding tank 40, the culture medium flow path 200, and the culture medium tank 60 shown in FIG. 3, the culture medium can be placed in the culture medium holding tank 40, the culture medium flow path 200, and the culture medium tank 60 by injecting the culture medium into the culture medium holding tank 40, the culture medium flow path 200, and the culture medium tank 60 from the supply port of the culture medium tank 60 while discharging the air from the culture medium holding tank 40, the culture medium flow path 200, and the culture medium tank 60. The air layer in the culture medium holding tank 40, the culture medium flow path 200, and the culture medium tank 60 may be completely eliminated, or an air layer may remain.

[0184] A supply device filled with culture medium and an empty discharge device may be connected to the culture medium flow path 200, and a fluid machine may be driven to introduce culture medium from the supply device into the culture medium flow path 200 and introduce air into the discharge device. In this case, the supply device may actively inject culture medium into the culture medium flow path 200, or the culture medium in the supply device may be sucked into the culture medium flow path 200, which has been made low-pressure by the driving of the fluid machine, thereby passively reducing the internal volume of the supply device. Furthermore, the discharge device may actively suck air from the culture medium flow path 200, or the air in the culture medium flow path 200, which has been made high-pressure by the driving of the fluid machine, may flow into the discharge device, thereby passively increasing the internal volume of the discharge device.

[0185] In addition, when culture medium is already contained in the culture medium holding tank 40, the culture medium flow path 200, and the culture medium tank 60, it is possible to replace the cell culture medium in the culture medium tank 60 by discharging the culture medium in the culture medium tank 60 from the outlet of the culture medium tank 60 while injecting the culture medium into the culture medium tank 60 from the supply port of the culture medium tank 60.

[0186] A supply device filled with medium and an empty discharge device may be connected to the medium flow path 200, and a fluid machine may be driven to introduce new medium from the supply device into the medium flow path 200 and introduce old medium into the discharge device. In this case, the supply device may actively inject new medium into the medium flow path 200, or the new medium in the supply device may be sucked into the medium flow path 200, which has been made low-pressure by the driving of the fluid machine, thereby passively reducing the internal volume of the supply device. Alternatively, the discharge device may actively suck old medium from the medium flow path 200, or the old medium in the medium flow path 200, which has been made high-pressure by the driving of the fluid machine, may flow into the discharge device, thereby passively increasing the internal volume of the discharge device.

[0187] The supply port for supplying the culture medium into the culture medium flow path 200 and the culture medium holding tank 40 and the outlet for discharging the air inside the culture medium flow path 200 and the culture medium holding tank 40 may be provided in a portion of the culture medium flow path 200 other than the portion where the culture medium tank 60 is provided. For example, the supply port for supplying the culture medium into the culture medium flow path 200 and the culture medium holding tank 40 and the outlet for discharging the air inside the culture medium flow path 200 and the culture medium holding tank 40 may be provided in the culture medium flow path 200.

[0188] The cell incubator according to the embodiment may include a temperature adjustment unit for heating and cooling at least one of the medium holding tank 40, the medium flow path 200, and the medium tank 60. The temperature of the medium can be adjusted by adjusting the temperature of any of the medium holding tank 40, the medium flow path 200, and the medium tank 60 with the temperature adjustment unit. The cell incubator according to the embodiment may further include a thermometer for measuring the temperature of the medium. The thermometer may measure the temperature of the medium based on the temperature of at least one of the medium holding tank 40, the medium flow path 200, and the medium tank 60 without contacting the medium, or may directly measure the temperature of the medium by contacting the medium. In this case, the temperature adjustment unit may be feedback-controlled so that the temperature of the medium is maintained at a predetermined temperature.

[0189] As shown in FIG. 3 , the culture medium holding tank 40, the culture medium flow path 200, the pump heads 151 and 152, and the culture medium tank 60 may be housed in a flow path case 70. Within the flow path case 70, the culture medium holding tank 40, the culture medium flow path 200, the pump heads 151 and 152, and the culture medium tank 60 may be completely embedded in a gas-impermeable material. The culture medium flow path 200 may be provided in the gas-impermeable material in the form of a tunnel. For example, the flow path case 70 may be provided with a hole for inserting a shaft into the pump head 151, a hole for inserting a shaft into the pump head 152, a hole for inserting a plug 61 into the supply port of the culture medium tank 60, and a hole for inserting a plug 62 into the outlet of the culture medium tank 60. The hole for inserting the plug 61 into the supply port of the culture medium tank 60 and the hole for inserting the plug 62 into the outlet of the culture medium tank 60 may be blockable.

[0190] 7, the drive unit 251 of the introduction fluid machine 51 and the drive unit 252 of the discharge fluid machine 52 may be disposed on a substrate-like drive unit holding member 80. The drive unit holding member 80 is provided with a hole for inserting a plug 61 into the supply port of the culture medium tank 60 and a hole 82 for inserting a plug 62 into the discharge port of the culture medium tank 60. The hole for inserting the plug 61 into the supply port of the culture medium tank 60 and the hole 82 for inserting the plug 62 into the discharge port of the culture medium tank 60 may be blockable.

[0191] The drive unit holding member 80 is brought into close contact with the flow path case 70 via a packing 90 shown in Fig. 1. The packing 90 prevents air from entering the flow path case 70 from the contact portion between the flow path case 70 and the drive unit holding member 80.

[0192] The fluid machine for introducing the culture medium into the culture medium retention tank 40 and discharging the culture medium from the culture medium retention tank 40 may be covered with an outside air blocking member for the fluid machine. For example, as shown in Fig. 7 , the outside air blocking member for the fluid machine includes an outside air blocking member for the introduction fluid machine 351 that covers the drive unit 251 of the introduction fluid machine 51 that is arranged in the drive unit holding member 80, and an outside air blocking member for the discharge fluid machine 352 that covers the drive unit 252 of the discharge fluid machine 52 that is arranged in the drive unit holding member 80.

[0193] The flow path case 70 and the drive unit holding member 80 are detachable. When the drive unit holding member 80 is tightly attached to the flow path case 70, the hole for inserting the plug 61 into the supply port of the culture medium tank 60 and the hole for inserting the plug 62 into the discharge port of the culture medium tank 60 are blocked, the drive unit 251 of the introduction fluid machine 51 is covered with the outside air blocking member 351 for the introduction fluid machine, and the drive unit 252 of the discharge fluid machine 52 is covered with the outside air blocking member 352 for the discharge fluid machine, the inside of the flow path case 70 is blocked from the outside air, and outside air cannot enter the flow path case 70. Therefore, gas exchange between the inside and outside of the flow path case 70 does not occur. Therefore, outside air does not enter the culture medium retention tank 40 and the culture medium flow path 200. By blocking the inside of the flow path case 70, which constitutes at least a part of the outside air blocking member for the culture medium flow path, from the outside air, it is possible to suppress fluctuations in the pH of the culture medium in the culture medium holding tank 40 and the culture medium flow path 200 and keep it within a specified range, even if the culture medium flow path 200 is a gas-permeable tube.

[0194] Furthermore, based on the findings of the present inventors, cells can be cultured in a completely sealed space, so there is no need to actively supply carbon dioxide gas, nitrogen gas, oxygen gas, etc. to the culture medium holding tank 40 and the culture medium flow path 200. Therefore, the culture medium holding tank 40 and the culture medium flow path 200 do not need to be placed in a CO2 incubator. Furthermore, since cells, microorganisms, viruses, dust, etc. present outside the culture medium holding tank 40 and the culture medium flow path 200 do not enter the sealed culture medium holding tank 40 and the culture medium flow path 200, the cleanliness of the culture medium holding tank 40 and the culture medium flow path 200 is maintained. Therefore, the culture medium holding tank 40 and the culture medium flow path 200 do not need to be placed in a clean room. The culture medium holding tank 40 may be embedded in a gas-impermeable material. In other words, the culture medium holding tank 40 may be embedded in a gas-impermeable material.

[0195] When the drive unit holding member 80 is removed from the flow path case 70, the hole in the flow path case 70 for inserting the plug 61 into the supply port of the culture medium tank 60 and the hole in the flow path case 70 for inserting the plug 62 into the discharge port of the culture medium tank 60 are blocked, thereby sealing the inside of the flow path case 70 and preventing substances inside the flow path case 70 from leaking out to the outside or outside air from entering the flow path case 70.

[0196] The channel case 70, which contains the culture medium channel 200 and the pump heads 151 and 152, is disposable. On the other hand, the drive unit holding member 80, which holds the drive units 251 and 252, can be reused repeatedly.

[0197] For example, the introduction fluid machine 51 and the discharge fluid machine 52 are controlled so that the amount of culture medium delivered into the culture medium holding tank 40 by the introduction fluid machine 51 shown in Fig. 2 is the same as the amount of culture medium discharged from the culture medium holding tank 40 by the discharge fluid machine 52. The introduction fluid machine 51 and the discharge fluid machine 52 may deliver the culture medium into the culture medium holding tank 40 at all times, or may deliver the culture medium at appropriate intervals.

[0198] When the culture medium is constantly fed into the culture medium holding tank 40, the flow rate of the culture medium fed into the culture medium holding tank 40 may or may not be constant. For example, the culture medium and the cell aggregates in the culture medium may be monitored with an imaging device, and the flow rate of the culture medium fed into the culture medium holding tank 40 may be increased or decreased depending on the state of the culture medium and the cell aggregates in the culture medium.

[0199] Furthermore, instead of constantly feeding the medium into the medium holding tank 40, the feeding of the medium may be started and stopped depending on, for example, the state of the medium, the state of the cell clumps in the medium, the number of cells, the number of cell clumps, the turbidity of the medium, and changes in pH. In this case, too, the flow rate of the medium being fed may be increased or decreased depending on the state of the medium and the cell clumps in the medium.

[0200] In agitated culture media, cells may randomly collide and combine to form cell clusters (colonies) of various sizes. This can result in a lack of uniformity among colonies. Furthermore, in colonies that are too large, nutrients and growth factors may not reach the colony interior, leading to differentiation and cell death from within. On the other hand, colonies that are too small may not be suitable for subculture. In contrast, in the culture tank 30 shown in Figure 2, the medium flow rate is slow or the medium does not flow, so cells rarely collide with each other. This makes it possible to maintain clonality within the colony. Therefore, for example, when the cells are stem cells such as iPS cells, it is possible to ensure the clonality of stem cells derived from a single cell. Furthermore, because the frequency of collisions between stem cells is low, it is possible to maintain uniformity in the size of stem cell colonies.

[0201] The cell culture vessel according to the embodiment may further include a photographing device such as a photo camera or video camera that photographs the cell-containing culture medium in the culture vessel 30 through the window 132 of the cover 32 of the culture vessel 30 .

[0202] According to the cell culture vessel of the embodiment, for example, cells are cultured in a completely closed system, which makes it possible to reduce the risk of cross-contamination due to cells leaking from the culture device. Furthermore, even if cells are infected with a virus such as HIV hepatitis virus, it is possible to reduce the risk of infection to the operator due to cell leakage. Furthermore, it is possible to reduce the risk of the medium in the cell culture vessel being contaminated by bacteria, viruses, mold, etc. in the air outside the cell culture vessel. Furthermore, according to the cell culture vessel of the embodiment, it is also possible to culture cells without using a CO2 incubator.

[0203] For example, if circulation of the culture medium is not required, the culture medium flow path 200 does not need to be connected to the culture medium holding tank 40 shown in FIG. 3. Furthermore, cells may be cultured in suspension or in adhesion culture in the culture tank 30. When cells are cultured in adhesion culture, the surface of the culture-side plate 21 shown in FIG. 1 may be cell-adhesive, or the surface of the culture component permeable member 10 may be cell-adhesive. Furthermore, cells may be guided while being cultured in the culture tank 30 of the cell culture device according to the embodiment. Furthermore, the culture medium flow path may be used without being connected to a culture medium holding tank or a culture tank, and cells may be cultured or guided in the culture medium flow path as a closed system.

[0204] The closed system is not limited to the cell culture vessels shown in Figures 1 to 7. For example, the closed system may be a container. The container may be a tube or a flask. The container may be made of resin or glass. To completely close the inside of the container, a film such as paraffin film may be wrapped around the cap or lid of the container. [Example]

[0205] Example 1 Stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher Scientific)) was gelled to prepare a gel medium. The pH of the gel medium was adjusted to between 4.0 and 10.0. 2 × 10 iPS cells, either single cells or cell clusters, were added to the gel medium. 5 15 mL of iPS cells / mL was added. The gel medium containing iPS cells was placed in 15 mL Falcon tubes (Corning, registered trademark). The caps of some of the Falcon tubes were then tightly fastened, and the Falcon tubes and caps were wrapped with paraffin film (Parafilm, registered trademark, Bemis) to isolate the Falcon tubes from the outside air and prevent complete exchange of gas (air) within the Falcon tubes with the outside air. The other Falcon tubes were simply capped, without wrapping with paraffin film.

[0206] The Falcon tubes without paraffin film were placed in a 37°C incubator with a 5% CO2 concentration to initiate suspension culture of iPS cells. The Falcon tubes wrapped in paraffin film were also placed in a 37°C incubator without a CO2 incubator to initiate suspension culture of iPS cells. Electronically controlled temperature-controlled bead baths, water baths, and incubators were used as incubators. The incubators were located in the laboratory and were not shielded from the laboratory air. Every two days, the caps of each Falcon tube were opened, and 2 mL of gel medium (pH between 4.0 and 10.0) was added to the Falcon tube. After adding the gel medium, the caps of the Falcon tubes to be placed in the incubator were tightened and wrapped around with paraffin film as described above.

[0207] Seven to 10 days after the start of culture in the Falcon tube, the cap of the Falcon tube was opened, and the iPS cell clumps formed in the gel medium were collected using a filter, washed with PBS, and placed in the Falcon tube. 500 μL of cell dissociation reagent (TrypLE Select®, ThermoFisher) was added to the cell clumps, and the cell clumps were incubated in a CO2 incubator for 5 minutes. Next, the Falcon tube was removed from the incubator, and 500 μL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR®, ThermoFisher SCIENTIFIC) was added to the Falcon tube to suspend the cell clumps and disaggregate the iPS cells into single cells. 2 mL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR®, ThermoFisher SCIENTIFIC) was added to the Falcon tube, and the Falcon tube was centrifuged at 200 g in a centrifuge. After centrifugation, the supernatant was removed from the Falcon tube, and the iPS cells were placed in the gel medium. The iPS cells were then cultured in suspension in the sealed Falcon tube for 7 to 10 days, with the gel medium being added every two days, as described above.

[0208] Thereafter, the same subculture and suspension culture for 7 to 10 days were repeated as above, and the iPS cells were cultured in suspension in a sealed Falcon tube for a total of more than one month.

[0209] When iPS cells cultured in Falcon tubes placed in an incubator and iPS cells cultured in Falcon tubes placed in a bead bath were observed under a microscope, it was confirmed that both had formed uniform cell masses, as shown in Figure 8. Similar results were obtained for iPS cells cultured in Falcon tubes placed in an incubator other than a bead bath.

[0210] During the passage, a portion of the single-cell iPS cells was aliquoted and fixed with 4% paraformaldehyde. The expression level of the cell surface antigen TRA-1-60 in the fixed iPS cells was measured using a flow cytometer. TRA-1-60 is a representative surface antigen of pluripotent stem cells, and its expression level is known to decrease in differentiated cells.

[0211] As shown in Figure 9, on days 8, 28, and 38 after the start of culture, nearly 100% of iPS cells cultured in Falcon tubes placed in an incubator and iPS cells cultured in Falcon tubes placed in a bead bath were TRA1-60 positive. Similar results were obtained with iPS cells cultured in Falcon tubes placed in a thermostatic chamber other than a bead bath. This demonstrates that by sealing the container in a closed system, stem cells can be cultured for long periods of time while maintaining pluripotency in an undifferentiated state, even without controlling the carbon dioxide concentration within the container.

[0212] Example 2 A gel medium was prepared in the same manner as in Example 1. 2 × 10 iPS cells were dissociated into single cells and placed in the gel medium. 51000 / mL of iPS cells were added. 2 mL of gel medium containing iPS cells was placed in a 2 mL gas-impermeable tube with a rubber packing, ensuring that no air layer remained inside. The tube's cap was then tightly fastened, and the tube and cap were wrapped in paraffin film to isolate the inside of the tube from the outside air and prevent outside air from entering the tube. This prevented the gel medium from coming into contact with the gas (air) layer during culture.

[0213] The tubes wrapped in paraffin film were placed inside a 37°C CO2 incubator and a 37°C incubator outside the CO2 incubator to initiate suspension culture of iPS cells. The incubators were electronically temperature-controlled bead baths, water baths, and incubators. The incubators were located in the laboratory and were not shielded from the laboratory air. No medium was added or replaced during the culture period. After 10 to 11 days of culture in the tubes, the caps were opened, and the iPS cell clumps formed in the gel medium were collected using a filter, washed with PBS, and placed in a tube. 500 μL of cell dissociation reagent (TrypLE Select, registered trademark, Thermo Fisher Scientific) was added to the cell clumps, and the cell clumps were incubated in a CO2 incubator for 5 minutes. Next, the tube was removed from the incubator, and 500 μL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) was added to the tube to suspend the cell clumps and break the iPS cells into single cells. 2 mL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) was added to the tube, and the tube was centrifuged at 200 g using a centrifuge. After centrifugation, the supernatant in the tube was removed, and the number of iPS cells was determined to be 2 × 10 5 The gel medium was poured into the tube so that the concentration of iPS cells was 1 / mL. Thereafter, the iPS cells were cultured in suspension in the sealed tube for 5 to 11 days without adding or replacing the gel medium, as described above.

[0214] Thereafter, the same subculture and suspension culture for 5 to 11 days were repeated as above, and the iPS cells were cultured in suspension in the sealed tube for a total of more than one month.

[0215] When the iPS cells cultured in the tubes placed inside the incubator were observed with a camera and a microscope, it was confirmed that they had formed uniform cell masses, as shown in Figure 10. Similar results were obtained with iPS cells cultured in tubes placed in a thermostatic bath outside the incubator.

[0216] During the passaging process, a portion of the single-cell iPS cells was aliquoted and fixed with 4% paraformaldehyde. The expression levels of the cell surface antigen TRA-1-60 in the fixed iPS cells were then measured using a flow cytometer. As shown in Figure 11, on days 10, 21, and 30 after the start of culture, over 90% of the iPS cells cultured in the incubator were TRA1-60 positive. Similar results were obtained with iPS cells cultured in a thermostatic chamber outside the incubator. This demonstrates that by sealing the container, stem cells can be cultured for long periods of time while maintaining their undifferentiated pluripotency, without controlling the carbon dioxide concentration inside the container or adding or replacing the medium.

[0217] Example 3 A gel medium was prepared in the same manner as in Example 1. Single-cell iPS cells were added to the gel medium. 2 mL of the gel medium containing iPS cells was placed in a 15 mL Falcon tube. The cap of the Falcon tube was then tightly fastened.

[0218] The Falcon tube was placed in a CO2 incubator at 37°C to initiate suspension culture of iPS cells. Every two days, the cap of the Falcon tube was opened and 2 mL of gel medium (pH 4.0 to 10.0) was added to the tube. After adding the gel medium, the cap was tightened as described above.

[0219] After 7 to 10 days of culture in the Falcon tube, the cap of the Falcon tube was opened, and the iPS cell clumps formed in the gel medium were collected using a filter, washed with PBS, and placed in the Falcon tube. 500 μL of cell dissociation reagent (TrypLE Select®, ThermoFisher) was added to the cell clumps, and the cell clumps were incubated in a CO2 incubator for 5 minutes. The Falcon tube was then removed from the incubator, and 500 μL of medium containing 20% ​​KnockOut SR® (ThermoFisher SCIENTIFIC), GlutaMAX® (ThermoFisher SCIENTIFIC), and non-essential amino acids (NEAA) was added to the Falcon tube to suspend the cell clumps and disaggregate the iPS cells into single cells. 2 mL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) was added to the Falcon tube, and the Falcon tube was centrifuged at 200 g using a centrifuge. After centrifugation, the supernatant in the Falcon tube was removed, and the number of iPS cells was determined to be 2 × 10 5 The gel medium was placed in a Falcon tube so that the iPS cells would reach a density of 100 cells / mL. The iPS cells were then cultured in suspension in the sealed Falcon tube for 7 to 10 days, with the gel medium being added every two days, as described above.

[0220] Thereafter, the iPS cells were subjected to the same subculture and suspension culture for 7 to 10 days as described above, and the cells were subjected to suspension culture in a sealed Falcon tube for a total of one month or more.

[0221] When the iPS cells cultured in the Falcon tubes were observed under a microscope, it was confirmed that all of them had formed cell clusters, as shown in Figure 12. Furthermore, when the expression level of the cell surface antigen TRA-1-60 in the iPS cells was measured using a flow cytometer in the same manner as in Example 3, as shown in Figure 13, almost 100% of the iPS cells were TRA-1-60 positive from days 7 to 21 after the start of culture.

[0222] Example 4 Growth factors were added to the medium (StemSpan H3000, registered trademark, STEMCELL Technologies Inc.), and deacylated gellan gum was further added to the medium to prepare a gel medium.

[0223] Place the prepared gel medium in a 15 mL tube and add 2 × 10 5 Blood cells (mononuclear cells) were seeded into the gel medium. The 15 mL tube was then placed in a CO2 incubator at 37°C and cultured for 7 days. A Sendai virus vector (CytoTune-iPS2.0, ID Pharma Co., Ltd.) carrying OCT3 / 4, SOX2, KLF4, and cMYC was then added to the gel medium at a multiplicity of infection (MOI) of 10.0, and the blood cells were infected with the Sendai virus.

[0224] After adding Sendai virus to the gel medium, 30 mL of stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) was added to the gel medium. The medium containing the Sendai virus-infected cells was then placed into the flask seeded with feeder cells. The flask was left for 15 days, and the Sendai virus-infected cells were cultured as adherents. There was no air space inside the flask. During this time, as shown in Figure 14, the flask cap was wrapped around the periphery with paraffin film to completely close the flask. No medium or gas exchange was performed, and the CO2 concentration inside the flask was not controlled.

[0225] After 15 days, the cells were observed under a microscope, and it was confirmed that ES cell-like colonies had formed, as shown in Figure 15. As shown in Figure 16, nearly 100% of the colonies were ES cell-like. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using a flow cytometer. As shown in Figure 17(a), nearly 100% of the cells before induction were TRA-1-60 negative. However, as shown in Figure 17(b), nearly 100% of the cells after induction were TRA-1-60 positive, confirming nearly complete reprogramming. This demonstrates that iPS cells can be induced from cells other than stem cells in a completely closed environment without medium or gas exchange.

[0226] Example 5 The gel medium prepared in the same manner as in Example 4 was placed in a 15 mL tube, and 2 × 10 5 Blood cells (mononuclear cells) were seeded into the gel medium. The 15 mL tube was then placed in a CO2 incubator at 37°C and cultured for 7 days. A Sendai virus vector (CytoTune-iPS2.0, ID Pharma Co., Ltd.) carrying OCT3 / 4, SOX2, KLF4, and cMYC was then added to the gel medium at a multiplicity of infection (MOI) of 10.0, and the blood cells were infected with the Sendai virus.

[0227] After adding Sendai virus to the gel medium, 15 mL of gelled stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) was added to the gel medium, and 15 mL of the medium containing the Sendai virus-infected cells was placed in a 15 mL tube. The 15 mL tube was left for 15 days, and the Sendai virus-infected cells were cultured in suspension. There was no air space inside the 15 mL tube. During this time, the 15 mL tube was completely closed, and no medium or gas exchange was performed, nor was the CO2 concentration inside the 15 mL tube controlled.

[0228] After 15 days, the cells were observed under a microscope, and it was confirmed that ES cell-like colonies had been formed, as shown in Figure 18. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using a flow cytometer. As shown in Figure 19, nearly 100% were TRA-1-60 positive, confirming nearly complete reprogramming. Therefore, it was demonstrated that iPS cells can be induced from cells other than stem cells in a completely closed environment without medium or gas exchange.

[0229] Example 6 As shown in Figures 20 and 21, a semipermeable membrane 110 (Asahi Kasei Corporation or SPECTRUM) was sandwiched between a culture side plate 21 and a medium side plate 22, and further, the semipermeable membrane 110, the culture side plate 21 and the medium side plate 22 were sandwiched between a culture tank 30 and a medium holding tank 40.

[0230] Gel medium was prepared by gelling stem cell medium (ReproCell) containing 20% ​​serum replacement (KnockOut SR, registered trademark, Gibco). 2 × 10 iPS cells were single-celled and placed in the gel medium. 5 Cells / mL were added to prepare a cell-containing medium.

[0231] The cell-containing medium was placed in a syringe, and the syringe was connected to the supply port 231 of the culture tank 30 via a plug 33. An empty syringe was also connected to the outlet 331 of the culture tank 30 via a plug 34. Next, the cell-containing medium in the syringe was injected into the culture tank 30 through the supply port 231 of the culture tank 30. Due to the increase in pressure within the culture tank 30, the piston of the syringe connected to the outlet 331 passively rose, and the air within the culture tank 30 moved into the syringe connected to the outlet 331 of the culture tank 30. The cell-containing medium was injected into the culture tank 30 until the air layer within the culture tank 30 was completely eliminated. Then, the supply port 231 and the outlet 331 of the culture tank 30 were covered.

[0232] The gel medium was placed in a syringe, and the syringe was connected to the inlet 240 of the culture medium holding tank 40 via plug 61. An empty syringe was connected to the outlet 340 of the culture medium holding tank 40 via plug 62. The gel medium in the syringe was then injected into the culture medium holding tank 40 via the inlet 240 of the culture medium holding tank 40. Due to the increase in pressure within the culture medium holding tank 40, the syringe connected to the outlet 340 of the culture medium holding tank 40 passively rose, and the air within the culture medium holding tank 40 moved into the syringe connected to the outlet 340 of the culture medium holding tank 40. The gel medium was injected into the culture medium holding tank 40 until the air layer within the culture medium holding tank 40 was completely eliminated. The inlet 240 and outlet 340 of the culture medium holding tank 40 were then sealed. This sealed the interiors of the culture tank 30 and the culture medium holding tank 40, completely preventing gas exchange between the interior and exterior of the culture tank 30 and the culture medium holding tank 40.

[0233] Suspension culture of iPS cells was initiated in the culture vessel 30. Subsequently, 2 mL of gel medium in the medium holding vessel 40 was replaced with 2 mL of fresh gel medium every two days. Seven to 10 days after the start of culture in the culture vessel 30, the cell-containing medium in the culture vessel 30 was removed with a syringe, and the iPS cell clumps formed in the gel medium were collected using a filter, washed with PBS, and placed in a Falcon tube. 500 μL of cell dissociation enzyme (TrypLE Select, Thermo Fisher) was added to the cell clumps, and the cell clumps were incubated in a CO2 incubator for 5 minutes. Next, the Falcon tube was removed from the incubator, and 500 μL of cell medium was added to the Falcon tube. The cell clumps were suspended and the iPS cells were dissociated into single cells. 2 mL of cell medium was added to the Falcon tube, and the Falcon tube was centrifuged at 200 g in a centrifuge. After centrifugation, the supernatant was removed, and the iPS cells and gel medium were placed in the Falcon tube to prepare cell-containing medium. Thereafter, in the same manner as above, the cell-containing medium was poured into the culture vessel 30, and the iPS cells were cultured in suspension for 7 to 10 days while replacing 2 mL of gel medium in the medium holding vessel 40 every two days.

[0234] Thereafter, the same subculture and suspension culture for 7 to 10 days were repeated as above, and the iPS cells were suspension cultured in the sealed culture vessel 30 for a total of one month or more.

[0235] When the iPS cells cultured in the culture vessel 30 were observed under a microscope, it was confirmed that all of them had formed uniform cell masses, as shown in FIG.

[0236] During the passaging process, a portion of the single-cell iPS cells was aliquoted and fixed using 4% paraformaldehyde. The expression level of the cell surface antigen TRA-1-60 in the fixed iPS cells was then measured using a flow cytometer. As shown in Figure 23, over 90% of the iPS cells were TRA-1-60 positive on day 39 of culture. This demonstrates that by sealing the container, stem cells can be cultured for long periods of time while maintaining pluripotency in an undifferentiated state, even without controlling the carbon dioxide concentration inside the container.

[0237] Example 7 A cell-containing medium was prepared in the same manner as in Example 6. A cell culture vessel similar to that shown in FIG. 2 was also prepared. The cell-containing medium was poured into the culture vessel 30 until the air layer inside the culture vessel 30 was completely eliminated. The supply port and outlet of the culture vessel 30 were then sealed. The culture medium holding vessel 40, the culture medium flow path 200, and the culture medium tank 60 were filled with gel culture medium. The inlet and outlet of the culture medium tank 60 were then sealed. This sealed the interiors of the culture vessel 30 and the culture medium holding vessel 40, completely preventing gas exchange between the inside and outside of the culture vessel 30 and the culture medium holding vessel 40.

[0238] Gel medium was circulated in the medium holding tank 40, the medium flow path 200, and the medium tank 60, and suspension culture of iPS cells was initiated in the culture tank 30. Thereafter, 10 mL of gel medium in the culture tank 60 was replaced with 10 mL of fresh gel medium every two to six days. Seven to ten days after the start of culture in the culture tank 30, the cell-containing medium in the culture tank 30 was discharged with a syringe, and the same subculture treatment as in Example 6 was carried out. The cell-containing medium was then injected into the culture tank 30 in the same manner as above, and the iPS cells were suspension cultured for seven to ten days while the 10 mL of gel medium in the culture tank 60 was replaced every four days.

[0239] Thereafter, the same subculture and suspension culture for 7 to 10 days were repeated as above, and the iPS cells were suspension cultured in the sealed culture vessel 30 for a total of one month or more.

[0240] When the iPS cells cultured in the culture vessel 30 were observed under a microscope, it was confirmed that all of them had formed uniform cell masses, as shown in Figure 24. Furthermore, when the expression level of the cell surface antigen TRA-1-60 in the iPS cells was measured using a flow cytometer in the same manner as in Example 6, as shown in Figure 25, almost 100% of the iPS cells on day 15 from the start of culture were TRA-1-60 positive.

[0241] Example 8 Growth factors were added to the medium (StemSpan H3000, registered trademark, STEMCELL Technologies Inc.), and deacylated gellan gum was further added to the medium to prepare a gel medium.

[0242] Place the prepared gel medium in a 15 mL tube and add 2 × 10 5 100 blood cells were seeded. The 15 mL tube was then placed in a CO2 incubator and the blood cells (mononuclear cells) were cultured for 7 days. A Sendai virus vector (CytoTune-iPS2.0, ID Pharma Co., Ltd.) carrying OCT3 / 4, SOX2, KLF4, and cMYC was then added to the gel medium at a multiplicity of infection (MOI) of 10.0, and the blood cells were infected with the Sendai virus.

[0243] After adding the Sendai virus to the gel medium, 15 mL of gelled stem cell medium (DMEM / F12 containing 20% ​​KnockOut SR (registered trademark, ThermoFisher SCIENTIFIC)) was added to the gel medium, and 15 mL of the medium containing cells infected with the Sendai virus was placed in the culture tank 30 shown in Figures 20 and 21, and the gel medium was poured into the culture medium holding tank 40. As in Example 6, the insides of the culture tank 30 and the culture medium holding tank 40 were sealed to completely prevent gas exchange between the inside and outside of the culture tank 30 and the culture medium holding tank 40.

[0244] Suspension culture of the cells into which the inducer was introduced was initiated in the culture vessel 30. Thereafter, 2 mL of gel medium in the medium holding vessel 40 was replaced with 2 mL of fresh gel medium every two days.

[0245] After 15 days, the cells were observed under a microscope, and it was confirmed that ES cell-like colonies had formed, as shown in Figure 26. Furthermore, the cells were fixed with 4% paraformaldehyde, and the expression level of the cell surface antigen TRA-1-60 in the fixed cells was measured using a flow cytometer. As shown in Figure 27, over 90% were TRA-1-60 positive, confirming almost complete reprogramming. Therefore, it was demonstrated that iPS cells can be induced from cells other than stem cells in a completely closed environment without medium or gas exchange. [Explanation of symbols]

[0246] 10...culture component permeable member, 21...culture side plate, 22...culture medium side plate, 30...culture tank, 31...casing, 32...cover, 33...plug, 34...plug, 40...culture medium holding tank, 41...rectifying plate, 51...inlet fluid machine, 52...discharge fluid machine, 60...culture medium tank, 61...plug, 62...plug, 70...flow path case, 80...drive unit holding member, 82...hole, 90...packing, 110...semipermeable membrane, 131...opening, 132...window, 140...opening, 145...discharge block, 151...po pump head, 152 pump head, 200 medium flow path, 231 supply port, 240 inlet port, 241 outlet port, 242 opening, 251 drive unit, 252 drive unit, 331 outlet port, 340 outlet port, 351 outside air blocking member for inlet fluid machine, 352 outside air blocking member for outlet fluid machine, 401 input device, 402 output device, 403 related storage device, 501 image processing unit, 511 contour definition unit, 512 cell evaluation unit, 513 statistical processing unit, 514 density calculation unit, 515 medium evaluation unit

Claims

1. The method comprises culturing mononuclear cells into which reprogramming factors have been introduced without changing the medium in a closed system without an air layer, in which gas exchange does not occur between the inside and the outside, until the mononuclear cells become positive for a cell marker indicating undifferentiated cells; In the culturing, the closed system is sealed, When the closed system is sealed, outside air does not enter the closed system. Cell culture methods.

2. The method of claim 1 further comprising controlling the temperature within the closed system.

3. The method according to claim 1 or 2, wherein the closed system is sealed and cells, microorganisms, viruses, and dust particles outside the closed system do not enter the closed system.

4. The method according to claim 1 , wherein when the closed system is sealed, substances in the closed system do not flow out of the closed system.

5. The method according to any one of claims 1 to 4, wherein at least one of carbon dioxide gas, nitrogen gas, and oxygen gas is not supplied into the closed system.

6. 6. The method of claim 1, wherein the pH of the medium in the closed system is maintained within a predetermined range.

7. 7. The method of claim 1, wherein at least a portion of the closed system is formed by embedding in a gas-impermeable material.

8. 7. The method of claim 1, wherein the closed system is made of a gas-impermeable material.

9. The method according to any one of claims 1 to 8, wherein the carbon dioxide concentration in the closed system is not controlled during the culturing.

10. The method according to any one of claims 1 to 9, wherein the carbon dioxide concentration outside the closed system is not controlled during the culturing.

11. The method of any one of claims 1 to 10, wherein the culture is a suspension culture.

12. The method of any one of claims 1 to 10, wherein the culture is an adherent culture.

13. 13. The method according to any one of claims 1 to 12, wherein the culturing is carried out in a gel medium within the closed system.

14. 13. The method according to any one of claims 1 to 12, wherein the culturing is carried out in a liquid medium in the closed system.

15. 15. The method of any one of claims 1 to 14, wherein the culture medium in the closed system is stirred.

16. 15. The method of any one of claims 1 to 14, wherein the culture medium in the closed system is not stirred.

17. The method of claim 1 , wherein the reprogramming factors are contained in a plasmid.

18. The method of claim 1 , wherein the reprogramming factor is RNA.

19. The method of claim 1 , wherein the reprogramming factor is contained in a Sendai virus.

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