Cell detachment method, cell detachment apparatus, and cell freezing storage system

Abstract

To collect cells from a culture container while reducing loss of the cells.SOLUTION: A cell detachment method according to an embodiment includes a detachment step and a collection step. In the detachment step, cells are detached from a surface of a culture container with which the cultured cells are in contact, by discharging a liquid used for freezing preservation of the cells onto the surface. In the collection step, the detached cells are collected together with the liquid used for freezing preservation that has been discharged.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The embodiments disclosed in the present specification and drawings relate to a cell detachment method, a cell detachment device, and a cell cryopreservation system. [Background technology]

[0002] After culturing cells in a culture vessel, the cells are detached from the culture vessel using physiological saline or a detachment solution. When the detached cells are to be stored for a long period of time without subculturing, the solvent in the suspension in which the detached cells are suspended (hereinafter referred to as the cell suspension detachment solution) is replaced with a cryopreservation solution, and the cryopreservation solution in which the cells are suspended (hereinafter referred to as the cell suspension preservation solution) is frozen and stored.

[0003] One method of liquid substitution is the use of a filter (hereinafter referred to as the filter liquid substitution method). In the filter liquid substitution method, cells are captured by passing the cell suspension detachment solution through a filter, and the cells are then recovered by passing a cryopreservation solution through the filter. However, with the filter liquid substitution method, it is not possible to recover many of the cells suspended in the cell suspension detachment solution, resulting in significant cell loss. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Publication No. 9-056813 Summary of the Invention [Problem to be solved by the invention]

[0005] One of the problems to be solved by the embodiments disclosed in this specification and the drawings is to recover cells from a culture vessel while reducing cell loss. However, the problems to be solved by the embodiments disclosed in this specification and the drawings are not limited to the above problem. Problems corresponding to the effects of each configuration shown in the embodiments described below can also be positioned as other problems. [Means for solving the problem]

[0006] The cell detachment method according to the embodiment includes a detachment step and a recovery step. In the detachment step, a solution used for cryopreservation of the cells is ejected toward a surface of a culture vessel in which the cells have been cultured, thereby detaching the cells from the surface. In the recovery step, the detached cells are recovered together with the ejected solution used for cryopreservation. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a diagram showing an example of the configuration of a cell cryopreservation system. [Figure 2] FIG. 2 is a diagram showing an example of the configuration of a cell detachment device. [Figure 3] FIG. 3 is a diagram showing a schematic external view of the peeling mechanism. [Figure 4] FIG. 4 is a diagram showing an example of a processing procedure for detached and frozen storage by the cell cryopreservation system. [Figure 5] FIG. 5 is a diagram schematically showing the processing procedure of steps S1 to S8 in FIG. [Figure 6] FIG. 6 is a diagram showing a schematic diagram of the processing procedure of the recovery step (S8). [Figure 7] FIG. 7 is a graph comparing the number of iPS cells after thawing and culturing between this example and the comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0008] Hereinafter, the cell detachment method, cell detachment device, and cell cryopreservation system according to the present embodiment will be described in detail with reference to the drawings.

[0009] Fig. 1 is a diagram showing an example of the configuration of a cell cryopreservation system 1 according to this embodiment. As shown in Fig. 1, the cell cryopreservation system 1 is a mechanical system that freezes and stores cells. The cells according to this embodiment are not particularly limited and may be any type of cell, but examples include iPS (induced pluripotent stem) cells, epithelial cells, endothelial cells, synovial cells, cardiomyocytes, myoblasts, fibroblasts, and neuroblasts.

[0010] As shown in FIG. 1, the cell cryopreservation system 1 includes a cell detachment device 10, a cryopreservation unit 20, and an incubator 30. The cell detachment device 10 is a mechanical device that detaches cells cultured in a culture vessel from the culture vessel and recovers the detached cells from the cell vessel. Specifically, the cell detachment device 10 includes a detachment mechanism 11, a recovery mechanism 13, and a control device 15. The detachment mechanism 11 is a mechanical device equipped with a tool for detaching cells cultured in the culture vessel from the culture vessel. The detachment mechanism 11 discharges a liquid used for cryopreservation of cells (hereinafter referred to as a cryopreservation liquid) toward the surface of the culture vessel in which the cells are cultured, with which the cells are in contact (hereinafter referred to as a culture surface), thereby detaching the cells from the culture surface. The recovery mechanism 13 is a mechanical device equipped with a tool for recovering the cells detached from the culture vessel. The recovery mechanism 13 recovers the cells detached by the detachment mechanism 11 together with the discharged cryopreservation liquid. The cryopreservation liquid in which the cells are suspended is referred to as a cell suspension preservation liquid. The control device 15 is a computer including a processor that performs overall control of the recovery mechanism 13 and the control device 15. The recovery mechanism 13 and the control device 15 operate in accordance with instructions from the control device 15.

[0011] The cryopreservation unit 20 is a mechanical system that freezes and stores the cells collected by the cell detachment device 100. Specifically, the cryopreservation unit 20 includes an ultra-low temperature freezer 21 and a cryopreservation container 22. The ultra-low temperature freezer 21 freezes the cell suspension preservation solution at a first temperature range at which the cells can metabolize. The ultra-low temperature freezer 21 includes an insulated freezer and a refrigeration circulation device that circulates a refrigerant through the freezer and cools the refrigerant. The first temperature range is, for example, approximately -90 to -50 degrees Celsius. The ultra-low temperature freezer 21 is also called a deep freezer. After the first freezing step by the ultra-low temperature freezer 21, the cryopreservation container 22 freezes and stores the cell suspension preservation solution at a second temperature range lower than the first temperature range at which the cells cannot metabolize. In this case, the second temperature range is approximately -200 to -150 degrees Celsius.

[0012] The incubator 30 includes a chamber that houses the culture vessel and a control circuit that maintains a constant temperature and / or humidity within the chamber. The incubator 30 may be configured separately from the cell detachment device 10, or the cell detachment device 10 may include the incubator 30. In the latter case, for example, all or some of the components of the detachment mechanism 11 and the recovery mechanism 13 may be provided within the chamber. With this configuration, cells can be cultured in the cell detachment device 10.

[0013] Fig. 2 is a diagram showing an example of the configuration of the cell detachment device 10. As shown in Fig. 2, the cell detachment device 10 has a detachment mechanism 11, a recovery mechanism 13, and a control device 15. The detachment mechanism 11, the recovery mechanism 13, and the control device 15 are connected via wired or wireless signal lines. The detachment mechanism 11 has a detachment liquid pump 111, a liquid supply pipe 112, a washing liquid pump 113, a liquid supply pipe 114, a cryopreservation liquid pump 115, a liquid supply pipe 116, a waste liquid pump 117, a suction pipe 118, a drive mechanism 119, and a support mechanism 121.

[0014] As shown in FIG. 2, a culture vessel 40 is installed in a position accessible to the detachment mechanism 11 and the recovery mechanism 13. Cells are cultured in the culture vessel 40. The shape of the culture vessel 40 is not particularly limited, but is envisioned as a dish, petri dish, flask, well plate, or the like having an opening through which the detachment mechanism 11 and the recovery mechanism 13 can access the inside of the culture vessel 40. The inner bottom surface of the culture vessel 40 is called the culture surface 41. Cultured cells are in contact with the culture surface 41. Before the cell detachment process by the detachment mechanism 11, multiple clumps (colonies) of cells are formed in the culture vessel 40 and adhere to the culture surface 41.

[0015] The detachment liquid pump 111 discharges the detachment liquid through the liquid supply pipe 112 in response to a drive signal from the drive device 119. The detachment liquid has the effect of weakening adhesion between cells and the culture surface 41 and adhesion between cells. As an example, the detachment liquid is a solution containing a protease and / or a chelating agent. The detachment liquid is stored in a detachment liquid tank (not shown). The detachment liquid pump 111 aspirates the detachment liquid from the detachment liquid tank and discharges the aspirated detachment liquid into the culture vessel 40 through the liquid supply pipe 112, thereby adding the detachment liquid to the cells contained in the culture vessel 40.

[0016] The liquid supply pipe 112 is a tubular structure, such as a tube, connected to the detachment liquid pump 111 and through which the detachment liquid flows. The liquid supply pipe 112 is supported by a support mechanism 121 so as to be movable up and down. The support mechanism 121 causes the liquid supply pipe 112 to descend toward the culture surface 41 to discharge the detachment liquid, and to ascend after the discharge of the detachment liquid is completed. The support mechanism 121 may be configured so as not to be movable up and down.

[0017] The washing liquid pump 113 discharges a washing liquid through the liquid supply pipe 114 in response to a drive signal from the drive device 119. The washing liquid is used to wash away the culture vessel 40 and impurities. For example, physiological saline or a liquid culture medium is used as the washing liquid. The washing liquid is stored in a washing liquid tank (not shown). The washing liquid pump 113 aspirates the washing liquid from the washing liquid tank and discharges the aspirated washing liquid into the culture vessel 40 through the liquid supply pipe 114, thereby washing the culture vessel 40 and / or cells. The impurities are, for example, dead cells floating in the washing liquid and calcium ions and magnesium ions present in the culture medium of the culture vessel 40. If the washing is insufficient, the chelating agent may not be able to sufficiently cleave the bonds between the cells. If the washing is insufficient, the chelating agent may react with calcium ions and magnesium ions present in the culture medium.

[0018] The liquid supply pipe 114 is a tubular structure, such as a tube, connected to the cleaning liquid pump 113 and through which the cleaning liquid flows. The liquid supply pipe 114 is supported by a support mechanism 121 so that it can move up and down. The support mechanism 121 causes the liquid supply pipe 114 to descend toward the culture surface 41 in order to discharge the cleaning liquid, and to ascend after the discharge of the cleaning liquid is completed.

[0019] The cryopreservation solution pump 115 discharges the cryopreservation solution through the liquid supply tube 114 in response to a drive signal from the drive device 119. The cryopreservation solution is used to reduce damage to cells when they are frozen for storage. The cryopreservation solution is a solution containing a cryoprotectant. The cryoprotectant promotes cell dehydration, slows the rate of ice crystallization, and inhibits ice formation. This action prevents ice crystals from damaging the cell membrane. For example, the cryoprotectant contains dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, propylene glycol, glycerin, polyvinylpyrrolidone, sorbitol, dextran, and / or trehalose. The cryopreservation solution may be a commercially available product such as STEM-CELLBANKER (registered trademark), or a solution in which the cryoprotectant is mixed with a culture medium. The cryopreservation solution is stored in a cryopreservation solution tank (not shown). The cryopreservation liquid pump 115 aspirates the cryopreservation liquid from the cryopreservation liquid tank and ejects the aspirated cryopreservation liquid toward the culture surface 41 of the culture vessel 40 via the liquid delivery pipe 116, thereby detaching cells in contact with the culture surface 41 from the culture surface 41.

[0020] Liquid supply pipe 116 is a tubular structure, such as a tube, connected to cryopreservation liquid pump 115 and through which the cryopreservation liquid flows. Liquid supply pipe 116 is supported by support mechanism 121 so that it can move up and down. Support mechanism 121 causes the tip of liquid supply pipe 116 to descend toward culture surface 41 when the cryopreservation liquid is being discharged, and to rise away from culture surface 41 after the discharge of the cryopreservation liquid has been completed.

[0021] The waste liquid pump 117 responds to a drive signal from a drive device 119 and aspirates various waste liquids contained in the culture vessel 40 through an aspirating tube 118. The waste liquids may include, for example, the stripping liquid and the washing liquid discharged into the culture vessel 40. The aspirated waste liquids are collected in a waste liquid tank through a waste tube (not shown).

[0022] The suction pipe 118 is a tubular structure, such as a tube, connected to the waste liquid pump 117 and through which the waste liquid flows. The suction pipe 118 is supported by a support mechanism 121 so that it can move up and down. The support mechanism 121 causes the suction pipe 118 to descend toward the culture surface 41 to suck the waste liquid, and then ascends after the suction of the waste liquid is completed.

[0023] The driving device 119 operates the stripping solution pump 111, the cleaning solution pump 113, the cryopreservation solution pump 115, and the waste solution pump 117 in accordance with the driving signals by individually supplying the driving signals to the stripping solution pump 111, the cleaning solution pump 113, the cryopreservation solution pump 115, and the waste solution pump 117 in accordance with the driving signals. The driving device 119 also operates the support mechanism 121 in accordance with the driving signals by supplying the driving signals to the support mechanism 121 in accordance with the driving signals, thereby moving the liquid feed tube 112, the liquid feed tube 114, the liquid feed tube 116, or the suction tube 118 up and down in accordance with the driving signals. As an example, a motor is used as the driving device 119.

[0024] 2, one driving device 119 drives all of the stripping solution pump 111, the cleaning solution pump 113, the cryopreservation solution pump 115, the waste solution pump 117, and the support mechanism 121, but this embodiment is not limited to this. A driving device 119 may be provided for each of the stripping solution pump 111, the cleaning solution pump 113, the cryopreservation solution pump 115, the waste solution pump 117, and the support mechanism 121, or a driving device 119 may be provided for any combination of the stripping solution pump 111, the cleaning solution pump 113, the cryopreservation solution pump 115, the waste solution pump 117, and the support mechanism 121.

[0025] The recovery mechanism 13 includes a recovery pump 131 , a suction tube 132 , a cryopreservation container 133 , a driving device 134 , a tilting mechanism 135 , and a driving device 136 .

[0026] In response to a drive signal from a drive device 134, the recovery pump 131 recovers the cells detached by the discharge of the cryopreservation solution together with the cryopreservation solution via a suction tube 132. As described above, the cryopreservation solution in which the cells are suspended is called a cell suspension preservation solution. The cell suspension preservation solution is stored in a cryopreservation container 133.

[0027] Specifically, the suction pipe 132 is a tubular structure connected to the recovery pump 131 and through which the cell suspension preservation solution flows. The suction pipe 132 has a suction branch pipe 141 for suctioning the cell suspension preservation solution from the culture vessel 40, a liquid delivery branch pipe 142 for delivering the suctioned cell suspension preservation solution to the cryopreservation vessel 133, and a valve 143 for delivering the suctioned cell suspension preservation solution from the suction branch pipe 141 to the liquid delivery branch pipe 142. The valve 143 is switched in response to a drive signal from the drive device 134.

[0028] The suction branch pipe 141 is supported by the support mechanism 121 so as to be movable up and down. The support mechanism 121 causes the tip of the suction branch pipe 141 to descend toward the culture surface 41 when aspirating the cell suspension preservation solution, and to ascend away from the culture surface 41 after the suction of the cell suspension preservation solution is completed.

[0029] A plurality of cryopreservation containers 133 are detachably connected to the liquid supply branch 142. The cell suspension preservation solution aspirated into the suction branch 141 flows through the liquid supply branch 142 and is sequentially stored in the plurality of cryopreservation containers 133. In FIG. 2, as an example, four cryopreservation containers 133 are connected. The cryopreservation containers 133 have a material and / or structure that is durable enough not to be damaged even when frozen and stored in the second temperature range by the cryopreservation container 22. As an example, the cryopreservation containers 133 may be cryotubes made of polypropylene or cryobags made of polyolefin-based materials. The number of cryopreservation containers 133 connected to the liquid supply branch 142 is not limited to a plurality, and may be one.

[0030] The driving device 134 operates the recovery pump 131 in response to a driving signal supplied to the recovery pump 131 in accordance with a command from the control device 15. As an example of the driving device 134, a motor is used.

[0031] The tilting mechanism 135 is a mechanical mechanism for tilting the culture vessel 40 relative to the horizontal in response to a drive signal from the drive device 136. Specifically, the tilting mechanism 135 tilts the culture vessel 40 relative to the horizontal so that the depth of the cell suspension storage at a first position in the culture vessel 40, where the suction tube 132 is inserted, is deeper than the depth of the cell suspension storage solution at another second position. The specific structure of the tilting mechanism 135 is not particularly limited.

[0032] The driving device 136 supplies a driving signal to the tilting mechanism 135 in accordance with a command from the control device 15, thereby operating the tilting mechanism 135 in accordance with the driving signal. As an example of the driving device 136, a motor is used.

[0033] The control device 15 controls the driving devices 119, 134, and 136 in a predetermined order to automatically perform cell detachment and recovery via the detachment solution pump 111, the washing solution pump 113, the cryopreservation solution pump 115, the waste liquid pump 117, the recovery pump 131, and the tilting mechanism 135. In this case, the control device 15 controls the flow rates of the discharge and suction of the detachment solution, the washing solution, the cryopreservation solution, and the cell suspension preservation solution, and controls the timing of the discharge and suction of the various liquids.

[0034] In the following description of the embodiment, the term "cells" refers to iPS cells. The detachment mechanism 11 detaches iPS cells from the culture surface 41 by spraying a cryopreservation solution onto the culture surface 41.

[0035] Fig. 3 is a diagram showing a schematic appearance of the peeling mechanism 11. As shown in Fig. 3, the peeling mechanism 11 has a support mechanism 121. The support mechanism 121 is a structure that supports the liquid feed tube 112, the liquid feed tube 114, the liquid feed tube 116, the suction tube 118, and the suction branch tube 141 so that they can each move up and down individually. As an example, the support mechanism 121 may be a ball screw, a linear guide, or the like.

[0036] A mounting surface 51 is provided below the liquid delivery tubes 112, 114, 116, suction tube 118 and suction branch tube 141, and a culture vessel 40 is placed on the mounting surface 51 directly below the liquid delivery tubes 114, 116, suction tube 118 and suction branch tube 141.

[0037] A nozzle 123 is provided at the tip of the liquid supply tube 116. The nozzle 123 is a mechanical component for discharging the cryopreservation solution delivered by the cryopreservation solution pump 115 through the liquid supply tube 116 as multiple droplets. Specifically, the nozzle 123 sprays the cryopreservation solution, i.e., converts it into multiple tiny droplets and discharges it in a mist. To convert the cryopreservation solution into a mist of tiny droplets, the nozzle 123 is formed with tiny holes. The number of holes may be one or more. The cryopreservation solution delivered to the nozzle 123 via the liquid supply tube 116 by the cryopreservation solution pump 115 passes through the tiny holes formed in the nozzle 123, forming tiny droplets and discharging them. As a result, the cryopreservation solution is sprayed toward the culture surface 41 of the culture vessel 40, and the mechanical action of the sprayed cryopreservation solution detaches cells in contact with the culture surface 41. The diameter, discharge speed, volume, range, etc. of the cryopreservation solution droplets can be set as desired.

[0038] In order to discharge the cryopreservation solution toward the entire surface of the culture surface 41, the liquid supply pipe 116 and the nozzle 123 are preferably mounted on the support mechanism 121 so as to be positioned approximately above the approximate center A1 of the culture surface 41. The liquid supply pipe 116 and the nozzle 123 may be formed integrally.

[0039] A tilting mechanism 135 is embedded below the mounting surface 51. The tilting mechanism 135 is assumed to be a balloon that expands and contracts by letting air in and out. In this case, the driving device 136 is assumed to be an air compressor, motor cylinder, air cylinder, or the like that lets air in and out of the balloon 135. The balloon 135 is installed below one end (hereinafter referred to as the rising end) 42 of the culture vessel 40. When an air compressor is used, the air compressor lets air into the balloon 135, causing it to expand, and the balloon 135 pushes up the rising end 42, thereby tilting the culture vessel 40. As a result, waste liquid, cell suspension preservation solution, etc. contained in the culture vessel 40 collect at the other end (hereinafter referred to as the fixed end) 43 of the culture vessel 40. The fixed end 43 refers to the area opposite the rising end 42 across approximately the center A1 of the culture surface 41.

[0040] To facilitate the suction of the solution from the culture vessel 40, the suction tube 118 and the suction branch tube 141 are preferably mounted on the support mechanism 121 so as to be positioned above the fixed side end portion 43. The positions of the liquid supply tube 112 and the liquid supply tube 114 are not particularly limited, and they may be provided at positions that do not interfere with the operation of the liquid supply tube 116, the suction tube 118, and the suction branch tube 141.

[0041] Next, the processing procedure for detachment and freezing storage by the cell cryopreservation system 1 will be described with reference to FIGS. 4 and 5. FIG. 4 is a diagram showing an example of the processing procedure for detachment and freezing storage by the cell cryopreservation system 1. Steps S1 to S8 in FIG. 4 are automatically performed by the control device 15 through sequence control and / or feedback control of the driving device 119 of the detachment mechanism 11 and the driving devices 134 and 136 of the collection mechanism 13. FIG. 5 is a diagram schematically showing the processing procedure for steps S1 to S8 in FIG. 4. In FIG. 5, only the components necessary for explaining each step are shown, and components not necessary for explanation are appropriately omitted. It is assumed that the incubator 30 and the cell detachment device 10 are integrally formed. Specifically, it is assumed that the liquid supply pipes 112, 114, 116, the suction pipe 118, and the nozzle 123 are provided in the chamber of the incubator 30.

[0042] 4 and 5, at the start of step S1, culture vessel 40 is placed on mounting surface 51. Culture vessel 40 contains a colony of cultured iPS cells and the medium used for the culture. The iPS cells may be cultured in incubator 30 or another incubator.

[0043] First, the culture medium is removed from the culture vessel 40 (step S1). Specifically, the control device 15 operates the waste liquid pump 117 via the driving device 119. The waste liquid pump 117 removes the culture medium from the culture vessel 40 by sucking the culture medium from the culture vessel 40 via the suction tube 118. Impurities such as the culture medium that are not sucked remain in the culture vessel 40.

[0044] After step S1 is performed, the iPS cells are washed with a washing solution (step S2). The culture medium contains impurities, including nutrients, in addition to the iPS cells. In step S2, the control device 15 operates the washing solution pump 113 via the drive device 119. The washing solution pump 113 discharges the washing solution into the culture vessel 40 via the liquid supply pipe 114. The discharged washing solution washes away the impurities. Thereafter, the waste liquid pump 117 sucks the washing solution containing the washed-out impurities from the culture vessel 40 via the suction pipe 118. This washes the culture vessel 40 and the iPS cells.

[0045] After step S2 is performed, a detachment solution is added (step S3). In step S3, the control device 15 activates the detachment solution pump 111 via the driving device 119. The detachment solution pump 111 discharges the detachment solution into the culture vessel 40 via the liquid supply pipe 112. The discharged detachment solution is added to the iPS cells.

[0046] After step S3, an immersion process is performed (step S4). The immersion process may also include incubation. If incubation is included, specifically, the control device 15 issues an incubation start command to the incubator 30. Upon receiving the start command, the incubator 30 maintains the environment of the culture vessel 40 containing the iPS cells to which the detachment solution has been added at a constant temperature and humidity for a predetermined constant temperature period. The constant temperature and humidity may be set to conditions suitable for culturing iPS cells, such as a temperature of approximately 37°C and a humidity of approximately 95%. During incubation, the detachment solution acts to reduce adhesion between the iPS cells and between the iPS cells and the culture surface 41. The constant temperature period may be set to an empirically determined value as the length of time required to sufficiently reduce adhesion. After the constant temperature period has elapsed, the incubator 30 terminates temperature and humidity control. If incubation is not included, the duration of the immersion process is managed. The immersion period may be set to an empirically determined value as the length of time required to sufficiently reduce adhesion. After the immersion period has elapsed, the process proceeds to step S5. If incubation is not performed in step S4, the incubator 30 may not be included in the cell cryopreservation system 1.

[0047] After step S4 is performed, the peeling liquid is removed (step S5). In step S5, the control device 15 operates the waste liquid pump 117 via the driving device 119. The waste liquid pump 117 aspirates the peeling liquid from the culture vessel 40 via the aspirating tube 118. Impurities such as the peeling liquid that have not been aspirated remain in the culture vessel 40.

[0048] After step S5 is performed, the iPS cells are washed with a washing solution (step S6). In step S6, the control device 15 activates the washing solution pump 113 via the drive device 119. The washing solution pump 113 discharges the washing solution into the culture vessel 40 via the liquid supply pipe 114. The discharged washing solution washes away impurities adhering to the culture vessel 40 and the iPS cells. Thereafter, the waste liquid pump 117 aspirates the washing solution containing the washed-out impurities from the culture vessel 40 via the suction pipe 118.

[0049] After step S6 is performed, the cryopreservation solution is discharged onto the culture surface 41 of the culture vessel 40 (step S7). In step S7, the control device 15 operates the cryopreservation solution pump 115 via the drive device 119. The cryopreservation solution pump 115 sprays the cryopreservation solution onto the culture surface 41 via the liquid supply pipe 116 and the nozzle 123. Because the adhesion between the iPS cells and the culture surface 41 has been weakened by the release agent, the iPS cells are detached from the culture surface 41 by the mechanical action of the sprayed droplets of the cryopreservation solution.

[0050] After step S7 is performed, the cryopreservation solution (cell suspension preservation solution) in which the iPS cells are suspended is collected into the cryopreservation container 133 (step S8). In step S7, the control device 15 operates the collection pump 131 via the drive device 134. The collection pump 131 aspirates the cell suspension preservation solution from the culture container 40 via the suction branch pipe 141. The aspirated cell suspension preservation solution is delivered to the cryopreservation container 133 via the liquid delivery branch pipe 142.

[0051] The recovery step (S8) will now be described in detail with reference to FIG. 6. FIG. 6 is a diagram schematically illustrating the processing procedure of the recovery step (S8). As shown in the left diagram of FIG. 6, spraying the cryopreservation solution generates bubbles of the cryopreservation solution in the culture vessel 40. The bubbles tend to form all over the culture surface 41 of the culture vessel 40. If iPS cells are placed in the cryopreservation vessel 133 together with the bubbles, there is a risk that the iPS cells will be damaged when the bubbles burst. To reduce this risk, the culture vessel 40 is tilted using the balloon 135, allowing the cell suspension preservation solution to be aspirated while avoiding the bubbles.

[0052] Specifically, as shown in the right diagram of FIG. 6 , the air compressor 136 inflates the balloon 135 to lift and tilt the rising end 42 of the culture vessel 40. By tilting the culture vessel 40, the cell suspension preservation solution collects at the fixed end 43. As a result, the depth of the cell suspension preservation solution is deeper at the fixed end 43 than at the rising end 42. Bubbles are light and form the supernatant. At the fixed end 43, the suction branch pipe 141 is lowered until the tip of the suction branch pipe 141 reaches a position deeper than the bubbles, and the cell suspension preservation solution is aspirated while avoiding the bubbles. By aspirating the cell suspension preservation solution while avoiding the bubbles, it is expected that approximately 95% of the cell suspension preservation solution sprayed onto the culture surface 41 can be recovered.

[0053] After step S8 is performed, the concentration of the cell suspension preservation solution is adjusted (step S9). Specifically, cryopreservation solution is added to the cryopreservation container 133 so that the concentration of the cell suspension preservation solution in the cryopreservation container 133 becomes a predetermined value. As an example, the predetermined value is when the number of iPS cells contained in 200 μl of cell suspension preservation solution becomes 3.0×10 5 pcs or more 7.0×10 5 The ratio is within the following range. The concentration of the cell suspension preservation solution may be measured by a flow cytometer, a cell counter, a turbidity method, or any other method. The cryopreservation solution may be injected into the cryopreservation container 133 via the liquid supply pipe 116 by the cryopreservation solution pump 115, or may be injected manually.

[0054] After step S9 is performed, the cryopreservation container 133 is placed in the ultra-low temperature freezer 21, and the cell suspension preservation solution is frozen (step S10). Specifically, the cryopreservation container 133 is disconnected from the liquid supply branch 142 by an operator or the like, and placed in the ultra-low temperature freezer 21. The ultra-low temperature freezer 21 maintains the temperature inside the container in a first temperature range of about -80°C. This makes it possible to freeze the iPS cells at about -80°C. The cells are stored in the ultra-low temperature freezer 21 for a relatively short, predetermined period (hereinafter referred to as the first storage period).

[0055] If iPS cells that have acclimatized to room temperature are suddenly frozen in a second temperature range that is lower than the first temperature range and where they cannot be metabolized, there is a risk that the iPS cells will be damaged, even if a cryoprotectant is contained in the cryopreservation solution. To reduce damage to iPS cells caused by this sudden drop in temperature, they are temporarily frozen in the first temperature range, where they can be metabolized. The first storage period is expected to be a relatively short period of about one day. As mentioned above, because iPS cells can metabolize in the first temperature range, the upper limit of the first storage period is about one month.

[0056] After step S10 is performed, the cryopreservation container 133 is placed in the cryopreservation container 22, and the cell suspension preservation solution is frozen and stored (step S11). As an example of the cryopreservation container 22, a liquid nitrogen storage container, which is an insulated container filled with liquid nitrogen, is used. In this case, the inside of the cryopreservation container 22 is maintained at a second temperature range of approximately -180°C. As described above, the iPS cells are frozen in the first temperature range and then frozen in the second temperature range, which makes it possible to reduce damage to the iPS cells due to freezing at the extremely low temperature of the second temperature range. The iPS cells are stored in the cryopreservation container 22 for a relatively long predetermined period (hereinafter referred to as the second storage period). In the second temperature range, the iPS cells cannot metabolize, and they can be stored for several years.

[0057] This completes the detached and frozen storage by the cell cryopreservation system 1.

[0058] Figure 7 compares the number of iPS cells after thawing and culturing between this example and a comparative example. In this example, as described above, the cells were detached by spraying with a cryopreservation solution and frozen without replacing the cell suspension with the cryopreservation solution. In the comparative example, the cells were manually detached using saline, and the saline containing the iPS cells was centrifuged for liquid replacement. The saline was replaced with the cryopreservation solution before freezing. The vertical axis of Figure 7 represents the number of iPS cells after thawing and culturing. The horizontal axis of Figure 7 represents the classification of culture conditions. "n=1, P3_Day7" indicates that iPS cells of the cell type labeled "1" were cultured for passage number "3" and culture period "7 days." Similarly, "n=1, P4_Day7" indicates that iPS cells of the cell type labeled "1" were cultured at passage number "4" for a culture period of "7 days," "n=2, P3_Day7" indicates that iPS cells of the cell type labeled "2" were cultured at passage number "3" for a culture period of "7 days," and "n=2, P4_Day7" indicates that iPS cells of the cell type labeled "2" were cultured at passage number "4" for a culture period of "7 days."

[0059] As shown in Figure 7, under all culture conditions, the present example produced a higher number of iPS cells than the comparative example. This is thought to be due in part to the fact that the present example does not require a liquid replacement step, thereby reducing damage to iPS cells compared to the comparative example. Therefore, it can be seen that the present example is superior to the comparative example in terms of the number of iPS cells after thawing.

[0060] The processing procedure for detaching and freezing the tissue shown in FIG. 4 is one example, and various elements can be deleted, added, and / or modified without departing from the spirit of the invention.

[0061] As one example, if the concentration of the cell suspension preservation solution is already appropriate or if concentration adjustment is not necessary, the adjustment step (S9) can be omitted. As another example, if there is no need to freeze the cell suspension preservation solution, the freezing step (S10) and the frozen storage step (S11) can be omitted. As another example, if the frozen preservation solution can be sprayed without adding a detachment solution, the addition step (S3), incubation step (S4), removal step (S5), and washing step (S6) can be omitted.

[0062] As another example, the mechanism 136 for tilting the culture vessel 40 is not limited to the method of inflating a balloon. As an example, the tilting mechanism 135 can also be realized by a mechanism that tilts the culture vessel 40 by pushing up the rising end 42 of the culture vessel 40 by extending a cylinder, or a mechanism that tilts the culture vessel 40 by lifting the rising end 42 with a wire or the like.

[0063] As another example, detachment of iPS cells is not limited to methods using spraying of a cryopreservation solution. For example, cryopreservation solution pump 115 may eject cryopreservation solution onto iPS cells via liquid delivery tube 116, and the iPS cells may be detached from culture surface 41 by the mechanical action of the ejected cryopreservation solution. As another example, cryopreservation solution pump 115 may deliver cryopreservation solution into culture vessel 40 via liquid delivery tube 116, and the iPS cells may be detached from culture surface 41 by the water flow generated by the liquid delivery. As another example, cryopreservation solution pump 115 may deliver cryopreservation solution into culture vessel 40 via liquid delivery tube 116, and the culture vessel 40 may be vibrated by tilting mechanism 135, and the iPS cells may be detached from culture surface 41 by the water flow generated by the vibration.

[0064] As another example, the tilting mechanism 135 can also be used when various waste liquids are aspirated by the waste liquid pump 117. Specifically, when the waste liquid pump 117 aspirates various waste liquids through the suction tube 118, the tilting mechanism 135 tilts the culture vessel 40 to increase the depth of the waste liquid at the position where the suction tube 118 is inserted. This makes it possible to easily collect various waste liquids from the culture vessel 40.

[0065] As another example, it is not necessary for all of steps S1 to S8 to be performed by detachment mechanism 11, and some or all of steps S1 to S8 can be performed manually by an operator. As an example, in the discharging step (S7), an operator may use a pipette or the like to discharge the cryopreservation solution onto culture surface 41, thereby detaching iPS cells from culture surface 41. In the recovering step (S8), an operator may use a pipette or the like to aspirate the cell suspension preservation solution from culture vessel 40, and then discharge the aspirated cell suspension preservation solution into cryopreservation container 133, thereby recovering the cell suspension preservation solution.

[0066] As described above, the cell detachment method according to this embodiment includes a detachment step and a recovery step. In the detachment step, a cell cryopreservation solution is ejected toward the culture surface of a culture vessel in which the iPS cells have been cultured, thereby detaching the iPS cells from the culture surface. In the recovery step, the detached iPS cells are recovered together with the ejected cryopreservation solution.

[0067] According to the above configuration, a cryopreservation solution (cell suspension preservation solution) in which cells are suspended can be recovered from a culture vessel. In other words, the recovered cell suspension preservation solution can be frozen or frozen and stored as is. This eliminates the need for a liquid replacement step, which is required when cells are detached from a culture surface using a detachment solution such as physiological saline. Therefore, this embodiment reduces the labor required for the liquid replacement step. While the liquid replacement step has the drawback of failing to recover many of the cells suspended in the cell suspension detachment solution, resulting in significant cell loss, this embodiment can directly freeze the cell suspension preservation solution recovered from the culture vessel, thereby eliminating cell loss associated with the liquid replacement step. Furthermore, since this embodiment does not require a liquid replacement step, cells recovered from the culture vessel can be rapidly frozen, thereby reducing damage to cells caused by exposure to room temperature compared to when a liquid replacement step is performed.

[0068] According to at least one of the embodiments described above, cells can be recovered from a culture vessel while reducing cell loss.

[0069] The term "processor" used in the above description refers to a circuit such as a CPU, a GPU, an application specific integrated circuit (ASIC), a programmable logic device (e.g., a simple programmable logic device (SPLD), a complex programmable logic device (CPLD), and a field programmable gate array (FPGA)). A processor realizes its function by reading and executing a program stored in a memory circuit. Note that instead of storing a program in a memory circuit, the program may be directly embedded in the processor circuit. In this case, the processor realizes its function by reading and executing the program embedded in the circuit. On the other hand, if the processor is, for example, an ASIC, the function is directly embedded in the processor circuit as a logic circuit instead of storing the program in a memory circuit. Note that each processor in this embodiment is not limited to being configured as a single circuit for each processor, but may be configured as a single processor by combining multiple independent circuits to realize its function. Furthermore, multiple components in FIGS. 1 and 2 may be integrated into a single processor to realize its function.

[0070] Although several embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, modifications, and combinations of embodiments can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims. [Explanation of symbols]

[0071] 1. Cell Cryopreservation System 10 Cell detachment device 11 Peeling mechanism 13. Recovery Mechanism 15 Control device 20 Cryo-Storage Unit 21 Ultra-low temperature freezer 22 Freezer storage container 30 Incubator 40 Culture vessel 41 Culture surface 42 rising end 43 Fixed side end 51 Placement surface 100 Cell detachment device 111 Stripping liquid pump 112 Liquid delivery pipe 113 Cleaning liquid pump 114 Liquid delivery pipe 115 Cryopreservation fluid pump 116 Liquid delivery pipe 117 Wastewater Pump 118 Suction tube 119 Drive equipment 121 Support mechanism 123 nozzle 131 Recovery pump 132 Suction tube 133 Cryopreservation container 134 Drive equipment 135 Tilting mechanism, balloon 136 Driving equipment, air compressors 141 Suction branch pipe 142 Liquid supply branch pipe 143 Valve

Claims

1. a detachment step of detaching the cells from a surface of a culture vessel in which the cells have been cultured by ejecting a liquid used for cryopreservation of the cells toward the surface on which the cells are in contact; a recovery step of recovering the detached cells together with the discharged cryopreservation liquid; A cell detachment method comprising:

2. The cell detachment method according to claim 1 , wherein the liquid used for cryopreservation is a solution containing a cryoprotectant and a culture medium.

3. The solution used for cryopreservation contains a cryoprotectant, The cell detachment method according to claim 1 , wherein the cryoprotectant comprises dimethyl sulfoxide, glycerol, polyethylene glycol, propylene glycol, glycerin, polyvinylpyrrolidone, sorbitol, dextran, and / or trehalose.

4. The cell detachment method according to claim 1 , wherein in the recovery step, the detached cells are recovered together with the discharged liquid used for cryopreservation, while avoiding bubbles generated by the discharge.

5. 2. The cell detachment method according to claim 1, wherein in the detachment step, the cells are detached from the surface by ejecting the liquid used for cryopreservation as a plurality of droplets toward the surface, ejecting the liquid used for cryopreservation onto the cells, or using a water flow generated by feeding or vibrating the liquid used for cryopreservation.

6. The cell detachment method according to claim 1 , further comprising an addition step of adding a detachment solution to the cells contained in the culture vessel in order to weaken adhesion of the cells to the surface prior to the detachment step.

7. The cell detachment method according to claim 6 , wherein the detachment solution contains a protease and / or a chelating agent.

8. The cell detachment method according to claim 1, further comprising a washing step, prior to the detachment step, of removing impurities from the culture vessel by washing the culture vessel, on whose surface the cells are adhered, with a washing solution.

9. The cell detachment method according to claim 8 , wherein the washing liquid is physiological saline or a liquid culture medium.

10. The cell detachment method according to claim 1, further comprising an additional step of adding the liquid used for cryopreservation to the suspension in order to adjust the concentration of the suspension of the recovered cells and the liquid used for cryopreservation to a predetermined value.

11. The cell detachment method according to claim 1 , further comprising a freezing step of freezing a suspension of the recovered cells suspended in the liquid used for cryopreservation.

12. The freezing step comprises: a first freezing step of freezing the suspension in a first temperature range at which the cells can metabolize; and a second freezing step of freezing and storing the suspension in a second temperature range lower than the first temperature range, at which the cells cannot metabolize the suspension after the first freezing step. The cell detachment method according to claim 11.

13. a detachment step of detaching the cells from a surface of a culture vessel in which the cells have been cultured by discharging a liquid toward the surface on which the cells are in contact; a recovery step of recovering the detached cells together with the ejected liquid; a freezing step of freezing the collected cells and the liquid; A cell detachment method comprising:

14. a detachment unit that detaches the cells from a surface of a culture vessel in which the cells have been cultured by ejecting a liquid used for cryopreservation of the cells toward the surface on which the cells are in contact; a recovery unit that recovers the detached cells together with the discharged liquid used for cryopreservation; A cell detachment device comprising:

15. The recovery unit includes: a suction tube through which the liquid used for cryopreservation flows; a freezing storage container detachably attached to the suction tube; a pump that aspirates a suspension of the detached cells in the discharged cryopreservation liquid through the aspirating tube and sends the liquid to the cryopreservation container; The cell detachment device according to claim 14.

16. The cell detachment device according to claim 15 , further comprising a freezing unit including a freezer in which the cryopreservation container is housed and which freezes the suspension contained in the cryopreservation container.

17. the recovery unit further includes a tilting mechanism that tilts the culture vessel relative to the horizontal so that the depth of the suspension at a first position in the culture vessel into which the suction tube is inserted is deeper than the depth of the suspension at another second position, The pump aspirates the suspension through the suction tube inserted at the first position. The cell detachment device according to claim 15.

18. The cell detachment device according to claim 14 , wherein the detachment unit has a nozzle that sprays the liquid used for cryopreservation onto the surface.

19. a discharge unit that discharges a liquid toward a surface of a culture vessel in which cells are cultured, the surface being in contact with the cells, thereby detaching the cells from the surface; a recovery unit that recovers the detached cells together with the discharged liquid; a freezing unit that freezes the collected cells and the liquid; A cell freezing storage system comprising:

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