JP2019088335A - Cell processing system and cell processing device - Google Patents

Abstract

To provide a cell processing system allowing processing of cells without contaminating the surroundings.SOLUTION: A cell processing system comprises: a housing 601; an outer housing 701 which contains the housing 601; an intake air purification filter 602 which is provided in the housing 601 and purifies gas sucked from the exterior of the housing 601; a circulation device which circulates the gas into and out of the housing 601 in the outer housing 701 so that the gas in the outer housing 701 is sucked into the housing 601 through the intake air purification filter 602 and so that the gas in the housing 601 is discharged into the outer housing 701; and a cell processing device which is disposed in the housing 601 and is for processing cells.SELECTED DRAWING: Figure 1

Description

  The present invention relates to cell technology, and relates to a cell processing system and a cell processing apparatus.

  Embryonic stem cells (ES cells) are stem cells established from human and mouse early embryos. ES cells have pluripotency that can differentiate into all cells present in a living body. Currently, human ES cells are available for cell transplantation therapy for many diseases, such as Parkinson's disease, juvenile diabetes, and leukemia. However, there are also obstacles to transplantation of ES cells. In particular, transplantation of ES cells can elicit an immune rejection similar to the rejection that occurs following unsuccessful organ transplantation. In addition, there are many criticisms and dissent from the ethical point of view on the use of ES cells established by destroying human embryos.

  Under such circumstances, Professor Shingo Yamanaka of Kyoto University has introduced induced pluripotent stem cells (iPS) by introducing four genes: Oct3 / 4, Klf4, c-Myc, and Sox2 into somatic cells. Succeeded in establishing cells). As a result, Professor Yamanaka received the 2012 Nobel Prize in Physiology and Medicine (see, for example, Patent Documents 1 and 2). iPS cells are ideal pluripotent cells free of rejection and ethical problems. Therefore, iPS cells are expected to be used for cell transplantation therapy.

Patent No. 4183742 JP, 2014-114997, A

  Induced stem cells such as iPS cells are established by introducing an inducer such as a gene into cells, and are expanded and cryopreserved. Conventionally, stem cells are manufactured manually by humans in a clean room. At this time, it is necessary to prevent human contact with blood and inducers used in producing stem cells. In addition, since cells may be infected with viruses and pathogens, it is necessary to contain cells so that they do not go outside. For example, the spread of cells infected with hepatitis virus or human immunodeficiency virus (HIV) has a lethal effect on humans and animals. Thus, blood, inducers, cells before induction and induced cells must also be prevented from being released out of the clean room. Furthermore, in order to prevent cross contamination with other people's iPS cells, iPS cells derived from only one person are produced in a clean room for a predetermined time.

  On the other hand, an object of the present invention is to provide a cell processing system and a cell processing apparatus capable of processing cells without contaminating the surroundings.

  According to the aspect of the present invention, the casing, the outer casing enclosing the casing, the intake purification filter provided on the casing for purifying the gas sucked from the outside of the casing, and the gas in the outer casing A circulation device that circulates the gas inside and outside the housing so as to be sucked into the housing through the intake air purification filter and the gas in the housing is exhausted into the outer housing; And a cell processing apparatus for processing cells.

  In the above-mentioned cell processing system, a pressure control hole may be provided in the outer casing.

  In the cell processing system described above, the circulation device may be provided with a gas discharge device provided in the housing for sucking gas from the inside of the housing and discharging the purified gas to the outside of the housing.

  In the above cell processing system, the gas discharge device may include an exhaust device that exhausts the gas in the housing to the outside of the housing, and an exhaust gas purification filter that purifies the gas sucked by the exhaust device.

  In the above cell processing system, the exhaust gas purification filter may be disposed upstream of the exhaust device.

  The above cell processing system may further include a shielding member that can be attached to the exhaust gas purification filter so as to shield the exhaust gas purification filter.

  In the above cell processing system, the shielding member can be attached to the exhaust gas purification filter so as to shield the exhaust gas purification filter from the inside of the casing, and the exhaust gas purification filter so as to shield the exhaust gas purification filter from the exhaust system And an exhaust device side shielding member that can be attached to the housing.

  The cell processing system described above may further include a sterilizer for sterilizing the exhaust gas purification filter.

  The cell processing system described above may further include a second exhaust gas purification filter disposed downstream of the exhaust system.

  The above cell processing system may further include a shielding member that can be attached to the second exhaust gas purification filter so as to shield the second exhaust gas purification filter.

  In the above cell processing system, the shielding member may include an exhaust device side shielding member that can be attached to the second exhaust gas purification filter so as to shield the second exhaust gas purification filter from the exhaust device.

  The cell processing system described above may further include a sterilizer for sterilizing the second exhaust gas purification filter.

  In the above-described cell processing system, the circulation device may be provided with an introduction device provided in the housing for suctioning the gas to be purified by the intake air purification filter from outside the housing.

  In the above cell processing system, the intake air purification filter may be disposed downstream of the introduction device.

  The cell processing system described above may further include a shielding member that can be attached to the intake air purification filter so as to shield the intake air purification filter.

  In the above cell processing system, the shielding member can be attached to the intake purification filter so as to shield the intake purification filter from the inside of the casing, and the intake purification filter to shield the intake purification filter from the introduction device And an introducer-side shield member that can be attached to the

  The cell processing system described above may further include a sterilizer for sterilizing the intake air purification filter.

  In the above cell processing system, the inside of the housing may be under negative pressure as compared to the outside of the housing.

  In the above cell processing system, stem cells may be cultured in the cell processing apparatus.

  The above cell processing system may further include a sterilizer for sterilizing a space in which the cell processing device in the housing is disposed.

  The cell processing system described above may further include a temperature management device that manages the temperature of the space in which the cell processing device in the housing is disposed.

  The cell processing system described above may further include a carbon dioxide concentration management device that manages the carbon dioxide concentration in the space where the cell processing device in the housing is disposed.

  In the above cell processing system, the inside of the housing may be divided into a plurality of regions.

  In the above cell processing system, the cell processing device is connected to the pre-introduction cell delivery channel through which the solution containing the cells passes and the pre-introduction cell delivery channel to introduce a pluripotency inducer into the cells to induce the induction factor. A factor introduction apparatus for producing introduced cells, and a cell mass producing apparatus for producing a plurality of cell souls consisting of stem cells by culturing inducer-induced cells may be provided.

  In the above cell processing system, a housing, an intake air purification filter provided on the housing for purifying gas sucked from outside the housing, and a gas for discharging the gas discharged from the housing are returned to the intake air purification filter Between the casing and the return member, the return member and the gas in the casing are discharged into the return member, and the gas in the return member is sucked into the casing through the intake air purification filter. And a cell processing apparatus disposed in the housing for processing cells.

  In the above-described cell processing system, the return member may have a shape that mates with the housing.

  In the cell processing system described above, the return member causes the base having a hollow inside to be in contact with the bottom surface of the casing, and the first opening provided in the base to communicate the ventilating portion provided in the first end face of the casing You may provide the 1st cover, and the 2nd cover which makes the ventilation part provided in the 2nd opening and the 2nd end face of a case which were provided in the base communicate.

  In the above cell processing system, the return member may be a duct.

  In the cell processing system described above, the circulation device may be provided with a gas discharge device provided in the housing for sucking gas from the inside of the housing and discharging the purified gas into the return member.

  In the cell processing system described above, the gas discharge device may include an exhaust device that exhausts the gas in the housing into the return member, and an exhaust gas purification filter that purifies the gas drawn by the exhaust device.

  In the above cell processing system, the exhaust gas purification filter may be disposed upstream of the exhaust device.

  The above cell processing system may further include a shielding member that can be attached to the exhaust gas purification filter so as to shield the exhaust gas purification filter.

  In the above cell processing system, the shielding member can be attached to the exhaust gas purification filter so as to shield the exhaust gas purification filter from the inside of the casing, and the exhaust gas purification filter so as to shield the exhaust gas purification filter from the exhaust system And an exhaust device side shielding member that can be attached to the housing.

  The cell processing system described above may further include a sterilizer for sterilizing the exhaust gas purification filter.

  The cell processing system described above may further include a second exhaust gas purification filter disposed downstream of the exhaust system.

  The above cell processing system may further include a shielding member that can be attached to the second exhaust gas purification filter so as to shield the second exhaust gas purification filter.

  In the above cell processing system, the shielding member may include an exhaust device side shielding member that can be attached to the second exhaust gas purification filter so as to shield the second exhaust gas purification filter from the exhaust device.

  The cell processing system described above may further include a sterilizer for sterilizing the second exhaust gas purification filter.

  In the above-described cell processing system, the circulation device may further include an introduction device provided in the housing for suctioning the gas to be purified by the intake air purification filter from the inside of the return member.

  In the above cell processing system, the intake air purification filter may be disposed downstream of the introduction device.

  The cell processing system described above may further include a shielding member that can be attached to the intake air purification filter so as to shield the intake air purification filter.

  In the above cell processing system, the shielding member can be attached to the intake purification filter so as to shield the intake purification filter from the inside of the casing, and the intake purification filter to shield the intake purification filter from the introduction device And an introducer-side shield member that can be attached to the

  The cell processing system described above may further include a sterilizer for sterilizing the intake air purification filter.

  In the above cell processing system, the inside of the housing may be under negative pressure as compared to the inside of the return member.

  In the cell processing device of the above-mentioned cell processing system, stem cells may be cultured.

  The above cell processing system may further include a sterilizer for sterilizing a space in which the cell processing device in the housing is disposed.

  The cell processing system described above may further include a temperature management device that manages the temperature of the space in which the cell processing device in the housing is disposed.

  The cell processing system described above may further include a carbon dioxide concentration management device that manages the carbon dioxide concentration in the space where the cell processing device in the housing is disposed.

  In the above cell processing system, the inside of the housing may be divided into a plurality of regions.

  In the above cell processing system, the cell processing device is connected to the pre-introduction cell delivery channel through which the solution containing the cells passes and the pre-introduction cell delivery channel to introduce a pluripotency inducer into the cells to induce the induction factor. A factor introduction apparatus for producing introduced cells, and a cell mass producing apparatus for producing a plurality of cell souls consisting of stem cells by culturing inducer-induced cells may be provided.

  Further, according to the aspect of the present invention, the cell processing device for processing cells, the embedding member for embedding the cell processing device, and the communication for connecting the outside of the embedding member and the cell processing device in the embedding member A cell processing apparatus is provided, comprising: a fluid delivery channel.

  In the above cell processing apparatus, the embedding member may be made of glass or resin.

  In the above cell processing apparatus, the embedding member may be made of metal.

  The above-described cell processing apparatus may further include a driving unit disposed outside the embedding member for feeding the liquid in the communicating liquid feeding passage.

  In the above cell processing apparatus, the drive unit may be connected to the outer wall of the embedding member.

  In the cell processing apparatus described above, a follower may be provided in the embedding member, to which the driving force is transmitted from the driver, and the follower may be connected to the fluid communication path.

  In the above cell processing apparatus, the cell processing device may include a mononuclear cell separation unit disposed inside the embedding member, which separates mononuclear cells from blood.

  The cell processing apparatus described above further includes a blood storage unit disposed outside the embedding member and configured to store at least one of blood and blood cells, and the communicating liquid passage includes the blood storage unit and the mononuclear cell separation unit. May be provided with a blood delivery channel that communicates the

  The cell processing apparatus described above further includes a separating agent storage unit disposed outside the embedding member and configured to store a separating agent for separating mononuclear cells, and the communicating liquid passage includes the separating agent storage unit and the separation agent storage unit. You may provide the separating agent liquid feeding path which makes a nuclear-sphere isolation | separation part connect.

  In the above-described cell processing device, the cell processing device may include a filter disposed inside the embedding member for isolating cells.

  In the above cell processing apparatus, the cell processing device may include a mononuclear cell purification filter disposed inside the embedding member.

  In the above-described cell processing apparatus, the cell processing device separates mononuclear cells from blood, the mononuclear cell separation unit disposed inside the embedding member, and the mononuclear cell purification disposed inside the embedding member The filter may be provided with a mononuclear cell liquid flow path disposed in the interior of the embedding member, which connects the mononuclear cell separation portion and the mononuclear cell purification filter.

  The above-described cell processing apparatus may further include a driving unit disposed outside the embedding member for feeding the liquid in the mononuclear cell liquid feeding channel.

  In the above cell processing apparatus, the drive unit may be connected to the outer wall of the embedding member.

  In the cell processing apparatus described above, a follower may be provided in the embedding member, to which the driving force is transmitted from the driver, and the follower may be connected to the mononuclear cell liquid transport path.

  In the above-described cell processing apparatus, the cell processing device may include a factor introducing unit disposed inside the embedding member, which introduces pluripotency inducer into cells to produce an inducer-introduced cell.

  The cell processing apparatus described above further includes a factor storage unit for storing the pluripotency inducing factor, which is disposed outside the embedding member, and the communicating fluid delivery path communicates the factor storage unit with the factor introducing unit. A fluid path may be provided.

  In the above-described cell processing apparatus, pluripotency inducer may be introduced into cells by RNA lipofection at the factor introduction site.

  In the above cell processing apparatus, the pluripotency inducer may be DNA, RNA or protein.

  In the above cell processing apparatus, a pluripotency inducer may be incorporated into a vector.

  In the above cell processing apparatus, the vector may be a Sendai virus vector.

  In the above cell processing apparatus, the cell processing device separates mononuclear cells from blood, a mononuclear cell separation unit disposed inside the embedding member, and a inducer by introducing a pluripotency inducer into the cells A factor introduction unit disposed inside the embedding member for producing the introduced cells, a pre-introduction cell liquid flow passage disposed inside the embedding member, which communicates the mononuclear cell separation unit and the factor introduction unit; May be provided.

  The above-described cell processing apparatus may further include a driving unit disposed outside the embedding member for feeding the liquid in the cell delivery channel before introduction.

  In the above cell processing apparatus, the drive unit may be connected to the outer wall of the embedding member.

  In the cell processing apparatus described above, a follower may be provided in the embedding member, to which the driving force is transmitted from the driver, and the follower may be connected to the cell liquid transport path before introduction.

  In the above cell processing apparatus, the cell processing device includes a mononuclear cell purification filter disposed inside the embedding member, and an embedding member for introducing a pluripotency inducer into the cells to produce an inducer-introduced cell. A factor introduction portion disposed inside the cell, and a pre-infusion cell liquid delivery path disposed inside the embedding member, which connects the mononuclear cell purification filter and the factor introduction portion to each other.

  The above-described cell processing apparatus may further include a driving unit disposed outside the embedding member for feeding the liquid in the cell delivery channel before introduction.

  In the above cell processing apparatus, the drive unit may be connected to the outer wall of the embedding member.

  In the cell processing apparatus described above, a follower may be provided in the embedding member, to which the driving force is transmitted from the driver, and the follower may be connected to the cell liquid transport path before introduction.

  In the above-described cell processing apparatus, the cell processing device may include a reprogramming incubator disposed inside the embedding member for culturing the inducer-introduced cells into which the pluripotency inducer has been introduced.

  The cell processing apparatus described above further includes a blood cell culture medium storage unit disposed outside the embedding member for storing the blood cell culture medium, and the communicating liquid flow path communicates the blood cell culture medium storage unit with the reprogramming incubator. It may be provided with a medium delivery channel.

  In the above-described cell processing apparatus, blood cell culture medium may be continuously supplied to the reprogramming incubator.

  In the above cell processing apparatus, the blood cell culture medium may be supplied to the reprogramming incubator at a predetermined timing.

  The above-described cell processing apparatus may further include a refrigerated storage unit disposed outside the embedding member that refrigerates the blood cell culture medium.

  The above-described cell processing apparatus further includes a stem cell culture medium storage unit disposed outside the embedding member and configured to store a stem cell culture medium, and the continuous liquid transfer path communicates the stem cell culture medium storage unit with the reprogramming culture unit. A fluid path may be provided.

  In the above-described cell processing apparatus, stem cell culture medium may be continuously supplied to the reprogramming incubator.

  In the above-described cell processing apparatus, the stem cell culture medium may be supplied to the reprogramming incubator at a predetermined timing.

  The above cell processing apparatus may further include a refrigerated storage unit disposed outside the embedding member, which stores the stem cell culture medium refrigerated.

  The above cell processing apparatus further includes a waste liquid storage unit, which is disposed outside the embedding member and stores the waste liquid, and the communication liquid feed path communicates the initialization incubator and the waste liquid storage portion. You may have.

  In the cell processing apparatus described above, the suspension incubator comprises a reprogramming incubator, a dialysis tube in which inducer-introduced cells and media are placed, and a container in which the dialysis tube is placed and the media is placed around the dialysis tube. May be provided.

  In the above-described cell processing apparatus, the cell processing device introduces a pluripotency inducer into cells to produce an inducer-introduced cell, a factor introducing unit disposed inside the embedding member, and an inducer-introduced cell It comprises: a reprogramming incubator placed inside the embedding member to be cultured; and a cell transfer line for introduced cells placed inside the embedding member that brings the factor introducing part and the reprogramming incubator into communication. It is also good.

  The above-described cell processing apparatus may further include a driving unit disposed outside the embedding member for sending the liquid in the introduced cell delivery channel.

  In the above cell processing apparatus, the drive unit may be connected to the outer wall of the embedding member.

  In the cell processing apparatus described above, a follower may be provided in the embedding member, to which the driving force is transmitted from the driver, and the follower may be connected to the introduced cell liquid transport path.

  In the above cell processing apparatus, the drive unit and the driven unit may be connected by magnetic force.

  In the above-described cell processing apparatus, the cell processing apparatus may include an expansion incubator disposed inside the embedding member for expanding and culturing a plurality of cell masses consisting of established stem cells.

  The above-mentioned cell processing apparatus further includes a stem cell culture medium storage unit arranged outside the embedding member for storing a stem cell culture medium, and the continuous liquid transfer path communicates the culture medium connecting the stem cell culture medium storage unit and the expansion incubator. It may have a way.

  In the above cell processing apparatus, stem cell culture medium may be continuously supplied to the expansion incubator.

  In the above cell processing apparatus, the stem cell culture medium may be supplied to the expansion incubator at a predetermined timing.

  The above cell processing apparatus may further include a refrigerated storage unit disposed outside the embedding member, which stores the stem cell culture medium refrigerated.

  The cell processing apparatus described above further includes a waste liquid storage unit, which is disposed outside the embedding member and stores the waste liquid, and the communication liquid feed path includes a waste liquid feed path that connects the expansion incubator and the waste liquid storage section. It may be

  In the above cell processing apparatus, a suspension incubator is provided, wherein the expansion incubator comprises: a dialysis tube in which the induction factor-introduced cells and the medium are placed; and a container in which the dialysis tube is placed and the culture medium is placed around the dialysis tube. You may have.

  In the above-mentioned cell processing apparatus, the cell processing device is adapted to culture the inducer-introduced cells, to expand and culture a plurality of cell masses consisting of established stem cells and a reprogramming culture vessel placed inside the embedding member, You may provide the expansion incubator arrange | positioned inside the embedding member, and the introduce | transduced cell liquid transport path arrange | positioned inside the embedding member which connects a reprogramming incubator and an expansion incubator.

  The above-described cell processing apparatus may further include a driving unit disposed outside the embedding member for sending the liquid in the introduced cell delivery channel.

  In the above cell processing apparatus, the drive unit may be connected to the outer wall of the embedding member.

  In the cell processing apparatus described above, a follower may be provided in the embedding member, to which the driving force is transmitted from the driver, and the follower may be connected to the introduced cell liquid transport path.

  In the above-described cell processing apparatus, the cell processing device may further include a cell splitter, which is provided in the introduced cell delivery channel, for dividing a cell mass and disposed inside the embedding member.

  In the above cell processing apparatus, the cell processing device may be provided with a solution displacing device that replaces the solution around cells.

  The above cell processing apparatus further includes a cryopreservation solution storage unit including a cryopreservation solution, which is disposed outside the embedding member, and the communicating liquid transport path connects the cryopreservation solution storage unit and the solution substitution device in communication. It may be equipped with a storage fluid delivery channel.

  The cell processing apparatus described above further includes a cryopreservation container, which is disposed outside the embedding member, for storing the cryopreservation solution in which the cell mass is dispersed, and the communicating liquid passage includes the solution substitution device and the cryopreservation You may provide the cell delivery channel for freezing which makes it connect with a container.

  In the cell processing apparatus described above, the cell processing apparatus expands and culture a plurality of cell clusters consisting of established stem cells, and the expansion incubator disposed inside the embedding member and the solution replacement for replacing the solution around the cells The apparatus may include a vessel, and an introduced cell delivery channel disposed in the interior of the embedding member, which communicates the expansion incubator with the solution replacer.

  The above-described cell processing apparatus may further include a driving unit disposed outside the embedding member for sending the liquid in the introduced cell delivery channel.

  In the above cell processing apparatus, the drive unit may be connected to the outer wall of the embedding member.

  In the cell processing apparatus described above, a follower may be provided in the embedding member, to which the driving force is transmitted from the driver, and the follower may be connected to the introduced cell liquid transport path.

  In the above-described cell processing apparatus, the cell processing device may further include a cell splitter, which is provided in the introduced cell delivery channel, for dividing a cell mass and disposed inside the embedding member.

  In the above cell processing apparatus, the cell processing device may be provided with a cell mass divider for dividing cell mass.

  According to the present invention, it is possible to provide a cell processing system and a cell processing apparatus capable of processing cells without contaminating the surroundings.

It is a typical perspective view of the cell processing system concerning an embodiment. It is a typical perspective view of the cell processing system concerning an embodiment. It is a typical perspective sectional view of a cell processing system concerning an embodiment. It is a typical sectional view of a cell processing system concerning an embodiment. It is a typical perspective sectional view of a cell processing system concerning an embodiment. It is a typical sectional view of a cell processing system concerning an embodiment. It is a typical perspective sectional view of a cell processing system concerning an embodiment. It is a typical sectional view of a cell processing system concerning an embodiment. It is a typical perspective view of the cell processing system concerning an embodiment. It is a typical perspective view of the cell processing system concerning an embodiment. It is a typical perspective sectional view of a cell processing system concerning an embodiment. It is a typical perspective view of the cell processing system concerning an embodiment. It is a typical perspective view of the cell processing system concerning an embodiment. It is a typical perspective view of the cell processing system concerning an embodiment. It is a typical sectional view of a cell processing system concerning an embodiment. It is a schematic diagram of the cell processing apparatus concerning embodiment. It is typical sectional drawing of an example of the introduction | transduction cell liquid flow path of the cell processing apparatus concerning embodiment. It is typical sectional drawing of an example of the introduction | transduction cell liquid flow path of the cell processing apparatus concerning embodiment. It is a schematic diagram of the culture | cultivation bag used with the cell processing apparatus which concerns on embodiment. It is a schematic diagram of the suspension incubator which concerns on embodiment. It is a schematic diagram of a replenishment medium feeding pump and a floating incubator concerning an embodiment. It is a schematic diagram of a replenishment medium feeding pump and a floating incubator concerning an embodiment. It is a schematic diagram of the suspension incubator and imaging device which concern on embodiment. It is a schematic diagram of the suspension incubator and imaging device which concern on embodiment. It is an example of the image of the cell which concerns on embodiment. It is a schematic diagram of a central processing unit concerning an embodiment. It is an example of the image of the cell mass which concerns on embodiment. It is an example of the image of the binarized cell mass which concerns on embodiment. It is an example of the image of the cell mass to which the high-pass filter concerning an embodiment was applied. It is an example of the image of the cell mass to which the water diversion algorithm which concerns on embodiment was applied. It is an example of the image of the cell mass to which the Distance Transform method which concerns on embodiment is applied. It is an example of the image of the cell mass to which the water diversion algorithm which concerns on embodiment was applied. It is an example of the image of the cell mass divided | segmented into the several area | region which concerns on embodiment. It is an example of the image of the cell mass from which the outline based on embodiment was extracted. It is an example of the image of the cell mass from which the outline based on embodiment was extracted. It is an example of the histogram of the size of the cell which concerns on embodiment. It is a schematic diagram of the suspension incubator and imaging device which concern on embodiment. It is an example of the graph which shows the relationship between pH of the culture medium which concerns on embodiment, and the hue of a culture medium. It is a schematic diagram of the cell mass dividing device which concerns on embodiment. It is a schematic diagram of the cell mass dividing device which concerns on embodiment. It is a schematic diagram of the cell mass dividing device which concerns on embodiment. It is a schematic diagram of the cell mass dividing device which concerns on embodiment. It is a schematic diagram of the cell mass dividing device which concerns on embodiment. It is a schematic diagram of the cell mass dividing device which concerns on embodiment. It is a schematic diagram of the cell mass dividing device which concerns on embodiment. It is a schematic diagram of the cell mass dividing device which concerns on embodiment. It is an example of the image of the divided cell mass which concerns on embodiment. It is a schematic diagram of the solution displacement device which concerns on embodiment. It is a schematic diagram of the cell processing apparatus concerning embodiment. It is a typical perspective view of the cell processing device concerning an embodiment. It is a typical perspective view of the cell processing device concerning an embodiment. It is a typical perspective view of the cell processing device concerning an embodiment. It is a typical perspective view of the drive part of the cell processing device concerning an embodiment. It is a typical perspective view of the drive part of the cell processing device concerning an embodiment. 5 is a fluorescence micrograph of Example 1. FIG. 7 is a graph showing the analysis results by the fluorescence activated flow cytometer according to Example 1. FIG. FIG. 7 is a photograph of iPS cell colonies according to Example 2. FIG. FIG. 7 is a photograph of iPS cell colonies according to Example 2. FIG. FIG. 7 is a photograph of iPS cell colonies according to Example 2. FIG. FIG. 6 is a graph showing the state of differentiation of iPS cell colonies according to Example 2. FIG. 7 is a photograph of a colony of iPS cells according to Example 3. 15 is a graph showing the results of Example 4; 7 is a photograph of an iPS cell mass according to Example 5. 15 is a graph showing the results of Example 5.

  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 symbols. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios among the drawings are included.

  The present disclosure includes inventions provisionally filed in the United States (62/356, 199) and for which a foreign application permit has already been issued.

  In the cell processing system according to the embodiment, as shown in FIG. 1 to FIG. 3, suction is performed from the outside of the casing 601 provided in the casing 601, the outer casing 701 containing the casing 601, and the casing 601. The gas inside the outer casing 701 is sucked into the casing 601 through the suction purification filter 602, and the gas inside the casing 601 is discharged into the outer casing 701. It includes a circulation device for circulating gas in and out of the housing 601 in the outer housing 701, and a cell processing device disposed in the housing 601 for processing cells.

  The housing 601 has, for example, a rectangular parallelepiped shape, but is not particularly limited thereto. The housing 601 is made of, for example, a material that can withstand heat sterilization or ultraviolet sterilization, but is not particularly limited thereto. At least a portion of the housing 601 may be transparent so that the inside can be observed from the outside. The housing 601 has an openable / closable structure for loading and unloading of the cell processing device disposed inside.

  The circulation device includes, for example, a gas discharge device 613 provided in the housing 601 for sucking a gas from the inside of the housing 601 and discharging the purified gas to the outside of the housing 601. The intake air purification filter 602 and the gas discharge device 613 are disposed to face each other, for example. For example, a High Efficiency Particulate Air (HEPA) filter and an Ultra Low Particulate Air (URPA) filter may be used as the intake air purification filter 602, but the invention is not limited thereto. Depending on the usage environment, for example, a Medium Efficiency Particulate Air (MEPA) filter may be used as the intake air purification filter 602. The intake purification filter 602 purifies the gas drawn into the casing 601 from the outside of the casing 601.

  As shown in FIG. 3, the gas discharge device 613 includes, for example, an exhaust device 605 for exhausting the gas in the housing 601 to the outside of the housing 601, and an exhaust gas purification filter 603 for purifying the gas sucked by the exhaust device 605. And. The exhaust device 605 includes, for example, a fan. The exhaust gas purification filter 603 may be disposed facing the inside of the housing 601 which is the upstream side of the exhaust device 605. It may be difficult to sterilize the exhaust device 605, which is an electrical device. On the other hand, by disposing the exhaust gas purification filter 603 on the upstream side of the exhaust device 605, contamination of the exhaust device 605 can be suppressed. The gas exhaust device 613 may further include a second exhaust gas purification filter 606 disposed downstream of the exhaust device 605.

  The materials of the exhaust gas purification filter 603 and the second exhaust gas purification filter 606 are, for example, the same as the intake gas purification filter 602. Even if the inside of the casing 601 is contaminated by the cell processing device, the gas purified by the exhaust gas purification filter 603 and the second exhaust gas purification filter 606 is exhausted to the outside of the casing 601. For example, even if the gas in the housing 601 contains a blood component or a virus, these impurities are captured by the exhaust gas purification filter 603. Further, even if dust or the like is mixed in the gas by the exhaust device 605, the dust or the like is captured by the second exhaust gas purification filter 606.

  As shown in FIGS. 1 to 3, the outer casing 701 has, for example, a rectangular shape, but is not particularly limited thereto. The outer housing 701 is made of, for example, a material that can withstand heat sterilization or ultraviolet sterilization, but is not particularly limited thereto. At least a portion of the outer housing 701 may be transparent so that the inside can be observed from the outside. The outer housing 701 has an openable / closable structure for the insertion and removal of the housing 601 disposed inside. However, it is preferable that the inside of the outer housing 701 be completely closed when the openable / closable structure and the pressure adjustment hole 702 described later are closed.

  As shown in FIG. 3, the outer casing 701 may be provided with a pressure adjustment hole 702 for adjusting the pressure in the outer casing 701. In addition, a closing member 703 capable of closing the pressure adjustment hole 702 may be attached to the outer housing 701. A filter is disposed in the pressure adjustment hole 702. The material of the filter disposed in the pressure adjustment hole 702 is, for example, the same as the intake purification filter 602.

  For example, in the case where the pressure inside the outer casing 701 is made the same as that outside the outer casing 701, gas can flow through the pressure adjustment hole 702. The gas exiting out of the outer housing 701 through the pressure adjustment hole 702 is purified by a filter disposed in the pressure adjustment hole 702. In addition, the gas coming out from the outside of the outer casing 701 through the pressure adjustment hole 702 is also purified by the filter disposed in the pressure adjustment hole 702.

  As shown in FIG. 4, the gas in the outer casing 701 is sucked into the casing 601 through the intake air purification filter 602. In addition, the gas in the housing 601 is discharged into the outer housing 701 by the gas discharge device 613. Therefore, the gas circulates inside and outside the housing 601 in the outer housing 701. The gas circulates inside and outside the casing 601 in the outer casing 701, whereby the intake purification filter 602, the exhaust purification filter 603 and the second exhaust purification filter 606 allow the casing 601 as well as the inside of the casing 601. The gas inside the outer housing 701 outside is also purified. For example, the cell processing system may be configured such that the cleanliness of air in the housing 601 and in the outer housing 701 is made into a class of ISO 1 to ISO 6 according to the ISO standard 14644-1.

  As shown in FIGS. 5 and 6, the second exhaust gas purification filter 606 may be omitted depending on the use environment. In addition, the circulation device may further include an introduction device 608 provided in the housing 601 for sucking the gas purified by the intake air purification filter 602 from the outside of the housing 601. The introduction device 608 includes, for example, a fan. The intake air purification filter 602 may be disposed on the downstream side of the introduction device 608 so as to face inside the housing 601.

  As shown in FIGS. 7 and 8, the cell processing system may include both the introduction device 608 and the second exhaust gas purification filter 606. The circulation device may include both of the introduction device 608 and the gas discharge device 613, or may have either.

  At least one of the exhaust device 605 and the introduction device 608 flows gas such that the inside of the housing 601 has a negative pressure compared to the outside of the housing 601. Thus, diffusion of impurities in the housing 601 to the outside of the housing 601 can be suppressed. However, depending on the use environment, the inside of the housing 601 may be positive pressure as compared to the outside of the housing 601.

  The cell processing system may include a cleanliness sensor that monitors the cleanliness of the gas in the housing 601.

The cell processing system may include a carbon dioxide concentration management device that manages the concentration of carbon dioxide (CO ₂ ) in the housing 601. For example, the carbon dioxide concentration management device manages the carbon dioxide concentration so that the concentration of carbon dioxide in the housing 601 becomes a predetermined value, for example, 5%. The carbon dioxide concentration management device may include a carbon dioxide concentration sensor that monitors the carbon dioxide concentration of the gas in the housing 601.

The cell processing system may include an oxygen concentration management device that manages the concentration of oxygen (O ₂ ) in the housing 601. For example, the oxygen concentration management device manages the oxygen concentration so that the oxygen concentration in the housing 601 is in a low oxygen state of 20% or less. The oxygen concentration management device may include an oxygen concentration sensor that monitors the oxygen concentration of the gas in the housing 601.

  The cell processing system may include a thermal management device that manages the temperature in the housing 601. For example, the temperature management device manages the temperature so that the temperature in the housing 601 becomes a predetermined value, for example, 37 ° C. The thermal management device comprises, for example, a Peltier element. The thermal management device may include a temperature sensor that monitors the temperature of the gas in the housing 601.

  The cell processing system may further include a sterilizer for sterilizing the inside of the housing 601. The sterilizer may be a dry heat sterilizer. Alternatively, the sterilization apparatus may sterilize the inside of the housing 601 by spraying or releasing a sterilizing gas such as ozone gas, hydrogen peroxide gas, or formalin gas or a sterilizing solution such as ethanol into the housing 601. Alternatively, the sterilizer may irradiate the inside of the housing 601 with ultraviolet (UV) or an electron beam to sterilize the inside of the housing 601. By sterilizing the inside of the housing 601, cells can be repeatedly cultured in the housing 601. In addition, contamination of the outside of the housing 601 or infection of the worker can be suppressed. The cell processing system may further include a sterilizer for sterilizing the outer casing 701.

  The inside of the housing 601 may be divided into a plurality of regions by a partition 604 or the like.

  The cell processing system may further include a shielding member 800 that can be attached to the exhaust gas purification filter 603 so as to shield the exhaust gas purification filter 603 as shown in FIGS. 9, 10 and 11. For example, the shielding member 800 shields the exhaust purification filter 603 from the exhaust device 605, and the housing side shielding member 801 that can be attached to the exhaust purification filter 603 so as to shield the exhaust purification filter 603 from the inside of the housing 601. And an exhaust system side shielding member 802 that can be attached to the exhaust gas purification filter 603.

  The cell processing system may further include a sterilizer for sterilizing the exhaust gas purification filter 603 shielded by the shielding member 800. The sterilizer sterilizes the exhaust gas purification filter 603 shielded by the shielding member 800 in the same manner as the method of sterilizing the inside of the housing 601. By sterilizing the exhaust gas purification filter 603 with a sterilizer, it is possible to prevent the worker from being contaminated when the exhaust gas purification filter 603 is replaced.

  By sterilizing the exhaust gas purification filter 603 shielded by the shielding member 800, the exhaust system 605 can be exposed to a sterilization environment, and the exhaust system 605 can be prevented from being damaged. When the sterilizer is a dry heat sterilizer, the shielding member 800 may be made of, for example, a heat insulating material. The shape of the shielding member 800 is not particularly limited, but may be plate-like or film-like.

  As shown in FIGS. 10 and 11, the case-side shielding member 801 may be movable by the guide 811. For example, the housing side shielding member 801 may be able to move between a place where the exhaust gas purification filter 603 is not shielded and a place where the exhaust gas purification filter 603 is shielded along the guide 811.

  The exhaust system shielding member 802 may be movable by a guide 812. For example, the exhaust system side shielding member 802 may be able to move along a guide 812 between a place where the exhaust gas purification filter 603 is not shielded and a place where the exhaust gas purification filter 603 is shielded.

  The housing side shielding member 801 and the exhaust device side shielding member 802 may be movable in the lateral direction with respect to the housing 601, or as shown in FIG. It may be

  Further, the cell processing system may further include a shielding member that can be attached to the second exhaust gas purification filter 606 so as to shield the second exhaust gas purification filter 606. For example, the shielding member that can be attached to the second exhaust gas purification filter 606 can be attached to the second exhaust gas purification filter 606 so as to shield the second exhaust gas purification filter 606 from the exhaust gas device 605 It has a member.

  The cell processing system may further include a sterilizer for sterilizing the second exhaust gas purification filter 606 shielded by the shielding member. The sterilizer sterilizes the second exhaust gas purification filter 606 shielded by the shielding member in the same manner as the method of sterilizing the inside of the housing 601. By sterilizing the second exhaust gas purification filter 606 with a sterilizer, it is possible to prevent the worker from being contaminated when the second exhaust gas purification filter 606 is replaced.

  By sterilizing the second exhaust gas purification filter 606 shielded by the shielding member, the exhaust system 605 can be exposed to the sterilization environment, and the exhaust system 605 can be prevented from being damaged. When the sterilizer is a dry heat sterilizer, the shielding member that shields the second exhaust gas purification filter 606 may be made of, for example, a heat insulating material. The shape of the shielding member for shielding the second exhaust gas purification filter 606 is not particularly limited, but may be plate-like or film-like.

  The shielding member shielding the second exhaust gas purification filter 606 may be movable by a guide. For example, the shielding member may be able to move along a guide between a place that does not shield the second exhaust gas purification filter 606 and a place that shields the second exhaust gas purification filter 606.

  The cell processing system may further include a shielding member that can be attached to the intake air purification filter 602 so as to shield the air intake purification filter 602. For example, the shielding member that can be attached to the intake air purification filter 602 introduces a housing side shielding member that can be attached to the intake air purification filter 602 so as to shield the intake air purification filter 602 from inside the housing 601; And an introduction device side shielding member that can be attached to the intake air purification filter 602 so as to shield the device 608.

  The cell processing system may further include a sterilizer for sterilizing the intake purification filter 602 shielded by the shielding member. The sterilizer sterilizes the intake air purification filter 602 shielded by the shielding member in the same manner as the method of sterilizing the inside of the housing 601. By sterilizing the intake air purification filter 602 with a sterilizer, it is possible to prevent the worker from being contaminated when the intake air purification filter 602 is replaced.

  By sterilizing the intake air purification filter 602 shielded by the shielding member, it is possible to prevent the introduction device 608 from being exposed to the sterilization environment and damage to the introduction device 608. When the sterilizer is a dry heat sterilizer, the shielding member that shields the intake air purification filter 602 may be made of, for example, a heat insulating material. The shape of the shielding member for shielding the intake air purification filter 602 is not particularly limited, but may be plate-like or film-like.

  The shielding member that shields the intake air purification filter 602 may be movable by a guide. For example, the shielding member may be able to move along a guide between a place that does not shield the intake air purification filter 602 and a place that shields the intake air purification filter 602.

  Alternatively, as shown in FIG. 13 to FIG. 15, the cell processing system according to the embodiment includes the casing 601 and the intake purification filter 602 provided on the casing 601 for purifying the gas sucked from the outside of the casing 601. And a return member 651 having a hollow inside for returning the gas discharged from the housing 601 to the intake air purification filter 602, and the gas in the housing 601 is discharged into the return member 651, and the return member A circulation device for circulating the gas between the housing and the return member so that the gas inside the housing 651 is sucked into the housing 601 through the intake air purification filter 602, and the housing 601 is disposed in the housing 601 And a cell processing device for processing cells.

  The housing 601 illustrated in FIGS. 13 to 15 may have the same configuration as the housing 601 described in FIGS. 1 to 9. Therefore, the housing 601 has, for example, a rectangular parallelepiped shape, but is not particularly limited thereto. The housing 601 is made of, for example, the material that can withstand the sterilization described above, but is not particularly limited thereto. At least a portion of the housing 601 may be transparent so that the inside can be observed from the outside. The housing 601 has an openable / closable structure for loading and unloading of the cell processing device disposed inside.

  The circulation device includes, for example, a gas discharge device 613 provided in the housing 601 for sucking a gas from the inside of the housing 601 and discharging the purified gas to the outside of the housing 601. The intake air purification filter 602 and the gas discharge device 613 are disposed to face each other, for example. For example, a High Efficiency Particulate Air (HEPA) filter and an Ultra Low Particulate Air (URPA) filter may be used as the intake air purification filter 602, but the invention is not limited thereto. Depending on the usage environment, for example, a Medium Efficiency Particulate Air (MEPA) filter may be used as the intake air purification filter 602. The intake purification filter 602 purifies the gas drawn into the casing 601 from the outside of the casing 601.

  Also in the cell processing system shown in FIGS. 13 to 15, as shown in FIG. 3, the gas discharge device 613 is, for example, an exhaust device 605 for exhausting the gas in the housing 601 into the return member 651, and And an exhaust gas purification filter 603 for purifying the gas sucked in 605. The exhaust device 605 includes, for example, a fan. The exhaust gas purification filter 603 may be disposed facing the inside of the housing 601 which is the upstream side of the exhaust device 605. It may be difficult to sterilize the exhaust device 605, which is an electrical device. On the other hand, by disposing the exhaust gas purification filter 603 on the upstream side of the exhaust device 605, contamination of the exhaust device 605 can be suppressed. The gas exhaust device 613 may further include a second exhaust gas purification filter 606 disposed downstream of the exhaust device 605.

  The materials of the exhaust gas purification filter 603 and the second exhaust gas purification filter 606 are, for example, the same as the intake gas purification filter 602. Even if the inside of the housing 601 is contaminated by the cell processing device, the gas purified by the exhaust gas purification filter 603 and the second exhaust gas purification filter 606 is exhausted into the return member 651. For example, even if the gas in the housing 601 contains a blood component or a virus, these impurities are captured by the exhaust gas purification filter 603. Further, even if dust or the like is mixed in the gas by the exhaust device 605, the dust or the like is captured by the second exhaust gas purification filter 606.

  As shown in FIGS. 13 to 15, the return member 651 has, for example, a shape fitted to the housing 601, but the present invention is not particularly limited thereto. The return member 651 communicates, for example, a base 652, which is hollow inside, in contact with the bottom surface of the housing 601, a first opening 653 provided in the base 652, and a ventilating part provided in the first end face of the housing 601. And a second cover 656 for communicating the second opening 655 provided in the base portion 652 and the ventilating portion provided in the housing 601.

  The first cover 654 covers, for example, the intake air purification filter 602 as a ventilation part provided on the first end face. The second cover 656 covers, for example, a gas discharge device 613 as a vent provided at the second end face.

  The return member 651 is made of, for example, a material that can withstand sterilization similarly to the housing 601, but is not particularly limited thereto. The return member may be a duct.

  As shown in FIG. 15, the gas in the housing 601 is discharged into the return member 651 by the gas discharge device 613. Further, the gas in the return member 651 is sucked into the casing 601 through the intake air purification filter 602. Accordingly, gas circulates between the housing 601 and the return member 651. By circulating the gas between the casing 601 and the return member 651, the intake purification filter 602, the exhaust purification filter 603, and the second exhaust purification filter 606 allow the casing not only in the casing 601 but also in the casing. The gas inside the return member 651 outside of 601 is also purified. For example, the cell processing system may be configured such that the cleanliness of the air in the housing 601 and in the return member 651 is made into a class of ISO1 to ISO6 according to the ISO standard 14644-1.

  Also in the cell processing system shown in FIGS. 13 to 15, as shown in FIGS. 5 and 6, the second exhaust gas purification filter 606 may be omitted depending on the use environment. In addition, the circulation device may further include an introduction device 608 provided in the housing 601 for sucking the gas purified by the intake air purification filter 602 from the inside of the return member 651. The introduction device 608 includes, for example, a fan. The intake air purification filter 602 may be disposed on the downstream side of the introduction device 608 so as to face inside the housing 601.

  Also in the cell processing system shown in FIGS. 13 to 15, as shown in FIGS. 7 and 8, both the introduction device 608 and the second exhaust gas purification filter 606 may be provided. The circulation device may include both of the introduction device 608 and the gas discharge device 613, or may have either.

  At least one of the exhaust device 605 and the introduction device 608 flows gas such that the inside of the housing 601 has a negative pressure compared to the outside of the housing 601. Thus, diffusion of impurities in the housing 601 to the outside of the housing 601 can be suppressed. However, depending on the use environment, the inside of the housing 601 may be positive pressure as compared to the outside of the housing 601.

Similar to the cell processing system described in FIGS. 1-9, the cell processing system shown in FIGS. 13-15 includes a cleanliness sensor for monitoring gas cleanliness in the housing 601, carbon dioxide in the housing 601. (CO ₂ ) concentration management device managing carbon dioxide concentration, oxygen concentration management device managing concentration of oxygen (O ₂ ) in housing 601, temperature management device managing temperature in housing 601, and housing At least one of the sterilizers for sterilizing the inside of the body 601 may be provided.

  The inside of the housing 601 may be divided into a plurality of regions by a partition 604 or the like.

  Also in the cell processing system shown in FIGS. 13 to 15, as shown in FIGS. 9, 10 and 11, a shielding member 800 which can be attached to the exhaust gas purification filter 603 to shield the exhaust gas purification filter 603 is further added. You may have. For example, the shielding member 800 shields the exhaust purification filter 603 from the exhaust device 605, and the housing side shielding member 801 that can be attached to the exhaust purification filter 603 so as to shield the exhaust purification filter 603 from the inside of the housing 601. And an exhaust system side shielding member 802 that can be attached to the exhaust gas purification filter 603.

  Also in the cell processing system shown in FIGS. 13 to 15, as shown in FIGS. 9, 10 and 11, a sterilizer for sterilizing the exhaust gas purification filter 603 shielded by the shielding member 800 may further be provided. . The sterilizer sterilizes the exhaust gas purification filter 603 shielded by the shielding member 800 in the same manner as the method of sterilizing the inside of the housing 601. By sterilizing the exhaust gas purification filter 603 with a sterilizer, it is possible to prevent the worker from being contaminated when the exhaust gas purification filter 603 is replaced.

  By sterilizing the exhaust gas purification filter 603 shielded by the shielding member 800, the exhaust system 605 can be exposed to a sterilization environment, and the exhaust system 605 can be prevented from being damaged. When the sterilizer is a dry heat sterilizer, the shielding member 800 may be made of, for example, a heat insulating material. The shape of the shielding member 800 is not particularly limited, but may be plate-like or film-like.

  Also in the cell processing system shown in FIGS. 13 to 15, as shown in FIGS. 10 and 11, the casing side shielding member 801 may be movable by the guide 811. For example, the housing side shielding member 801 may be able to move between a place where the exhaust gas purification filter 603 is not shielded and a place where the exhaust gas purification filter 603 is shielded along the guide 811.

  The exhaust system shielding member 802 may be movable by a guide 812. For example, the exhaust system side shielding member 802 may be able to move along a guide 812 between a place where the exhaust gas purification filter 603 is not shielded and a place where the exhaust gas purification filter 603 is shielded.

  The housing side shielding member 801 and the exhaust device side shielding member 802 may be movable in the lateral direction with respect to the housing 601, or as shown in FIG. It may be

  Further, the cell processing system shown in FIGS. 13 to 15 further includes a shielding member that can be attached to the second exhaust gas purification filter 606 so as to shield the second exhaust gas purification filter 606 shown in FIG. It is also good. For example, the shielding member that can be attached to the second exhaust gas purification filter 606 can be attached to the second exhaust gas purification filter 606 so as to shield the second exhaust gas purification filter 606 from the exhaust gas device 605 It has a member.

  Also in the cell processing system shown in FIGS. 13 to 15, as shown in FIG. 12, a sterilizer for sterilizing the second exhaust gas purification filter 606 shielded by the shielding member may be provided. The sterilizer sterilizes the second exhaust gas purification filter 606 shielded by the shielding member in the same manner as the method of sterilizing the inside of the housing 601. By sterilizing the second exhaust gas purification filter 606 with a sterilizer, it is possible to prevent the worker from being contaminated when the second exhaust gas purification filter 606 is replaced.

  By sterilizing the second exhaust gas purification filter 606 shielded by the shielding member, the exhaust system 605 can be exposed to the sterilization environment, and the exhaust system 605 can be prevented from being damaged. When the sterilizer is a dry heat sterilizer, the shielding member that shields the second exhaust gas purification filter 606 may be made of, for example, a heat insulating material. The shape of the shielding member for shielding the second exhaust gas purification filter 606 is not particularly limited, but may be plate-like or film-like.

  The shielding member shielding the second exhaust gas purification filter 606 may be movable by a guide. For example, the shielding member may be able to move along a guide between a place that does not shield the second exhaust gas purification filter 606 and a place that shields the second exhaust gas purification filter 606.

  Further, as shown in FIG. 12, the cell processing system shown in FIG. 13 to FIG. 15 may further include a shielding member that can be attached to the intake air purification filter 602 so as to shield the intake air purification filter 602. . For example, the shielding member that can be attached to the intake air purification filter 602 introduces a housing side shielding member that can be attached to the intake air purification filter 602 so as to shield the intake air purification filter 602 from inside the housing 601; And an introduction device side shielding member that can be attached to the intake air purification filter 602 so as to shield the device 608.

  Also in the cell processing system shown in FIGS. 13 to 15, a sterilizer for sterilizing the intake air purification filter 602 shielded by the shielding member may be provided. The sterilizer sterilizes the intake air purification filter 602 shielded by the shielding member in the same manner as the method of sterilizing the inside of the housing 601. By sterilizing the intake air purification filter 602 with a sterilizer, it is possible to prevent the worker from being contaminated when the intake air purification filter 602 is replaced.

  By sterilizing the intake air purification filter 602 shielded by the shielding member, it is possible to prevent the introduction device 608 from being exposed to the sterilization environment and damage to the introduction device 608. When the sterilizer is a dry heat sterilizer, the shielding member that shields the intake air purification filter 602 may be made of, for example, a heat insulating material. The shape of the shielding member for shielding the intake air purification filter 602 is not particularly limited, but may be plate-like or film-like.

  The shielding member that shields the intake air purification filter 602 may be movable by a guide. For example, the shielding member may be able to move along a guide between a place that does not shield the intake air purification filter 602 and a place that shields the intake air purification filter 602.

  The cell processing device disposed in the housing 601 is, for example, a separation device 10 for separating cells from blood and a pre-introduction cell through which a solution containing cells separated by the separation device 10 passes as shown in FIG. It is connected to a fluid delivery channel 20, an induction factor fluid delivery mechanism 21 for sending a pluripotency inducing factor into the pre-infusion cell fluid delivery channel 20, and a pre-infusion cell fluid delivery channel 20 to introduce the pluripotency inducing factor into cells. Then, the factor introduction device 30 for producing induction factor-introduced cells, the cell mass production device 40 for producing a plurality of cell souls consisting of stem cells by culturing the induction factor-introduced cells, and packaging each of the plurality of cell masses in sequence And a package device 100.

  The separation device 10 receives, for example, a vial containing human blood. The separation device 10 may be, for example, an anticoagulant tank for storing an anticoagulant such as ethylenediaminetetraacetic acid (EDTA), heparin, and biological preparation standard blood storage solution A (ACD-A solution, Terumo Corporation). Have. The separation device 10 adds an anticoagulant to human blood from an anticoagulant tank using a pump or the like.

  In addition, the separation device 10 includes, for example, a separation reagent tank for storing a mononuclear cell separation reagent such as Ficoll-Paque PREMIUM (registered trademark, GE Healthcare Japan Co., Ltd.). The separation apparatus 10 dispenses, for example, 5 mL of the mononuclear cell separation reagent from the separation reagent tank to, for example, two 15 mL tubes using a pump or the like. A resin bag may be used instead of the tube.

  Furthermore, the separation device 10 includes a buffer tank that stores a buffer such as phosphate buffered saline (PBS). The separation apparatus 10 adds and dilutes 5 mL of buffer solutions to 5 mL of human blood, for example, from a buffer solution tank using a pump or the like. Furthermore, the separation apparatus 10 adds diluted human blood in 5 mL each on the mononuclear cell separation reagent in the tube using a pump or the like.

  The separation device 10 further includes a temperature settable centrifuge. The centrifuge is set to, for example, 18 ° C. The separation device 10 uses a transfer device or the like to place a tube containing a mononuclear cell separation reagent, human blood and the like into the holder of the centrifuge. The centrifuge centrifuges the solution in the tube, for example, at 400 × g for 30 minutes. Instead of the tube, the resin bag may be centrifuged.

  After centrifugation, the separation device 10 collects, with a pump or the like, the white turbid intermediate layer of mononuclear cells of the solution in the tube. The separation device 10 sends out the collected suspension of mononuclear cells to the cell fluid delivery channel 20 before introduction using a pump or the like. Alternatively, further, the separation device 10 adds, for example, 12 mL of PBS to 2 mL of the recovered mononuclear cell solution, and places the tube in the holder of the centrifuge. The centrifuge centrifuges the solution in the tube, for example at 200 × g for 10 minutes.

  After centrifugation, the separator 10 aspirates and removes the supernatant of the solution in the tube using a pump or the like, and 3 mL of mononuclear cell culture medium such as X-VIVO 10 (registered trademark, Ronza Japan Co., Ltd.) is used as a tube. Add to the mononuclear cell solution and suspend. The separation apparatus 10 sends out the mononuclear cell suspension to the cell delivery line 20 before introduction using a pump or the like. The separation device 10 may separate mononuclear cells from blood using a dialysis membrane. When using somatic cells such as fibroblasts previously separated from the skin etc., the separation device 10 may be omitted.

  The separation device 10 may separate cells suitable for induction by methods other than centrifugation. For example, if the cells to be separated are T cells, cells positive for any of CD3, CD4 and CD8 may be separated by panning. If the cells to be separated are vascular endothelial precursor cells, cells positive for CD34 may be separated by panning. If the cells to be separated are B cells, cells positive for any of CD10, CD19 and CD20 may be separated by panning. Moreover, you may isolate | separate not only by panning but the magnetic cell separation method (MACS), flow cytometry, etc. Also, cells suitable for induction are not limited to cells derived from blood.

  The induction factor delivery mechanism 21 includes an induction factor introduction reagent tank for storing an induction factor introduction reagent solution and the like. An inducer-introducing reagent solution such as a gene transfer reagent solution includes, for example, an electroporation solution such as a Human T Cell Nucleofector (registered trademark, Lonza Japan Co., Ltd.) solution, a supplement solution, and a plasmid set. The plasmid set contains, for example, 0.83 μg of pCXLE-hOCT3 / 4-shp53-F, 0.83 μg of pCXLE-hSK, 0.83 μg of pCE-hUL, and 0.5 μg of pCXWB-EBNA1. The induction factor delivery mechanism 21 uses the micropump or the like to suspend the mononuclear cell suspension in the induction factor delivery reagent solution, so that the induction factor delivery reagent solution is delivered to the cell delivery path 20 before introduction. Send out.

  The inner wall of the cell delivery channel 20 before introduction may be coated with poly-HEMA (poly 2-hydroxyethyl methacrylate) to make it noncell-adhesive so that cells do not adhere. Alternatively, a material to which cells do not easily adhere may be used as the material of the cell delivery line 20 before introduction. In addition, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the cell delivery line 20 before introduction, the conditions in the cell delivery line 20 before introduction are managed in the housing 601. It becomes equivalent to temperature and carbon dioxide concentration. Furthermore, from the viewpoint of contamination prevention, the backflow prevention valve may be provided in the cell liquid-sending path 20 before introduction.

  The factor introduction device 30 connected to the cell delivery line 20 before introduction is, for example, an electroporator, receives a mixed solution of an induction factor introduction reagent solution and a mononuclear cell suspension, and Perform electroporation. The factor introduction device 30 adds the mononuclear cell culture medium to a solution containing mononuclear cells electroporated with the plasmid after performing the electroporation. The factor introduction device 30 sends out a solution containing mononuclear cells (hereinafter referred to as “induction factor introduced cells”) into which the plasmid has been electroporated to the introduced cell delivery channel 31 using a pump or the like.

  The factor introducing device 30 is not limited to the electroporator. The factor introduction device 30 may introduce RNA encoding a reprogramming factor into cells by lipofection. The lipofection method is a method in which a complex of a negatively charged nucleic acid and a positively charged lipid is formed by electrical interaction, and the complex is taken into a cell by endocytosis or membrane fusion. . The lipofection method has advantages such as less damage to cells, excellent introduction efficiency, easy operation, and less time-consuming. In addition, in the lipofection method, since there is no possibility that the reprogramming factor is inserted into the genome of the cells, it is not necessary to sequence the whole genome of the obtained stem cells to confirm the presence or absence of the insertion of the foreign gene. Reprogramming factor RNAs as pluripotency inducers include, for example, Oct3 / 4 mRNA, Sox2 mRNA, Klf4 mRNA, and c-Myc mRNA.

  For example, small interfering RNA (siRNA) or lipofection reagent is used for lipofection of reprogramming factor RNA. As the lipofection reagent for RNA, lipofection reagent for siRNA and lipofection reagent for mRNA can be used. More specifically, Lipofectamine (registered trademark) RNAiMAX (Thermo Fisher Scientific), Lipofectamine (registered trademark) MessengerMAX (Thermo Fisher Scientific), Lipofectamin (registered trademark) 2000, Lipofectamin (registered trademark) 3000 as a lipofection reagent for RNA. Neon Transfection System (Thermo Fisher scientific), Stemfect RNA Transfection Reagent (Stemfect), NextFect (R) RNA Transfection Reagent (Bioo Scientific), Amaxa Registered product) Human T cell Nucleofector (registered product) kit (Lonza, VAPA-1002), Amaxa (registered product) Human CD34 cell Nucleofector (registered product) kit (Lonza, VAPA-1003), and ReproRNA (registered trademark) A transfection reagent (STEMCELL Technologies) etc. can be used.

  When the factor introducing device 30 introduces the reprogramming factor into cells by the lipofection method, the reprogramming factor RNA, the reagent and the like are sent out to the pre-introduced cell delivery line 20 by the induction factor delivery mechanism 21.

  The inner wall of the introduced cell delivery channel 31 may be coated with poly-HEMA to be non-adhesive so that cells do not adhere. Alternatively, a material to which cells do not easily adhere may be used as the material of the introduced cell delivery channel 31. Further, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the introduced cell delivery channel 31, the conditions in the introduced cell delivery channel 31 are the controlled temperature in the housing 601 and the like. It becomes equivalent to carbon dioxide concentration. Furthermore, from the viewpoint of contamination prevention, the backflow prevention valve may be provided in the introduced cell delivery channel 31. Also, after electroporation, a large number of cells may die, resulting in a cell mass of dead cells. Therefore, a filter for removing dead cell mass may be provided in the introduced cell delivery channel 31. Alternatively, as shown in FIG. 17, one or more weirs may be provided inside the introduced cell fluid delivery channel 31 so as to intermittently change the inner diameter. Alternatively, as shown in FIG. 18, the inner diameter of the introduced cell liquid feed channel 31 may be changed intermittently.

  As shown in FIG. 16, the cell mass production device 40 connected to the introduced cell liquid feed channel 31 comprises an initialization culture device 50 for culturing the induction factor introduced cells produced by the factor introduction device 30 and an initialization culture device. 50. A first dividing mechanism 60 for dividing a cell mass (cell colony) consisting of stem cells established in 50 into a plurality of cell masses, and an expansion culture for expanding and culturing a plurality of cell masses divided by the first dividing mechanism 60 A device 70, a second dividing mechanism 80 for dividing a cell mass consisting of stem cells expanded and cultured by the expansion culture device 70 into a plurality of cell masses, and a cell soul transport mechanism 90 for sequentially transmitting a plurality of cell souls to the package device 100. And.

  The reprogramming culture device 50 can store a well plate inside. In addition, the reprogramming culture apparatus 50 includes a pipetting machine. The reprogramming culture apparatus 50 receives the solution containing the inducer-introduced cells from the introduced cell delivery channel 31, and distributes the solution to the wells with a pipetting machine. The reprogramming culture apparatus 50 adds stem cell culture media such as StemFit (registered trademark, Ajinomoto Co., Ltd.) on the third, fifth, seventh days, for example, after distributing the induction factor-introduced cells into the wells. The medium may be supplemented with basic fibroblast growth factor (basic FGF) as a supplement. Note that sustained release beads such as StemBeads FGF2 (funakoshi) that continue to supply FGF-2 (basic FGF, bFGF, FGF-b) to the medium may be added to the medium. Also, because FGF may be unstable, heparin mimetic polymers may be conjugated to FGF to stabilize FGF. Furthermore, transforming growth factor beta (TGF-β), activin (Activin) and the like may be added to the medium. The reprogramming culture apparatus 50 performs culture medium exchange, for example, on the 9th day after distributing inducer-introduced cells in the wells, and thereafter culture medium every two days until the cell mass (colony) of iPS cells exceeds 1 mm. Make a replacement. Note that changing the medium also includes partially replacing the medium and supplying the medium.

  When a cell mass is formed, the reprogramming culture apparatus 50 recovers the cell mass with a pipetting machine, and adds a trypsin alternative recombinant enzyme such as TrypLE Select (registered trademark, Life Technologies) to the recovered cell mass. Furthermore, the reprogramming culture apparatus 50 places the container containing the recovered cell mass in an incubator, and causes the cell mass to react with the trypsin alternative recombinant enzyme for 10 minutes at 37 ° C. and 5% carbon dioxide. When the cell mass is physically broken, the trypsin alternative recombinant enzyme may not be present. For example, the reprogramming culture apparatus 50 breaks cell clumps by pipetting with a pipetting machine. Alternatively, the reprogramming culture apparatus 50 may be provided with cells through the cell mass through a pipe provided with a filter or a pipe whose internal diameter is intermittently changed as in the introduced cell delivery channel 31 shown in FIG. 17 or 18. The mass may be broken. Thereafter, the reprogramming culture apparatus 50 adds a pluripotent stem cell culture medium such as StemFit (registered trademark, Ajinomoto Co., Ltd.) to a solution containing the crushed cell mass.

  The culture in the reprogramming culture apparatus 50 may be performed in a carbon dioxide permeable bag instead of the well plate. The culture may be adhesion culture or suspension culture. In the case of suspension culture, agitation culture may be performed. Also, the medium may be in the form of agar. Agar-like media include gellan gum polymers and deacylated gellan gum polymers. When using an agar-like medium, since the cells do not settle or adhere, there is no need to stir even in suspension culture, and it is possible to produce a single cell mass derived from one cell. The culture in the reprogramming culture apparatus 50 may be a hanging drop culture.

  The reprogramming culture apparatus 50 may be provided with a first medium replenishing apparatus for replenishing a well plate or a carbon dioxide permeable bag with a medium containing a culture solution. The first medium supply device collects the culture solution in the well plate or carbon dioxide permeable bag, filters the culture solution using a filter or a dialysis membrane, and reuses the purified culture solution. Good. At that time, a growth factor or the like may be added to the culture solution to be recycled. In addition, the reprogramming culture device 50 may further include a temperature control device that controls the temperature of the culture medium, a humidity control device that controls the humidity near the culture medium, and the like.

  In the reprogramming culture apparatus 50, for example, cells are placed in a culture solution permeable bag 301 such as a dialysis membrane as shown in FIG. 19, and the culture solution permeable bag 301 is treated as a culture solution non-permeable carbon dioxide. The culture solution may be put in the bags 301, 302 in the sexual bag 302. The reprogramming culture apparatus 50 prepares a plurality of bags 302 containing fresh culture fluid, and for every predetermined period, the bag 302 containing the bag 301 containing cells is a bag containing fresh culture fluid. It may be replaced with 302.

  Moreover, the culture method in the reprogramming culture apparatus 50 is not limited to the above-mentioned method, and a floating incubator as shown in FIG. 20 may be used. The suspension incubator shown in FIG. 20 comprises a dialysis tube 75 in which the induction factor-introduced cells and the gel medium are placed, and a container 76 in which the dialysis tube 75 is placed and the gel medium is placed around the dialysis tube 75. The suspension incubator may also be equipped with a pH sensor around the dialysis tube 75 to measure the hydrogen ion index (pH) of the gel medium.

  The dialysis tube 75 is made of a semipermeable membrane, and is permeable to, for example, a ROCK inhibitor. The molecular weight cut-off of the dialysis tube 75 is 0.1 KDa or more, 10 KDa or more, or 50 KDa or more. The dialysis tube 75 may be, for example, cellulose ester, ethyl cellulose, cellulose esters, regenerated cellulose, polysulfone, polyacrylonitrile, polymethyl methacrylate, ethylene vinyl alcohol copolymer, polyester polymer alloy, polycarbonate, polyamide, cellulose acetate, cellulose di It comprises acetate, cellulose triacetate, copper ammonium rayon, saponified cellulose, hemophane membrane, phosphatidylcholine membrane, vitamin E coating membrane and the like.

  As the container 76, a conical tube such as a centrifugal tube can be used. The container 76 is made of, for example, polypropylene. The container 76 may be carbon dioxide permeable. As a carbon dioxide permeable container 76, G-Rex (registered trademark, Wilson Wolf) can be used.

  Inducer-transduced cells are placed in dialysis tubing 75. The gel medium is not agitated. Also, the gel medium does not contain feeder cells. The dialysis tube 75 may be connected to a fluid delivery path for delivering a medium containing cells into the dialysis tube 75. In addition, the dialysis tube 75 may be connected to a fluid delivery path for delivering the medium containing the cells in the dialysis tube 75 to the outside of the container.

  The gel medium may be, for example, a blood cell culture medium or a culture medium for stem cells with a final concentration of 0.5% to 0.001% by weight, 0.1% to 0.005% by weight, or 0.05% by weight of deacylated gellan gum. It is prepared by adding so that it becomes weight%-0.01 weight%. For example, at the beginning of reprogramming culture, a gel culture medium prepared from a blood cell culture medium is used, and thereafter, a gel culture medium manufactured from a stem cell culture medium is used.

  As a medium for stem cells, for example, human ES / iPS medium such as Primate ES Cell Medium (ReproCELL) can be used.

  However, the medium for stem cells is not limited thereto, and various stem cell cultures can be used. For example, Primate ES Cell Medium, Reprostem, ReproFF, ReproFF2, ReproXF (Reprocell), mTeSR1, TeSR2, TeSRE8, ReproTeSR (STEMCELL Technologies), PluriSTEM (registered trademark) Human ES / iPS Medium (Merck), NutriStem (registered trademark) XF / FF Culture Medium for Human iPS and ES Cells, Pluriton reprogramming medium (Stemgent), PluriSTEM (registered trademark), Stemfit AK02N, Stemfit AK03 (Ajinomoto), ESC-Sure (registered trademark) erum and feeder free medium for hESC / iPS (Applied StemCell), and L7 may be utilized (registered trademark) hPSC Culture System (LONZA) or the like.

  Gel culture media include gellan gum, hyaluronic acid, rhamsan gum, dayutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparitin sulfate, kerato sulfate, chondroitin sulfate, delta man sulfate, rhamnan sulfate, You may contain at least 1 sort (s) of high molecular compound selected from the group which consists of those salts. Also, the gel medium may contain methylcellulose. By including methylcellulose, aggregation of cells is further suppressed.

  Alternatively, the gel medium may be poly (glycerol monomethacrylate) (PGMA), poly (2-hydroxypropyl methacrylate) (PHPMA), poly (N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimized terminated, N-hydroxysuccinimide NHS) ester terminated, triethoxysilane terminated, Poly (N-isopropylacrylamide-co-acylamide), Poly (N-isopropylacrylamide-co-acrylic acid), Poly (N-isopropylacrylamide-co-butylacrylate), Poly (N-isopropylacrylamide-co-co- It may comprise a minor temperature sensitive gel selected from methacrylic acid), Poly (N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-Isopropylacrylamide.

  The gel medium contained in the dialysis tube 75 may not contain the ROCK inhibitor. The gel medium to be placed around the dialysis tube 75 in the container 76 has, for example, a ROCK inhibitor at a final concentration of 1000 μmol / L or more and 0.1 μmol / L or less, 100 μmol / L or more and 1 μmol / L or less, or 5 μmol / L It is added daily so as to be at least 20 μmol / L. By adding the ROCK inhibitor to the gel medium surrounding the dialysis tube 75, the ROCK inhibitor penetrates into the dialysis tube 75 to promote colony formation by cells.

  The gel culture medium may or may not contain a growth factor such as basic fibroblast growth factor (bFGF) or TGF-β, for example.

  During suspension culture of the cells in the dialysis tube 75, the gel medium around the dialysis tube 75 in the container 76 is replaced. Note that replacing the medium also includes partially replacing and replenishing the medium. In this case, the gel medium may not be supplied into the dialysis tube 75. Alternatively, gel culture medium is replenished in the dialysis tube 75 during suspension culture of cells in the dialysis tube 75. In this case, the gel medium may not be supplied around the dialysis tube 75 in the container 76.

  The cell processing apparatus according to the embodiment uses, as shown in FIG. 21, the replacement medium supply pump 77 as a medium supply apparatus to replace or replenish the gel medium around the dialysis tube 75 in the container 76. . As the feeding medium feeding pump 77, a pump or the like used for dripping can be used. The feed medium feed pump 77 and the container 76 of the floating incubator are connected by a feed tube 78. The feeding medium feeding pump 77 feeds the gel medium into the container 76 of the suspension incubator via the feeding tube 78. A waste liquid tube 79 is connected to the container 76 of the floating incubator. The gel medium in the container 76 of the floating incubator is drained through the drainage tube 79. The gel medium in the container 76 of the floating culture vessel may be drained by, for example, the pressure of fresh gel culture medium supplemented by the replenishment medium feed pump 77, or may be evacuated using gravity. It may be discharged by a discharge pump.

  The temperature of the gel medium which is fed to the incubator from the supplementary medium delivery pump 77 is set, for example, so that the temperature of the gel medium in the incubator does not change rapidly. For example, when the temperature of the gel medium in the incubator is 37 ° C., the temperature of the gel medium fed to the incubator is also set to 37 ° C. However, the culture medium before being sent to the incubator may be stored refrigerated at a low temperature such as 4 ° C. in the refrigerated storage unit.

  For example, the amount of gel medium fed into the container 76 of the suspension incubator by the replenishment medium feed pump 77 and the amount of gel medium discharged from the container 76 of the suspension incubator are the same. The culture medium delivery pump 77 is controlled. The feeding medium feeding pump 77 may always feed the gel culture medium into the container 76 of the suspension incubator, or may feed the gel culture medium at appropriate intervals.

  When the gel medium is constantly fed, the flow rate of the gel medium to be fed may or may not be constant. For example, as described later, the medium and the cell mass in the medium are monitored by a photographing device, and the flow rate of the gel medium to be fed is increased or decreased according to the state of the cell mass in the medium and the medium You may

  In addition, the gel medium may be started and ended according to the state of the medium and the cell mass in the medium without constantly supplying the gel medium. Also in this case, the flow rate of the gel medium to be fed may be increased or decreased depending on the medium and the state of cell mass in the medium.

  If the flow rate of the gel medium fed to the incubator is too large, cells in the incubator may be damaged by the pressure of the gel medium. Therefore, the flow rate of the gel medium sent to the incubator is set so that the cells are not damaged.

  If the culture of cells is continued without changing the medium, accumulation of waste products such as lactic acid excreted by the cells and change in pH may adversely affect cell culture. In addition, various proteins such as bFGF and recombinant proteins contained in the medium may be degraded, and components necessary for cell culture may be lost from the medium.

  On the other hand, fresh medium is fed into the incubator by the feeding medium feed pump 77 and the old medium is discharged from the incubator to remove wastes from the incubator and to properly adjust the pH in the medium. It is possible to keep the concentration within the range and to supply the necessary components for cell culture. This makes it possible to keep the state of the culture medium close to a constant state.

  Note that FIG. 21 shows an example in which the feeding medium feed pump 77 and the container 76 of the floating incubator are connected by the feed tube 78. On the other hand, as shown in FIG. 22, the feeding medium feed pump 77 and the inside of the dialysis tube 75 in the vessel 76 of the floating incubator may be connected by the feeding tube 78. By sending fresh gel medium into the dialysis tube 75, wastes contained in the medium in the dialysis tube 75 are discharged out of the dialysis tube 75. In addition, it is possible to keep the pH of the medium in the dialysis tube 75 within an appropriate range, and to supply the medium in the dialysis tube 75 with components necessary for culturing the cells.

  The cell processing system may further include an initialization culture imaging device such as a photo camera or a video camera for imaging the culture in the initialization culture device 50 shown in FIG. Here, when a colorless medium is used as the medium used in the reprogramming culture apparatus 50, it is possible to suppress diffuse reflection and autofluorescence that may occur when using a colored medium. However, in order to confirm the pH of the culture medium, a pH indicator such as phenol red may be included. In addition, since the shape and size of the cells are different between the induced cells and the non-induced cells, the cell processing system is configured by imaging the cells in the reprogramming culture apparatus 50. You may further provide the guidance state monitoring apparatus which calculates a ratio. Alternatively, the induction status monitoring device may specify the proportion of cells induced by antibody immunostaining or RNA extraction. Furthermore, the cell processing system may be equipped with an uninduced cell removal device that removes uninduced cells by magnetic cell separation methods, flow cytometry, etc.

  Here, when the cells are cultured on a flat dish such as a petri dish, the existing range of the cells spreads in a plane. Therefore, if the photographing device and the petri dish are arranged such that the optical axis of the lens of the photographing device is orthogonal to the dish surface, it is possible to focus on almost all cells on the petri dish.

  However, when the cells are suspended in a culture medium and suspension culture is performed, the range of the cells is three-dimensionally expanded, so that the distance in the optical axis direction from the imaging device to each cell varies. As such, it can be difficult to focus on all cells without the lens.

  On the other hand, the depth of field can be increased by using a bright lens (a lens with a small F value) or by irradiating a bright illumination to the object to be measured and narrowing the aperture of the lens as much as possible. It is possible.

  Alternatively, a plurality of images may be captured while changing the focal position of the lens little by little, and the plurality of captured images may be combined to obtain a pseudo-deeply focused image. In addition, each of the plurality of images is an image in which the in-focus cells and the out-of-focus blur cells are mixed. Therefore, in-focus partial images may be collected from a plurality of images to generate one composite image.

  Alternatively, as shown in FIG. 23, a telecentric lens 172 may be disposed between the reprogramming culture photographing device 171 and a subject such as a cell in the floating incubator. Since the telecentric lens 172 makes the principal ray passing through the center of the lens diaphragm from the subject such as a cell parallel to the lens optical axis, the distance from the initialization culture photographing device 171 to each of the plurality of cells in the suspension incubator is one. Even if it does not, the size of the cell to be imaged does not change according to the distance.

  FIG. 24 is a schematic view of the floating incubator and the like shown in FIG. 23 as viewed from above. In FIG. 24, the container 76 shown in FIG. 23 is omitted. When imaging cells with the initial culture imaging device 171, the illumination light source 173 for cell observation is disposed in a direction perpendicular to the optical axis of the initial culture imaging device 171 or in a direction near the imaging device from the vertical direction; It is preferable to use a scattered light illumination method in which illumination light is emitted from the cell observation illumination light source 173 to the cells. Thereby, the scattered light by the illumination light which hit the cells reaches the initialization culture photographing device 171, but the illumination light which does not hit the cells permeates the culture medium and does not reach the initialization culture photographing device 171. Therefore, in the image, the portion of the medium becomes relatively dark and the portion of the cell becomes relatively bright. However, the illumination method is not limited to this as long as cells can be recognized in the image. FIG. 25 is an example of an image of cells imaged by the scattered light illumination method. The part of the medium is relatively dark and the part of the cell is relatively bright.

  The cell processing system according to the embodiment may include a central processing unit (CPU) 500 including an image processing unit 501 that performs image processing on an image captured by the initialization culture imaging apparatus 171, as shown in FIG. . The CPU 500 may be connected to an input device 401 such as a keyboard and a mouse, and an output device 402 such as a monitor. The CPU 500 receives an image from the initialization culture imaging apparatus 171 via a bus, an image interface, and the like.

  The image processing unit 501 may include a contour definition unit 511 that defines contours of cells or cell clusters in an image of cells. FIG. 27 is an example of an image of an iPS cell mass captured by magnifying through a macro zoom lens or the like. In the image shown in FIG. 27, the part that looks like a white mass is an iPS cell mass, and the dark part of the background is a culture medium.

  Here, when the image shown in FIG. 27 is an 8-bit gray scale image, the value of the luminance of a pixel having a luminance value equal to or higher than a predetermined threshold is set to the value of the highest luminance such as 255 and is less than the predetermined threshold. If a binarization process is applied to the image in which the luminance value of the pixel having the luminance value of 0 is replaced with the lowest luminance value such as 0, as shown in FIG. The inside also becomes black with the lowest brightness, and a portion in which the inside of the cell mass and the portion of the culture medium are connected may occur. Therefore, in binarization processing, cells or cell masses may not be extracted.

  On the other hand, the contour definition unit 511 of the cell processing system according to the embodiment shown in FIG. 26 passes high frequency components higher than or equal to a predetermined frequency included in the spatial frequency to the image of cells and transmits low frequencies lower than the predetermined frequency. Apply a high pass filter to block the components and to make the luminance value the lowest value, for example 0. In the cell image, in the cell or cell mass part, the high frequency component included in the spatial frequency is large, and in the medium part, the high frequency component included in the spatial frequency is small. Therefore, as shown in FIG. 29, in the image of the cell to which the high-pass filter is applied, the brightness value of the medium portion is, for example, the lowest value such as 0, and in the cell or cell cluster portion, the brightness The value is left as it is. Therefore, it is possible to regard a portion where the brightness does not reach the lowest value as a cell or a cell mass.

  Here, in the image shown in FIG. 29, even if a portion where the brightness does not reach the minimum value is detected by blob analysis as a blob, for example, two cell masses in contact with each other are one cell mass It may be recognized as

  On the other hand, the contour definition unit 511 of the cell processing system according to the embodiment shown in FIG. 26 applies the water drop algorithm to the image to which the high pass filter is applied. The watershed algorithm considers the brightness gradient of the image as mountain relief, and makes the area of water flowing from the high position of the mountain (high position of the brightness value) to the low position (low position of the brightness value) one area. To divide the image.

  For example, the contour definition unit 511 of the cell processing system according to the embodiment converts the image into the image by the Distance Transform method before applying the watershed algorithm to the image. The Distance Transform method is a method of image conversion in which the value of the luminance of each pixel of an image is replaced according to the distance to the nearest background pixel. For example, as shown in FIG. 30 (a), in the image to which the high-pass filter is applied, the luminance value of the area of the culture medium is collectively converted to 255 which is the highest luminance value, as shown in FIG. Make it a white background. Furthermore, the value of luminance of each pixel inside the cell area is converted to 0 or more and less than 255 according to the distance to the nearest background pixel. For example, as the distance from the nearest background pixel is increased, the luminance value is decreased.

  Next, the contour definition unit 511 of the cell processing system according to the embodiment applies a watershed algorithm to the image transformed by the Distance Transform method. In the image shown in FIG. 30 (b), when the dark part with low luminance is regarded as the ridge of a mountain and water is dropped from above the image in the vertical direction, as shown by the arrow in FIG. 30 (c) Guess how the water flows and, as shown by the broken line in Fig. 30 (c), regard the place where the water flowing from various directions collides as a valley, and divide the cell area at the bottom of the valley. Do.

  If the pixel of the cell area | region of the image shown in FIG. 29 is transformed by the Distance Transform method, the image shown in FIG. 31 will be obtained. When the watershed algorithm is applied to the image shown in FIG. 31, the image shown in FIG. 32 is obtained. When the obtained dividing line is superimposed on the original image shown in FIG. 27, the image shown in FIG. 33 is obtained. In FIG. 33, it is possible to consider that the cell mass present in each region divided by the dividing line is not a mass in contact with a plurality of cell masses but one cell mass. Therefore, in each region, as shown in FIG. 34, by extracting the contour of the cell mass, it is possible to accurately extract one cell mass.

The image processing unit 501 of the cell processing system according to the embodiment shown in FIG. 26 may further include a cell evaluation unit 512. The cell evaluation unit 512 evaluates the size and the like of one cell mass extracted by the contour definition unit 511. For example, the cell evaluation unit 512 calculates the area of one cell mass extracted by the contour definition unit 511. Furthermore, for example, when the shape of one cell mass can be regarded as circular, the cell evaluation unit 512 calculates the diameter of one cell mass from the area using the following equation (1).
D = 2 (s / π) ^1/2 (1)
Here, D represents a diameter and S represents an area.

  If the cell mass grows too large, the nutrients and hormones contained in the culture medium may not reach the inside and cells may differentiate. In addition, if the cell mass is too small, transition to expansion culture without using a ROCK inhibitor may result in cell death or karyotype abnormality. Therefore, the cell evaluation unit 512 may issue a warning when the size of each cell mass is out of an appropriate range. In addition, the cell evaluation unit 512 may output that it is the timing to shift to the expansion culture when the size of each cell mass becomes equal to or larger than a predetermined threshold. Furthermore, the supply rate of the culture medium in the reprogramming culture apparatus 50 may be changed according to the calculated size of the cell mass. For example, as the size of the cell mass increases, the supply rate of the culture medium may be increased.

  The image processing unit 501 of the cell processing system according to the embodiment may further include a statistical processing unit 513 that performs statistical processing on information obtained from the image subjected to the image processing. FIG. 35 shows an example in which the image shown in FIG. 25 is subjected to image processing to extract and outline the cell mass. FIG. 36 is an example of a cell mass size histogram created based on the image shown in FIG. As described above, by continuously and periodically acquiring cell information, it is possible to quantitatively grasp the growth degree, the number, the confluence degree, etc. of the cell mass, and stabilize the culture result. It is possible. Also, the supply rate of the culture medium in the reprogramming culture apparatus 50 may be changed according to the calculated number of cell masses. For example, as the number of cell masses increases, the media delivery rate may be increased.

  The image processing unit 501 of the cell processing system according to the embodiment shown in FIG. 26 calculates the turbidity of the culture medium from the image of the culture medium, and calculates the density of cell clusters in the culture medium based on the turbidity of the culture medium 514 may be further provided.

  For example, the CPU 500 is connected with a related storage device 403 provided with a volatile memory, a non-volatile memory, or the like. The relation storage device 403 stores, for example, the relation between the turbidity of the culture medium and the density of cell clusters in the culture medium, which is acquired in advance. The density calculation unit 514 reads out the relationship between the turbidity and the density from the relationship storage device 403. Furthermore, the density calculation unit 514 calculates the density of the value of the cell mass in the medium based on the value of the turbidity of the medium calculated from the image of the medium, and the relationship between the turbidity and the density. Thereby, it is possible to nondestructively measure the density of the cell mass without collecting the cell mass from the medium.

  In addition, the density calculation unit 514 may output that it is the timing to shift to the expansion culture when the density of the cell mass is equal to or more than a predetermined threshold. Furthermore, the density calculation unit 514 may calculate the density of the cell mass in the culture medium over time to calculate the growth rate of the cell mass. An abnormal growth rate may indicate that the cell is abnormal. For example, the density calculation unit 514 issues a warning when the abnormal growth rate is calculated. In this case, cell culture may be discontinued.

  When the density of cell clusters in the culture medium becomes high and the distance between cell clusters is too short, a plurality of cell clusters may adhere to each other to form one large cell cluster. In a large cell mass, the nutrients and hormones contained in the culture medium may not reach inside, and the cells inside may differentiate. On the other hand, if the density of the cell mass in the medium is lower than the preferred range, the growth rate of the cell mass and the ability to form the cell mass may be significantly reduced.

  On the other hand, according to the density calculation unit 514, since it is possible to calculate the density of the cell mass, it is possible to easily determine whether the density of the cell mass is within a suitable range. . If the density of cell mass falls below a suitable range, for example, it may be determined to stop the culture. Furthermore, the supply rate of the culture medium in the reprogramming culture apparatus 50 may be changed according to the calculated density of cell masses. For example, as the density of cell mass increases, the media delivery rate may be increased.

  In addition, as shown in FIG. 37, the illumination light source 174 for observing the culture medium is placed at a position facing the reset culture photographing device 171 with the floating incubator interposed, in order to observe a change in color of the culture medium accompanying cell metabolism and the like. May be arranged. As the culture light for observation of culture medium 174, for example, a surface light source can be used, and the illumination light for observation of culture medium 174 emits, for example, white parallel light. The illumination light emitted from the culture light for observation of culture medium 174 passes through the culture medium and enters the initialization culture imaging device 171, so that the color of the culture medium can be imaged by the initialization culture imaging device 171.

  Generally, when performing cell culture, the pH of the medium is brought to a constant value of around 6.8 to 7.2. When measuring the pH of the medium, a pH reagent such as phenol red is added to the medium. Phenol red changes with the pH of the medium. If the concentration of carbon dioxide in the gas in contact with the medium is insufficient, the medium becomes alkaline because the carbon dioxide derived from bicarbonate and the carbon dioxide in the gas do not equilibrate, and the color of the medium becomes reddish purple. Become. Also, when waste products mainly composed of lactic acid excreted by cells accumulate, the medium becomes acidic and the color of the medium becomes yellow. The acidification of the medium also indicates the depletion of nutrients in the medium.

  The image processing unit 501 of the cell processing system according to the embodiment shown in FIG. 26 may further include a culture medium evaluation unit 515 for evaluating the culture medium based on the image of the culture medium illuminated by the culture light for observation of culture medium. . The culture medium evaluation unit 515, for example, image-processes the image of the culture medium, and expresses the color of the culture medium by HSV, using three parameters of hue (Hue), saturation (Saturation), and lightness (Value). Among these, hue is a parameter corresponding to the concept generally called "color tone" or "color tone". Hue is generally expressed in units of angles.

  FIG. 38 is an example of a graph showing the relationship between the change in the color of the medium and the change in the pH of the medium when cells are cultured for a long period of time without changing the medium. Immediately after the start of culture, the pH of the medium was close to 7.7, but with the passage of time, the pH of the medium dropped to near 7.1. Along with this, the color of the culture medium, which was close to 40 immediately after the start of culture, rose to near 60 with the passage of time. Thus, the color of the medium correlates with the culture time and the pH of the medium. Therefore, culture medium evaluation unit 515 shown in FIG. 26 determines the state of the culture medium by monitoring the hue of the culture medium.

  For example, the relation storage device 403 stores the relation between the color of the culture medium and the pH of the culture medium, which is acquired in advance. The culture medium evaluation unit 515 reads out the relationship between the hue and the pH from the relationship storage device 403. Furthermore, the culture medium evaluation unit 515 calculates the value of the pH of the photographed culture medium based on the value of the hue 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 obtain an image of the culture medium over time, and may calculate the value of the pH of the culture medium.

  As shown in FIG. 22, the pH of the culture medium may be measured by a pH sensor 271. In addition, the temperature of the culture medium may be measured by a thermometer 272. In this case, the medium evaluation unit 515 may receive the value of the pH of the medium from the pH sensor 271 and may receive the value of the temperature of the medium from the thermometer 272.

  When the color of the culture medium or the pH of the culture medium is out of the predetermined range, the culture medium evaluation unit 515 may judge to promote the exchange of the culture medium or may judge that contamination occurs in the culture medium. Note that replacing the medium also includes partially replacing and replenishing the medium.

  Chemically analyzing the components of the medium is costly, and removing the medium outside the system for chemical analysis of the medium may not maintain the sterility of the medium. In contrast, monitoring the condition of the medium by monitoring the color of the medium is low cost and does not affect the sterility of the medium.

  If the culture medium evaluation unit 515 determines that the color of the culture medium or the pH of the culture medium is outside the predetermined range, the medium around the dialysis tube 75 of the floating incubator is replaced by the replenishment medium feed pump 77 shown in FIG. Be done. Alternatively, if medium exchange is constantly performed, the exchange rate of the medium around the dialysis tube 75 of the floating incubator by the supply medium feed pump 77 is increased, and the flow rate of the medium to be exchanged is increased. This makes it possible to maintain the pH of the medium in a range suitable for cell culture and to supply the medium with sufficient nutrients.

  Furthermore, the medium evaluation unit 515 may calculate the growth rate of cells from the change rate of the hue of the medium. For example, the relation storage device 403 stores the relation between the change rate of the hue of the culture medium and the growth rate of the cells, which are acquired in advance. The culture medium evaluation unit 515 reads out the relationship between the change rate of hue and the growth rate from the relationship storage device 403. Furthermore, the culture medium evaluation unit 515 calculates the value of the cell growth rate based on the calculated value of the change rate of the hue and the relationship between the change rate of the hue and the growth rate.

  In addition, if the medium evaluation unit 515 determines that the temperature of the medium is outside the predetermined range, the medium management unit 515 may control the temperature management device to change the temperature around the incubator or the temperature of the supplied medium. Good. For example, when the temperature of the culture medium is lower than a predetermined range, the culture medium evaluation unit 515 controls the temperature management device so that the temperature of the culture medium rises. In addition, when the temperature of the culture medium is higher than a predetermined range, the culture medium evaluation unit 515 controls the temperature management device so that the temperature of the culture medium is lowered.

  A first cell mass delivery channel 51 is connected to the reprogramming culture apparatus 50 shown in FIG. The reprogramming culture apparatus 50 sends the liquid containing the trypsin alternative recombinant enzyme and the cell mass to the first cell mass supply channel 51 using a pump or the like. When the cell mass is physically broken, the trypsin alternative recombinant enzyme may not be present. In addition, the first cell mass delivery channel 51 has an inner diameter that allows only induced cells smaller than a predetermined size to pass, and is connected to a branch channel that removes uninduced cells of a predetermined size or more. It may be As described above, in the case of using a gel culture medium, it is possible to recover a cell mass by sucking up the gel culture medium.

  The pump for pumping out the solution containing the cell mass to the first cell mass feed channel 51 is, for example, the case where the value of the cell mass size calculated by the cell evaluation unit 512 shown in FIG. May be driven. Alternatively, in the pump for pumping out the solution containing the cell mass to the first cell mass delivery channel 51 shown in FIG. 16, for example, the value of the cell mass density calculated by the density calculation unit 514 shown in FIG. It may be driven when it becomes more than.

  The inner wall of the first cell mass delivery channel 51 shown in FIG. 16 may be coated with poly-HEMA so as not to adhere to the cells, to be noncell-adhesive. Alternatively, a material to which cells do not easily adhere may be used as the material of the first cell mass delivery channel 51. In addition, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the first cell mass delivery channel 51, the conditions in the first cell mass delivery channel 51 can be controlled in the housing 601. Equivalent to the temperature and carbon dioxide concentration. Furthermore, a backflow prevention valve may be provided in the first cell mass delivery channel 51 from the viewpoint of contamination prevention.

  The first cell mass delivery channel 51 is connected to the first dividing mechanism 60. The first dividing mechanism 60 includes, for example, a mesh. The cell mass contained in the solution is divided into a plurality of cell masses of the size of each pore of the mesh when passing through the mesh by water pressure. For example, if the size of each pore of the mesh is uniform, the sizes of the plurality of cell masses after division will be substantially uniform. Alternatively, the first dividing mechanism 60 may include a nozzle. For example, by finely processing the inside of the substantially conical nozzle in a step-like manner, the cell mass contained in the solution is divided into a plurality of cell masses when passing through the nozzle.

  Alternatively, as shown in FIG. 39, the first dividing mechanism 60 may include a cell mass divider including the end block 61, the connection block 62, and the tip block 63. Each of the end block 61, the connection block 62, and the tip block 63 is provided with a through hole through which a medium containing a cell mass flows. As shown in FIGS. 40 and 41, the end block 61, the connection block 62, and the tip block 63 are connected. The cell mass divider may comprise one connection block 62 or may comprise a plurality of connection blocks 62.

  As shown in FIG. 39, a recess 62a is provided at a first end of the connection block 62, and a protrusion 62b is provided at a second end opposite to the first end of the connection block 62. The convex portion 62 b has, for example, a cylindrical shape. As shown in FIGS. 40 and 41, in the case where there are a plurality of connection blocks 62, the convex portions 62b engage with the recesses 62a of the adjacent connection blocks 62. The side wall of the convex portion 62b shown in FIG. 39 may be smooth or may be provided with an external thread. The inner wall of the recess 62a may be smooth or may be provided with a female screw.

  The through holes provided in the connection block 62 communicate with the first large diameter portion 62c communicating with the recess 62a and the first large diameter portion 62c, and have a smaller diameter than the first large diameter portion 62c. 62d and a second large diameter portion 62e communicating with the small diameter portion 62d, having a larger diameter than the small diameter portion 62d, and having an opening at the tip of the convex portion 62b.

  The cross-sectional shape of each of the first large diameter portion 62c, the small diameter portion 62d, and the second large diameter portion 62e is, for example, a circle. The hole diameter of the first large diameter portion 62c and the hole diameter of the second large diameter portion 62e are, for example, the same. Thereby, as shown in FIGS. 40 and 41, when there are a plurality of connection blocks 62 and the plurality of connection blocks 62 are connected, the second large diameter portion 62e is the first large diameter of the adjacent connection block 62. It smoothly communicates with the portion 62c.

  Although the hole diameter of the 1st and 2nd large hole diameter parts 62c and 62e shown in FIG. 39 is 2.0 mm or more and 4.0 mm or less, for example, it is not specifically limited. Although the hole diameter of the small hole diameter part 62d is 0.4 mm or more and 1.2 mm or less, for example, it is not specifically limited. A step is formed in a portion continuing from the first large diameter portion 62c to the small diameter portion 62d. Further, steps are also formed in a portion continuing from the small diameter portion 62d to the second large diameter portion 62e. The side walls of the steps may be perpendicular to the central axis of the through hole or may be inclined less than 90 °.

  The central axes of the first and second large diameter portions 62c and 62e of the connection block 62 and the central axis of the small diameter portion 62d may coincide with each other. Alternatively, as shown in FIG. 42, the central axes of the first and second large diameter portions 62c and 62e of the connection block 62 and the central axis of the small diameter portion 62d may be offset.

  A recessed portion 63a is provided at a first end of the distal end block 63 shown in FIG. 39, and a nozzle portion 63b is provided at a second end opposed to the first end of the distal end block 63. When connecting the tip block 63 and the connection block 62, the recess 63 a of the tip block 63 engages with the projection 62 b of the connection block 62. The inner wall of the recess 63a may be smooth or may be provided with a female screw.

  The through hole provided in the tip block 63 communicates with the large hole portion 63c communicating with the recess 63a and the large hole portion 63c, has a smaller hole diameter than the large hole portion 63c, and has an opening at the tip of the nozzle portion 63b. And a portion 63d.

  Each cross-sectional shape of the large hole diameter portion 63c and the small hole diameter portion 63d is, for example, a circle. The hole diameter of the large hole diameter portion 63c of the tip block 63 and the hole diameter of the second large hole diameter portion 62e of the connection block 62 are, for example, the same. Thereby, as shown in FIGS. 40 and 41, when the connection block 62 and the distal end block 63 are connected, the second large pore diameter portion 62e of the connected block 62 is the large pore diameter of the adjacent distal end block 63. It smoothly communicates with the portion 63c.

  The hole diameter of the large hole diameter portion 63c shown in FIG. 39 is, for example, 2.0 mm or more and 4.0 mm or less, but is not particularly limited. The hole diameter of the small hole diameter portion 63d is, for example, 0.4 mm or more and 1.2 mm or less, but is not particularly limited. A step is formed in a portion continuing from the large diameter portion 63c to the small diameter portion 63d. The side walls of the steps may be perpendicular to the central axis of the through hole or may be inclined less than 90 °.

  A recess 61 a is provided at a first end of the end block 61, and a protrusion 61 b is provided at a second end opposite to the first end of the end block 61. When the end block 61 and the connection block 62 are connected, the convex portion 61 b of the end block engages with the recess 62 a of the connection block 62. The side wall of the convex portion 61b of the end block may be smooth or may be provided with an external thread.

  The through hole provided in the end block 61 communicates with the recess 61 a and has at least a large hole diameter portion 61 c having an opening at the tip of the protrusion 61 b.

  Each cross-sectional shape of the recessed part 61a and the large hole diameter part 61c is, for example, a circle. The hole diameter of the large hole diameter portion 61c of the end block 61 and the hole diameter of the second large hole diameter portion 62e of the connection block 62 are, for example, the same. Thereby, as shown in FIGS. 40 and 41, when the end block 61 and the connection block 62 are connected, the large hole diameter portion 61c of the end block 61 and the large hole diameter portion 62c of the adjacent connection block 62 It communicates smoothly.

  Although the hole diameter of the large hole diameter part 61c shown in FIG. 39 is 2.0 mm or more and 4.0 mm or less, for example, it is not specifically limited.

  The material of each of the end block 61, the connection block 62, and the tip block 63 includes, but is not limited to, a resin such as polypropylene.

  As shown in FIGS. 40, 41 and 42, for example, an insertion nozzle 64 is inserted into the recess 61a of the end block 61. The insertion nozzle 64 is connected to a suction / discharge device that sucks and discharges the medium containing the cell mass directly or through a tube or the like. The end block 61, the connection block 62, and the tip block 63 are connected, and the nozzle portion 63b of the tip block 63 is pierced with the medium containing the cell mass, and suction and discharge of the medium is performed once with a suction and discharge device, or When the aspiration and discharge are repeated, the culture medium containing the cell mass reciprocates in the connection block 62 and the through hole in the tip block 63. Since the through holes in the connection block 62 and the tip block 63 are provided with steps, cell clusters in the medium are efficiently divided into small cell clusters.

  Conventionally, division of cell mass has been performed by a technician using a pipetman or the like. However, as shown in FIG. 47 (a), in the conventional method, the sizes of the divided cell masses were uneven. Moreover, the size of the cell mass obtained by the technician was various. If the cell mass after division is too large, the nutrients and hormones contained in the culture medium may not reach the inside and cells may differentiate. In addition, if the cell mass is too small, cell death or karyotype abnormality may occur if the ROCK inhibitor is not used. On the other hand, when using a cell mass divider as shown in FIG. 40, FIG. 41 and FIG. 42, the cell mass is divided into cell masses of uniform size as shown in FIG. 47 (b) It is possible. When the cell mass is divided using a cell mass divider, the medium is trypsin, TrypLE Express (registered trademark, ThermoFisher SCIENTIFIC), TrypLE Select (registered trademark, ThermoFisher SCIENTIFIC), or TrypLE Select (registered trademark) An enzyme such as ThermoFisher SCIENTIFIC may be included. In addition, it is also possible to disintegrate the cell mass into single cells by increasing the connecting block 62 or increasing the pressure at which the culture medium is aspirated and discharged.

  In addition, when the number of repetitions of a large pore size part and a small pore size part, length, etc. are determined, a cell mass splitter does not need to be comprised from several blocks. For example, as shown in FIG. 43, the cell mass divider has an integral columnar shape having a through hole inside, and the through holes through which the medium containing the cell mass flows have large pore sizes 65a, 65c, 65e, It may be in communication with 65 g and the large diameter portions 65a, 65c, 65e and 65g, and may alternately have small diameter portions 65b, 65d and 65f having a smaller diameter than the large diameter portions 65a, 65c, 65e and 65g. . Also in this case, as shown in FIG. 44, the central axes of the large diameter portions 65a, 65c, 65e, 65g and the central axes of at least a part of the small diameter portions 65b, 65d, 65f may be offset. .

  Alternatively, the cell mass in the medium may be divided into small cell masses by passing the medium only once through the cell mass divider. In that case, as shown in FIG. 45, insertion portions 66a and 66b into which tubes and the like can be inserted may be provided at both ends of the cell mass divider. The culture medium is discharged from the insertion part 66a through the through hole and discharged from the insertion part 66b, while the cell mass contained in the culture medium is divided. Also in this case, as shown in FIG. 46, the central axes of the large diameter portions 65a, 65c, 65e, 65g and the central axes of at least a part of the small diameter portions 65b, 65d, 65f may be offset. .

  The expansion culture apparatus 70 is connected to the first dividing mechanism 60 shown in FIG. The solution containing the cell mass divided by the first dividing mechanism 60 is sent to the expansion culture apparatus 70.

  The expansion culture apparatus 70 can store a well plate inside. Moreover, the expansion culture apparatus 70 is equipped with a pipetting machine. The expansion culture apparatus 70 receives the solution containing the plurality of cell masses from the first dividing mechanism 60, and distributes the solution to the wells with a pipetting machine. After distributing the cell mass to the wells, the expansion culture apparatus 70 cultures the cell mass at, for example, about 8 days at 37 ° C. and 5% carbon dioxide. Moreover, the expansion culture apparatus 70 performs culture medium exchange suitably.

  Thereafter, the expansion culture apparatus 70 adds trypsin alternative recombinant enzyme such as TrypLE Select (registered trademark, Life Technologies) to the cell mass. Furthermore, the expansion culture apparatus 70 puts the container containing the cell mass in an incubator, and makes the cell mass react with the trypsin alternative recombinant enzyme at 37 ° C. and 5% carbon dioxide for 1 minute. When the cell mass is physically broken, the trypsin alternative recombinant enzyme may not be present. For example, the expansion culture apparatus 70 breaks cell clumps by pipetting with a pipetting machine. Alternatively, the expansion culture apparatus 70 may pass a cell mass to a pipe provided with a filter or a pipe that changes the inner diameter intermittently like the introduced cell delivery channel 31 shown in FIG. 17 or 18. You may break it. Thereafter, the expansion culture apparatus 70 adds a medium such as a maintenance culture medium to the solution containing the cell mass. Furthermore, in the case of the adhesion culture, the expansion culture apparatus 70 peels the cell mass from the container with an automatic cell scraper or the like, and the solution containing the cell mass is transferred to the first dividing mechanism 60 via the expansion culture liquid flow channel 71. Send to

  The culture in the expansion culture apparatus 70 may be performed in a carbon dioxide permeable bag instead of the well plate. The culture may be adhesion culture, suspension culture or hanging drop culture. In the case of suspension culture, agitation culture may be performed. Also, the medium may be in the form of agar. Agar-like media include gellan gum polymers and deacylated gellan gum polymers. When the agar-like medium is used, the cells do not sink or adhere, and therefore, it is not necessary to stir even in suspension culture.

  The expansion culture apparatus 70 may include a second medium replenishing apparatus for replenishing the well plate or the carbon dioxide permeable bag with the culture solution. The second medium supply device collects the culture solution in the well plate or carbon dioxide permeable bag, filters the culture solution using a filter or a dialysis membrane, and reuses the purified culture solution. Good. At that time, a growth factor or the like may be added to the culture solution to be recycled. Further, the expansion culture apparatus 70 may further include a temperature control apparatus that controls the temperature of the culture medium, a humidity management apparatus that controls the humidity near the culture medium, and the like.

  Also in the expansion culture apparatus 70, for example, cells are placed in a culture solution permeable bag 301 such as a dialysis membrane as shown in FIG. 19, and the culture solution permeable bag 301 is treated as a culture solution impermeable carbon dioxide. The culture solution may be put in the bags 301, 302 in the sexual bag 302. The reprogramming culture apparatus 50 prepares a plurality of bags 302 containing fresh culture fluid, and for every predetermined period, the bag 302 containing the bag 301 containing cells is a bag containing fresh culture fluid. It may be replaced with 302.

  Further, the culture method in the expansion culture apparatus 70 is not limited to the above-described method, and a floating incubator as shown in FIG. 20 may be used as in the culture method in the reprogramming culture apparatus 50. In the expansion culture apparatus 70, a plurality of cell masses are placed in the dialysis tube 75 of the suspension incubator shown in FIG. Details of the suspension incubator are as described above. Further, also in the expansion culture apparatus 70, as shown in FIG. 21, the gel culture medium around the dialysis tube 75 in the container 76 is replaced or replenished by using the replenishment medium feed pump 77. Alternatively, as shown in FIG. 22, the supply medium feed pump 77 and the inside of the dialysis tube 75 in the vessel 76 of the suspension incubator are connected by the feed tube 78, and the medium in the dialysis tube 75 is connected. The components necessary for culture may be supplied.

  The cell processing system may further include an expansion culture imaging device for imaging the culture in the expansion culture device 70 shown in FIG. Here, when a colorless medium is used as the medium used in the expansion culture apparatus 70, it is possible to suppress diffuse reflection and autofluorescence which may occur when a colored medium is used. However, in order to confirm the pH of the culture medium, a pH indicator such as phenol red may be included. In addition, since the shape and size of the cells are different between the induced cells and the non-induced cells, the cell processing system measures the proportion of cells induced by photographing the cells in the expansion culture apparatus 70. You may further provide the guidance state monitoring apparatus which calculates these. Alternatively, the induction status monitoring device may specify the proportion of cells induced by antibody immunostaining or RNA extraction. Furthermore, the cell processing system may be equipped with an uninduced cell removal device that removes uninduced cells by magnetic cell separation methods, flow cytometry, etc.

  The expansion culture imaging apparatus is the same as the initialized culture imaging apparatus 171 shown in FIG. 23, and the culture in the expansion culture apparatus 70 may be imaged through the telecentric lens 172. The illumination method and the like at the time of imaging by the expansion culture imaging device is also the same as the illumination method and the like at the time of imaging by the initialized culture imaging device 171, and is as described above.

  The expansion culture imaging apparatus is also connected to the CPU 500 including the image processing unit 501 shown in FIG. The image processing unit 501 including the contour definition unit 511, the cell evaluation unit 512, the statistical processing unit 513, the density calculation unit 514, and the culture medium evaluation unit 515 is similar to the image captured by the initialization culture imaging device 171. Image processing is performed on the image captured by the device. The details of the image processing unit 501 are as described above.

  For example, in the expansion culture, when the cell mass grows too large, the nutrients and hormones contained in the culture medium may not reach inside, and the cells may differentiate. In addition, if passage is performed while the cell mass is too small, cell death or karyotype abnormality may occur if the ROCK inhibitor is not used. Therefore, the cell evaluation unit 512 may issue a warning when the size of each cell mass is out of an appropriate range. In addition, the cell evaluation unit 512 may output that it is a pass-through timing when the size of each cell mass becomes equal to or more than a predetermined threshold. In this case, cell clusters may be disrupted to reduce individual cell clusters, and the culture may be again passaged again in the incubator. Furthermore, it is also possible to determine whether or not the disruption was properly performed by calculating the sizes of individual cell clusters after disrupting the cell clusters at passage. Furthermore, the supply rate of the culture medium in the expansion culture apparatus 70 may be changed according to the calculated size of the cell mass. For example, as the size of the cell mass increases, the supply rate of the culture medium may be increased.

  Further, the supply rate of the culture medium in the expansion culture apparatus 70 may be changed according to the number of cell clusters calculated by the statistical processing unit 513. For example, as the number of cell masses increases, the media delivery rate may be increased.

  The density calculation unit 514 may output that it is a pass-through timing when the density of the cell mass is equal to or more than a predetermined threshold. If the density of the cell mass is higher than the suitable range, it is possible to adjust the density of the cell mass within the suitable range, for example, by passaging. Furthermore, it is also possible to determine whether or not the disruption was properly performed by calculating the density of the cell mass after disrupting the cell mass at passage. Furthermore, the supply rate of the culture medium in the expansion culture apparatus 70 may be changed according to the calculated density of cell masses. For example, as the density of cell mass increases, the media delivery rate may be increased.

  If the culture medium evaluation unit 515 determines that the color of the culture medium or the pH of the culture medium is outside the predetermined range, the dialysis culture tube of the suspension incubator is also supplied by the supplementary culture medium feed pump 77 shown in FIG. The medium around 75 is replaced. Alternatively, if medium exchange is constantly performed, the exchange rate of the medium around the dialysis tube 75 of the floating incubator by the supply medium feed pump 77 is increased, and the flow rate of the medium to be exchanged is increased. This makes it possible to maintain the pH of the medium in a range suitable for cell culture and to supply the medium with sufficient nutrients.

  The cell mass divided by the first dividing mechanism 60 shown in FIG. 16 is again cultured in the expansion culture apparatus 70. The division of the cell mass in the first dividing mechanism 60 and the culture of the cell mass in the expansion culture apparatus 70 are repeated until the required amount of cells is obtained.

  The pump for pumping out the solution containing the cell mass in the expansion culture apparatus 70 to the first dividing mechanism 60 via the expansion culture water supply channel 71 is, for example, a cell mass calculated by the cell evaluation unit 512 shown in FIG. It may be driven when the value of the value of に becomes equal to or more than a predetermined threshold. Alternatively, in the pump for pumping out the solution containing the cell mass to the first cell mass delivery channel 51 shown in FIG. 16, for example, the value of the cell mass density calculated by the density calculation unit 514 shown in FIG. It may be driven when it becomes more than.

  A second cell mass delivery channel 72 is connected to the expansion culture apparatus 70. The expansion culture apparatus 70 is used to pump the liquid containing the cell mass, which has been expanded and peeled off from the container, to the second cell mass feed channel 72 using a pump or the like. However, in the case of suspension culture, exfoliation is unnecessary. The second cell mass delivery channel 72 has an inner diameter that allows only induced cells smaller than a predetermined size to pass, and is connected to a branch channel that removes uninduced cells of a predetermined size or more. It is also good.

  The inner wall of the second cell mass delivery channel 72 may be coated with poly-HEMA to be non-cell-adhesive so that cells do not adhere. Alternatively, a material to which cells do not easily adhere may be used as the material of the second cell mass delivery channel 72. In addition, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the second cell mass delivery channel 72, the conditions in the second cell mass delivery channel 72 can be managed in the housing 601. Equivalent to the temperature and carbon dioxide concentration. Furthermore, a backflow prevention valve may be provided in the second cell mass delivery channel 72 from the viewpoint of contamination prevention.

  The second cell mass delivery channel 72 is connected to the second dividing mechanism 80. The second dividing mechanism 80 includes, for example, a mesh. The cell mass contained in the solution is divided into a plurality of cell masses of the size of each pore of the mesh when passing through the mesh by water pressure. For example, if the size of each pore of the mesh is uniform, the sizes of the plurality of cell masses after division will be substantially uniform. Alternatively, the second dividing mechanism 80 may include a nozzle. For example, by finely processing the inside of the substantially conical nozzle in a step-like manner, the cell mass contained in the solution is divided into a plurality of cell masses when passing through the nozzle.

  Alternatively, like the first dividing mechanism 60, the second dividing mechanism 80 is a cell mass dividing device including the end block 61, the connecting block 62, and the tip block 63 shown in FIGS. 39 to 42, or An integrated cell splitter shown in FIG. 46 may be provided. Details of the cell mass divider are as described above.

  The second dividing mechanism 80 shown in FIG. 16 is connected to a cellular soul transport mechanism 90 for sequentially sending a plurality of cellular souls to the package device 100. A pre-package cell flow channel 91 is connected between the cellular soul transport mechanism 90 and the packaging device 100. The cellular soul transport mechanism 90 sequentially sends each of the cell masses divided by the second division mechanism 80 to the package device 100 via the pre-package cell channel 91 using a pump or the like.

  The pre-package cell channel 91 may be coated with poly-HEMA so that cells do not adhere. Alternatively, a material to which cells do not easily adhere may be used as the material of the pre-package cell flow channel 91. In addition, by using a material with good temperature conductivity and carbon dioxide permeability as the material of the pre-package cell flow channel 91, the conditions in the pre-package cell flow channel 91 are the controlled temperature in the housing 601 and It becomes equivalent to carbon dioxide concentration. The pre-package cell flow channel 91 may be provided with a backflow prevention valve from the viewpoint of contamination prevention.

  The pre-package cell flow channel 91 is connected to a cryopreservation liquid delivery mechanism 110. The cryopreservation solution delivery mechanism 110 delivers the cell cryopreservation solution to the pre-package cell channel 91. Thereby, the cell mass is suspended in the cell cryopreservation solution in the pre-package cell channel 91.

  The packaging apparatus 100 sequentially freezes each of the plurality of cell masses sent via the pre-package cell flow channel 91. For example, each time the packaging apparatus 100 receives a cell mass, the cell mass is placed in a cryopreservation container such as a cryotube, and the cell mass solution is instantaneously frozen at, for example, -80 ° C or less. If a cryopreservation container with a small surface area per volume tends to take a long time to freeze, it is preferable to use a cryopreservation container with a large surface area per volume. By using a cryopreservation container having a large surface area per volume, it is possible to increase cell viability after thawing. The shape of the cryopreservation container includes, but is not limited to, capillary and spherical shapes. Also, depending on the required cell viability after thawing, it is not always necessary to do the snap freezing.

  For freezing, for example, a vitrification method is used. In this case, DAP 213 (Cosmo Bio Inc.) and Freezing Medium (Reprocel Co., Ltd.) can be used as a cell cryopreservation solution. Freezing may be carried out by a conventional method other than the vitrification method. In this case, as a cell cryopreservation solution, CryoDefend-Stem Cell (R & D system company), STEM-CELL BANKER (registered trademark, Nippon Zenyaku Kogyo Co., Ltd.), etc. can be used. The freezing may be performed by liquid nitrogen or by a Peltier device. Use of a Peltier element makes it possible to control temperature change and to suppress temperature unevenness. If the frozen cells are for clinical use, the cryopreservation container is preferably a completely closed system. However, the packaging apparatus 100 may be packaged in a storage container without freezing stem cells.

  Alternatively, in the packaging apparatus 100, the solution of cell mass may be replaced with the cryopreservation solution from the medium using a solution substitution device 101 as shown in FIG. Inside the solution displacing device 101, a filter 102 is provided on the bottom surface with pores through which cell clusters can not pass. In addition, in the solution displacing device 101, a cell mass introduction hole to which a first liquid delivery channel 103 for feeding a medium containing a cell mass is connected on an internal filter 102, and a cell mass on an internal filter 102. A substitution solution introduction hole to which a second liquid transfer flow path 104 for transferring a non-containing freeze liquid is connected, and a first discharge flow path 105 for discharging a frozen liquid containing cell aggregates from the filter 102 inside is connected. Cell mass outlet holes are provided. Furthermore, the solution displacing device 101 is provided with a waste liquid discharge port to which a second discharge flow path 106 for discharging the solution that has permeated the filter 102 is connected. A tube or the like can be used for each of the first liquid transfer flow path 103, the second liquid transfer flow path 104, the first discharge flow path 105, and the second discharge flow path 106.

  First, as shown in FIGS. 48 (a) and 48 (b), with the flow of the solution in the second discharge channel 106 stopped, the inside of the solution displacing device 101 from the first liquid delivery channel 103 is started. Add the medium containing the cell mass. Next, as shown in FIG. 48 (c), the flow of the solution in the second discharge channel 106 is allowed, and the culture medium is discharged from the solution displacement device 101. At this time, as shown in FIG. 48 (d), cell masses remain on the filter 102. Furthermore, as shown in FIGS. 48E and 48F, with the flow of the solution in the second discharge channel 106 stopped, the inside of the solution displacing device 101 from the second liquid delivery channel 104 Add the cryopreservation solution, and disperse the cell mass in the cryopreservation solution. Thereafter, as shown in FIG. 48 (g), the cryopreservation solution containing the cell mass is drained from the first drainage channel 105. The cryopreservation solution containing the cell mass is sent to a cryopreservation container or the like via the first discharge channel 105.

  In addition, the solution substitution device 101 shown in FIG. 48 can be used not only for the replacement of the medium with the cryopreservation solution but also for the replacement of the old medium with the new medium. In this case, the second fluid delivery channel 104 delivers fresh media. Alternatively, the solution displacing device 101 can also be used to replace the medium with a solution containing a cell mass splitting enzyme when dividing cell masses. Examples of cell mass dividing enzymes include trypsin and trypsin alternative recombinant enzymes such as TrypLE Select (registered trademark, Life Technologies). In this case, the second delivery channel 104 delivers a solution containing a cell mass dividing enzyme.

  The cell processing system may further include a package process imaging device for imaging the package process in the packaging device 100 shown in FIG.

  The cell processing system includes the operation records of the separation device 10, the cell delivery channel 20 before introduction, the induction factor delivery mechanism 21, the factor delivery device 30, the cell mass preparation device 40, the package device 100, etc., and the imaging device The image may be transmitted to an external server by wire or wirelessly. Also, in an external server, conditions such as induction factor introduction conditions, culture conditions, and freezing conditions, neural networks, incomplete initialization of stem cells, failure of stem cell differentiation and proliferation, and chromosomal abnormalities etc. The relationship between the result and the result may be analyzed, the conditions leading to the result may be extracted, or the result may be predicted. Furthermore, the external server controls the separation device 10, the induction factor delivery mechanism 21, the factor introduction device 30, the cell mass production device 40, the package device 100, etc. of the cell processing system based on the standard operation procedure (SOP). It may be monitored whether each device is operating based on SOP, and operation records of each device may be generated automatically.

  According to the cell processing system described above, it is possible to perform induction, establishment, expansion culture, and cryopreservation of stem cells such as iPS cells at once in a fully automatic manner.

  In addition, the cell processing apparatus with which the cell processing system which concerns on embodiment is equipped is not limited to the structure shown in FIG. For example, in the cell processing apparatus shown in FIG. 49, blood is sent from the blood storage unit 201 to the mononuclear cell separation unit 203 via the blood delivery channel 202. As the blood storage unit 201 and the mononuclear cell separation unit 203, for example, a tube can be used. The blood delivery channel 202 is, for example, a resin tube, a silicon tube, or the like. The same applies to the other liquid feed paths described later. The blood storage unit 201 may be provided with an identifier such as a bar code to manage blood information. The pump 204 is used for liquid feeding. As the pump 204, 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. Examples of tubing pumps include Perister Pump (registered trademark, Atto Co., Ltd.), and RP-Q1 and RP-TX (Takasago Electric Co., Ltd.). Examples of piezoelectric pumps include SDMP 304, SDP 306, SDM 320, and APP-20KG (Takasago Electric Co., Ltd.). In addition, a microfluidic chip module (Takasago Electric Industry Co., Ltd.) in which various types of pumps are combined may be used. By using a closed pump such as Perister Pump (registered trademark), a tubing pump, and a diaphragm pump, it is possible to pump blood without direct contact with the blood inside the blood delivery channel 202. The same applies to the other pumps described later. Alternatively, a syringe pump may be used as the pump 204 and the pump 207, the pump 216, the pump 222, the pump 225, the pump 234, the pump 242, and the pump 252 described later. Even pumps other than sealed pumps can be reused by heat sterilization and the like.

  An erythrocyte coagulating agent is sent to the mononuclear cell separation unit 203 from the separation agent storage unit 205 via the liquid feeding path 206 and the pump 207. For example, a tube can be used as the separating agent storage unit 205. An identifier such as a barcode may be attached to the separating agent storage unit 205 to manage information of the separating agent. As the erythrocyte coagulating agent, for example, HetaSep (registered trademark, STEMCELL Technologies) or an erythrocyte coagulating agent (Nipro) can be used. In the mononuclear cell separation unit 203, red blood cells are sedimented by the red blood cell coagulant, and mononuclear cells are separated. The supernatant containing the mononuclear cells in the mononuclear cell separation unit 203 is sent to the mononuclear cell purification filter 210 via the mononuclear cell liquid flow path 208 and the pump 209. In the mononuclear cell purification filter 210, components other than mononuclear cells are removed to obtain a solution containing mononuclear cells. As the mononuclear cell purification filter 210, Purecell (registered trademark, PALL), Cell soba E (Asahi Kasei Corporation), Sepacel PL (Asahi Kasei Corporation), Adacolumn (registered trademark, JIMRO), and separation bag (Nipro Corporation) Etc. can be used.

  In FIG. 49, the mononuclear cell separation unit 203, the separating agent storage unit 205, the mononuclear cell purification filter 210, the pumps 204, 207, 209, and the like constitute a separation device.

  The solution containing mononuclear cells is sent to the factor introduction unit 213 via the cell delivery channel 211 before introduction and the pump 212. For example, a tube can be used as the factor introducing unit 213. The pluripotency inducer is sent to the factor introducing unit 213 from the factor storage unit 214 including the pluripotency inducer via the factor liquid flow path 215 and the pump 216. For example, a tube can be used as the factor storage unit 214. The factor storage unit 214 may be provided with an identifier such as a barcode to manage information on the pluripotency inducing factor. The factor storage unit 214, the pump 216, and the like constitute an induction factor delivery mechanism. In the factor introduction unit 213 as a factor introduction device, a pluripotency inducer is introduced into cells by, for example, an RNA lipofection method to produce inducer-introduced cells. However, the method of transfection of the inducer is not limited to the RNA lipofection method. For example, Sendai virus vectors containing pluripotency inducer may be used. Alternatively, the pluripotency inducer may be a protein.

  The inducer-introduced cells are sent to the reprogramming incubator 219 as a part of the cell mass production apparatus through the introduced cell delivery channel 217 and the pump 218. The introduced cell delivery channel 217 is, for example, temperature permeable and carbon dioxide permeable. As the reprogramming incubator 219, a floating incubator shown in FIG. 20 may be used. In this case, inducer-transduced cells are placed in dialysis tubing. From the blood cell culture medium storage unit 220 containing blood cell culture medium for the first few days after reintroduction of pluripotency inducing factor into the reprogramming incubator 219 shown in FIG. The blood cell culture medium is supplemented via The medium delivery channel 221 is, for example, temperature permeable and carbon dioxide permeable. An identifier such as a barcode may be attached to the blood cell culture medium storage unit 220 to manage information of the blood cell culture medium. The blood cell culture medium storage unit 220, the culture medium delivery channel 221, and the pump 222 constitute a culture medium supply device. The pump 222 may continuously supply the blood cell culture medium according to the instruction of the CPU 500 shown in FIG. 26, or may supply the blood cell culture medium at a predetermined timing.

  Thereafter, the stem cell culture medium is replenished from the stem cell culture medium storage unit 223 including the stem cell culture medium to the reprogramming culture incubator 219 shown in FIG. 49 via the culture medium delivery channel 224 and the pump 225. An identifier such as a barcode may be attached to the stem cell medium storage unit 223 to manage information on the stem cell medium. The medium delivery channel 224 is, for example, temperature permeable and carbon dioxide permeable. The stem cell culture medium storage unit 223, the culture medium delivery channel 224, and the pump 225 constitute a culture medium supply device. The pump 225 may continuously replenish the stem cell culture medium according to the instruction of the CPU 500 shown in FIG. 26, or may replenish the stem cell culture medium at a predetermined timing.

  The blood cell culture medium storage unit 220 and the stem cell culture medium storage unit 223 may be cold stored, for example, at a low temperature such as 4 ° C. in the cold storage unit 260. The culture medium sent from the blood cell culture medium storage unit 220 and the stem cell culture medium storage unit 223 may be sent to the incubator after the temperature is raised to 37 ° C. by a heater outside the cold storage unit 260, for example. Alternatively, the temperature of the surrounding medium of the liquid-feeding channel may be set so that the temperature of the culture medium which has been cryopreserved is raised to 37 ° C. while advancing the liquid-feeding channel. The aged culture medium in the reprogramming incubator 219 is sent to the waste fluid storage section 228 via the waste fluid delivery channel 226 and the pump 227. An identifier such as a bar code may be attached to the waste liquid storage unit 228 to manage information on the waste liquid.

  The cell mass cultured in the reprogramming culture vessel 219 is transferred to the first expansion incubator 232 as a part of the cell mass production apparatus through the introduced cell delivery channel 229, the pump 230, and the cell mass divider 231. Sent. The cell mass divider 231 may have, for example, the configuration shown in FIG. 45 or FIG. By passing through the cell mass divider 231, the cell mass is divided into smaller cell masses. As the first expansion incubator 232 shown in FIG. 49, a floating incubator shown in FIG. 20 may be used. In this case, the cell mass is placed in a dialysis tube. The stem cell culture medium is replenished to the first expansion incubator 232 shown in FIG. 49 from the stem cell culture medium storage unit 223 including the stem cell culture medium via the culture medium delivery channel 233 and the pump 234. The introduced cell delivery channel 229 and the medium delivery channel 233 are, for example, temperature permeable and carbon dioxide permeable. The stem cell medium storage unit 223, the medium delivery path 233, and the pump 234 constitute a medium supply device. The pump 234 may continuously replenish the stem cell culture medium according to the instruction of the CPU 500 shown in FIG. 26, or may replenish the stem cell culture medium at a predetermined timing.

  The aged culture medium in the first expansion incubator 232 shown in FIG. 49 is sent to the waste fluid storage section 228 via the waste fluid delivery channel 235 and the pump 236.

  The cell mass cultured in the first expansion incubator 232 passes through the introduced cell feed path 237, the pump 238, and the cell mass divider 239 to the second expansion incubator 240 as a part of the cell mass production apparatus. Sent. The cell mass divider 239 may have, for example, the configuration shown in FIG. 45 or FIG. By passing through cell mass divider 239, the cell mass is divided into smaller cell masses. As the second expansion incubator 240 shown in FIG. 49, a floating incubator shown in FIG. 20 may be used. In this case, the cell mass is placed in a dialysis tube. In the second expansion incubator 240 shown in FIG. 49, the stem cell culture medium is replenished from the stem cell culture medium storage unit 223 including the stem cell culture medium via the culture medium delivery channel 241 and the pump 242. The introduced cell delivery line 237 and the medium delivery line 241 are, for example, temperature permeable and carbon dioxide permeable. The stem cell culture medium storage unit 223, the culture medium delivery channel 241, and the pump 242 constitute a culture medium replenishment device. The pump 242 may continuously replenish the stem cell culture medium according to the instruction of the CPU 500 shown in FIG. 26, or may replenish the stem cell culture medium at a predetermined timing.

  The aged culture medium in the second expansion incubator 240 shown in FIG. 49 is sent to the waste fluid storage section 228 via the waste fluid delivery channel 243 and the pump 244.

  The cell mass cultured in the second expansion culture device 240 is sent to the solution substitution device 247 through the introduced cell delivery channel 245 and the pump 246. The solution displacer 247 may have, for example, the configuration shown in FIG. In the solution replacer 247 shown in FIG. 49, the cell mass is held by the filter, and the culture medium is sent to the waste fluid storage section 228 via the waste fluid delivery channel 248 and the pump 249.

  After stopping the flow of the solution in the waste liquid feed path 248 by stopping the operation of the pump 249 or after closing the waste liquid feed path 248 with a valve or the like, the solution displacing unit 247 contains a frozen stock solution containing a frozen stock solution. From the storage unit 250, a cryopreservation solution is introduced via the liquid delivery path 251 and the pump 252. This disperses the cell mass in the cryopreservation solution.

  The cryopreservation solution in which the cell mass is dispersed is sent into the cryopreservation container 255 via the liquid feed path 253 and the pump 254 as a part of the package device. The cryopreservation container 255 is stored in the low temperature storage 256. For example, liquid nitrogen at −80 ° C. is sent from the liquid nitrogen storage 257 to the low temperature storage 256 via the liquid feed path 258. Thereby, the cell mass in the cryopreservation container 255 is frozen. However, freezing of the cell mass may not depend on liquid nitrogen. For example, the low temperature storage 256 may be a freezer such as a compression freezer, an absorption freezer, or a Peltier freezer.

  A backflow prevention valve may be provided as appropriate in the above-described liquid feed path. The above-described liquid delivery channel, mononuclear cell separation unit 203, mononuclear cell purification filter 210, factor introduction unit 213, initialization incubator 219, first expansion incubator 232, second expansion incubator 240, and solution replacement The vessel 247 or the like is disposed, for example, on the member 259. The material of the member 259 is, for example, a resin, but is not limited thereto. The member 259 is made of, for example, a sterilizable heat resistant material. The shape of the member 259 may be plate-like or cassette-like. Also, the member 259 may be a flexible bag. The liquid flow path through which the culture medium flows is made of, for example, a carbon dioxide permeable material. In addition, the above-mentioned liquid flow path, mononuclear cell separation unit 203, mononuclear cell purification filter 210, factor introduction unit 213, reprogramming incubator 219, first expansion incubator 232, second expansion incubator 240, and The solution displacing device 247 or the like may be divided into a plurality of cases and stored.

  The member 259 and the housing 601 illustrated in FIG. 1 include, for example, fitting portions that fit together. Therefore, the member 259 shown in FIG. 49 is disposed at a predetermined position in the housing 601 shown in FIG. In the housing 601, the pump shown in FIG. 49, the blood storage unit 201, the separating agent storage unit 205, the factor storage unit 214, the blood cell culture medium storage unit 220, the stem cell culture medium storage unit 223, the waste liquid storage unit 228, cryopreservation The container 255, the low temperature storage 256, and the liquid nitrogen storage 257 are placed at predetermined positions. When the member 259 is disposed at a predetermined position in the housing 601 shown in FIG. 1, the liquid delivery path in the member 259 shown in FIG. 49 is a pump, a blood storage unit 201, a separating agent storage unit 205, and a factor storage unit. 214, a blood cell culture medium storage unit 220, a stem cell culture medium storage unit 223, a waste liquid storage unit 228, a cryopreservation container 255, a low temperature storage 256, and a liquid nitrogen storage 257;

  For example, the members 259 and the channels provided in the members 259 are disposable, and may be discarded and replaced with new ones after freezing of the cell mass is completed. Alternatively, in the case where the member 259 and the channel provided in the member 259 are reused, an identifier such as a bar code may be attached to the member 259 to manage the number of times of use and the like.

  According to the cell processing system according to the embodiment described above, it is possible to automatically process frozen preservation products of stem cells such as iPS cells from blood without human intervention.

  Also, for example, as shown in FIGS. 50, 51 and 52, the cell processing apparatus includes a cell culture device 900, an embedding member 2000 for embedding the cell culture device 900, and the outside of the embedding member 2000 and the embedding member. And a fluid communication channel for communicating with the cell culture device 900 in the filling member 2000. In addition, in FIG. 51 and FIG. 52, the embedding member 2000 is abbreviate | omitted.

  As a material of the embedding member 2000, resin, glass, metal and the like can be used. While the embedding member 2000 is transparent, it can observe the cell culture device 900 disposed inside the embedding member 2000, but may be opaque.

  For example, in the cell processing apparatus, the blood storage channel 1201 disposed outside the embedding member 2000 to the mononuclear cell separation unit 1203 disposed inside the embedding member 2000 from the blood storage unit 1201 for storing blood. Blood is delivered via 1202. As the blood storage unit 1201 and the mononuclear cell separation unit 1203, for example, a tube can be used. The blood delivery channel 1202 is, for example, a resin tube or a silicon tube. The same applies to the other liquid feed paths described later. The blood storage unit 1201 may be provided with an identifier such as a bar code to manage blood information.

  For liquid transfer, a driving unit 1204 such as a pump that can be attached to the outer wall of the embedding member 2000 is used. As the drive unit 1204, 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. Examples of tubing pumps include Perister Pump (registered trademark, Atto Co., Ltd.), and RP-Q1 and RP-TX (Takasago Electric Co., Ltd.). Examples of piezoelectric pumps include SDMP 304, SDP 306, SDM 320, and APP-20KG (Takasago Electric Co., Ltd.). In addition, a microfluidic chip module (Takasago Electric Industry Co., Ltd.) in which various types of pumps are combined may be used. By using a sealed pump such as Perister Pump (registered trademark), a tubing pump, and a diaphragm pump, it is possible to pump blood without direct contact with the blood inside the blood delivery channel 1202. The same applies to the other pumps described later. Alternatively, a syringe pump may be used as the drive unit 1204 and a drive unit described later. Even pumps other than sealed pumps can be reused by heat sterilization and the like.

  For example, as shown in FIGS. 53 and 54, a driven unit 2204 to which the driving force is transmitted from the drive unit 1204 is provided inside the embedding member 2000, and the driven unit 2204 is connected to the blood supply path 1202. It may be connected. The drive unit 1204 and the follower unit 2204 may be connected by magnetic force. The drive unit 1204 may include an electromagnet that generates a magnetic force. The same applies to the driving units other than the driving unit 1204.

  In the mononuclear cell separation unit 1203 disposed in the interior of the embedding member 2000 shown in FIGS. 50, 51 and 52, the separation agent supply channel from the separation agent storage unit 1205 disposed outside the embedding member 2000. A separating agent for separating mononuclear cells such as a red blood cell coagulant is sent through a drive unit 1207 that can be attached to the outer wall of the embedding member 2000 and 1206. For example, a tube can be used as the separating agent storage unit 1205. An identifier such as a barcode may be attached to the separating agent storage unit 1205 to manage information of the separating agent. As the erythrocyte coagulating agent, for example, HetaSep (registered trademark, STEMCELL Technologies) or an erythrocyte coagulating agent (Nipro) can be used.

  In the mononuclear cell separation unit 1203, red blood cells are sedimented by the red blood cell coagulant, and mononuclear cells are separated. The supernatant containing the mononuclear cells in the mononuclear cell separation unit 1203 is supplied via the mononuclear cell liquid flow path 1208 disposed inside the embedding member 2000 and the driving unit 1209 attachable to the outer wall of the embedding member 2000, It is sent to a mononuclear cell purification filter 1210. Sedimented components and the like generated in the mononuclear cell separation unit 1203 are sent to a waste liquid storage unit 4228 disposed outside the embedding member 2000 via a waste liquid supply passage communicating with the outside from the embedding member 2000. .

  In the mononuclear cell purification filter 1210, components other than mononuclear cells are removed to obtain a solution containing mononuclear cells. As the mononuclear cell purification filter 1210, Purecell (registered trademark, PALL), Cell soba E (Asahi Kasei Corporation), Sepacel PL (Asahi Kasei Corporation), Adacolumn (registered trademark, JIMRO), and separation bag (Nipro Corporation) Etc. can be used.

  In FIG. 52, the mononuclear cell separation unit 1203, the separating agent storage unit 1205, the mononuclear cell purification filter 1210, the drive units 1204, 1207, 1209 and the like constitute a separation device. The solution containing mononuclear cells separated by the mononuclear cell purification filter 1210 is passed through a mononuclear cell channel 2001 disposed inside the embedding member 2000 connected to the mononuclear cell purification filter 1210 to obtain a mononuclear cell. It may be sent to a mononuclear cell storage unit 2002 disposed inside the embedding member 2000 connected to the ball flow channel 2001. The solution containing components other than mononuclear cells separated by the mononuclear cell purification filter 1210 passes through the other component flow path 2003 disposed inside the embedding member 2000 connected to the mononuclear cell purification filter 1210, It may be sent to the other component storage unit 2004 disposed inside the embedding member 2000, which is connected to the other component flow path 2003.

  The solution containing mononuclear cells is disposed in the interior of the embedding member 2000 via the pre-introduction cell liquid flow path 1211 disposed inside the embedding member 2000 and the drive unit 1212 attachable to the outer wall of the embedding member 2000. It is sent to the factor introduction unit 1213 that has been The mononuclear cells separated by the mononuclear cell separation unit 1203 may be sent to the factor introduction unit 1213 without passing through the mononuclear cell purification filter 1210. For example, a tube can be used as the factor introducing unit 1213. In the factor introducing unit 1213, a factor storage path 1215 at least a part of which is provided inside the embedding member 2000 from a factor storage unit 1214 including a pluripotency inducing factor, which is disposed outside the embedding member 2000. The pluripotency inducer is sent via the drive unit 1216 disposed outside the embedding member 2000. For example, a tube can be used as the factor storage unit 1214. The factor storage unit 1214 may be provided with an identifier such as a barcode to manage information on the pluripotency inducing factor. The factor storage unit 1214, the drive unit 1216, and the like constitute an induction factor liquid transfer mechanism.

  In the factor introducing unit 1213 as at least a part of the factor introducing device, a pluripotency inducer is introduced into a cell by, for example, an RNA lipofection method, and an inducer-transduced cell is produced. However, the method of transfection of the inducer is not limited to the RNA lipofection method. For example, Sendai virus vectors containing pluripotency inducer may be used. Alternatively, the pluripotency inducer may be a protein. In addition, transfection methods include transfection using an episomal plasmid, and electroporation. The waste fluid generated in the factor introducing unit 1213 is sent to a waste fluid storage unit 5228 disposed outside the embedding member 2000 via a waste solution delivery path communicating with the outside of the embedding member 2000.

  The inducer-introduced cells can be used as part of the cell mass production apparatus through the introduced cell delivery channel 1217 provided inside the embedding member 2000 and the drive unit 1218 disposed outside the embedding member 2000. It is sent to the reprogramming incubator 1219. The reprogramming incubator 1219 is embedded in the embedding member 2000. As the reprogramming incubator 1219, a floating incubator shown in FIG. 20 may be used. In this case, inducer-transduced cells are placed in dialysis tubing. In the reprogramming incubator 1219 shown in FIG. 52, a blood cell culture medium storage unit 1220 including a blood cell culture medium disposed outside the embedding member 2000 for the first few days after the pluripotency inducing factor is introduced into the cells. Then, the blood cell culture medium is replenished via the medium delivery channel 1221 at least a part of which is provided inside the embedding member 2000, and the driving unit 1254 disposed outside the embedding member 2000. An identifier such as a barcode may be attached to the blood cell culture medium storage unit 1220 to manage information of the blood cell culture medium. The blood cell culture medium storage unit 1220, the culture medium delivery channel 1221, and the drive unit 1254 constitute a culture medium supply device. The driving unit 1254 may continuously supply the blood cell culture medium according to the instruction of the CPU 500 shown in FIG. 26, or may supply the blood cell culture medium at a predetermined timing.

  Thereafter, in the reprogramming incubator 1219 shown in FIG. 52, a medium delivery channel provided in the interior of the embedding member 2000 from the stem cell medium storage unit 1223 including the stem cell medium disposed outside the embedding member 2000, The stem cell culture medium is replenished via a driving unit disposed outside the embedding member 2000. An identifier such as a barcode may be attached to the stem cell medium storage unit 1223 to manage information on the stem cell medium. The stem cell culture medium storage unit 1223, the culture medium delivery channel, and the drive unit constitute a culture medium supply device. The driving unit may continuously replenish the stem cell culture medium according to the instruction of the CPU 500 shown in FIG. 26, or may replenish the stem cell culture medium at a predetermined timing.

  The blood cell culture medium storage unit 1220 and the stem cell culture medium storage unit 1223 may be cold stored, for example, at a low temperature such as 4 ° C. in the cold storage unit. The culture medium sent from the blood cell culture medium storage unit 1220 and the stem cell culture medium storage unit 1223 may be sent to the incubator after being heated to 37 ° C. by a heater outside the cold storage unit, for example. Alternatively, the temperature of the surrounding medium of the liquid-feeding channel may be set so that the temperature of the culture medium which has been cryopreserved is raised to 37 ° C. while advancing the liquid-feeding channel. The aged culture medium in the initialization incubator 1219 is sent to the waste fluid storage 1228 disposed outside the embedding member 2000 through the waste liquid delivery channel 1226 communicating with the outside from the embedding member 2000. . An identifier such as a bar code may be attached to the waste liquid storage unit 1228 to manage information on the waste liquid.

  The cell mass cultured in the reprogramming incubator 1219 is introduced into a cell transfer channel 1229 provided inside the embedding member 2000, a drive unit 1230 disposed outside the embedding member 2000, and the inside of the embedding member 2000. Of the cell mass producing apparatus through the cell mass divider 1231 provided in the drive unit, the drive unit 1218 disposed outside the embedding member 2000, and the introduced cell liquid feeding path 2229 provided inside the embedding member 2000. It is sent to an expansion incubator 1232 as a part. The cell mass divider 1231 provided inside the embedding member 2000 may have, for example, the configuration shown in FIG. 45 or 46. By passing through the cell mass divider 1231, the cell mass is divided into smaller cell masses.

  As the expansion incubator 1232 shown in FIG. 52, a floating incubator shown in FIG. 20 may be used. In this case, the cell mass is placed in a dialysis tube. In the expansion incubator 1232 shown in FIG. 52, a medium delivery channel 1224, which communicates with the outside of the embedding member 2000 from the stem cell medium storage unit 1223 including the stem cell medium, which is disposed outside the embedding member 2000. The stem cell culture medium is replenished via a driving unit 1222 disposed outside the embedding member 2000. The stem cell culture medium storage unit 1223, the culture medium delivery channel 1224, and the drive unit 1222 constitute a culture medium supply device. The driving unit may continuously replenish the stem cell culture medium according to the instruction of the CPU 500 shown in FIG. 26, or may replenish the stem cell culture medium at a predetermined timing.

  The aged culture medium in the expansion incubator 1232 shown in FIG. 52 is disposed to the outside of the embedding member 2000 via the drainage liquid feeding path 1235 which communicates with the inside of the embedding member 2000. Sent to

  The cell mass cultured in the expansion incubator 1232 is provided in a cell transfer channel 1245 provided inside the embedding member 2000, a drive unit 1246 attachable to the outer wall of the embedding member 2000, and the embedding member 2000. It is sent to a solution displacing device 1247 provided inside the embedding member 2000 via the cell mass divider 2231. The solution displacer 1247 may have, for example, the configuration shown in FIG. In the solution replacer 1247 shown in FIG. 52, the cell mass is held by the filter, and the culture medium is disposed outside the embedding member 2000 via the waste liquid delivery channel 1248 that communicates with the inside of the embedding member 2000. It is sent to the waste storage section 3228.

  After closing the waste liquid delivery channel 1248 with a valve or the like, the solution displacing unit 1247 is disposed outside the embedding member 2000, from the cryopreservation solution storage unit 1250 containing the cryopreservation solution, outside the embedding member 2000. The cryopreservation fluid is introduced via the cryopreservation fluid delivery channel 1251 and the drive unit 1252 attachable to the outer wall of the embedding member 2000, which are in communication with each other. This disperses the cell mass in the cryopreservation solution.

  The cryopreservation solution in which the cell mass is dispersed is transferred to the cryopreservation container 1255 via the cell delivery channel for freezing 1253 communicating with the inside of the embedding member 2000 and the driving unit 1209 attachable to the outer wall of the embedding member 2000. Sent inside. The liquid delivery path 1253, the drive unit 1254, and the cryopreservation container 1255 form a part of the package device. The cryopreservation container 1255 is then transferred into a cryogenic storage.

  In the cell processing apparatus shown in FIGS. 50 to 52, after connecting the factor introducing unit 1213, the reprogramming incubator 1219, the expansion incubator 1232 and the like included in the above-mentioned cell culture instrument 900, they are embedded, The embedding member 2000 is shaped and processed. Alternatively, the cell processing apparatus may form the factor introducing unit 1213, the reprogramming incubator 1219, the expansion incubator 1232, and the like provided in the cell culture instrument 900 inside the embedding member 2000 by an apparatus such as a 3D printer. . The factor introduction unit 1213, the reprogramming incubator 1219, the expansion incubator 1232 and the like included in the cell culture device 900 are embedded in the embedding member 2000, and the induction is performed on the cells being processed and the cells. It becomes possible to suppress the diffusion of factors and the like to the outside. Also, conversely, it is possible to inhibit external contaminants from coming into contact with the cells being treated.

  As mentioned above, although the present invention has been described by the embodiments, it should not be understood that the description and the drawings which form a part of this disclosure limit the present invention. Various alternative embodiments, embodiments and operation techniques should be apparent to those skilled in the art from this disclosure. For example, the factor introduction device 30 may induce cells by transfection using a virus vector such as retrovirus, lentivirus, and Sendai virus, plasmid, or protein transfection instead of electroporation or RNA lipofection. Good. Alternatively, cells may be induced by the introduction of a compound as a factor. The cell delivery channel 20 before introduction, the introduced cell delivery channel 31, the cell mass delivery channel 51, the expansion culture delivery channel 71, the cell mass delivery channel 72, and the prepackaged cell channel 91 are substrates by the microfluidics technology. It may be provided on top. As such, it should be understood that the present invention includes various embodiments and the like not described herein.

Example 1
(Preparation)
Human blood cells were obtained from healthy adult men. In addition, modified mRNA (TriLink), non-adhesion dish, 15 mL tube, 50 mL tube, Ficoll, cytoflow meter (BD), antibody against CD34 (Miltenyi Biotec), antibody against CD3 (Miltenyi Biotec), MACS (registered trademark) Buffer (Miltenyi Biotec), T cell culture medium, low serum culture medium (Opti-MEM®, Gibco), siRNA transfection reagent (Lipofectamine® RNAiMAX, ThermoFisher Science), and antibody to TRA-1-60 (BD) Prepared.

  The T cell (CD3 positive cells) medium was a mixture of A medium and B medium below. A medium was a mixture of 15 mL of X vivo-10 (Lonza, 04-743Q) and IL-2 (10 μg / mL). B medium is mixed with 50 μL of X vivo-10 and Dynabeads CD3 / CD28 (Life Technologies, 111-31D) in a 1.5 mL tube, vortexed for 5 seconds, spun down, and allowed to stand in DynaMag-2 (Thermo fisher Science) After standing for one minute, the supernatant was removed.

  Also, 10 μL of IL-6 (100 μg / mL), 10 μL of SCF (300 μg / mL), 10 μL of TPO (300 μg / mL), 10 μL of Flt3 ligand (300 μg) in 10 mL of serum-free medium (StemSpan H3000, STEMCELL Technologies) Blood cell culture medium (blood stem / progenitor cell culture medium) was prepared by adding 10 μL of IL-3 (10 μg / mL) and

  In addition, OCT3 / 4 mRNA containing solution, SOX2 mRNA containing solution, KLF4 mRNA containing solution, c-MYC mRNA containing solution, LIN28A mRNA containing solution, and green fluorescent protein (GFP) each having a concentration of 100 ng / μL, respectively. MRNA containing solution was prepared. Next, 385 μL of OCT3 / 4 mRNA containing solution, 119 μL of SOX2 mRNA containing solution, 156 μL of KLF4 mRNA containing solution, 148 μL of c-MYC mRNA containing solution, 83 μL of LIN28A mRNA containing solution, and 110 μL of The mRNA-containing solution of GFP was mixed to obtain a reprogramming factor mixed solution. The resulting reprogramming factor mixture was dispensed in 50 μL portions into 1.5 mL RNase-Free tubes (Eppendorf Tube®, Eppendorf) and stored in a −80 ° C. freezer.

(Preparation of mononuclear cells)
The centrifuge was set to 18 ° C. Blood was drawn from 5 to 50 mL and EDTA was added to the blood and mixed gently. In addition, 5 mL of a medium for human lymphocyte separation (Ficoll-Paque PREMIUM, GE Healthcare Japan) was dispensed into two 15 mL tubes. 5 mL of PBS was added to the blood to dilute, and then 5 mL was overlaid on the medium for human lymphocyte separation in a tube. At this time, diluted blood was slowly added to the medium along the tube wall so as not to disturb the interface.

  The solution in the tube was centrifuged at 400 × g at 18 ° C. for 30 minutes. At this time, both acceleration and deceleration were performed slowly. After centrifugation, a white cloudy intermediate layer appeared in the tube. This white cloudy intermediate layer contains mononuclear cells. The white cloudy intermediate layer in the tube was slowly collected with a pipetman and transferred to a fresh 15 mL tube. At this time, the lower layer was kept from sucking up. About 1 mL of the cloudy white intermediate layer could be collected from one tube. The two middle layers were put together and transferred to one tube.

  To the collected mononuclear cells, 12 mL PBS was added, and the solution was further centrifuged at 200 × g and 18 ° C. for 10 minutes. Thereafter, the supernatant of the solution is aspirated and removed using an aspirator, and 3 mL of a chemically-defined serum-free hematopoietic cell culture medium (X-VIVO (registered trademark) 10, Lonza) is added and suspended to give a mononuclear cell suspension. I got Among them, 10 μL of mononuclear cell suspension was stained with trypan blue and counted with a hemocytometer.

(Separation of CD34 or CD3 positive cells)
1 × 10 ⁷ mononuclear cells and CD34 antibody or CD3 antibody were reacted in 100 μL of a 4 ° C. solution for 15 minutes. After the reaction, 5 mL of MACS (registered trademark) buffer (Miltenyi Biotec) was added to the solution and centrifuged at 270 g. After centrifugation, the supernatant was removed and 1 mL of MACS buffer was added. Then, using a separation program of an automatic magnetic cell separation device (autoMACS, Miltenyi Biotec), CD34 positive cells or CD3 positive cells were separated among mononuclear cells.

(Culture of separated cells)
The isolated 5 × 10 ⁶ mononuclear cells were suspended in 1 mL of T cell culture medium or blood stem / progenitor cell culture medium, seeded in a 12-well plate and cultured. The culture conditions were 5% CO ₂ concentration, 19% oxygen concentration, and 37 ° C. temperature.

(Lipofection of reprogramming factor)
100 μL of low serum medium (Opti-MEM (registered trademark), Gibco) and 25 μL of the reprogramming factor mixture were mixed to form a first mixture. In addition, 112.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco) and 12.5 μL of siRNA introduction reagent (Lipofectamine (registered trademark) RNAiMAX, ThermoFisher Science) were mixed, and used as a second mixed solution. Thereafter, the first mixed solution and the second mixed solution were mixed, and allowed to stand at room temperature for 15 minutes to make a lipofection reaction solution.

Of the resulting lipofection reaction solution, 60 μL was gently added to a 12-well plate in which mononuclear cells were cultured, and subsequently mononuclear cells were cultured feeder-free for 18 hours at 37 ° C. The culture conditions were 5% CO ₂ concentration, 19% oxygen concentration, and 37 ° C. temperature. The density of mononuclear cells when the lipofection reaction solution was added was 3 × 10 ⁶ . After 18 hours, mononuclear cells were collected in a 15 mL tube, centrifuged at 300 g, and the supernatant was removed. The mononuclear cell suspension was then returned to the same 12-well plate by adding 1.25 mL of CD34 hemocyte culture medium to a 15 mL tube, and mononuclear cells were cultured feeder-free at 37 degrees overnight. The culture conditions were 5% CO ₂ and 19% oxygen. The above steps were repeated once every two days for seven days.

(Confirmation of GFP expression)
On the seventh day after the start of lipofection, the density of cells subjected to a total of 4 times of lipofection was 3 × 10 ⁶ . When a part of the cells were removed from the 12-well plate and GFP expression was confirmed by a fluorescence microscope, as shown in FIG. 55, GFP expression was confirmed. This confirmed that mRNA was transfected into mononuclear cells, and that protein was synthesized from the transfected mRNA.

(Confirmation of TRA-1-60 expression)
Seven days after the start of lipofection, a part of the cells was removed from the 12-well plate, and the removed cells were against TRA-1-60, a surface antigen specifically expressed in iPS cells that began to be reprogrammed. The antibody was stained with Allophycocyanin (APC) fluorescent dye-labeled antibody. After that, by confirming the proportion of TRA-1-60 positive cells with fluorescence activated cell sorter (FACS (registered trademark), BD), reprogramming is started in the cells, iPS cell gene is expressed, and iPS cells are I confirmed that I could do it.

  As shown in FIG. 56, a dot plot was created in which the intensity of autofluorescence was taken on the x-axis and the fluorescence intensity of the fluorescently labeled anti-TRA-1-60 antibody on the y-axis. TRA-1-60 positive cells were not detected in the negative control where no gene was introduced. In contrast, TRA-1-60 positive cells were detected in experiments 1, 2 and 3. In Experiment 1, the results were derived from whole mononuclear cells not separated by the marker, and Experiment 2 was the results derived from cells separated by CD3 positive, and Experiment 3 was separated as CD34 positive. It is the result derived from cells. Therefore, it has been shown that lipofection of reprogramming factor RNA can be used to introduce reprogramming factor into blood-derived cells to induce iPS cells.

(Example 2)
500 mL of Primate ES Cell Medium (ReproCELL) and 0.2 mL of bFGF (Gibco PHG0266) at a concentration of 10 μg / mL were mixed to prepare bFGF-containing human iPS medium.

  In addition, deacylated gellan gum (Nissan Chemical) was added to bFGF-containing human iPS medium to a concentration of 0.02% by weight to prepare bFGF-containing human iPS gel medium. Furthermore, 5 mL of trypsin at a concentration of 2.5 wt%, 5 mL of collagenase IV at a concentration of 1 mg / mL, 0.5 mL of a concentration of 0.1 mol / L of CaCl2, 10 mL of KnockOut serum substitute (registered trademark, Invitrogen 10828) -028) and 30 mL of purified water were mixed to prepare a stripping solution generally called CTK solution.

300 μL of CTK solution was added to a 6-well dish (Thermoscientific 12-556-004) in which iPS cells are cultured on feeder cells, and incubated for 3 minutes in a CO ₂ incubator. After 3 minutes, the dish was removed from the incubator, and it was confirmed that only the feeder cells were detached, and the CTK solution was removed using an aspirator. After removing the CTK solution, add 500 μL of PBS (Santa Cruz Biotech sc-362183) to a 6-well dish, wash the iPS cells, remove the PBS from the 6-well dish, and remove 0.3 mL of scraping solution (Accutase®) Was added to a 6-well dish, placed in a CO ₂ incubator, and incubated for 5 minutes. Thereafter, 0.7 mL of bFGF-containing iPS medium was added to a 6-well dish to suspend the iPS cells into single cells.

After suspending the iPS cells, 4 mL of bFGF-containing human iPS medium was added to a 15 mL centrifuge tube, and the iPS cell suspension was centrifuged at 270 g using a centrifuge. After centrifugation, the supernatant was removed, 1 mL of bFGF-containing human iPS medium was added to a 15 mL centrifuge tube, and the cell count was calculated using a hemocytometer. After cell counting, 5 × 10 ⁵ cells of iPS cells were seeded in 15 mL falcon tube (registered trademark, Corning 352096) or non-adherent dishes, and then cultured in suspension without agitation.

  In a 15 mL tube, 2 mL of bFGF-containing human iPS gel medium was used. In a non-adherent dish, 2 mL of ungelled bFGF-containing human iPS medium was used. To each culture medium, ROCK inhibitor (Selleck S1049) was added at 10 μmol / L. Thereafter, 500 μL of bFGF-containing human iPS gel medium was added daily to a 15 mL tube and non-adherent dish, and 500 μL of bFGF-containing human iPS medium was added daily to the non-adhered dish. In addition, ROCK inhibitor was added to a 15 mL tube and a non-adherent dish daily to a final concentration of 10 μmol / L, and suspension culture was continued for 7 days.

  The results are shown in FIG. As shown in FIG. 57 (b), when iPS cells were cultured using bFGF-containing human iPS medium which was not gelled in a non-adherent dish, aggregation of iPS cell colonies was remarkably observed. In contrast, as shown in FIG. 57 (a), when iPS cells were cultured in a 15 mL tube using bFGF-containing human iPS gel medium, no noticeable aggregation was observed. Fig. 58 (a) is a photograph of day 1 when iPS cells were cultured using bFGF-containing human iPS gel medium in a 15 mL tube, and Fig. 58 (b) is a human iPS containing bFGF in a 15 mL tube. It is a photograph of the 9th day when iPS cells are cultured using gel culture medium. From the photographs of FIG. 58 (a) and FIG. 58 (b), it was confirmed that iPS cells of different strains did not aggregate and formed colonies.

  FIG. 59 (a) is a photograph immediately before re-seeding a colony of iPS cells suspended and cultured in gel medium for 7 days on feeder cells. FIG. 59 (b) is a photograph showing the morphology of colonies three days after that. As a result, as shown in FIG. 60, it was confirmed that 95% or more of the colonies were undifferentiated. Thus, it was shown that iPS cells can be cultured in gel medium while maintaining an undifferentiated state.

(Example 3)
The same bFGF-containing human iPS medium as in Example 2 and bFGF-containing human iPS gel medium were prepared. 300 μL of CTK solution was added to a 6-well dish in which iPS cells were cultured on feeder cells, and incubated in a CO ₂ incubator for 3 minutes. After 3 minutes, the dish was removed from the incubator, and it was confirmed that only the feeder cells had been detached, and the CTK solution was removed using an aspirator. After removing the CTK solution, 500 μL of PBS was added to the dish, iPS cells were washed, PBS was removed from the dish, 0.3 mL of Accumax was added to the dish, the dish was placed in a CO ₂ incubator, and incubated for 5 minutes . Thereafter, 0.7 mL of bFGF-containing iPS medium was added to the dish, and the iPS cells were suspended until they became single cells.

After suspending the iPS cells, 4 mL of bFGF-containing human iPS medium was added to a 15 mL centrifuge tube, and the iPS cell suspension was centrifuged at 270 g using a centrifuge. After centrifugation, the supernatant was removed, 1 mL of bFGF-containing human iPS medium was added to a 15 mL centrifuge tube, and the cell count was calculated using a hemocytometer. After cell counting, 5 × 10 ⁵ cells of iPS cells were seeded in 15 mL tubes and suspended in culture without agitation.

  In a 15 mL tube, 2 mL of bFGF-containing human iPS gel medium was used. Each medium was added with 10 μmol / L of ROCK inhibitor. Thereafter, 500 μL of bFGF-containing human iPS gel medium was added daily to a 15 mL tube. In addition, 500 μL of gel culture medium contained 0.5 μL of ROCK inhibitor. In addition, as a control, iPS cells were suspended and cultured for 7 days under the same conditions except that the ROCK inhibitor was not added.

  As shown in FIG. 61 (a), iPS cells did not form colonies when the ROCK inhibitor was not added to the bFGF-containing human iPS medium. On the other hand, as shown in FIG. 61 (b), when a ROCK inhibitor was added to bFGF-containing human iPS medium, iPS cells formed colonies. From this result, it was shown that the ROCK inhibitor is effective when the iPS cells are suspended and cultured from a single cell.

(Example 4)
Using a Falcon conical tube 50 mL (registered trademark) which is a non-carbon dioxide permeable container and a G-Rex (registered trademark, Wilson Wolf) which is a carbon dioxide permeable container as a container for storing a dialysis tube, The cells were suspended and cultured under the same conditions except for the container. As a result, as shown in FIG. 62, cell viability was higher when cultured using a carbon dioxide permeable container.

(Example 5)
The gel medium containing iPS cells was placed in each of two dialysis modules (Spectrum G235035) equipped with dialysis tubes with a molecular weight cut off of 100 kDa. Furthermore, the dialysis modules were placed in 50 mL centrifuge tubes, respectively, and the gel medium was placed around the dialysis tubes in the centrifuge tubes. Also, the gel medium containing iPS cells was directly put into another 50 mL centrifuge tube.

  After that, the pump was connected as shown in FIG. 21 to one of the two centrifuge tubes containing a dialysis tube inside for several days, and the gel medium in the centrifuge tube was continuously exchanged. The gel medium was stored at 4 ° C. and set to 37 ° C. when it reached the centrifuge tube. The pump was not connected to the other of the two centrifuge tubes which contained the dialysis tube inside, and the gel medium in the centrifuge tube was not replaced. Also, the gel medium in the centrifuge tube without the dialysis tube inside was not replaced.

  After culturing for the same period, when the cells cultured in each container were observed, as shown in FIGS. 63 and 64, the cell mass was cultured in the dialysis tube, and the gel medium around the dialysis tube was pumped. When replaced continuously, a large number of cell masses were formed. Also, there were very few differentiated cells. However, when the cell mass was cultured in the dialysis tube and the gel medium around the dialysis tube was not continuously replaced with a pump, the cell mass decreased and differentiated cells increased. Also, when the cell mass was cultured without using a dialysis tube and the gel medium was not continuously replaced with a pump, almost no cell mass was formed.

  2 ... tube, 10 ... separation device, 20 ... cell delivery path before introduction, 21 ... induction factor delivery mechanism, 30 ... factor introduction device, 31 ... introduction cell delivery Path 40, cell mass preparation device 50: initialization culture device 51: cell mass feed channel 60: division mechanism 61: end block 61a: concave portion 61b: convex part, 61c: large pore size part, 62: connection block, 62a: concave part, 62b: convex part, 62c: large pore size part, 62d: small pore size part , 62e: large diameter portion, 63: tip block, 63a: concave portion, 63b: nozzle portion, 63c: large diameter portion, 63d: small diameter portion, 64: insertion Nozzle, 65a: large diameter portion, 65b: small diameter portion, 66a: insertion portion, 66b: insertion portion, 70 · · · · · · · · Expansion culture apparatus, 71 · · · · · · · expansion culture fluid delivery channel, · · · · · cell mass delivery channel, 75 · · · · · · · · · · · · · · · · · · · · · · · container, 77 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ... ··· Liquid transfer tube, 79 · · · Waste liquid tube, 80 · · · Split mechanism, 90 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · pre-package cell flow path, 100 · · · package device, 101 · · · · Solution displacing unit · 102 · · · · · · · · · · · · · · · · · · · · solution substitution device, 102 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 103 Storage liquid delivery mechanism, 171 ... initialization culture photographing device, 172 ... telecentric lens, 173 ... illumination light source for cell observation, 174 ... illumination light source for culture medium observation, 201 ... blood storage section , 202 ... blood supply channel, 203 ... mononuclear cell separation unit 204: pump, 205: separating agent storage unit, 206: liquid feed path, 207: pump, 208: mononuclear cell liquid feed path, 209: pump, 210: 210 Monocyte purification filter, 211 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Factor storage portion, 215 · · · · · · · · ·. · · Pumps 217 · · · · · · · · · Introduction cell feeding channel, 218 · · · · · · · · · · · · · · · · · · initialization incubator, 220 · · · blood cell culture medium storage unit, 221 · · · · · · · · · · · Pump · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · stem cell culture medium storage unit 224 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · , 229: introduced cell delivery channel, 230: pump, 231: cell mass Divider, 232 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 2 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Cell mass divider, 240 · · · expansion incubator, 241 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · Pump · · · · · · · · · · · · Introduction cell feeding channel 246 · · · · · · · · 247 · · · solution replacement, 248 · · · waste liquid delivery channel 249 · · · · · · · · · · · · · frozen stock Storage part, 251 ... liquid feeding path, 252 ... pump, 253 ... liquid feeding path, 254 ... pump, 255 ... frozen storage container, 256 ... low temperature storage, 257 ...・ Liquid nitrogen storage, 258 ・ ・ ・ Liquid transfer path, 259 ・ ・ ・ Members, 260 ・ ・ ・ Cold storage Sections 271: sensor 272: thermometer 301: bag 302: bag 401: input device 402: output device 403: relation 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, 601 ... An enclosure, an intake purification filter, an exhaust purification filter, an exhaust device, an exhaust purification filter, an introduction device, an introduction device, and a gas discharge device. 651 ... return member, 652 ... base, 653 ... opening, 654 ... cover, 655 ... opening, 656 ... cover, 701 ... outer case, 702 ... pressure Adjustment hole, 703 ... closed part , 800: shielding member, 801: housing side shielding member, 802: exhaust device side shielding member, 811: guide, 812: guide, 900: cell culture device, 1201. · Blood storage unit, 1202 ··· Blood feeding channel, 1203 · · · mononuclear cell separating unit, 1204 · · · driving unit, 1205 · separating agent storage unit, 1206 · · · separating agent feeding channel, 1207 · · · drive unit, 1208 · · · mononuclear cell liquid flow path, 1210 · · · mononuclear cell purification filter, 1211 · · · · cell liquid flow path before introduction, 1212 · · · drive portion, 1213 · · · Factor introduction part, 1214 ... factor storage part, 1215 ... factor liquid transport path, 1216 ... driver, 1217 ... introduced cell liquid transport path, 1218 ... driver, 1219 ... initial stage Culture incubator, 1220 ... blood cell culture medium storage unit, 12 21 ··· Medium transfer channel, 1222 · · · Drive unit, 1223 · · · stem cell culture medium storage unit, 1224 · · · culture medium transfer channel, 1226 · · · waste liquid transfer channel, 1228 · · · waste storage section , 1229: introduced cell delivery channel, 1230: driving unit, 1231: cell mass divider, 1232: enlargement incubator, 1235: waste liquid delivery channel, 1245: introduced cells Liquid feed path, 1246: drive unit, 1247: solution substitution unit, 1248: waste liquid feed path, 1250: frozen storage liquid storage section, 1251: frozen storage liquid transport path, 1252 ... drive unit, 1253 ... cell delivery channel for freezing, 1253 ... delivery channel, 1254 ... drive unit, 1255 ... cryopreservation container, 2000 ... embedding member, 2001 · · Mononuclear cell channel, 2002 · · · mononuclear cell storage unit, 2003 · · Other component flow channel, 2004 · · · Other component storage unit, 2204 · · · · · follower unit, 2228 · · · waste liquid storage unit, 2229 · · · · Introduction cell flow path, 2231 · · · cell mass divider, 3228 · · · waste liquid storage unit, 4228 · · · waste liquid storage unit, 5228 · · · waste liquid storage unit

Claims (116)

And
An outer housing enclosing the housing;
An intake purification filter provided in the casing for purifying gas sucked from the outside of the casing;
The casing is disposed in the outer casing such that the gas in the outer casing is sucked into the casing through the intake air purification filter and the gas in the casing is discharged into the outer casing. A circulation device that circulates inside and outside the body,
A cell processing device for processing cells, disposed in the housing;
, A cell processing system.   The cell processing system according to claim 1, wherein the outer casing is provided with a pressure control hole.   The cell processing system according to claim 1, wherein the circulation device includes a gas discharge device provided in the housing for sucking a gas from inside the housing and discharging the purified gas to the outside of the housing. The gas discharge device is
An exhaust device for exhausting the gas in the casing to the outside of the casing;
An exhaust gas purification filter for purifying the gas sucked by the exhaust device;
The cell processing system according to claim 3, comprising   The cell processing system according to claim 4, wherein the exhaust gas purification filter is disposed upstream of the exhaust device.   The cell processing system according to claim 5, further comprising a shielding member attachable to the exhaust gas purification filter so as to shield the exhaust gas purification filter. The shielding member is
A housing side shielding member that can be attached to the exhaust gas purification filter so as to shield the exhaust gas purification filter from the inside of the housing;
An exhaust system side shielding member which can be attached to the exhaust gas purification filter so as to shield the exhaust gas purification filter from the exhaust system;
The cell processing system according to claim 6, comprising   The cell processing system according to claim 6, further comprising a sterilizer for sterilizing the exhaust gas purification filter.   The cell processing system according to claim 5, further comprising a second exhaust gas purification filter disposed downstream of the exhaust device.   The cell processing system according to claim 9, further comprising a shielding member attachable to the second exhaust gas purification filter to shield the second exhaust gas purification filter.   The cell processing system according to claim 10, wherein the shielding member comprises an exhaust device side shielding member that can be attached to the second exhaust gas purification filter so as to shield the second exhaust gas purification filter from the exhaust device. .   The cell processing system according to claim 10, further comprising a sterilizer for sterilizing the second exhaust gas purification filter.   The cell processing according to any one of claims 1 to 12, wherein the circulation device includes an introduction device provided in the casing for suctioning the gas purified by the intake air purification filter from outside the casing. system.   The cell processing system according to claim 13, wherein the intake air purification filter is disposed downstream of the introduction device.   15. The cell processing system according to claim 14, further comprising a shielding member attachable to the intake air purification filter to shield the intake air purification filter. The shielding member is
A case-side shielding member that can be attached to the intake air purification filter so as to shield the intake air purification filter from the inside of the case;
An introduction device side shielding member that can be attached to the intake air purification filter so as to shield the intake air purification filter from the introduction device;
The cell processing system according to claim 15, comprising   The cell processing system according to claim 15, further comprising a sterilizer for sterilizing the intake air purification filter.   The cell processing system according to any one of claims 1 to 17, wherein the inside of the case has a negative pressure as compared to the outside of the case.   The cell processing system according to any one of claims 1 to 18, wherein stem cells are cultured in the cell processing apparatus.   The cell processing system according to any one of claims 1 to 19, further comprising a sterilizer for sterilizing a space in the housing in which the cell processing device is disposed.   The cell processing system according to any one of claims 1 to 20, further comprising a temperature management device that manages a temperature of a space in the housing in which the cell processing device is disposed.   The cell processing system according to any one of claims 1 to 21, further comprising a carbon dioxide concentration management device that manages a carbon dioxide concentration in a space in which the cell processing device in the housing is disposed.   The cell processing system according to any one of claims 1 to 22, wherein the inside of the housing is divided into a plurality of areas. The cell processing apparatus
A pre-introduction cell delivery channel through which the solution containing cells passes;
A factor introducing device connected to the pre-introduction cell fluid delivery channel for introducing a pluripotency inducer into the cells to produce an inducer-introduced cell;
A cell mass producing device for producing a plurality of cell souls consisting of stem cells by culturing the induction factor-introduced cells;
The cell processing system according to any one of claims 1 to 23, comprising: And
An intake purification filter provided in the casing for purifying gas sucked from the outside of the casing;
A return member for returning the gas discharged from the housing to the intake air purification filter;
The casing, the casing, and the return member are arranged such that the gas in the casing is discharged into the return member, and the gas in the return member is drawn into the casing through the intake air purification filter. And a circulating device for circulating between
A cell processing device for processing cells, disposed in the housing;
, A cell processing system.   26. The cell processing system of claim 25, wherein the return member has a shape that mates with the housing. The return member is
A base having a hollow inside, which is in contact with the bottom surface of the housing;
A first cover communicating the first opening provided in the base and the ventilating portion provided on the first end face of the housing;
A second cover communicating the second opening provided in the base and the ventilating portion provided on the second end face of the housing;
The cell processing system according to claim 25 or 26, comprising   26. The cell processing system of claim 25, wherein the return member is a duct.   29. The apparatus according to any one of claims 25 to 28, wherein the circulation device is provided in the housing and is provided with a gas discharge device for sucking gas from the inside of the housing and discharging the purified gas into the return member. Cell processing system as described in. The gas discharge device is
An exhaust device for exhausting the gas in the casing into the return member;
An exhaust gas purification filter for purifying the gas sucked by the exhaust device;
30. The cell processing system of claim 29, comprising:   31. The cell processing system according to claim 30, wherein the exhaust purification filter is disposed upstream of the exhaust system.   The cell processing system according to claim 31, further comprising a shielding member attachable to the exhaust gas purification filter to shield the exhaust gas purification filter. The shielding member is
A housing side shielding member that can be attached to the exhaust gas purification filter so as to shield the exhaust gas purification filter from the inside of the housing;
An exhaust system side shielding member which can be attached to the exhaust gas purification filter so as to shield the exhaust gas purification filter from the exhaust system;
33. The cell processing system of claim 32, comprising:   34. The cell processing system according to claim 32, further comprising a sterilizer for sterilizing the exhaust gas purification filter.   The cell processing system according to claim 31, further comprising a second exhaust gas purification filter disposed downstream of the exhaust device.   36. The cell processing system of claim 35, further comprising a shielding member attachable to the second exhaust gas purification filter to shield the second exhaust gas purification filter.   37. The cell processing system according to claim 36, wherein the shielding member comprises an exhaust device side shielding member that can be attached to the second exhaust gas purification filter so as to shield the second exhaust gas purification filter from the exhaust device. .   The cell processing system according to claim 36, further comprising a sterilizer for sterilizing the second exhaust gas purification filter.   39. The apparatus according to any one of claims 25 to 38, wherein the circulation device further comprises an introduction device provided in the housing for suctioning the gas purified by the intake air purification filter from within the return member. Cell processing system.   40. The cell processing system according to claim 39, wherein the intake air purification filter is disposed downstream of the introduction device.   41. The cell processing system according to claim 40, further comprising a shielding member attachable to the intake air purification filter to shield the intake air purification filter. The shielding member is
A case-side shielding member that can be attached to the intake air purification filter so as to shield the intake air purification filter from the inside of the case;
An introduction device side shielding member that can be attached to the intake air purification filter so as to shield the intake air purification filter from the introduction device;
42. The cell processing system of claim 41, comprising:   43. The cell processing system according to claim 41, further comprising a sterilizer for sterilizing the intake air purification filter.   44. The cell processing system according to any one of claims 25 to 43, wherein the inside of the housing is under negative pressure as compared to the inside of the return member.   45. The cell processing system according to any one of claims 25 to 44, wherein stem cells are cultured in the cell processing apparatus.   The cell processing system according to any one of claims 25 to 45, further comprising a sterilizer for sterilizing a space in the housing in which the cell processor is disposed.   The cell processing system according to any one of claims 25 to 46, further comprising a temperature management device that manages the temperature of the space in the housing in which the cell processing device is disposed.   48. The cell processing system according to any one of claims 25 to 47, further comprising a carbon dioxide concentration management device that manages a carbon dioxide concentration in a space in which the cell processing device in the housing is disposed.   The cell processing system according to any one of claims 25 to 48, wherein the inside of the housing is divided into a plurality of regions. The cell processing apparatus
A pre-introduction cell delivery channel through which the solution containing cells passes;
A factor introducing device connected to the pre-introduction cell fluid delivery channel for introducing a pluripotency inducer into the cells to produce an inducer-introduced cell;
A cell mass producing device for producing a plurality of cell souls consisting of stem cells by culturing the induction factor-introduced cells;
50. The cell processing system of any one of claims 25-49, comprising: A cell processing device for processing cells;
An embedding member for embedding the cell processing device;
A fluid communication passage that brings the outside of the embedding member into communication with the cell processing device in the embedding member;
A cell processing apparatus comprising:   52. The cell processing device according to claim 51, wherein the embedding member is made of glass or resin.   52. The cell processing device according to claim 51, wherein the embedding member is made of metal.   54. The cell processing apparatus according to any one of claims 51 to 53, further comprising a drive unit disposed outside the embedding member for feeding the liquid in the communication fluid delivery passage.   55. The cell processing device according to claim 54, wherein the drive unit is connected to the outer wall of the embedding member.   The cell according to claim 54 or 55, wherein a follower is provided in the embedding member, to which driving force is transmitted from the driver, and the follower is connected to the communication liquid transfer path. Processing unit.   57. The cell processing device according to any one of claims 51 to 56, wherein the cell processing device comprises a mononuclear cell separation part disposed inside the embedding member for separating mononuclear cells from blood. It further comprises a blood storage unit arranged outside the embedding member for storing blood and / or blood cells.
58. The cell processing apparatus according to claim 57, wherein the communication fluid delivery passage comprises a blood delivery passage that brings the blood storage unit and the mononuclear cell separation unit into communication. It further comprises a separating agent storage unit for storing a separating agent for separating mononuclear cells, which is arranged outside the embedding member,
59. The cell processing apparatus according to claim 57, wherein the communication liquid supply path includes a separation agent liquid supply path that causes the separation agent storage unit and the mononuclear cell separation unit to communicate with each other.   57. The cell processing device of any of claims 51-56, wherein the cell processing device comprises a filter disposed inside the embedding member that isolates cells.   57. The cell processing device according to any one of claims 51 to 56, wherein the cell processing device comprises a mononuclear cell purification filter disposed inside the embedding member. The cell processing device is
A mononuclear cell separation portion disposed inside the embedding member for separating mononuclear cells from blood;
A mononuclear cell purification filter disposed inside the embedding member;
A mononuclear cell liquid delivery path disposed in the interior of the embedding member, which connects the mononuclear cell separation unit and the mononuclear cell purification filter;
57. The cell processing device of any one of claims 51-56, comprising:   The cell processing apparatus according to claim 62, further comprising: a driving unit disposed outside the embedding member for feeding a liquid in the mononuclear cell liquid delivery channel.   64. The cell processing device according to claim 63, wherein the drive unit is connected to the outer wall of the embedding member.   65. The embedded member according to claim 63, wherein a follower is provided in the driving member to which the driving force is transmitted from the driver, and the follower is connected to the mononuclear cell liquid transfer path. Cell processing equipment.   57. The factor introducing unit according to any one of claims 51 to 56, wherein the cell processing device comprises a factor introducing unit disposed inside the embedding member for introducing a pluripotency inducer into cells to produce an inducer-transduced cell. The cell processing apparatus as described in a term. The system further comprises a factor storage unit, which stores pluripotency inducer, disposed outside the embedding member
67. The cell processing apparatus according to claim 66, wherein the communication liquid feed path includes a factor liquid feed path that brings the factor storage unit and the factor introduction portion into communication.   68. The cell processing apparatus according to claim 66 or 67, wherein the pluripotency inducer is introduced into the cell by RNA lipofection in the factor introduction part.   69. The cell processing device according to claim 68, wherein the pluripotency inducer is DNA, RNA or protein.   68. The cell processing device according to claim 66 or 67, wherein the pluripotency inducer is incorporated into a vector.   71. The cell processing device according to claim 70, wherein the vector is a Sendai virus vector. The cell processing device is
A mononuclear cell separation portion disposed inside the embedding member for separating mononuclear cells from blood;
A factor introduction unit disposed inside the embedding member, which introduces pluripotency inducer into cells to produce inducer-introduced cells;
A pre-introduction cell fluid delivery channel disposed in the interior of the embedding member, which brings the mononuclear cell separation part into communication with the factor introduction part;
57. The cell processing device of any one of claims 51-56, comprising:   73. The cell processing apparatus according to claim 72, further comprising a drive unit disposed outside the embedding member for transporting the liquid in the cell delivery channel before introduction.   74. The cell processing device according to claim 73, wherein the drive unit is connected to the outer wall of the embedding member.   The drive unit according to claim 73 or 74, wherein the embedded member is provided with a driven unit to which driving force is transmitted from the drive unit, and the driven unit is connected to the cell liquid transfer path before introduction. Cell processing equipment. The cell processing device is
A mononuclear cell purification filter disposed inside the embedding member;
A factor introduction unit disposed inside the embedding member, which introduces pluripotency inducer into cells to produce inducer-introduced cells;
A pre-introduction cell flow channel disposed in the interior of the embedding member, which brings the mononuclear cell purification filter into communication with the factor introduction unit;
57. The cell processing device of any one of claims 51-56, comprising:   The cell processing apparatus according to claim 76, further comprising: a driving unit disposed outside the embedding member for feeding the liquid in the cell delivery channel before introduction.   78. The cell processing device according to claim 77, wherein the drive unit is connected to the outer wall of the embedding member.   78. The enveloping member according to claim 76 or 77, further comprising: a follower to which driving force is transmitted from the driver, the follower being connected to the pre-introduction cell liquid flow path. Cell processing equipment.   57. The apparatus according to any one of claims 51 to 56, wherein the cell processing device comprises a reprogramming incubator disposed inside the embedding member for culturing an inducer-transduced cell into which a pluripotency inducer has been introduced. Cell processing apparatus as described. The blood cell culture medium storage unit, further comprising a blood cell culture medium, disposed outside the embedding member.
81. The cell processing apparatus according to claim 80, wherein the communication liquid feed path includes a culture medium liquid feed path that brings the blood cell culture medium storage unit into communication with the initialization incubator.   82. The cell processing device according to claim 81, wherein the blood cell culture medium is continuously supplied to the reprogramming incubator.   The cell processing apparatus according to claim 81, wherein the blood cell culture medium is replenished to the reprogramming incubator at a predetermined timing.   The cell processing apparatus according to claim 81, further comprising: a refrigerated storage unit disposed outside the embedding member that refrigerates the blood cell culture medium. It further comprises a stem cell culture medium storage unit for storing a stem cell culture medium, which is disposed outside the embedding member,
81. The cell processing apparatus according to claim 80, wherein the communication liquid transfer path includes a culture medium flow path that brings the stem cell culture medium storage unit and the initialization incubator into communication with each other.   86. The cell processing device according to claim 85, wherein the stem cell culture medium is continuously supplied to the reprogramming incubator.   86. The cell processing apparatus according to claim 85, wherein the stem cell culture medium is replenished to the reprogramming incubator at a predetermined timing.   86. The cell processing apparatus according to claim 85, further comprising a refrigerated storage unit disposed outside the embedding member, which stores the stem cell culture medium refrigerated. It further comprises a waste liquid storage unit for storing waste liquid disposed outside the embedding member,
81. The cell processing apparatus according to claim 80, wherein the communication liquid feed path includes a waste liquid feed path that brings the initialization incubator and the waste liquid storage unit into communication. The reprogramming incubator is
A dialysis tube in which the inducer-introduced cells and the medium are placed;
A container into which the dialysis tubing is placed and in which media is placed around the dialysis tubing;
Equipped with a floating incubator, equipped with a
81. A cell processing apparatus according to claim 80. The cell processing device is
A factor introduction unit disposed inside the embedding member, which introduces pluripotency inducer into cells to produce inducer-introduced cells;
A reprogramming incubator placed inside the embedding member for culturing the inducer-introduced cells;
A channel for introducing introduced cells disposed in the interior of the embedding member, which brings the factor introducing unit into communication with the reprogramming incubator;
57. The cell processing device of any one of claims 51-56, comprising:   92. The cell processing apparatus according to claim 91, further comprising: a driving unit disposed outside the embedding member for feeding a liquid in the introduced cell delivery channel.   The cell processing device according to claim 92, wherein the drive unit is connected to an outer wall of the embedding member.   93. The embedded member according to claim 91 or 92, further comprising: a driven unit to which a driving force is transmitted from the driving unit, the driven unit being connected to the liquid transfer path for introduced cells. Cell processing equipment.   95. The method of claim 94, wherein the drive and the follower are magnetically connected.   57. An expansion incubator according to any one of claims 51 to 56, wherein the cell processing device comprises an expansion incubator disposed inside the embedding member for expanding and culturing a plurality of cell clusters consisting of established stem cells. Cell processing equipment. It further comprises a stem cell culture medium storage unit for storing a stem cell culture medium, which is disposed outside the embedding member,
97. The cell processing apparatus according to claim 96, wherein the communication liquid transfer path comprises a culture medium flow path connecting the stem cell culture medium storage unit and the expansion incubator.   100. The cell processing device according to claim 97, wherein the stem cell culture medium is continuously supplied to the expansion incubator.   The cell processing device according to claim 97, wherein the stem cell culture medium is replenished to the expansion incubator at a predetermined timing.   The cell processing apparatus according to claim 97, further comprising: a refrigerated storage unit disposed outside the embedding member, which stores the stem cell culture medium refrigerated. It further comprises a waste liquid storage unit for storing waste liquid disposed outside the embedding member,
97. The cell processing apparatus according to claim 96, wherein the communication liquid feed path includes a waste liquid feed path that brings the expansion incubator into communication with the waste liquid storage unit. The expansion incubator is
A dialysis tube in which the inducer-introduced cells and the medium are placed;
A container into which the dialysis tubing is placed and in which media is placed around the dialysis tubing;
Equipped with a floating incubator, equipped with a
97. A cell processing apparatus according to claim 96. The cell processing device is
A reprogramming incubator placed inside the embedding member for culturing the inducer-introduced cells;
An expansion incubator disposed inside the embedding member for expanding and culturing a plurality of cell masses consisting of established stem cells;
An introduced cell delivery channel disposed in the interior of the embedding member, which connects the reprogramming incubator and the expansion incubator;
57. The cell processing device of any one of claims 51-56, comprising:   104. The cell processing apparatus according to claim 103, further comprising: a driving unit disposed outside the embedding member for feeding a liquid in the introduced cell delivery channel.   The cell processing device according to claim 104, wherein the drive unit is connected to the outer wall of the embedding member.   106. The embedded member according to claim 104 or 105, further comprising: a driven unit to which a driving force is transmitted from the driving unit is provided in the embedding member, and the driven unit is connected to the liquid transfer path for introduced cells. Cell processing equipment.   105. The cell processing device according to any one of claims 103 to 104, further comprising: a cell splitter, disposed in the embedding member, for dividing cell mass, provided in the transduced cell liquid flow path. The cell processing apparatus according to item 1.   57. The cell processing device of any of claims 51-56, wherein the cell processing device comprises a solution displacing device that replaces the solution around cells. It further comprises a cryopreservation solution storage unit containing a cryopreservation solution, which is disposed outside the embedding member,
109. The cell processing apparatus according to claim 108, wherein the communication liquid feed path includes a cryopreservation liquid feed path that brings the cryopreservation liquid storage unit into communication with the solution replacement unit. The cryopreservation container further comprises a cryopreservation container, which is disposed outside the embedding member, for storing a cryopreservation solution in which cell aggregates are dispersed,
110. The cell processing apparatus according to claim 109, wherein the communicating fluid delivery passage comprises a cell delivery fluid passage for freezing which brings the solution substitution device into communication with the cryopreservation container. The cell processing device is
An expansion incubator disposed inside the embedding member for expanding and culturing a plurality of cell masses consisting of established stem cells;
A solution displacing device for displacing the solution around the cells;
An introduced cell delivery channel disposed in the interior of the embedding member, which communicates the expansion incubator with the solution replacer;
57. The cell processing device of any one of claims 51-56, comprising:   The cell processing apparatus according to claim 111, further comprising: a driving unit disposed outside the embedding member for feeding the liquid in the introduced cell delivery channel.   113. The cell processing device according to claim 112, wherein the drive unit is connected to the outer wall of the embedding member.   114. The embedded member according to claim 112 or 113, further comprising: a driven unit to which driving force is transmitted from the driving unit, the driven unit being connected to the liquid transfer path for introduced cells. Cell processing equipment.   115. The cell processing apparatus according to any one of claims 111 to 114, further comprising: a cell splitter, disposed in the embedding member, for dividing cell mass, provided in the transduced cell liquid flow path. The cell processing apparatus according to item 1.   57. The cell processing device of any one of claims 51 to 56, wherein the cell processing device comprises a cell mass divider that divides cell mass.