JP2008079554A - Closed-system cell recovery device and cell culture apparatus, and recovering method and culturing method for cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a closed-system cell recovery device enabling automated recovery of cultured cells and a method for the recovery of cells, and a cell culture apparatus enabling efficient subculture and a method of culturing the cells. <P>SOLUTION: The closed-system cell recovery device includes a culture vessel 1 housing a culture cell group; a reagent supplying means 3 connected to the culture vessel and supplying the culture vessel with a peeling liquid for lowering the cell adhesion of at least the culture cell group; a peeling means 2 for peeling the culture cell group adhering to the inner wall surface of the culture vessel from the inner wall surface by making a fluid content, such as the peeling liquid in the culture vessel, flow in the surface direction; and a flowing means 4 for the culture cell group connected to the culture vessel. The cell culture apparatus includes a waste liquid recovery means 6 connected to the culture vessel in parallel with the flowing means and connected to the culture vessel in series with the reagent supplying means, and a cell recovery vessel 5 provided connectably at the downstream end of the flowing means and enabling the recultivation of the cultured cell. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  In particular, the present invention relates to a cell culture apparatus and a cell detachment / dispersion method capable of repeatedly subculturing adhesion-dependent cells forming animal tissues and organs.

  Collecting cultured cells such as the above-mentioned adhesion-dependent cells supplies proteolytic enzymes such as trypsin and collagenase to the cultured cells in a petri dish or the like that has been treated so that the cells are likely to adhere, and serves as an auxiliary agent. After adding EDTA or the like to cut off the integrin or other adhesive factor that attaches the cells to the inner wall of the culture vessel, and after cutting off the cadherin or other adhesive factor that adheres the cells together, the lid of the culture vessel is removed. This is done by opening and peeling the cultured cells from the culture vessel by pipetting.

  Furthermore, culture cells can be recovered by pipetting together with the cultured cells because the proteolytic enzyme degrades and destroys cell surface markers, channels, receptors, etc. other than the above-mentioned adhesion factors, and adversely affects the cultured cells. Processing such as centrifugation is performed to replace the proteolytic enzyme with a buffer solution such as PBS.

As described above, the collection of the cultured cells is an open-type manual operation such as opening the lid of the culture container, and thus there is a risk of contamination, and it does not endure bothering. Furthermore, a proteolytic enzyme is a protein derived from a living body, and there is a possibility that a prion, a virus, or the like is mixed therein. When using cultured cells for medical treatment, it is desirable to avoid the use of a proteolytic enzyme.
Therefore, although it is desired to automate the collection of cultured cells with a closed system so that it can be easily performed, it is automated because the work process is complicated, such as pipetting and centrifugation. It has been difficult.

  On the other hand, as shown in Patent Document 1, for example, a reagent supply means for supplying a stripping solution such as a proteolytic enzyme, a cleaning solution such as a buffer solution, and a medium, and a plurality of these connected in parallel to these reagent supply means Proposed a cell culture apparatus having a culture container, a treatment container connected to these culture containers and provided with a filter at the bottom for preventing passage of cultured cells, and a medium supply means connected to the bottom of the treatment solution Has been.

According to this cell culture device, the cultured cell group in one culture vessel can be peeled off from the inner wall surface of the culture vessel by the peeling solution supplied from the reagent supply means, and supplied to the processing vessel.
Next, the peeled cultured cell group is supplied to the processing container with a constant flow rate along with the stripping solution, and only the cultured cell group is accumulated without passing through the filter, and the stripping solution and the like are discharged from the processing container. Then, the medium is supplied from the bottom of the processing container by the medium supply means, and the cultured cell group on the filter is re-supplied to the plurality of culture containers together with the medium.
Thus, when detaching the cultured cell group from the culture container, pipetting and centrifugation are not required, so that it can be automated and the cultured cell group can be easily recovered.

However, since the above-described cell culture apparatus peels the cultured cell group from the inside of the culture vessel only by reaction with the stripping solution, it is necessary to use a strong stripping solution. For this reason, as described above, there is a risk that even cell surface markers other than the adhesion factor are destroyed and the collected cells are adversely affected.
Furthermore, the collected cultured cell group is supplemented by a filter, and even if it is supplied again to a new culture container, there is a problem that the recovery rate of cells by subculture becomes extremely low. In addition to this, there is no means for releasing the adhesion between the cells, the cell density is high while many cells remain adhered to each other, and the cells cannot be divided, and the cells are supplied from the bottom. Therefore, there is a problem in that it cannot be evenly distributed to the culture vessel and cannot be subcultured efficiently.

JP 2005-198626 A

  The present invention has been made in view of such circumstances, and does not require pipetting or the like while suppressing adverse effects on the cultured cells due to the stripping solution, and is a closed cell that can automate the collection of the cultured cells. It is an object of the present invention to provide a collection apparatus, a cell collection method, and a cell culture apparatus and a cell culture method that can be efficiently subcultured.

  A closed-system cell recovery device according to the first aspect of the present invention includes a culture container in which a cultured cell group is accommodated, and a stripping solution that is connected to the culture container and reduces at least the cell adhesion of the cultured cell group. Reagent supply means for supplying to the culture vessel and fluid containing material such as the stripping solution in the culture vessel are allowed to flow in the surface direction, and the cultured cell group attached to the inner wall surface of the culture vessel is removed from the inner wall surface It is characterized by having a peeling means for peeling from the culture vessel and a distribution means for the cultured cell group connected to the culture vessel.

  Here, the cell adhesiveness means cell adhesiveness in a broad sense including adhesiveness such as bonding between cells and cell-matrix adhesiveness such as inner walls of cells and culture vessels.

  According to a second aspect of the present invention, in the closed system cell recovery device according to the first aspect, the peeling means is a vibration device that vibrates the culture vessel in a horizontal direction.

  According to a third aspect of the present invention, there is provided the closed-system cell recovery device according to the first aspect, wherein the peeling means supplies the air into the culture container, and the culture container includes the fluid container. And a rocking mechanism that moves the seesaw so as to flow in the surface direction.

  According to a fourth aspect of the present invention, there is provided the closed-system cell recovery device according to any one of the first to third aspects, wherein the distribution means is provided with a thin tube for releasing the adhesion of the cells of the cultured cell group. In addition, a suction mechanism for allowing the cultured cell group to pass through is provided in the thin tube.

  According to a fifth aspect of the present invention, in the closed-system cell recovery device according to the fourth aspect, the inner diameter of the thin tube is 0.2 mm or more and 1.0 mm or less.

  The invention described in claim 6 is a cell culture device for re-culturing the cultured cells using the closed cell recovery device according to any one of claims 1 to 5, comprising: A waste liquid collecting means connected in parallel to the culture container in parallel with the reagent supply means and connected to the downstream end of the flow means, and connected to the culture container in parallel. And a cell collection container to be re-cultured.

  The invention according to claim 7 is the cell culture device according to claim 6, wherein the distribution means is provided with a dilution mechanism for diluting and dispersing the cultured cells in a culture medium, and the cell collection container comprises: A plurality are provided, and each is provided so as to be connectable in parallel to the downstream end of the circulation means.

  The invention according to claim 8 is the cell culture apparatus according to claim 7, wherein the culture container, the reagent supply means, the peeling means, the flow means, the waste liquid collection means, and the cell collection container are disposed in an incubator. It is characterized by being housed in.

  In the method for recovering cells according to the invention of claim 9, a stripping solution for reducing cell adhesion of the cultured cell group is supplied into a culture container in which the cultured cell group is housed, A reaction step of reacting the cultured cell group, and after the reaction step, supplying a cleaning solution into the culture vessel, and washing off the stripping solution while the cultured cell group is attached to the inner wall surface of the culture vessel A step of replacing the stripping solution and the cleaning solution, and a fluid container such as the cleaning solution stored in the culture vessel in a plane direction to cause the cultured cell group to move inside the culture vessel. And a peeling step for peeling from the wall surface, and the cultured cell group peeled from the inner wall surface is discharged to a distribution means connected to the discharge port of the culture vessel.

  The invention described in claim 10 is characterized in that, in the cell recovery method according to claim 9, the stripping solution is a divalent metal chelating agent.

  The invention according to claim 11 is the method for recovering cells according to claim 9 or 10, wherein after the washing step, a calcium-containing solution such as a medium is supplied into the culture container, and the A replacement step is provided in which a part of the cleaning solution is discharged to the outside, and a part of the cleaning solution in the culture vessel is replaced with the calcium-containing solution, and the calcium-containing solution in the culture vessel, the cleaning solution, etc. The fluid container is characterized by flowing in the surface direction in the peeling step.

  A twelfth aspect of the invention is characterized in that, in the cell recovery method according to any one of the ninth to eleventh aspects, in the detaching step, the culture vessel is vibrated in a horizontal direction.

  The invention according to claim 13 is the cell recovery method according to any one of claims 9 to 11, wherein in the detaching step, air is supplied into the culture container, and the culture container is The seesaw motion is performed so that the fluid container flows in the surface direction.

  The invention according to claim 14 is the method for recovering cells according to any one of claims 9 to 13, wherein the cultured cell group detached from the inner wall surface of the culture container is the It is characterized by having a cell dispersion step in which the cells of the cultured cell group are released from each other by being distributed through a thin tube interposed in the distribution means, and the cultured cells are dispersed in the fluid container.

  The invention according to claim 15 is characterized in that, in the method for collecting cells according to claim 14, the cultured cells are dispersed in a fluid container by passing the cultured cell group through the capillary tube a plurality of times. Yes.

  The invention described in claim 16 is a cell culturing method for re-culturing cultured cells using the method for recovering cells according to claim 14 or 15, wherein the cultured cell group is provided after the cell dispersion step. A seeding step of supplying the cultured cells in which the adhesion between the cells is released to a plurality of cell recovery containers, and a culture step of reculturing the cultured cells in the cell recovery containers.

  The invention according to claim 17 is the cell culturing method according to claim 16, wherein a dilution step for diluting and dispersing the cultured cells in a medium is provided after the cell dispersion step. The cell liquid is supplied to the cell collection container in the seeding step.

  According to the closed-system cell recovery device according to any one of claims 1 to 5, the peeling solution is supplied from the reagent supply means to the culture vessel, and the culture vessel of the culture cell group accommodated in the culture vessel By reducing the adhesiveness to the inner wall surface and the adhesion between cells and causing the fluid container in the culture vessel to flow in the surface direction by the peeling means, the cultured cell group attached to the culture vessel and its inner wall surface During this period, a shearing stress can be applied to detach the cultured cell group.

  As the peeling means, a vibration device that vibrates the culture vessel in the horizontal direction as in the invention described in claim 2 can be suitably used. Further, as in the invention described in claim 3, an air supply mechanism and a swing mechanism can be used. In this case, the culture cell group is supplied by supplying air into the culture container by the air supply mechanism, moving the culture container to the seesaw by the swinging mechanism, and causing the fluid container to flow in the surface direction every time the seesaw movement is performed. Can be peeled off.

For this reason, stripping solution should just be what can peel a cultured cell group from the inner wall face of the said culture container by a peeling means. As a result, by using a strong peeling agent that significantly reduces cell adhesion performance, the reaction between the peeling solution and cultured cells can suppress adverse effects on the cells, increasing the cell survival rate, Cultured cell populations can be collected.
In addition, as described above, since the cultured cell group does not need to be collected by a pipette or the like, the entire apparatus can be automated.

  Next, the separated cultured cell group is discharged to the distribution means, and the collection container containing the culture medium is connected to the downstream end of the distribution means, so that contamination can be mixed in the closed system. The cell culture group can be easily recovered.

In that case, according to the invention described in claim 4, the cultured cell group can be passed through the thin tube interposed in the distribution means by the suction mechanism, and the adhesion of the cells of the cultured cell group can be released. It can be collected as cells. For this reason, stripping solution should just be what can cancel | release the adhesion | attachment of cells with a thin tube, and can suppress having a bad influence on a cultured cell by reaction with stripping solution.
In addition, it is possible to efficiently proliferate in the cell collection container by inoculating the cell collection container with the cultured cells whose adhesion has been released.

  In particular, as in the invention described in claim 5, by setting the inner diameter of the thin tube to 0.2 mm or more and 1.0 mm or less, cultured cells having a diameter of several tens of μm to several hundreds of μm remain in a lump shape. Even if it is supplied as a cultured cell group, it adheres to the outer periphery of the cell group in order to release the cell-to-cell adhesion by applying shear stress between the cells due to the flow velocity difference between the central part of the capillary and the vicinity of the inner wall. It is possible to prevent the cells from being damaged. For this reason, killing of cultured cells can be prevented and the survival rate of cultured cells can be increased. At that time, the linear velocity of the cultured cell group or the like can be kept uniform by decreasing the flow rate when the inner diameter of the narrow tube is small, and increasing the flow rate when the inner diameter is large.

According to the invention described in claim 6, the waste liquid recovery means is connected in series with the reagent supply means, connected in parallel with the flow means, and cell recovery is performed at the downstream end of the flow means. Since the container is provided so that it can be connected, the stripping solution contained in the culture vessel is supplied to the waste liquid recovery means connected in series with the reagent supply means, for example, by supplying the cleaning liquid from the reagent supply means to the culture container. It can be discharged. Thereby, since the cultured cell group can be separated from the stripping solution and supplied to the distribution means, automation of the entire apparatus can be made realistic, and the cultured cell group can be temporarily placed on a filter or the like. Therefore, it is possible to prevent the cultured cells from being supplemented by an obstacle such as a filter.
As a result, subculture can be performed simply, and the survival rate of the cultured cells to be planted in the cell recovery container can be increased, thereby increasing the cell recovery rate by subculture.

  Furthermore, according to the invention described in claim 8, the cultured cells are always kept in a constant temperature condition by accommodating the culture container, the reagent supply means, the peeling means, the flow means, and the waste liquid collecting means in an incubator. Therefore, it is possible to reliably prevent the death of cultured cells due to poor temperature conditions.

  On the other hand, according to the method for culturing cells according to any one of claims 9 to 15, in the reaction step, a peeling solution is supplied into the culture container and reacted with the cultured cell group accommodated in the culture container. Thereby, the adhesiveness with respect to the inner wall surface of the culture container of a cultured cell group and the adhesiveness of cells can be reduced.

  Next, in the cleaning step, a cleaning solution is supplied into the culture vessel, the stripping solution is washed away, and the stripping solution and the cleaning solution are replaced. It is possible to suppress destruction and adverse effects on cultured cells.

  Thereafter, according to the invention described in claim 11, in the replacement step, by adding a calcium-containing solution such as a medium to the washing liquid in the culture vessel, the calcium lost by the stripping solution is replenished, and the film strength is increased. Cells that fall and become fragile can be strengthened and protected.

  Next, in the peeling step, a shearing stress is applied between the cultured cells adhering to the culture vessel and the adhering surface by flowing a fluid container such as a washing solution in the surface direction, and the cultured cell group is It can peel from the inner wall surface of the said culture container.

  Specifically, as in the invention described in claim 12, the cultured cell group can be separated by the action of the shear stress by vibrating the culture container in the horizontal direction. In addition, as in the invention described in claim 13, since air is supplied into the culture vessel and the culture vessel is caused to perform seesaw motion, the fluid container flows in the plane direction every time the seesaw motion is performed. The cell group can be detached by the action of the shear stress.

For this reason, the cultured cells are not limited as long as the cultured cell group can be detached in the detaching step, as in the above-described cell collection device, and can suppress adverse effects on the cultured cells in the reaction step. There is no need to collect the sample with a pipette or the like, and the collection method can be automated.
Subsequently, the cultured cell group can be recovered from the downstream end of the distribution means by discharging the detached cultured cell group to the distribution means connected to the discharge port of the culture container.

In that case, according to the invention described in claim 14, in the cell dispersion step after the peeling step, the cultured cell group is passed through a thin tube interposed in the distribution means, and the cells of the cultured cell group are adhered to each other. It can be unwound and the cultured cells can be dispersed in the fluid container.
For this reason, as above-mentioned, as long as it can peel | exfoliate adhesion | attachment of cells by a thin tube while a cultured cell group can be peeled in a peeling process as mentioned above, It is what is described in Claim 10. A divalent chelating agent can be suitably used as in the invention. As a result, adverse effects on the cultured cells due to the reaction with the stripping solution can be suppressed.
In addition, the cells can be efficiently propagated by seeding the cultured cells uniformly dispersed in the fluid container in the cell collection container.

  In particular, according to the invention described in claim 15, even if the tubule is designed to be short by passing the cultured cell group through the tubule interposed in the flow means together with the stripping solution or the like, it is a multiple of the length. The cells can be evenly dispersed in the fluid container with the same efficiency as in the case of designing in the above. For this reason, the length of a thin tube can be earned by letting a cultured cell group pass through a thin tube several times, and the proliferation of a cell after cell seeding can be performed remarkably efficiently.

  Next, according to the invention described in claim 16, in the seeding step, the cultured cells in which the adhesion of the cells of the cultured cells is released are supplied to the cell recovery container, and then in the cell recovery container in the culture step. Subculturing can be performed by culturing the above cultured cells.

  According to the invention described in claim 7 or 17, the cultured cells can be diluted and dispersed in the culture medium by a dilution mechanism or a dilution process, and the cultured cells can be evenly distributed to a plurality of cell recovery containers. Can be repeated multiple times, and subculture can be performed efficiently.

An embodiment of a cell culture device according to the present invention will be described with reference to FIGS. 1 and 2.
As shown in FIG. 1, the cell culture device of this embodiment includes a mounting table (vibration device) 2 on which a culture vessel 1 in which cultured cell groups are accommodated, and a reagent supply that supplies a reagent to the culture vessel 1. Means 3, distribution means 4 for discharging the cultured cell group from the culture container 1, cell recovery container 5 provided to be connectable to the downstream end of the distribution means 4, and culture container 1 in parallel with the distribution means 4 And a waste liquid recovery means 6 connected to. The culture container 1, the mounting table 2, the reagent supply means 3, the distribution means 4, the cell recovery container 5, and the waste liquid recovery means 6 are all housed in a carbon dioxide incubator (not shown). The system is operated by a control system (not shown) for operating the entire apparatus.

  The culture vessel 1 and the cell collection vessel 5 are both formed in a rectangular shape in plan view made of a transparent synthetic resin, and a hollow 11 having a substantially rhombus shape in plan view is formed at the center thereof, and the cultured cell group is accommodated. Has been. And the flow path 12 and 13 connected to the both-ends corner | angular part of the longitudinal direction of the container 1 in this cavity 11 is formed, and these flow paths 12 and 13 mutually face toward the one side surface 1a of the longitudinal direction of the container 1. They are formed in parallel.

As shown in FIG. 2, the mounting table 2 on which the culture vessel 1 is mounted includes a mounting plate 21 that is horizontally disposed and a vertical bar 22 that is integrally provided at a lower portion of the mounting plate 21. And an eccentric cam 23 having a rotational speed of 600 rpm to 4200 rpm, which is provided integrally with the lower portion of the vertical bar 22 and has two horizontal disks arranged in the vertical direction and connected to each other, and the eccentric cam 23 And a motor 24 for rotating the motor.
The motor 24 rotates a disk-like wheel 25 disposed horizontally at the upper part thereof, and an endless belt 26 disposed around the outer periphery of the wheel 25 and the lower horizontal disk in the eccentric cam 23. By moving in the circumferential direction, the eccentric cam 23 is rotated at a rotational speed of 2000 rpm or more for detachment of the cultured cell group in the culture vessel 1. Then, the eccentric cam 23 moves the mounting plate 21 horizontally on the support frame 27 by moving the vertical bar 22 integrally provided outside the center of the upper horizontal disk around the rotation center. It is supposed to let you.
In addition, the mounting plate 21 causes the channel 13 to incline the bottom of the vessel 1 at 45 degrees with the central portion between the channel 12 and the channel 13 of the culture vessel 1 as an axis by an inclination mechanism (not shown). The side is provided to be movable upward.

The reagent supply means 3 includes a calcium supply syringe 31 filled with a calcium-containing solution, a release solution supply syringe 32 filled with a release solution such as EDTA, and a cleaning solution supply syringe filled with a cleaning solution such as PBS. 33 three syringes are provided.
Here, a medium or a calcium chloride solution is used as the calcium-containing solution, preferably a 0.01 to 40 mM calcium chloride solution, and more preferably a 1 to 4 mM calcium chloride solution.

Moreover, as stripping solution, 0, 5 mM to 10 mM EDTA, divalent metal chelating agents such as EGTA, DTPA, NTA, TTHA, or proteolytic enzymes such as trypsin and collagenase, integrin, cadherin, etc. Peptides that competitively inhibit adhesion of adhesion proteins are used. Preferably, a bivalent metal chelating agent such as EDTA, which can suppress adverse effects on the cultured cell by reaction with the cultured cell, is used. Mainly, a partly proteolytic enzyme or a mixture of the above peptides is used.
As the washing solution, a solution that does not contain calcium and does not adversely affect cultured cells is used. For example, an isotonic buffer solution such as PBS, HEPES, tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution (Tris), etc. Alternatively, an isotonic solution such as a sucrose solution, a saline solution or an aqueous potassium chloride solution having the same osmotic pressure as that of the cultured cells is used.

  Each of the syringes 31, 32, and 33 has a tube 34 connected to a filler outlet, and tube pumps 31a, 32a, and 33a are interposed in the tubes 34 corresponding to the syringes. Further, the downstream end of each tube 34 is connected to a pipe connector 36 having four connection ports, and a connection tube 37 disposed horizontally toward the culture vessel 1 is connected to the remaining connection port. . A three-pronged pipe connector 38 is interposed in the connection tube 37, and the tube 37 on the downstream side of the pipe connector 38 is arranged vertically and integrated with one side of the culture vessel 1 in the longitudinal direction. It is connected to the flow path 12 through the thin tube 10 provided for this purpose.

  Then, the pump 32a is operated for a predetermined time by the control system, and the peeling solution in the syringe 32 is supplied into the culture vessel 1 through the tube 34, the pipe connector 36, the connection tube 37, and the thin tube 10, and then the pump The operation of 32a is stopped, and the peeling solution and the cultured cell group react in the culture vessel 1. Next, the pump 33a is operated for a predetermined time by the control system, and after the cleaning liquid in the syringe 33 is similarly supplied into the culture vessel 1, the pump 33a is stopped, and then the pump 31a is operated for a predetermined time. Similarly, the calcium-containing solution in 31 is supplied into the culture vessel 1 and the pump 31a is stopped.

  Next, the control system operates the motor 24 of the mounting table 2 described above, the eccentric cam 23 rotates at 2000 rpm to 4200 rpm, and the mounting plate 21 moves horizontally, so that the cleaning solution and calcium-containing solution in the culture vessel 1 are moved. The fluid containing material such as the fluid flows, a shear stress acts between the cultured cell group adhering to the culture vessel 1 and its attachment surface, and the cultured cell group is peeled off.

  On the other hand, a vertically arranged tube 41 is connected to the remaining connection port of the pipe connector 38. The tube 41 is provided with a pinch valve 41a, and its downstream end is a pipe. The connection tool 42 is connected to the arrangement opening at the upper end. The pipe connector 42 is disposed in the vertical direction, and the discharge port of the syringe pump 44 disposed directly below the tube 41 is connected to the disposition port at the lower end.

And by the said control system, while the mounting plate 21 of the said mounting plate 2 is inclined, the pinch valve 41a is opened, the plunger of the syringe pump 44 is pulled at a predetermined | prescribed speed, The detached cultured cell group is It is supplied to the narrow tube 10 from the flow path 12 together with the fluid container.
The syringe pump 44 constitutes a suction mechanism together with the tube 41 and the pipe connector 42.

  The narrow tube 10 has an inner diameter of 0.2 mm or more and 1.0 mm or less, more preferably an inner diameter of 0.3 mm or more and 0.8 mm or less. When the inner diameter is less than 0.2 mm, the cultured cell group having a diameter of about several tens of μm to several hundreds of μm circulates in a lump shape, and the cells attached to the outer periphery may be damaged and killed. On the other hand, if the thickness exceeds 1.0 mm, the cultured cell group circulates only in the central part of the tubule, so that the adhesion between the cells cannot be released. This is because the cells are covered with cells, the proliferation efficiency is lowered, and the seeding cannot be performed uniformly.

  On the other hand, when the inner diameter is 0.2 mm or more and 1.0 mm or less, the flow rate is decreased when the inner diameter is smaller, and the flow rate is increased when the inner diameter is larger. Thus, due to the difference in flow rate between the central portion and the outer peripheral portion, shear stress can be applied between the cells, the adhesion between the cells can be released, and the cultured cell group can be dispersed in the fluid container. In particular, when the inner diameter is 0.3 mm, the flow rate can be 0.18 ml / s, and when the inner diameter is 0.5 mm, the flow rate can be 0.45 ml / s. In the case of 8 mm, since the flow rate can be 1.28 ml / s, when the inner diameter is 0.3 mm or more and 0.8 mm or less, the flow rate is 0.18 to 1.28 ml / s. Because it can be made practical.

  Next, by pushing the plunger of the syringe pump 44 at a predetermined speed by the control system, the cultured cells and the fluid contained in the thin tube 10 are circulated and re-supplied into the culture vessel 1. Yes. Thereafter, the mounting plate 21 is tilted as described above, and the syringe pump 44 is pulled at a predetermined speed, whereby cultured cells and the like are circulated through the thin tube 10, and the operation of the syringe pump 44 and the like is repeated a predetermined number of times. As a result, the cultured cells are uniformly dispersed in the fluid container and become a cell turbid solution.

  On the other hand, the pipe connector 42 has four connection ports. Of the remaining two ports provided on the side portion, the upper connection port is arranged toward the cell collection container 5. The tube 43 is connected. Moreover, the discharge port of the syringe pump 45 filled with the culture medium is connected to the lower arrangement port. Note that a pinch valve 43 a is interposed in the tube 43.

Then, the control system closes the pinch valve 41a and alternately pushes out the plungers of the syringe pumps 44 and 45 at a predetermined speed for a predetermined number of times, whereby the syringe pump 45 of the cultured cells in the syringe pump 44 is connected via the connector 42. And the supply of the medium in the syringe pump 45 to the syringe pump 44 are alternately and repeatedly performed. As a result, the cell turbid solution is diluted and dispersed by the medium to form a cell solution, which is filled in the syringe of the syringe pump 45. At this time, since the pinch valves 41a and 43a are closed, outflow of cultured cells and the like to the tubes 41 and 43 is prevented.
The syringe pumps 44 and 45, together with the pinch valves 41a and 43a and the connection tool 42, constitute a dilution mechanism. The thin tube 10 to a part of the tube 37, the pipe connection tool 38, the tube 41, the syringe pump 44, 45 and the tube 43 constitute the circulation means 4.

On the other hand, a plurality of cell collection containers 5 are stacked in the shelf 50 in the vertical direction, and the downstream ends of the tubes 43 are provided to be connectable.
Then, the control system opens the pinch valve 43a and pushes the plunger of the syringe pump 45, so that a predetermined amount of cell fluid is seeded in each container 5 at a predetermined speed.

On the other hand, the waste liquid recovery means 6 includes a tube 61 connected to one side surface 1a in the longitudinal direction of the culture vessel 1 so as to communicate with the flow path 13, and a recovery bottle 62 in which the downstream end of the tube 61 is disposed. And an outside air supply pipe 63 for supplying outside air into the collection bottle 62 via a sterile filter 63a.
Then, when the pump 33 a is operated by the control system, the stripping solution in the culture vessel 1 is discharged to the collection bottle 62 through the tube 61. The control system includes an interface for inputting each predetermined value such as the temperature and the number of times described above.

[Other Embodiments]
The cultured cell group can be detached from the inner wall surface of the culture vessel 1 by using an air supply mechanism and a swing mechanism (not shown) including the waste liquid collecting means 6 instead of the mounting table 2 described above.
The waste liquid collecting means 6 pulls the syringe pump 44 so that the external air in the collection bottle 62 supplied by the outside air supply pipe 63 via the aseptic filter 63a passes from the flow path 13 to the inside of the culture container 1 via the tube 61. The air supply mechanism is configured together with the syringe pump 44.

  The swing mechanism is configured to place the culture vessel 1 on a placement plate, as with the placement table 2. The mounting plate can hold the culture vessel 1 at an angle of 30 degrees with the central portion between the flow channel 12 and the flow channel 13 as an axis, and the flow channel 12 side downward. Thus, the culture vessel 1 can be held at an inclination of 30 degrees. Further, the placing plate causes the seesaw motion of the culture vessel 1 at a speed of once / second so that the channel 12 side and the channel 13 side are alternately positioned below. Further, similarly to the mounting table 2, an inclination mechanism is provided, and after the seesaw motion, the culture vessel 1 is inclined 45 degrees with the flow channel 12 side downward.

  For this reason, after the syringe pump 44 is pulled by the control system and air is supplied into the culture container 1, the culture container 1 is subjected to a seesaw motion by a rocking mechanism, so that the washing liquid and calcium in the culture container 1 are contained. A fluid container such as a solution flows, and a shearing stress acts between the cultured cell group adhering to the culture vessel 1 and its adhering surface, so that the cultured cells are separated. Thereafter, by the above control system, the culture vessel 1 is inclined 45 degrees with the flow channel 12 side down, and the detached cultured cell group is discharged from the flow channel 12 to the thin tube 10 together with the fluid container. In FIG. 10, the adhesion between cells is gradually released.

Next, a cell culture method according to the present invention using the above-described cell culture apparatus will be described.
The cell culturing method of the present embodiment includes a washing step of supplying a stripping solution such as EDTA into the culture vessel 1 and supplying a washing solution such as PBS into the culture vessel 1 after the reaction step of reacting with the cultured cell group. A replacement step of replacing a part of the washing solution with a calcium-containing solution, a peeling step of vibrating the culture vessel 1 in the horizontal direction to peel the cultured cell group from the culture vessel 1, and a capillary tube for the peeled cultured cell group A cell dispersion step for releasing the adhesion between the cultured cells and dispersing the cultured cells with a calcium-containing solution, etc., a dilution step for diluting and dispersing the cultured cells with the adhesion removed in the medium, and this dilution A seeding step of supplying the dispersed cell solution to the cell recovery container; and a culture step of culturing the cultured cells in the cell recovery container.
Here, the stripping solution, the cleaning solution, and the calcium-containing solution are the same as those described in the above cell culture device.

In the reaction step, in the state where the pinch valves 41a and 43a are closed, the stripping solution supply syringe 32 is operated and the tube pump 32a is operated to supply the stripping solution into the culture vessel 1. Then, the culture medium filled with the cultured cell group in the culture container 1 is discharged from the tube 61 to the collection bottle 62, whereby the culture container 1 is replaced with a stripping solution.
Then, the cell adhesion of the cultured cell group is lowered by keeping the temperature and carbon dioxide concentration constant for a predetermined time with a carbon dioxide incubator and reacting the cultured cell group with the stripping solution.

Next, in the cleaning step, the cleaning liquid supply syringe 33 is operated and the tube pump 33a is operated to supply the same amount of cleaning liquid as that of the stripping liquid, whereby about 90% of the stripping liquid in the culture vessel 1 is cleaned. And the stripping solution is discharged to the collection bottle 62. At that time, the cultured cell group remains attached to the inner wall surface of the culture vessel 1.
Thereby, it is prevented that even the cell surface marker other than the adhesion factor is destroyed by the peeling solution, and the adverse effect on the cultured cell group is prevented.

  Next, in the replacement step, the calcium supply syringe 31 and the tube pump 31a are operated to replace a part of the cleaning liquid in the culture vessel 1 with the calcium-containing solution and collect a part of the cleaning liquid. Drain into bottle 62. Thereby, nutrients required for cells such as proteins decomposed by the stripping solution are supplemented, and the cells are strengthened and protected.

  In the peeling step, immediately after the calcium-containing solution is supplied in the replacement step (that is, by supplying the calcium-containing solution, the cell adhesion of the cultured cells is reproduced, and the cultured cell group becomes the culture container. The motor 24 of the mounting table 2 is operated on the mounting plate 21 on which the cultured cell group is stored together with a fluid container such as a washing solution and a culture medium. Thus, the cell is horizontally moved toward the combined vector direction in the plane in which the X-axis direction and the Y-axis direction are combined, and the cultured cell group is separated from the inner wall surface of the culture vessel 1.

  In addition, after replacing the mounting table 2 and using the air supply mechanism and the rocking mechanism to pull the syringe pump 44 by the air supply mechanism and supplying the air into the culture vessel 1 in the same manner as the cell culture apparatus described above. It is also possible to cause the culture container 1 to perform a seesaw motion by a swing mechanism and to peel the cultured cell group from the culture container 1.

  Next, in the cell separation step, the mounting plate 21 is moved upward on the flow path 13 side so that the bottom of the container 1 is inclined at 45 degrees with the central portion between the flow paths 12 and 13 of the culture container 1 as an axis. At the same time, by opening the pinch valve 41a and pulling the plunger of the syringe pump 44, the cultured cell group is supplied from the flow path 12 to the thin tube 10 together with the fluid container. At that time, the flow rate in the narrow tube 10 is adjusted according to the pulling condition of the plunger, the adhesion between the cells is released in the narrow tube 10, and the cultured cells are dispersed in the fluid container.

  Subsequently, the plunger of the syringe pump 44 is pushed out, so that the cultured cell group and the like are resupplied into the culture container 1 together with the fluid container. At that time, in the cultured cell group, when the flow direction is changed in the thin tube 10, a large shear stress is generated between the cells, so that the adhesion between the cells is efficiently released, and the cultured cells become the fluid container. Distributed. Then, the mounting plate 21 is tilted again to supply cultured cells and the like to the thin tube 10.

As described above, the separation step is repeated a predetermined number of times (in this embodiment, 10 times), and the cultured cells are distributed (20 times) to the capillary tube 10 to reliably release the adhesion between the cells and to ensure the cultured cells. Is dispersed in a fluid container to form a cell turbid solution.
Thereafter, the cell turbid liquid is filled into the syringe of the syringe pump 44 from the pipe connector 38 disposed below in the vertical direction through the tube 41 and the pipe connector 42 by pulling the plunger of the syringe pump 44. Is done.

Next, in the dilution step, the pinch valve 41a is closed, the syringe pumps 44 and 45 are alternately operated four times, and the culture turbid solution filled in the syringe of the syringe pump 44 is preliminarily stored in the syringe of the syringe pump 45. After diluting and dispersing in the medium filled in the cell to obtain a cell solution, the cell solution is filled in the syringe of the syringe pump 45 by operating the pump 44.
In addition, the process from the supply of the calcium-containing solution to the dilution step in the above-described peeling step is completed within 5 minutes.

  Next, the seeding step closes the valve 41a, opens the pinch valve 43a, and pushes out the plunger of the syringe pump 45, thereby supplying the tube 43 with the cell fluid filled in the syringe of the pump 45, By sequentially connecting the downstream end of the tube 43 to each cell collection container 5, the cell solution is evenly distributed to each cell collection container 5. Then, the air in each cell collection container 5 is discharged and supplied into the collection bottle 62 through the connection pipe 51.

  In the culturing step, the cells in each cell collection container 5 are cultured by keeping the temperature and carbon dioxide concentration constant in the carbon dioxide incubator for a predetermined period. Meanwhile, when the medium is changed after an appropriate number of days for each cell, the cultured cells in each cell collection container 5 have the target cell density. Thereafter, each cell collection container 5 is taken out and sequentially placed on the mounting table 2 as the culture container 1, and a plurality of new cell collection containers 5 are stacked in the shelf 50, whereby repeated subculture is performed. .

  Subsequently, as an example, a human liver cancer-derived cell line (HepG2) and a Syrian hamster pancreatic β cell-derived cell line (HIT-T15) were subcultured.

[Example 1 (in the case of HepG2 cells)]
First, HepG2 cells were diluted with a medium so as to be 12.8 × 10 ⁴ cells / ml, and 4.5 ml was seeded in a culture vessel having a plasma treatment on the bottom, and cultured for 3 days. Thereafter, the culture vessel 1 was placed on the placement plate 21 of the placement table 2.
In addition, while using a peristaltic pump as the tube pumps 31a, 32a, and 33a, all set the liquid feeding speed to 3 ml / min, the syringe 31 and the syringe pump 45 have a 1.8 mM calcium chloride-containing medium, and the syringe 32 has Each 4 ml of 4 mM sodium edetate (EDTA) diluted with phosphate buffered saline (PBS) and syringe 33 were filled with 50 ml of PBS.

  Then, PBS-diluted EDTA is supplied from the syringe 32 to the culture vessel 1 at 3 ml / min for 3 minutes, replaced with the culture medium in the culture vessel 1, and incubated at 25 ° C. for 20 minutes. 1 was fed at 3 ml / min for 3 minutes to replace EDTA. Next, the medium is supplied from the syringe 31 at a rate of 3 ml / min for 1 minute. Immediately, a vibration of 2600 rpm by the mounting table 2 is applied for 20 seconds to separate the cultured cell group from the culture vessel 1, and the culture vessel 1 is inclined at 45 degrees. At the same time, the pinch valve 41a was opened and the plunger of the syringe pump 44 was pulled. Thus, after the cultured cell group is discharged from the culture vessel 1 together with the medium and the like and distributed to the thin tube 10, the cultured cell group is again distributed to the thin tube 10 by pressing the plunger, and the culture vessel 1 Re-supplied. Then, again, the culture vessel 1 was tilted by the mounting table 2 and the cultured cell group was circulated through the tubule 10 for a total of 10 sets and supplied to the syringe pump 44.

Next, the tip of the syringe pump 44 is removed from the pipe connector 42, 1 ml of the cell turbid solution is collected from the syringe pump 44, and the number of cells is counted. As a result, 2.81 × 10 ⁶ cells are recovered, which will be described later. A high recovery rate of 98% was obtained with respect to the number of cells in Reference Example 1.

  Next, after the tip of the syringe pump 44 is connected to the pipe connector 42 again, the pinch valve 41a is closed, and the plungers of the syringe pumps 44 and 45 are alternately pushed out four times each, thereby being supplied into the syringe pump 44. The obtained cell turbid solution was diluted and dispersed with the medium of the syringe pump 45 to obtain a cell solution, and then the plunger of the syringe pump 44 was pushed and accommodated in the syringe pump 45. Thereafter, the plunger of the syringe pump 45 was pushed out, whereby the cell solution was supplied to the tube 43 and distributed to the 12 cell collection containers 5.

  Next, immediately after the distribution, the number of cultured cells in the 12 cell collection containers 5 was counted and found to be about the same number.

[Example 2 (in the case of HIT-T15 cells)]
Next, HIT-T15 cells are diluted with a medium to 5 × 10 ⁴ cells / ml, seeded with 4.5 ml in a culture vessel having a plasma treatment on the bottom, and cultured for 6 days. The container was mounted on the mounting plate 21 of the mounting table 2. In addition, the types of tube pumps 31a, 32a, and 33a, the liquid feeding speed, and the fillers in the syringes of the syringes 31, 32, and 33 and the syringe pump 45 were the same as those in Example 1.

  Next, as in Example 1, the medium in the culture vessel 1 was replaced with PBS-diluted EDTA supplied from the syringe 32, and then incubated at 37 ° C for 20 minutes. Next, in the same manner as in Example 1, the medium is supplied from the syringe 31 and immediately vibrated, the cultured cell group is peeled off, discharged from the culture container 1 and circulated through the capillary tube 10. The refeeding was repeated 10 sets, and the syringe pump 44 was fed.

Next, 1 ml of the cell turbid solution was collected from the syringe pump 44 and the number of cells was counted. As a result, 1.14 × 10 ⁶ cells were recovered, which was 106% of the number of cells in Reference Example 2 described later. A high recovery rate was obtained.

  Next, the tip of the syringe pump 44 is connected to the pipe connector 42, diluted and dispersed by the syringe pumps 44 and 45, and immediately after being distributed to the 12 cell collection containers 5 in the same manner as in Example 1, When the number of cultured cells in the cell collection container 5 was counted, it was about the same number.

[Example 3 (in the case of HepG2 cells)]
First, HepG2 cells are diluted with a medium so as to be 12.8 × 10 ⁴ cells / ml, and after culturing for 3 days in the same manner as in Example 1, the culture vessel 1 containing the cells is placed on the mounting plate 21. Placed on top. In addition, the types of tube pumps 31a, 32a, 33a, the liquid feeding speed, and the filler in the syringes 31, 32, 33 syringe pump 45 were the same as those in Example 1.
Thereafter, PBS-diluted EDTA was fed from the syringe 32 and incubated at 37 ° C. for 20 minutes.

  Next, as in Example 1, after supplying the PBS 33 and the medium from the syringe 31 from the syringe 33, 2.4 ml of the syringe pump 44, which is an air supply mechanism, is drawn, and about 2 ml of air is drawn from the collection bottle 62 into the tube 61. To the inside of the culture vessel 1. Next, the culture vessel 1 was moved by seesaw motion (10 reciprocations) for 20 seconds at a rate of once / second by a rocking mechanism, and the cultured cells were detached. Thereafter, in the same manner as in Example 1, the culture vessel 1 is tilted at 45 degrees, the cultured cell group and the like are circulated through the thin tube 10, and then the plunger of the syringe pump 44 is pushed, whereby the cultured cell group is moved to the thin tube 10. It was distributed and re-supplied to the culture vessel 1. In the same manner as described above, the culture vessel 1 was tilted at 45 degrees, and the culture cell group was similarly circulated through the thin tube 10 for a total of 10 sets and supplied to the syringe pump 44.

Next, when the tip of the syringe pump 44 is removed from the pipe connector 42, 1 ml of the cell turbid solution is collected from the syringe pump 44, and the number of cells is counted. As a result, 3.05 × 10 ⁶ cells are recovered, which will be described later. A high recovery rate of 103% was obtained with respect to the number of cells in Reference Example 3.

[Reference Example 1]
In the same manner as in Example 1, HepG2 cells were diluted with a medium so as to be 12.8 × 10 ⁴ cells / ml, and 4.5 ml was seeded in a sealed container having a plasma treatment on the bottom, and cultured for 3 days. .
Next, discard the medium of HepG2 cells using a syringe, disassemble the sealed container, take out only the bottom, wash once with PBS, add 1 ml of 0.25% trypsin, 1 mM EDTA, and 37 ° C. The reaction was allowed for 10 minutes.
After confirming that the cells were detached, the cells were completely detached from the bottom surface by pipetting, collected in a 10 ml centrifuge tube, centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the cells were dispersed by pipetting. . When 2 ml of the medium was added to the cell turbid solution and the cultured cells were counted, 2.86 × 10 ⁶ cells were recovered.

[Reference Example 2]
In the same manner as in Example 2, HIT-T15 cells were diluted with a medium so as to be 5 × 10 ⁴ cells / ml, and 4.5 ml was seeded in a sealed container having a plasma treatment on the bottom, and cultured for 6 days. .
Next, the medium of HIT-T15 cells is discarded using a syringe, and after disassembling the sealed container, only the bottom surface portion is taken out. Similarly to Reference Example 1, HIT-T15 cells are reacted with a stripping solution containing trypsin and EDTA. The cells were detached from the bottom of the sealed container, and the cells were completely detached from the bottom by pipetting, collected in a 10 ml centrifuge tube, centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the cells were dispersed by pipetting. Subsequently, 2 ml of a medium was added to this cell turbid solution, and the cultured cells were counted. As a result, 1.07 × 10 ⁶ cells were recovered.

[Reference Example 3]
In the same manner as in Example 3, HepG2 cells were diluted with a medium so as to be 12.8 × 10 ⁴ cells / ml, seeded with 4.5 ml in a sealed container having a plasma treatment on the bottom, and cultured for 6 days.
Next, the medium of HepG2 cells is discarded using a syringe, and after the sealed container is disassembled, only the bottom surface is taken out, and in the same manner as in Reference Example 1, HepG2 cells are reacted with a stripping solution containing trypsin and EDTA. The cell was completely detached from the bottom surface by pipetting, collected in a 10 ml centrifuge tube, centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and the cells were dispersed by pipetting. Subsequently, 2 ml of a medium was added to the cell turbid solution, and the cultured cells were counted. As a result, 2.95 × 10 ⁶ cells were recovered.

  According to the above-described cell culture apparatus or cell culture method, the peeling liquid is supplied from the peeling liquid supply syringe 32 to the culture container, and the adhesion of the cultured cell group accommodated in the culture container to the inner wall surface of the culture container and By reducing the adhesiveness between cells and then supplying the cleaning liquid from the cleaning liquid supply syringe 33, it is possible to suppress the peeling liquid from adversely affecting the cultured cells.

  Next, after supplying the calcium-containing solution from the calcium supply syringe 31, the culture vessel 1 is horizontally moved by the mounting table 2, so that the cultured cell group attached to the bottom inner wall surface of the culture vessel 1 and its adhesion surface In between, the shear stress can be applied in the direction of the combined vector in the X-axis direction and the Y-axis direction, and all the cultured cell groups attached in all directions can be efficiently detached.

  Next, the mounting plate 21 is tilted by 45 degrees by the tilt mechanism, the pinch valve 41a is opened, and the plunger of the syringe pump 44 is pulled, so that the cultured cell group together with the calcium-containing solution and the like are drawn from the flow path 12 into the narrow tube 10. And the shear stress is applied between the cells by the flow rate difference between the central portion in the narrow tube 10 and the vicinity of the inner wall, and the adhesion between the cells can be released.

  Subsequently, the plunger of the syringe pump 44 is pushed, and the cultured cell group of the thin tube 10 is supplied to the culture container 1. Then, when the flow direction changes, a large shear stress is generated between the cultured cells, the cells are released from each other, and the cultured cell group can be efficiently dispersed in the fluid container.

Then, by again tilting the mounting plate 21 at 45 degrees and repeatedly circulating the cultured cells through the capillary tube 10, the adhesion between the cells can be gradually released.
For this reason, compared with the case where the cultured cell group is once circulated and dispersed in the tubules, the inner diameter of the tubules is increased, the length of the tubules is shortened, and the adhesion between the cells is released even if the circulation speed is reduced. At the same time, it can be dispersed in a calcium-containing solution or the like. As a result, the survival rate of cultured cells can be increased, and the cell recovery rate by subculture can be significantly increased.

  Next, the cultured cells can be simply diluted and dispersed in the medium by supplying the cultured cells to the syringe pump 44 and alternately operating the syringe pumps 44 and 45. For this reason, it is possible to supply the cultured cells evenly to the cell collection container 5, and as a result, it is possible to efficiently perform subculture.

  On the other hand, since the waste liquid collecting means 6 is constituted by a tube 61 or the like communicating with the flow path 13 and provided in parallel with the flow means 4, the cleaning liquid is cultured from the flow path 12 in a pressurized manner using the syringe 33 or the tube pump 33 a. By supplying it to the container, the stripping solution can be easily discharged from the flow path 13 to the collection bottle 62, and automation of the entire apparatus can be made realistic.

Further, the culture vessel 1, the reagent supply means 3, the flow means 4, the cell collection container 5 and the waste liquid collection means 6 are hermetically connected by the tubes 34, 37, 41, 43, 61 to constitute a closed cell culture apparatus. Therefore, it is possible to prevent contaminants from entering the system and contaminating bacteria.
Furthermore, the tubes 34, 37, 41, 43, 61, in which the devices such as the syringes 31, 32, 33 and the tube pumps 31 a, 32 a, 33 a in the reagent supply means 3, the distribution means 4 and the waste liquid collection means 6 are all disposable, Since they are connected by the pinch valves 41a, 43a and the pipe connectors 36, 38, 42, it is possible to prevent contamination from being mixed due to repeated use.

  Note that the present invention is not limited to the above-described embodiment. For example, the reagent supply unit 3 includes syringes 31, 32, 33, tubes 34, 37, tube pumps 31a, 32a, 33a, and pipe connectors. It may be constituted by other than 36. Moreover, the thin tube 10 does not need to be integrally provided on one side of the culture vessel 1, and only needs to be interposed in the distribution means 4.

It is a conceptual explanatory view showing one embodiment of the cell culture device of the present invention. It is explanatory drawing of the mounting base 2, (a) is a front view, (b) is a longitudinal cross-sectional view.

Explanation of symbols

1 culture vessel 2 mounting table (vibration device)
3 Reagent supply means 4 Distribution means 5 Cell collection container

Claims (17)

  A culture container in which the cultured cell group is housed, a reagent supply means connected to the culture container and supplying at least a peeling solution for reducing the cell adhesiveness of the cultured cell group to the culture container, and the above in the culture container A peeling means for causing a fluid container such as a peeling solution to flow in a plane direction and peeling the cultured cell group adhering to the inner wall surface of the culture vessel from the inner wall surface, and the cultured cell connected to the culture vessel A closed-system cell recovery device comprising: a group distribution means.   The closed cell recovery device according to claim 1, wherein the peeling means is a vibration device that vibrates the culture vessel in a horizontal direction.   The stripping means has an air supply mechanism for supplying air into the culture vessel, and a rocking mechanism for causing the culture vessel to perform a seesaw motion so that the fluid container flows in a plane direction. Item 2. The closed cell recovery apparatus according to Item 1. 2. The distribution means is provided with a thin tube for releasing adhesion between cells of the cultured cell group, and a suction mechanism for allowing the cultured cell group to pass through the thin tube. 4. The closed-system cell recovery device according to any one of items 1 to 3.   The closed cell recovery device according to claim 4, wherein the narrow tube has an inner diameter of 0.2 mm or more and 1.0 mm or less. A cell culture device for re-culturing the cultured cells using the closed system cell recovery device according to any one of claims 1 to 5,
Connected to the culture vessel in parallel with the flow means, waste liquid recovery means connected to the culture vessel in series with the reagent supply means, provided to be connectable to the downstream end of the flow means, A cell culture apparatus comprising a cell collection container for re-culturing the cultured cells.   The distribution means is provided with a dilution mechanism for diluting and dispersing the cultured cells in the medium, and a plurality of the cell recovery containers are provided, each of which can be connected in parallel to the downstream end of the distribution means. The cell culture device according to claim 6, which is provided.   The cell culture apparatus according to claim 7, wherein the culture container, the reagent supply means, the peeling means, the distribution means, the waste liquid recovery means, and the cell recovery container are accommodated in an incubator. In the culture vessel in which the cultured cell group is accommodated, a stripping solution that reduces the cell adhesion of the cultured cell group is supplied, and a reaction step of reacting the stripped solution and the cultured cell group;
After this reaction step, a cleaning solution is supplied into the culture vessel, and the peeling solution is washed away with the cultured cell group attached to the inner wall surface of the culture vessel, thereby replacing the peeling solution with the cleaning solution. A cleaning process;
A peeling step of peeling the cultured cell group from the inner wall surface of the culture container by causing the fluid container such as the washing liquid contained in the culture container to flow in the surface direction;
A method for recovering cells, characterized in that the cultured cell group separated from the inner wall surface is discharged to a distribution means connected to the discharge port of the culture container.   10. The method for recovering cells according to claim 9, wherein the stripping solution is a divalent metal chelating agent.   After the washing step, a calcium-containing solution such as a medium is supplied into the culture vessel, a part of the washing solution in the culture vessel is discharged to the outside, and a part of the washing solution in the culture vessel is taken out of the calcium A substitution step for substitution with a contained solution is provided, and fluid containing materials such as the calcium-containing solution and the washing solution in the culture vessel are caused to flow in a plane direction in the peeling step. 10. The method for recovering cells according to 10.   The cell collection method according to any one of claims 9 to 11, wherein in the peeling step, the culture vessel is vibrated in a horizontal direction.   12. In the peeling step, air is supplied into the culture vessel, and the culture vessel is caused to perform a seesaw motion so that the fluid container flows in a plane direction. The method for recovering cells according to Item.   After the detachment step, the cultured cell group separated from the inner wall surface of the culture vessel is circulated through the thin tube interposed in the distribution means, thereby releasing the adhesion of the cells of the cultured cell group, and the culturing. The cell collection method according to any one of claims 9 to 13, further comprising a cell dispersion step of dispersing the cells in the fluid container.   The method for recovering cells according to claim 14, wherein the cultured cells are dispersed in a fluid container by passing the cultured cell group through the capillary tube a plurality of times. A cell culture method for re-culturing a cultured cell using the cell recovery method according to claim 14 or 15,
After the cell dispersion step, a seeding step of supplying the cultured cells in which the cells of the cultured cell group are released to each other to a plurality of cell recovery containers;
A method for culturing cells, comprising the step of culturing the cultured cells in the cell collection container.   A dilution step for diluting and dispersing the cultured cells in the medium is provided after the cell dispersion step, and the diluted and dispersed cell solution is supplied to the cell collection container in the seeding step. The method for culturing cells according to claim 16.

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