Cell incubator

Abstract

To provide devices capable of efficiently culturing a variety of cells in addition to iPS cells.SOLUTION: A cell incubator comprises: 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 is configured for holding a cell-containing medium and culturing cells; and a medium-holding vessel 40 that covers the other side of the culture component permeable member 10 and configured for holding the medium.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to cell technology and relates to a cell incubator.

Background Art

[0002] Embryonic stem cells (ES cells) are stem cells established from early embryos of humans and mice. ES cells have pluripotency that can differentiate into all cells existing in the living body. Currently, human ES cells can be used for cell transplantation therapy for many diseases such as Parkinson's disease, juvenile diabetes, and leukemia. However, there are also obstacles to the transplantation of ES cells. In particular, the transplantation of ES cells can cause an immune rejection reaction similar to the rejection reaction that occurs following unsuccessful organ transplantation. In addition, there are many criticisms and opposing opinions from an ethical perspective regarding the use of ES cells established by destroying human embryos.

[0003] Under such background circumstances, 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. As a result, Professor Yamanaka received the Nobel Prize in Physiology or Medicine in 2012 (see, for example, Patent Documents 1 and 2). iPS cells are ideal pluripotent cells without rejection reactions or ethical problems. Therefore, iPS cells are expected to be used for cell transplantation therapy.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] There is a demand for an apparatus capable of efficiently culturing various cells, not limited to iPS cells. One object of the present invention is to provide a cell culture apparatus capable of efficiently culturing cells.

Means for Solving the Problems

[0006] According to an aspect of the present invention, there is provided a cell culture apparatus including a culture component permeable member through which culture components can permeate, a culture tank that holds a cell-containing medium and covers one surface of the culture component permeable member for culturing cells, and a medium holding tank that holds a medium and covers the other surface of the culture component permeable member.

[0007] In the above cell culture apparatus, the culture component permeable member may be a semipermeable membrane.

[0008] The above cell culture apparatus may further include a culture-side plate and a medium-side plate each provided with an opening and sandwiching the culture component permeable member, wherein the cell-containing medium in the culture tank can contact the culture component permeable member through the opening of the culture-side plate, and the medium in the medium holding tank can contact the culture component permeable member through the opening of the medium-side plate.

[0009] In the above cell culture apparatus, the culture-side plate may be dark-colored.

[0010] In the above cell culture apparatus, the culture tank and the medium holding tank may be detachable.

[0011] In the above cell culture apparatus, the surface of the culture-side plate may be non-cell-adhesive.

[0012] In the above cell culture apparatus, the surface of the culture-side plate may be non-protein-adhesive.

[0013] In the above cell culture apparatus, the surface of the culture-side plate may be cell-adhesive.

[0014] In the above cell culture apparatus, the surface of the culture component permeable member may be non-cell-adhesive.

[0015] In the above cell culture device, the surface of the culture component permeation member may be non-protein adhesive.

[0016] In the above cell culture device, the surface of the culture component permeation member may be cell adhesive.

[0017] In the above cell culture device, the culture component permeation member may be cell adhesive.

[0018] In the above cell culture device, the culture component permeation member may be non-cell adhesive.

[0019] In the above cell culture device, even when the culture tank and the medium holding tank are in a sealed state, outside air may not need to enter the culture tank and the medium holding tank.

[0020] In the above cell culture device, even when the culture tank and the medium holding tank are in a sealed state, cells, microorganisms, viruses, and dust outside the culture tank and the medium holding tank may not need to enter the culture tank and the medium holding tank.

[0021] In the above cell culture device, even when the culture tank and the medium holding tank are in a sealed state, substances in the culture tank and the medium holding tank may not need to flow out of the culture tank and the medium holding tank.

[0022] In the above cell culture device, even when the culture tank and the medium holding tank are in a sealed state, gas exchange may not need to occur between the inside and outside of the culture tank and the medium holding tank.

[0023] In the above cell culture device, even when the culture tank and the medium holding tank are in a sealed state, at least any one of carbon dioxide gas, nitrogen gas, and oxygen gas may not need to be supplied into the culture tank and the medium holding tank.

[0024] In the above cell culture device, the pH of the medium in the culture tank and the medium holding tank may be maintained within a predetermined range.

[0025] In the above-described cell culture apparatus, the culture tank may be embedded in a gas-impermeable substance.

[0026] In the above-described cell culture apparatus, the medium holding tank may be embedded in a gas-impermeable substance.

[0027] In the above-described cell culture apparatus, it may further include a packing disposed between the culture component permeable member and the culture tank.

[0028] In the above-described cell culture apparatus, the packing may be disposed between the outer periphery of the culture component permeable member and the culture tank.

[0029] In the above-described cell culture apparatus, the outer diameter of the packing may be larger than the outer diameter of the culture component permeable member.

[0030] The above-described cell culture apparatus may further include a packing disposed between the culture component permeable member and the medium holding tank.

[0031] In the above-described cell culture apparatus, the packing may be disposed between the outer periphery of the culture component permeable member and the medium holding tank.

[0032] In the above-described cell culture apparatus, the outer diameter of the packing may be larger than the outer diameter of the culture component permeable member.

[0033] In the above-described cell culture apparatus, the inside of the culture tank may be non-cell-adhesive.

[0034] In the above-described cell culture apparatus, the inside of the culture tank may be non-protein-adhesive.

[0035] In the above-described cell culture apparatus, the inside of the culture tank may be cell-adhesive.

[0036] Also, according to an aspect of the present invention, there is provided a sealable culture tank for culturing cells, the culture tank being provided with a supply port for supplying fluid into the culture tank and a discharge port for discharging the fluid in the culture tank, and the supply port and the discharge port being sealable.

[0037] In the above culture tank, the feeder for supplying fluid to the supply port may be detachable.

[0038] In the above culture tank, the feeder may be connected to the supply port via a supply needleless connector.

[0039] In the above culture tank, the discharger for discharging fluid to the discharge port may be detachable.

[0040] In the above culture tank, the discharger may be connected to the discharge port via a discharge needleless connector.

[0041] In the above culture tank, the feeder for supplying fluid to the supply port is detachable, the discharger for discharging fluid to the discharge port is detachable, and when fluid is supplied from the feeder into the culture tank, the fluid in the culture tank may move into the discharger.

[0042] In the above culture tank, when a medium is supplied from the feeder into the culture tank, the air in the culture tank may move into the discharger.

[0043] In the above culture tank, when a medium is supplied from the feeder into the culture tank, the medium in the culture tank may move into the discharger.

[0044] In the above culture tank, the medium may contain cells.

[0045] In the above culture tank, when supplying fluid from the feeder into the culture tank, outside air may not enter the culture tank.

[0046] In the above-mentioned culture tank, a window may be provided.

[0047] In the above-mentioned culture tank, a transparent heater may be provided on the window.

[0048] The above-mentioned culture tank may be provided with a temperature control unit for adjusting the temperature inside the culture tank.

[0049] The above-mentioned culture tank may be provided with a thermometer for measuring the temperature inside the culture tank.

[0050] In the above-mentioned culture tank, even when the culture tank is in a sealed state, outside air may not enter the culture tank.

[0051] In the above-mentioned culture tank, even when the culture tank is in a sealed state, cells, microorganisms, viruses, and dust outside the culture tank may not enter the culture tank.

[0052] In the above-mentioned culture tank, even when the culture tank is in a sealed state, substances inside the culture tank may not flow out of the culture tank.

[0053] In the above-mentioned culture tank, even when the culture tank is in a sealed state, gas exchange may not occur between the inside and outside of the culture tank.

[0054] In the above-mentioned culture tank, even when the culture tank is in a sealed state, at least any one of carbon dioxide gas, nitrogen gas, and oxygen gas may not be supplied into the culture tank.

[0055] In the above-mentioned culture tank, the pH of the medium inside the culture tank may be maintained within a predetermined range.

[0056] In the above-mentioned culture tank, the culture tank may be embedded in a gas-impermeable substance.

[0057] In the above-mentioned culture tank, the inclination of the culture tank may be adjustable.

[0058] In the above culture tank, stem cells may be expanded and cultured.

[0059] In the above culture tank, the stem cells may be iPS cells, ES cells, or somatic stem cells.

[0060] In the above culture tank, cells into which an inducer has been introduced may be cultured and induced into stem cells.

[0061] In the above culture tank, an inducer may be added to the medium in the culture tank, and the inducer may be introduced into the cells being cultured in the culture tank.

[0062] In the above culture tank, cells into which an inducer has been introduced may be induced into stem cells.

[0063] In the above culture tank, the stem cells may be iPS cells.

[0064] In the above culture tank, the cells may be blood cells.

[0065] In the above culture tank, cells into which an inducer has been introduced in the culture tank may be cultured and induced into different types of cells.

[0066] In the above culture tank, an inducer may be added to the medium in the culture tank, the inducer may be introduced into the cells being cultured in the culture tank, and the cells may be induced into different types of cells.

[0067] In the above culture tank, the inducer may be RNA.

[0068] In the above culture tank, the inducer may be contained in Sendai virus.

[0069] In the above culture tank, the inducer may be contained in a plasmid.

[0070] In the above culture tank, the cells may be cultured.

[0071] In the above-mentioned culture tank, the cells may be at least one selected from blood cells, nerve cells, cardiomyocytes, epithelial cells, mesenchymal cells, and hepatocytes.

[0072] In the above-mentioned culture tank, the cells may be cultured in suspension in the culture tank.

[0073] In the above-mentioned culture tank, the cells may be cultured adherently in the culture tank.

[0074] In the above-mentioned culture tank, the cells may be cultured in the gel medium in the culture tank.

[0075] In the above-mentioned culture tank, the interior may be non-cell-adhesive.

[0076] In the above-mentioned culture tank, the interior may be non-protein-adhesive.

[0077] In the above-mentioned culture tank, the interior may be cell-adhesive.

[0078] Moreover, according to an aspect of the present invention, there is provided a culture device including the above-mentioned culture tank and an imaging device that images at least one of the medium and cells in the culture tank.

[0079] In the above-mentioned culture device, the imaging device may image the cells through a telecentric lens.

[0080] The above-mentioned culture device may further include an image processing unit that applies a high-pass filter to the image obtained by the imaging device.

[0081] In the above-mentioned culture device, the image processing unit may apply a watershed algorithm to the image to which the high-pass filter has been applied to extract cells or cell clusters in the image.

[0082] In the above-described culture apparatus, the image processing unit may apply the Distance Transform method to the image before applying the watershed algorithm to the image.

[0083] In the above-described culture apparatus, the image processing unit may calculate the size of the extracted cells or cell clusters.

[0084] In the above-described culture apparatus, the image processing unit may calculate the number of the extracted cells or cell clusters.

[0085] The above-described culture apparatus may further include a relationship storage device that stores the relationship between the turbidity of the culture medium and the density of cells or cell clusters in the culture medium, calculate the value of the turbidity of the culture medium in the culture tank based on the image obtained by the imaging device, and further include an image processing unit that calculates the value of the density of the photographed cells or cell clusters based on the calculated turbidity value and the relationship.

[0086] The above-described culture apparatus may further include an image processing unit that calculates the value of the density of cells or cell clusters in the culture medium from the number of the extracted cells or cell clusters and the ratio of the volume of the region photographed by the imaging device to the volume of the entire culture tank.

[0087] The above-described culture apparatus may further include a relationship storage device that stores the relationship between the color of the culture medium and the pH of the culture medium, calculate the value of the color of the culture medium in the culture tank in the image obtained by the imaging device, and further include an image processing unit that calculates the value of the pH of the photographed culture medium based on the calculated color value and the relationship.

[0088] Further, according to an aspect of the present invention, there is provided a sealable culture medium holding tank for holding a culture medium, in which an inlet for supplying a fluid into the culture medium holding tank and an outlet for discharging the fluid in the culture medium holding tank are provided in the culture medium holding tank, and the inlet and the outlet are sealable.

[0089] In the above-described culture medium holding tank, the feeder for supplying a fluid to the inlet may be detachable.

[0090] In the above-mentioned medium holding tank, the feeder may be connected to the inlet through a supply needleless connector.

[0091] In the above-mentioned medium holding tank, the discharger for discharging fluid from the outlet may be detachable.

[0092] In the above-mentioned medium holding tank, the discharger may be connected to the outlet through a discharge needleless connector.

[0093] In the above-mentioned medium holding tank, the feeder for supplying fluid to the inlet is detachable, the discharger for discharging fluid from the outlet is detachable, and when fluid is supplied from the feeder into the medium holding tank, the fluid in the medium holding tank may move into the discharger.

[0094] In the above-mentioned medium holding tank, when medium is supplied from the feeder into the medium holding tank, the air in the medium holding tank may move into the discharger.

[0095] In the above-mentioned medium holding tank, when medium is supplied from the feeder into the medium holding tank, the medium in the medium holding tank may move into the discharger.

[0096] In the above-mentioned medium holding tank, when fluid is supplied from the feeder into the medium holding tank, outside air may not enter the medium holding tank.

[0097] The above-mentioned medium holding tank may be provided with a temperature adjusting unit for adjusting the temperature inside the medium holding tank.

[0098] The above-mentioned medium holding tank may be provided with a thermometer for measuring the temperature inside the medium holding tank.

[0099] In the above-mentioned medium holding tank, even when the medium holding tank is in a sealed state, outside air may not enter the medium holding tank.

[0100] In the above-mentioned medium holding tank, when the medium holding tank is in a sealed state, it is not necessary for cells, microorganisms, viruses, and dust outside the medium holding tank to enter the medium holding tank.

[0101] In the above-mentioned medium holding tank, when the medium holding tank is in a sealed state, it is not necessary for substances inside the medium holding tank to flow out of the medium holding tank.

[0102] In the above-mentioned medium holding tank, when the medium holding tank is in a sealed state, it is not necessary for gas exchange to occur inside and outside the medium holding tank.

[0103] In the above-mentioned medium holding tank, when the medium holding tank is in a sealed state, it is not necessary to supply at least any one of carbon dioxide gas, nitrogen gas, and oxygen gas into the medium holding tank.

[0104] In the above-mentioned medium holding tank, the pH of the medium inside the medium holding tank may be maintained within a predetermined range.

[0105] In the above-mentioned medium holding tank, the medium holding tank may be embedded in a gas-impermeable substance.

[0106] In the above-mentioned medium holding tank, a rectifying plate disposed inside the medium holding tank may be provided.

[0107] In the above-mentioned medium holding tank, one or more discharge ports for supplying the medium into the medium holding tank, and one or more discharge ports communicating with the inlet may be provided.

[0108] In the above-mentioned medium holding tank, it may be possible to insert a discharge block provided with one or more discharge ports for supplying the medium into the medium holding tank, and one or more discharge ports capable of communicating with the inlet.

[0109] In the above-mentioned medium holding tank, an opening for discharging the air inside the medium holding tank to the outside when introducing the medium into the medium holding tank may be provided.

[0110] In the above-described medium holding tank, a medium flow path may be connected to the inlet and the outlet of the medium holding tank.

[0111] In the above-described medium holding tank, the medium may be circulated in the medium holding tank and the medium flow path.

[0112] In the above-described medium holding tank, the interior of the medium holding tank may be sealed when the medium holding tank is connected to the medium flow path.

[0113] In the above-described medium holding tank, the medium holding tank may be connected to a culture tank for culturing cells.

[0114] In the above-described medium holding tank, the interior of the medium holding tank may be sealed when the medium holding tank is connected to the culture tank.

[0115] Further, according to an aspect of the present invention, there is provided a sealable medium flow path, in which a supply port for supplying a fluid into the medium flow path and a discharge port for discharging the fluid in the medium flow path are provided in the medium flow path, and the supply port and the discharge port are sealable.

[0116] In the above-described medium flow path, a supplier for supplying a fluid to the supply port may be detachable.

[0117] In the above-described medium flow path, the supplier may be connected to the supply port via a supply needleless connector.

[0118] In the above-described medium flow path, a discharger for discharging a fluid to the discharge port may be detachable.

[0119] In the above-described medium flow path, the discharger may be connected to the discharge port via a discharge needleless connector.

[0120] In the above-described culture medium flow path, a feeder for supplying fluid to the supply port is detachable, a discharger for discharging fluid to the discharge port is detachable, and when fluid is supplied from the feeder into the culture medium flow path, the fluid in the culture medium flow path may move into the discharger.

[0121] In the above-described culture medium flow path, the culture medium flow path may further include a fluid machine provided therein for moving fluid.

[0122] In the above-described culture medium flow path, a feeder for supplying fluid to the supply port is detachable, a discharger for discharging fluid to the discharge port is detachable, and when the fluid machine is driven, fluid may be supplied from the feeder to the culture medium flow path, and the fluid in the culture medium flow path may move into the discharger.

[0123] In the above-described culture medium flow path, when culture medium is supplied from the feeder into the culture medium flow path, the air in the culture medium flow path may move into the discharger.

[0124] In the above-described culture medium flow path, when culture medium is supplied from the feeder into the culture medium flow path, the culture medium in the culture medium flow path may move into the discharger.

[0125] In the above-described culture medium flow path, when fluid is supplied from the feeder into the culture medium flow path, outside air does not necessarily have to enter the culture medium flow path.

[0126] The above-described culture medium flow path may include a temperature adjustment unit for adjusting the temperature inside the culture medium flow path.

[0127] The above-described culture medium flow path may include a thermometer for measuring the temperature inside the culture medium flow path.

[0128] In the above-described culture medium flow path, even when the culture medium flow path is in a sealed state, outside air does not necessarily have to enter the culture medium flow path.

[0129] In the above-described culture medium flow path, even when the culture medium flow path is in a sealed state, cells, microorganisms, viruses, and dust outside the culture medium flow path do not need to enter the culture medium flow path.

[0130] In the above-described culture medium flow path, even when the culture medium flow path is in a sealed state, substances within the culture medium flow path do not need to flow out to the outside of the culture medium flow path.

[0131] In the above-described culture medium flow path, even when the culture medium flow path is in a sealed state, gas exchange does not need to occur between the inside and outside of the culture medium flow path.

[0132] In the above-described culture medium flow path, even when the culture medium flow path is in a sealed state, at least any one of carbon dioxide gas, nitrogen gas, and oxygen gas does not need to be supplied into the culture medium flow path.

[0133] In the above-described culture medium flow path, the pH of the culture medium within the culture medium flow path may be maintained within a predetermined range.

[0134] The above-described culture medium flow path may be blocked from the outside air by an outside air blocking member for the culture medium flow path.

[0135] The above-described culture medium flow path may be embedded in a gas-impermeable substance.

[0136] The above-described culture medium flow path may be at least partially a hole provided in a member.

[0137] In the above-described culture medium flow path, the fluid machine may be blocked from the outside air by an outside air blocking member for the fluid machine.

[0138] In the above-described culture medium flow path, the culture medium flow path is blocked from the outside air by an outside air blocking member for the culture medium flow path, the culture medium flow path and the pump head of the fluid machine are arranged within the outside air blocking member for the culture medium flow path, and the drive unit connected to the pump head of the fluid machine may be arranged outside the outside air blocking member for the culture medium flow path.

[0139] In the above-described culture medium flow path, the pump head and the drive unit may be detachable.

[0140] In the above-described medium flow path, a hermetic member for the medium flow path including a medium flow path and a pump head inside may be disposable.

[0141] In the above-described medium flow path, the drive unit may be held by a drive unit holding member, and the hermetic member for the medium flow path and the drive unit holding member may be detachable.

[0142] In the above-described medium flow path, when the drive unit holding member is removed from the hermetic member for the medium flow path, gas exchange may not occur between the inside and outside of the hermetic member for the medium flow path.

[0143] In the above-described medium flow path, the medium flow path may be connected to a culture tank for culturing cells.

[0144] In the above-described medium flow path, the medium may be circulated between the medium flow path and the inside of the culture tank.

[0145] In the above-described medium flow path, when the medium flow path is connected to the culture tank, the inside of the medium flow path may be sealed.

[0146] In the above-described medium flow path, the medium flow path may be connected to a medium holding tank for holding the medium.

[0147] In the above-described medium flow path, the medium may be circulated between the medium flow path and the inside of the medium holding tank.

[0148] In the above-described medium flow path, when the medium flow path is connected to the medium holding tank, the inside of the medium flow path may be sealed.

[0149] In the above-described medium flow path, cells may be cultured inside the medium flow path.

[0150] In the above-described medium flow path, the inside may be non-cell adhesive.

[0151] In the above-described culture medium flow path, the interior may be non-adhesive to proteins.

[0152] In the above-described culture medium flow path, the interior may be cell-adhesive.

[0153] Also, according to an aspect of the present invention, there is provided a culture device including the above-described culture medium flow path and an imaging device that images at least one of the culture medium and cells in the culture medium flow path.

[0154] In the above-described culture device, the imaging device may image cells via a telecentric lens.

[0155] The above-described culture device may further include an image processing unit that applies a high-pass filter to an image obtained by the imaging device.

[0156] In the above-described culture device, the image processing unit may apply a watershed algorithm to the image to which the high-pass filter has been applied to extract cells or cell clusters in the image.

[0157] In the above-described culture device, the image processing unit may apply a Distance Transform method to the image before applying the watershed algorithm to the image.

[0158] In the above-described culture device, the image processing unit may calculate the size of the extracted cells or cell clusters.

[0159] In the above-described culture device, the image processing unit may calculate the number of the extracted cells or cell clusters.

[0160] The above-described culture device may further include a relationship storage device that stores the relationship between the turbidity of the culture medium and the density of cells or cell clusters in the culture medium, calculate the value of the turbidity of the culture medium in the culture medium flow path based on the image obtained by the imaging device, and further include an image processing unit that calculates the value of the density of the imaged cells or cell clusters based on the calculated turbidity value and the relationship.

[0161] The above-described culture device may further include an image processing unit that calculates the density value of cells or cell aggregates in the culture medium flow path from the number of extracted cells or cell aggregates and the ratio of the volume of the region imaged by the imaging device to the volume of the entire culture medium flow path.

[0162] The above-described culture device may further include a relationship storage device that stores the relationship between the color of the culture medium and the pH of the culture medium, calculates the color value of the culture medium in the culture medium flow path in the image obtained by the imaging device, and further includes an image processing unit that calculates the pH value of the imaged culture medium based on the calculated color value and the relationship.

[0163] Further, according to an aspect of the present invention, there is provided a plate used when culturing cells, which is used in a stacked manner with a culture component permeable member that is a membrane permeable to culture medium components and has an opening.

[0164] The above-described plate may be dark in color.

[0165] In the above-described plate, the size of the portion where no opening is provided may be larger than the size of cells or cell aggregates composed of cells.

[0166] In the above-described plate, the surface may be cell-adhesive.

Effect of the Invention

[0167] According to the present invention, it is possible to provide a cell culture device capable of efficiently culturing cells.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0169] Hereinafter, embodiments of the present invention will be described. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Of course, there are also parts where the dimensional relationships and ratios are different between the drawings.

[0170] As shown in FIG. 1, the cell incubator according to the embodiment includes a culture component permeable member 10 through which culture components can permeate, a culture tank 30 that holds a cell-containing medium and cultures cells, covering one surface of the culture component permeable member 10, and a medium holding tank 40 that holds a medium, covering the other surface of the culture component permeable member 10. The cell-containing medium in the culture tank 30 can contact the culture component permeable member 10. Also, the medium in the medium holding tank 40 can contact the culture component permeable member 10. The medium in the medium holding tank 40 does not contain cells.

[0171] The culture component permeable member 10 permeates the active ingredients of the medium in the medium holding tank 40 into the cell-containing medium in the culture tank 30. Also, the culture component permeable member 10 may permeate the waste products in the cell-containing medium in the culture tank 30 into the medium in the medium holding tank 40. As the culture component permeable member 10, for example, a semipermeable membrane and a mesh can be used. The semipermeable membrane includes a dialysis membrane.

[0172] When the culture component permeation member 10 is a semipermeable membrane, the molecular weight cut-off of the semipermeable membrane is, for example, 0.1 KDa or more, 10 KDa or more, or 50 KDa or more. The semipermeable membrane is composed of, for example, cellulose ester, ethyl cellulose, cellulose esters, regenerated cellulose, polysulfone, polyacrylonitrile, polymethyl methacrylate, ethylene vinyl alcohol copolymer, polyester-based polymer alloy, polycarbonate, polyamide, cellulose acetate, cellulose diacetate, cellulose triacetate, cuprammonium rayon, saponified cellulose, hemophan membrane, phosphatidylcholine membrane, and vitamin E coating membrane, etc.

[0173] When the culture component permeation member 10 is a mesh, the mesh has pores smaller than the cells or cell aggregates cultured in the culture tank 30. Thereby, the cells or cell aggregates in the culture tank 30 are prevented from moving into the culture medium holding tank 40. The material of the mesh is, for example, resin and metal, but is not particularly limited. The surface of the culture component permeation member 10 may be non-cell adhesive.

[0174] The cell incubator according to the embodiment may further include a culture-side plate 21 and a medium-side plate 22 each provided with an opening, sandwiching the culture component permeable member 10. The culture-side plate 21 and the medium-side plate 22 sandwich the culture component permeable member 10 so as to suppress the fluctuation of the culture component permeable member 10 due to the pressure of the cell-containing medium in the culture tank 30 and the medium in the medium holding tank 40, thereby holding the culture component permeable member 10. Thereby, it is possible to prevent the culture component permeable member 10 from coming into contact with the inner wall of the culture tank 30 or the medium holding tank 40 due to pressure fluctuations. The culture-side plate 21 and the medium-side plate 22 have a hardness that does not vary with the pressure received from the cell-containing medium in the culture tank 30 and the medium in the medium holding tank 40. The materials of the culture-side plate 21 and the medium-side plate 22 are, for example, resin and metal, but are not particularly limited. The surface of the culture-side plate 21 may be non-cell adhesive. When the culture component permeable member 10 does not vary due to the pressure received from the cell-containing medium in the culture tank 30 and the medium in the medium holding tank 40, the culture-side plate 21 and the medium-side plate 22 may be omitted.

[0175] The culture-side plate 21 is provided with an opening so that the cell-containing medium in the culture tank 30 can contact the culture component permeable member 10. Further, the medium-side plate 22 is provided with an opening so that the medium in the medium holding tank 40 can contact the culture component permeable member 10. Through the opening of the culture-side plate 21, the components of the cell-containing medium in the culture tank 30 and the components of the medium in the medium holding tank 40 can permeate through the culture component permeable member 10. The shape of the opening provided in each of the culture-side plate 21 and the medium-side plate 22 is, for example, a circle, but is not particularly limited. The opening provided in each of the culture-side plate 21 and the medium-side plate 22 has a size within a range that can suppress the fluctuation of the culture component permeable member 10. The openings are provided in each of the culture-side plate 21 and the medium-side plate 22, for example, in a lattice pattern or randomly.

[0176] The culture side plate 21 may have a dark color such as black, for example. When the culture side plate 21 has a dark color, it is possible to visually recognize or image the cells in the cell-containing culture medium with high contrast against the background of the culture side plate 21. If the size, such as the area of the portion of the culture side plate 21 where no opening is provided, is larger than that of the cells or cell aggregates, it becomes easier to visually recognize or image the cells or cell aggregates with high contrast against the background of the portion of the culture side plate 21 where no opening is provided. However, by adjusting the light irradiated onto the cells or cell aggregates, it is also possible to visually recognize or image the cells or cell aggregates even if the culture component permeable member 10 and the culture side plate 21 are transparent.

[0177] The culture tank 30 and the culture medium holding tank 40 may be fixed with screws, pins, electromagnets, or the like. The contact portion of the culture tank 30 and at least a part of one surface of the culture side plate 21 are in close contact. At least a part of the other surface of the culture side plate 21 and at least a part of one surface of the culture component permeable member 10 are in close contact. At least a part of the other surface of the culture component permeable member 10 and at least a part of one surface of the culture medium side plate 22 are in close contact. At least a part of the other surface of the culture medium side plate 22 and the contact portion of the culture medium holding tank 40 are in close contact. When making them in close contact, 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 tank 30. The packing may be disposed between the outer periphery of the culture component permeable member 10 and the culture tank 30. The outer diameter of the packing disposed between the culture component permeable member 10 and the culture tank 30 may be larger than the outer diameter of the culture component permeable member 10. Also, 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.

[0178] The culture tank 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 tank 30 may be coated with a cell non - adhesive substance such as poly - HEMA (poly 2 - hydroxyethyl methacrylate) so that cells do not adhere, rendering the inner wall of the culture tank 30 non - adhesive to cells. The housing 31 is provided with an opening 131 for exposing the culture component permeation member 10 through the opening of the culture - side plate 21. As shown in FIG. 2, the cover 32 of the culture tank 30 is provided with a window 132 through which the cell - containing medium in the culture tank 30 can be observed. As the material of the window 132, for example, glass and resin can be used.

[0179] 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 arranged on the window 132 and may be a transparent heater such as a transparent conductive film that heats the window. Alternatively, the cell incubator according to the embodiment may include a temperature control unit for heating and cooling the housing 31 or the cover 32 of the culture tank 30. By temperature - regulating any one of the housing 31, the cover 32, and the window 132 with the temperature control unit, it is possible to regulate the temperature of the cell - containing medium in the culture tank 30. The cell incubator according to the embodiment may further include a thermometer for measuring the temperature of the cell - containing medium in the culture tank 30. The thermometer may measure the temperature of the cell - containing medium based on the temperature of the culture tank 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 reaches a predetermined temperature. The temperature of the cell - containing medium is adjusted, for example, to be between 20°C and 45°C.

[0180] As shown in Fig. 1, the culture tank 30 is provided with a supply port 231 for supplying fluid into the culture tank 30 and a discharge port 331 for discharging the fluid in the culture tank 30. For example, a plug 33 shown in Fig. 2, which can be connected to a feeder such as a bag, bellows, and syringe for supplying fluid, is inserted into the supply port 231. The feeder 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 feeder is detachable from the supply port 231. When the feeder is not connected to the supply port 231, the supply port 231 can be sealed, and no fluid exchange occurs between the inside and outside of the culture tank 30 through the supply port 231.

[0181] The plug 33 may be a needleless connector. The needleless connector may be of a split septum type or a mechanical valve type. When the plug 33 is a split septum type needleless connector, the plug 33 includes a disk valve provided with a slit. When supplying fluid into the culture tank 30, a feeder or a flow path connected to the feeder is inserted into the slit of the disk valve. When no feeder or flow path connected to the feeder is inserted into the slit, the slit is sealed. When a feeder or a flow path connected to the feeder is inserted into the slit, the disk valve adheres to the outer periphery of the feeder or the flow path connected to the feeder. Therefore, even when a feeder or a flow path connected to the feeder is inserted into the plug 33, outside air does not enter the culture tank 30 through the plug 33. However, the plug 33 may be a connector into which a needle is inserted.

[0182] Also, for example, a plug 34 shown in Fig. 2, to which a discharger such as a bag, bellows, and syringe for discharging the fluid in the culture tank 30 can be connected, is inserted into the discharge port 331. The discharger may be a fluid machine such as a pump. However, the discharger may be directly connected to the discharge port 331 shown in Fig. 1. The discharger may actively suck the fluid in the culture tank 30. Alternatively, the discharger may passively increase the internal volume according to the pressure in the culture tank 30 and receive the fluid extruded from the culture tank 30. The discharger is detachable from the discharge port 331. When the discharger is not connected to the discharge port 331, the discharge port 331 can be sealed, and no fluid exchange occurs between the inside and outside of the culture tank 30 through the discharge port 331. The plug 34 may be a needleless connector. The needleless connector may be of a split septum type or a mechanical valve type. Even when a discharger or a flow path connected to the discharger 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.

[0183] For example, when the culture tank 30 is in a state of being in close contact with the culture medium holding tank 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 tank 30, while discharging the air in the culture tank 30 from the discharge port 331, by injecting a cell-containing culture medium into the culture tank 30 from the supply port 231, it is possible to put the cell-containing culture medium into the culture tank 30 shown in Fig. 2. Also, it is possible to completely eliminate the air layer in the culture tank 30. However, an air layer may remain in the culture tank 30. When a cell-containing culture medium is already present in the culture tank 30, by discharging the cell-containing culture medium in the culture tank 30 from the discharge port 331 shown in Fig. 1 and injecting another cell-containing culture medium into the culture tank 30 from the supply port 231, it is possible to replace at least a part of the cell-containing culture medium in the culture tank 30 shown in Fig. 2.

[0184] The cell incubator according to the embodiment may further include a culture tank holding member that can hold the culture tank 30 and can adjust the inclination of the culture tank 30. By adjusting the inclination of the culture tank 30, it becomes easier to discharge gases such as air in the culture tank 30.

[0185] The supply port 231 and the discharge port 331 of the culture tank 30 can be closed by a stopper or the like. Alternatively, the plugs 33 and 34 respectively connected to the supply port 231 and the discharge port 331 of the culture tank 30 can be closed. Or, alternatively, the supply port 231 of the culture tank 30 can be shielded from the outside by being connected to a feeder, and the discharge port 331 of the culture tank 30 can be shielded from the outside by being connected to a discharger. When the supply port 231 and the discharge port 331 are closed and the culture tank 30 is brought into close contact with 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. Thereby, the entry of outside air into the culture tank 30 is suppressed, the change in the pH of the cell-containing culture medium in the culture tank 30 is suppressed, and it is maintained within a predetermined range. The predetermined range of the pH of the cell-containing culture medium is, for example, from 6.0 to 9.0. According to the findings of the present inventors, since cells can be cultured in a completely closed enclosed space, it is not necessary to actively supply carbon dioxide gas, nitrogen gas, oxygen gas, etc. into the culture tank 30. Therefore, the culture tank 30 2 does not have to be placed in an incubator. Also, since cells, microorganisms, viruses, dust, etc. existing outside the culture tank 30 do not enter the sealed culture tank 30, the cleanliness inside the culture tank 30 is maintained. Therefore, the culture tank 30 does not have to be placed in a clean room. The culture tank 30 may be embedded in a gas-impermeable substance. In other words, the culture tank 30 may be embedded in a gas-impermeable substance.

[0186] The cells cultured in the culture tank 30 may be stem cells such as induced pluripotent stem (iPS) cells, embryonic stem cells (ES cells), and somatic stem cells, or may be cells other than stem cells. The somatic stem cells may be mesenchymal stem cells. In the culture tank 30, the stem cells may be expanded and cultured. The cells cultured in the culture tank 30 may be cells into which an inducer has been introduced and are cells induced into stem cells. Alternatively, the cells cultured in the culture tank 30 may be cells into which an inducer has not been introduced, and an inducer may be added to the cell-containing medium in the culture tank 30 to introduce the inducer into the cells and induce the cells into stem cells. Inducing cells into stem cells is sometimes referred to as reprogramming. The cells induced into stem cells may be blood cells. Also, the cells cultured in the culture tank 30 may be cells into which an inducer has been introduced and are cells induced into different types of cells. Alternatively, the cells cultured in the culture tank 30 may be cells into which an inducer has not been introduced, and an inducer may be added to the cell-containing medium in the culture tank 30 to introduce the inducer into the cells and induce the cells into different types of cells. Inducing differentiated cells into different types of differentiated cells is sometimes referred to as transdifferentiation or lineage reprogramming or cell fate reprogramming. Also, in the culture tank 30, the stem cells may be induced into cells. The inducer may be RNA or may be a protein. The inducer may be contained, for example, in a plasmid, Sendai virus, adenovirus, lentivirus, and retrovirus. The cells cultured in the culture tank 30 may be non-induced cells. Examples of the cells cultured in the culture tank 30 include blood cells, nervous system cells, myocardial cells, epithelial cells, vascular endothelial cells, mesenchymal cells, fibroblasts, hepatocytes, insulin-producing cells, retinal pigment epithelial cells, and corneal cells. Examples of blood cells include T cells, B cells, NK cells, NKT cells, megakaryocytes, macrophages, granulocytes, neutrophils, eosinophils, hematopoietic stem cells, blood stem / progenitor cells, erythrocytes, leukocytes, and platelets. Examples of nervous system cells include neurons and glial cells, oligodendrocytes, and neural stem cells.Examples of cardiomyogenic cells include cardiac stem cells, cardiomyocytes, and pacemaker cells. Examples of epithelial cells include keratinocytes, intestinal epithelial cells, oral epithelium, and corneal epithelial cells. Examples of mesenchymal cells include dermal cells, osteoblasts, adipocytes, muscle cells, and chondrocytes. In the cell-containing medium, the cells may form cell clusters (colonies). The cells may be animal cells including humans, insect cells, or plant cells.

[0187] The cell-containing medium may be in a gel form. In this case, the cell-containing medium may contain at least one polymer compound selected from the group consisting of gellan gum, deacylated gellan gum, hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectinic acid, pectininic acid, heparan sulfate, heparin, heparitin sulfate, keratan sulfate, chondroitin sulfate, delta-mann sulfate, laminan sulfate, and salts thereof. Further, the cell-containing medium may contain methylcellulose. By containing methylcellulose, aggregation of cells is more suppressed.

[0188] Alternatively, the cell-containing medium may contain at least a temperature-responsive 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.

[0189] In the present disclosure, the gel-like medium or the gel medium includes a polymer medium.

[0190] The medium holding tank 40 shown in FIG. 1 is provided with an opening 140 shown in FIG. 3 for exposing the culture component permeation member 10 through the opening of the medium-side plate 22. The opening 140 is covered with the culture component permeation member 10 shown in FIG. 1. Further, the medium holding tank 40 shown in FIG. 3 is provided with an inlet 240 for introducing fluid into the medium holding tank 40 and an outlet 340 for discharging the fluid in the medium holding tank 40. Furthermore, a plurality of flow rectifying plates 41 may be arranged in the medium holding tank 40. The plurality of flow rectifying plates 41 are arranged, for example, so as to alternately project from the opposing inner walls of the medium holding tank 40.

[0191] For example, when the medium holding tank 40 is in a state of being in close contact with the culture tank 30 with the 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 medium holding tank 40, while discharging the air in the medium holding tank 40 from the discharge port 340 shown in FIG. 3, by injecting the cell medium into the medium holding tank 40 from the inlet 240, it is possible to put the cell medium into the medium holding tank 40. Further, when the medium is already present in the medium holding tank 40, while discharging the cell medium in the medium holding tank 40 from the discharge port 340, by injecting the cell medium into the medium holding tank 40 from the inlet 240, it is possible to flow the cell medium in the medium holding tank 40.

[0192] When a plurality of flow rectifying plates 41 are arranged in the medium holding tank 40, in the medium holding tank 40, the medium flows along the plurality of flow rectifying plates 41 from the inlet 240 toward the discharge port 340. Therefore, an opportunity for the components of the medium to contact the culture component permeable member 10 is ensured.

[0193] Alternatively, as shown in FIG. 4, one or a plurality of discharge ports 241 communicating with the inlet 240 shown in FIG. 3 may be provided on the inner wall of the medium holding tank 40. The plurality of discharge ports 241 shown in FIG. 4 are provided, for example, in a horizontal row. The number and arrangement of the plurality of discharge ports 241 may be arranged evenly or randomly. The number and arrangement of the plurality of discharge ports 241 are set according to characteristics such as the viscosity of the medium. As shown in FIG. 5, by discharging the medium from the plurality of discharge ports 241, it is possible to improve the uniformity of the medium contacting the culture component permeable member 10 in the medium holding tank 40.

[0194] A discharge block 145 provided with one or a plurality of discharge ports 241 may be insertable into the inner wall of the medium holding tank 40. For example, discharge blocks 145 having different patterns such as the number and arrangement of the plurality of discharge ports 241 may be prepared and used appropriately according to the characteristics of the medium and the cells to be cultured. The upper side of the inner wall of the 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 medium holding tank 40 with respect to gravity may be bent or curved upward or downward.

[0195] As shown in FIGS. 4 and 5, openings 242 may be provided near a plurality of discharge ports 241 on the inner wall of the culture medium holding tank 40. As the culture medium discharged from the plurality of discharge ports 241 accumulates in the culture medium holding tank 40, the air in the culture medium holding tank 40 flows out to the outside through the openings 242. After the culture medium is put into the culture medium holding tank 40, the openings 242 may be sealed.

[0196] As shown in FIG. 3, the inlet 240 and the outlet 340 of the culture medium holding tank 40 may be connected by a culture medium flow path 200, and the culture medium may be circulated 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 substance. In other words, the culture medium flow path 200 may be embedded in a gas-impermeable substance. For example, the culture medium flow path 200 may be a hole provided in a member made of resin, glass, metal, or the like. In this case, for example, the culture medium flow path 200 is formed by bonding members provided with recesses. 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 may be provided in the culture medium flow path 200. The fluid machine includes, 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.

[0197] As the introduction fluid machine 51 and the discharge fluid machine 52 shown in FIG. 1, a positive displacement pump can be used. Examples of positive displacement pumps include reciprocating pumps including piston pumps, plunger pumps, and diaphragm pumps, or rotary pumps including gear pumps, vane pumps, and screw pumps. Examples of diaphragm pumps include tubing pumps and piezoelectric (piezo) pumps. A tubing pump is sometimes called a peristaltic pump. Also, a microfluidic chip module combining various types of pumps may be used.

[0198] When using a sealed pump such as a peristaltic pump (registered trademark), a tubing pump, or a diaphragm pump, it is possible to pump the medium inside the medium flow path 200 shown in FIG. 3 without the pump directly contacting the medium. Alternatively, a syringe pump may be used as the fluid machine 51 for introduction and the fluid machine 52 for discharge. Even a pump other than a sealed pump can be reused by heat sterilization or the like.

[0199] When the fluid machine 51 for introduction is a sealed pump, as shown in FIG. 1, the fluid machine 51 for introduction 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 a roller that squeezes a medium flow path such as a tube from the outside. The drive unit 251 rotates the roller of the pump head 151. When the fluid machine 52 for discharge is a sealed pump, the fluid machine 52 for discharge 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 a roller that squeezes a medium flow path such as a tube from the outside. The drive unit 252 rotates the roller of the pump head 152.

[0200] As shown in FIG. 3, a medium tank 60 into which the medium can enter may be provided in the medium flow path 200. The medium that has entered the medium tank 60 from the medium flow path 200 flows out again into the medium flow path 200. By providing the medium tank 60, it is possible to increase the volume of the medium circulating between the medium flow path 200 and the medium holding tank 40.

[0201] The culture medium tank 60 may be provided with a supply port for supplying fluid into the culture medium tank 60 and a discharge port for discharging the fluid in the culture medium tank 60. For example, a plug 61 shown in FIG. 6 that can connect a feeder such as a bag, bellows, and syringe for supplying fluid is inserted into the supply port of the culture medium tank 60. The feeder may be a fluid machine such as a pump. However, the feeder may be directly connected to the supply port of the culture medium tank 60. The feeder is detachable from the supply port. When the feeder is not connected to the supply port, the supply port can be sealed, and no fluid exchange occurs between the inside and outside of the culture medium flow path 200 through the supply port. Alternatively, the supply port is shielded from the outside when connected to the feeder. The plug 61 may be a needleless connector. The needleless connector may be of a split septum type or a mechanical valve type. Even when a feeder or a flow path connected to the feeder 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 be a connector into which a needle is inserted.

[0202] Also, for example, a plug 62 into which a discharger such as a bag, bellows, and syringe for discharging the fluid in the culture medium tank 60 can be connected is inserted into the discharge port of the culture medium tank 60. The discharger may be a fluid machine such as a pump. However, the discharger may be directly connected to the discharge port of the culture medium tank 60. The discharger may actively suck the fluid in the culture medium flow path. Alternatively, the discharger may passively increase the internal volume according to the pressure in the culture medium flow path and receive the fluid extruded from the culture medium flow path. The discharger is detachable from the discharge port. When the discharger is not connected to the discharge port, the discharge port can be sealed, and no fluid exchange occurs between the inside and outside of the culture medium flow path 200 through the discharge port. Alternatively, the discharge port is shielded from the outside when connected to the discharger. The plug 62 may be a needleless connector. The needleless connector may be of a split septum type or a mechanical valve type. Even when a discharger or a flow path connected to the discharger 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.

[0203] For example, when the culture medium holding tank 40 shown in FIG. 1 is in a state of being 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, while discharging the air in the culture medium holding tank 40, the culture medium flow path 200, and the culture medium tank 60 from the discharge port of 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, it is possible to put the culture medium into 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 the air layer may remain.

[0204] A feeder filled with a culture medium and an empty discharger may be connected to the culture medium flow path 200, and a fluid machine may be driven to introduce the culture medium from the feeder into the culture medium flow path 200 and introduce air into the discharger. At this time, the feeder may actively inject the culture medium into the culture medium flow path 200, or the culture medium in the feeder may be sucked into the culture medium flow path 200 that has become low pressure due to the driving of the fluid machine, and the internal volume of the feeder may passively decrease. Also, the discharger may actively suck the air in the culture medium flow path 200, or the air in the culture medium flow path 200 that has become high pressure due to the driving of the fluid machine may flow into the discharger, and the internal volume of the discharger may passively increase.

[0205] Also, when the culture medium is already 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 injecting the culture medium into the culture medium tank 60 from the supply port of the culture medium tank 60 while discharging the culture medium in the culture medium tank 60 from the discharge port of the culture medium tank 60.

[0206] A feeder filled with a fresh culture medium and an empty discharger may be connected to the culture medium flow path 200, and a fluid machine may be driven to introduce the fresh culture medium from the feeder into the culture medium flow path 200 and introduce the old culture medium into the discharger. At this time, the feeder may actively inject the fresh culture medium into the culture medium flow path 200, or the fresh culture medium in the feeder may be sucked into the culture medium flow path 200 that has become low pressure due to the driving of the fluid machine, and the internal volume of the feeder may passively decrease. Also, the discharger may actively suck the old culture medium in the culture medium flow path 200, or the old culture medium in the culture medium flow path 200 that has become high pressure due to the driving of the fluid machine may flow into the discharger, and the internal volume of the discharger may passively increase.

[0207] The supply port for supplying the culture medium into the culture medium flow path 200 and the culture medium holding tank 40 and the discharge port for discharging the air in the culture medium flow path 200 and the culture medium holding tank 40 may be provided outside the portion of the culture medium flow path 200 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 discharge port for discharging the air in the culture medium flow path 200 and the culture medium holding tank 40 may be provided in the culture medium flow path 200.

[0208] The cell incubator according to the embodiment may include a temperature control unit for heating and cooling at least any one of the medium holding tank 40, the medium flow path 200, and the medium tank 60. By adjusting the temperature of any one of the medium holding tank 40, the medium flow path 200, and the medium tank 60 with the temperature control unit, it is possible to adjust the temperature of the medium. 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 any 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 control unit may be feedback-controlled so that the temperature of the medium becomes a predetermined temperature. The temperature of the medium is adjusted to, for example, 4°C or higher and 45°C or lower, or 20°C or higher and 45°C or lower.

[0209] As shown in FIG. 3, the medium holding tank 40, the medium flow path 200, the pump head 151, the pump head 152, and the medium tank 60 may be stored in the flow path case 70. Inside the flow path case 70, the medium holding tank 40, the medium flow path 200, the pump head 151, the pump head 152, and the medium tank 60 may be completely embedded in a gas-impermeable substance. The medium flow path 200 may be provided in a tunnel shape in the gas-impermeable substance. For example, the flow path case 70 is 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 the plug 61 into the supply port of the medium tank 60, and a hole for inserting the plug 62 into the discharge port of the medium tank 60. The hole for inserting the plug 61 into the supply port of the medium tank 60 and the hole for inserting the plug 62 into the discharge port of the medium tank 60 may be capable of being closed.

[0210] As shown in FIG. 7, the drive unit 251 of the introduction fluid machine 51 and the drive unit 252 of the discharge fluid machine 52 may be arranged on the substrate-like drive unit holding member 80. The drive unit holding member 80 is provided with a hole for inserting the plug 61 into the supply port of the culture medium tank 60 and a hole 82 for inserting the 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 capable of being closed.

[0211] The drive unit holding member 80 is brought into close contact with the flow path case 70 via the packing 90 shown in FIG. 1. The packing 90 suppresses the entry of air into the flow path case 70 from the contact portion between the flow path case 70 and the drive unit holding member 80.

[0212] The 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 may be covered with an external air blocking member for the fluid machine. The external air blocking member for the fluid machine includes, for example, as shown in FIG. 7, an external air blocking member 351 for the introduction fluid machine that covers the drive unit 251 of the introduction fluid machine 51 arranged on the drive unit holding member 80, and an external air blocking member 352 for the discharge fluid machine that covers the drive unit 252 of the discharge fluid machine 52 arranged on the drive unit holding member 80.

[0213] The flow path case 70 and the drive unit holding member 80 are detachable. When the drive unit holding member 80 is brought into close contact with the flow path case 70, holes for inserting the plug 61 into the supply port of the culture medium tank 60 and holes for inserting the plug 62 into the discharge port of the culture medium tank 60 are blocked, the drive unit 251 of the fluid machine 51 for introduction is covered with the outside air blocking member 351 for the fluid machine for introduction, and the drive unit 252 of the fluid machine 52 for discharge is covered with the outside air blocking member 352 for the fluid machine for discharge, the inside of the flow path case 70 is blocked from the outside air, and the outside air cannot enter the flow path case 70. Therefore, the gas exchange between the inside and outside of the flow path case 70 does not occur. Accordingly, the outside air does not enter the culture medium holding 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, even if the culture medium flow path 200 is a gas-permeable tube, it is possible to suppress the fluctuation of 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 predetermined range. The predetermined range of the pH of the culture medium is, for example, from 6.0 to 9.0. According to the findings of the present inventors, since cells can be cultured in a completely closed space, it is not necessary to actively supply carbon dioxide gas, nitrogen gas, oxygen gas, etc. into the culture medium holding tank 40 and the culture medium flow path 200. Therefore, it is not necessary to place the culture medium holding tank 40 and the culture medium flow path 200 in an incubator. Also, since cells, microorganisms, viruses, dust, etc. existing 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 inside the culture medium holding tank 40 and the culture medium flow path 200 is maintained. Therefore, it is not necessary to place the culture medium holding tank 40 and the culture medium flow path 200 in a clean room. The culture medium holding tank 40 may be embedded in a gas-impermeable substance. In other words, the culture medium holding tank 40 may be embedded in a gas-impermeable substance. 2 It is not necessary to place it in an incubator. Also, since cells, microorganisms, viruses, dust, etc. existing 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 inside the culture medium holding tank 40 and the culture medium flow path 200 is maintained. Therefore, it is not necessary to place the culture medium holding tank 40 and the culture medium flow path 200 in a clean room. The culture medium holding tank 40 may be embedded in a gas-impermeable substance. In other words, the culture medium holding tank 40 may be embedded in a gas-impermeable substance.

[0214] When the drive unit holding member 80 is removed from the flow path case 70, by closing the holes in the flow path case 70 for inserting the plug 61 into the supply port of the medium tank 60 and the holes in the flow path case 70 for inserting the plug 62 into the discharge port of the medium tank 60, the inside of the flow path case 70 can be sealed, suppressing the outflow of substances inside the flow path case 70 to the outside and the entry of outside air into the flow path case 70.

[0215] The flow path case 70 containing the medium flow path 200 and the pump heads 151, 152 inside is disposable. On the other hand, the drive unit holding member 80 holding the drive units 251, 252 can be reused.

[0216] For example, the introduction fluid machine 51 and the discharge fluid machine 52 are controlled so that the amount of the medium sent into the medium holding tank 40 by the introduction fluid machine 51 shown in FIG. 2 is the same as the amount of the medium discharged from the medium holding tank 40 by the discharge fluid machine 52. The introduction fluid machine 51 and the discharge fluid machine 52 may constantly send the medium into the medium holding tank 40, or may send the medium at appropriate intervals.

[0217] When constantly sending the medium into the medium holding tank 40, the flow rate of the medium sent into the medium holding tank 40 may or may not be constant. For example, the medium and the cell mass in the medium are monitored by an imaging device, and the flow rate of the medium sent into the medium holding tank 40 may be increased or decreased according to the state of the medium and the cell mass in the medium.

[0218] Also, without constantly sending the medium into the medium holding tank 40, for example, according to the state of the medium, the state of the cell mass in the medium, the number of cells, the number of cell masses, the turbidity of the medium, and the change in pH, the start and end of the medium feeding may be performed. Also in this case, the flow rate of the fed medium may be increased or decreased according to the state of the medium and the cell mass in the medium.

[0219] In a stirred medium, cells may randomly collide and bind to form cell clusters (colonies) of various sizes. Therefore, the homogeneity between colonies may not be maintained. Furthermore, in colonies that are too large, nutrients and growth factors may not reach the interior of the colony, and differentiation and cell death may occur from the inside. On the other hand, colonies that are too small may not be suitable for subculture. In contrast, in the culture tank 30 shown in FIG. 2, since the flow rate of the medium is slow or the medium does not flow, the frequency of cell collisions is low. Therefore, it is possible to maintain clonality in the colonies. Thus, 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 somatic cell. Also, since the frequency of collisions between stem cells is low, it is possible to keep the size of the stem cell colonies uniform.

[0220] The cell culturing apparatus according to the embodiment may further include an imaging device such as a photographic camera or a video camera that photographs the cell-containing medium in the culture tank 30 through the window 132 of the cover 32 of the culture tank 30. Here, when a colorless medium is used, it is possible to suppress specular reflection and autofluorescence that may occur when a colored medium is used. However, it may contain a pH indicator such as phenol red to confirm the pH of the medium. Also, since the shape and size of cells differ between stem cells maintaining an undifferentiated state and differentiated cells, the cell culturing apparatus may further include a differentiation state monitoring device that monitors the differentiation state of the cells by photographing the cells in the culture tank 30.

[0221] When culturing cells on a flat dish such as a petri dish, the area where the cells are present spreads out planar. Therefore, if the imaging device and the petri dish are arranged so that the optical axis of the lens of the imaging device is orthogonal to the dish surface, it is possible to focus on almost all the cells on the petri dish.

[0222] However, when cells are suspended in a culture medium in the culture tank 30 for suspension culture, since the range of cell presence expands three-dimensionally, there is variation in the distance in the optical axis direction from the imaging device to each cell. Therefore, it may be difficult to focus on all cells.

[0223] On the other hand, it is possible to increase the depth of field by using a bright lens (a lens with a small F-number), or by imaging while irradiating the object to be measured with bright illumination and closing the aperture of the lens as much as possible.

[0224] Alternatively, a plurality of images may be captured while gradually changing the focal position of the lens, and the captured plurality of images may be synthesized to obtain a pseudo-deeply focused image. Also, each of the plurality of images will be an image in which in-focus cells and out-of-focus blurred cells are mixed. Therefore, in-focus partial images may be collected from the plurality of images to generate a single composite image.

[0225] Also, for example, cells may be imaged through a telecentric lens. A telecentric lens makes the chief ray passing through the center of the lens aperture from the subject such as a cell parallel to the optical axis of the lens. Therefore, even if the distances from the imaging device to each of the plurality of cells in the culture tank 30 are not uniform, the size of the cells to be imaged does not change according to the distance.

[0226] When imaging the cells in the culture tank 30 with the imaging device, it is advisable to use the scattered light illumination method in which the illumination light source is arranged in a direction perpendicular to the optical axis of the imaging device, or in a direction close to the imaging device from the perpendicular direction, and the illumination light is irradiated from the illumination light source to the cells in the culture tank 30. As a result, the scattered light due to the illumination light hitting the cells reaches the imaging device, but the illumination light that did not hit the cells does not reach the imaging device. Therefore, in the image, the part of the culture medium becomes relatively dark and the part of the cells becomes relatively bright. However, as long as the cells can be recognized in the image, the illumination method is not limited to this.

[0227] As shown in FIG. 8, the cell incubator according to the embodiment may include a central processing unit (CPU) 500 having an image processing unit 501 that processes an image captured by an imaging device disposed in front of the culture tank 30. An input device 401 such as a keyboard and a mouse, and an output device 402 such as a monitor may be connected to the CPU 500. The CPU 500 receives an image from an imaging device disposed in front of the culture tank 30 shown in FIG. 2 via a bus and an image interface or the like.

[0228] The image processing unit 501 shown in FIG. 8 may include a contour definition unit 511 that defines the contour of a cell or a cell mass in the cell image. FIG. 9 is an example of an image of an iPS cell mass captured by magnification through a macro zoom lens or the like. In the image shown in FIG. 9, the portion that appears as a white mass is the iPS cell mass, and the dark portion of the background is the culture medium.

[0229] Here, when the image shown in FIG. 9 is an 8-bit grayscale image, a binarization process is applied to the image in which the luminance value of a pixel having a luminance value equal to or higher than a predetermined threshold is set to the highest luminance value such as 255, and the luminance value of a pixel having a luminance value lower than the predetermined threshold is set to the lowest luminance value such as 0. As shown in FIG. 10, not only the portion of the culture medium but also the inside of the cell or the cell mass becomes black with the lowest luminance, and a portion where the inside of the cell or the cell mass and the portion of the culture medium are connected may occur. Therefore, in the binarization process, there may be cases where cells or cell masses cannot be extracted.

[0230] In contrast, the contour definition unit 511 shown in FIG. 8 applies a high-pass filter to the image of the cell, which passes high-frequency components of a predetermined frequency or higher included in the spatial frequency and blocks low-frequency components of less than the predetermined frequency, and sets the luminance value to the lowest value such as 0, for example. In the image of the cell, there are many high-frequency components included in the spatial frequency in the part of the cell or cell mass, and there are few high-frequency components included in the spatial frequency in the part of the culture medium. Therefore, as shown in FIG. 11, in the image of the cell to which the high-pass filter is applied, the luminance value of the part of the culture medium becomes the lowest value such as 0, for example, and in the part of the cell or cell mass, the luminance value becomes a relatively large value compared to the part of the culture medium. Therefore, it is possible to regard the part where the luminance does not become the lowest value as a cell or cell mass.

[0231] Here, in the image shown in FIG. 11, even if the part where the luminance does not become the lowest value is detected as a blob by blob analysis, for example, two cells or cell masses in contact with each other may be recognized as one cell or cell mass.

[0232] In contrast, the contour definition unit 511 shown in FIG. 8 applies the watershed algorithm to the image to which the high-pass filter is applied. The watershed algorithm regards the luminance gradient of the image as the undulation of mountains, and divides the image so that the area formed by the water flowing from a high position (a position with a large or small luminance value) of the mountain to a low position (a position with a small or large luminance value) is regarded as one region.

[0233] For example, before applying the watershed algorithm to the image, the contour definition unit 511 converts the image by the Distance Transform method. The Distance Transform method is an image conversion method that replaces the luminance value of each pixel in the image according to the distance to the nearest background pixel. For example, as shown in Fig. 12(a), in the image to which the high-pass filter has been applied, the luminance values of the medium regions are uniformly converted to the maximum luminance value of 255, and a white background is created as shown in Fig. 12(b). Further, the luminance value of each pixel inside the cell region is converted to a value between 0 and less than 255 according to the distance to the nearest background pixel. For example, the farther away from the nearest background pixel, the lower the luminance value.

[0234] Next, the contour definition unit 511 applies the watershed algorithm to the image converted by the Distance Transform method. In the image shown in Fig. 12(b), the dark parts with low luminance are regarded as the ridges of the mountains, and when water is dropped from above in the vertical direction of the image, it is estimated how the water flows as shown by the arrows in Fig. 12(c). As shown by the dashed line in Fig. 12(c), the places where the water flowing from various directions collides are regarded as valleys, and at the bottom of the valleys, the cell regions are divided.

[0235] When the pixels in the cell regions of the image shown in Fig. 11 are converted by the Distance Transform method, the image shown in Fig. 13 is obtained. When the watershed algorithm is applied to the image shown in Fig. 13, the image shown in Fig. 14 is obtained. When the obtained dividing line is overlaid on the original image shown in Fig. 9, the image shown in Fig. 15 is obtained. In Fig. 15, it is possible to regard the cells or cell clusters existing in each region divided by the dividing line as not a cluster where a plurality of cells or cell clusters are in contact, but a single cell or cell cluster. Therefore, in each region, as shown in Fig. 16, by extracting the contours of the cells or cell clusters, it becomes possible to accurately extract a single cell or cell cluster.

[0236] The image processing unit 501 shown in FIG. 8 may further include a cell evaluation unit 512. The cell evaluation unit 512 evaluates the size and the like of one cell or cell mass extracted by the contour definition unit 511. For example, the cell evaluation unit 512 calculates the area of one cell or cell mass extracted by the contour definition unit 511. Further, for example, when the shape of one cell mass can be regarded as substantially circular, the cell evaluation unit 512 calculates the diameter of one cell or cell mass from the area using the following formula (1). D = 2(s / π) 1 / 2 (1) Here, D represents the diameter and S represents the area.

[0237] If the cell mass grows too large, nutrients and hormones contained in the culture medium may not reach the inside, and the cells may differentiate. Also, if the cell mass is cultured in a state where it is too small, cell death or karyotype abnormalities may occur. Therefore, when the size of an individual cell mass is outside an appropriate range, the cell evaluation unit 512 may issue a warning. Further, when the size of an individual cell mass is equal to or greater than a predetermined threshold, the cell evaluation unit 512 may output that it is the timing for subculturing. Furthermore, according to the calculated size of the cell mass, the fluid machinery of the cell incubator may be controlled to change the circulation rate of the culture medium in the culture medium flow path. For example, as the size of the cell mass increases, the circulation rate of the culture medium may be increased.

[0238] The image processing unit 501 may further include a statistical processing unit 513 that statistically processes information obtained from the image-processed image. The statistical processing unit 513 may, for example, calculate the frequency distribution of the size or create a histogram for the cell mass extracted by the contour definition unit 511. Also, the statistical processing unit 513 may calculate the growth rate, number, and density, etc. of the cell mass by continuously and periodically acquiring cell information. Thereby, it is possible to quantitatively grasp the state of the cell mass and to stabilize the culture result. Further, according to the calculated number of cell masses, the fluid machinery of the cell incubator may be controlled to change the circulation rate of the culture medium in the culture medium flow path. For example, as the number of cell masses increases, the circulation rate of the culture medium may be increased.

[0239] The image processing unit 501 may further include a density calculation unit 514 that calculates the turbidity of the culture medium from the image of the culture medium and calculates the density of cells or cell aggregates in the culture medium based on the turbidity of the culture medium.

[0240] For example, a relational storage device 403 including a volatile memory or a non-volatile memory is connected to the CPU 500. The relational storage device 403 stores, for example, the relationship between the turbidity of the culture medium and the density of cells or cell aggregates in the culture medium, which has been acquired in advance. The density calculation unit 514 reads out the relationship between the turbidity and the density from the relational storage device 403. Further, the density calculation unit 514 calculates the density of cells or cell aggregates in the culture medium based on the value of the turbidity of the culture medium calculated from the image of the culture medium and the relationship between the turbidity and the density. Thereby, it is possible to non-destructively measure the density of cells or cell aggregates in the culture tank 30 without collecting cell aggregates from the culture medium. Alternatively, the density calculation unit 514 may calculate the density value of cells or cell aggregates in the culture medium from the number of extracted cells or cell aggregates and the ratio of the volume of the region actually photographed by the photographing device to the volume of the entire culture tank 30 without using the turbidity of the culture medium.

[0241] In addition, when the density of cells or cell aggregates becomes equal to or higher than a predetermined threshold value, the density calculation unit 514 may output that it is the timing for subculture. Further, the density calculation unit 514 may calculate the density of cells or cell aggregates in the culture medium over time and calculate the growth rate of the cell aggregates. An abnormal growth rate may indicate that the cells are abnormal. For example, when an abnormal growth rate is calculated, the density calculation unit 514 issues a warning. In this case, the cell culture may be terminated.

[0242] When the density of cells or cell aggregates in the culture medium increases and the distance between cell aggregates becomes too close, a plurality of cells or cell aggregates may adhere to each other to form one large cell aggregate. In a large cell aggregate, nutrients and hormones contained in the culture medium may not reach the inside, and the internal cells may differentiate. On the contrary, when the density of cells or cell aggregates in the culture medium is lower than the suitable range, the growth rate of the cell aggregates and the ability to form cell aggregates may be significantly reduced.

[0243] On the other hand, according to the density calculation unit 514, since it is possible to calculate the density of cells or cell aggregates, it is possible to easily determine whether the density of cells or cell aggregates is within a suitable range. When the density of cells or cell aggregates is lower than the suitable range, for example, a decision to discontinue the culture may be made. Further, according to the calculated density of cells or cell aggregates, the fluid machinery of the cell incubator may be controlled to change the circulation rate of the culture medium in the culture medium flow path. For example, as the density of cells or cell aggregates increases, the circulation rate of the culture medium may be increased.

[0244] The image processing unit 501 may further include a culture medium evaluation unit 515 that evaluates the culture medium based on an image of the culture medium in which cells are being cultured in the culture tank 30 of the cell incubator. The culture medium evaluation unit 515, for example, performs image processing on the image of the culture medium and represents the color of the culture medium in terms of three parameters: hue, saturation, and value (HSV). Among these, the hue is a parameter corresponding to a concept generally referred to as "color tone" or "color shade". The hue is generally expressed in units of angles.

[0245] For example, the relational memory device 403 stores the relationship between the hue of the culture medium and the pH of the culture medium, which has been acquired in advance. The culture medium evaluation unit 515 reads out the relationship between the hue and the pH from the relational memory device 403. Further, the culture medium evaluation unit 515 calculates the pH value of the photographed culture medium based on the hue value of the culture medium calculated from the image of the culture medium and the relationship between the hue and the pH. For example, the culture medium evaluation unit 515 may acquire an image of the culture medium over time and calculate the pH value of the culture medium.

[0246] When the hue of the culture medium or the pH of the culture medium in which the cells in the culture tank 30 shown in FIG. 2 are being cultured is outside a predetermined range, the culture medium evaluation unit 515 may determine to promote the replacement of the culture medium held in the culture medium holding tank 40, or may determine that contamination has occurred in the culture medium. Note that replacing the culture medium includes partially replacing the culture medium and replenishing it.

[0247] Furthermore, the medium evaluation unit 515 shown in FIG. 8 may calculate the cell growth rate from the rate of change in the hue of the medium in which the cells in the culture tank 30 are being cultured. For example, the relational memory device 403 stores the relationship between the rate of change in the hue of the medium and the cell growth rate, which has been acquired in advance. The medium evaluation unit 515 reads out the relationship between the rate of change in hue and the growth rate from the relational memory device 403. Furthermore, the medium evaluation unit 515 calculates the value of the cell growth rate based on the calculated value of the rate of change in hue and the relationship between the rate of change in hue and the growth rate.

[0248] According to the cell incubator according to the embodiment, for example, since cells are cultured in a completely closed system, it is possible to reduce the risk of cross-contamination due to leakage of cells from the culture device. Also, for example, even when the cells are infected with a virus such as the HIV hepatitis virus, it is possible to reduce the risk of infection to the operator due to leakage of the cells. Furthermore, it is possible to reduce the risk of contamination of the medium in the cell incubator by bacteria, viruses, mold, etc. in the air outside the cell incubator. Moreover, according to the cell incubator according to the embodiment, it is also possible to culture cells without using an 2 incubator.

[0249] As described above, the present invention has been described by way of embodiments, but the description and drawings forming part of this disclosure should not be understood as limiting the invention. Various alternative embodiments, embodiments and operation techniques should be apparent to those skilled in the art from this disclosure. For example, when circulation of the culture medium is not required, the culture medium flow path 200 does not have to be connected to the culture medium holding tank 40 shown in FIG. 3. Further, in the culture tank 30 of the cell culture device according to the embodiment, differentiated cells such as blood cells may be reprogrammed into stem cells, or stem cells may be induced into differentiated cells such as nerve cells. Further, the cells may be cultured in suspension or adherently in the culture tank 30. When the cells are cultured adherently, 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. Further, the culture medium flow path may be used without being connected to the culture medium holding tank or the culture tank, and cells may be cultured in the culture medium flow path. In this case, the cells and the culture medium cultured in the culture medium flow path may be observed. Thus, it should be understood that the present invention encompasses various embodiments and the like not described herein.

Example

[0250] (Example 1) As shown in FIGS. 17 and 18, a semipermeable membrane 110 (Asahi Kasei Corporation or SPECTRUM) was sandwiched between the culture side plate 21 and the culture medium side plate 22, and further, the semipermeable membrane 110, the culture side plate 21 and the culture medium side plate 22 were sandwiched between the culture tank 30 and the culture medium holding tank 40.

[0251] DMEM / F12 containing 20% alternative serum (KnockOut SR, registered trademark, Gibco) was gelled to prepare a gel culture medium. Single-cell iPS cells were added to the gel culture medium at 2×10 5 cells / mL to prepare a cell-containing culture medium.

[0252] 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 the plug 33. Also, an empty syringe was connected to the discharge port 331 of the culture tank 30 via the plug 34. Next, the cell-containing medium in the syringe was injected into the culture tank 30 from the supply port 231 of the culture tank 30. Due to the pressure increase in the culture tank 30, the piston of the syringe connected to the discharge port 331 rose passively, and the air in the culture tank 30 moved into the syringe connected to the discharge port 331 of the culture tank 30. The cell-containing medium was injected into the culture tank 30 until the air layer in the culture tank 30 completely disappeared. Thereafter, the supply port 231 and the discharge port 331 of the culture tank 30 were shielded.

[0253] The gel medium was placed in a syringe, and the syringe was connected to the inlet 240 of the medium holding tank 40 via the plug 61. Also, an empty syringe was connected to the discharge port 340 of the medium holding tank 40 via the plug 62. Next, the gel medium in the syringe was injected into the medium holding tank 40 from the inlet 240 of the medium holding tank 40. Due to the pressure increase in the medium holding tank 40, the syringe connected to the discharge port 340 of the medium holding tank 40 rose passively, and the air in the medium holding tank 40 moved into the syringe connected to the discharge port 340 of the medium holding tank 40. The gel medium was injected into the medium holding tank 40 until the air layer in the medium holding tank 40 completely disappeared. Thereafter, the inlet 240 and the discharge port 340 of the medium holding tank 40 were shielded. Thereby, the interiors of the culture tank 30 and the medium holding tank 40 were sealed so that gas exchange did not occur completely between the interiors and exteriors of the culture tank 30 and the medium holding tank 40.

[0254] Suspension culture of iPS cells was started in the culture tank 30. Thereafter, once every two days, 2 mL of the gel medium in the medium holding tank 40 was replaced with 2 mL of fresh gel medium. Seven to ten days after the start of the culture in the culture tank 30, the cell-containing medium in the culture tank 30 was discharged with a syringe, and the cell mass of iPS cells formed in the gel medium was collected using a filter, washed with PBS, and placed in a Falcon tube. Further, 500 μL of cell dissociation enzyme (TrypLE Select, Thermo Fisher) was added to the cell mass, and CO 2The cell mass was incubated in the incubator for 5 minutes. Next, the Falcon tube was taken out of the incubator, 500 μL of cell culture medium was put into the Falcon tube, and the cell mass was suspended to make the iPS cells into single cells. 2 mL of cell culture medium 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 iPS cells and gel medium were put into the Falcon tube to prepare a cell-containing medium. Then, in the same manner as above, the cell-containing medium was injected into the culture tank 30, and the iPS cells were cultured in suspension for 7 to 10 days while replacing 2 mL of the gel medium in the medium holding tank 40 once every two days.

[0255] Thereafter, in the same manner as above, subculture and suspension culture for 7 to 10 days were repeated, and in total, the iPS cells were cultured in suspension in the sealed culture tank 30 for more than one month.

[0256] When the iPS cells cultured in the culture tank 30 were observed under a microscope, as shown in Fig. 19, it was confirmed that all of them formed uniform cell masses.

[0257] Also, at the time of subculture, some single-cell iPS cells were dispensed and fixed using 4%-paraformaldehyde. Furthermore, using a flow cytometer, the expression level of the cell surface antigen TRA-1-60 in the fixed iPS cells was measured. TRA-1-60 is a representative surface antigen of pluripotent stem cells, and it is known that the expression level decreases in differentiated cells.

[0258] As a result, as shown in Fig. 20, the iPS cells on the 39th day from the start of culture were more than 90% TRA-1-60 positive. Therefore, it was shown that when the container is sealed, the stem cells can be cultured in an undifferentiated state while maintaining pluripotency over a long period without controlling the carbon dioxide concentration in the container.

[0259] (Reference Example) A gel medium was prepared in the same manner as in Example 1. Single-cell iPS cells were added to the gel medium. The gel medium containing the iPS cells was placed in a 15 mL Falcon tube. Then, the cap of the Falcon tube was tightened firmly.

[0260] The Falcon tube was placed in an incubator with a carbon dioxide concentration of 5% to initiate suspension culture of the iPS cells. Then, once every two days, the cap of the Falcon tube was opened, and 2 mL of the gel medium was added into the Falcon tube. After adding the gel medium, the cap was tightened as described above.

[0261] Seven to ten days after the start of culture in the Falcon tube, the cap of the Falcon tube was opened, and the cell clusters of iPS cells formed in the gel medium were collected using a filter, washed with PBS, and placed in the Falcon tube. Further, 500 μL of cell dissociation enzyme was added to the cell clusters, and CO 2 The cell clusters were incubated in the incubator for 5 minutes. Next, the Falcon tube was taken out from the incubator, 500 μL of stem cell medium was put into the Falcon tube, the cell clusters were suspended to make the iPS cells into single cells. 2 mL of iPS medium was added into 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 iPS cells and the gel medium were put into the Falcon tube. Then, in the same manner as above, while adding the gel medium once every two days, the iPS cells were suspended and cultured in the sealed Falcon tube for 7 to 10 days.

[0262] Thereafter, in the same manner as above, passage and suspension culture for 7 to 10 days were repeated, and in total, the iPS cells were suspended and cultured in the sealed Falcon tube for more than one month.

[0263] When the iPS cells cultured in a Falcon tube were observed under a microscope, as shown in Fig. 21, it was confirmed that all of them formed cell clusters. Also, in the same manner as in Example 1, when the expression level of the cell surface antigen TRA-1-60 in the iPS cells was measured using a flow cytometer, as shown in Fig. 22, the iPS cells were almost 100% TRA-1-60 positive. The size and number of cell clusters of the iPS cells cultured in the Falcon tube were measured. The results are shown in Fig. 23.

[0264] (Example 2) A cell-containing medium was prepared in the same manner as in Example 1. Also, a cell culture apparatus similar to the cell culture apparatus shown in Fig. 2 was prepared. The cell-containing medium was injected into the culture tank 30 until the air layer in the culture tank 30 completely disappeared. Then, the supply port and the discharge port of the culture tank 30 were shielded. Also, the medium holding tank 40, the medium flow path 200, and the medium tank 60 were filled with a gel medium. Then, the inlet and the outlet of the medium tank 60 were shielded. Thereby, the interiors of the culture tank 30 and the medium holding tank 40 were sealed so that gas exchange did not occur completely between the interiors and the exteriors of the culture tank 30 and the medium holding tank 40.

[0265] The gel medium was circulated in the medium holding tank 40, the medium flow path 200, and the medium tank 60, and the suspension culture of the iPS cells was started in the culture tank 30. Then, once every 2 to 6 days, 10 mL of the gel medium in the medium tank 60 was replaced with 10 mL of fresh gel medium. 7 to 10 days after the start of the 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 1 was performed. In the same manner as above, the cell-containing medium was injected into the culture tank 30, and the iPS cells were suspension-cultured for 7 to 10 days while replacing 10 mL of the gel medium in the medium tank 60 once every 4 days.

[0266] Thereafter, in the same manner as above, subculture and suspension culture for 7 to 10 days were repeated, and in total, the iPS cells were suspension-cultured in the sealed culture tank 30 for more than 1 month.

[0267] When the iPS cells cultured in the culture tank 30 were observed under a microscope, as shown in Fig. 24, it was confirmed that all of them formed uniform cell masses. Also, in the same manner as in Example 1, when the expression level of the cell surface antigen TRA-1-60 in the iPS cells was measured using a flow cytometer, as shown in Fig. 25, the iPS cells on the 15th day from the start of culture were almost 100% TRA-1-60 positive.

[0268] The size and number of cell masses of iPS cells cultured in a cell culture device similar to the cell culture device shown in Fig. 2 were measured. The results are shown in Fig. 23. It was confirmed that the number of clamps was larger compared to the reference example. Therefore, it was shown that using the cell culture device improved the cell survival rate and promoted the growth rate.

[0269] (Example 3) A growth factor was added to the medium (StemSpan H3000, registered trademark, STEMCELL Technologies Inc.), and further deacylated gellan gum was added to the medium to prepare a gel medium.

[0270] The prepared gel medium was placed in a 15 mL tube, and 2×10 5 blood cells were seeded. Then, the 15 mL tube was placed in a CO 2 incubator, and blood cells (mononuclear cells) were cultured for 7 days. Then, a Sendai virus vector (CytoTune-iPS2.0, ID Pharma Co., Ltd.) carrying OCT3 / 4, SOX2, KLF4, and cMYC was added to the gel medium so that the multiplicity of infection (MOI) was 10.0, and the blood cells were infected with the Sendai virus.

[0271] 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. Among them, the medium containing cells infected with 15 mL of Sendai virus was placed in the culture tank 30 shown in FIGS. 17 and 18, and the gel medium was injected into the medium holding tank 40. Similar to Example 1, the interiors of the culture tank 30 and the medium holding tank 40 were sealed so that gas exchange did not occur completely between the inside and outside of the culture tank 30 and the medium holding tank 40.

[0272] Suspension culture of the cells introduced with the initialization factor was started in the culture tank 30. Then, once every two days, 2 mL of the gel medium in the medium holding tank 40 was replaced with 2 mL of fresh gel medium.

[0273] After 15 days, when the cells were observed under a microscope, as shown in FIG. 26, it was confirmed that ES cell-like colonies were formed. Also, 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 FIG. 27, it was confirmed that more than 90% were TRA-1-60 positive and almost completely reprogrammed. Therefore, it was shown that iPS cells can be induced from somatic cells other than stem cells without medium exchange and gas exchange in a completely closed environment.

Explanation of reference numerals

[0274] 10 ··· culture component permeable member, 21 ··· culture side plate, 22 ··· culture medium side plate, 30 ··· culture tank, 31 ··· housing, 32 ··· cover, 33 ··· plug, 34 ··· plug, 40 ··· culture medium holding tank, 41 ··· flow rectifying plate, 51 ··· fluid machine for introduction, 52 ··· fluid machine for discharge, 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 ··· pump head, 152 ··· pump head, 200 ··· culture medium flow path, 231 ··· supply port, 240 ··· introduction port, 241 ··· discharge port, 242 ··· opening, 251 ··· drive unit, 252 ··· drive unit, 331 ··· discharge port, 340 ··· discharge port, 351 ··· external air blocking member for fluid machine for introduction, 352 ··· external air blocking member for fluid machine for discharge, 401 ··· input device, 402 ··· output device, 403 ··· relationship storage device, 501 ··· image processing unit, 511 ··· contour definition unit, 512 ··· cell evaluation unit, 513 ··· statistical processing unit, 514 ··· density calculation unit, 515 ··· culture medium evaluation unit

Claims

1. A culture component permeable member through which a culture component can permeate; A sealable culture tank that holds a cell-containing culture medium and cultures cells, covering one surface of the culture component permeable member; A culture medium holding tank that holds a culture medium, covering the other surface of the culture component permeable member; Comprising: A supply port for supplying a fluid into the culture tank and a discharge port for discharging the fluid in the culture tank are provided in the culture tank; The supply port and the discharge port are sealable; A supply device for supplying a fluid to the supply port is detachable; A discharge device for discharging a fluid to the discharge port is detachable; When a fluid is supplied from the supply device into the culture tank, the internal volume of the discharge device increases and the fluid in the culture tank moves into the discharge device; A cell incubator.

2. The cell incubator according to claim 1, wherein the supply device is connected to the supply port via a supply needleless connector.

3. The cell incubator according to claim 1, wherein the discharge device is connected to the discharge port via a discharge needleless connector.

4. The cell incubator according to any one of claims 1 to 3, wherein when a culture medium is supplied from the supply device into the culture tank, the air in the culture tank moves into the discharge device.

5. The cell incubator according to claim 3, wherein when a culture medium is supplied from the supply device into the culture tank, the culture medium in the culture tank moves into the discharge device.

6. The cell incubator according to claim 4 or 5, wherein the culture medium contains cells.

7. The cell incubator according to any one of claims 1 to 6, wherein when a fluid is supplied from the supply device into the culture tank, outside air does not enter the culture tank.

8. The cell incubator according to any one of claims 1 to 7, provided with a window.

9. The cell incubator according to claim 8, wherein a transparent heater is provided on the window.

10. The cell incubator according to any one of claims 1 to 9, comprising a temperature adjustment unit for adjusting the temperature inside the culture tank.

11. The cell incubator according to any one of claims 1 to 10, comprising a thermometer for measuring the temperature inside the culture tank.

12. The cell incubator according to any one of claims 1 to 11, wherein outside air does not enter the culture tank in a sealed state of the culture tank.

13. The cell culturing apparatus according to any one of claims 1 to 12, wherein, with the culture tank being in a sealed state, cells, microorganisms, viruses, and dust outside the culture tank do not enter the culture tank.

14. The cell culturing apparatus according to any one of claims 1 to 13, wherein, with the culture tank being in a sealed state, substances inside the culture tank do not flow out to the outside of the culture tank.

15. The cell culturing apparatus according to any one of claims 1 to 14, wherein, with the culture tank being in a sealed state, gas exchange does not occur between the inside and outside of the culture tank.

16. The cell culturing apparatus according to any one of claims 1 to 15, wherein, with the culture tank being in a sealed state, at least any one of carbon dioxide gas, nitrogen gas, and oxygen gas is not supplied into the culture tank.

17. The cell culturing apparatus according to any one of claims 1 to 16, wherein the pH of the culture medium in the culture tank is maintained within a predetermined range.

18. The cell culturing apparatus according to any one of claims 1 to 17, wherein the culture tank is embedded in a gas-impermeable substance.

19. The cell culturing apparatus according to any one of claims 1 to 18, wherein the inclination of the culture tank is adjustable.

20. The cell culturing apparatus according to any one of claims 1 to 19, wherein stem cells are expanded and cultured in the culture tank.

21. The cell culturing apparatus according to claim 20, wherein the stem cells are iPS cells, ES cells, or somatic stem cells.

22. The cell culturing apparatus according to any one of claims 1 to 19, wherein cells into which an inducer has been introduced are cultured in the culture tank and induced into stem cells.

23. The cell culturing apparatus according to any one of claims 1 to 19, wherein an inducer is added to the culture medium in the culture tank, and the inducer is introduced into the cells being cultured in the culture tank.

24. The cell culturing apparatus according to claim 23, wherein the cells into which the inducer has been introduced are induced into stem cells.

25. The cell culturing apparatus according to claim 22 or 24, wherein the stem cells are iPS cells.

26. The cell culturing apparatus according to any one of claims 22 or 23, wherein the cells into which the inducer is introduced are blood cells.

27. The cell culturing apparatus according to any one of claims 1 to 19, wherein cells into which an inducer has been introduced are cultured in the culture tank and induced into different types of cells.

28. An inducer is added to the medium in the culture tank, the inducer is introduced into the cells being cultured in the culture tank, and the cells are induced into different types of cells. The cell culture device according to any one of claims 1 to 19.

29. The cell culture device according to any one of claims 22 to 28, wherein the inducer is RNA.

30. The cell culture device according to any one of claims 22 to 29, wherein the inducer is contained in Sendai virus.

31. The cell culture device according to any one of claims 22 to 28, wherein the inducer is contained in a plasmid.

32. The cell culture device according to any one of claims 1 to 19, wherein cells are cultured in the culture tank.

33. The cell culture device according to claim 32, wherein the cells are blood cells.

34. The cell culture device according to any one of claims 1 to 33, wherein cells are suspension-cultured in the culture tank.

35. The cell culture device according to any one of claims 1 to 33, wherein cells are adherently cultured in the culture tank.

36. The cell culture device according to any one of claims 1 to 34, wherein cells are cultured in the gel medium in the culture tank.

37. The cell culture device according to any one of claims 1 to 36, wherein the interior is non-cell adhesive.

38. The cell culture device according to any one of claims 1 to 37, wherein the interior is non-protein adhesive.

39. The cell culture device according to any one of claims 1 to 36, wherein the interior is cell adhesive.

40. The cell culture device according to any one of claims 1 to 39, and an imaging device that images at least one of the medium and cells in the culture tank. A culture device comprising the same.

41. The culture device according to claim 40, wherein the imaging device images the cells through a telecentric lens.

42. The culture device according to claim 40 or 41, further comprising an image processing unit that applies a high-pass filter to the image obtained by the imaging device.

43. The culture device according to claim 42, wherein the image processing unit applies a watershed algorithm to the image to which the high-pass filter has been applied to extract cells or cell clusters in the image.

44. The culture device according to claim 43, wherein the image processing unit applies the Distance Transform method to the image before applying the watershed algorithm to the image.

45. The culture device according to claim 43 or 44, wherein the image processing unit calculates the size of the extracted cell or cell aggregate.

46. The culture device according to any one of claims 43 to 45, wherein the image processing unit calculates the number of the extracted cells or cell aggregates.

47. The culture device further includes a relationship storage device that stores the relationship between the turbidity of the culture medium and the density of cells or cell aggregates in the culture medium. The culture device according to any one of claims 40 to 46, further comprising an image processing unit that calculates the value of the turbidity of the culture medium in the culture tank based on the image obtained by the imaging device, and calculates the value of the density of the photographed cells or cell aggregates based on the calculated turbidity value and the relationship.

48. The culture device according to claim 43 or 44, further comprising an image processing unit that calculates the value of the density of cells or cell aggregates in the culture medium from the number of the extracted cells or cell aggregates and the ratio of the volume of the region photographed by the imaging device to the volume of the entire culture tank.

49. The culture device further includes a relationship storage device that stores the relationship between the color of the culture medium and the pH of the culture medium. The culture device according to any one of claims 40 to 47, further comprising an image processing unit that calculates the value of the color of the culture medium in the culture tank in the image obtained by the imaging device, and calculates the value of the pH of the photographed culture medium based on the calculated color value and the relationship.

Images