JP7090337B2 - Cell processing system and cell processing equipment - Google Patents

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 early human or mouse embryos. ES cells have pluripotency that allows them to differentiate into all cells existing in the 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 ES cell transplantation. In particular, ES cell transplantation can elicit immune rejection similar to the rejection that follows unsuccessful organ transplantation. In addition, there are many criticisms and dissenting opinions from an ethical point of view regarding the use of ES cells established by destroying human embryos.

Under these circumstances, Professor Shinya Yamanaka of Kyoto University introduced four genes: Oct3 / 4, Klf4, c-Myc, and Sox2 into somatic cells to induce pluripotent stem cells (iPS). Succeeded in establishing cells). As a result, Professor Yamanaka received the 2012 Nobel Prize in Physiology or Medicine (see, for example, Patent Documents 1 and 2). iPS cells are ideal pluripotent cells without rejection or ethical issues. Therefore, iPS cells are expected to be used for cell transplantation therapy.

Japanese Patent No. 4183742 Japanese Unexamined Patent Publication No. 2014-114997

Induced stem cells such as iPS cells are established by introducing an inducing factor such as a gene into the cells, and are expanded-cultured and cryopreserved. Traditionally, stem cells have been manually produced in a clean room by humans. At this time, it is necessary to prevent the blood and inducing factors used in producing stem cells from coming into contact with humans. In addition, since cells may be infected with viruses and pathogens, containment is required to prevent the cells from being exposed to the outside. For example, when cells infected with hepatitis virus or human immunodeficiency virus (HIV) spread to the outside, they have a fatal effect on humans and animals. Therefore, blood, inducers, pre-inducible cells, 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 human being are produced in a clean room over a predetermined time.

On the other hand, one of the objects 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 housing, the outer housing including the housing, the intake purification filter provided in the housing for purifying the gas sucked from the outside of the housing, and the gas in the outer housing are used. , A circulation device that circulates gas inside and outside the housing inside the outer housing and is arranged inside the housing so that the gas is sucked into the housing through the intake purification filter and discharged into the outer housing. A cell processing system comprising a cell processing apparatus for processing cells is provided.

In the above cell treatment system, a pressure adjusting hole may be provided in the outer housing.

In the above cell processing system, the circulation device may include a gas discharge device provided in the housing, which sucks gas from the inside of the housing and discharges 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 inside the housing to the outside of the housing, and an exhaust purification filter that purifies the gas sucked by the exhaust device.

In the above cell treatment system, the exhaust purification filter may be arranged on the upstream side of the exhaust device.

In the above cell treatment system, a shielding member that can be attached to the exhaust purification filter so as to shield the exhaust purification filter may be further provided.

In the above cell treatment system, the shielding member is a housing-side shielding member that can be attached to the exhaust purification filter so as to shield the exhaust purification filter from the inside of the housing, and an exhaust purification filter so as to shield the exhaust purification filter from the exhaust device. It may be provided with an exhaust device side shielding member that can be attached to.

In the above cell treatment system, a sterilizer for sterilizing the exhaust gas purification filter may be further provided.

In the above cell treatment system, a second exhaust purification filter arranged on the downstream side of the exhaust device may be further provided.

In the above cell treatment system, a shielding member that can be attached to the second exhaust purification filter so as to shield the second exhaust purification filter may be further provided.

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

In the above cell treatment system, a sterilizer for sterilizing the second exhaust gas purification filter may be further provided.

In the above cell processing system, the circulation device may include an introduction device provided in the housing for sucking the gas purified by the intake air purification filter from the outside of the housing.

In the above cell processing system, the intake purification filter may be arranged on the downstream side of the introduction device.

In the above cell processing system, a shielding member that can be attached to the intake purification filter so as to shield the intake purification filter may be further provided.

In the above cell processing system, the shielding member is a housing-side shielding member that can be attached to the intake purification filter so as to shield the intake purification filter from the inside of the housing, and an intake purification filter so as to shield the intake purification filter from the introduction device. It may be provided with an introduction device-side shielding member that can be attached to the device.

In the above cell treatment system, a sterilizer for sterilizing the inspiratory purification filter may be further provided.

In the above cell processing system, the inside of the housing may have a negative pressure as compared with the outside of the housing.

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

In the above-mentioned cell processing system, a sterilizing device for sterilizing the space in which the cell processing device is arranged in the housing may be further provided.

In the above-mentioned cell processing system, a temperature control device for controlling the temperature of the space in which the cell treatment device in the housing is arranged may be further provided.

In the above-mentioned cell processing system, a carbon dioxide concentration management device for controlling the carbon dioxide concentration in the space in which the cell processing device in the housing is arranged may be further provided.

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 apparatus is connected to the pre-introduction cell delivery channel through which the solution containing the cells passes and the pre-introduction cell delivery channel, and introduces a pluripotent inducer into the cell to induce the cell. It may be provided with a factor introduction device for producing introduced cells and a cell mass producing device for producing a plurality of cell souls composed of stem cells by culturing the inducer-introduced cells.

In the above cell processing system, the housing, the intake purification filter provided in the housing for purifying the gas sucked from the outside of the housing, and the gas discharged from the housing are returned to the intake purification filter. The gas is between the housing and the return member so that the return member and the gas in the housing are discharged into the return member and the gas in the return member is sucked into the housing through the intake purification filter. It may be provided with a circulation device for circulating cells and a cell processing device for processing cells, which is arranged in a housing.

In the above cell processing system, the return member may have a shape that fits into the housing.

In the above cell processing system, the return member communicates a base having a hollow inside, which is in contact with the bottom surface of the housing, and a first opening provided at the base and a ventilation portion provided at the first end surface of the housing. A first cover and a second cover for communicating the second opening provided in the base portion and the ventilation portion provided in the second end surface of the housing may be provided.

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

In the above cell processing system, the circulation device may include a gas discharge device provided in the housing, which sucks gas from the inside of the housing and discharges the purified gas into the return member.

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

In the above cell treatment system, the exhaust purification filter may be arranged on the upstream side of the exhaust device.

In the above cell treatment system, a shielding member that can be attached to the exhaust purification filter so as to shield the exhaust purification filter may be further provided.

In the above cell treatment system, the shielding member is a housing-side shielding member that can be attached to the exhaust purification filter so as to shield the exhaust purification filter from the inside of the housing, and an exhaust purification filter so as to shield the exhaust purification filter from the exhaust device. It may be provided with an exhaust device side shielding member that can be attached to.

In the above cell treatment system, a sterilizer for sterilizing the exhaust gas purification filter may be further provided.

In the above cell treatment system, a second exhaust purification filter arranged on the downstream side of the exhaust device may be further provided.

In the above cell treatment system, a shielding member that can be attached to the second exhaust purification filter so as to shield the second exhaust purification filter may be further provided.

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

In the above cell treatment system, a sterilizer for sterilizing the second exhaust gas purification filter may be further provided.

In the above cell processing system, the circulation device may further include an introduction device provided in the housing, which sucks the gas purified by the intake air purification filter from the inside of the return member.

In the above cell processing system, the intake purification filter may be arranged on the downstream side of the introduction device.

In the above cell processing system, a shielding member that can be attached to the intake purification filter so as to shield the intake purification filter may be further provided.

In the above cell processing system, the shielding member is a housing-side shielding member that can be attached to the intake purification filter so as to shield the intake purification filter from the inside of the housing, and an intake purification filter so as to shield the intake purification filter from the introduction device. It may be provided with an introduction device-side shielding member that can be attached to the device.

In the above cell treatment system, a sterilizer for sterilizing the inspiratory purification filter may be further provided.

In the above cell processing system, the inside of the housing may have a negative pressure as compared with the inside of the return member.

Stem cells may be cultured in the cell processing apparatus of the above cell processing system.

In the above-mentioned cell processing system, a sterilizing device for sterilizing the space in which the cell processing device is arranged in the housing may be further provided.

In the above-mentioned cell processing system, a temperature control device for controlling the temperature of the space in which the cell treatment device in the housing is arranged may be further provided.

In the above-mentioned cell processing system, a carbon dioxide concentration management device for controlling the carbon dioxide concentration in the space in which the cell processing device in the housing is arranged may be further provided.

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 apparatus is connected to the pre-introduction cell delivery channel through which the solution containing the cells passes and the pre-introduction cell delivery channel, and introduces a pluripotent inducer into the cell to induce the cell. It may be provided with a factor introduction device for producing introduced cells and a cell mass producing device for producing a plurality of cell souls composed of stem cells by culturing the inducer-introduced cells.

Further, according to the aspect of the present invention, the cell processing instrument for processing cells, the embedding member for embedding the cell processing instrument, and the cell processing instrument outside the embedding member and inside the embedding member are communicated with each other. A cell processing apparatus comprising a liquid delivery channel is provided.

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 cell processing apparatus may further include a drive unit arranged outside the embedding member for delivering the liquid in the communication liquid passage.

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

In the above-mentioned cell processing apparatus, a driven portion to which a driving force is transmitted from the driving portion is provided in the embedding member, and the driven portion may be connected to a communication liquid passage.

In the above cell processing apparatus, the cell processing instrument may include a mononuclear cell separating portion arranged inside an embedding member that separates mononuclear cells from blood.

In the above cell processing apparatus, a blood storage unit that stores at least one of blood and blood cells, which is arranged outside the embedding member, is further provided, and a communication liquid passage is provided as a blood storage unit and a mononuclear cell separation unit. It may be provided with a blood delivery channel for communicating with the blood.

In the above-mentioned cell processing apparatus, a separating agent storage unit for storing a separating agent for separating mononuclear cells, which is arranged outside the embedding member, is further provided, and a communication liquid passage is simply provided with the separating agent storage unit. It may be provided with a separating agent liquid feeding path for communicating the nuclear ball separating portion.

In the cell processing apparatus described above, the cell processing instrument may include a filter placed inside an embedding member that isolates the cells.

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

In the above cell processing apparatus, the cell processing apparatus separates the mononuclear cells from the blood, the mononuclear cell separating portion arranged inside the embedding member, and the mononuclear cell purification arranged inside the embedding member. It may be provided with a filter and a monocyte delivery channel arranged inside the embedding member, which communicates the mononuclear cell separation part and the mononuclear cell purification filter.

The cell processing apparatus may further include a drive unit arranged outside the embedding member for delivering the liquid in the mononuclear ball delivery path.

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

In the above-mentioned cell processing apparatus, a driven portion to which a driving force is transmitted from the driving portion is provided in the embedding member, and the driven portion may be connected to a mononuclear ball feeding path.

In the above-mentioned cell processing apparatus, the cell processing instrument may include a factor introduction portion arranged inside an embedding member, which introduces a pluripotent inducer into a cell to prepare an inducer-introduced cell.

In the above cell processing apparatus, a factor storage unit for storing pluripotent inducing factors is further provided outside the embedding member, and a communication liquid passage communicates the factor storage unit and the factor introduction unit. It may be provided with a liquid channel.

In the above cell processing apparatus, the pluripotent inducer may be introduced into cells by RNA lipofection at the factor introduction section.

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

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

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

In the above cell processing apparatus, the cell processing instrument introduces a pluripotent inducer into the cell and the mononuclear cell separator arranged inside the embedding member, which separates the mononuclear cells from the blood. A factor introduction section arranged inside the embedding member for producing the introduction cells, and a pre-introduction cell delivery path arranged inside the embedding member for communicating the monocyte separation section and the factor introduction section. May be provided.

The above-mentioned cell processing apparatus may further include a drive unit arranged outside the embedding member for delivering the liquid in the pre-introduction cell delivery path.

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

In the above-mentioned cell processing apparatus, a driven portion to which a driving force is transmitted from the driving portion is provided in the embedding member, and the driven portion may be connected to the pre-introduction cell delivery path.

In the above cell processing apparatus, the cell processing instrument prepares an inducing factor-introduced cell by introducing a pluripotent inducing factor into the cell and a monocyte purification filter arranged inside the embedding member. It may be provided with a factor introduction section arranged inside the cell and a pre-introduction cell delivery channel arranged inside the embedding member that communicates the monocyte purification filter and the factor introduction section.

The above-mentioned cell processing apparatus may further include a drive unit arranged outside the embedding member for delivering the liquid in the pre-introduction cell delivery path.

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

In the above-mentioned cell processing apparatus, a driven portion to which a driving force is transmitted from the driving portion is provided in the embedding member, and the driven portion may be connected to the pre-introduction cell delivery path.

In the above cell processing apparatus, the cell processing instrument may include an reprogramming incubator arranged inside the embedding member for culturing the inducing factor-introduced cells into which the pluripotent inducing factor has been introduced.

In the above cell processing apparatus, a blood cell medium storage unit for storing blood cell medium, which is arranged outside the embedding member, is further provided, and a communication liquid passage communicates between the blood cell medium storage unit and the initialization incubator. It may be provided with a medium feeding channel to allow the cells to be fed.

In the above cell processing apparatus, the blood cell medium may be continuously replenished to the reprogramming incubator.

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

The above-mentioned cell processing apparatus may further include a refrigerated storage unit arranged outside the embedding member for refrigerating and storing the blood cell medium.

In the above-mentioned cell processing apparatus, a stem cell medium storage section for storing the stem cell medium, which is arranged outside the embedding member, is further provided, and a communication medium feeding channel communicates the stem cell medium storage section and the reprogramming incubator. It may be provided with a liquid channel.

In the above cell processing apparatus, the stem cell medium may be continuously replenished to the reprogramming incubator.

In the above cell processing apparatus, the stem cell medium may be replenished to the reprogramming incubator at a predetermined timing.

The cell processing apparatus may further include a refrigerated storage unit arranged outside the embedding member for refrigerating and storing the stem cell medium.

In the above cell treatment apparatus, a waste liquid storage unit for storing waste liquid, which is arranged outside the embedding member, is further provided, and a communication liquid supply path is provided as a waste liquid delivery path for communicating the initialization incubator and the waste liquid storage unit. You may have it.

In the above cell processing apparatus, the reprogramming incubator comprises a dialysis tube containing inducing factor-introduced cells and a medium, and a container in which the dialysis tube is placed and the medium is placed around the dialysis tube. May be provided.

In the above-mentioned cell processing apparatus, the cell processing instrument introduces a pluripotent inducing factor into the cell to prepare an inducing factor-introduced cell, and the factor-introducing portion arranged inside the embedding member and the inducing factor-introduced cell. It is equipped with an initialization incubator arranged inside the embedding member for culturing, and an introduced cell delivery channel arranged inside the embedding member for communicating the factor introduction unit and the initialization incubator. May be good.

The cell processing apparatus may further include a drive unit arranged outside the embedding member for delivering the liquid in the introduced cell delivery path.

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

In the above-mentioned cell processing apparatus, a driven portion to which a driving force is transmitted from the driving portion is provided in the embedding member, and the driven portion may be connected to the introduction cell liquid feeding path.

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

In the above cell processing apparatus, the cell processing instrument may include an expansion incubator arranged inside an embedding member for expanding and culturing a plurality of cell clusters composed of established stem cells.

In the above-mentioned cell processing apparatus, a culture medium storage unit for storing the stem cell medium, which is arranged outside the embedding member, is further provided, and a communication medium feeding channel communicates the stem cell medium storage unit and the expansion incubator. It may have a road.

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

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

The cell processing apparatus may further include a refrigerated storage unit arranged outside the embedding member for refrigerating and storing the stem cell medium.

In the above cell treatment apparatus, a waste liquid storage unit for storing waste liquid, which is arranged outside the embedding member, is further provided, and a communication liquid supply channel is provided with a waste liquid delivery path for communicating the expansion incubator and the waste liquid storage unit. You may have.

In the above cell processing apparatus, the expansion incubator comprises a suspension incubator including a dialysis tube in which inducing factor-introduced cells and a medium are placed, and a container in which the dialysis tube is placed and the medium is placed around the dialysis tube. You may be prepared.

In the above cell processing apparatus, the cell processing instrument expands and cultures a plurality of cell clusters consisting of an reprogramming incubator arranged inside an embedding member and established stem cells for culturing inducing factor-introduced cells. It may be provided with an expansion incubator arranged inside the embedding member and an introduced cell delivery channel arranged inside the embedding member that communicates the initialization incubator and the expansion incubator.

The cell processing apparatus may further include a drive unit arranged outside the embedding member for delivering the liquid in the introduced cell delivery path.

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

In the above-mentioned cell processing apparatus, a driven portion to which a driving force is transmitted from the driving portion is provided in the embedding member, and the driven portion may be connected to the introduction cell liquid feeding path.

In the above cell processing apparatus, the cell processing instrument may further include a cell dividing device provided in the introduction cell delivery channel and arranged inside the embedding member for dividing the cell mass.

In the above cell processing apparatus, the cell processing instrument may include a solution replacement device that replaces the solution around the cell.

In the above cell processing apparatus, a cryopreservation solution storage unit containing a cryopreservation solution, which is arranged outside the embedding member, is further provided, and a communication liquid passage communicates the cryopreservation solution storage unit and the solution replacement device. It may be provided with a storage liquid delivery path.

In the above cell processing apparatus, a cryopreservation container for storing a cryopreservation solution in which cell mass is dispersed is further provided outside the embedding member, and a communication liquid passage is provided with a solution exchanger and cryopreservation. It may be provided with a cell delivery channel for freezing that communicates with the container.

In the above cell processing apparatus, the cell processing instrument replaces the pericellular solution with the expansion incubator placed inside the embedding member, which expands and cultures a plurality of cell clusters consisting of established stem cells. It may be provided with a vessel and an introduced cell delivery channel arranged inside an embedding member that communicates the expansion incubator and the solution replacement device.

The cell processing apparatus may further include a drive unit arranged outside the embedding member for delivering the liquid in the introduced cell delivery path.

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

In the above-mentioned cell processing apparatus, a driven portion to which a driving force is transmitted from the driving portion is provided in the embedding member, and the driven portion may be connected to the introduction cell liquid feeding path.

In the above cell processing apparatus, the cell processing instrument may further include a cell dividing device provided in the introduction cell delivery channel and arranged inside the embedding member for dividing the cell mass.

In the above-mentioned cell processing apparatus, the cell processing instrument may include a cell mass dividing device that divides a 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 schematic perspective view of the cell processing system which concerns on embodiment. It is a schematic perspective view of the cell processing system which concerns on embodiment. It is a schematic perspective sectional view of the cell processing system which concerns on embodiment. It is a schematic sectional view of the cell processing system which concerns on embodiment. It is a schematic perspective sectional view of the cell processing system which concerns on embodiment. It is a schematic sectional view of the cell processing system which concerns on embodiment. It is a schematic perspective sectional view of the cell processing system which concerns on embodiment. It is a schematic sectional view of the cell processing system which concerns on embodiment. It is a schematic perspective view of the cell processing system which concerns on embodiment. It is a schematic perspective view of the cell processing system which concerns on embodiment. It is a schematic perspective sectional view of the cell processing system which concerns on embodiment. It is a schematic perspective view of the cell processing system which concerns on embodiment. It is a schematic perspective view of the cell processing system which concerns on embodiment. It is a schematic perspective view of the cell processing system which concerns on embodiment. It is a schematic sectional view of the cell processing system which concerns on embodiment. It is a schematic diagram of the cell processing apparatus which concerns on embodiment. It is a schematic cross-sectional view of an example of the introduction cell liquid feeding path of the cell processing apparatus which concerns on embodiment. It is a schematic cross-sectional view of an example of the introduction cell liquid feeding path of the cell processing apparatus which concerns on embodiment. It is a schematic diagram of the culture bag used in 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 the supplementary culture medium feeding pump and the suspension incubator which concerns on embodiment. It is a schematic diagram of the supplementary culture medium feeding pump and the suspension incubator which concerns on embodiment. It is a schematic diagram of the suspension incubator and the imaging apparatus which concerns on embodiment. It is a schematic diagram of the suspension incubator and the imaging apparatus which concerns on embodiment. It is an example of an image of a cell according to an embodiment. It is a schematic diagram of the central processing unit processing unit which concerns on embodiment. It is an example of an image of a cell mass according to an embodiment. It is an example of the image of the binarized cell mass according to the embodiment. It is an example of an image of a cell mass to which a high-pass filter according to an embodiment is applied. It is an example of an image of a cell mass to which the watershed algorithm according to the embodiment is applied. It is an example of an image of a cell mass to which the Distance Transform method according to the embodiment is applied. It is an example of an image of a cell mass to which the watershed algorithm according to the embodiment is applied. It is an example of an image of a cell mass divided into a plurality of regions according to an embodiment. It is an example of the image of the cell mass from which the contour according to the embodiment was extracted. It is an example of the image of the cell mass from which the contour according to the embodiment was extracted. It is an example of the histogram of the cell size which concerns on embodiment. It is a schematic diagram of the suspension incubator and the imaging apparatus which concerns on embodiment. It is an example of the graph which shows the relationship between the pH of the culture medium and the hue of a culture medium which concerns on embodiment. It is a schematic diagram of the cell mass divider which concerns on embodiment. It is a schematic diagram of the cell mass divider which concerns on embodiment. It is a schematic diagram of the cell mass divider which concerns on embodiment. It is a schematic diagram of the cell mass divider which concerns on embodiment. It is a schematic diagram of the cell mass divider which concerns on embodiment. It is a schematic diagram of the cell mass divider which concerns on embodiment. It is a schematic diagram of the cell mass divider which concerns on embodiment. It is a schematic diagram of the cell mass divider which concerns on embodiment. It is an example of the image of the divided cell mass according to the embodiment. It is a schematic diagram of the solution replacement device which concerns on embodiment. It is a schematic diagram of the cell processing apparatus which concerns on embodiment. It is a schematic perspective view of the cell processing apparatus which concerns on embodiment. It is a schematic perspective view of the cell processing apparatus which concerns on embodiment. It is a schematic perspective view of the cell processing apparatus which concerns on embodiment. It is a schematic perspective view of the drive part of the cell processing apparatus which concerns on embodiment. It is a schematic perspective view of the drive part of the cell processing apparatus which concerns on embodiment. It is a fluorescence microscope photograph which concerns on Example 1. FIG. It is a graph which shows the analysis result by the fluorescence activation flow cytometer which concerns on Example 1. FIG. It is a photograph of the colony of iPS cells according to Example 2. It is a photograph of the colony of iPS cells according to Example 2. It is a photograph of the colony of iPS cells according to Example 2. It is a graph which shows the state of the differentiation of the colony of the iPS cell which concerns on Example 2. FIG. It is a photograph of the colony of iPS cells according to Example 3. It is a graph which shows the result of Example 4. It is a photograph of the iPS cell mass according to Example 5. It is a graph which shows the result of Example 5.

An embodiment of the present invention will be described below. In the description of the following drawings, the same or similar parts are represented by the same or similar reference numerals. However, the drawings are schematic. Therefore, the specific dimensions and the like should be determined in light of the following explanations. In addition, it goes without saying that parts having different dimensional relationships and ratios are included between the drawings.

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

As shown in FIGS. 1 to 3, the cell processing system according to the embodiment sucks from the housing 601, the outer housing 701 containing the housing 601 and the outside of the housing 601 provided in the housing 601. The intake purification filter 602 that purifies the gas and the gas inside the outer housing 701 are sucked into the housing 601 via the intake purification filter 602, and the gas inside the housing 601 is discharged into the outer housing 701. The gas is circulated inside and outside the housing 601 inside the outer housing 701, and a cell processing device for processing cells arranged inside the housing 601 is provided.

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 and ultraviolet sterilization, but is not particularly limited thereto. At least a part of the housing 601 may be transparent so that the inside can be observed from the outside. The housing 601 has a structure that can be opened and closed for taking in and out the cell processing device arranged inside.

The circulation device includes, for example, a gas discharge device 613 provided in the housing 601 that sucks gas from the inside of the housing 601 and discharges the purified gas to the outside of the housing 601. The intake air purification filter 602 and the gas discharge device 613 are arranged so as to face each other, for example. As the intake air purification filter 602, for example, a HEPA (High Efficiency Particulate Air) filter and a URPA (Ultra Low Particulate Air) filter can be used, but the present invention is not limited thereto. Depending on the usage environment, for example, a MEPA (Medium Efficiency Particulate Air) filter may be used as the intake air purification filter 602. The intake air purification filter 602 purifies the gas sucked into the housing 601 from the outside of the housing 601.

As shown in FIG. 3, the gas discharge device 613 includes, for example, an exhaust device 605 that exhausts the gas inside the housing 601 to the outside of the housing 601 and an exhaust purification filter 603 that purifies the gas sucked by the exhaust device 605. , Equipped with. The exhaust device 605 includes, for example, a fan. The exhaust purification filter 603 may be arranged so as to face the inside of the housing 601 on the upstream side of the exhaust device 605. It can be difficult to sterilize the exhaust device 605, which is an electrical device. On the other hand, by arranging the exhaust purification filter 603 on the upstream side of the exhaust device 605, it is possible to prevent the exhaust device 605 from being contaminated. The gas discharge device 613 may further include a second exhaust purification filter 606 arranged on the downstream side of the exhaust device 605.

The materials of the exhaust gas purification filter 603 and the second exhaust gas purification filter 606 are the same as those of the intake air purification filter 602, for example. 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 to the outside of the housing 601. For example, even if the gas in the housing 601 contains blood components or viruses, 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 purification filter 606.

As shown in FIGS. 1 to 3, the outer housing 701 has, for example, a rectangular parallelepiped shape, but is not particularly limited thereto. The outer housing 701 is made of, for example, a material that can withstand heat sterilization and ultraviolet sterilization, but is not particularly limited thereto. At least a part of the outer housing 701 may be transparent so that the inside can be observed from the outside. The outer housing 701 has a structure that can be opened and closed for taking in and out the housing 601 arranged inside. However, it is preferable that the inside of the outer housing 701 is completely closed in a state where the structure can be opened and closed and the pressure adjusting hole 702 described later is closed.

As shown in FIG. 3, the outer housing 701 may be provided with a pressure adjusting hole 702 for adjusting the pressure in the outer housing 701. Further, a closing member 703 capable of closing the pressure adjusting hole 702 may be attached to the outer housing 701. A filter is arranged in the pressure adjusting hole 702. The material of the filter arranged in the pressure adjusting hole 702 is, for example, the same as that of the intake air purifying filter 602.

For example, when the air pressure inside the outer housing 701 is the same as that outside the outer housing 701, the gas can flow through the pressure adjusting hole 702. The gas that goes out from the inside of the outer housing 701 through the pressure adjusting hole 702 is purified by the filter arranged in the pressure adjusting hole 702. Further, the gas flowing out from the outside of the outer housing 701 through the pressure adjusting hole 702 is also purified by the filter arranged in the pressure adjusting hole 702.

As shown in FIG. 4, the gas in the outer housing 701 is sucked into the housing 601 via the intake air purifying filter 602. Further, the gas in the housing 601 is discharged into the outer housing 701 by the gas discharge device 613. Therefore, in the outer housing 701, the gas circulates inside and outside the housing 601. As the gas circulates inside and outside the housing 601 inside the outer housing 701, the intake purification filter 602, the exhaust purification filter 603, and the second exhaust purification filter 606 cause not only the inside of the housing 601 but also the housing 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 the air in the housing 601 and the outer housing 701 is in the ISO 1 to ISO 6 class according to ISO standard 14644-1.

As shown in FIGS. 5 and 6, the second exhaust gas purification filter 606 may be omitted depending on the usage environment. Further, 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 arranged on the downstream side of the introduction device 608, facing the inside of the housing 601.

As shown in FIGS. 7 and 8, the cell treatment system may include both the introduction device 608 and the second exhaust gas purification filter 606. The circulation device may be provided with both an introduction device 608 and a gas discharge device 613, or may be provided with either one.

At least one of the exhaust device 605 and the introduction device 608 flows gas so that the inside of the housing 601 has a negative pressure as compared with the outside of the housing 601. This makes it possible to prevent impurities in the housing 601 from diffusing out of the housing 601. However, depending on the usage environment, the inside of the housing 601 may have a positive pressure as compared with 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 controls 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 controls 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 temperature control device that controls the temperature inside the housing 601. For example, the temperature control device controls the temperature so that the temperature in the housing 601 becomes a predetermined value, for example, 37 ° C. The temperature control device includes, for example, a Pelche element. The temperature control 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 sterilizer may spray or release a sterilizing gas such as ozone gas, hydrogen peroxide gas, or formalin gas or a sterilizing solution such as ethanol into the housing 601 to sterilize the inside of the housing 601. Alternatively, the sterilizer may sterilize the inside of the housing 601 by irradiating the inside of the housing 601 with ultraviolet rays (UV) or an electron beam. By sterilizing the inside of the housing 601 it is possible to repeatedly culture the cells in the housing 601. Further, it is possible to prevent the outside of the housing 601 from being contaminated or the worker from being infected. The cell treatment system may further include a sterilizer for sterilizing the outer housing 701.

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

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

The cell treatment 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 operator from being contaminated when the exhaust gas purification filter 603 is replaced.

By sterilizing the exhaust purification filter 603 shielded by the shielding member 800, it is possible to prevent the exhaust device 605 from being exposed to the sterilization environment and the exhaust device 605 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-shaped or film-shaped.

As shown in FIGS. 10 and 11, the housing-side shielding member 801 may be movable by the guide 811. For example, the housing-side shielding member 801 may be able to move back and forth between a place that does not shield the exhaust gas purification filter 603 and a place that shields the exhaust gas purification filter 603 along the guide 811.

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

The housing-side shielding member 801 and the exhaust device-side shielding member 802 may be movable in the horizontal direction with respect to the housing 601 or, as shown in FIG. 12, can be moved in the vertical direction with respect to the housing 601. May be.

Further, the cell treatment 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 purification filter 606 can be attached to the second exhaust purification filter 606 so as to shield the second exhaust purification filter 606 from the exhaust device 605. It is equipped with a member.

The cell treatment system may further include a sterilizer for sterilizing the second exhaust gas purification filter 606 shielded by a shielding member. The sterilizer sterilizes the second exhaust gas purification filter 606 shielded by the shielding member by the same method 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 operator from being contaminated when the second exhaust gas purification filter 606 is replaced.

By sterilizing the second exhaust purification filter 606 shielded by the shielding member, it is possible to prevent the exhaust device 605 from being exposed to the sterilized environment and the exhaust device 605 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 that shields the second exhaust gas purification filter 606 is not particularly limited, but may be plate-shaped or film-shaped.

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

Further, the cell processing system may further include a shielding member that can be attached to the intake purification filter 602 so as to shield the intake purification filter 602. For example, as the shielding member that can be attached to the intake air purification filter 602, 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 the inside of the housing 601 and an intake air purification filter 602 are introduced. It is provided with an introduction device-side shielding member that can be attached to the intake air purification filter 602 so as to shield from the device 608.

The cell treatment system may further include a sterilizer for sterilizing the inspiratory purification filter 602 shielded by a shielding member. The sterilizer sterilizes the intake air purification filter 602 shielded by the shielding member by the same method 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 operator 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 the introduction device 608 from being damaged. 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 that shields the intake air purification filter 602 is not particularly limited, but may be plate-shaped or film-shaped.

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 back and forth between a place where the intake air purification filter 602 is not shielded and a place where the intake air purification filter 602 is shielded along the guide.

Alternatively, as shown in FIGS. 13 to 15, the cell processing system according to the embodiment has an intake purification filter 602 for purifying the gas sucked from the outside of the housing 601 provided in the housing 601 and the housing 601. A return member 651 having a hollow inside for returning the gas discharged from the housing 601 to the intake purification filter 602, and a return member 651 in which the gas inside the housing 601 is discharged into the return member 651. A circulation device for circulating gas between the housing and the return member and a housing 601 are arranged so that the gas in 651 is sucked into the housing 601 through the intake purification filter 602. , A cell processing apparatus for processing cells.

The housing 601 shown in FIGS. 13 to 15 may have the same configuration as the housing 601 described with reference to 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, a material that can withstand the above-mentioned sterility, but is not particularly limited thereto. At least a part of the housing 601 may be transparent so that the inside can be observed from the outside. The housing 601 has a structure that can be opened and closed for taking in and out the cell processing device arranged inside.

The circulation device includes, for example, a gas discharge device 613 provided in the housing 601 that sucks gas from the inside of the housing 601 and discharges the purified gas to the outside of the housing 601. The intake air purification filter 602 and the gas discharge device 613 are arranged so as to face each other, for example. As the intake air purification filter 602, for example, a HEPA (High Efficiency Particulate Air) filter and a URPA (Ultra Low Particulate Air) filter can be used, but the present invention is not limited thereto. Depending on the usage environment, for example, a MEPA (Medium Efficiency Particulate Air) filter may be used as the intake air purification filter 602. The intake air purification filter 602 purifies the gas sucked into the housing 601 from the outside of the housing 601.

In the cell treatment system shown in FIGS. 13 to 15, 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 into the return member 651 and an exhaust device. The exhaust gas purification filter 603 for purifying the gas sucked by the 605 is provided. The exhaust device 605 includes, for example, a fan. The exhaust purification filter 603 may be arranged so as to face the inside of the housing 601 on the upstream side of the exhaust device 605. It can be difficult to sterilize the exhaust device 605, which is an electrical device. On the other hand, by arranging the exhaust purification filter 603 on the upstream side of the exhaust device 605, it is possible to prevent the exhaust device 605 from being contaminated. The gas discharge device 613 may further include a second exhaust purification filter 606 arranged on the downstream side of the exhaust device 605.

The materials of the exhaust gas purification filter 603 and the second exhaust gas purification filter 606 are the same as those of the intake air purification filter 602, for example. Even if the inside of the housing 601 is contaminated by the cell processing device, the gas purified by the exhaust purification filter 603 and the second exhaust purification filter 606 is exhausted into the return member 651. For example, even if the gas in the housing 601 contains blood components or viruses, 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 purification filter 606.

As shown in FIGS. 13 to 15, the return member 651 has a shape that fits into, for example, the housing 601 but is not particularly limited thereto. The return member 651 communicates with, for example, a base portion 652 having a hollow inside, which is in contact with the bottom surface of the housing 601 and a first opening 653 provided in the base portion 652 and a ventilation portion provided in the first end surface of the housing 601. It is provided with a first cover 654 for communicating with the second opening 655 provided in the base portion 652 and a second cover 656 for communicating the ventilation portion provided in the housing 601.

The first cover 654 covers, for example, the intake air purifying filter 602 as a ventilation portion provided on the first end surface. The second cover 656 covers, for example, the gas discharge device 613 as a ventilation portion provided on the second end surface.

The return member 651 is made of, for example, a material that can withstand sterility like 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 housing 601 through the intake air purifying filter 602. Therefore, the gas circulates between the housing 601 and the return member 651. As the gas circulates between the housing 601 and the return member 651, the intake purification filter 602, the exhaust purification filter 603, and the second exhaust purification filter 606 cause not only the inside of the housing 601 but also the housing. The gas inside the return member 651 outside the 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 in the ISO 1 to ISO 6 class according to ISO standard 14644-1.

In the cell treatment 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 usage environment. Further, 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 arranged on the downstream side of the introduction device 608, facing the inside of the housing 601.

The cell treatment system shown in FIGS. 13 to 15 may also include both the introduction device 608 and the second exhaust gas purification filter 606, as shown in FIGS. 7 and 8. The circulation device may be provided with both an introduction device 608 and a gas discharge device 613, or may be provided with either one.

At least one of the exhaust device 605 and the introduction device 608 flows gas so that the inside of the housing 601 has a negative pressure as compared with the outside of the housing 601. This makes it possible to prevent impurities in the housing 601 from diffusing out of the housing 601. However, depending on the usage environment, the inside of the housing 601 may have a positive pressure as compared with the outside of the housing 601.

Similar to the cell treatment system described with reference to FIGS. 1 to 9, the cell treatment system shown in FIGS. 13 to 15 is a cleanliness sensor for monitoring the cleanliness of gas in the housing 601 and carbon dioxide in the housing 601. A carbon dioxide concentration control device that controls the concentration of (CO ₂ ), an oxygen concentration control device that controls the concentration of oxygen (O ₂ ) in the housing 601, a temperature control device that controls the temperature inside the housing 601 and a housing. It may be equipped with at least one of sterilizers for sterilizing the inside of the body 601.

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

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

The cell treatment system shown in FIGS. 13 to 15 may further include a sterilizer for sterilizing the exhaust gas purification filter 603 shielded by the shielding member 800, as shown in FIGS. 9, 10 and 11. .. 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 operator from being contaminated when the exhaust gas purification filter 603 is replaced.

By sterilizing the exhaust purification filter 603 shielded by the shielding member 800, it is possible to prevent the exhaust device 605 from being exposed to the sterilization environment and the exhaust device 605 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-shaped or film-shaped.

In the cell treatment system shown in FIGS. 13 to 15, as shown in FIGS. 10 and 11, the housing-side shielding member 801 may be movable by the guide 811. For example, the housing-side shielding member 801 may be able to move back and forth between a place that does not shield the exhaust gas purification filter 603 and a place that shields the exhaust gas purification filter 603 along the guide 811.

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

The housing-side shielding member 801 and the exhaust device-side shielding member 802 may be movable in the horizontal direction with respect to the housing 601 or, as shown in FIG. 12, can be moved in the vertical direction with respect to the housing 601. May be.

Further, the cell treatment system shown in FIGS. 13 to 15 is further provided with 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. May be good. For example, the shielding member that can be attached to the second exhaust purification filter 606 can be attached to the second exhaust purification filter 606 so as to shield the second exhaust purification filter 606 from the exhaust device 605. It is equipped with a member.

The cell treatment system shown in FIGS. 13 to 15 may also be provided with a sterilizer for sterilizing the second exhaust gas purification filter 606 shielded by the shielding member, as shown in FIG. The sterilizer sterilizes the second exhaust gas purification filter 606 shielded by the shielding member by the same method 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 operator from being contaminated when the second exhaust gas purification filter 606 is replaced.

By sterilizing the second exhaust purification filter 606 shielded by the shielding member, it is possible to prevent the exhaust device 605 from being exposed to the sterilized environment and the exhaust device 605 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 that shields the second exhaust gas purification filter 606 is not particularly limited, but may be plate-shaped or film-shaped.

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

Further, the cell processing system shown in FIGS. 13 to 15 may further include a shielding member that can be attached to the intake purification filter 602 so as to shield the intake purification filter 602, as shown in FIG. .. For example, as the shielding member that can be attached to the intake air purification filter 602, 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 the inside of the housing 601 and an intake air purification filter 602 are introduced. It is provided with an introduction device-side shielding member that can be attached to the intake air purification filter 602 so as to shield from the device 608.

The cell treatment system shown in FIGS. 13 to 15 may also be provided with 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 by the same method 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 operator 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 the introduction device 608 from being damaged. 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 that shields the intake air purification filter 602 is not particularly limited, but may be plate-shaped or film-shaped.

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 back and forth between a place where the intake air purification filter 602 is not shielded and a place where the intake air purification filter 602 is shielded along the guide.

As shown in FIG. 16, the cell processing device arranged in the housing 601 is, for example, a separation device 10 for separating cells from blood and pre-introduction cells through which a solution containing the cells separated by the separation device 10 passes. A pluripotent inducer is introduced into cells by being connected to a liquid delivery path 20, an inducer liquid delivery mechanism 21 that sends a pluripotent inducer into the pre-introduction cell delivery path 20, and a pre-introduction cell delivery path 20. A factor introduction device 30 for producing inducing factor-introduced cells, a cell mass producing device 40 for culturing inducing factor-introduced cells to produce a plurality of cell souls consisting of stem cells, and a plurality of cell masses are sequentially packaged. The package device 100 is provided.

Separator 10 receives, for example, a vial containing human blood. The separator 10 has an anticoagulant tank for storing anticoagulants such as ethylenediaminetetraacetic acid (EDTA), heparin, and biopharmaceutical standard blood storage solution A (ACD-A solution, Terumo Corporation). I have. The separating device 10 adds the anticoagulant to human blood from the anticoagulant tank using a pump or the like.

Further, the separation device 10 is provided with a separation reagent tank for storing a reagent for separating mononuclear cells such as Ficoll-Paque PREMIUM (registered trademark, GE Healthcare Japan Co., Ltd.). The separation device 10 dispenses 5 mL each of the mononuclear cell separation reagent from the separation reagent tank into, for example, two 15 mL tubes using a pump or the like. A resin bag may be used instead of the tube.

Further, the separation device 10 is provided with a buffer solution tank for storing a buffer solution such as phosphate buffered saline (PBS). The separation device 10 uses a pump or the like to dilute 5 mL of the buffer solution by adding 5 mL of the buffer solution to, for example, 5 mL of human blood from the buffer solution tank. Furthermore, the separation device 10 adds 5 mL each of diluted human blood onto the mononuclear cell separation reagent in the tube using a pump or the like.

The separation device 10 further includes a centrifuge whose temperature can be set. The centrifuge is set to, for example, 18 ° C. The separation device 10 uses a moving device or the like to put a tube containing a reagent for separating mononuclear cells, human blood, or the like into a holder of a centrifuge. The centrifuge centrifuges the solution in the tube at, for example, 400 xg for 30 minutes. Instead of the tube, the resin bag may be centrifuged.

After centrifugation, the separation device 10 collects the white turbid intermediate layer of the mononuclear cells of the solution in the tube by a pump or the like. The separation device 10 sends the recovered suspension of mononuclear cells to the pre-introduction cell delivery channel 20 using a pump or the like. Alternatively, the separator 10 further adds, for example, 12 mL of PBS to 2 mL of the recovered mononuclear cell solution and puts the tube in the holder of the centrifuge. The centrifuge centrifuges the solution in the tube at, for example, 200 xg for 10 minutes.

After centrifugation, the separator 10 sucks and removes the supernatant of the solution in the tube using a pump or the like, and tubes 3 mL of mononuclear cell medium such as X-VIVO 10 (registered trademark, Lonza Japan Co., Ltd.). Suspend in addition to the mononuclear cell solution inside. The separation device 10 sends the mononuclear cell suspension to the pre-introduction cell delivery channel 20 by using a pump or the like. The separation device 10 may use a dialysis membrane to separate mononuclear cells from blood. Further, when using somatic cells such as fibroblasts previously separated from the skin or the like, the separation device 10 may be omitted.

The separation device 10 may separate cells suitable for induction by a method 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 progenitor cells, the 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. Further, the separation is not limited to panning, and separation may be performed by a magnetic cell separation method (MACS), flow cytometry, or the like. Further, the cells suitable for induction are not limited to cells derived from blood.

The inducing factor liquid feeding mechanism 21 includes an inducing factor introducing reagent tank for storing the inducing factor introducing reagent solution and the like. Inducing factor-introducing reagent solutions such as gene-introducing reagent solutions include, for example, electroporation solutions such as Human T Cell Nucleofector® solutions, supplement solutions, and plasmid sets. 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 inducer-introducing reagent solution 21 uses a micropump or the like to transfer the inducer-introducing reagent solution into the pre-introduction cell delivery path 20 so that the mononuclear cell suspension is suspended in the inducing factor-introducing reagent solution. send out.

The inner wall of the pre-introduction cell delivery channel 20 may be coated with poly-HEMA (poly 2-hydroxyethyl meshrylate) to prevent cells from adhering to the cells. Alternatively, a material that does not easily adhere to cells may be used as the material for the pre-introduction cell delivery channel 20. Further, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the pre-introduction cell liquid supply passage 20, the conditions in the pre-introduction cell liquid supply passage 20 were controlled in the housing 601. Equivalent to temperature and carbon dioxide concentration. Further, the pre-introduction cell delivery passage 20 may be provided with a check valve from the viewpoint of preventing contamination.

The factor introduction device 30 connected to the pre-introduction cell delivery channel 20 is, for example, an electroporator, which receives a mixed solution of an inducing factor introduction reagent solution and a mononuclear cell suspension, and is used as a plasmid for mononuclear cells. Perform electroporation. After electroporation, the factor introduction device 30 adds the mononuclear cell medium to the solution containing the mononuclear cells electroporated with the plasmid. The factor introduction device 30 uses a pump or the like to deliver a solution containing mononuclear cells electroporated with a plasmid (hereinafter referred to as “inducing factor introduction cells”) to the introduction cell delivery channel 31.

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

For lipofection of reprogramming factor RNA, for example, small interfering RNA (siRNA) or lipofection reagent is used. As the RNA lipofection reagent, siRNA lipofection reagent and mRNA lipofection reagent can be used. More specifically, RNA lipofection reagents include Lipofectamine (registered trademark) RNAiMAX (Thermo Fisher Scientific), Lipofectamine (registered trademark) Transfection MAX (Thermo Fisher Scientific), and Lipto (registered trademark) , Neon Transfection System (Thermo Transfection Scientific), Stemfect RNA transfection reagent (Stemfect), NextFect (registered trademark) RNA Transfection Reagent (BioSient ), Amaxa (registered product) Human CD34 cell Nucleofector (registered product) kit (Lonza, VAPA-1003), ReproRNA (registered trademark) transfection reagent (STEMCELL Technologies) and the like can be used.

When the factor introduction device 30 introduces a reprogramming factor into cells by the lipofection method, the reprogramming factor RNA, reagents and the like are delivered to the pre-introduction cell delivery path 20 by the inducing factor delivery mechanism 21.

The inner wall of the introduced cell delivery path 31 may be coated with poly-HEMA to prevent cells from adhering to each other to make them non-adhesive. Alternatively, a material that is difficult for cells to adhere to may be used as the material of the introduction cell delivery path 31. Further, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the introduction cell liquid delivery path 31, the conditions in the introduction cell delivery path 31 are the controlled temperature in the housing 601 and the controlled temperature. Equivalent to carbon dioxide concentration. Further, the introduced cell liquid feeding path 31 may be provided with a check valve from the viewpoint of preventing contamination. Also, after electroporation, many cells may die and a cell mass of dead cells may form. Therefore, a filter for removing dead cell clumps may be provided in the introduced cell delivery path 31. Alternatively, as shown in FIG. 17, one or a plurality of folds whose inner diameter is intermittently changed may be provided inside the introduction cell liquid supply passage 31. Alternatively, as shown in FIG. 18, the inner diameter of the introduced cell delivery path 31 may be changed intermittently.

As shown in FIG. 16, the cell mass producing apparatus 40 connected to the introduced cell delivery path 31 includes an initialization culture apparatus 50 for culturing the inducing factor-introduced cells produced by the factor introduction apparatus 30 and an initialization culture apparatus. A first division mechanism 60 that divides a cell mass (cell colony) consisting of stem cells established in 50 into a plurality of cell masses, and an expansion culture that expands and cultures a plurality of cell masses divided by the first division mechanism 60. The device 70, a second division mechanism 80 that divides a cell mass composed 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 that sequentially sends a plurality of cell souls to a package device 100. And.

The initialization culture device 50 can store a well plate inside. Further, the initialization culture apparatus 50 includes a pipetting machine. The reprogramming culture apparatus 50 receives the solution containing the inducing factor-introduced cells from the introduction cell delivery path 31, and distributes the solution to the wells by a pipetting machine. The reprogramming culture apparatus 50 adds a stem cell medium such as StemFit (registered trademark, Ajinomoto Co., Inc.), for example, on the 3rd, 5th, and 7th days after distributing the inducer-introduced cells to the wells. Basic fibroblast growth factor (basic FGF) may be added to the medium as a supplement. In addition, 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, since FGF may be unstable, a heparin-mimicking polymer may be conjugated to FGF to stabilize FGF. Further, transforming growth factor beta (TGF-β), activin, etc. may be added to the medium. The reprogramming culture apparatus 50 exchanges the medium, for example, on the 9th day after distributing the inducer-introduced cells to the wells, and thereafter, the medium is used every two days until the cell mass (colony) of the iPS cells exceeds 1 mm. Make a replacement. It should be noted that exchanging the medium includes partially replacing the medium or replenishing the medium.

When the cell mass is formed, the reprogramming culture apparatus 50 collects the cell mass with a pipetting machine and adds a trypsin alternative recombinant enzyme such as TrypLE Select (registered trademark, Life Technologies) to the collected cell mass. Further, the reprogramming culture apparatus 50 puts the container containing the recovered cell mass in an incubator and reacts the cell mass with the trypsin alternative recombinant enzyme at 37 ° C. and 5% carbon dioxide for 10 minutes. If the cell mass is physically disrupted, the trypsin alternative recombinant enzyme may not be present. For example, the initialization culture apparatus 50 breaks a cell mass by pipetting with a pipetting machine. Alternatively, the reprogramming culture apparatus 50 passes the cell mass through a pipe provided with a filter or a pipe whose inner diameter is intermittently changed, similar to the introduced cell delivery path 31 shown in FIG. 17 or FIG. You may crush the lumps. After that, the reprogramming culture apparatus 50 adds a medium for pluripotent stem cells such as StemFit (registered trademark, Ajinomoto Co., Inc.) to the solution containing the crushed cell mass.

The culture in the initialization culture apparatus 50 may be performed in a carbon dioxide permeable bag instead of a well plate. Further, the culture may be an adhesive culture or a suspension culture. In the case of suspension culture, stirring culture may be performed. Further, the medium may be agar-like. Examples of the agar-like medium include gellan gum polymers and deacylated gellan gum polymers. When agar-like medium is used, cells do not sink or adhere, so it is possible to prepare a single cell mass derived from a single cell without the need for stirring even in a suspension culture. The culture in the initialization culture apparatus 50 may be a hanging drop culture.

The initialization culture device 50 may include a first culture medium replenishment device for replenishing a medium containing a culture medium in a well plate or a carbon dioxide permeable bag. The first medium replenishing device may collect the culture medium in a well plate or a carbon dioxide permeable bag, filter the culture medium using a filter or a dialysis membrane, and reuse the purified culture solution. good. At that time, a growth factor or the like may be added to the reused culture solution. Further, the initialization culture device 50 may further include a temperature control device for controlling the temperature of the medium, a humidity control device for controlling the humidity in the vicinity of the 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 permeable to carbon dioxide permeable to the culture solution. The culture solution may be put in the sex bags 302 and the bags 301 and 302. The initialization culture apparatus 50 prepares a plurality of bags 302 containing a fresh culture solution, and at predetermined intervals, a bag 302 containing a bag 301 containing cells is used as a bag containing a fresh culture solution. It may be replaced with 302.

Further, the culture method in the initialization culture apparatus 50 is not limited to the above method, and a suspension incubator as shown in FIG. 20 may be used. The suspension incubator shown in FIG. 20 includes a dialysis tube 75 in which the inducer-introduced cells and a 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. Further, the suspension incubator may be provided with a pH sensor for measuring the hydrogen ion index (pH) of the gel medium around the dialysis tube 75.

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 is, for example, cellulose ester, ethyl cellulose, cellulose ester, regenerated cellulose, polysulfone, polyacrylic nitrile, polymethylmethacrylate, ethylene vinyl alcohol copolymer, polyester polymer alloy, polycarbonate, polyamide, cellulose acetate, cellulose di It is composed of acetate, cellulose triacetate, copper ammonium rayon, saponified cellulose, hemophan film, phosphatidylcholine film, vitamin E coating film 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 the carbon dioxide permeable container 76, G-Rex (registered trademark, Wilson Wolf) can be used.

The inducer-introduced cells are placed in the dialysis tube 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 liquid feeding path for feeding a medium containing cells in the dialysis tube 75. Further, the dialysis tube 75 may be connected to a liquid feeding path for feeding the medium containing the cells in the dialysis tube 75 to the outside of the container.

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

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

However, the medium for stem cells is not limited to this, and various stem cell media can be used. For example, Prime ES Cell Medium, Reprotem, ReproFF, ReproFF2, ReproXF (Reprocell), mTeSR1, TeSR2, TeSRE8, ReproTeSR (STEMCELL Technologies), Pluristem (registered trademark) FF Culture Medium for Human iPS and ES Cells, Puriton reprogramming medium (Stemment), PluristEM (registered trademark), Stemfit AK02N, Stemfit AK02N, Stemfit AK03 (Ajinomoto) StemCell), L7 (registered trademark) hPSC Culture System (LONZA) and the like may be used.

Gel media include gellan gum, hyaluronic acid, lambzan gum, daiyutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectinic acid, pectinic acid, heparan sulfate, heparan, heparitin sulfate, keratosulfate, chondroitin sulfate, deltaman sulfate, ramnan sulfate, and It may contain at least one polymer compound selected from the group consisting of those salts. Further, the gel medium may contain methyl cellulose. By including methyl cellulose, aggregation between cells is further suppressed.

Alternatively, the gel medium is poly (glycerol monomethacrylate) (PGMA), poly (2-hydroxypropylcrylic) (PHPMA), Poly (N-isopropylacrylamide) (PNIPAM), amine terminated, carboxylic acid terminated, maleimide terminated, N-hydroxysuccinimide (N-hydroxysuccinimide ( NHS) ester terminated, triethoxysilane terminated, Poly (N-isopropylacrylamide-co-acrylamide), Poly (N-isopropylacrylamide-co-acrylic acid), Poly (N-isopropylacrylamide-co-butylacrylate), Poly (N-isopropylacrylamide-co- It may contain a small amount of temperature sensitive gel selected from methacrylic acid), Poly (N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-Isopropylacrylamide.

The gel medium placed in the dialysis tube 75 may not contain a ROCK inhibitor. In the gel medium placed around the dialysis tube 75 in the container 76, for example, the final concentration of the ROCK inhibitor is 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. Add daily so that the concentration is 20 μmol / L or less. By adding the ROCK inhibitor to the gel medium around the dialysis tube 75, the ROCK inhibitor permeates into the dialysis tube 75 and promotes colonization by cells.

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

During the suspension culture of the cells in the dialysis tube 75, the gel medium around the dialysis tube 75 in the container 76 is replaced. It should be noted that exchanging the medium includes partially replacing the medium or replenishing the medium. In this case, it is not necessary to replenish the gel medium in the dialysis tube 75. Alternatively, the gel medium is replenished in the dialysis tube 75 while the cells are suspended and cultured in the dialysis tube 75. In this case, it is not necessary to replenish the gel medium around the dialysis tube 75 in the container 76.

As shown in FIG. 21, the cell processing apparatus according to the embodiment uses a supplementary medium delivery pump 77 as a medium supplement device to replace or replenish the gel medium around the dialysis tube 75 in the container 76. .. As the supplementary medium delivery pump 77, a pump or the like used for infusion can be used. The replenishment medium feed pump 77 and the container 76 of the suspension incubator are connected by a feed tube 78. The replenishment medium feed pump 77 feeds the gel medium into the container 76 of the suspension incubator via the feed 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 suspension incubator is discharged through the waste liquid tube 79. The gel medium in the vessel 76 of the suspension incubator may be discharged by the pressure of the fresh gel medium replenished by the replenishment medium feed pump 77, or may be discharged by using gravity. It may be discharged by an discharge pump.

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

For example, the amount of gel medium sent into the container 76 of the suspension incubator by the supply medium feed pump 77 and the amount of gel medium discharged from the container 76 of the suspension incubator are replenished so as to be the same. The medium feed pump 77 is controlled. The replenishment medium feed pump 77 may constantly feed the gel medium into the container 76 of the suspension incubator, or may feed the gel medium at appropriate intervals.

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

Further, instead of constantly feeding the gel medium, the gel medium may be started and stopped depending on the state of the medium and the cell mass in the medium. In this case as well, the flow rate of the gel medium to be fed may be increased or decreased depending on the state of the medium and the cell mass in the medium.

If the flow rate of the gel medium sent to the incubator is too large, the 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 cells are continuously cultured without changing the medium, the accumulation of waste products such as lactic acid excreted by the cells and the change in pH may adversely affect the cell culture. In addition, various proteins such as bFGF and recombinant proteins contained in the medium are decomposed, and the components necessary for cell culture may be damaged from the medium.

On the other hand, by sending the fresh medium into the incubator with the supplementary medium delivery pump 77 and discharging the old medium from the incubator, waste products are removed from the incubator and the pH in the medium is appropriately adjusted. It is possible to keep the range within the range and supply the components necessary for cell culture. This makes it possible to keep the state of the medium close to a constant state.

Note that FIG. 21 shows an example in which the supplementary medium feed pump 77 and the container 76 of the suspension incubator are connected by the feed tube 78. On the other hand, as shown in FIG. 22, the replenishment medium feeding pump 77 and the inside of the dialysis tube 75 in the container 76 of the suspension incubator may be connected by the liquid feeding tube 78. By sending the fresh gel medium into the dialysis tube 75, the waste products contained in the medium in the dialysis tube 75 are discharged to the outside of the dialysis tube 75. Further, 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 the components necessary for cell culture.

The cell processing system may further include an initialization culture imaging device such as a photographic camera or a video camera for photographing the culture in the initialization culture apparatus 50 shown in FIG. Here, if a colorless medium is used as the medium used in the initialization culture apparatus 50, it is possible to suppress diffuse reflection and autofluorescence that may occur when a colored medium is used. However, a pH indicator such as phenol red may be contained to confirm the pH of the medium. Further, since the shape, size, etc. of the induced cells and the non-induced cells are different, the cell processing system captures the cells in the reprogramming culture apparatus 50 to obtain the induced cells. Further, a guidance state monitoring device for calculating the ratio may be provided. Alternatively, the induction condition monitoring device may specify the proportion of induced cells by antibody immunostaining method or RNA extraction method. Further, the cell treatment system may include an uninduced cell removing device that removes uninduced cells by a magnetic cell separation method, flow cytometry, or the like.

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

However, when the cells are suspended in a medium for suspension culture, the range of existence of the cells expands three-dimensionally, so that the distance from the imaging device to each cell in the optical axis direction varies. Therefore, it can be difficult to focus on all cells without using a lens.

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

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

Alternatively, as shown in FIG. 23, a telecentric lens 172 may be arranged between the initialization culture imaging device 171 and a subject such as a cell in a suspension incubator. Since the telecentric lens 172 makes the main light ray passing through the center of the lens diaphragm parallel to the optical axis of the lens from a subject such as a cell, the distance from the initialization culture imaging device 171 to each of a plurality of cells in the suspension incubator is one. Even if it does not look like this, the size of the cells imaged does not change with 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 initialization culture imaging device 171, a cell observation illumination light source 173 is arranged in a direction perpendicular to the optical axis of the initialization culture imaging device 171 or in a direction close to the imaging device from a direction perpendicular to the optical axis. It is preferable to use a scattered light illumination method for irradiating cells with illumination light from an illumination light source for cell observation 173. As a result, the scattered light from the illumination light that hits the cells reaches the reprogramming culture imaging device 171 but the illumination light that does not hit the cells passes through the medium and does not reach the reprogramming culture imaging device 171. Therefore, in the image, the medium portion becomes relatively dark and the cell portion becomes relatively bright. However, the illumination method is not limited to this as long as the cells can be recognized in the image. FIG. 25 is an example of an image of cells captured by the scattered light illumination method. The medium part is relatively dark and the cell part is relatively bright.

As shown in FIG. 26, the cell processing system according to the embodiment may include a central processing unit (CPU) 500 including an image processing unit 501 that processes an image captured by the initialization culture imaging device 171. .. An input device 401 such as a keyboard and a mouse, and an output device 402 such as a monitor may be connected to the CPU 500. The CPU 500 receives an image from the initialization culture imaging device 171 via a bus, an image interface, or the like.

The image processing unit 501 may include a contour defining unit 511 that defines the contour of the cell or cell mass in the image of the cell. FIG. 27 is an example of an image of an iPS cell mass imaged magnified through a macro zoom lens or the like. In the image shown in FIG. 27, the portion that looks like a white mass is the iPS cell mass, and the portion with a dark background is the medium.

Here, when the image shown in FIG. 27 is an 8-bit grayscale image, the luminance value of a pixel having a luminance value equal to or higher than a predetermined threshold value is set to the maximum luminance value such as 255, and is less than the predetermined threshold value. When a binarization process is added to the image in which the luminance value of the pixel having the luminance value of is replaced with the lowest luminance value such as 0, as shown in FIG. 28, not only the portion of the medium but also the cell mass The interior also becomes the lowest brightness black, and there may be a portion where the interior of the cell mass and the portion of the medium are connected. Therefore, it may not be possible to extract cells or cell masses by the binarization treatment.

On the other hand, the contour definition unit 511 of the cell processing system according to the embodiment shown in FIG. 26 passes a high frequency component having a predetermined frequency or higher included in the spatial frequency through the image of the cell, and has a low frequency lower than the predetermined frequency. A high-pass filter is applied that blocks the components and sets the brightness value to the lowest value, for example 0. In the image of the cell, the high frequency component contained in the spatial frequency is large in the cell or cell mass portion, and the high frequency component contained in the spatial frequency is small in the medium portion. Therefore, as shown in FIG. 29, in the image of the cell to which the high-pass filter is applied, the luminance value of the medium portion becomes the lowest value such as 0, and in the cell or cell mass portion, the luminance value becomes. The value remains the same. Therefore, the portion where the brightness does not reach the minimum value can be regarded as a cell or a cell mass.

Here, in the image shown in FIG. 29, even if the portion where the brightness does not become the minimum value is detected as a blob by blob analysis, for example, two cell masses in contact with each other are regarded as 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 watershed algorithm to the image to which the high-pass filter is applied. The diversion ridge algorithm considers the brightness gradient of the image as the undulations of the mountain, and treats the area where water flows from the high position (high brightness value position) to the low position (low brightness value position) of the mountain as one area. The image is divided.

For example, the contour definition unit 511 of the cell processing system according to the embodiment converts the image into an image by the Distance Transform method before applying the watershed algorithm to the image. The Distance Transfer method is an image conversion method in which the luminance value 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 region of the medium is collectively converted into the maximum luminance value of 255, and as shown in FIG. 30 (b). Make it a white background. Further, the brightness value of each pixel inside the cell region is converted to 0 or more and less than 255 according to the distance to the nearest background pixel. For example, the farther away from the nearest background pixel, the lower the luminance value.

Next, the contour definition unit 511 of the cell processing system according to the embodiment applies the watershed algorithm to the image converted by the Distance Transform method. In the image shown in FIG. 30 (b), when the dark part with low brightness is regarded as a mountain ridge and water is dropped from above in the direction perpendicular to the image, water is shown as shown by the arrow in FIG. 30 (c). As shown by the broken line in FIG. 30 (c), the place where water flowing from various directions collides with is regarded as a valley, and the cell region is divided at the bottom of the valley. do.

When the pixels of the cell region of the image shown in FIG. 29 are converted by the Distance Transform method, the image shown in FIG. 31 is obtained. When the watershed algorithm is applied to the image shown in FIG. 31, the image shown in FIG. 32 is obtained. By superimposing the obtained dividing line on the original image shown in FIG. 27, the image shown in FIG. 33 is obtained. In FIG. 33, the cell mass existing in each region divided by the dividing line can be regarded as one cell mass, not a mass in which a plurality of cell masses are in contact with each other. Therefore, in each region, as shown in FIG. 34, by extracting the outline 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. Further, for example, when the shape of one cell mass can be regarded as a circle, 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 medium may not reach the inside and the cells may differentiate. In addition, if the cell mass is too small and the culture is expanded without using a ROCK inhibitor, cell death or karyotype abnormality may occur. Therefore, the cell evaluation unit 512 may issue a warning when the size of an individual cell mass is out of an appropriate range. Further, the cell evaluation unit 512 may output that it is the timing to shift to the expanded culture when the size of each cell mass exceeds a predetermined threshold value. Further, the supply rate of the medium in the reprogramming culture apparatus 50 may be changed according to the calculated cell mass size. For example, the supply rate of the medium may be increased as the size of the cell mass increases.

The image processing unit 501 of the cell processing system according to the embodiment may further include a statistical processing unit 513 that statistically processes information obtained from the image processed image. FIG. 35 is an example in which the image shown in FIG. 25 is subjected to image processing to extract a portion of the cell mass and give an outline. FIG. 36 is an example of a histogram of cell mass size created based on the image shown in FIG. 35. In this way, by continuously and regularly acquiring cell information, it is possible to quantitatively grasp the growth degree, number, density, etc. of the cell mass, and stabilize the culture result. It is possible. Further, the supply rate of the medium in the reprogramming culture apparatus 50 may be changed according to the calculated number of cell clusters. For example, as the number of cell clusters increases, the feed rate of the medium 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 medium from the image of the medium, and calculates the density of the cell mass in the medium based on the turbidity of the medium. 514 may be further provided.

For example, a related storage device 403 including a volatile memory, a non-volatile memory, or the like is connected to the CPU 500. The relationship storage device 403 stores, for example, the relationship between the turbidity of the medium and the density of the cell mass in the medium, which is acquired in advance. The density calculation unit 514 reads out the relationship between turbidity and density from the relationship storage device 403. Further, 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. This makes it possible to measure the density of the cell mass non-destructively without collecting the cell mass from the medium.

Further, 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 becomes equal to or higher than a predetermined threshold value. Further, the density calculation unit 514 may calculate the density of the cell mass in the medium over time and calculate the growth rate of the cell mass. Abnormal growth rates may indicate that the cells are abnormal. For example, the density calculation unit 514 issues a warning when an abnormal growth rate is calculated. In this case, the cell culture may be stopped.

If the density of cell masses in the medium becomes high and the cell masses are too close to each other, a plurality of cell masses may adhere to each other to form one large cell mass. In a large cell mass, nutrients and hormones contained in the medium may not reach the 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 preferable 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, it is possible to calculate the density of the cell mass, so that it is possible to easily determine whether or not the density of the cell mass is within a suitable range. .. If the density of the cell mass becomes lower than the preferable range, it may be decided to stop the culture, for example. Further, the supply rate of the medium in the reprogramming culture apparatus 50 may be changed according to the calculated density of the cell mass. For example, the feed rate of the medium may be increased as the density of the cell mass increases.

Further, in order to observe the change in the color of the medium due to the metabolism of cells and the like, as shown in FIG. 37, a light source for observing the medium 174 is located at a position facing the initialization culture imaging device 171 with the suspension incubator in between. May be placed. As the medium observation illumination light source 174, for example, a surface light source can be used, and the medium observation illumination light source 174 emits, for example, white parallel light. Since the illumination light emitted by the illumination light source 174 for observing the medium passes through the medium and is incident on the initialization culture imaging apparatus 171, it is possible to image the color of the medium with the initialization culture imaging apparatus 171.

Normally, when cell culture is performed, the pH of the medium is set to a constant value in the vicinity of 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 varies with the pH of the medium. If the concentration of carbon dioxide in the gas in contact with the medium is not sufficient, the carbon dioxide derived from bicarbonate contained in the medium and the carbon dioxide in the gas do not balance, so the medium becomes alkaline and the color of the medium becomes magenta. Become. In addition, when waste products such as lactic acid excreted by cells accumulate, the medium becomes acidic and the color of the medium becomes yellow. The acidity of the medium also indicates that the nutrients in the medium are depleted.

The image processing unit 501 of the cell processing system according to the embodiment shown in FIG. 26 may further include a medium evaluation unit 515 that evaluates the medium based on the image of the medium illuminated by the illumination light source for medium observation. .. For example, the medium evaluation unit 515 processes an image of the medium and expresses the color of the medium by HSV with three parameters of hue, saturation, and brightness. Of these, hue is a parameter that corresponds to the concept generally called "hue" or "hue". Hue is generally expressed in units of angle.

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

For example, the relationship storage device 403 stores a previously acquired relationship between the hue of the medium and the pH of the medium. The medium evaluation unit 515 reads out the relationship between hue and pH from the relationship storage device 403. Further, the medium evaluation unit 515 calculates the pH value of the photographed medium based on the hue value of the medium calculated from the image of the medium and the relationship between the hue and the pH. For example, the medium evaluation unit 515 may acquire an image of the medium over time and calculate the pH value of the medium.

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

When the hue of the medium or the pH of the medium is out of the predetermined range, the medium evaluation unit 515 may determine that the exchange of the medium is promoted, or may determine that contamination has occurred in the medium. It should be noted that exchanging the medium includes partially replacing the medium or replenishing the medium.

Chemically analyzing the components of the medium is costly and may not maintain the sterility of the medium if the medium is removed from the system for chemical analysis. On the other hand, monitoring the state of the medium by monitoring the hue of the medium is low cost and does not affect the aseptic state of the medium.

When the medium evaluation unit 515 determines that the hue of the medium or the pH of the medium is outside the predetermined range, the medium around the dialysis tube 75 of the suspension incubator is replaced by, for example, the supplementary medium delivery pump 77 shown in FIG. Will be done. Alternatively, when the medium is constantly replaced, the rate of medium replacement around the dialysis tube 75 of the suspension incubator by the supplementary medium feed pump 77 is increased, and the flow rate of the replaced medium 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.

Further, the medium evaluation unit 515 may calculate the cell growth rate from the rate of change in the hue of the medium. For example, the relationship storage device 403 stores a previously acquired relationship between the rate of change in hue of the medium and the rate of cell proliferation. The medium evaluation unit 515 reads out the relationship between the hue change rate and the growth rate from the relationship storage device 403. Further, the medium evaluation unit 515 calculates the value of the cell growth rate based on the calculated value of the hue change rate and the relationship between the hue change rate and the growth rate.

Further, even if the medium evaluation unit 515 controls the temperature control device so as to change the temperature around the incubator or the temperature of the supplied medium when it is determined that the temperature of the medium is outside the predetermined range. good. For example, when the temperature of the medium is lower than a predetermined range, the medium evaluation unit 515 controls the temperature control device so that the temperature of the medium rises. When the temperature of the medium is higher than a predetermined range, the medium evaluation unit 515 controls the temperature control device so that the temperature of the medium drops.

The first cell mass feeding passage 51 is connected to the reprogramming culture apparatus 50 shown in FIG. The reprogramming culture apparatus 50 sends the trypsin alternative recombinant enzyme and the cell mass-containing solution to the first cell mass delivery channel 51 using a pump or the like. If the cell mass is physically disrupted, the trypsin alternative recombinant enzyme may not be present. Further, the first cell mass delivery channel 51 has an inner diameter through which only induced cells smaller than a predetermined size pass, and is connected to a branch flow path for removing uninduced cells larger than a predetermined size. May be. As described above, when a gel medium is used, the cell mass can be recovered by sucking up the gel medium.

The pump that sends out the solution containing the cell mass to the first cell mass delivery path 51 is, for example, when the value of the cell mass size calculated by the cell evaluation unit 512 shown in FIG. 26 becomes equal to or larger than a predetermined threshold value. May be driven. Alternatively, in the pump that sends the solution containing the cell mass to the first cell mass delivery path 51 shown in FIG. 16, for example, the value of the cell mass density calculated by the density calculation unit 514 shown in FIG. 26 has a predetermined threshold value. When the above is reached, it may be driven.

The inner wall of the first cell mass delivery channel 51 shown in FIG. 16 may be coated with poly-HEMA so that cells do not adhere to the inner wall, so that the cells do not adhere to each other. Alternatively, a material that is difficult for cells to adhere to may be used as the material of the first cell mass delivery passage 51. Further, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the first cell mass liquid delivery path 51, the conditions in the first cell mass delivery path 51 are controlled in the housing 601. Equivalent to the temperature and carbon dioxide concentration. Further, the first cell mass feeding passage 51 may be provided with a check valve from the viewpoint of preventing contamination.

The first cell mass delivery passage 51 is connected to the first partitioning mechanism 60. The first partitioning mechanism 60 includes, for example, a mesh. As the cell mass contained in the solution passes through the mesh by water pressure, it is divided into a plurality of cell masses having the size of each pore of the mesh. For example, if the size of each pore of the mesh is uniform, the size of the plurality of cell masses after division is also substantially uniform. Alternatively, the first partitioning mechanism 60 may include a nozzle. For example, by finely processing the inside of a substantially conical nozzle in a stepped shape, the cell mass contained in the solution is divided into a plurality of cell masses as it passes through the nozzle.

Alternatively, as shown in FIG. 39, the first partitioning mechanism 60 may include a cell mass divider having a terminal block 61, a connecting block 62, and an distal block 63. The terminal block 61, the connecting block 62, and the advanced block 63 are each provided with a through hole through which a medium containing a cell mass flows. As shown in FIGS. 40 and 41, the terminal block 61, the connecting block 62, and the tip block 63 are connected. The cell mass divider may include one connecting block 62 or a plurality of connecting blocks 62.

As shown in FIG. 39, a concave portion 62a is provided at the first end of the connecting block 62, and a convex portion 62b is provided at the second end facing the first end of the connecting block 62. The convex portion 62b is, for example, a columnar shape. As shown in FIGS. 40 and 41, when there are a plurality of connecting blocks 62, the convex portion 62b is fitted with the concave portion 62a of the adjacent connecting block 62. The side wall of the convex portion 62b shown in FIG. 39 may be smooth or may be provided with a male screw. The inner wall of the recess 62a may be smooth or may be provided with a female screw.

The through hole provided in the connecting block 62 communicates with the first large hole diameter portion 62c communicating with the recess 62a and the first large hole diameter portion 62c, and the small hole diameter portion having a smaller hole diameter than the first large hole diameter portion 62c. It has a second large hole diameter portion 62e that communicates with the small hole diameter portion 62d, has a larger hole diameter than the small hole diameter portion 62d, and has an opening at the tip of the convex portion 62b.

The cross-sectional shapes of the first large hole diameter portion 62c, the small hole diameter portion 62d, and the second large hole diameter portion 62e are, for example, circular. The hole diameter of the first large hole diameter portion 62c and the hole diameter of the second large hole diameter portion 62e are, for example, the same. As a result, as shown in FIGS. 40 and 41, there are a plurality of connecting blocks 62, and when the plurality of connecting blocks 62 are connected, the second large hole diameter portion 62e has the first large hole diameter of the adjacent connecting block 62. Smoothly communicates with the portion 62c.

The hole diameters of the first and second large hole diameter portions 62c and 62e shown in FIG. 39 are, for example, 2.0 mm or more and 4.0 mm or less, but are not particularly limited. The hole diameter of the small hole diameter portion 62d 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 continuous from the first large hole diameter portion 62c to the small hole diameter portion 62d. Further, a step is also formed in a portion continuous from the small hole diameter portion 62d to the second large hole diameter portion 62e. The side wall of the step may be perpendicular to the central axis of the through hole or may be inclined to less than 90 °.

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

A recess 63a is provided at the first end of the tip block 63 shown in FIG. 39, and a nozzle portion 63b is provided at the second end facing the first end of the tip block 63. When connecting the tip block 63 and the connecting block 62, the concave portion 63a of the tip block 63 fits with the convex portion 62b of the connecting 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 diameter portion 63c communicating with the recess 63a and the large hole diameter portion 63c, has a smaller hole diameter than the large hole diameter portion 63c, and has a small hole diameter having an opening at the tip of the nozzle portion 63b. It has a portion 63d and.

The cross-sectional shape of each 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 connecting block 62 are, for example, the same. As a result, as shown in FIGS. 40 and 41, when the connecting block 62 and the tip block 63 are connected, the second large hole diameter portion 62e of the connecting block 62 has a large hole diameter of the adjacent tip block 63. 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 continuous from the large hole diameter portion 63c to the small hole diameter portion 63d. The side wall of the step may be perpendicular to the central axis of the through hole or may be inclined to less than 90 °.

A concave portion 61a is provided at the first end of the terminal block 61, and a convex portion 61b is provided at the second end facing the first end of the terminal block 61. When connecting the terminal block 61 and the connecting block 62, the convex portion 61b of the terminal block fits with the concave portion 62a of the connecting block 62. The side wall of the convex portion 61b of the end block may be smooth or may be provided with a male screw.

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

The cross-sectional shape of each of the recess 61a and the large hole diameter portion 61c is, for example, a circle. The hole diameter of the large hole diameter portion 61c of the terminal block 61 and the hole diameter of the second large hole diameter portion 62e of the connecting block 62 are, for example, the same. As a result, as shown in FIGS. 40 and 41, when the terminal block 61 and the connecting block 62 are connected, the large hole diameter portion 61c of the terminal block 61 becomes the large hole diameter portion 62c of the adjacent connecting block 62. Communicate smoothly.

The hole diameter of the large hole diameter portion 61c shown in FIG. 39 is, for example, 2.0 mm or more and 4.0 mm or less, but is not particularly limited.

Examples of the materials of the terminal block 61, the connecting block 62, and the tip block 63 include, but are 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 terminal block 61. A suction discharger for sucking and discharging a medium containing a cell mass is connected to the insertion nozzle 64 directly or via 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 into the medium containing the cell mass, and the medium is sucked and discharged once with a suction discharger, or the medium is sucked and discharged once. When the suction and discharge are repeated, the medium containing the cell mass reciprocates in the through hole in the connecting block 62 and the tip block 63. Since the through holes in the connecting block 62 and the tip block 63 are provided with steps, the cell mass in the medium is efficiently divided into small cell masses.

Conventionally, the cell mass has been divided by a technician using a pipette man or the like. However, as shown in FIG. 47 (a), in the conventional method, the size of the divided cell mass was non-uniform. In addition, the size of the cell mass obtained by the technician varied. If the cell mass after division is too large, the nutrients and hormones contained in the medium may not reach the inside, and the cells may differentiate. In addition, if the cell mass is too small, cell death or karyotype abnormality may occur when the ROCK inhibitor is not used. On the other hand, when a cell mass divider as shown in FIGS. 40, 41, and 42 is used, the cell mass is divided into cell masses of uniform size as shown in FIG. 47 (b). It is possible. When dividing the cell mass using the cell mass divider, the medium may be trypsin, Trypsin Express (registered trademark, Thermo Fisher SCIENTIFIC), TrypLE Select (registered trademark, Thermo Fisher SCIENTIFIC), or TrypLE (registered trademark). It may contain an enzyme such as Thermo Fisher SCIENTIFIC). It is also possible to decompose the cell mass into a single cell by increasing the number of connecting blocks 62 or increasing the pressure at the time of suctioning and discharging the medium.

When an appropriate number of repetitions, length, etc. of the large pore diameter portion and the small pore diameter portion are determined, the cell mass divider does not have to be composed of a plurality of blocks. For example, the cell mass divider has an integrated columnar shape having a through hole inside as shown in FIG. 43, and the through hole through which the medium containing the cell mass flows has large pore diameter portions 65a, 65c, 65e. 65 g and the small hole diameter portions 65b, 65d, 65f which communicate with the large pore diameter portions 65a, 65c, 65e, 65g and have a smaller hole diameter than the large pore diameter portions 65a, 65c, 65e, 65g may be alternately provided. .. Also in this case, as shown in FIG. 44, the central axis of the large hole diameter portions 65a, 65c, 65e, 65g and the central axis of at least a part of the small hole diameter portions 65b, 65d, 65f may be offset. ..

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

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

The expansion culture device 70 can store a well plate inside. Further, the expansion culture device 70 includes a pipetting machine. The expansion culture apparatus 70 receives a solution containing a plurality of cell masses from the first partitioning mechanism 60, and distributes the solution to the wells by a pipetting machine. The expansion culture apparatus 70 distributes the cell mass to the wells, and then incubates the cell mass at 37 ° C. and 5% carbon dioxide, for example, for about 8 days. In addition, the expansion culture device 70 appropriately exchanges the medium.

After that, the expansion culture apparatus 70 adds a trypsin alternative recombinant enzyme such as TrypLE Select (registered trademark, Life Technologies) to the cell mass. Further, the expansion culture apparatus 70 puts a container containing the cell mass in an incubator and reacts the cell mass with the trypsin alternative recombinant enzyme at 37 ° C. and 5% carbon dioxide for 1 minute. If the cell mass is physically disrupted, the trypsin alternative recombinant enzyme may not be present. For example, the expansion culture apparatus 70 breaks a cell mass by pipetting with a pipetting machine. Alternatively, the expansion culture apparatus 70 passes the cell mass through a pipe provided with a filter or a pipe whose inner diameter is intermittently changed, similar to the introduced cell delivery path 31 shown in FIG. 17 or FIG. May be crushed. After that, the expansion culture apparatus 70 adds a medium such as a maintenance culture medium to the solution containing the cell mass. Further, in the case of adhesive 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 subjected to the first division mechanism 60 via the expansion culture feeding path 71. Send to.

The culture in the expansion culture device 70 may be performed in a carbon dioxide permeable bag instead of a well plate. Further, the culture may be an adhesive culture, a suspension culture, or a hanging drop culture. In the case of suspension culture, stirring culture may be performed. Further, the medium may be agar-like. Examples of the agar-like medium include gellan gum polymers and deacylated gellan gum polymers. When the agar-like medium is used, the cells do not sink or adhere, so that it is not necessary to stir even though it is a suspension culture.

The expansion culture device 70 may include a second medium replenishment device for replenishing the culture solution in a well plate or a carbon dioxide permeable bag. The second medium replenisher may collect the culture medium in the well plate or carbon dioxide permeable bag, filter the culture medium using a filter or dialysis membrane, and reuse the purified culture solution. good. At that time, a growth factor or the like may be added to the reused culture solution. Further, the expansion culture device 70 may further include a temperature control device for controlling the temperature of the medium, a humidity control device for controlling the humidity in the vicinity of the 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 permeable to carbon dioxide permeable to the culture solution. The culture solution may be put in the sex bags 302 and the bags 301 and 302. The initialization culture apparatus 50 prepares a plurality of bags 302 containing a fresh culture solution, and at predetermined intervals, a bag 302 containing a bag 301 containing cells is used as a bag containing a fresh culture solution. It may be replaced with 302.

Further, the culture method in the expansion culture device 70 is not limited to the above method, and a suspension incubator as shown in FIG. 20 may be used in the same manner as the culture method in the initialization culture device 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. The details of the suspension incubator are as described above. Further, also in the expansion culture apparatus 70, as shown in FIG. 21, the gel medium around the dialysis tube 75 in the container 76 is replaced or replenished by using the supplementary medium feed pump 77. Alternatively, as shown in FIG. 22, the supplementary medium feeding pump 77 and the inside of the dialysis tube 75 in the container 76 of the suspension incubator are connected by the liquid feeding tube 78, and the cells are connected to the medium in the dialysis tube 75. Ingredients necessary for culturing may be supplemented.

The cell processing system may further include an expansion culture imaging device for photographing the culture in the expansion culture apparatus 70 shown in FIG. Here, if a colorless medium is used as the medium used in the expansion culture device 70, it is possible to suppress diffuse reflection and autofluorescence that may occur when a colored medium is used. However, a pH indicator such as phenol red may be contained to confirm the pH of the medium. Further, 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 ratio of the induced cells by photographing the cells in the expansion culture apparatus 70. It may be further provided with a guidance state monitoring device for calculating. Alternatively, the induction condition monitoring device may specify the proportion of induced cells by antibody immunostaining method or RNA extraction method. Further, the cell treatment system may include an uninduced cell removing device that removes uninduced cells by a magnetic cell separation method, flow cytometry, or the like.

The expansion culture imaging device is the same as the initialization culture imaging device 171 shown in FIG. 23, and the culture in the expansion culture imaging device 70 may be imaged via the telecentric lens 172. The lighting method and the like when the magnifying culture imaging apparatus takes an image are the same as the lighting method and the like when the initialization culture imaging apparatus 171 takes an image, and are as described above.

The expanded culture imaging device is also connected to the CPU 500 including the image processing unit 501 shown in FIG. 26. 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 medium evaluation unit 515 is used for magnified culture imaging in the same manner as the image captured by the initialization culture imaging apparatus 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 expanded culture, if the cell mass grows too large, the nutrients and hormones contained in the medium may not reach the inside and the cells may differentiate. In addition, if the cell mass is passaged in a state of being 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 an individual cell mass is out of an appropriate range. Further, the cell evaluation unit 512 may output that it is the timing of passage when the size of each cell mass exceeds a predetermined threshold value. In this case, the cell mass may be crushed to make the individual cell mass smaller, and the passage may be resumed in the incubator again. Furthermore, it is also possible to determine whether or not the disruption was performed appropriately by calculating the size of each cell mass after disrupting the cell mass in the passage. Furthermore, the supply rate of the medium in the expansion culture apparatus 70 may be changed according to the calculated cell mass size. For example, the supply rate of the medium may be increased as the size of the cell mass increases.

Further, the supply rate of the 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 clusters increases, the feed rate of the medium may be increased.

When the density of the cell mass becomes equal to or higher than a predetermined threshold value, the density calculation unit 514 may output that it is the timing of passage. When the density of the cell mass becomes higher than the suitable range, it is possible to adjust the density of the cell mass within the suitable range, for example, by subculture. Furthermore, it is also possible to determine whether or not the disruption was performed appropriately by calculating the density of the cell mass after disrupting the cell mass in the passage. Further, the supply rate of the medium in the expansion culture apparatus 70 may be changed according to the calculated density of the cell mass. For example, the feed rate of the medium may be increased as the density of the cell mass increases.

When the medium evaluation unit 515 determines that the hue of the medium or the pH of the medium is outside the predetermined range, the dialysis tube of the suspension incubator is also used in the expansion culture device 70 by, for example, the supplementary medium delivery pump 77 shown in FIG. The medium around 75 is replaced. Alternatively, when the medium is constantly replaced, the rate of medium replacement around the dialysis tube 75 of the suspension incubator by the supplementary medium feed pump 77 is increased, and the flow rate of the replaced medium 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 division mechanism 60 shown in FIG. 16 is again cultured in the expansion culture apparatus 70. The division of the cell mass in the first division 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 that sends the solution containing the cell mass in the expansion culture device 70 to the first division mechanism 60 via the expansion culture delivery path 71 is, for example, the cell mass calculated by the cell evaluation unit 512 shown in FIG. 26. It may be driven when the value of the magnitude of is equal to or greater than a predetermined threshold value. Alternatively, in the pump that sends the solution containing the cell mass to the first cell mass delivery path 51 shown in FIG. 16, for example, the value of the cell mass density calculated by the density calculation unit 514 shown in FIG. 26 has a predetermined threshold value. When the above is reached, it may be driven.

A second cell mass feeding passage 72 is connected to the expansion culture apparatus 70. The expansion culture apparatus 70 sends out the liquid containing the cell mass that has been expanded and exfoliated from the container to the second cell mass delivery channel 72 by using a pump or the like. However, in the case of suspension culture, peeling is not necessary. The second cell mass delivery channel 72 has an inner diameter that allows only induced cells smaller than a predetermined size to pass through, and is connected to a branch flow path that removes uninduced cells larger than a predetermined size. May be good.

The inner wall of the second cell mass delivery channel 72 may be coated with poly-HEMA to prevent cells from adhering to the cells so that they are non-adhesive. Alternatively, a material that is difficult for cells to adhere to may be used as the material of the second cell mass delivery passage 72. Further, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the second cell mass liquid supply passage 72, the conditions in the second cell mass liquid feed passage 72 are controlled in the housing 601. Equivalent to the temperature and carbon dioxide concentration. Further, the second cell mass feeding passage 72 may be provided with a check valve from the viewpoint of preventing contamination.

The second cell mass delivery channel 72 is connected to the second division mechanism 80. The second dividing mechanism 80 includes, for example, a mesh. As the cell mass contained in the solution passes through the mesh by water pressure, it is divided into a plurality of cell masses having the size of each pore of the mesh. For example, if the size of each pore of the mesh is uniform, the size of the plurality of cell masses after division is also substantially uniform. Alternatively, the second split mechanism 80 may include a nozzle. For example, by finely processing the inside of a substantially conical nozzle in a stepped shape, the cell mass contained in the solution is divided into a plurality of cell masses as it passes through the nozzle.

Alternatively, the second partitioning mechanism 80, like the first partitioning mechanism 60, is a cell mass divider having the terminal block 61, the connecting block 62, and the tip block 63 shown in FIGS. 39 to 42, or from FIG. 43. It may be equipped with the integrated cell divider shown in FIG. Details of the cell mass divider are as described above.

A cell soul transport mechanism 90 that sequentially sends a plurality of cell souls to the packaging device 100 is connected to the second division mechanism 80 shown in FIG. A pre-packaged cell flow path 91 is connected between the cell soul transport mechanism 90 and the package device 100. The cell 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 flow path 91 by using a pump or the like.

The pre-packaged cell flow path 91 may be coated with poly-HEMA to prevent cells from adhering. Alternatively, a material that makes it difficult for cells to adhere may be used as the material of the pre-packaged cell flow path 91. Further, by using a material having good temperature conductivity and carbon dioxide permeability as the material of the pre-package cell flow path 91, the conditions in the pre-package cell flow path 91 can be controlled by the controlled temperature in the housing 601 and. Equivalent to carbon dioxide concentration. The cell flow path 91 in front of the package may be provided with a check valve from the viewpoint of preventing contamination.

A cryopreservation liquid delivery mechanism 110 is connected to the pre-package cell flow path 91. The cryopreservation liquid delivery mechanism 110 feeds the cell cryopreservation liquid into the pre-packaged cell flow path 91. As a result, the cell mass is suspended in the cell cryopreservation solution in the pre-packaged cell flow path 91.

The packaging device 100 sequentially freezes each of the plurality of cell masses sent through the pre-packaged cell flow path 91. For example, the packaging device 100 puts the cell mass in a cryopreservation container such as a cryotube each time it receives the cell mass, and momentarily freezes the cell mass solution at, for example, −80 ° C. or lower. If a cryopreservation container having a small surface area per volume is used, it tends to take a long time to freeze. Therefore, it is preferable to use a cryopreservation container having a large surface area per volume. By using a cryopreservation container having a large surface area per volume, it is possible to increase the survival rate of cells after thawing. The shape of the cryopreservation container includes, but is not limited to, a capillary shape and a spherical shape. Further, depending on the required survival rate of cells after thawing, it is not always necessary to perform instantaneous freezing.

For freezing, for example, a vitrification method is used. In this case, DAP213 (Cosmo Bio Co., Ltd.) and Freezing Medium (Reprocell Co., Ltd.) can be used as the cell cryopreservation solution. Freezing may be carried out by a usual method other than the vitrification method. In this case, as the cell cryopreservation solution, CryoDefend-Stem Cell (R & D System Co., Ltd.), STEM-CELLBANKER (registered trademark, Nippon Zenyaku Kogyo Co., Ltd.) and the like can be used. Freezing may be performed by liquid nitrogen or by a Pelche element. By using the Pelche element, it is possible to control the temperature change and suppress the temperature unevenness. If the frozen cells are for clinical use, the cryopreservation vessel is preferably a completely closed system. However, the packaging device 100 may package the stem cells in a storage container without freezing them.

Alternatively, in the packaging device 100, the solution of the cell mass may be replaced with a cryopreservation solution from the medium by using the solution substitution device 101 as shown in FIG. 48. Inside the solution substituent 101, a filter 102 provided with pores on the bottom surface through which cell clumps do not permeate is provided. Further, the solution exchanger 101 has a cell mass introduction hole to which a first liquid feeding flow path 103 for feeding a medium containing a cell mass is connected on the internal filter 102, and the cell mass is placed on the internal filter 102. A replacement solution introduction hole to which a second liquid feeding flow path 104 for sending a freezing liquid not contained is connected, and a first discharging flow path 105 for discharging the frozen liquid containing a cell mass is connected from the internal filter 102. A cell mass outflow hole is provided. Further, the solution exchanger 101 is provided with a waste liquid outflow hole to which a second discharge flow path 106 for discharging the solution that has passed through the filter 102 is connected. A tube or the like can be used for each of the first liquid feed flow path 103, the second liquid feed 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), in a state where the flow of the solution in the second discharge flow path 106 is stopped, the inside of the solution exchanger 101 is entered from the first liquid supply flow path 103. Put the medium containing the cell mass in. Next, as shown in FIG. 48 (c), the solution is allowed to flow in the second discharge flow path 106, and the medium is discharged from the solution exchanger 101. At this time, as shown in FIG. 48 (d), the cell mass remains on the filter 102. Further, as shown in FIGS. 48 (e) and 48 (f), in a state where the flow of the solution in the second discharge flow path 106 is stopped, the inside of the solution replacer 101 is entered from the second liquid supply flow path 104. The cryopreservation solution is put into the cryopreservation solution, and the cell mass is dispersed in the cryopreservation solution. Then, as shown in FIG. 48 (g), the cryopreservation solution containing the cell mass is discharged from the first discharge flow path 105. The cryopreservation solution containing the cell mass is sent to a cryopreservation container or the like via the first discharge flow path 105.

The solution separator 101 shown in FIG. 48 can be used not only for the replacement of the medium with the cryopreservation solution but also with the replacement of the old medium with the new medium. In this case, the second liquid feed flow path 104 feeds a new medium. Alternatively, the solution replacement device 101 can also be used to replace the medium with a solution containing a cell mass dividing enzyme when dividing the cell mass. 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 liquid feed flow path 104 feeds a solution containing a cell mass-dividing enzyme.

The cell processing system may further include a packaging process photographing apparatus for photographing the packaging process in the packaging apparatus 100 shown in FIG.

The cell treatment system was photographed by an operation record of a separation device 10, a pre-introduction cell delivery path 20, an inducer factor delivery mechanism 21, a factor introduction device 30, a cell mass preparation device 40, a package device 100, and the like, and an imaging device. The image may be transmitted to an external server by wire or wirelessly. In addition, on an external server, using a neural network, for example, conditions such as inducer introduction conditions, culture conditions, and freezing conditions, incomplete reprogramming of stem cells, failure of stem cell differentiation and proliferation, chromosomal abnormalities, etc. The relationship between the result and the result may be analyzed to extract the conditions that lead to the result, or the result may be predicted. Further, the external server controls the separation device 10, the inducing factor delivery mechanism 21, the factor introduction device 30, the cell mass preparation device 40, the packaging device 100, etc. of the cell processing system based on the standard working procedure (SOP). , Whether or not each device is operating may be monitored based on SOP, and an operation record of each device may be automatically generated.

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

The cell processing apparatus included in the cell processing system according to the embodiment is not limited to the configuration shown in FIG. For example, in the cell processing apparatus shown in FIG. 49, blood is sent from the blood storage section 201 to the mononuclear cell separation section 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 path 202 is, for example, a resin tube, a silicon tube, or the like. The same applies to the other liquid feeding channels described later. An identifier such as a barcode may be attached to the blood storage unit 201 to manage blood information. Pump 204 is used for liquid delivery. 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 the tubing pump include a perista pump (registered trademark, ATTO Co., Ltd.), and RP-Q1 and RP-TX (Takasago Electric Co., Ltd.). Examples of piezoelectric pumps include SDMP304, SDP306, SDM320, and APP-20KG (Takasago Electric Co., Ltd.). Further, a microfluidic chip module (Takasago Electric Co., Ltd.) in which various types of pumps are combined may be used. By using a closed pump such as a perista pump (registered trademark), a tubing pump, and a diaphragm pump, it is possible to feed the blood without the pump directly contacting the blood inside the blood feeding channel 202. The same applies to other pumps described later. Alternatively, a syringe pump may be used as the pump 204, and the pump 207, pump 216, pump 222, pump 225, pump 234, pump 242, and pump 252, which will be described later. Even pumps other than closed pumps can be reused by heat sterilization or the like.

The erythrocyte coagulant is sent from the separating agent storage unit 205 to the mononuclear cell separating unit 203 via the liquid feeding path 206 and the pump 207. As the separating agent storage unit 205, for example, a tube can be used. An identifier such as a barcode may be attached to the separating agent storage unit 205 to manage the information on the separating agent. As the erythrocyte coagulant, for example, HetaSep (registered trademark, STEMCELL Technologies), erythrocyte coagulant (Nipro) and the like can be used. In the mononuclear cell separation unit 203, erythrocytes are settled by the erythrocyte coagulant, and the 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 ball feeding path 208 and the pump 209. In the mononuclear cell purification filter 210, components other than mononuclear cells are removed, and a solution containing mononuclear cells is obtained. Examples of the mononuclear cell purification filter 210 include Purecell (registered trademark, PALL), Cellsorber E (Asahi Kasei Co., Ltd.), Sepacell PL (Asahi Kasei Co., Ltd.), Adacolumn (registered trademark, JIMRO), and Separation bag (Nipro Co., Ltd.). Etc. can be used.

In FIG. 49, a mononuclear cell separation unit 203, a separator storage unit 205, a mononuclear cell purification filter 210, 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 pre-introduction cell delivery path 211 and the pump 212. As the factor introduction unit 213, for example, a tube can be used. The pluripotent inducer is sent to the factor introduction unit 213 from the factor storage unit 214 containing the pluripotent inducer via the factor delivery channel 215 and the pump 216. As the factor storage unit 214, for example, a tube can be used. An identifier such as a barcode may be attached to the factor storage unit 214 to manage information on the pluripotent inducer. The factor storage unit 214, the pump 216, and the like constitute an inducing factor liquid feeding mechanism. In the factor introduction unit 213 as a factor introduction device, a pluripotent inducer is introduced into a cell by, for example, an RNA lipofection method, and an inducer-introduced cell is produced. However, the method of transfecting the inducing factor is not limited to the RNA lipofection method. For example, a Sendai virus vector containing a pluripotent inducer may be used. Alternatively, the pluripotent inducer may be a protein.

The inducer-introduced cells are sent to the reprogramming incubator 219 as part of the cell mass-producing apparatus via the introduced cell delivery path 217 and the pump 218. The introduced cell delivery path 217 is, for example, temperature permeable and carbon dioxide permeable. As the initialization incubator 219, the suspension incubator shown in FIG. 20 may be used. In this case, the inducer-introduced cells are placed in a dialysis tube. In the reprogramming incubator 219 shown in FIG. 49, the medium delivery channel 221 and the pump 222 were introduced from the blood cell medium storage unit 220 containing the blood cell medium for the first few days after the pluripotency-inducing factor was introduced into the cells. The blood cell medium is replenished through. The culture 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 medium storage unit 220 to manage information on the blood cell medium. The blood cell medium storage unit 220, the medium delivery path 221 and the pump 222 constitute a medium replenishment device. The pump 222 may be continuously replenished with the blood cell medium or may be replenished with the blood cell medium at a predetermined timing according to the instruction of the CPU 500 shown in FIG.

Then, the reprogramming incubator 219 shown in FIG. 49 is replenished with the stem cell medium from the stem cell medium storage unit 223 containing the stem cell medium via the 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 culture 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 replenishment device. The pump 225 may be continuously replenished with the stem cell medium or may be replenished with the stem cell medium at a predetermined timing according to the instruction of the CPU 500 shown in FIG.

The blood cell medium storage unit 220 and the stem cell medium storage unit 223 may be refrigerated and stored at a low temperature such as 4 ° C. in the refrigerated storage unit 260, for example. The medium sent from the blood cell medium storage unit 220 and the stem cell medium storage unit 223 may be, for example, heated to 37 ° C. by a heater outside the refrigerated storage unit 260 and then sent to the incubator. Alternatively, the medium stored at a low temperature may be set to a temperature around the liquid feeding passage so that the temperature rises to 37 ° C. while proceeding through the liquid feeding passage. The old medium in the initialization incubator 219 is sent to the waste liquid storage unit 228 via the waste liquid delivery passage 226 and the pump 227. An identifier such as a barcode may be attached to the waste liquid storage unit 228 to manage the waste liquid information.

The cell mass cultured in the reprogramming incubator 219 is sent to the first expansion incubator 232 as a part of the cell mass producing apparatus via the introduction cell delivery path 229, the pump 230, and the cell mass divider 231. Sent. The cell mass divider 231 may include, for example, the configuration shown in FIG. 45 or FIG. 46. 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, the suspension incubator shown in FIG. 20 may be used. In this case, the cell mass is placed in a dialysis tube. The first expanded incubator 232 shown in FIG. 49 is replenished with the stem cell medium from the stem cell medium storage unit 223 containing the stem cell medium via the medium delivery channel 233 and the pump 234. The introduced cell delivery path 229 and the medium delivery path 233 are, for example, temperature permeable and carbon dioxide permeable. The stem cell culture medium storage unit 223, the culture medium delivery channel 233, and the pump 234 constitute a culture medium replenishment device. The pump 234 may be continuously replenished with the stem cell medium or may be replenished with the stem cell medium at a predetermined timing according to the instructions of the CPU 500 shown in FIG.

The old medium in the first expansion incubator 232 shown in FIG. 49 is sent to the waste liquid storage unit 228 via the waste liquid delivery passage 235 and the pump 236.

The cell mass cultured in the first expansion incubator 232 passes through the introduced cell delivery path 237, the pump 238, and the cell mass divider 239 to the second expansion incubator 240 as a part of the cell mass preparation device. Sent. The cell mass divider 239 may include, for example, the configuration shown in FIG. 45 or FIG. By passing through the cell mass divider 239, the cell mass is divided into smaller cell masses. As the second expansion incubator 240 shown in FIG. 49, the suspension incubator shown in FIG. 20 may be used. In this case, the cell mass is placed in a dialysis tube. The second expanded incubator 240 shown in FIG. 49 is replenished with the stem cell medium from the stem cell medium storage unit 223 containing the stem cell medium via the medium delivery channel 241 and the pump 242. The introduced cell delivery path 237 and the medium delivery path 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 be continuously replenished with the stem cell medium or may be replenished with the stem cell medium at a predetermined timing according to the instruction of the CPU 500 shown in FIG.

The old medium in the second expanded incubator 240 shown in FIG. 49 is sent to the waste liquid storage unit 228 via the waste liquid delivery passage 243 and the pump 244.

The cell mass cultured in the second expansion incubator 240 is sent to the solution replacement device 247 via the introduction cell delivery path 245 and the pump 246. The solution substituent 247 may include, for example, the configuration shown in FIG. 48. In the solution exchanger 247 shown in FIG. 49, the cell mass is held by a filter, and the medium is sent to the waste liquid storage unit 228 via the waste liquid delivery path 248 and the pump 249.

After stopping the flow of the solution in the waste liquid feed passage 248 by stopping the drive of the pump 249, or after closing the waste liquid feed passage 248 with a valve or the like, the solution exchanger 247 contains a cryopreservation liquid containing a cryopreservation liquid. The cryopreservation liquid is put into the storage unit 250 via the liquid feed path 251 and the pump 252. As a result, the cell mass is dispersed in the cryopreservation solution.

The cryopreservation solution in which the cell mass is dispersed is sent into the cryopreservation container 255 via the liquid delivery path 253 and the pump 254 as a part of the packaging device. The cryopreservation container 255 is stored in a low temperature storage 256. Liquid nitrogen at, for example, −80 ° C. is sent from the liquid nitrogen storage 257 to the low temperature storage 256 via the liquid supply passage 258. As a result, the cell mass in the cryopreservation container 255 is frozen. However, the freezing of the cell mass does not have to depend on liquid nitrogen. For example, the low temperature storage 256 may be a freezer such as a compression type freezer, an absorption type freezer, or a Pelche type freezer.

A check valve may be appropriately provided in the above-mentioned liquid feed path. The above-mentioned liquid feed path, mononuclear cell separation section 203, mononuclear cell purification filter 210, factor introduction section 213, initialization incubator 219, first expansion incubator 232, second expansion incubator 240, and solution replacement. The vessel 247 and the like are arranged on the member 259, for example. The material of the member 259 is, for example, a resin, but the material is not limited thereto. The member 259 is made of, for example, a heat-resistant material that can be sterilized. The shape of the member 259 may be a plate shape or a cassette shape. Further, the member 259 may be a flexible bag. The liquid delivery channel through which the medium flows is made of, for example, a carbon dioxide permeable material. Further, the above-mentioned liquid feeding path, mononuclear cell separation section 203, mononuclear cell purification filter 210, factor introduction section 213, initialization incubator 219, first expansion incubator 232, second expansion incubator 240, and The solution substituent 247 and the like may be divided and stored in a plurality of cases.

The member 259 and the housing 601 shown in FIG. 1 include, for example, a fitting portion that fits each other. Therefore, the member 259 shown in FIG. 49 is arranged at a predetermined position in the housing 601 shown in FIG. Further, 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 medium storage unit 220, the stem cell medium storage unit 223, the waste liquid storage unit 228, and the cryopreservation unit are cryopreserved. The container 255, the low temperature storage 256, and the liquid nitrogen storage 257 are arranged in predetermined positions. When the member 259 is arranged at a predetermined position in the housing 601 shown in FIG. 1, the liquid feeding passage 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. It is in contact with 214, a blood cell medium storage section 220, a stem cell medium storage section 223, a waste liquid storage section 228, a cryopreservation container 255, a low temperature storage section 256, and a liquid nitrogen storage section 257.

For example, the members 259 and the flow paths provided in the members 259 are disposable, and after the cell mass has been frozen, they may be discarded and replaced with new ones. Alternatively, when the flow path or the like provided in the member 259 and the member 259 is reused, an identifier such as a barcode may be attached to the member 259 to manage the number of times of use and the like.

According to the cell treatment system according to the embodiment described above, it is possible to automatically treat a frozen storage product of stem cells such as iPS cells from blood without human intervention.

Further, 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 an outer and outer embedding member 2000. It may be provided with a communication liquid passage for communicating with the cell culture device 900 in the embedding member 2000. In addition, in FIG. 51 and FIG. 52, the embedding member 2000 is omitted.

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

For example, in a cell processing apparatus, a blood delivery path is provided from a blood storage unit 1201 that stores blood, which is arranged outside the embedding member 2000, to a mononuclear cell separation unit 1203, which is arranged inside the embedding member 2000. 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 path 1202 is, for example, a resin tube, a silicon tube, or the like. The same applies to the other liquid feeding channels described later. Blood information may be managed by attaching an identifier such as a barcode to the blood storage unit 1201.

For liquid feeding, a drive unit 1204 such as a pump that can be attached to the outer wall of the embedding member 2000 is used. A positive displacement pump can be used as the drive unit 1204. 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 the tubing pump include a perista pump (registered trademark, ATTO Co., Ltd.), and RP-Q1 and RP-TX (Takasago Electric Co., Ltd.). Examples of piezoelectric pumps include SDMP304, SDP306, SDM320, and APP-20KG (Takasago Electric Co., Ltd.). Further, a microfluidic chip module (Takasago Electric Co., Ltd.) in which various types of pumps are combined may be used. By using a closed pump such as a perista pump (registered trademark), a tubing pump, and a diaphragm pump, it is possible to feed the blood without the pump directly contacting the blood inside the blood feeding channel 1202. The same applies to other pumps described later. Alternatively, a syringe pump may be used as the drive unit 1204 and the drive unit described later. Even pumps other than closed pumps can be reused by heat sterilization or the like.

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

The mononuclear cell separation unit 1203 arranged inside the embedding member 2000 shown in FIGS. 50, 51 and 52 has a separating agent liquid feed path from the separating agent storage unit 1205 arranged outside the embedding member 2000. A separating agent for separating mononuclear cells such as a red blood cell coagulant is sent via a driving unit 1207 that can be attached to the outer wall of 1206 and the embedding member 2000. As the separating agent storage unit 1205, for example, a tube can be used. Information on the separating agent may be managed by attaching an identifier such as a barcode to the separating agent storage unit 1205. As the erythrocyte coagulant, for example, HetaSep (registered trademark, STEMCELL Technologies), erythrocyte coagulant (Nipro) and the like can be used.

In the mononuclear cell separator 1203, erythrocytes are settled by the erythrocyte coagulant, and the mononuclear cells are separated. The supernatant containing the mononuclear cells in the mononuclear ball separation unit 1203 is via a mononuclear ball feeding path 1208 arranged inside the embedding member 2000 and a drive unit 1209 that can be attached to the outer wall of the embedding member 2000. It is sent to the mononuclear cell purification filter 1210. Sedimentation components and the like generated in the mononuclear cell separation unit 1203 are sent from the embedding member 2000 to the waste liquid storage unit 4228 arranged outside the embedding member 2000 via a waste liquid delivery path communicating with the outside. ..

In the mononuclear cell purification filter 1210, components other than mononuclear cells are removed, and a solution containing mononuclear cells is obtained. Examples of the mononuclear cell purification filter 1210 include Purecell (registered trademark, PALL), Cellsorber E (Asahi Kasei Co., Ltd.), Sepacell PL (Asahi Kasei Co., Ltd.), Adacolumn (registered trademark, JIMRO), and Separation bag (Nipro Co., Ltd.). Etc. can be used.

In FIG. 52, a mononuclear cell separation unit 1203, a separator storage unit 1205, a mononuclear cell purification filter 1210, a drive unit 1204, 1207, 1209, etc. constitute a separation device. The solution containing the mononuclear cells separated by the mononuclear cell purification filter 1210 passes through the mononuclear cell flow path 2001 arranged inside the embedding member 2000 connected to the mononuclear cell purification filter 1210 and is mononuclear. It may be sent to the mononuclear cell storage unit 2002 arranged inside the embedding member 2000 connected to the sphere flow path 2001. The solution containing the components other than the mononuclear cells separated by the mononuclear cell purification filter 1210 passes through the other component flow path 2003 arranged inside the embedding member 2000 connected to the mononuclear cell purification filter 1210. It may be sent to the other component storage unit 2004 arranged inside the embedding member 2000 connected to the other component flow path 2003.

The solution containing mononuclear cells is placed inside the embedding member 2000 via a pre-introduction cell delivery path 1211 arranged inside the embedding member 2000 and a drive unit 1212 that can be attached to the outer wall of the embedding member 2000. It is sent to the factor introduction unit 1213. 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. As the factor introduction unit 1213, for example, a tube can be used. In the factor introduction unit 1213, at least a part of the factor storage unit 1214 containing the pluripotent inducing factor, which is arranged outside the embedding member 2000, is provided inside the embedding member 2000. , And the pluripotency inducing factor is sent via the drive unit 1216 located outside the embedding member 2000. As the factor storage unit 1214, for example, a tube can be used. An identifier such as a barcode may be attached to the factor storage unit 1214 to manage information on the pluripotent inducer. The factor storage unit 1214, the drive unit 1216, and the like constitute an inducing factor liquid feeding mechanism.

In the factor introduction unit 1213 as at least a part of the factor introduction device, the pluripotent inducer is introduced into the cell by, for example, the RNA lipofection method, and the inducer introduction cell is produced. However, the method of transfecting the inducing factor is not limited to the RNA lipofection method. For example, a Sendai virus vector containing a pluripotent inducer may be used. Alternatively, the pluripotent inducer may be a protein. In addition, examples of the transfection method include a transfection method using an episomal plasmid and an electroporation method. The waste liquid generated in the factor introduction unit 1213 is sent from the inside of the embedding member 2000 to the waste liquid storage unit 5228 arranged outside the embedding member 2000 via a waste liquid delivery path communicating with the outside.

The inducer-introduced cell is used as a part of the cell mass producing apparatus via the introduced cell delivery path 1217 provided inside the embedding member 2000 and the driving unit 1218 arranged outside the embedding member 2000. It is sent to the initialization incubator 1219. The initialization incubator 1219 is embedded in the embedding member 2000. As the initialization incubator 1219, the suspension incubator shown in FIG. 20 may be used. In this case, the inducer-introduced cells are placed in a dialysis tube. In the reprogramming incubator 1219 shown in FIG. 52, a blood cell medium storage unit 1220 containing a blood cell medium placed outside the embedding member 2000 for the first few days after the pluripotency-inducing factor was introduced into the cells. The blood cell medium is replenished from the medium through a medium delivery channel 1221 provided at least partially inside the embedding member 2000 and a drive unit 1254 arranged outside the embedding member 2000. An identifier such as a barcode may be attached to the blood cell medium storage unit 1220 to manage information on the blood cell medium. The blood cell medium storage unit 1220, the medium delivery path 1221, and the drive unit 1254 constitute a medium replenishment device. The drive unit 1254 may continuously replenish the blood cell medium or may replenish the blood cell medium at a predetermined timing according to the instruction of the CPU 500 shown in FIG. 26.

After that, in the initialization incubator 1219 shown in FIG. 52, a medium delivery path provided inside the embedding member 2000 from the stem cell medium storage unit 1223 containing the stem cell medium arranged outside the embedding member 2000, And the stem cell medium is replenished via a drive unit located 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 path, and the drive unit constitute a culture medium replenishment device. The driving unit may continuously replenish the stem cell medium or may replenish the stem cell medium at a predetermined timing according to the instruction of the CPU 500 shown in FIG. 26.

The blood cell medium storage unit 1220 and the stem cell medium storage unit 1223 may be refrigerated and stored at a low temperature such as 4 ° C. in the refrigerated storage unit, for example. The medium sent from the blood cell medium storage unit 1220 and the stem cell medium storage unit 1223 may be, for example, heated to 37 ° C. by a heater outside the refrigerating storage unit and then sent to the incubator. Alternatively, the medium stored at a low temperature may be set to a temperature around the liquid feeding passage so that the temperature rises to 37 ° C. while proceeding through the liquid feeding passage. The old medium in the initialization incubator 1219 is sent from the embedding member 2000 to the waste liquid storage unit 1228 arranged outside the embedding member 2000 via the waste liquid delivery passage 1226 communicating with the outside. .. The waste liquid storage unit 1228 may be assigned an identifier such as a barcode to manage waste liquid information.

The cell mass cultured in the reprogramming incubator 1219 has an introduced cell delivery path 1229 provided inside the embedding member 2000, a drive unit 1230 arranged outside the embedding member 2000, and the inside of the embedding member 2000. One of the cell mass producing devices via the cell mass divider 1231 provided in the cell mass dividing device 1231, the driving unit 1218 arranged outside the embedding member 2000, and the introduced cell liquid supply path 2229 provided inside the embedding member 2000. It is sent to the 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 FIG. 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, the suspension 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 is arranged outside the embedding member 2000 and communicates from the outside to the inside of the embedding member 2000 from the stem cell medium storage unit 1223 containing the stem cell medium, And the stem cell medium is replenished via the drive unit 1222 arranged 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 replenishment device. The driving unit may continuously replenish the stem cell medium or may replenish the stem cell medium at a predetermined timing according to the instruction of the CPU 500 shown in FIG. 26.

The old medium in the expansion incubator 1232 shown in FIG. 52 is arranged outside the embedding member 2000 via the waste liquid feeding passage 1235 communicating from the inside of the embedding member 2000 to the outside. Will be sent to.

The cell mass cultured in the expansion incubator 1232 is provided inside the introduced cell delivery path 1245 provided inside the embedding member 2000, the drive unit 1246 that can be attached to the outer wall of the embedding member 2000, and the embedding member 2000. It is sent to the solution replacer 1247 provided inside the embedding member 2000 via the cell mass divider 2231. The solution substituent 1247 may include, for example, the configuration shown in FIG. 48. In the solution substituent 1247 shown in FIG. 52, the cell mass is held by the filter, and the medium is placed outside the embedding member 2000 via the waste liquid delivery path 1248 communicating from the inside of the embedding member 2000 to the outside. It is sent to the waste liquid storage unit 3228.

After closing the waste liquid feed path 1248 with a valve or the like, the solution exchanger 1247 is placed outside the embedding member 2000 from the cryopreservation liquid storage unit 1250 containing the cryopreservation liquid, which is arranged outside the embedding member 2000. The cryopreservation liquid is put into the cryopreservation liquid through the cryopreservation liquid delivery passage 1251 and the drive unit 1252 that can be attached to the outer wall of the embedding member 2000. As a result, the cell mass is dispersed in the cryopreservation solution.

The cryopreservation solution in which the cell mass is dispersed is placed in the cryopreservation container 1255 via a freezing cell delivery path 1253 that communicates from the inside of the embedding member 2000 to the outside and a drive unit 1209 that can be attached to the outer wall of the embedding member 2000. Will be sent within. The liquid delivery path 1253, the drive unit 1254, and the cryopreservation container 1255 form a part of the packaging device. The cryopreservation container 1255 is then transferred into a cold storage.

The cell processing apparatus shown in FIGS. 50 to 52 connects the factor introduction unit 1213, the reprogramming incubator 1219, the expansion incubator 1232, etc. provided in the above-mentioned cell culture device 900, and then embeds them. The embedding member 2000 is molded and processed. Alternatively, the cell processing apparatus may form a factor introduction unit 1213, an initialization incubator 1219, an expansion incubator 1232, etc. provided in the cell culture apparatus 900 inside the embedding member 2000 by an apparatus such as a 3D printer. .. By embedding the factor introduction unit 1213, the initialization incubator 1219, the expansion incubator 1232, etc. included in the cell culture apparatus 900 with the embedding member 2000, the cells being treated and the induction introduced into the cells. It is possible to suppress the diffusion of factors and the like to the outside. On the contrary, it is possible to prevent external contaminants from coming into contact with the cells being treated.

As mentioned above, the invention has been described by embodiment, but the description and drawings that form part of this disclosure should not be understood to limit the invention. This disclosure should reveal to those skilled in the art various alternative embodiments, embodiments and operational techniques. For example, the factor introduction device 30 may induce cells by transfection using a viral vector such as retrovirus, lentivirus, and Sendaivirus, transfection using a plasmid, or protein transfection instead of electroporation or RNA lipofection. good. In addition, cells may be induced by introducing a compound as a factor. The pre-introduction cell delivery path 20, the introduced cell delivery path 31, the cell mass delivery path 51, the expanded culture delivery path 71, the cell mass delivery path 72, and the pre-package cell flow path 91 are substrates by microfluidics technology. It may be provided on the top. As described above, it should be understood that the present invention includes various embodiments not described here.

(Example 1)
(Preparation)
Human blood cells were obtained from healthy adult males. In addition, modified mRNA (TriLink), non-adherent dish, 15 mL tube, 50 mL tube, Ficol, cytoflow meter (BD), antibody against CD34 (Miltenyi Biotec), antibody against CD3 (Miltenyi Biotec), MACS (registered trademark). Antibodies (BD) to buffers (Miltenyi Biotec), T cell media, low serum media (Opti-MEM®, Gibco), siRNA-introducing reagents (Lipofectamine® RNAiMAX, Thermo Fisher Science), and TRA-1-60. I prepared.

The T cell (CD3 positive cell) medium was a mixture of A medium and B medium below. Medium A was a mixture of 15 mL of X vivo-10 (Lonza, 04-743Q) and IL-2 (10 μg / mL). For B medium, mix X vivo-10 and 50 μL of Dynabeads CD3 / CD28 (Life Technologies, 111-31D) in a 1.5 mL tube, vortex for 5 seconds, spin down, and place in DynaMag-2 (Thermo Fisher Science). After allowing to stand for 1 minute, the supernatant was removed.

Also, 10 μL IL-6 (100 μg / mL), 10 μL SCF (300 μg / mL), 10 μL TPO (300 μg / mL), and 10 μL Flt3 ligand (300 μg) in 10 mL serum-free medium (StemSpan H3000, STEMCELL Technologies). / ML) and 10 μL of IL-3 (10 μg / mL) were added to prepare a blood cell medium (blood stem / progenitor cell medium).

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) having a concentration of 100 ng / μL, respectively. The mRNA-containing solution of 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 mixture. The obtained reprogramming factor mixture was dispensed into RNase-Free tubes (Eppendorf tube (registered trademark), Eppendorf) having a volume of 1.5 mL each in an amount of 50 μL, and stored in a freezer at -80 ° C.

(Preparation of mononuclear cells)
The centrifuge was set to 18 ° C. 5 mL to 50 mL of blood was collected, EDTA was added to the blood and mixed gently. In addition, 5 mL each of a medium for separating human lymphocytes (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 layered 5 mL each on a medium for human lymphocyte separation in a tube. At this time, diluted blood was slowly added onto the medium along the tube wall of the tube so as not to disturb the interface.

The solution in the tube was centrifuged at 400 xg at 18 ° C. for 30 minutes. At this time, both acceleration and deceleration were carried out slowly. After centrifugation, a cloudy white intermediate layer appeared in the tube. This cloudy middle layer contains mononuclear cells. The white turbid middle layer in the tube was slowly recovered with Pipetman and transferred to a new 15 mL tube. At this time, the lower layer was not sucked. About 1 mL of the white turbid middle layer could be recovered from one tube. The two intermediate layers were transferred together into one tube.

To the recovered mononuclear cells, 12 mL of PBS was added, and the solution was further centrifuged at 200 × g at 18 ° C. for 10 minutes. Then, the supernatant of the solution is aspirated and removed using an aspirator, and 3 mL of a serum-free hematopoietic cell medium (X-VIVO® 10, Ronza) having a known composition is added and suspended, and the monocyte suspension is added. Got A 10 μL mononuclear cell suspension was stained with trypan blue and counted on a hemocytometer.

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

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

(Initialization factor lipofection)
A 100 μL low serum medium (Opti-MEM®, Gibco) and 25 μL of the reprogramming factor mixture were mixed to obtain a first mixture. In addition, 112.5 μL of low serum medium (Opti-MEM (registered trademark), Gibco) and 12.5 μL of siRNA introducing reagent (Lipofectamine (registered trademark) RNAiMAX, Thermo Fisher Science) were mixed to prepare a second mixed solution. Then, the first mixed solution and the second mixed solution were mixed and allowed to stand at room temperature for 15 minutes to prepare a lipofection reaction solution.

60 μL of the obtained lipofection reaction solution was gently added to a 12-well plate in which mononuclear cells were cultured, and then the mononuclear cells were cultured at 37 ° C. for 18 hours in a feeder-free manner. The culture conditions were a CO ₂ concentration of 5%, an oxygen concentration of 19%, and a temperature of 37 ° C. 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 and centrifuged at 300 g to remove the supernatant. Then, 1.25 mL of blood cell medium for CD34 was added to a 15 mL tube, the mononuclear cell suspension was returned to the same 12-well plate, and the mononuclear cells were cultured overnight at 37 ° C. in a feeder-free manner. The culture conditions were a CO ₂ concentration of 5% and an oxygen concentration of 19%. The above process was repeated once every 2 days for 7 days.

(Confirmation of GFP expression)
On the 7th 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 was taken out from the 12-well plate and the expression of GFP was confirmed with a fluorescence microscope, the expression of GFP was confirmed as shown in FIG. 55. This confirmed that mRNA was transfected into mononuclear cells and that protein was synthesized from the transfected mRNA.

(Confirmation of expression of TRA-1-60)
On the 7th day after the start of lipofection, a part of the cells was removed from the 12-well plate, and the removed cells were subjected to TRA-1-60, which is a surface antigen specifically expressed in iPS cells that had begun to be reprogrammed. The antibody was stained with an antibody labeled with an Allophycocyanin (APC) fluorescent dye. Then, by confirming the proportion of TRA-1-60 positive cells with a fluorescence activated cell sorter (FACS®, BD), reprogramming is started in the cells, the iPS cell gene is expressed, and the iPS cells are expressed. I confirmed that it would be possible.

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 was taken on the y-axis. No TRA-1-60 positive cells were detected in the negative control without gene transfer. On the other hand, in Experiments 1, 2 and 3, TRA-1-60 positive cells were detected. In Experiment 1, the result was derived from the whole mononuclear sphere that was not separated by the marker, Experiment 2 was the result derived from the cells isolated in CD3 positive, and Experiment 3 was the result in which the cells were isolated in CD34 positive. It is the result derived from cells. Therefore, it was shown that the lipofection of reprogramming factor RNA can be used to introduce reprogramming factors into blood-derived cells and induce iPS cells.

(Example 2)
A human iPS medium containing bFGF was prepared by mixing 500 mL of Primate ES Cell Medium (ReproCELL) and bFGF (Gibco PHG0266) having a concentration of 0.2 mL of 10 μg / mL.

Further, deacylated gellan gum (Nissan Chemical Industries, Ltd.) was added to the bFGF-containing human iPS medium so as to have a concentration of 0.02% by weight to prepare a bFGF-containing human iPS gel medium. In addition, 5 mL of trypsin at a concentration of 2.5 wt%, 5 mL of Collagenase IV at a concentration of 1 mg / mL, CaCl2 at a concentration of 0.5 mL of 0.1 mol / L2, and 10 mL of KnockOut serum replacement (registered trademark, Invitrogen 10828). -028) and 30 mL of purified water were mixed to prepare a stripping solution generally called a CTK solution.

300 μL of CTK solution was added to 6 well dishes (Thermoscientific 12-556-004) in which iPS cells were cultured on feeder cells, and the cells were incubated in a CO ₂ incubator for 3 minutes. After 3 minutes, the dish was removed from the incubator, it was confirmed that only the feeder cells were peeled off, and the CTK solution was removed using an aspirator. After removing the CTK solution, 500 μL of PBS (Santa Cruz Biotech sc-362183) was added to the 6-well dish, the iPS cells were washed, the PBS was removed from the 6-well dish, and 0.3 mL of the stripping solution (Incubator, registered trademark) was added. ) Was added to a 6-well dish, placed in a CO ₂ incubator and incubated for 5 minutes. Then, 0.7 mL of bFGF-containing iPS medium was added to the 6-well 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 number of cells was calculated using a hemocytometer. After cell calculation, 5 × 10 ⁵ iPS cells were seeded in a 15 mL Falcon tube (registered trademark, Corning 352096) or a non-adhesive dish, and subsequently cultured in suspension without stirring.

In the 15 mL tube, 2 mL of bFGF-containing human iPS gel medium was used. For non-adhesive dishes, 2 mL of non-gelled bFGF-containing human iPS medium was used. A ROCK inhibitor (Selleck S1049) was added to each medium so as to be 10 μmol / L. Then, 500 μL of bFGF-containing human iPS gel medium was added daily to the 15 mL tube and non-adhesive dish, and 500 μL of bFGF-containing human iPS medium was added daily to the non-adhesive dish. In addition, a ROCK inhibitor was added to a 15 mL tube and a non-adhesive dish every day so that the final concentration was 10 μmol / L, and suspension culture was continued for 7 days.

The result is shown in FIG. 57. As shown in FIG. 57 (b), when iPS cells were cultured using bFGF-containing human iPS medium which was not gelled with a non-adhesive dish, aggregation of iPS cell colonies was remarkably observed. On the other hand, as shown in FIG. 57 (a), when iPS cells were cultured using bFGF-containing human iPS gel medium in a 15 mL tube, no conspicuous aggregation was observed. FIG. 58 (a) is a photograph of the first day when iPS cells were cultured in a 15 mL tube using a bFGF-containing human iPS gel medium, and FIG. 58 (b) shows a bFGF-containing human iPS in a 15 mL tube. It is a photograph of the 9th day when iPS cells were cultured using a gel medium. From the photographs of FIGS. 58 (a) and 58 (b), it was confirmed that iPS cells of different strains did not aggregate and formed colonies.

FIG. 59 (a) is a photograph of iPS cell colonies suspended and cultured in a gel medium for 7 days immediately before re-seed on feeder cells. FIG. 59 (b) is a photograph when the morphology of the colony was confirmed 3 days later. As a result, as shown in FIG. 60, it was confirmed that 95% or more of the colonies were undifferentiated. Therefore, it was shown that iPS cells can be cultured in a gel medium while maintaining an undifferentiated state.

(Example 3)
The same bFGF-containing human iPS medium and bFGF-containing human iPS gel medium as in Example 2 were prepared. 300 μL of CTK solution was added to a 6-well dish in which iPS cells were cultured on feeder cells, and the cells were incubated in a CO ₂ incubator for 3 minutes. After 3 minutes, the dish was removed from the incubator, it was confirmed that only the feeder cells were peeled off, 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, and the dish was placed in a CO ₂ incubator and incubated for 5 minutes. .. Then, 0.7 mL of bFGF-containing iPS medium was added to the dish, and 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 number of cells was calculated using a hemocytometer. After cell calculation, 5 × 10 ⁵ iPS cells were seeded in 15 mL tubes and then suspended-cultured without stirring.

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

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

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

(Example 5)
Gel medium containing iPS cells was placed in each of two dialysis modules (Spectrem G235035) equipped with a dialysis tube having a molecular weight cut off of 100 kDa. Further, the dialysis module was placed in each 50 mL centrifuge tube, and the gel medium was placed around the dialysis tube in the centrifuge tube. Also, the gel medium containing the iPS cells was placed directly in another 50 mL centrifuge tube.

Then, for several days, a pump was connected to one of the two centrifugal tubes containing the dialysis tube inside, as shown in FIG. 21, and the gel medium in the centrifugal tube was continuously replaced. The gel medium was stored at 4 ° C and set to 37 ° C when reaching the centrifuge tube. No pump was connected to the other centrifuge tube of the two centrifuge tubes containing 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, 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 exchanged continuously, a large number of cell clusters were formed. In addition, 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 by a pump, the cell mass decreased and the differentiated cells increased. In addition, when the cell mass was cultured without using the dialysis tube and the gel medium was not continuously exchanged by the pump, almost no cell mass was formed.

2 ... Tube, 10 ... Separation device, 20 ... Pre-introduction cell delivery path, 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 delivery path, 60 ... division mechanism, 61 ... terminal block, 61a ... recess, 61b ... Convex part, 61c ... Large hole diameter part, 62 ... Connecting block, 62a ... Concave part, 62b ... Convex part, 62c ... Large hole diameter part, 62d ... Small hole diameter part , 62e ... Large hole diameter part, 63 ... Tip block, 63a ... Recessed part, 63b ... Nozzle part, 63c ... Large hole diameter part, 63d ... Small hole diameter part, 64 ... Insertion Nozzle, 65a ... Large pore diameter, 65b ... Small pore diameter, 66a ... Insert, 66b ... Insert, 70 ... Expansion culture device, 71 ... Expansion culture feeding channel, 72 ... Cell mass delivery channel, 75 ... Dialysis tube, 76 ... Container, 77 ... Supplementary medium delivery pump, 78 ... Liquid delivery tube, 79 ... Waste liquid tube, 80.・ ・ Division mechanism, 90 ・ ・ ・ cell soul transport mechanism, 91 ・ ・ ・ pre-package cell flow path, 100 ・ ・ ・ package device, 101 ・ ・ ・ solution replacer, 102 ・ ・ ・ filter, 103 ・ ・ ・Liquid flow path, 104 ... Liquid flow path, 105 ... Discharge flow path, 106 ... Discharge flow path, 110 ... Cryopreservation liquid delivery mechanism, 171 ... Initialization culture imaging device, 172 ... Telecentric lens, 173 ... Illuminated light source for cell observation, 174 ... Illuminated light source for medium observation, 201 ... Blood storage unit, 202 ... Blood delivery channel, 203 ... Mononuclear Sphere separation unit, 204 ... pump, 205 ... separator storage unit, 206 ... liquid supply path, 207 ... pump, 208 ... mononuclear ball delivery path, 209 ... pump, 210 ... Mononuclear cell purification filter, 211 ... Cell delivery path before introduction, 212 ... Pump, 213 ... Factor introduction section, 214 ... Factor storage section, 215 ... Factor delivery section Route, 216 ... Pump, 217 ... Introduced cell delivery path, 218 ... Pump, 219 ... Initialization incubator, 220 ... Blood cell medium storage unit, 221 ... Medium delivery Road, 222 ... Pump, 223 ... Stem cell medium storage unit, 224 ... Media delivery path, 225 ... Pump, 226 ... Waste liquid delivery path, 227 ... Pump, 228 ...・ Waste liquid storage unit, 229 ... introduced cell delivery path, 230 ... pump, 231 ... cell mass Divider, 232 ... Expansion incubator, 233 ... Medium fluid delivery path, 234 ... Pump, 235 ... Waste fluid delivery path, 236 ... Pump, 237 ... Introduction cell delivery channel , 238 ... Pump, 239 ... Cell mass divider, 240 ... Expansion incubator, 241 ... Medium fluid delivery path, 242 ... Pump, 243 ... Waste fluid delivery channel, 244.・ ・ Pump, 245 ・ ・ ・ Introduced cell liquid supply path, 246 ・ ・ ・ Pump, 247 ・ ・ ・ Solution replacement device, 248 ・ ・ ・ Waste liquid delivery path, 249 ・ ・ ・ Pump, 250 ・ ・ ・ Cryopreservation liquid Storage unit, 251 ... Liquid transfer path, 252 ... Pump, 253 ... Liquid transfer path, 254 ... Pump, 255 ... Freeze storage container, 256 ... Low temperature storage, 257 ... -Liquid nitrogen storage, 258 ... liquid supply path, 259 ... members, 260 ... refrigerated storage, 271 ... sensor, 272 ... thermometer, 301 ... bag, 302 ... -Bag, 401 ... Input device, 402 ... Output device, 403 ... Related storage device, 501 ... Image processing unit, 511 ... Contour definition unit, 512 ... Cell evaluation unit, 513 ... Statistical processing unit, 514 ... Density calculation unit, 515 ... Medium evaluation unit, 601 ... Housing, 602 ... Intake purification filter, 603 ... Exhaust purification filter, 605 ... Exhaust device, 606 ... Exhaust purification filter, 608 ... Introduction device, 613 ... Gas discharge device, 651 ... Return member, 652 ... Base, 653 ... Opening, 654 ... Cover , 655 ... Opening, 656 ... Cover, 701 ... Outer housing, 702 ... Pressure adjusting hole, 703 ... Closing member, 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 delivery path 1203 ... Mononuclear ball separation unit, 1204 ... Drive unit, 1205 ... Separator storage unit, 1206 ... Separator liquid feed path, 1207 ... Drive unit, 1208 ... Mononuclear ball feed Liquid channel, 1210 ... Mononuclear cell purification filter, 1211 ... Cell delivery path before introduction, 1212 ... Drive section, 1213 ... Factor introduction section, 1214 ... Factor storage section, 1215 ... -Factor delivery path, 1216 ... Drive section, 1217 ... Introduction cell delivery path, 1218 ... Drive section, 1219 ... Initialization incubator, 1220 ... Blood cell medium storage section, 12 21. , 1229 ... Introduced cell flow path, 1230 ... Drive unit, 1231 ... Cell mass divider, 1232 ... Expansion incubator, 1235 ... Waste liquid delivery path, 1245 ... Introduced cell Liquid transfer path, 1246 ... Drive unit, 1247 ... Solution replacement, 1248 ... Waste liquid transfer path, 1250 ... Freeze storage solution storage unit, 1251 ... Freeze storage solution transfer path, 1252 ... Drive unit, 1253 ... Cell delivery path for freezing, 1253 ... Liquid delivery path, 1254 ... Drive section, 1255 ... Freezing storage container, 2000 ... Embedding member, 2001. .. Mononuclear cell flow path, 2002 ... Mononuclear cell storage section, 2003 ... Other component flow path, 2004 ... Other component storage section, 2204 ... Driven section, 2228 ... Waste liquid storage section , 2229 ... Introduced cell medium, 2231 ... Cell mass divider, 3228 ... Waste liquid storage unit, 4228 ... Waste liquid storage unit, 5228 ... Waste liquid storage unit

Claims (21)

Cell processing equipment that processes cells and
An embedding member for embedding the cell processing instrument and
A communication liquid passage for communicating the outside of the embedding member and the cell processing instrument inside the embedding member,
A drive unit arranged outside the embedding member for sending the liquid in the communication liquid passage, and a drive unit.
It is a cell processing device equipped with
A driven portion to which a driving force is transmitted from the driving portion is provided in the embedded member, and the driven portion is connected to the communication liquid passage.
The cells are cultured in the cell processing instrument and
The cell processing apparatus further includes a waste liquid storage unit for storing waste liquid, which is arranged outside the embedding member.
The communication liquid supply passage includes a waste liquid liquid supply passage that communicates the cell processing instrument and the waste liquid storage unit.
Cell processing device. The cell processing apparatus according to claim 1, wherein the embedding member is made of glass or resin. The cell processing apparatus according to claim 1, wherein the embedding member is made of metal. The cell processing apparatus according to claim 1, wherein the driving unit is connected to an outer wall of the embedding member. Cell processing equipment that processes cells and
An embedding member for embedding the cell processing instrument and
A communication liquid passage for communicating the outside of the embedding member and the cell processing instrument inside the embedding member,
It is a cell processing device equipped with
The cell processing instrument comprises a mononuclear cell separator disposed inside the embedding member that separates the mononuclear cells from the blood.
The cell processing apparatus further includes a waste liquid storage unit for storing waste liquid, which is arranged outside the embedding member.
The communication liquid supply passage includes a waste liquid liquid supply passage that communicates the cell processing instrument and the waste liquid storage unit.
Cell processing device. Further comprising a blood storage unit located outside the embedding member to store at least one of blood and blood cells.
The cell processing apparatus according to claim 5, wherein the communication liquid passage includes a blood liquid passage for communicating the blood storage unit and the mononuclear cell separation unit. Cell processing equipment that processes cells and
An embedding member for embedding the cell processing instrument and
A communication liquid passage for communicating the outside of the embedding member and the cell processing instrument inside the embedding member,
A tubing pump arranged inside the embedding member for sending the liquid in the communication liquid passage, and a tubing pump.
It is a cell processing device equipped with
The cell processing instrument comprises a filter placed inside the embedding member that isolates the cells.
The cell processing apparatus further includes a waste liquid storage unit for storing waste liquid, which is arranged outside the embedding member.
The communication liquid supply passage includes a waste liquid liquid supply passage that communicates the cell processing instrument and the waste liquid storage unit.
Cell processing device. Cell processing equipment that processes cells and
An embedding member for embedding the cell processing instrument and
A communication liquid passage for communicating the outside of the embedding member and the cell processing instrument inside the embedding member,
A tubing pump arranged inside the embedding member for sending the liquid in the communication liquid passage, and a tubing pump.
It is a cell processing device equipped with
The cell processing instrument comprises a mononuclear cell purification filter disposed within the embedding member.
The cell processing apparatus further includes a waste liquid storage unit for storing waste liquid, which is arranged outside the embedding member.
The communication liquid supply passage includes a waste liquid liquid supply passage that communicates the cell processing instrument and the waste liquid storage unit.
Cell processing device. Cell processing equipment that processes cells and
An embedding member for embedding the cell processing instrument and
A communication liquid passage for communicating the outside of the embedding member and the cell processing instrument inside the embedding member,
It is a cell processing device equipped with
The cell processing instrument
A mononuclear cell separator arranged inside the embedding member, which separates mononuclear cells from blood,
A mononuclear cell purification filter arranged inside the embedding member,
A mononuclear cell liquid feeding path arranged inside the embedding member, which allows the mononuclear cell separation unit and the mononuclear cell purification filter to communicate with each other.
Equipped with
The cell processing apparatus further includes a waste liquid storage unit for storing waste liquid, which is arranged outside the embedding member.
The communication liquid supply passage includes a waste liquid liquid supply passage that communicates the cell processing instrument and the waste liquid storage unit.
Cell processing device. The cell processing apparatus according to claim 9, further comprising a driving unit arranged outside the embedding member for feeding the liquid in the mononuclear ball feeding path. The cell processing apparatus according to claim 10, wherein the driving unit is connected to an outer wall of the embedding member. The tenth or eleventh aspect of the present invention, wherein a driven portion to which a driving force is transmitted from the driving portion is provided in the embedded member, and the driven portion is connected to the mononuclear ball feeding path. Cell processing device. Cell processing equipment that processes cells and
An embedding member for embedding the cell processing instrument and
A communication liquid passage for communicating the outside of the embedding member and the cell processing instrument inside the embedding member,
It is a cell processing device equipped with
The cell processing instrument comprises a factor-introducing portion located inside the embedding member that introduces an inducing factor into the cell to produce an inducing factor-introduced cell.
The cell processing apparatus further includes a waste liquid storage unit for storing waste liquid, which is arranged outside the embedding member.
The communication liquid supply passage includes a waste liquid liquid supply passage that communicates the cell processing instrument and the waste liquid storage unit.
Cell processing device. Further, a factor storage unit for storing the inducing factor, which is arranged outside the embedding member, is provided.
The cell processing apparatus according to claim 13, wherein the communication liquid passage includes a factor liquid supply passage that communicates the factor storage unit and the factor introduction unit. The cell processing apparatus according to claim 13 or 14, wherein the inducer is introduced into the cell by RNA lipofection in the factor introduction unit. The cell processing apparatus according to claim 15, wherein the inducing factor is DNA, RNA, or protein. The cell processing apparatus according to claim 13 or 14, wherein the inducing factor is incorporated in a vector. The cell processing apparatus according to claim 17, wherein the vector is a Sendai virus vector. The cell processing apparatus according to any one of claims 1 to 4, wherein the cell processing instrument comprises an incubator arranged inside the embedding member for culturing the inducing factor-introduced cells into which the inducing factor has been introduced. .. The invention according to any one of claims 1 to 4, wherein the cell processing instrument comprises an expansion incubator arranged inside the embedding member, which expands and cultures a plurality of cell clusters composed of established stem cells. Cell processing device. The cell processing apparatus according to any one of claims 1 to 4, wherein the cell processing instrument comprises a solution substituent that replaces a solution around cells.

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