Cell manufacturing apparatus and system

Abstract

This cell production device comprises: a cell preparation plate that comprises a fluid circuit having a plurality of functional parts integrated therein; and a plurality of closed connectors that connect the fluid circuit to an external space in a closed manner. The fluid circuit comprises, as the plurality of functional parts: a plurality of injection and discharge parts through which a plurality of types of fluids can be injected into the fluid circuit or can be discharged to outside of the fluid circuit via the plurality of closed connectors; a plurality of fluid retention parts which can store the plurality of types of fluids which should be injected or discharged; and a cell induction and culture part which, on the basis of the types of stored fluids, can carry out at least one of inducing or culturing cells.

Description

Technical Field

[0001] The present invention relates to a cell manufacturing apparatus and its system, and particularly to a cell manufacturing apparatus and its system that simplify the fluid injection / discharge process.

Background Art

[0002] Embryonic stem cells (ES cells) are stem cells established from the early cups of humans and mice, and have the pluripotency to differentiate into all cells existing in the living body. Human ES cells are considered to be available for cell transplantation methods for many diseases such as Parkinson's disease, juvenile diabetes, and leukemia. However, transplantation of ES cells has a problem of causing rejection reactions, similar to organ transplantation. In addition, there are many opposing opinions from an ethical perspective regarding the use of ES cells established by destroying human cups.

[0003] On the other hand, Professor Shinya Yamanaka of Kyoto University succeeded in establishing induced pluripotent stem cells (iPS cells) by introducing four genes: Oct3 / 4, Klf4, c-Myc, and Sox2 into somatic cells, and won the Nobel Prize in Physiology or Medicine in 2012 (see, for example, Patent Document 1). iPS cells are ideal pluripotent cells without rejection reactions or ethical problems, and are expected to be used in cell transplantation methods.

[0004] Induced stem cells such as iPS cells are established by introducing induction factors such as genes into cells, and are expanded in culture and cryopreserved. However, for example, to produce clinical iPS cells (GLP, GMP grade), a very cleanly maintained clean room is required, and high maintenance costs are incurred. For industrialization, the issue has been how to improve the operation method of the clean room to reduce costs.

[0005] Furthermore, while the creation of iPS cells relies heavily on manual labor, there are few technicians capable of producing iPS cells for clinical use. The entire process, from stem cell establishment to storage, is complex. Clinical cell culture requires three steps: verification of the Standard of Process (SOP), adherence to the SOP, and confirmation that the procedure was followed correctly. Performing these steps manually is highly unproductive. Cell culture requires 24 / 7 monitoring, and stem cell storage can last for decades, making it impossible to manage the process solely through human intervention.

[0006] Therefore, closed-system cell manufacturing equipment has been developed that eliminates the need for highly cleanrooms and can be operated in normally controlled areas (for example, where at least one of microorganisms and particulate matter is Grade D or higher according to WHO-GMP standards) (see, for example, Patent Document 2). Furthermore, cell manufacturing systems equipped with robots to assist in cell manufacturing have also been developed to automate complex cell manufacturing processes and eliminate the need for human labor. The following documents are known prior art regarding such cell manufacturing equipment.

[0007] Patent Document 3 discloses a somatic cell manufacturing system that packages a pre-transfer cell delivery channel, a factor introduction device for introducing somatic cell-inducing factors into pre-transfer cells to produce factor-inducing cells, and a cell production device for culturing factor-inducing cells to produce somatic cells, all within a single housing.

[0008] Patent Document 4 discloses a cell culture vessel in which the culture vessel and the flow channel are in a closed system, and the growth state of the cell culture can be clearly observed by holding the second vessel eccentrically inside the first vessel.

[0009] Patent Document 5 discloses a cell culture apparatus in which a culture medium storage means, a cell inoculation means, and a culture vessel are configured in a closed system. The cell culture apparatus reduces the operator's workload by determining the cell culture status from an image of the cells in the culture vessel and performing the culture operation based on this determination.

[0010] Patent Document 6 discloses a cell culture apparatus comprising a main body having side walls surrounding the volume of a cell culture chamber, a lid covering the cell culture chamber, and a bottom plate located at the bottom of the main body, wherein the main body has an integrated microfluidic conduit for fluid communication between an inlet / outlet connector and the cell culture chamber.

[0011] Patent Document 7 discloses a microchip reaction apparatus equipped with a bubble removal means for moving bubbles in the internal space of a microchip to the external space. Patent Document 8 discloses a cell culture apparatus equipped with a vent that allows gas to be discharged from a first culture medium storage chamber and a second culture medium storage chamber, and an air filter is provided in the vent.

[0012] Patent Document 9 discloses a cell culture apparatus equipped with a channel-integrated plate and a base plate. The channel-integrated plate comprises a channel plate forming channels for the culture medium and a pump section equipped with a group of peristaltic pumps for supplying and discharging the culture medium, while the base plate is equipped with a drive source such as a motor. [Prior art documents] [Patent Documents]

[0013] [Patent Document 1] Patent No. 4183742 [Patent Document 2] Japanese Patent Publication No. 2018-019685 [Patent Document 3] International Publication No. 2018 / 154788 [Patent Document 4] International Publication No. 2014 / 049701 [Patent Document 5] International Publication No. 2007 / 052716 [Patent Document 6] Special Publication No. 2014-514926 [Patent Document 7] Japanese Patent Publication No. 2014-226623 [Patent Document 8] International Publication No. 2017 / 154880 [Patent Document 9] Japanese Patent Publication No. 2017-221166 [Overview of the Initiative] [Problems that the invention aims to solve]

[0014] Conventional cell culture devices often integrate only cell culture functions such as a culture medium reservoir, culture medium supply and discharge channels, and cell culture vessels. Few devices integrate cell induction functions such as cell reprogramming, reprogramming, fate reversal, direct reprogramming, differentiation reversal, differentiation induction, and transformation through the introduction of inducible factors. In devices where these various functional parts are configured in a closed system, it is necessary to inject or discharge various fluids into or out of the device in predetermined amounts at the appropriate timing, making the injection and discharge process cumbersome. Furthermore, if cell production using such devices is to be fully automated, the injection and discharge operations of the robot also become complicated.

[0015] Therefore, there is a need for technology to simplify the fluid injection and discharge process in cell manufacturing devices that integrate multiple functional parts. [Means for solving the problem]

[0016] One aspect of the present disclosure comprises a cell preparation plate having a fluid circuit integrating a plurality of functional parts, and a plurality of closed connectors that connect the fluid circuit to an external space in a closed manner, wherein the fluid circuit comprises a plurality of injection / discharge sections capable of injecting a plurality of types of fluids into or out of the fluid circuit via the plurality of closed connectors, a plurality of fluid reservoirs capable of storing the plurality of types of fluids to be injected or discharged, a plurality of transfer sections capable of transferring each of the stored plurality of types of fluids, and a cell induction culture section that performs cell induction and culture based on the stored plurality of types of fluids, wherein the plurality of fluid reservoirs contain fluids including source cells, Inducing factor introduction reagents, reprogramming or induction media, The present invention provides a cell manufacturing apparatus that stores a fluid containing target cells, respectively. Another aspect of the present disclosure provides a cell production plate including a fluid circuit integrating a plurality of functional parts, the fluid circuit including, as the plurality of functional parts, a plurality of fluid reservoirs capable of storing a plurality of types of fluids, a plurality of transfer parts capable of transferring each of the plurality of types of stored fluids, and a cell induction and culture part configured to perform cell induction and culture based on the plurality of types of stored fluids, wherein the plurality of fluid reservoirs store a fluid containing original cells, Inducing factor introduction reagents, reprogramming or induction media, and a fluid containing target cells, respectively, and provides a cell production apparatus. Another aspect of the present disclosure provides a cell production system including a cell production plate including a fluid circuit integrating a plurality of functional parts, a cell production apparatus including a plurality of closed connectors configured to connect the fluid circuit to an external space in a closed manner, and a fluid container connectable to at least one of the plurality of closed connectors, the fluid circuit including a plurality of injection / discharge parts configured to inject a plurality of types of fluids into or discharge the plurality of types of fluids from the fluid circuit via the plurality of closed connectors, the fluid container including a fluid reservoir part capable of storing the plurality of types of fluids, body and a discharge / suction part configured to discharge the fluid into the fluid circuit and suction the fluid from the fluid circuit via at least one of the plurality of closed connectors, the fluid circuit or the fluid container further including a plurality of transfer parts capable of transferring the stored fluid into the fluid circuit, wherein the plurality of fluid reservoirs store a fluid containing original cells, multiple and a fluid containing target cells, respectively. Inducing factor introduction reagents, reprogramming or induction media, and provides a cell production system.

Advantages of the Invention

[0017] According to an aspect of the present disclosure, since the cell production plate can store a plurality of types of fluids or the fluid container can discharge the stored fluid into the cell production plate, even in a cell production apparatus integrating a plurality of functional parts, it is possible to use a specific fluid by a predetermined amount at an appropriate timing, and the process of injecting / discharging the fluid by a person or a robot can be simplified.

Brief Description of the Drawings

[0018] [Figure 1]This is a diagram showing the configuration of a cell manufacturing apparatus in one embodiment. [Figure 2] This is a disassembled perspective view showing an example of a cell preparation plate. [Figure 3A] This is an enlarged cross-sectional view showing an example of a method for fastening a flat plate to a lid. [Figure 3B] This is an enlarged cross-sectional view showing an example of a method for fastening a flat plate to a lid. [Figure 4A] This is a diagram showing an example of a fluid reservoir section provided within a cell preparation plate. [Figure 4B] This is a diagram showing an example of a fluid reservoir section installed inside a fluid container. [Figure 4C] This is a diagram showing an example of a fluid reservoir section installed inside a fluid container. [Figure 5A] This is a front perspective view showing an example of a base plate. [Figure 5B] This is a rear perspective view showing an example of a base plate. [Modes for carrying out the invention]

[0019] Embodiments of this disclosure will be described in detail below with reference to the attached drawings. In each drawing, identical or similar components are denoted by the same or similar reference numerals. Furthermore, the embodiments described below do not limit the technical scope of the invention as described in the claims or the meaning of the terms used. In this document, the term "closed" means that sources of contamination such as microorganisms and viruses do not enter the device and cause biological contamination, and / or that fluids inside the device (including substances such as cells, microorganisms, virus particles, proteins, and nucleic acids) do not leak out and cause cross-contamination, and / or that handling fluids from a donor infected with a pathogen inside the device does not cause a biohazard. However, the device in this document may be configured to allow fluids that are not sources of contamination, such as carbon dioxide, nitrogen, and oxygen, to enter the device or leak out of the device.

[0020] Figure 1 shows the configuration of the cell manufacturing apparatus 1 in this embodiment. The cell manufacturing apparatus 1 is a cell transformation device equipped with cell induction functions that perform cell reprogramming, reprogramming, fate reversal, direct reprogramming, differentiation reversal, differentiation induction, transformation, etc., by introducing inducible factors, but it may also be a cell culture device equipped only with cell culture functions that perform culture, expansion culture, etc. The cell manufacturing apparatus 1 injects a fluid containing source cells (for example, somatic cells such as blood cells and fibroblasts, or stem cells such as ES cells and iPS cells), manufactures target cells (for example, stem cells, progenitor cells, terminally differentiated cells) from the source cells, and discharges the fluid containing the target cells. Differentiated cells such as fibroblasts, nerve cells, retinal epithelial cells, hepatocytes, β cells, kidney cells, mesenchymal stem cells, blood cells, megakaryocytes, T cells, chondrocytes, cardiomyocytes, muscle cells, vascular cells, epithelial cells, or other somatic cells may be produced as target cells. The cell manufacturing apparatus 1 is a closed-system cell processing device in which all parts that need to be highly clean are concentrated internally, and can be used in normally controlled areas. The enclosed space inside the device is configured to prevent the exchange of gases, viruses, microorganisms, impurities, etc., with the outside. However, by adding a fluid exchange filter, etc., as described later, to the device, it may be configured to allow the exchange of non-contaminating fluids between the inside and outside of the device.

[0021] As shown in Figure 1, the cell manufacturing apparatus 1 includes a cell manufacturing plate 2 and a closed connector 3. The cell manufacturing plate 2 is equipped with a closed fluid circuit 4 isolated from the external space S, and the fluid circuit 4 has a flow path that highly integrates multiple functional parts. The closed connector 3 is a connector for injecting fluid into the fluid circuit 4 or discharging fluid from the fluid circuit 4, and is attached to the cell manufacturing plate 2. The closed connector 3 is a connector that connects the fluid circuit 4 and the fluid container in a closed manner to the external space S, and may be, for example, a sterile connection connector, a needleless connector, a needle connector, a heat-sealed tube, etc. The needleless connector may be a split septum type or a mechanical valve type. The fluid container is a syringe, a variable volume bag, etc., and when the closed connector 3 is connected to the fluid container, the fluid circuit 4 is in fluid communication with the fluid container, while when the closed connector 3 and the fluid container are not connected, the fluid circuit 4 is isolated from the external space S. This prevents biological contamination, cross-contamination, and biohazards in the fluid circuit 4. Preferably, the cell manufacturing apparatus 1 is equipped with multiple closed connectors 3 to allow for the injection or discharge of multiple types of fluids.

[0022] Figure 2 shows an example of a cell preparation plate 2. As shown in Figure 2, the cell preparation plate 2 comprises a plate 20 and a lid 21. The plate 20 can be molded from, for example, a biologically safe resin, metal, etc. Preferably, the plate 20 is molded using a mold, such as injection molding or compression molding, but it may also be molded using a 3D printer. Various molding methods such as stereolithography, fused deposition modeling, powder sintering, and inkjet can be used as 3D printers. A groove 20a is provided on at least one of the front and back surfaces of the plate 20 as a channel for fluid flow, and a fluid circuit 4 is formed by combining multiple grooves 20a. In addition, a portion of the fluid circuit 4 is provided with a section where the width or depth of the groove 20a is relatively larger to form a storage tank 20b for temporarily storing fluid. The walls of the grooves 20a and storage tank 20b may be coated with poly-HEMA (poly 2-hydroxyethyl methacrylate) to make them non-adherent to cells. Conversely, if cells have difficulty entering the groove 20a or the storage tank 20b, the walls of the groove 20a and the storage tank 20b may be made to have low protein adsorption properties. At least a portion of the groove 20a and the storage tank 20b is preferably white or black in order to observe changes in fluid, cells, cell aggregates, etc. over time using an image recognition sensor, an ultrasonic recognition sensor, etc.

[0023] The lid 21 may be made of, for example, a biologically safe resin, quartz glass, etc. The lid 21 (i.e., at least a portion of the cell production plate 2) is preferably transparent so that the changes in the fluid, cells, cell aggregates, etc., within the fluid circuit 4 over time can be observed using an image recognition sensor, an ultrasonic recognition sensor, etc. This observation allows for transition to the next cell production process at an appropriate time. The lid 21 is fixed to the plate 20 so as to cover the fluid circuit 4 by a biologically safe fixing method, such as chemical bonding, welding, or adhesive bonding, in order to isolate the fluid circuit 4 from the external space. For chemical bonding, silane coupling agents, plasma irradiation, etc., may be used. For welding, laser welding, ultrasonic welding, etc., may be used, and for adhesive bonding, ultraviolet-curing adhesives, etc., may be used. After the plate 20 and the lid 21 are fixed together, the cell production plate 2 is subjected to sterilization treatment, such as heat sterilization, gamma ray sterilization, ultraviolet sterilization, or electron beam sterilization, to make the fluid circuit 4 highly clean.

[0024] Figures 3A and 3B show an example of a method for fixing the plate and the lid. To isolate the fluid circuit 4 from the external space, embankments 20c may be formed in advance on both sides of the groove 20a and the storage tank 20b, the plate 20 may be covered with the lid 21, and the embankments 20c may be heated by irradiating or applying laser light, ultrasonic waves, etc. to at least the embankments 20c, thereby sealing the groove 20a and the storage tank 20b by welding the plate 20 and the lid 21 together. Alternatively, the plate 20 including the groove 20a and the storage tank 20b may be covered with the lid 21, and the parts other than the groove 20a and the storage tank 20b may be heated by irradiating or applying laser light, ultrasonic waves, etc., thereby sealing the groove 20a and the storage tank 20b by welding the plate 20 and the lid 21 together. In this case, since all parts other than the groove 20a and the storage tank 20b are fixed together, the fixing strength is increased.

[0025] Referring again to Figure 1, the fluid circuit 4 comprises at least an injection / discharge unit 10 and a cell induction culture unit 13 as multiple functional parts. The fluid circuit 4 may optionally also include a first volume variable unit 11, a transfer unit 12, a fluid reservoir unit 14, a fluid mixing unit 15, a cell separation unit 16, and a cell aggregate disruption unit 17. Since these various functional parts are integrated into a single cell production plate 2, manufacturing processes such as connecting separate components in a closed manner via tubes, pumps, connectors, etc., are eliminated, reducing the manufacturing man-hours and manufacturing costs of the cell production device 1.

[0026] The injection / discharge unit 10 is equipped with injection / discharge channels for injecting or discharging fluid into or out of the fluid circuit 4 via a closed connector 3. To enable injection or discharge of multiple types of fluids, it is preferable that the injection / discharge unit 10 is equipped with multiple injection / discharge channels 10a-10f. For example, the first injection / discharge channel 10a can inject or discharge fluids containing original cells, the second injection / discharge channel 10b can inject or discharge fluids such as reagents for separating original cells, anticoagulants, and phosphate-buffered saline. The third injection / discharge channel 10c can inject or discharge fluids such as reagents for introducing inducible factors, and the fourth injection / discharge channel 10d can inject or discharge fluids such as various culture media for initialization or induction, cell detachment reagents such as trypsin-alternative recombinant enzymes, and single-cell separation reagents. Induction media include initialization media, reprogramming media, fate reversal media, direct programming media, differentiation conversion media, differentiation induction media, and transformation media. Furthermore, the fifth injection / discharge channel 10e allows a fluid containing cells that have undergone at least one of induction and / or culture to be discharged or injected as a sample into a fluid container 19, and the sixth injection / discharge channel 10f allows a fluid containing target cells to be discharged or injected into a fluid container. The fluid container 19 for sample discharge may be a closed connector 3, for example, a variable volume bag connected to a heat-sealable tube. Alternatively, when discharging the fluid containing target cells, a coolant such as liquid nitrogen may be supplied around the sixth injection / discharge channel 10f to freeze the fluid containing the target cells and seal the cell preparation plate, or the fluid container discharged from the sixth injection / discharge channel 10f via the closed connector 3 may be frozen with a coolant such as liquid nitrogen.

[0027] The first volume variable section 11 includes a physical or chemical volume variable material that stores the fluid that is pushed out or drawn out by the injected or discharged fluid. A fluid relief channel is provided to release the fluid that was originally in the fluid circuit 4, and the physical volume variable material is connected to the fluid relief channel, or a pressure valve that opens and closes at a constant pressure is provided in the fluid relief channel and the chemical volume variable material is stored in the storage tank, thereby enabling the movement of fluid while maintaining the airtightness of the fluid circuit 4. The physical volume variable material may be, for example, a flexible bag or syringe. The chemical volume variable material may include, for example, a fluid absorbent such as soda lime or silica gel, and a fluid release agent stored in a storage tank separate from the storage tank in which the fluid absorbent is stored. The variable volume material keeps the internal pressure of the closed fluid circuit 4 approximately constant, and the fluid extruded or drawn out by the injected or discharged fluid is confined within the cell preparation plate 2. Therefore, there is no need to release fluid to the outside of the cell preparation plate 2 or to take in fluid from the outside, making it possible to form the cell preparation plate 2 into a plate shape while maintaining the airtightness of the fluid circuit 4. Such a cell preparation plate 2 is easy for robots to handle.

[0028] The transfer unit 12 is equipped with a pump that transfers fluid within the fluid circuit 4. The pump may be a positive displacement pump with controllable flow rate, such as a rotary pump or a reciprocating pump. A peristaltic pump is preferred as the rotary pump. In the case of a peristaltic pump, a flexible tube is sealed and connected to a connector provided at the end of the flow path, and the fluid is transferred by handling the tube with rollers. Since the tube is blocked by the rollers, when the pump stops, the fluid flow is blocked, and flow rate control is possible. A diaphragm pump is also preferred as the reciprocating pump. However, in the case of a diaphragm pump, since the diaphragm does not block the flow path, flow rate control is possible by using a flow path shut-off valve in combination.

[0029] To transfer fluid to the appropriate functional part at the appropriate time, it is preferable that the transfer unit 12 is equipped with multiple pumps P1-P8. For example, the first pump P1-third pump P3 transfer the fluid stored in fluid reservoirs A1-A3 at the appropriate time, and the fourth pump P4 and eighth pump P8 transfer the fluid stored in the cell separation unit 16 at the appropriate time. The fifth pump P5 transfers the fluid stored in fluid reservoir A4 at the appropriate time, and the sixth pump P6-seventh pump P7 transfer the fluid stored in the cell induction culture unit 13 at the appropriate time. If, for example, a peristaltic pump is used as the pump, a rotary encoder capable of detecting the amount of rotation may be provided on the rotating shaft of the pump to obtain information on whether the pump is operating normally, such as whether the pump has rotated reliably or by the appropriate angle. Alternatively, for example, a visual marker may be provided on the end of the rotating shaft of the pump, and the rotational movement of the marker may be directly captured in an image by an image recognition sensor. To ensure that the pump transfer is carried out reliably, a flow rate measuring unit (not shown) may be further provided either before or after the pump. The flow rate measuring unit may be, for example, a flow sensor provided adjacent to at least one of the flow path communicating with the transfer unit and the storage tank, or an image recognition sensor that captures the change in the fluid over time in at least one of the flow path communicating with the transfer unit and the storage tank. Various measurement methods that do not adversely affect cells can be used for the flow sensor, such as Karman vortex type, impeller type, or diaphragm type, and directly acquire fluid flow rate information. The image recognition sensor acquires flow rate information from the movement of the fluid by performing image recognition from an external camera or the like through the transparent lid 21. The image recognition sensor may be one of the other image recognition sensors described in this document, thereby reducing the number of parts and manufacturing costs.

[0030] The cell induction culture unit 13 comprises a cell induction culture tank 13a that performs at least one of cell induction and culture based on the transferred fluid, and a culture medium circulation path 13b that is in fluid communication with the cell induction culture tank 13a and circulates the culture medium. The cell induction culture tank 13a is heated to a predetermined culture temperature, for example, 37°C, by a heating element. The culture medium circulation path 13b is cooled to a predetermined culture medium quality maintenance temperature, for example, 4°C-8°C, by a cooling element. The cell induction culture tank 13a is in a sealed state and does not need to be supplied with fluids such as carbon dioxide, nitrogen, or oxygen, but at least one of the cell induction culture tank 13a and the culture medium circulation path 13b may further be equipped with a fluid exchange filter that exchanges fluids such as carbon dioxide, nitrogen, or oxygen between the inside and outside of the device. Furthermore, the cell induction culture tank 13a may be a three-dimensional culture tank for cell suspension culture, or it may be a two-dimensional culture tank for adherent culture. For adherent culture, the cell induction culture tank 13a may be coated with cell adhesion coatings such as Matrigel, collagen, polylysine, fibronectin, vitronectin, gelatin, and laminin, laminin fragments, or it may be filled with hollow fibers. Furthermore, the cell induction culture tank 13a may integrally comprise a culture tank 30 and a culture medium tank 31 for supplying culture medium to the culture tank 30. In this case, it is preferable that the cell induction culture tank 13a is equipped with a specific component permeable member 32, such as a semipermeable membrane, that allows only specific components to pass between the culture tank 30 and the culture medium tank 31. The specific component permeable member 32 allows specific components such as various culture media, cell adhesion coatings, and cell detachment reagents to pass through.

[0031] The cell induction culture unit 13 may further include a pH measuring unit 13c for measuring the pH value of the culture medium used. The pH measuring unit 13c is preferably installed in the culture medium circulation path 13b or the cell induction culture tank 13a to measure the pH value of the culture medium used. The pH measuring unit 13c may be, for example, an image recognition sensor or an electrode measurement sensor. The image recognition sensor measures the pH value using a color measurement with an external camera or the like via a transparent lid. The electrode measurement sensor measures the pH value using the glass electrode method. In the case of color measurement, by making at least a part of the culture medium circulation path 13b (for example, the bottom surface of the color measurement area) white, the color can be accurately detected. This pH value makes it possible to quantitatively understand the state of the culture medium. Furthermore, to sharpen the outlines of the cell images in the image, it is preferable to further include an illumination unit that illuminates the cell induction culture tank 13a from at least one of the following directions: front, circumferential (for example, perpendicular to the observation surface), and rear. The lighting unit may include, for example, LED lighting and may be embedded inside the cell preparation plate 2, or the cell induction culture vessel 13a may be made more convex than the cell preparation plate 2 and provided outside the cell preparation plate 2. The culture vessel 30 may be covered with a light-transmitting transparent material so that the lighting reaches the cells.

[0032] The fluid reservoir 14 includes a storage tank for storing fluids to be injected into or discharged outside the fluid circuit 4. Preferably, the fluid reservoir 14 includes multiple storage tanks A1-A4 to enable the storage of multiple types of fluids. Storage tanks A1-A4 are formed as areas with relatively large widths or depths in the flow path, allowing for the use of various fluids in predetermined amounts at appropriate timings. For example, the first storage tank A1 stores fluids containing source cells, the second storage tank A2 stores fluids such as source cell separation reagents, anticoagulants, and phosphate-buffered saline, the third storage tank A3 stores fluids such as inducing factor introduction reagents, and the fourth storage tank A4 stores fluids such as various culture media, cell adhesion coatings, and cell detachment reagents. A storage tank for storing fluids containing target cells may also be included.

[0033] Figure 4A shows an example of a fluid reservoir 14 provided in the cell preparation plate 2. As described above, multiple injection / discharge sections 10 and multiple fluid reservoir sections 14 are provided in the cell preparation plate 2, allowing multiple types of fluids to be injected from the fluid container 19 to the fluid circuit 4 or discharged from the fluid circuit 4 to the fluid container 19 via multiple closed connectors 3, and also allowing them to be stored in the fluid reservoir section 14. In addition, multiple transfer sections 12 are provided in the fluid circuit 4, allowing the stored multiple types of fluids to be transferred within the fluid circuit 4, making it possible to transfer a specific stored fluid in a predetermined amount at an appropriate timing. This simplifies the fluid injection / discharge process performed by human or robotic personnel. Furthermore, by connecting these fluid reservoirs 14 to the first variable volume section 11, the fluid originally contained in the fluid reservoir 14 is pushed out to the first variable volume section 11, or the fluid originally contained in the first variable volume section 11 is drawn out to the fluid reservoir 14, thereby enabling the movement of fluid within the fluid circuit 4 while maintaining the airtightness of the fluid circuit 4.

[0034] Figures 4B-4C show an example of a fluid reservoir 14 provided in a fluid container 19. As an alternative embodiment, a fluid reservoir 14 and a discharge / suction unit 40 may be provided in the fluid container 19 separately from the cell preparation plate 2, and the fluid container 19 may be connected to at least one of a plurality of closed connectors 3, so that the fluid can be discharged to the fluid circuit 4 or drawn in from the fluid circuit 4 via the closed connector 3. Alternatively, the fluid container 19 may include a fluid reservoir 14 capable of storing at least one of a plurality of fluids, and a discharge / suction unit 40 capable of discharging the fluid to the fluid circuit 4 or drawing it in from the fluid circuit 4. One fluid container 19 equipped with a plurality of discharge / suction units 40 may be connected to a plurality of closed connectors 3, in which case it is preferable to attach the plurality of closed connectors 3 to one side of the cell preparation plate 2. Furthermore, by providing a transfer unit 12 in the fluid circuit 4 or the fluid container 19, and making it possible to transfer the stored fluid into the fluid circuit 4, it becomes possible to transfer a predetermined amount of a specific stored fluid at an appropriate timing. This simplifies the process of injecting and discharging fluid by human or robotic means. The fluid reservoir 14 may be composed of a variable-volume member, such as a syringe or flexible bag, as shown in Figure 4B, or a constant-volume member, such as a rigid container, as shown in Figure 4C. In the latter case, a second variable-volume section 41 is provided inside the fluid container 19 that communicates with the fluid reservoir 14. By drawing the fluid originally contained in the second variable-volume section 41 into the fluid reservoir 14 or pushing the fluid originally contained in the fluid reservoir 14 into the second variable-volume section 41, fluid movement within the fluid container 19 is made possible while maintaining the airtightness of the fluid container 19.

[0035] The fluid mixing section 15 is equipped with a mixing channel for mixing multiple fluids that are immiscible with each other. Preferably, the mixing channel includes a fluid confluence channel and a mixed flow generation channel. The fluid confluence channel is a channel that brings together immiscible fluids into a single channel, and the mixed flow generation channel is a channel that generates a mixed flow in the combined fluids. For example, the mixed flow generation channel may be a spiral channel that penetrates from the front surface to the back surface of the flat plate. In order to return fluid from the back surface to the front surface of the flat plate, two spiral channels are provided that penetrate the flat plate, and a communication passage is provided on the back surface of the flat plate to allow fluid communication between these spiral channels.

[0036] The cell separation unit 16 is equipped with separation tanks D1-D2 for separating cells or cell aggregates. For example, separation tanks D1-D2 are storage tanks formed by relatively increasing the width or depth of the flow path. The first separation tank D1 separates the fluid containing the original cells into a fluid containing only the original cells, and the second separation tank D2 separates the other cell aggregates by allowing only relatively large cell aggregates to settle. Methods for separating the original cells include reagents for original cell separation, panning, magnetic cell separation (MACS), flow cytometry, etc.

[0037] The cell aggregate disruption section 17 is equipped with a disruption channel for further disrupting the separated cell aggregates (clumps of one or more cells). The disruption channel has a relatively smaller channel area compared to the upstream channel and is preferably meandering. By making the channel meander, an undercurrent is generated, applying shear stress to the cell aggregate and breaking down the large, grown cell aggregate into smaller cell aggregates. An undercurrent refers to, for example, any of the following: a flow that generates vortices, turbulence, reverse flow, a flow that creates parts with different flow velocities, a flow that generates shear force, or a flow that creates parts where flows with different directions of propagation collide.

[0038] Preferably, the cell manufacturing apparatus 1 further includes a base plate that is detachably connected to the back side of the cell production plate 2. Figures 5A and 5B show an example of a base plate. The base plate 5 controls the fluid, temperature, etc., of the cell production plate 2. The cell production plate 2 is positioned to face the hazardous area side 70, where robots and the like will act, while the base plate 5 is positioned to face the safe area side 71, opposite to the hazardous area side 70. From the viewpoint of preventing biological contamination, the cell production plate 2 may be disposable, and the base plate 5 may be reusable. In addition, the cell manufacturing apparatus 1 is configured to be maintainable from the safe area side 71. By having such a dual structure in the cell manufacturing apparatus 1, robots and the like can interact with multiple cell manufacturing apparatuses 1 in a one-to-many manner.

[0039] The cell manufacturing apparatus 1 may further include a positioning member 72 and a plate sealing member 73 on the connecting surface of the cell manufacturing plate 2 and the base plate 5. The positioning member 72 may be a convex and concave portion that fits together with each other, and it positions the connection position between the cell manufacturing plate 2 and the base plate 5. The plate sealing member 73 may be a gasket, packing, etc., attached to the outer circumference of the connecting surface, and by connecting the cell manufacturing plate 2 and the base plate 5, the inside of the plate sealing member 73 is sealed from the outside space, and gas permeation from the back surface of the cell manufacturing plate 2 is suppressed. The base plate 5 is equipped with a drive unit 74 that drives the transfer unit 12. The drive unit 74 is equipped with, for example, a motor that drives a peristaltic pump. Furthermore, it is preferable to provide electrical contacts 75 on the connecting surface of the cell manufacturing plate 2 and the base plate 5 that supply power to electrical elements placed on the cell manufacturing plate 2, such as a heating element, a cooling element, a flow sensor, etc.

[0040] According to the above embodiment, since the cell production plate 2 can store multiple types of fluids, or the fluid container 19 can discharge the stored fluids to the cell production plate 2, even in a cell manufacturing apparatus 1 that integrates multiple functional parts, it becomes possible to use a specific fluid in a predetermined amount at an appropriate time, and the fluid injection and discharge process by human or robot can be simplified.

[0041] Although various embodiments have been described herein, it should be noted that the present invention is not limited to the embodiments described above, and various modifications can be made within the scope of the following claims. [Explanation of symbols]

[0042] 1 Cell production equipment 2-cell preparation plate 3. Closed-type connector 4 Fluid circuit 5 Base plate 10 Injection / discharge section 10a-10f 1st-6th injection / discharge channels 11. First volume variable section 12 Transfer section 13 Cell induction culture department 13a Cell induction culture tank 13b Culture medium circulation pathway 13c pH measuring section 14 Fluid reservoir 15 Fluid mixing section 16 Cell separation section 17 Cell aggregate disruption section 19 Fluid containers 20 flat plate 20a groove 20b Reservoir 20c Embankment 21 Lid 21a Front cover 21b Back cover 30 Culture tank 31 culture tanks 32. Specific component permeable material 40 Discharge suction section 41 Second volume variable section 70 Dangerous Area 71 Safety area side 72 Positioning member 73 Plate sealing member 74 Drive Unit 75 Electrical contacts A1-A4 1st-4th storage tank D1-D2 1st-2nd separation tank P1-P8 Pumps 1-8 S External space

Claims

1. A cell preparation plate equipped with a fluid circuit that integrates multiple functional sites, Multiple closed connectors that connect the fluid circuit to the external space in a closed manner, Equipped with, The fluid circuit comprises the following multiple functional parts: Multiple injection / discharge units capable of injecting multiple types of fluids into the fluid circuit or discharging them outside the fluid circuit via the multiple closed connectors, Multiple fluid reservoirs capable of storing the multiple types of fluids to be injected or discharged, Multiple transfer units capable of transferring each of the multiple types of fluids stored, A cell induction and culture unit that performs cell induction and culture based on the multiple types of fluids stored therein, Equipped with, A cell manufacturing apparatus in which the plurality of fluid reservoirs each store a fluid containing source cells, an inducer introduction reagent, a culture medium for reprogramming or induction, and a fluid containing target cells, respectively.

2. It includes a cell preparation plate equipped with a fluid circuit that integrates multiple functional sites, The fluid circuit comprises the following multiple functional parts: Multiple fluid reservoirs capable of storing multiple types of fluids, Multiple transfer units capable of transferring each of the multiple types of fluids stored, A cell induction and culture unit that performs cell induction and culture based on the multiple types of fluids stored therein, Equipped with, A cell manufacturing apparatus in which the plurality of fluid reservoirs each store a fluid containing source cells, an inducer introduction reagent, a culture medium for reprogramming or induction, and a fluid containing target cells, respectively.

3. The cell manufacturing apparatus according to claim 1 or 2, further comprising a first volume variable section for storing fluids extruded or drawn out by the aforementioned plurality of fluids.

4. The cell manufacturing apparatus according to claim 3, wherein the first volume variable section comprises a physical or chemical volume variable material.

5. The cell manufacturing apparatus according to claim 4, wherein the physical volume-adjustable material comprises a flexible bag or a syringe.

6. The cell manufacturing apparatus according to claim 1, wherein each of the plurality of fluid reservoirs is located between each of the plurality of closed connectors and each of the plurality of transfer units.

7. The cell manufacturing apparatus according to claim 1, wherein the plurality of closed connectors are attached to one side of the cell manufacturing plate and are connectable to a fluid container having a plurality of discharge and suction sections.

8. A cell production plate equipped with a fluid circuit integrating multiple functional parts, and a cell production apparatus equipped with multiple closed connectors that connect the fluid circuit to the external space in a closed manner, A fluid container that can be connected to at least one of the plurality of closed connectors, Equipped with, The aforementioned fluid circuit is The system includes multiple injection and discharge sections that can inject multiple types of fluids into the fluid circuit or discharge them outside the fluid circuit via the multiple closed connectors, The aforementioned fluid container is Multiple fluid reservoirs capable of storing multiple types of fluids, A discharge / suction unit that discharges the fluid into the fluid circuit and draws it out from the fluid circuit via at least one of the plurality of closed connectors, Equipped with, The fluid circuit or the fluid container further comprises a plurality of transfer units capable of transferring the stored fluid into the fluid circuit, A cell manufacturing system in which the plurality of fluid reservoirs each store a fluid containing source cells, an inducer reagent, a culture medium for reprogramming or induction, and a fluid containing target cells, respectively.

9. The cell manufacturing system according to claim 8, wherein the fluid circuit further comprises a first volume variable section for storing fluid that is extruded or drawn out by the injected or discharged fluid.

10. The cell manufacturing system according to claim 9, wherein the first volume variable section comprises a physical or chemical volume variable material.

11. The cell manufacturing system according to any one of claims 8 to 10, wherein the fluid container further comprises a second variable volume section for storing the fluid drawn out and extruded by the discharged and aspirated fluid.

12. The cell manufacturing system according to claim 11, wherein the second volume variable section comprises a physical or chemical volume variable material.

13. The cell manufacturing apparatus according to claim 10 or 12, wherein the physical volume variable material comprises a flexible bag or a syringe.

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