WO2004027014A1

WO2004027014A1 - Cell dispersing apparatus, cell dispersion cassette and tissue retention part

Info

Publication number: WO2004027014A1
Application number: PCT/JP2003/011705
Authority: WO
Inventors: Ken-Ichiro Hata; Kunihiko Okada
Original assignee: Japan Tissue Engineering Co.,Ltd
Priority date: 2002-09-17
Filing date: 2003-09-12
Publication date: 2004-04-01
Also published as: AU2003262095A1; JPWO2004027014A1; JP4520855B2

Abstract

Control unit (60) charges a trypsin solution through supply pipe (40) into liquid pooling part (20) so as to impregnate the living tissue retained in tissue retention part (12) with the trypsin solution, and rotates the liquid pooling part (20) at low speed by means of motor (32). As a result, cells are slowly detached from the epidermal tissue by the action of solution stream and trypsin. Subsequently, a dispersion terminating liquid is charged through the supply pipe (40) into the liquid pooling part (20) to thereby terminate the action of trypsin, and the liquid pooling part (20) is rotated at high speed by means of the motor (32). As a result, violent stream occurs at the trypsin solution pooled in the liquid pooling part (20), so that the cells are exposed to outward centrifugal force due to the stream and thus accumulated in cell accumulation part (26). Thereafter, the solution pooled in the liquid pooling part (20) is discharged outside through discharge pipe (50). At that time, as the cells are accumulated in the cell accumulation part (26), only waste liquid is discharged.

Description

Description ^: Cell dispersion apparatus, cell dispersion cassette and tissue holding member

The present invention relates to a cell dispersion device for separating and dispersing cells from a living tissue, a cell dispersion cassette and a tissue holding member used for the device. Background art

In recent years, cell culture technology has been used in various fields, such as culturing cells in vitro (in vitro), administering the cultured cells to patients for treatment, using them for toxicity testing of pharmaceuticals, etc. It is becoming. Although such cell culture techniques have been used for a long time, most of the processes are manual operations by hand, and the burden on workers is large, and automation is desired. However, establishing such automation technology requires not only engineering knowledge, but also cytology, biological knowledge, and even medical knowledge. It has not been established. On the other hand, in the cell dispersion step, which is the initial step of cell culture, the tissue collected from the patient or donor is placed in a liquid containing enzymes such as dispase ゃ trypsin, and then separated and dispersed in a cell state by pipetting. An enzyme treatment is performed to recover the cell suspension of the supernatant. However, since these enzymes are susceptible to cell damage, the enzyme treatment must be repeated for a relatively short period of time and the cells dispersed by the enzyme treatment must be recovered little by little, placing a heavy burden on workers. . Also, the more manual operations performed by such operators, the higher the possibility of contamination, which is not preferable. Japanese Patent Application Laid-Open No. 2000-161614 discloses an apparatus in which the cell dispersion step is automated, but this apparatus automates a method for mechanically separating cells. However, the method for separating cells using an enzyme such as dispase-trypsin as a dispersant is not automated. For general methods of mechanical or enzymatic cell separation, see, for example, the well-known literature (Yutaka Matsutani, “Introduction to Animal Cell Culture Methods, 29, Experimental Methods for Biological Chemistry”; Please refer to pp. 81-88, 1959, February 25, p.

An object of the present invention is to provide a cell dispersing device that can easily perform a process of separating and dispersing cells from a living tissue using a dispersant. Another object is to provide a cell dispersion device that can reduce the possibility of contamination. Further, another object of the present invention is to provide a cell dispersion cassette / tissue holding member suitable for such a cell dispersion device.

In order to solve at least one of the above-mentioned objects, the present inventors present the following cell dispersion device and cell dispersion cassette.

A first aspect of the present invention is a cell dispersion device for separating and dispersing cells from living tissue,

A tissue holding unit that has a plurality of cell passage holes sized to allow the cells to pass through at least in part, and that can hold the living tissue;

A liquid storage unit configured to surround the tissue holding unit and capable of storing an amount of the dispersant-containing liquid in which the living tissue held by the tissue holding unit is immersed;

The dispersant-containing liquid flows through the cell holding hole of the tissue holding unit so that cells separated from the living tissue pass through the cell passage hole of the tissue holding unit to the outside of the tissue holding unit by the action of the dispersant-containing liquid stored in the liquid storage unit. Water flow generation part to generate liquid When

It is provided with.

In this cell dispersion device, a living tissue is held in a tissue holding section, a liquid containing a dispersant is stored in a liquid storage section, and the living tissue is immersed in the liquid containing the dispersant. A water flow is generated in the liquid containing the dispersant such that cells separated from the tissue pass outward from the cell passage hole of the tissue holding unit. In other words, this cell dispersion device substitutes for a manual operation of adding and dispersing a liquid containing a dispersant to living tissue to separate and disperse cells from the living tissue. Therefore, according to the cell dispersing device, the process of separating and dispersing cells from the living tissue using the dispersant can be easily performed.

The water flow generating unit may generate a water flow that allows cells separated from the living tissue to pass through the cell passage hole of the tissue holding unit to the outside of the tissue holding unit. It may be generated by rotating or shaking the tissue holding unit holding the liquid, or may be generated by rotating or shaking the liquid storage unit storing the liquid. The tissue holding unit may be, for example, a bottomed cylindrical member having a filter surface that allows cells to pass through but does not pass through biological tissue, or a bag-like member having a similar filter surface. . These may be fixed to the liquid storage part, or may be fixed to a member independent of the liquid storage part. In addition, it may be fixed detachably or may be fixed so that it is difficult to attach and detach. Furthermore, it may be fixed with an adhesive, a fastener, or the like, or may be suspended in the liquid storage unit by a string or the like.

The living tissue in the present invention is a tissue piece composed of one or more types of adhesion-dependent cells, and may be any tissue piece that can be dispersed in cells by an enzyme treatment. In particular, epidermis, epithelium, dermis, cartilage, bone, Tissue pieces composed of adhesion-dependent cells such as periosteum, blood vessels, liver, and kidney can be mentioned as preferable subjects. Tissue excerpts should be obtained from humans or from warm-blooded animals such as mice, rats, guinea pigs, hamsters, chickens, egrets, pigs, sheep, pigs, pomas, dogs, cats, monkeys, etc. it can. In addition, the cells constituting the tissue piece include spleen cells, nerve cells, knees] 3 cells, Langerhans cells, epidermal cells, epithelial cells (including skin epithelium, oral mucosal epithelium, corneal epithelium, amniotic epithelium, etc.), endothelial cells , Fibroblasts, myoblasts, adipocytes, synovial cells, chondrocytes, osteoblasts, mammary cells, liver cells, periosteum-derived cells, or stromal cells, or precursors of these cells, as well as embryos Examples include stem cells such as sex stem cells and mesenchymal stem cells and adhesion-dependent cancer cells.

The cell dispersion device of the present invention may include a control unit that controls the operation of the water flow generation unit based on a predetermined set value. This makes it possible to automate the process of separating and dispersing the cells from the living tissue, thereby reducing the burden on the operation. Here, the “set value” is a value of a parameter relating to the water flow, and includes, for example, the momentum of the water flow and the time for generating the water flow.

The cell dispersion device according to the present invention may further include a dispersant-containing liquid supply unit configured to supply the dispersant-containing liquid to the liquid storage unit, and a dispersant-containing liquid after acting on the living tissue from the liquid storage unit. And a suitable dispersant-containing liquid discharge section. In this case, the operation of supplying and discharging the dispersant-containing liquid to and from the liquid storage section can be easily performed.

As described above, the cell dispersion device of the present invention including the dispersant-containing liquid supply unit and the dispersant-containing liquid discharge unit operates the dispersant-containing liquid supply unit to store the dispersant-containing liquid in the liquid storage unit. Then, the operation of the water flow generation unit is controlled based on a predetermined set value, and then the dispersant-containing liquid discharge is performed. A control unit may be provided for operating the outlet unit to discharge the liquid containing the dispersant after acting on the living tissue from the liquid storage unit. This makes it possible to automate the process of supplying the dispersant-containing liquid to the liquid reservoir, the process of separating cells from living tissue, and the process of discharging the dispersant-containing liquid to the liquid reservoir. It is reduced.

In addition, the cell dispersion device of the present invention including a control unit includes a dispersion stop liquid supply unit that can supply a dispersion stop liquid that stops the action of the dispersant to the liquid storage unit. The control unit includes the water flow In controlling the operation of the generating unit, the dispersant-containing water flow that controls the operation of the water flow generating unit based on a predetermined set value to assist in separating cells from the biological tissue is included. The dispersion stop liquid supply unit is operated to supply a predetermined amount of the dispersion stop liquid to the liquid storage unit, and then the operation of the water flow generation unit is controlled based on a predetermined set value. Then, a water flow may be generated in the dispersant-containing liquid such that cells separated from the living tissue pass outward from the cell passage hole of the tissue holding unit. In this way, the cell flow is controlled so that the cells can be easily separated from the living tissue (for example, a gentle water flow) before the suspension liquid is injected. It is possible to control the water flow so that it can easily pass through the passage hole (for example, a steep water flow).

In addition, the cell dispersion device of the present invention including a control unit includes a cleaning liquid supply unit that can supply a cleaning liquid for cleaning the living tissue to the liquid storage unit, and a cleaning liquid supply unit that cleans the living tissue from the liquid storage unit. A cleaning liquid discharge unit capable of discharging a cleaning liquid, wherein the control unit activates the cleaning liquid supply unit to supply a predetermined amount of the cleaning liquid to the liquid storage unit before performing the process using the dispersant-containing liquid. After that, the operation of the water flow generation unit is controlled based on a predetermined set value. A washing step of draining the washing solution after washing the living tissue may be performed. In this way, the cleaning process can be automated and the burden on the operator can be reduced.

In addition, the cell dispersion device of the present invention including a control unit includes: a suspension liquid supply unit capable of supplying a suspension liquid for suspending cells separated from the living tissue to the liquid storage unit; A cell suspension discharge unit capable of discharging a cell suspension from a storage unit, wherein the control unit activates the suspension liquid supply unit after the treatment using the dispersant-containing liquid. A predetermined amount of the suspension liquid is supplied to the liquid storage unit, and then the operation of the water flow generation unit is controlled based on a predetermined set value to convert the cells in the liquid storage unit to the suspension liquid. The suspension may be suspended to form a cell suspension, and then the cell suspension discharge section may be operated to execute the cell collection step of discharging the cell suspension from the liquid storage section. In this way, the cell collection process can be automated, and the burden on the operator is reduced.

Further, in the cell dispersion device of the present invention provided with a control unit, the control unit controls the operation of the water flow generation unit such that the forward and reverse directions of the water flow generated in the dispersant-containing liquid are switched every predetermined time. May be. In this case, the cells can be efficiently separated from the living tissue as compared with the water flow in only one direction, and can be effectively passed from the cell passage hole of the tissue holding portion to the outside of the tissue holding portion. Here, the direction of the water flow is switched every time a predetermined time elapses. The predetermined time at this time may be set as appropriate according to the momentum of the water flow. For example, if the water flow is a vortex, 50 to 50 At 0 rpm, it is preferably set in the range of 0.2 to 2 seconds.

In the cell dispersion device according to the aspect of the invention, the liquid storage unit may be configured to move from the cell passage hole of the tissue holding unit to the outside of the tissue holding unit when the water flow generating unit generates the water flow in the dispersant-containing liquid. A cell that accumulates cells that have passed It may have a cell accumulation part. In this case, the cells separated from the living tissue can be easily collected. Here, the cell passage hole is provided in at least a part of a side surface of the tissue holding unit, and the cell accumulation unit is configured to generate the water flow when the water flow generation unit generates the water flow in the dispersant-containing liquid. Centrifugal force may act on the cells separated from the living tissue, and the centrifugal force may be used to accumulate the cells that have passed through the cell passage holes of the tissue holding unit. This makes it possible to easily recover the cells used from the living tissue by using the centrifugal force. At this time, the cell accumulating section may be formed into a sharp shape along the direction of the centrifugal force of the liquid storage section. In this way, the cells accumulated in the cell accumulating portion are likely to remain in the cell accumulating portion even after the centrifugal force stops working.

In the cell dispersion device of the present invention, at least the tissue holding unit and the liquid storage unit may be detachably attached to the device main body. In this case, the tissue holding section and the liquid storing section can be removed from the main body of the apparatus and sterilized or disposable.

In the cell dispersion device of the present invention, at least the tissue holding unit and the liquid storage unit may be arranged in a closed space. In this way, the possibility of contamination of the tissue holding unit and the liquid storage unit is reduced. At this time, the sealed space may be filled with a sterilizing gas. In this way, the enclosed space can be sterilized, and the possibility of contamination of the tissue holding unit and the liquid storage unit is further reduced.

In the cell dispersion device of the present invention, the water flow generation unit generates a vortex in the dispersant-containing liquid, and the tissue holding unit is disposed at a position eccentric from a rotation center of the vortex. Is also good. With this configuration, compared to the case where the tissue holding unit is arranged at the center of rotation of the vortex, cells separated from the living tissue can be effectively directed to the outside of the tissue holding unit from the cell passage hole of the tissue holding unit. Can be passed.

In the cell dispersion device of the present invention, the cell passage hole is provided on at least a part of a side surface of the tissue holding unit, and the water flow generating unit rotates the liquid storage unit or the tissue holding unit. The liquid storage unit may be integrated with the tissue holding unit or provided independently. When the liquid storage unit is integrated with the tissue holding unit, both rotate integrally, and when the liquid storage unit is independent of the tissue holding unit, the liquid storage unit or the tissue holding unit is rotated. Only rotate.

A second aspect of the present invention is a cell dispersing cassette in which the tissue holding unit and the liquid storage unit in the cell dispersing device described above are integrated and detachable from the cell dispersing device. By using the cell dispersion cassette, the tissue holding unit and the liquid storage unit can be easily sterilized or disposable.

According to a third aspect of the present invention, in the above-described cell dispersion device, a bottomed cylindrical shape is formed to function as the tissue holding unit, and a discharge pipe capable of discharging liquid from the liquid storage unit penetrates the bottom. It is a tissue holding member. By using this tissue holding member, the cell dispersion device can be easily assembled. The discharge pipe functions as the dispersant-containing liquid discharge section, the cleaning liquid discharge section, and the cell suspension discharge section described above.

A fourth aspect of the present invention is a cell dispersion device for separating and dispersing cells from living tissue,

A liquid storage unit capable of storing an amount of the dispersant-containing liquid in which the living tissue is immersed; a water flow generation unit configured to generate a water flow in the dispersant-containing liquid by rotating the liquid storage unit;

A control unit that controls the operation of the water flow generation unit so that the forward and reverse directions of the water flow generated in the dispersant-containing liquid are switched every time a predetermined time elapses; It is provided with.

In this cell dispersion device, after a liquid containing a dispersant is stored in a liquid storage part and a living tissue is immersed in the liquid containing the dispersant, a predetermined time elapses in the forward and reverse directions of the water flow generated in the liquid containing the dispersant. Switch every time. In other words, this cell dispersion device substitutes for a manual operation of adding and adding a dispersant-containing liquid to living tissue to separate and disperse cells from the living tissue. Therefore, according to the cell dispersion device, the process of separating and dispersing cells from the living tissue using the dispersant can be easily performed.

The cell dispersing device may be configured such that, when the dispersant-containing liquid that has acted on the living tissue is discharged from the liquid reservoir, the cells dispersed in the dispersant-containing liquid pass through, but the living tissue does not pass. And a discharge unit capable of discharging the dispersant-containing liquid via the liquid. In this case, the cells can be collected separately from the living tissue. In addition, the discharge section may discharge the dispersant-containing liquid after adding the dispersion-stopping liquid to the dispersant-containing liquid. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic explanatory view of a cell dispersing apparatus according to an embodiment, FIG. 2 is a flowchart of a cell dispersing process, and FIGS. 3 to 5 are explanatory views of main parts of a cell dispersing apparatus according to another embodiment. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, a preferred embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a schematic explanatory view of the cell dispersion device of the present embodiment. FIG. 1 shows a cross section of the tissue holding unit 12, the liquid storage unit 20, and the cradle 34. The cell dispersing device 10 is a cylindrical tissue holding unit 1 with a bottom capable of holding a living tissue. 2, a liquid storage unit 20 configured to surround the tissue holding unit 12, a water flow generation unit 30 for rotating the liquid storage unit 20, and the respective units 12, 20, 30 sealed. A storage container 36 that can store the liquid, a supply pipe 40 that supplies various liquids from outside the storage container to the liquid storage unit 20, and a discharge that discharges the liquid stored in the liquid storage unit 20 to the outside of the storage container. A pipe 50 and a control unit 60 that controls the operation of the water flow generation unit 30 and the supply and discharge of liquid to and from the liquid storage unit 20 are provided. In the present embodiment, the size of the storage container 36 is Is a rectangular parallelepiped with a base of approximately 25 cm and a height of 40 cm.

The tissue holding portion 12 is formed in a cylindrical shape with a bottom using a mesh member such as nylon, stainless steel, steel, or the like. The mesh member allows the cells to be dispersed and the living tissue to pass through. It has an unusually large hole diameter. Therefore, the tissue holding part 12 has a large number of cell passage holes 14 on the side and bottom surfaces, and the inside and the outside of the tissue holding part 12 communicate with each other through the cell passage holes 14. ing. A vertically extending portion of the discharge tube 50 is fixed to the bottom surface of the tissue holding portion 12 so as to penetrate therethrough. The tissue holding portion 12 is fixed by the discharge tube 50 to the bottom surface of the liquid storage portion 20. It is supported in a floating state.

The liquid storage section 20 is a container having a shape such that the openings of two centrifuge tubes are joined to face each other, in other words, a substantially spindle-shaped container that is flat in the horizontal direction. The liquid storage section 20 has an opening 22 in the upper center, and the tissue holding section 12 is arranged inside the liquid storage section 20 from the opening 22. Further, a plurality of convex fins 24 are provided on the bottom surface of the liquid storage section 20 from the center outward. Further, a cell accumulating portion 26 having a shape that is sharpened outward is provided on the periphery of the liquid storing portion 20.

The water flow generating section 30 includes a motor 32 and a cradle 34 fixed to a rotating shaft 32 a of the motor 32. The cradle 3 4 is made of thick and elastic resin It is formed in a shape that can wrap up more than half of the liquid storage part 20, and is wrapped around the periphery of the liquid storage part 20 by using the resin properties of the cradle 34. The table 34 securely holds the liquid storage section 20. When the rotation shaft 32a of the motor 32 rotates while the liquid storage unit 20 is held on the receiving table 34, the liquid storage unit 20 rotates with the receiving table 34 accordingly. The tissue holding section 12 does not rotate because it is provided independently of the liquid storage section 20. In addition, in order to remove the liquid storage section 20 held by the cradle 34, it is sufficient to remove the cradle 34 while pushing and spreading it using resin elasticity.

The storage container 36 is a container having a sealable opening / closing lid (not shown) on the upper part, and a sterile gas supply having a supply pipe 40, a discharge pipe 50, and an opening / closing valve 37a on a side wall. A sterilizing gas discharge section 38 having a section 37 and an on-off valve 38a is penetrated while maintaining a sealed state.

The supply pipe 40 supplies various liquids arranged outside the storage container to the liquid storage unit 20. Specifically, the first to fourth pinch valves 41, 42, which are small solenoid valves, are provided. Washing solution, phosphate buffer solution (PBS) with antibiotics, and separation and dispersion of cells by switching between 4, 3 and 4 Stop trypsin solution containing trypsin as a dispersing agent and trypsin An appropriate solution is selected from a dispersion stop solution containing a trypsin inhibitor and a liquid medium such as Dulbecco's modified Eagle's medium (DMEM), and supplied to the liquid reservoir 20 by a pump 45. The supply pipe 40 is fixed by a clamp 55 fixed inside the storage container 36. In addition, examples of the dispersion stop solution include a serum-containing medium. At this time, the serum becomes trypsin inhibitory.

The discharge pipe 50 discharges the liquid stored in the liquid storage section 20 out of the container. Specifically, the discharge pinch valve 5 which is a small electromagnetic valve is used. The washing liquid, the trypsin solution, the dispersion stop solution, and the cell suspension after the dispersion treatment in the liquid storage unit 20 are discharged by opening and closing the 2 and driving the pump 54. The discharge pipe 50 penetrates the bottom surface of the tissue holding portion 12 and is fixed to the tissue holding portion 12 at the penetrating portion, and is fixed by a clamp 56 fixed inside the storage container 36. Have been. In addition, the suction port of the discharge pipe 50 is located below the bottom surface of the tissue holding unit 12 and slightly floating from the bottom surface of the liquid storage unit 20.

The control unit 60 reads the program stored in the storage unit 62 in a timely manner, measures the time with the built-in timer 64 based on the program, and is attached to the supply pipe 40 and the discharge pipe 50 according to the timing. It controls the operation of the pumps 45, 54 and the pinch valves 41 to 44, 52 and controls the drive of the motor 32 of the water flow generator 30. Further, the control unit 60 stores various set values input by the operator via the input operation unit 66 in the storage unit 62, and executes a program based on the set values.

Next, a cell dispersion process using the cell dispersion device 10 of the present embodiment will be described. Here, a human epidermal tissue is taken as an example of a living tissue, and an example in which human epidermal cells (keratinocytes) are separated and dispersed will be described.

First, the operator cuts the collected epidermal tissue into small pieces that are suitable for cell dispersion. Usually, cells are cut as finely as possible so that cells can be collected more efficiently. Then, the minced epidermal tissue is put into the tissue holding unit 12 of the cell dispersion device 10. Although the epidermal tissue in the tissue holding portion 12 is mince-shaped, it is larger than the cell passage hole 14 of the tissue holding portion 12, and is retained by the tissue holding portion 12 without passing through the cell passage hole 14. You. Subsequently, the operator inputs various setting values via the input operation unit 66 of the control unit 60. Here, the step of washing the epidermal tissue with the washing solution, the details of separating and dispersing cells from the epidermal tissue In order to perform the cell dispersion step and the cell collection step of collecting the dispersed cells, set the liquid volume, motor rotation speed, motor rotation time, and motor rotation direction in each step.

Next, the operator operates the input operation unit 66 of the control unit 60 to instruct the start of the cell dispersion processing program. Then, the control unit 60 reads out the cell dispersion processing program from the storage unit 62 and executes the program. FIG. 2 is a flowchart of the cell dispersion processing. When this program is started, the controller 60 opens the first pinch valve 41, closes the second to fourth pinch valves 42 to 44, and then drives the pump 45 to supply the supply pipe 40. The cleaning liquid is supplied to the liquid storage unit 20 through the pump. When the input amount of the cleaning liquid reaches a preset value while calculating the input amount of the cleaning liquid based on the discharge amount and the discharge time of the pump 45, the pump 45 Is stopped and the first pinch valve 41 is closed (step S100). The set value of the amount of the cleaning solution is determined so that the epidermal tissue held in the tissue holding section 12 is completely immersed.

Subsequently, the control unit 60 rotates the motor 32 of the water flow generation unit 30 by a predetermined rotation speed and rotation time (step S110). As a result, the liquid storage unit 20 integrally supported by the cradle 34 rotates, and a water flow (swirl) is generated in the washing liquid to wash the epidermis in the tissue holding unit 12. The water flow is effectively generated by the fins 24 provided on the bottom surface of the liquid storage section 20. At this time, the motor rotation speed is set to 50 to 200 rpm, and the motor rotation time is set to 5 to 30 minutes. The motor rotation speed and the motor rotation time are set to appropriate values according to the site from which the epidermal tissue was collected, the size of the epidermal tissue held in the tissue holding unit 12, and the like. Further, the motor may be rotated only in the forward direction, but it is preferable to alternately perform the forward and reverse rotations in consideration of the cleaning effect. In addition, turn the motor 3 2 When stopping the rotation, the rotation may be stopped suddenly, or the rotation speed may be gradually reduced before stopping.

Subsequently, the control unit 60 opens the discharge pinch valve 52 and drives the pump 54 to discharge the cleaning liquid stored in the liquid storage unit 20 to the outside through the discharge pipe 50 (Step S 1 2 0). In the process of step S110, the cells are not separated and dispersed from the epidermal tissue, so that the discharged washing solution contains almost all unnecessary substances other than the cells. Note that the end point of the cleaning liquid discharge may be, for example, a point in time when a predetermined liquid amount is discharged based on the discharge amount and discharge time of the pump 54, or that the pump 54 starts to suck gas. May be detected. The above steps S100 to S120 are the cleaning process.

Next, the control section 60 opens the second pinch valve 42 and closes the first, third and fourth pinch valves 41, 43, and 44, and then drives the pump 45 to supply the supply pipe. The trypsin solution was injected into the liquid storage unit 20 through 40, and the input amount reached the preset value while calculating the input amount of the trypsin solution based on the discharge amount and the discharge time of the pump 45. At this time, the pump 45 is stopped, and the second pinch pulp 42 is closed (step S130). The set value of the amount of the trypsin solution is determined such that the epidermal tissue held in the tissue holding unit 12 is completely immersed and that the dispersion stopping liquid can be added later.

Subsequently, the control unit 60 rotates the motor 32 of the water flow generation unit 30 for a predetermined low rotation speed and rotation time (step S140). As a result, the liquid storage unit 20 integrally supported by the receiving table 34 rotates. At this time, the motor rotation speed is set to 50 to 500 rpm, and the motor rotation time is set to 1 to 30 minutes. By this low-speed rotation operation, a gentle water flow is generated in the trypsin solution stored in the liquid storage unit 20, and the The cells are gradually separated from the epidermal tissue by the action of the water flow and trypsin. This water flow is effectively generated by the fins 24 provided on the bottom surface of the liquid storage section 20. The cells separated in this manner float in the solution, and a part of the cells pass from the inside of the tissue holding unit 12 through the cell passage hole 14 to the outside of the tissue holding unit 12 by a water flow or the like. But the rest stays inside the tissue holding unit 12. The motor rotation speed and the motor rotation time are determined according to the area where the epidermal tissue was collected and the size of the epidermal tissue placed in the tissue holding part 12, etc. Is set to occur. Although the motor may be rotated only in the forward direction, it is preferable to alternately rotate in the forward and reverse directions. For example, the forward / reverse direction of the water flow generated in the trypsin solution may be switched by switching the forward / reverse rotation in the rotation direction at an interval of 0.2 to 2 seconds. Further, when stopping the rotation of the motor 32, the rotation may be stopped suddenly, or the rotation speed may be gradually reduced before stopping.

Subsequently, the control section 60 opens the third pinch valve 43 and closes the first, second and fourth pinch valves 41, 42, 44, and then drives the pump 45 to supply the supply pipe 4. The dispersion stop liquid is injected into the liquid storage unit 20 through 0, and the injection amount reaches the preset value while calculating the injection amount of the dispersion stop liquid based on the discharge amount and the discharge time of the pump 45. Then, the pump 45 is stopped and the third pinch valve 43 is closed (step S150). This stops the action of trypsin, so that the separated cells are not likely to be subsequently damaged by trypsin. The set value of the liquid amount of the dispersion stopping liquid is set to an amount sufficient to stop the action of trypsin and to such an extent that the dispersion stop liquid does not overflow from the liquid reservoir 20.

Subsequently, the control unit 60 rotates the motor 32 of the water flow generation unit 30 for a predetermined high rotation speed and rotation time (Step S160). This Thereby, the liquid storage part 20 integrally supported by the receiving base 34 rotates. At this time, the rotation speed is set to 100 to 200 rpm, and the rotation time is set to 1 to 10 minutes. In addition, the motor rotation direction is only one direction of forward rotation. By this high-speed rotation operation, a vigorous water flow is generated in the trypsin solution stored in the liquid storage unit 20, and this water flow is effectively generated by the fins 24 provided on the bottom surface of the liquid storage unit 20. . Of the cells separated from the epidermal tissue, those remaining inside the tissue holding part 12 are subjected to an outward centrifugal force from the center by the water flow, and the tissue holding part 12 is passed through the cell passage hole 14. After being dispersed outside the cell, it accumulates in the cell accumulator 26 of the liquid reservoir 20, and the cells separated from the epidermal tissue that have come out of the tissue holder 12 The water flows receive centrifugal force outward from the center and accumulates in the cell accumulator 26 of the liquid reservoir 20. Since the cell accumulating portion 26 is formed in a sharp shape along the direction of the centrifugal force, the cells subjected to the centrifugal force easily accumulate. After this high-speed rotation operation, a tissue mass larger than the cell passage hole 14 remains inside the tissue holding portion 12. The rotation speed and the rotation time are set to values that allow cells separated from the epidermal tissue to be sufficiently accumulated in the cell accumulation unit 26. In addition, when stopping the rotation of the motor 32, it is preferable to gradually reduce the rotation speed and then stop because of high-speed rotation.

Subsequently, the control unit 60 opens the discharge pinch valve 52 and drives the pump 54 to discharge the solution stored in the liquid storage unit 20 to the outside through the discharge pipe 50 (Step S). 170). At this time, since the cells are accumulated in the cell accumulation section 26, only the waste liquid is discharged outside. The end point of the waste liquid discharge may be, for example, a time point when a predetermined liquid amount is discharged based on the discharge amount and the discharge time of the pump 54, as in the case of the cleaning liquid, or the pump 54 suctions gas. It may be a point in time when it is detected that it has begun. Less than The above steps S130 to S170 are the cell dispersion process.

Next, the control section 60 opens the fourth pinch valve 44 and closes the first to third pinch valves 41 to 43, then drives the pump 45 to drive the liquid storage section 2 through the supply pipe 40. Inject 0% FBS-containing DMEM as medium into 0, calculate the medium injection amount based on the discharge amount and discharge time of the pump 45, and when the input amount reaches the preset value set in advance, the pump 4 5 is stopped and the fourth pinch pulp 44 is closed (step S 180). The set value of the volume of the medium is determined to be almost the same as that of the washing solution.

Subsequently, the control unit 60 rotates the motor 32 of the water flow generation unit 30 by a predetermined rotation speed and rotation time (Step S190). As a result, the liquid storage unit 20 integrally supported by the cradle 34 rotates, and a water flow is generated in the culture medium stored in the liquid storage unit 20 and accumulated in the cell accumulation unit 26. Cells are suspended in the medium. At this time, since the purpose is to suspend the cells accumulated in the cell accumulating unit 26 in the medium, the cells are also rotated at a low speed during the high-speed rotation operation in step S160 in the cell dispersion process. Preferably, the rotation speed is set to 50 to 200 rpm, and the rotation is preferably alternately performed in the forward and reverse directions.

Subsequently, the control section 60 opens the discharge pinch valve 52 and drives the pump 54 to discharge the solution stored in the liquid storage section 20 to the outside through the discharge pipe 50 (Step S). 200). The discharged liquid at this time is recovered as a cell suspension in a recovery container (not shown) provided at the outlet of the discharge pipe 50. The end point of the recovery of the cell suspension may be, for example, the time when a predetermined amount of liquid is discharged based on the discharge amount and the discharge time of the pump 54, as in the case of the washing liquid. It may be the time when the start of gas suction is detected. The above steps S180 to S200 are the cell collection process, and the program ends with the processing of step S200. You. However, the program may be terminated after executing steps S130 to S200 several times.

After the cell dispersion processing is completed in this manner, the operator opens the opening / closing lid (not shown) of the storage container 36, loosens the clamp 55, removes the supply pipe 40, and loosens the clamp 56 to discharge the discharge pipe 5. Remove the tissue holding unit 12 together with 0, and remove the liquid storage unit 20 from the cradle 34 of the water flow generation unit 30. Washing and sterilizing the supply pipe 40, discharge pipe 50, tissue holding section 12 and liquid storage section 20 removed in this way enables the next use and suppresses the contamination. be able to. In addition, if disposable (disposable) is used without washing / sterilization and new products are used next time, contamination can be surely suppressed. Further, the container 36 is closed, and the on-off valve 38a is closed, the on-off valve 37a is opened, the sterilizing gas is supplied from the sterilizing gas supply section 37, the on-off valve 37a is closed, and then the on-off valve 37a is closed. Sterilization can be performed by opening the valve 38a and discharging the sterilizing gas from the sterilizing gas discharge section 38, so that the contamination can be suppressed in this respect as well.

Now, the obtained cell suspension is transferred to the seeding step and cultured after adjusting the cell density or as it is without adjusting the cell density. This seeding step may be performed manually by an operator, but it can be performed by connecting it to an automated cell culture device (for example, refer to Japanese Patent Application Laid-Open No. 2001-2756559) and culturing it. Intervention may be effectively prevented by minimizing intervention. In addition, microcarrier culture is performed by introducing scaffolding materials such as microphone mouth beads into the liquid reservoir after the cell dispersion step and culturing while rotating the motor at a low speed, instead of collecting as a cell suspension. Can be. By doing so, the steps from cell dispersion to culturing can be performed only by the present apparatus. In the cell dispersing device 10 of the present embodiment described in detail above, the tissue holding unit 12 holds the minced epidermal tissue, and the liquid storage unit 20 stores the dispersant-containing liquid. After immersion, a high-speed water flow that allows cells separated from the epidermal tissue to pass outward through the cell passage hole 14 of the tissue holding part 12 by the action of the dispersant-containing liquid or the low-speed water flow It is generated in the liquid containing the agent. That is, the cell dispersing device 10 performs a manual operation of adding the dispersant-containing solution to the epidermal tissue and separating and dispersing the cells from the epidermal tissue by an operator by pipetting. Therefore, according to the cell dispersion device 10, the process of separating and dispersing the cells from the epidermal tissue using the dispersant can be easily performed.

In addition, the cell dispersion device 10 includes a liquid supply unit including first to fourth pinch valves 41 to 44, a pump 45, and a supply pipe 40 capable of supplying a trypsin solution or the like, and trypsin acts on epidermal tissue. It has a liquid discharge section consisting of a discharge pinch valve 52, a pump 54, and a discharge pipe 50 that can discharge waste liquid etc. after cleaning, so that various liquids such as trypsin solution are supplied to the liquid storage section 20. The discharging operation can be easily performed. In addition, the control unit 60 operates the first to fourth pinch valves 41 to 44 and the pump 45 to supply a predetermined amount of various liquids to the liquid storage unit 20, and then sets the predetermined set value. To control the motor 32 of the water flow generation unit 30 based on this, and then to operate the discharge pinch valve 52 and the pump 54 to discharge the waste liquid and cell suspension from the liquid storage unit 20. Since various processes can be automated, the burden on the operator is reduced.

Furthermore, since the tissue holding section 12 employs a bottomed cylindrical body formed of a mesh member, a configuration having a plurality of cell passage holes 14 on the side and bottom surfaces can be easily realized.

Furthermore, the cells separated from the epidermal tissue are subjected to centrifugal force and subjected to liquid storage. Since the cells accumulate in the cell accumulating unit 26 provided in the cell 0, they can be easily collected. Since the cell accumulation part 26 is formed in a sharp shape along the direction of the centrifugal force in the liquid storage part 20, the cells accumulated in the cell accumulation part 26 stop after the centrifugal force stops working. Also tend to stay in the cell accumulation section 26. Further, in the cell dispersion device of the present invention, a fin 24 extending in a direction intersecting the direction of the generated water flow is provided on the bottom surface of the liquid storage portion 20. This effectively generates a water flow that allows cells separated from the tissue holding portion 12 to pass through the cell passage hole 14 of the tissue holding portion 12 to the outside of the tissue holding portion 12.

Further, since the tissue holding section 12 and the liquid storage section 20 are detachably attached to the apparatus main body, the tissue holding section 12 and the liquid storage section 2 are removed from the apparatus main body after the cell dispersion processing is completed. It can be removed and sterilized or disposable, and the potential for contamination is low. Also, the containment vessel 36 can be sterilized, further reducing the possibility of contamination. In addition, in FIG. 2, if steps S130 to S200 are repeatedly performed to collect cells sequentially, cells can be recovered at a high recovery rate while suppressing cell damage. .

It should be noted that the present invention is not limited to the above-described embodiment at all, and it is needless to say that the present invention can be implemented in various forms without departing from the technical scope of the present invention.

For example, in the above-described embodiment, the tissue holding unit 12 and the liquid storage unit 20 are provided independently. However, as shown in FIG. 3, the upper periphery of the tissue holding unit 12 and the liquid storage unit 20 are separated. The cell dispersing cassette 70 in which the openings 22 are bonded to each other with an adhesive or the like and the both are integrated may be used. At this time, as shown in FIG. 3 (a), the discharge pipe 50 may be fixed so as to penetrate the bottom surface of the tissue holding portion 12. In that case, a bearing is used instead of the clamp 56. Connected connector Evening 57 is arranged, and discharge pipe 50 is divided into movable part 50a (tissue holding part 12 side) and stationary part 50b, and movable part 50a and stationary part 5 are connected by connector 57. 0 b and may be connected. In this way, when the liquid storage section 20 rotates together with the tissue holding section 12 by rotating the motor 32, the movable section 50a of the discharge pipe 50 fixed to the tissue holding section 12 also rotates. However, since the connector 57 has a bearing function, the connector 57 can rotate independently of the stationary portion 50b of the discharge pipe 50. Alternatively, as shown in FIG. 3 (b), the discharge tube 50 is configured not to be fixed while penetrating the bottom surface of the tissue holding portion 12, and the tissue holding portion 12 can rotate around the discharge tube 50. In that case, the discharge pipe 50 may be fixed by the clamp 56. Whichever configuration is employed, the same operation and effect as those of the above-described embodiment can be obtained. In addition, since the cell dispersion cassette 70 in which the tissue holding unit 12 and the liquid storage unit 20 are integrated is used, the assembly and sterilization of the cell dispersion device 10 are compared with the case where both are independent. Work load is reduced. The cell dispersion cassette 70 may be disposable.

In the above-described embodiment, the supply pipe 40 is described as a single supply pipe. However, a supply pipe may be provided for each type of liquid supplied to the liquid storage unit 20, and a pump and a pinch valve may be provided for each supply pipe. Good. In addition, although the single discharge pipe 50 has been described, a discharge pipe may be provided for each type of liquid discharged from the liquid storage unit 20, and a pump and a pinch valve may be provided for each discharge pipe. Alternatively, a supply pipe and a discharge pipe may be provided for each combination by appropriately combining various liquids.

In the above-described embodiment, the liquid stored in the liquid storage unit 20 is rotated by the motor 32 to generate a water flow in the liquid stored in the liquid storage unit 20. Similar water flow may be generated by rotating the unit 12. Or, instead of rotating, May be shaken in the left-right direction to generate a water flow.

In the above-described embodiment, the cells separated from the living tissue are moved from the cell passage hole 14 of the tissue holding unit 12 to the tissue holding unit 1 by rotating the liquid storage unit 20 around the central axis in a substantially spindle shape. A water flow was generated to allow the cells to separate from the living tissue through the cell passage hole of the tissue holder by rotating the liquid reservoir 20 around an eccentric axis. May be generated such that the water flows to the outside.

In the above-described embodiment, the fins 24 are provided on the bottom surface of the liquid storage unit 20. However, in addition to or instead of this, similar fins may be provided on the bottom surface of the tissue holding unit 12. .

Further, in the above-described embodiment, the tissue holding unit 12 is disposed at the rotation center of the liquid storage unit 20, that is, at the center of the vortex generated in the dispersant-containing liquid. However, as shown in FIG. The part 12 may be detachably attached to the upper part of the liquid storage part 20 by a hook or a screw so as to be disposed at a position eccentric from the center of the vortex. In this way, compared to the case where the tissue holding unit 12 is arranged at the center of rotation of the vortex, the cells separated from the living tissue are more effectively applied to the outside of the tissue holding unit 12 from the cell passage hole 14 of the tissue holding unit 12. Can be passed through.

Further, in the above-described embodiment, the tissue holding portion 12 is formed in a cylindrical shape with a bottom using a mesh member. However, the tissue holding portion 12 is formed in a bag shape (tea bag shape) using a mesh member. May be used. Further, whether the tissue holding section 12 is cylindrical or bag-shaped with a bottom, the tissue holding section 12 may be suspended from above the liquid storage section 20 with a string.

In the above-described embodiment, the tissue holding unit 12 is provided in the liquid storage unit 20. However, as shown in FIG. 5, the tissue holding unit 12 is not provided and the living tissue is left as it is. May be in 20. In the cell dispersion device in Fig. 5, At the tip of the suction port of the tube 50, a filter 50f that allows cells to pass through but does not pass through biological tissues is attached. In this cell dispersion device, a trypsin solution is stored in a liquid storage unit 20, and a living tissue is immersed in the trypsin solution. Then, cells are separated and dispersed from the living tissue by a water flow generated in the trypsin solution. Here, it is preferable that the water flow (vortex flow) generated in the trypsin solution is switched in the forward and reverse directions every time a predetermined time elapses. When the trypsin solution that has acted on the living tissue is discharged from the liquid reservoir 20, the trypsin solution is discharged via the filter 50 #, so that the cells can be separated and collected from the living tissue. Industrial potential

INDUSTRIAL APPLICABILITY The present invention can be used in a cell culture technique for culturing cells in vitro (in vitro).

Claims

The scope of the claims

1. A cell dispersion device for separating and dispersing cells from a living tissue, wherein the tissue holding unit has a plurality of cell passage holes at least partially large enough to allow the cells to pass through, and is capable of holding the living tissue. ,

The dispersant-containing liquid flows through the cell holding hole of the tissue holding unit so that cells separated from the living tissue pass through the cell passage hole of the tissue holding unit to the outside of the tissue holding unit by the action of the dispersant-containing liquid stored in the liquid storage unit. A water flow generating section for generating a liquid;

A cell dispersion device comprising:

2. The cell dispersion device according to claim 1, wherein

A control unit for controlling the operation of the water flow generation unit

A cell dispersion device comprising:

3. The cell dispersion device according to claim 1, wherein

A dispersant-containing liquid supply unit capable of supplying the dispersant-containing liquid to the liquid storage unit;

A dispersant-containing liquid discharge unit capable of discharging the dispersant-containing liquid after acting on the living tissue from the liquid storage unit;

A cell dispersion device comprising:

4. The cell dispersion device according to claim 3, wherein

Activating the dispersant-containing liquid supply unit to supply a predetermined amount of the dispersant-containing liquid to the liquid storage unit, and then controlling the operation of the water flow generation unit based on a predetermined set value; A control unit that activates the dispersant-containing liquid discharge unit to discharge the dispersant-containing liquid after acting on the living tissue from the liquid storage unit A cell dispersion device comprising:

5. The cell dispersion device according to claim 2 or 4, wherein

A dispersion stopping liquid supply unit capable of supplying a dispersion stopping liquid for stopping the action of the dispersant to the liquid storage unit;

In controlling the operation of the water flow generation unit, the control unit controls the operation of the water flow generation unit based on a predetermined set value to assist in separating cells from the living tissue. Is generated in the dispersant-containing liquid, and thereafter, the dispersion stopping liquid supply section is operated to supply the dispersion stopping liquid to the liquid storage section in a predetermined amount, and thereafter, the water flow generating section is performed based on a predetermined set value. Generating a water flow in the dispersant-containing liquid such that the cells separated from the living tissue pass outward from the cell passage holes of the tissue holding unit by controlling the operation of

Cell dispersion device.

6. The cell dispersion device according to claim 2, 4, or 5, wherein

A cleaning liquid supply unit capable of supplying a cleaning liquid for cleaning the living tissue to the liquid storage unit;

A washing solution discharging unit capable of discharging a washing solution after washing the living tissue from the liquid storage unit,

With

The control unit activates the cleaning liquid supply unit to supply a predetermined amount of the cleaning liquid to the liquid storage unit before the processing using the dispersant-containing liquid, and thereafter, based on a predetermined set value. A cell dispersion device for controlling the operation of the water flow generation unit, and then performing a cleaning step of operating the cleaning liquid discharge unit to discharge the cleaning liquid after cleaning the living tissue from the liquid storage unit.

7. The cell dispersion device according to claim 2, 4, 5, or 6, wherein A suspension liquid supply unit capable of supplying a suspension liquid for suspending cells separated from the living tissue to the liquid storage unit;

A cell suspension discharge unit capable of discharging a cell suspension from the liquid storage unit,

The control unit, after the treatment using the dispersant-containing liquid, activates the suspension liquid supply unit to supply a predetermined amount of the suspension liquid to the liquid storage unit, and then determines a predetermined amount. The operation of the water flow generation unit is controlled based on the set value, and the cells in the liquid storage unit are suspended in the suspension liquid to form a cell suspension, and then the cell suspension discharge unit is operated. Performing a cell collection step of discharging the cell suspension from the liquid storage unit.

Cell dispersion device.

8. The control unit controls the operation of the water flow generation unit such that the forward / reverse direction of the water flow generated in the dispersant-containing liquid is switched every time a predetermined time elapses. The cell dispersion device according to item 1. “9. The liquid storage unit is configured to store cells that have passed through the cell passage hole of the tissue holding unit to the outside of the tissue holding unit when the water flow generation unit generates the water flow in the dispersant-containing liquid. Having a cell accumulating portion for accumulating,

The cell dispersion device according to claim 1.

10. The cell passage hole is provided in at least a part of a side surface of the tissue holding unit,

The cell accumulating portion is configured such that a centrifugal force acts on cells separated from the living tissue when the water flow generating portion generates the water flow in the dispersant-containing liquid, and the centrifugal force causes the cells to pass through the cell passage hole of the tissue holding portion. A portion that accumulates cells that have passed outside the tissue holding portion,

The cell dispersion device according to claim 9.

1 1. The cell accumulating section is formed in the liquid storage section in the direction of the centrifugal force. It is formed along a pointed shape,

The cell dispersion device according to claim 10.

12. At least the tissue holding unit and the liquid storage unit are detachably attached to the apparatus main body.

The cell dispersion device according to any one of claims 1 to 11.

13. At least the tissue holding unit and the liquid storage unit are arranged in a closed space,

The cell dispersion device according to any one of claims 1 to 12.

14. The enclosed space is capable of being filled with sterile gas.

14. The cell dispersion device according to claim 13.

15. The water flow generating section is for generating a vortex in the dispersant-containing liquid,

The cell dispersion device according to any one of claims 1 to 14, wherein the tissue holding unit is disposed at a position eccentric from a center of the vortex.

16. The cell passage hole is provided in at least a part of a side surface of the tissue holding unit, and the water flow generating unit rotates the liquid storage unit or the tissue holding unit, and the liquid storage unit Is integrated with or independent of the tissue holding unit,

The cell dispersion device according to any one of claims 1 to 15.

17. The cell dispersion device according to any one of claims 1 to 16, wherein the tissue holding unit and the liquid storage unit are integrated or configured to be integrated. Removable cassette for cell dispersion.

18. A discharge tube formed in a bottomed cylindrical shape so as to function as the tissue holding portion in the cell dispersion device according to any one of claims 1 to 16, and capable of discharging a liquid from the liquid storage portion. The tissue retaining member has a penetrating bottom.

1 9. A cell dispersion device for separating and dispersing cells from living tissue, A liquid storage unit capable of storing an amount of the dispersant-containing liquid in which the living tissue is immersed; a water flow generation unit configured to generate a water flow in the dispersant-containing liquid by rotating the liquid storage unit;

A control unit that controls the operation of the water flow generation unit such that the forward and reverse directions of the water flow generated in the dispersant-containing liquid are switched every time a predetermined time elapses;

A cell dispersion device comprising:

20. The cell dispersion device according to claim 19, wherein

When the dispersant-containing liquid after acting on the living tissue is discharged from the liquid reservoir, the cells dispersed in the dispersant-containing liquid pass through the filter but do not pass through the living tissue through the filter. Discharge unit that can discharge the contained liquid

A cell dispersion device comprising: