Cell extraction methods, cell extraction systems, subculture methods, subculture systems

Abstract

There was a need for a technique that could distinguish between cells with small differences in adhesive strength within a container and extract the target cells with a high selectivity ratio. [Solution] A cell extraction method comprising: a release agent injection step of injecting a first liquid containing a release agent into a culture vessel to make the adhesion force of the first cells lower than that of the second cells; a first detachment step of applying a first physical force to the contents of the culture vessel via the first liquid to detach at least a portion of the first cells and discharging the first liquid containing the detached first cells from the culture vessel; a second detachment step of supplying a second liquid to the culture vessel and having a second detachment mechanism apply a second physical force greater than the first physical force to the contents of the culture vessel via the second liquid to detach the second cells; and a recovery step of recovering the second liquid containing the second cells from the culture vessel.

Description

Technical Field

[0001] The present invention relates to a cell extraction method for selectively peeling and extracting cells from inside a container, a cell extraction system, and the like.

Background Art

[0002] iPS cells are pluripotent stem cells having the ability to differentiate into cells of various tissues and organs and the ability to proliferate almost infinitely. By introducing an initializing factor into monocytes, initialization of the genetic information of the cells is started. By culturing in a dedicated medium for several days, the initialization of the cells is completed, and iPS cells can be established. As a method for introducing the initializing factor, a method using a Sendai virus vector is known.

[0003] As the process until iPS cell establishment, first, monocytes are infected with a virus vector. The cells infected with the virus vector start to be initialized and change from floating cells to adherent cells. The cells that have changed to adherent cells proliferate on the bottom surface of the culture container, and the cells that have completed initialization become iPS cells and proliferate while forming colonies. On the other hand, the adherent cells during initialization also proliferate and grow in proximity to the iPS cell colonies. Therefore, the inside of the culture container becomes a state in which iPS cells and cells during initialization containing an initializing factor are mixed.

[0004] Cells during initialization do not show pluripotency and may inhibit differentiation induction. In addition, undifferentiated cells during initialization have a risk of tumorigenesis in vivo, and thus are unnecessary cells that should be removed before differentiation induction.

[0005] Cells undergoing reprogramming can be identified in the following ways. The first method is identification using green fluorescent protein (GFP). By using a viral vector programmed to produce GFP within the cell, infected cells that have begun reprogramming will fluoresce green. Once reprogramming is complete and the cells become iPS cells, they will no longer fluoresce green. On the other hand, cells undergoing reprogramming still fluoresce green because genes derived from reprogramming factors remain within the cell. Therefore, cells expressing GFP are cells undergoing reprogramming that should be removed. Cells undergoing reprogramming can also be identified using surface antigen markers. One example is the SSEA-1 surface antigen marker. Cells that are positive for SSEA-1 are cells that do not exhibit pluripotency like iPS cells. Therefore, similar to GFP, it is possible to identify cells undergoing reprogramming using this method.

[0006] Traditionally, cells in the process of reprogramming were removed by manual colony picking. By detaching and collecting the central cell of an iPS cell colony using a manipulator and then subculturing it, only iPS cells could be propagated. However, colony picking carries the risk of contamination of cells in the process of reprogramming, contamination by microorganisms, and damage to cells due to mechanical stress, depending on the skill level of the operator. Therefore, it is necessary to ensure and maintain a sterile environment and for skilled technicians to perform the detachment work with the utmost care, which has been practically difficult.

[0007] Patent Document 1 describes a cell culture apparatus for co-culturing human iPS cells and non-human cells, such as mouse fibroblasts, as feeder cells, and for separately harvesting each type of cell. Specifically, it discloses an apparatus that applies a first agitation followed by a second agitation to the culture vessel in order to detach the cells from the vessel, with the first agitation having a larger number of agitations, a higher agitation frequency, and a larger agitation angle compared to the second agitation. It is expected that by using an automated apparatus, it will be possible to harvest the desired cells without depending on the skill level of the operator.

[0008] Patent Document 2 discloses a culture method and a subculturing method that apply high-frequency vibrations to a culture vessel to selectively detach cells from the cell adhesion surface. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Japanese Patent Publication No. 2017-201946 [Patent Document 2] Japanese Patent Publication No. 2014-18185 [Overview of the project] [Problems that the invention aims to solve]

[0010] The apparatus disclosed in Patent Document 1 may be somewhat effective in systems where non-human cells, such as mouse fibroblasts, are co-cultured as feeder cells.

[0011] However, when creating iPS cells from mononuclear cells, as mentioned above, separating iPS cells from cells undergoing reprogramming and recovering them with high yield was difficult using the method described in Patent Document 1. This is because, when creating iPS cells from mononuclear cells, the difference in adhesion between the two types of cells to be separated, namely iPS cells and cells undergoing reprogramming, is small. Therefore, as disclosed in Patent Document 1, if a large agitation is initially applied to the culture vessel, both iPS cells and cells undergoing reprogramming may detach and be recovered together without separation.

[0012] Furthermore, Patent Document 1 controls the immersion time in a release agent and the number of incubation cycles to control the adhesion between human iPS cells and feeder cells, which are mouse fibroblasts. As a result, the iPS cells are immersed in the release agent for a long time or multiple times, which can cause the proteins on the surface of the iPS cells to denature, potentially degrading the quality of the cells. For example, this could lead to a decrease in the adhesion of iPS cells to the culture vessel used for subculturing, making subculturing impossible.

[0013] The method described in Patent Document 2 involves physically selecting a region within a culture vessel and applying a detaching force to detach cells. Therefore, if the cells to be separated are unevenly distributed within the culture vessel in a manner that makes them easily distinguishable, it may be possible to selectively detach the cells.

[0014] However, if, for example, iPS cells and cells in the process of reprogramming are mixed in a culture vessel, selectively detaching the cells is not easy. Even if localized areas are selected and detached sequentially, the processing time from immersion in the detaching agent to completion of detachment and subculturing becomes long, which may lead to a decrease in the quality of the iPS cells. For example, the adhesion of iPS cells to the culture vessel used for subculturing may decrease, resulting in the problem of not being able to subculture them.

[0015] Therefore, there was a need for a technology that could distinguish cells with small differences in adhesive strength within a container and extract the target cells with a high selectivity ratio. [Means for solving the problem]

[0016] A first aspect of the present invention is a cell extraction method characterized by comprising: a release agent injection step of injecting a first liquid containing a release agent from a first injection unit into a culture vessel containing a mixture of first cells and second cells, thereby lowering the adhesion force of the first cells to that of the second cells; a first detachment step of, after the release agent injection step, in which a first detachment mechanism applies a first physical force to the contents of the culture vessel via the first liquid to detach at least a portion of the first cells, and discharges the first liquid containing the detached first cells from the culture vessel; a second detachment step of, after the first detachment step, in which a second liquid is supplied to the culture vessel from a second injection unit, and a second detachment mechanism applies a second physical force greater than the first physical force to the contents of the culture vessel via the second liquid to detach the second cells that were present inside the culture vessel; and a recovery step of, in which a recovery mechanism recovers the second liquid containing the detached second cells from the culture vessel.

[0017] Furthermore, a second aspect of the present invention is a cell extraction system characterized in that a first liquid containing a release agent is injected from a first injection port into a culture vessel containing a mixture of first cells and second cells having greater adhesion than the first cells, thereby immersing the first cells and the second cells; after the release agent injection port, a first release mechanism performs a first release step in which it applies a first physical force to the contents of the culture vessel via the first liquid to release at least a portion of the first cells, and then discharges the first liquid containing the released first cells from the culture vessel; after the first release step, a second liquid is injected from a second injection port into the culture vessel, and a second release mechanism performs a second release step in which it applies a second physical force greater than the first physical force to the contents of the culture vessel via the second liquid to release the second cells that were present inside the culture vessel; and a recovery mechanism performs a recovery step in which it recovers the second liquid containing the released second cells from the culture vessel. [Effects of the Invention]

[0018] According to the present invention, it is possible to provide a technique advantageous for distinguishing cells with small differences in adhesive force present in a container and extracting target cells with a high selection ratio.

Brief Description of Drawings

[0019] [Figure 1] A block diagram showing the configuration of a cell detachment and sorting system including a cell detachment device according to an embodiment. [Figure 2] A schematic diagram illustrating the configuration of a cell detachment device according to an embodiment. [Figure 3] A flowchart showing a procedure for detaching cells from a culture container and selectively collecting second cells by a cell detachment and sorting system. [Figure 4] (a) A schematic diagram for explaining step S1. (b) A schematic diagram for explaining steps S2 and S3. (c) A schematic diagram for explaining steps S4 and S5. [Figure 5] (a) A schematic diagram for explaining step S6. (b) A schematic diagram for explaining step S7. (c) A schematic diagram for explaining steps S8 and S9. [Figure 6] (a) A schematic diagram for explaining step S10. (b) A schematic diagram for explaining step S11.

Embodiments for Carrying Out the Invention

[0020] Referring to the drawings, a cell extraction method, a cell extraction system, etc. according to an embodiment of the present invention will be described. The following embodiments are illustrative, and for example, those skilled in the art can appropriately modify and implement the details of the configuration without departing from the gist of the present invention.

[0021] In the drawings referred to in the following description of the embodiments and examples, unless otherwise specified, elements denoted by the same reference numerals have the same functions. In the drawings, when a plurality of the same elements are arranged, the assignment of reference numerals and their descriptions may be omitted.

[0022] Furthermore, since drawings may be schematically represented for the convenience of illustration and explanation, the shape, size, and arrangement of elements shown in the drawings may not strictly correspond to those of actual objects. In addition, notations such as "XX or greater and YY or less" or "XX~YY" that indicate a numerical range mean a numerical range that includes the endpoints XX (lower limit) and YY (upper limit). When numerical ranges are described in steps, the upper and lower limits of each numerical range can be combined in any way.

[0023] [Embodiment] (Configuration of the cell extraction system) Figure 1 is a block diagram showing the configuration of the cell detachment and sorting system 1 as a cell extraction system according to the embodiment. The cell detachment and sorting system 1 shown in Figure 1 is a cell extraction system capable of performing the cell extraction method according to the embodiment, and selectively detaches target cells from two or more types of cells and extracts them from the culture vessel. As shown in Figure 1, the cell detachment and sorting system 1 has a cell detachment device 10 and a constant temperature incubator 20.

[0024] The cells used in this embodiment are not particularly limited as long as they are cells that can be used for adherent culture using a culture vessel. For example, they can be mammalian cells such as primate cells or rodent cells, and preferably, they can be cells of humans, monkeys, mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, or goats, or cells derived from these.

[0025] The cells handled by the cell extraction system according to this embodiment are preferably pluripotent stem cells, and examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells or induced pluripotent stem cells), Muse cells (Multilineage-differentiating Stress Enduring Cells), embryonic tumor cells (EC cells), or embryonic germ stem cells (EG cells). In this embodiment, the cells to be extracted are preferably pluripotent stem cells of mammals such as primates or rodents. More preferably, they are iPS (induced Pluripotent Stem) cells of mammals such as primates or rodents, and most preferably, they are human iPS cells. In this embodiment, either cells that proliferate forming a monolayer on the adhesion surface or cells that proliferate forming a multilayer may be used.

[0026] The cell detachment device 10 is a mechanical device that detaches and collects cells cultured in a culture vessel from the culture vessel, and comprises a control device 11 and a detachment mechanism 12. The control device 11 is a computer that comprehensively controls the operation of the detachment mechanism 12, and the detachment mechanism 12 operates according to commands from the control device 11. The control device 11 may also be part of a control unit that controls the entire cell detachment and sorting system 1, including the incubator 20.

[0027] The detachment mechanism 12 is a mechanical mechanism that has the function of detaching cells cultured in a culture vessel from the culture vessel. As will be described in detail later, the detachment mechanism 12 can accelerate cell detachment by applying a physical force to the contents of the culture vessel via a liquid. When detaching a first cell from the culture vessel, the detachment mechanism 12 can apply a first physical force to the contents of the culture vessel via a flowing first liquid. When detaching a second cell from the culture vessel, the detachment mechanism 12 can apply a second physical force, which is greater than the first physical force, to the contents of the culture vessel via a flowing second liquid. The detachment mechanism 12 may apply the first and second physical forces by different types of mechanisms, or it may apply them by changing the operating conditions using the same type of mechanism.

[0028] The incubator 20 is equipped with a chamber capable of housing a culture vessel, and the temperature and / or humidity inside the chamber can be controlled to culture the cells contained in the culture vessel. The incubator 20 and the cell detachment device 10 may be configured separately or as a single unit. In the latter case, for example, if all or some of the components of the detachment mechanism 12 are provided inside the chamber of the incubator 20, it will be possible to culture cells in the cell detachment device 10.

[0029] Figure 2 is a schematic diagram illustrating the configuration of a cell detachment device 10 according to an embodiment. A culture vessel 30 in which cells are cultured is placed inside the detachment mechanism 12. The shape of the culture vessel 30 is not particularly limited, but a dish, petri dish, flask, well plate, etc., having an opening that allows the detachment mechanism 12 to access the inside of the culture vessel 30 is preferably used. The inner bottom surface of the culture vessel 30 is called the culture surface 31. Cultured cells are in contact with the culture surface 31. Before the cell detachment process by the detachment mechanism 12, multiple cell clumps (colonies) are formed in the culture vessel 30 and are adhered to the culture surface 31.

[0030] The culture vessel 30 is an open or closed culture vessel having a culture surface 31 suitable for adherent cell culture. The structure and material of the culture surface 31 are not particularly limited as long as it is a surface suitable for cell culture, but it is preferable that it is coated for cell adhesion in order to improve the efficiency of cell adhesion. Examples of cell adhesion coatings include extracellular matrix (ECM) coatings such as gelatin, collagen, laminin, fibronectin, entactin, hyaluronic acid, adhesive oligopeptides, polylactic acid, polyglycolic acid, polylysine, or polyornithine, or polymer coatings. In particular, for a culture vessel 30 for pluripotent stem cells, it is preferable to use, for example, CORNING®'s 35mm dish 430165 as the culture vessel and to form the culture surface 31 by coating its surface with, for example, iMatrix®-551 manufactured by MATRIXOME.

[0031] The cell detachment device 10 sequentially detaches two or more types of cells that have been adherently cultured in the culture vessel 30 from the culture vessel 30. In this embodiment, for example, cells in the process of reprogramming, which have been initiated using a viral vector, and iPS cells can be treated as two or more types of cells and detached sequentially. When comparing cells in the process of reprogramming with iPS cells, the adhesion strength of the latter is greater than that of the former, but as will be described in detail later, this embodiment utilizes this difference in adhesion strength to detach and extract iPS cells with a high selectivity ratio.

[0032] In the following description of the embodiment, the cells cultured in the culture vessel 30 are cells obtained when single-donor mononuclear cells are reprogrammed, with the first cells being GFP-positive cells that have not yet been reprogrammed, and the second cells being iPS cells that have been reprogrammed.

[0033] As shown in Figure 2, the cell detachment apparatus 10 comprises a control device 11 and a detachment mechanism 12. The detachment mechanism 12 includes various pumps such as a washing solution pump 121, a detachment solution pump 123, a detachment solution pump for second cells 125, a waste liquid discharge pump 127, and a cell suspension recovery pump 129, as well as a drive mechanism 132. The control device 11 is connected to the detachment mechanism 12 via wired or wireless communication and controls the operation of the various pumps and the drive mechanism 132 of the detachment mechanism 12.

[0034] The washing solution pump 121 responds to a control signal from the control device 11 by distributing the washing solution into the culture vessel 30 via the washing solution delivery pipe 122. The washing solution delivery pipe 122 is a cylindrical structure (e.g., a tube) through which the washing solution flows. The washing solution is used to wash away impurities attached to the culture surface 31 of the culture vessel 30 and to the cells present in the culture vessel 30. As the washing solution, for example, physiological saline, liquid culture medium, or cryopreservation solution stored in a washing solution tank (not shown) may be used.

[0035] The washing solution pump 121 draws washing solution from the washing solution tank and delivers it into the culture vessel 30 via the washing solution delivery pipe 122. This allows the culture surface 31 and / or cells adhering to the culture surface 31 to be washed. Impurities removed by washing include, for example, dead cells floating in the culture vessel 30, or calcium ions and magnesium ions present in the culture medium of the culture vessel 30. If washing is insufficient, when a delaminating agent is injected, the calcium ions and magnesium ions remaining in the culture medium may react with the chelating agent, potentially preventing the chelating agent from adequately cleaving the cell bonds. In this embodiment, sufficient washing is performed by the washing unit equipped with the washing solution pump 121 and the washing solution delivery pipe 122.

[0036] The delamination pump 123, in response to a control signal from the control device 11, discharges a liquid containing the delamination agent (first liquid) into the culture vessel 30 via the delamination agent delivery pipe 124 (first injection section). The delamination agent delivery pipe 124 is a cylindrical structure (e.g., a tube) through which the liquid containing the delamination agent flows. The delamination agent has the effect of weakening the adhesion between cells and the culture surface 31, and the adhesion between cells. As the liquid containing the delamination agent (first liquid), for example, a solution containing proteolytic enzymes and / or chelating agents stored in a delamination agent tank (not shown) may be used. As will be described later, the delamination agent is used, for example, to delaminate GFP-positive cells, which are the first cells, from the culture vessel 30.

[0037] The stripping solution pump 123 draws the stripping solution from the stripping solution tank and delivers it to the culture vessel 30 via the stripping solution delivery pipe 124. As a result, the cells adhering to the culture surface 31 are immersed in the solution containing the stripping solution (first solution).

[0038] The second cell detachment solution pump 125 responds to a control signal from the control device 11 by discharging the second cell detachment solution (second solution) into the culture vessel 30 via the second cell detachment solution discharge tube 126 (second injection section). The second cell detachment solution discharge tube 126 is a structure that flows and discharges the second cell detachment solution, and for example, a tube, pipette, or spray nozzle can be used. The second cell detachment solution is used to detach the second cells in contact with the culture surface 31 from the culture surface 31. As the second cell detachment solution, a solution that does not contain proteolytic enzymes or chelating agents is used, and preferably physiological saline or liquid culture medium can be used.

[0039] The second cell detachment solution pump 125 draws the second cell detachment solution from the second cell detachment solution tank (not shown) and discharges it into the culture vessel 30 via the second cell detachment solution discharge tube 126. The second cell detachment solution pump 125 and the second cell detachment solution discharge tube 126 constitute a discharge mechanism that operates in response to a control signal from the control device 11.

[0040] The second cell in contact with the culture surface 31 is detached from the culture surface 31 by the action of a physical force applied by the second cell detachment solution. The detachment mechanism 12 can apply a first physical force via the first solution when detaching the first cell from the culture vessel, and a second physical force greater than the first physical force via the second solution when detaching the second cell from the culture vessel. In this example, by appropriately setting the operating conditions of the second cell detachment solution pump 125 (e.g., discharge pressure, flow rate, etc.) and the structure of the outlet of the second cell detachment solution discharge tube 126, a second physical force greater than the first physical force can be applied to the cells in the culture vessel via the second solution. In this example, the second detachment mechanism is composed of a discharge mechanism having a second cell detachment solution pump 125 and a second cell detachment solution discharge tube 126.

[0041] The waste liquid discharge pump 127 responds to a control signal from the control device 11 by sucking the liquid to be discharged from the culture vessel 30 (waste liquid) through the waste liquid suction tube 128. The waste liquid may be, for example, a liquid containing a release agent (release solution) remaining in the culture vessel 30, a washing solution, or a liquid culture medium. The waste liquid suction tube 128 is a cylindrical structure that sucks up and circulates the waste liquid; for example, a tube is used. The waste liquid sucked up from the culture vessel 30 is discharged into a waste liquid tank (not shown).

[0042] The cell suspension recovery pump 129 responds to a control signal from the control device 11 by aspirating the cell suspension from the culture vessel 30 via the cell suspension suction tube 130 and recovering it in a recovery tank (not shown). The cell suspension is a second cell detachment solution containing second cells detached from the culture surface 31. The recovery mechanism consists of the cell suspension recovery pump 129, the cell suspension suction tube 130, and a recovery tank (not shown).

[0043] The drive mechanism 132 is a mechanical mechanism capable of changing the orientation of the culture vessel 30 in response to a control signal input from the control device 11 via the signal line 131. The specific form of the drive mechanism 132 is not particularly limited, but it may consist of a drive source such as a motor or air cylinder and a movable part that supports the culture vessel 30 in a manner that allows the orientation to be changed by combining links and cams. By appropriately setting the driving conditions of the drive mechanism 132 (e.g., the tilt angle of the culture vessel in the oscillating motion, the period and number of oscillating motions, etc.), a first physical force can be applied to the cells in the culture vessel via the first liquid. In this example, the drive mechanism 132 constitutes the first detachment mechanism.

[0044] The control device 11 executes a control program pre-recorded on a recording medium to control the operation of each of the pumps and drive mechanisms 132 described above, thereby automatically performing the process of detaching and collecting desired cells from the culture vessel. For example, the control device 11 can control the sequence of discharging or aspirating various liquids, control the flow rate, control the timing, and so on.

[0045] According to the cell detachment sorting system 1 of this embodiment, cells with small differences in adhesion strength present in the culture vessel can be selectively recovered with high yield. For example, iPS cells can be recovered with a high selectivity ratio from a culture vessel containing a mixture of mononuclear cells that have undergone reprogramming with reprogramming factors introduced and iPS cells that have completed reprogramming with the introduction of reprogramming factors introduced into mononuclear cells. For example, cells that have undergone reprogramming using a viral vector and iPS cells can be detached sequentially, and iPS cells can be recovered with a high selectivity ratio.

[0046] (Methods for cell detachment and cell extraction) The procedure for selective cell detachment and recovery using the cell detachment and sorting system 1 will be explained with reference to the drawings. Figure 3 is a flowchart showing the procedure for a cell extraction method in which cells are detached from a culture vessel and selectively extracted as a second cell using the cell detachment and sorting system 1. Figures 4(a) to 6(b) are schematic diagrams illustrating each step of the processing procedure. Each figure shows only the components necessary for explaining the process, and components not necessary for explanation are omitted as appropriate.

[0047] In the following example, the incubator 20 and the cell detachment device 10 are assumed to be integrally formed. Specifically, within the chamber of the incubator 20, the washing solution delivery tube 122, the detachment agent delivery tube 124, the second cell detachment solution discharge tube 126, the waste liquid suction tube 128, the cell suspension suction tube 130, and the drive mechanism 132 are assumed to be arranged so as to be accessible to the culture vessel.

[0048] Before starting the cell extraction process, the culture vessel 30 contains GFP-positive cells, which are the first cultured cells, colonies of iPS cells, which are the second cultured cells, and the culture medium used for the first culture. The cells in the culture vessel 30 may have been cultured in the incubator 20 or in another incubator.

[0049] When the cell extraction process is initiated to selectively detach and recover the second cells from the first and second cells mixed in the culture vessel 30, first, in step S1, the liquid culture medium is removed from the culture vessel 30 as shown in Figure 4(a). Specifically, the control device 11 drives the waste liquid discharge pump 127. The waste liquid discharge pump 127 sucks the liquid culture medium from the culture vessel 30 via the waste liquid suction tube 128 and discharges it outside the culture vessel 30. The discharged waste liquid is collected in a waste liquid tank (not shown). The culture vessel 30 remains with the first cells, the second cells, and the culture medium that was not aspirated.

[0050] Next, in step S2, the first washing process (first washing step) is performed. As shown in Figure 4(b), the control device 11 drives the washing solution pump 121 and delivers the washing solution into the culture vessel 30 via the washing solution delivery pipe 122. In addition to the adherent cultured cells, the culture vessel 30 contains residual culture medium that was not aspirated in step S1, as well as impurities such as nutrients contained in the culture medium. These impurities are washed away from the cells by the washing solution discharged from the washing solution delivery pipe 122.

[0051] Next, in step S3, as shown in Figure 4(b), the control device 11 drives the waste liquid discharge pump 127, sucks up the washing liquid containing impurities through the waste liquid suction pipe 128, discharges it from the culture vessel 30, and collects it in a waste liquid tank (not shown). Steps S2 and S3 perform the first washing process, washing the cells in the culture vessel 30 and the culture surface 31 of the culture vessel 30.

[0052] Next, in step S4, which is the stripping agent injection process, as shown in Figure 4(c), the control device 11 drives the stripping agent pump 123 and discharges the stripping agent (first solution) into the culture vessel 30 via the stripping agent delivery pipe 124. A sufficient amount of stripping agent to immerse the first and second cells is discharged into the culture vessel 30.

[0053] Next, in step S5, as shown in Figure 4(c), an immersion process (immersion step) is performed in which the first cells and the second cells are immersed in the release agent for a predetermined time in order to allow the release agent to act. Due to the action of the release agent, the adhesive strength of both the GFP-positive cells (first cells) and the iPS cells (second cells) may decrease. The immersion process is performed so that the adhesive strength of the first cells becomes smaller than that of the second cells, making it easier for the first cells to be selectively detached in the first detachment process described later.

[0054] It is also possible to perform incubation in parallel with the immersion treatment. If incubation is performed, the control device 11 notifies the incubator 20 of the command to start incubation. Upon receiving the command, the incubator 20 maintains the environment in which the culture vessel 30 to which the detachment solution has been added is placed at a predetermined temperature and humidity for a predetermined period. The predetermined temperature and humidity should be set to conditions suitable for culturing iPS cells; for example, the temperature should be set to about 37°C and the humidity to about 95%. During incubation, the detachment solution acts on the cells, reducing the adhesion between GFP-positive cells (first cells) and the culture surface 31, the adhesion between iPS cells (second cells), and the adhesion between iPS cells (second cells) and the culture surface 31. The period for maintaining the predetermined temperature (constant temperature period) should be set to a length determined by experiment, for example, the time required to reduce the adhesion to a predetermined level. After the constant temperature period has elapsed, the control device 11 terminates the temperature and humidity control by the incubator 20.

[0055] If incubation is not performed in parallel with the immersion treatment, the control device 11 immerses the first and second cells in the release agent for a predetermined immersion period. The immersion period can be set to a length determined, for example, by experiment, as the time required to reduce the adhesive strength to a predetermined level.

[0056] Following the immersion treatment, in step S6, which is the first detachment step, a first detachment treatment is performed to selectively detach GFP-positive cells (first cells). In the first detachment treatment, as shown in Figure 5(a), the orientation of the culture vessel 30 is changed by the drive mechanism 132, and a first physical force is applied to the contents of the culture vessel 30 via the detachment agent solution (first liquid) that flows due to the change in orientation, thereby detaching at least a portion of the first cells. Note that Figure 5(a) is a schematic diagram and the liquid surface does not appear to be tilted, but in reality the liquid surface can be tilted. For example, by repeatedly tilting the culture vessel 30 and allowing the detachment solution to flow, the first cells, which are GFP-positive cells, can be selectively detached from the culture vessel or the second cells.

[0057] Furthermore, it is preferable to perform step S6 immediately after completing step S5. This is because it prevents the second cells from being unnecessarily immersed in the detachment solution, thereby reducing the possibility of damage to the second cells from the detachment solution.

[0058] The first delamination process is preferably performed in a closed culture vessel 30. If the culture vessel 30 is closed, even if the liquid level is shaken, the liquid will not overflow outside the culture vessel 30 and contaminate the equipment. Also, any liquid that overflows from the culture vessel 30 will not come into contact with the external environment and then recirculate back into the culture vessel 30, thus preventing contamination from being introduced into the culture vessel 30. However, if the flow of liquid inside the culture vessel 30 is controlled with high precision, the first delamination process may be performed in an open culture vessel without a lid, for example.

[0059] The conditions for the first delamination process are set so that the first cells are sufficiently delaminated, while the delamination of the second cells is suppressed. For example, the tilt angle θ of the bottom surface of the culture vessel relative to the horizontal plane may be set to 5°, the number of oscillations to 20 times, the time required for one tilting motion to 0.3 seconds, and the interval between tilting motions to 0.5 seconds. For example, the tilting motion is controlled so that the average flow velocity of the delamination solution moving inside the culture vessel 30 is 0.24 [m / sec].

[0060] The first peeling process is preferably carried out under peeling conditions that peel off more than 70% of the first cells that were present inside the culture vessel 30 at the start of the peeling agent injection process (step S4).

[0061] For example, the state of the culture surface 31 during or after the first detachment process can be observed using a camera or the like, and the tilt angle, tilt speed, number of tilt movements, interval between tilt movements, etc., can be adjusted so that the first cells are sufficiently detached, but the detachment of the second cells is suppressed. These conditions can be set in advance through experiments and stored as operating parameters in the control program of the control device 11.

[0062] In the subsequent step S7, the control device 11 drives the waste liquid discharge pump 127 and, as shown in Figure 5(b), recovers the release agent from the culture vessel 30 via the waste liquid suction pipe 128 and discharges it into a first container (not shown). The recovered release agent contains cells (mainly the first cells) that were detached in the first release treatment, but if the recovered release agent is treated as waste liquid, the first container may be a waste liquid tank. In addition to the second cells, which are the target of extraction, impurities such as release agent and first cells that were not aspirated may remain in the culture vessel 30.

[0063] In the following step S8, a second washing process (second washing step) is performed. As shown in Figure 5(c), the control device 11 drives the washing solution pump 121 and discharges the washing solution into the culture vessel 30 via the washing solution delivery pipe 122. The washing solution pump 121 and the washing solution delivery pipe 122 constitute the washing unit.

[0064] The contents of the culture vessel 30 include not only the second cells that have not yet been detached, but also impurities such as the detachment agent that remained after being aspirated in step S7, the first cells present in the detachment agent, or the first cells attached to the second cells. The discharged washing solution washes the second cells that have not yet been detached, and the impurities are washed away from the second cells.

[0065] To effectively perform the second washing process, the control device 11 can activate the drive mechanism 132 to agitate the culture vessel 30 simultaneously with or after the discharge of the washing solution. This allows for efficient removal of impurities such as first cells adhering to the culture vessel 30 and the second cells. The operation of the drive mechanism 132 is carried out under conditions that allow sufficient washing away of first cells (GFP-positive cells) adhering to the second cells (iPS cells) and any remaining stripping agent in the culture vessel 30. For example, the tilt angle θ of the bottom surface of the culture vessel relative to the horizontal plane should be set to 5°, the number of agitations to 20, the time required for one tilting motion to be 0.3 seconds, and the interval between tilting motions to be 0.5 seconds. For example, the tilting motion is controlled so that the average flow velocity of the washing solution moving inside the culture vessel 30 due to the tilting motion is 0.24 [m / sec].

[0066] The second washing process should preferably be carried out under washing conditions that remove more than 70% of the first cells present inside the culture vessel 30 at the start of the release agent injection process (step S4). Furthermore, it is desirable that the second washing process be carried out under washing conditions that leave more than 70% of the second cells present inside the culture vessel 30 at the start of the release agent injection process (step S4) inside the culture vessel 30 at the end of the second washing process.

[0067] For example, the state of the culture surface 31 during or after the second washing process can be observed using a camera or the like, and the tilt angle, tilt speed, number of tilt movements, interval between tilt movements, etc., can be adjusted so that impurities are thoroughly washed away, but the detachment of the second cells is suppressed. These washing conditions can be set in advance through experiments and stored as operating parameters in the control program of the control device 11.

[0068] Step S8 is preferably performed immediately after completing Step S7. This prevents the second cells from unnecessarily continuing to come into contact with the detachment solution remaining in the culture vessel 30, thereby reducing the possibility of damage to the second cells from the detachment solution.

[0069] Then, in step S9, as shown in Figure 5(c), the control device 11 drives the waste liquid discharge pump 127, sucks up the washing liquid containing impurities through the waste liquid suction pipe 128, discharges it from the culture vessel 30, and collects it in a second container (not shown). If the recovered washing liquid is to be treated as waste liquid, the second container may be a waste liquid tank.

[0070] Steps S8 and S9 are performed to clean the second cells that have not yet been detached from the culture vessel 30, as well as the culture surface 31 of the culture vessel 30. To effectively remove any remaining detachment agent in the culture vessel 30 and any first cells adhering to the second cells, steps S8 and S9 of the flowchart in Figure 3 may be repeated multiple times (for example, twice).

[0071] In the following step S10, a second detachment process (second detachment step) is performed. In the second detachment process, a physical force is applied to the contents of the culture vessel 30 via a second cell detachment solution (second liquid) to detach the second cells. In the first detachment process, a first physical force was applied to the contents of the culture vessel 30 via a detachment agent (first liquid) that flows due to changes in the posture of the culture vessel 30. In the second detachment process, a second physical force, greater than the first physical force in the first detachment process, is applied to the second cells via the second cell detachment solution (second liquid) to detach the second cells. As the second cell detachment solution, a liquid that does not adversely affect the characteristics of the second cells is used, such as physiological saline or liquid culture medium. That is, a liquid that does not contain detachment agents such as proteolytic enzymes or chelating agents is used.

[0072] Specifically, as shown in Figure 6(a), the control device 11 drives the second cell detachment solution pump 125 and discharges the second cell detachment solution (second solution) in a mist form toward the culture surface 31 via the second cell detachment solution discharge tube 126. The second cell detachment solution is discharged under conditions that allow it to detach the second cells without physically damaging them.

[0073] Preferably, the tip of the second cell detachment solution discharge tube 126 is a spray nozzle, and the second cell detachment solution is discharged as a plurality of minute droplets with a predetermined initial velocity. The average particle size of the minute droplets is, for example, 50 μm or more and 600 μm or less. In other words, "mist-like" does not refer to the shape of the spatial area in which the droplets are discharged (e.g., conical, linear), but rather to the fact that the droplets are minute in size.

[0074] The droplets of the second cell detachment solution may be discharged continuously from the second cell detachment solution discharge tube 126, or they may be discharged pulsed or intermittently. For example, the second cells may be detached from the culture vessel 30 by discharging a group of droplets of the second cell detachment solution from the second cell detachment solution discharge tube 126 in a single discharge. In this case, the amount of the second cell detachment solution discharged in a single discharge, i.e., the total amount of droplets discharged in a single discharge, is preferably 3.0 mL or less. The discharge time for a single discharge of the second cell detachment solution is preferably 0.2 seconds or less. The discharge angle from the second cell detachment solution discharge tube 126 can be set so that the discharged group of droplets of the second cell detachment solution contacts the entire surface (preferably 95% or more of the area) of the second cell colony in a plan view. Typically, the second cell detachment solution discharge tube 126 discharges the second cell detachment solution in a conical area, but it may also discharge in a straight line, for example.

[0075] Due to the mechanical action of the droplets of the discharged second cell detachment solution, a stronger shear force is directly applied to the second cells (iPS cells) than during the first detachment process (step S6), causing the second cells (iPS cells) to detach from the culture surface 31. As a result, the detached iPS cells are suspended in the second cell detachment solution discharged into the culture vessel 30, and the second cell detachment solution becomes a cell suspension.

[0076] The second detachment process is preferably carried out under conditions that detach more than 90% of the second cells that were present inside the culture vessel 30 at the end of the second washing process (step S10) from the culture vessel 30 and include them in the cell suspension.

[0077] The droplet dispensing conditions (e.g., droplet size, dispensing frequency, initial droplet velocity, etc.) may be adjusted by observing the state of the culture surface 31 during or after the second detachment process using, for example, a camera, to ensure that the second cells are properly detached. These dispensing conditions may be set in advance through experiments and stored as operating parameters of the control program of the control device 11 (e.g., driving conditions for the second cell detachment solution pump 125).

[0078] Once the second detachment step (step S10) is completed, the process moves to step S11, which is the recovery step, and the cell suspension containing the second cells (iPS cells), which are the target of extraction, is recovered from the culture vessel 30. Specifically, as shown in Figure 6(b), the control device 11 drives the cell suspension recovery pump 129, aspirates the cell suspension from the culture vessel 30 via the cell suspension suction tube 130, and injects it into a third container (not shown) that is detachably attached to the cell detachment sorting system 1. There may be one third container into which the cell suspension is injected, or the cell suspension may be divided and injected into multiple third containers. The third containers containing the cell suspension of the target iPS cells are removed from the cell detachment sorting system 1 and transported to the next process (e.g., subculturing) according to the intended use of the iPS cells.

[0079] By performing each of the above steps, the cell detachment process by the cell detachment sorting system 1 is completed. According to the cell detachment method of this embodiment, cells with small differences in adhesion strength present in the culture vessel can be distinguished and recovered selectively with a high yield. For example, iPS cells can be recovered with a high selectivity ratio from a culture vessel containing a mixture of cells in the process of reprogramming, in which reprogramming factors have been introduced into mononuclear cells, and iPS cells in which reprogramming has been completed, in which reprogramming factors have been introduced into mononuclear cells. For example, cells in the process of reprogramming, in which reprogramming has been initiated using a viral vector, and iPS cells can be detached sequentially, and iPS cells can be recovered with a high selectivity ratio. [Examples]

[0080] In the following examples, a cell extraction device and cell extraction method according to the above-described embodiment were used in a subculture system for creating and subculturing iPS cells from mononuclear cells. The cells cultured in the culture vessel were cells obtained by reprogramming mononuclear cells from a single donor; the first cells were GFP-positive cells, and the second cells were iPS cells.

[0081] In the example, a cell suspension containing the second type of cell, iPS cells, was recovered by the method described with reference to Figures 3 and 4(a) to 6(b). In the comparative example, the first detachment step in step S6 described in Figure 3 was omitted, and only the second detachment step in step S10 was performed to recover the cell suspension from the culture vessel.

[0082] For the examples and comparative examples, cell suspensions recovered from the culture vessels were subcultured from P0 to P1. First, for P0, cells were detached using the methods of the examples and comparative examples, and the ratio of GFP-positive cells to the total cells contained in the cell suspension recovered in step S11 was measured. The recovered cell suspension was used to reseed cells into a new culture vessel, and the cells were cultured in the culture section for 7 days. Subsequently, cells were detached and recovered from the new culture vessel using the methods of the examples and comparative examples, and the ratio of GFP-positive cells to the total cells was measured. The rate of decrease in GFP-positive cells for the examples and comparative examples was calculated using the GFP-positive cell rate at the time of P0 cell recovery and the GFP-positive cell rate at the time of P1 cell recovery. The rate of decrease is defined by the following formula 1.

[0083] (Formula 1) Reduction rate = (Percentage of GFP-positive cells in P1) / (Percentage of GFP-positive cells in P0)

[0084] Table 1 shows the percentage decrease in GFP-positive cells after passage from P0 to P1 for the examples and comparative examples. [Table 1]

[0085] As shown in Table 1, in subculture, this example shows a significantly higher rate of reduction in the GFP-positive cell percentage compared to the comparative example. In this example, in the first detachment process, GFP-positive cells with relatively low adhesion are selectively removed from the culture vessel, and then the iPS cells with relatively high adhesion remaining in the culture vessel are recovered by the second detachment process. For this reason, the rate of reduction in the GFP-positive cell percentage is higher compared to the comparative example.

[0086] This embodiment allows for the removal of non-pluripotent cells that are in the reprogramming stage before differentiation induction with a high selectivity ratio. As a result, the purity of iPS cells, which are the target of subculturing, can be increased, and it can be seen that the quality of the extracted iPS cells in this embodiment is significantly superior to that of the comparative example.

[0087] [Other embodiments] It should be noted that the present invention is not limited to the embodiments and examples described above, and many modifications are possible within the technical concept of the present invention. For example, all or part of the different embodiments and examples described above may be combined and implemented. For example, the first cell is not limited to GFP-positive cells, but may be, for instance, an SSEA-1-positive cell.

[0088] The washing solution recovered from the culture vessel in step S3, the release agent recovered from the culture vessel in step S7, and the washing solution recovered from the culture vessel in step S9 may be collected in separate containers depending on the post-recovery processing method, or they may be collected in a common container (e.g., a waste liquid tank).

[0089] In the first detachment step (step S6), it is sufficient to apply a weak shear force (first physical force) to the vicinity of the culture surface 31 via the first liquid, such that the first cells detach but the second cells do not. Therefore, the first detachment step is not limited to a method / mechanism for tilting the culture vessel 30. For example, it may be a mechanism that moves the culture surface 31 of the culture vessel 30 in a horizontal plane (e.g., linear reciprocating motion or circular motion in a plan view) without tilting it with respect to the horizontal direction. Alternatively, it may be a mechanism that moves the culture vessel 30 up and down in the vertical direction. Alternatively, a combination of tilting, horizontal movement, vertical movement, and vibration application to the culture vessel (e.g., ultrasonic vibration) may be used to apply a weak shear force (first physical force).

[0090] Alternatively, the culture vessel 30 may be configured to flow the first liquid within the culture vessel 30, thereby applying a weak shear force (first physical force) near the culture surface 31, by means of a fluidization mechanism or stirring mechanism within the culture vessel 30, or by using a pump installed outside the culture vessel 30. For example, the stripping agent may be discharged from the stripping agent pump 123 via the stripping agent delivery pipe 124, while the waste liquid discharge pump 127 is operated to suck the stripping agent through the waste liquid suction pipe 128, thereby generating a flow of the stripping agent within the culture vessel 30.

[0091] The second peeling mechanism may apply a second physical force using a mechanism different from the first peeling mechanism, or it may apply a second physical force by changing the operating conditions using the same mechanism as the first peeling mechanism. For example, after injecting the second liquid into the culture vessel, the same mechanism as the first peeling mechanism may shake the culture vessel with greater intensity in the second peeling step than in the first peeling step.

[0092] The control device 11 is a computer that comprehensively controls the peeling mechanism 12 and includes a processor. The processor may consist of, for example, a CPU, a GPU, an Application Specific Integrated Circuit (ASIC), a programmable logic device (for example, a Simple Programmable Logic Device (SPLD), a Complex Programmable Logic Device (CPLD), and a Field Programmable Gate Array (FPGA)). The processor performs its functions by reading and executing a program stored in a memory circuit. Alternatively, instead of storing the program in a memory circuit, the program may be directly incorporated into the processor's circuitry. In this case, the processor performs its functions by reading and executing the program incorporated into the circuitry. On the other hand, if the processor is, for example, an ASIC, the program is not stored in a memory circuit, but the function is directly incorporated as a logic circuit into the processor's circuitry. The processor can perform control of some or all of the peeling process described with reference to Figure 3 by executing a control program recorded on a recording medium that can be read by the computer. The control device 11 can perform control of part or all of the peeling process by means of, for example, sequence control, feedback control, or a combination of sequence control and feedback control.

[0093] The present invention can also be realized by supplying a program that implements one or more of the functions of the embodiments to a system or device via a network or storage medium, and by having one or more processors in the computer of that system or device read and execute the program. It can also be realized by a circuit (e.g., an ASIC) that implements one or more functions.

[0094] This specification discloses at least the following: [Item 1] A release agent injection step involves injecting a first solution containing a release agent into a culture vessel containing a mixture of first cells and second cells from a first injection port, thereby lowering the adhesion strength of the first cells to that of the second cells. Following the release agent injection step, the first release mechanism applies a first physical force to the contents of the culture vessel via the first liquid to release at least a portion of the first cells, and the first liquid containing the released first cells is discharged from the culture vessel in a first release step. A second peeling step is performed in which, after the first peeling step, a second liquid is supplied from the second injection unit to the culture vessel, and the second peeling mechanism applies a second physical force greater than the first physical force to the contents of the culture vessel via the second liquid, thereby peeling off the second cells that were present inside the culture vessel. The recovery mechanism includes a recovery step of recovering the second liquid containing the detached second cells from the culture vessel. A cell extraction method characterized by the following features. [Matter 2] In the first peeling step, the first peeling mechanism changes the orientation of the culture vessel. The cell extraction method described in item 1, characterized by the following: [Matter 3] In the second peeling step, the second peeling mechanism causes the culture vessel to shake. A cell extraction method according to item 1 or 2, characterized by the above. [Matter 4] In the second peeling step, the dispensing mechanism of the second peeling mechanism dispenses the second liquid into the contents of the culture vessel. A cell extraction method according to any one of items 1 to 3, characterized by the following: [Matter 5] The discharge mechanism includes a spray nozzle for discharging droplets of the second liquid. The cell extraction method described in item 4, characterized by the features described above. [Matter 6] In the second peeling step, the stirring mechanism of the second peeling mechanism causes the second liquid to flow in the culture vessel. A cell extraction method according to any one of items 1 to 5, characterized by the following: [Matter 7] In the first peeling step, more than 70% of the first cells that were present inside the culture vessel at the start of the peeling agent injection step are peeled off. A cell extraction method according to any one of items 1 to 6, characterized by the following: [Matter 8] Between the first peeling step and the second peeling step, there is a washing step in which a washing solution is injected from the washing unit into the culture container to wash the contents, and then the washing solution is discharged from the culture container. A cell extraction method according to any one of items 1 to 7, characterized by the following: [Matter 9] At the end of the washing step, more than 70% of the first cells that were present inside the culture vessel at the start of the release agent injection step have been discharged from the culture vessel. The cell extraction method according to item 8, characterized by the following: [Matter 10] At the end of the washing step, more than 70% of the second cells that were present inside the culture vessel at the start of the release agent injection step are left inside the culture vessel. A cell extraction method according to item 8 or 9, characterized by the above. [Matter 11] In the second peeling step, more than 90% of the second cells that were present inside the culture vessel at the end of the washing step are peeled off. A cell extraction method according to any one of items 8 to 10, characterized by the following: [Matter 12] The first liquid is a solution containing a proteolytic enzyme and / or a chelating agent. The second liquid is physiological saline or liquid culture medium. A cell extraction method according to any one of items 1 to 11, characterized by the features described herein. [Matter 13] The washing solution is one of the following: physiological saline, liquid culture medium, or cryopreservation solution. A cell extraction method according to any one of items 8 to 12, characterized by the following: [Matter 14] The first cell is a mononuclear cell in which a reprogramming factor has been introduced, The second cell is an iPS cell in which a reprogramming factor has been introduced into a mononuclear cell and the reprogramming process is complete. A cell extraction method according to any one of items 1 to 13, characterized by the following: [Matter 15] The second cells contained in the second liquid recovered in the recovery step of the cell extraction method described in any one of items 1 to 14 are subcultured. A method for subculturing culture characterized by the following features. [Matter 16] A release agent injection step is performed in which a first liquid containing a release agent is injected from a first injection port into a culture vessel containing a mixture of first cells and second cells having greater adhesion than the first cells, thereby immersing the first cells and the second cells. After the release agent injection step, the first release mechanism performs a first release step in which it applies a first physical force to the contents of the culture vessel via the first liquid to release at least a portion of the first cells, and then discharges the first liquid containing the released first cells from the culture vessel. After the first peeling step, the second liquid is injected into the culture vessel from the second injection section, and the second peeling mechanism applies a second physical force greater than the first physical force to the contents of the culture vessel via the second liquid, thereby performing a second peeling step in which the second cells present inside the culture vessel are peeled off. The recovery mechanism performs a recovery step of recovering the second liquid containing the detached second cells from the culture vessel. A cell extraction system characterized by the following features. [Matter 17] In the first peeling step, the first peeling mechanism changes the orientation of the culture vessel. A cell extraction system as described in item 16, characterized by the features described herein. [Matter 18] In the second peeling step, the second peeling mechanism causes the culture vessel to shake. A cell extraction system according to item 16 or 17, characterized by the above. [Matter 19] In the second peeling step, the dispensing mechanism of the second peeling mechanism dispenses the second liquid into the contents of the culture vessel. A cell extraction system according to any one of claims 16 to 18, characterized by the above. [Matter 20] The discharge mechanism includes a spray nozzle for discharging droplets of the second liquid. A cell extraction system according to item 19, characterized by the features described above. [Matter 21] In the second peeling step, the stirring mechanism of the second peeling mechanism causes the second liquid to flow in the culture vessel. A cell extraction system according to any one of claims 16 to 20, characterized by the above. [Matter 22] In the first peeling step, more than 70% of the first cells that were present inside the culture vessel at the start of the peeling agent injection step are peeled off. A cell extraction system according to any one of items 16 to 21, characterized by the above. [Matter 23] Between the first peeling step and the second peeling step, a washing step is performed in which a washing solution is injected from the washing unit into the culture container to wash the contents, and then the washing solution is discharged from the culture container. The cell extraction system according to item 22, characterized in that [Matter 24] At the end of the washing step, more than 70% of the first cells that were present inside the culture vessel at the start of the release agent injection step have been discharged from the culture vessel. A cell extraction system according to item 23, characterized by the features described above. [Matter 25] At the end of the washing step, more than 70% of the second cells that were present inside the culture vessel at the start of the release agent injection step are left inside the culture vessel. A cell extraction system according to item 23 or 24, characterized by the above. [Matter 26] In the second peeling step, more than 90% of the second cells that were present inside the culture vessel at the end of the washing step are peeled off. A cell extraction system according to item 23, characterized by the features described above. [Matter 27] The first liquid is a solution containing a proteolytic enzyme and / or a chelating agent. The second liquid is physiological saline or liquid culture medium. A cell extraction system according to any one of items 16 to 21, characterized by the above. [Matter 28] The washing solution is one of the following: physiological saline, liquid culture medium, or cryopreservation solution. A cell extraction system according to item 23, characterized by the features described above. [Matter 29] The first cell is a mononuclear cell in which a reprogramming factor has been introduced, The second cell is an iPS cell in which a reprogramming factor has been introduced into a mononuclear cell and the reprogramming process is complete. A cell extraction system according to any one of claims 16 to 28, characterized by the above. [Matter 30] The cell extraction system described in any one of items 16 to 29 comprises a culture section for subculturing the second cells contained in the second liquid recovered in the recovery step. A subculture system characterized by the following features. [Explanation of Symbols]

[0095] 1···Cell detachment sorting system / 10···Cell detachment device / 11···Control device / 12···Detachment mechanism / 20···Incubator / 30···Culture vessel / 31···Culture surface / 121···Washing solution pump / 122···Washing solution delivery tube / 123··Detachment solution pump / 124··Detachment agent delivery tube / 125···Detachment solution pump for second cells / 126···Detachment solution discharge tube for second cells / 127···Waste liquid discharge pump / 128···Waste liquid suction tube / 129···Cell suspension recovery pump / 130···Cell suspension suction tube / 131···Signal line / 132···Drive mechanism

Claims

1. A release agent injection step involves injecting a first solution containing a release agent into a culture vessel containing a mixture of first cells and second cells from a first injection port, thereby lowering the adhesion strength of the first cells to that of the second cells. Following the release agent injection step, the first release mechanism applies a first physical force to the contents of the culture vessel via the first liquid to release at least a portion of the first cells, and the first liquid containing the released first cells is discharged from the culture vessel in a first release step. A second peeling step is performed in which, after the first peeling step, a second liquid is supplied from the second injection unit to the culture vessel, and the second peeling mechanism applies a second physical force greater than the first physical force to the contents of the culture vessel via the second liquid, thereby peeling off the second cells that were present inside the culture vessel. The recovery mechanism includes a recovery step of recovering the second liquid containing the detached second cells from the culture vessel. A cell extraction method characterized by the following features.

2. In the first peeling step, the first peeling mechanism changes the orientation of the culture vessel. The cell extraction method according to feature 1.

3. In the second peeling step, the second peeling mechanism shakes the culture vessel. The cell extraction method according to feature 1.

4. In the second peeling step, the dispensing mechanism of the second peeling mechanism dispenses the second liquid into the contents of the culture vessel. The cell extraction method according to feature 1.

5. The discharge mechanism includes a spray nozzle for discharging droplets of the second liquid. The cell extraction method according to feature 4.

6. In the second peeling step, the stirring mechanism of the second peeling mechanism causes the second liquid to flow in the culture vessel. The cell extraction method according to feature 1.

7. In the first peeling step, more than 70% of the first cells that were present inside the culture vessel at the start of the peeling agent injection step are peeled off. The cell extraction method according to any one of claims 1 to 6.

8. Between the first peeling step and the second peeling step, there is a washing step in which a washing solution is injected from the washing unit into the culture container to wash the contents, and then the washing solution is discharged from the culture container. The cell extraction method according to feature 1.

9. At the end of the washing step, more than 70% of the first cells that were present inside the culture vessel at the start of the release agent injection step have been discharged from the culture vessel. The cell extraction method according to feature 8.

10. At the end of the washing step, more than 70% of the second cells that were present inside the culture vessel at the start of the release agent injection step are left inside the culture vessel. The cell extraction method according to feature 8.

11. In the second peeling step, more than 90% of the second cells that were present inside the culture vessel at the end of the washing step are peeled off. The cell extraction method according to feature 8.

12. The first liquid is a solution containing a proteolytic enzyme and / or a chelating agent. The second liquid is physiological saline or liquid culture medium. The cell extraction method according to any one of claims 1 to 6.

13. The washing solution is one of the following: physiological saline, liquid culture medium, or cryopreservation solution. The cell extraction method according to feature 8.

14. The first cell is a mononuclear cell in which a reprogramming factor has been introduced, The second cell is an iPS cell in which a reprogramming factor has been introduced into a mononuclear cell and the reprogramming process is complete. The cell extraction method according to any one of claims 1 to 6.

15. The second cells contained in the second liquid recovered in the recovery step of the cell extraction method according to any one of claims 1 to 6 are subcultured. A method for subculturing culture characterized by the following features.

16. A release agent injection step is performed in which a first liquid containing a release agent is injected from a first injection port into a culture vessel containing a mixture of first cells and second cells having greater adhesion than the first cells, thereby immersing the first cells and the second cells. After the peeling agent injection step, the first peeling mechanism performs a first peeling step in which it applies a first physical force to the contents of the culture vessel via the first liquid to peel off at least a portion of the first cells, and then discharges the first liquid containing the peeled first cells from the culture vessel. After the first peeling step, the second liquid is injected into the culture vessel from the second injection unit, and the second peeling mechanism applies a second physical force greater than the first physical force to the contents of the culture vessel via the second liquid, thereby performing a second peeling step in which the second cells present inside the culture vessel are peeled off. The recovery mechanism performs a recovery step of recovering the second liquid containing the detached second cells from the culture vessel. A cell extraction system characterized by the following features.

17. In the first peeling step, the first peeling mechanism changes the orientation of the culture vessel. The cell extraction system according to claim 16, characterized in that it is as described above.

18. In the second peeling step, the second peeling mechanism shakes the culture vessel. The cell extraction system according to claim 16, characterized in that it is as described above.

19. In the second peeling step, the dispensing mechanism of the second peeling mechanism dispenses the second liquid into the contents of the culture vessel. The cell extraction system according to claim 16, characterized in that it is as described above.

20. The discharge mechanism includes a spray nozzle for discharging droplets of the second liquid. The cell extraction system according to claim 19, characterized in that it is as described above.

21. In the second peeling step, the stirring mechanism of the second peeling mechanism causes the second liquid to flow in the culture vessel. The cell extraction system according to claim 16, characterized in that it is as described above.

22. In the first peeling step, more than 70% of the first cells that were present inside the culture vessel at the start of the peeling agent injection step are peeled off. A cell extraction system according to any one of claims 16 to 21, characterized by the above.

23. Between the first peeling step and the second peeling step, a washing step is performed in which a washing solution is injected from the washing unit into the culture container to wash the contents, and then the washing solution is discharged from the culture container. The cell extraction system according to claim 22, characterized in that it is a cell extraction system.

24. At the end of the washing step, more than 70% of the first cells that were present inside the culture vessel at the start of the release agent injection step have been discharged from the culture vessel. The cell extraction system according to claim 23, characterized in that it is as follows.

25. At the end of the washing step, more than 70% of the second cells that were present inside the culture vessel at the start of the release agent injection step are left inside the culture vessel. The cell extraction system according to claim 23, characterized in that it is as follows.

26. In the second peeling step, more than 90% of the second cells that were present inside the culture vessel at the end of the washing step are peeled off. The cell extraction system according to claim 23, characterized in that it is as follows.

27. The first liquid is a solution containing a proteolytic enzyme and / or a chelating agent. The second liquid is physiological saline or liquid culture medium. A cell extraction system according to any one of claims 16 to 21, characterized by the above.

28. The washing solution is one of the following: physiological saline, liquid culture medium, or cryopreservation solution. The cell extraction system according to claim 23, characterized in that it is as follows.

29. The first cell is a mononuclear cell in which a reprogramming factor has been introduced, The second cell is an iPS cell in which a reprogramming factor has been introduced into a mononuclear cell and the reprogramming process is complete. A cell extraction system according to any one of claims 16 to 21, characterized by the above.

30. The cell extraction system according to any one of claims 16 to 21 comprises a culture section for subculturing the second cells contained in the second liquid recovered in the recovery step, A subculture system characterized by the following features.

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