JP5103003B2 - Cell processing equipment - Google Patents

Description

  The present invention relates to a cell processing apparatus. More specifically, the present invention relates to a cell processing apparatus for performing predetermined processing on cells such as a human body using a filtration membrane.

  Usually, cytology is used as a screening method for the cervix. Here, for cytology of the cervix, the surface of the cervix is scraped with a special sampling instrument such as a cotton swab or scraper, and the scraped cells are immediately smeared on a slide glass to prepare a sample and observed with a microscope or the like. Diagnosis is performed by Alternatively, when it is necessary to process a large amount of specimens as in a health checkup, the group of cells collected by rubbing is stored in a fixative solution and transported to an inspection center, and a smear is prepared at the inspection center. Is diagnosed with Papanicolaou and observed with a microscope to obtain diagnostic information on the lesion.

  In any case, since smears must be prepared for each specimen and observed under a microscope, the processing is complicated and time consuming, and the diagnosis requires a high level of skill. Automation attempts are being considered.

  An example of an automated cytodiagnosis method is flow cytometry. Flow cytometry immunizes cells with any fluorescently labeled antibody, flows the cell suspension through the flow cell and irradiates it with laser light, and measures fluorescence information and scattered light information emitted from individual cells. Thus, a plurality of pieces of measurement information are obtained from a large amount of cells in a short time, and correlation analysis and statistical analysis are performed. In cytodiagnosis, an arbitrary protein that depends on a lesion such as cancer is selected as a marker, and a suspension of cells treated with a fluorescent-labeling specific antibody against this marker is measured with a flow cytometer. The presence or absence can be measured. Such a cell staining step requires a cell-reagent reaction step and a cell washing step by solution exchange.

  As a method for preparing a measurement sample for a flow cytometer through such steps, there is a centrifugal separation method using a difference in specific gravity between cells and a solution. However, the centrifugal separation method is not a technique that is very suitable for automation from the viewpoint of increasing the size of the cell processing apparatus and safety. On the other hand, as a method other than the centrifugation method, there is a filtration method using the size of cells.

  Patent Document 1 describes a technique of filtering and collecting cells by using a filter membrane made of an elastically deformable material and changing the pore diameter using elastic deformation. That is, in the technique of Patent Document 1, cells are captured with a filtration membrane having a small pore diameter in a compressed state, and then the pore diameter of the filtration membrane is increased in a non-compressed state and captured with a liquid supplied from the opposite direction. The formed cells are detached from the filtration membrane.

  However, the technique described in Patent Document 1 requires a device for stretching and compressing the filtration membrane, and uses a plurality of filtration membranes, and thus has a drawback that the process is complicated and costly. In addition, since a large amount of a collecting solution for collecting cells is required and the cell content in the collecting solution is low, there is a problem that it is not suitable for preparing a measurement sample for a flow cytometer.

JP-A-10-137557

  This invention is made | formed in view of such a situation, it processes with respect to the cell on the said filtration membrane using one filtration membrane, and it can collect | recover the said processed cell efficiently. An object of the present invention is to provide a cell processing apparatus.

The cell treatment device of the present invention is a cell treatment device for treating cells on a filtration membrane, wherein a placement part for placing a holder having the filtration membrane, and a cell containing cells in the holder placed in the placement part A first dispensing unit for dispensing the suspension, a second dispensing unit for dispensing the reagent to the holder arranged in the arrangement unit, and a liquid for releasing the cells on the filtration membrane are supplied. A liquid supply unit, and an air pressure source for applying pressure to the back side of the cell-existing surface of the filtration membrane, and dispensing a cell suspension on the filtration membrane of the holder by the first dispensing unit, The second dispensing unit dispenses a reagent to cells on the filtration membrane, and the liquid supply unit supplies the liquid so that the cells treated with the reagent are collected from the filtration membrane. .

The cell treatment apparatus of the present invention dispenses a cell suspension onto a filter membrane of a holder by a first dispensing unit, dispenses a reagent to cells on the filtration membrane by a second dispensing unit, and supplies a liquid supply unit Since the liquid is supplied so that the cells treated with the reagent can be collected from the filtration membrane, the reagent treatment and the collection processing are automatically performed on the cells dispensed on one filtration membrane. Can be done.
In addition, when the recovered liquid is supplied onto the filtration membrane in order to release the cells on the filtration membrane, the liquid supplied on the filtration membrane may become a leak flow that permeates the filtration membrane due to its supply pressure. By providing an air pressure source that applies pressure to the back side of the cell membrane surface of the filtration membrane, air is applied to the back side of the cell membrane surface of the filtration membrane to prevent deformation and leakage flow of the filtration membrane. Can do. As a result, the liquid can be efficiently and uniformly supplied to the cell existence region of the filtration membrane, and the cells on the filtration membrane can be efficiently recovered.

  It is preferable to further include a suction part that sucks the liquid dispensed in the holder placed in the placement part through the filtration membrane. In this case, it is possible to efficiently replace the liquid on the filtration membrane by sucking the liquid dispensed on the filtration membrane.

  It is preferable that the suction part sucks so that a predetermined liquid remains on the filtration membrane. In this case, since the predetermined liquid remains on the filtration membrane, the cells do not adhere or adhere strongly to the filtration membrane, so that the cells are not damaged or deformed.

  It is preferable to provide a liquid level sensor for detecting the liquid on the filtration membrane. In this case, when sucking the liquid on the filtration membrane through the filtration membrane by the suction unit, the liquid on the filtration membrane is detected by the liquid level sensor, and the suction operation of the suction unit is controlled based on the signal. can do. As a result, the liquid can be sucked by the suction portion so that a predetermined amount of liquid remains on the filtration membrane.

  A liquid supply member is provided on the opposite surface disposed opposite to the cell existence region on the filtration membrane, on the opposite surface, a spacer portion for defining a distance between the filtration membrane and the opposite surface, and a supply port for supplying liquid It is preferable to provide. In this case, the cell has a relatively simple configuration of a facing surface arranged to face the cell existing region on the filtration membrane, and a spacer portion that is provided on the facing surface and defines a distance between the filtration membrane and the facing surface. It is possible to supply the liquid so as to apply a force that releases the filter from the filtration membrane.

  It is preferable that the flow path formed by the cell existence region, the opposing surface and the spacer portion is spiral. When the flow path is spiral, the flow resistance is small, so that the liquid can be smoothly supplied to the cell existence region of the filtration membrane.

  It is preferable that the pneumatic pressure source is provided with valve means for blocking the communication state with the back side of the cell existing surface of the filtration membrane. After applying a predetermined pressure to the back side of the cell membrane surface of the filtration membrane, the communication between the air pressure source and the back side of the cell membrane surface of the filtration membrane is blocked by the valve means, thereby This space can be a sealed closed space, and the permeate flow of the filtration membrane when the recovered liquid is supplied onto the filtration membrane can be further reduced.

  It is preferable that the holder arrangement portion is configured to be able to arrange the holder in an annular shape and to be able to rotate. By making the holder arrangement part in which the holder is arranged annularly rotatable, it is possible to easily realize a configuration for automatically processing a plurality of objects to be processed by rotating the holder arrangement part.

  A filtration membrane can be made into 2 layer structure of the membrane filter in which a cell is mounted, and the mesh filter for supporting this membrane filter. By adopting such a two-layer structure, it is possible to prevent damage to the filtration membrane in the processing or recovery process of cells on the filtration membrane and to reduce deformation. Thereby, the function of a filtration membrane can fully be exhibited.

The cell treatment device of the present invention is a cell treatment device for treating cells on a filtration membrane,
A holder arrangement part capable of arranging a plurality of holders having the filtration membrane;
A first dispensing unit for dispensing a cell suspension containing cells into a holder disposed in the holder placement unit;
A second dispensing unit for dispensing a reagent to the holder disposed in the holder arranging unit;
A suction part capable of sucking the liquid dispensed into the holder through a filtration membrane;
A control unit for controlling the suction operation of the suction unit;
A recovery liquid supply unit that supplies a recovery liquid for releasing the cells on the filtration membrane treated with the reagent, and in the process of processing the cells on the filtration membrane, a predetermined amount of liquid is applied to the filtration membrane. It is characterized in that the suction of the liquid by the suction part can be controlled so as to remain.

  The cell treatment apparatus of the present invention includes a suction unit that can suck the liquid dispensed in the holder through the filtration membrane, and a control unit that controls the suction operation of the suction unit, and the cells are formed on the filtration membrane. In the process of processing the liquid, the suction of the liquid by the suction part can be controlled so that a predetermined amount of liquid remains on the filtration membrane. Accordingly, in the treatment process, the cells do not strongly adhere to or adhere to the filtration membrane due to the lack of the liquid content, so that the treatment such as reagent treatment or washing treatment is performed on one filter without damaging or deforming the cells. be able to. As a result, since a large amount of reagent is required to ensure the reagent replacement rate as in the prior art, the apparatus can be simplified and the processing speed can be increased.

  A cell processing apparatus for processing cells on a filtration membrane, wherein a placement part for placing a holder having the filtration membrane, and a cell suspension containing cells in the holder placed in the placement part A first dispensing unit, a second dispensing unit that dispenses a reagent to the holder disposed in the placement unit, a facing surface disposed to face the filtration membrane, and provided on the facing surface, It is characterized by comprising a spacer section for defining a distance from the facing surface, and a liquid supply section for supplying a liquid for releasing the cells on the filtration membrane.

  The cell treatment device of the present invention comprises: a facing surface disposed facing the filtration membrane; a spacer portion provided on the facing surface; defining a distance between the filtration membrane and the facing surface; and a supply port for supplying the liquid. And providing a liquid supply unit for supplying a liquid for releasing the cells on the filtration membrane, the cells can be efficiently released from the filtration membrane.

  According to the cell treatment device of the present invention, the reagent on the cells on the filtration membrane is continuously exchanged using one filtration membrane, and the treated cells are collected, thereby simplifying the device and The processing speed can be increased.

Hereinafter, embodiments of the cell treatment apparatus of the present invention will be described in detail with reference to the accompanying drawings.
FIG. 1 is an explanatory plan view of a cell processing apparatus according to an embodiment of the present invention. The cell treatment apparatus 1 filters cells in a cell suspension with a filtration membrane, and after the cells on the filtration membrane are subjected to reagent treatment such as staining and washing treatment with a washing solution, the cells on the filtration membrane are then treated. Is a device for recovering the product in the recovery solution.

  As shown in FIGS. 1 and 10, the cell treatment apparatus 1 dispenses a cell suspension containing cells into a holder arrangement unit 10 in which a plurality of holders 2 having filters (filtration membranes) are arranged, and the holder 2. A sample dispensing unit 20 that is a first dispensing unit, a reagent dispensing unit 30 that is a second dispensing unit that dispenses a reagent to the holder 2, and a cell for releasing a reagent-treated cell on a filtration membrane A recovery liquid supply unit 40 that is a recovery liquid supply unit that supplies the recovery liquid, a sample recovery unit 50 that recovers a sample in the recovery liquid, a holder detachment unit 60 that removes the used holder 2 from the holder placement unit 10, and a recovery liquid supply A washing spitz 80 for washing the unit 40, a vial transfer part 71 for transferring a vial containing a cell suspension, and a chip for containing a specimen recovered from the recovered liquid by the recovery unit 40. It is mainly constituted by a tube feeder 130, the rotation driving unit 110 for rotating the holder placement portion, the suction unit 120 and the control unit 101 for sucking the liquid in the holder 2 filter for transferring over drive. The control unit 101 includes a sample dispensing unit 20, a reagent dispensing unit, a collection liquid supply unit 40, a sample collection unit 50, a rotation drive unit 110, a suction unit 120, a holder detachment unit 60, a washing spitz 80, and a tube transfer unit. 130 and the vial transfer section 71 are electrically connected.

  The holder placement portion 10 has a ring shape, and a plurality of recesses 11 (see FIGS. 3 to 4) are formed at predetermined intervals along the circumferential direction. And the holder 2 which has a filter in each recess 11 is arrange | positioned freely. Thereby, the holder 2 is annularly arranged at a predetermined interval. The holder placement unit 10 can be rotated at a predetermined pitch by a drive unit (not shown) (rotation drive unit 110), whereby each holder 2 is subjected to necessary processing such as a sample dispensing position and a reagent dispensing position. Can be moved to. By making the holder arrangement part 10 in which the holder 2 is arranged in a ring shape rotatable, a configuration for automatically processing a plurality of objects to be processed by rotating the holder arrangement part 10 can be easily realized.

  As shown in FIGS. 3 to 4, the holder 2 has a substantially short cylindrical shape and is made of a synthetic resin such as polypropylene. For reinforcement, about 10% by weight of glass fiber may be mixed in the synthetic resin. A filter 3 on which cells in the cell suspension are placed is stretched near one end surface 2a of the holder 2 (an end surface on the side inserted into the recess 11). On the outer peripheral surface in the vicinity of the other end surface of the holder 2, a ring-shaped locking portion 4 having a substantially L-shaped cross section is formed. The locking portion 4 includes a flange portion 4a that protrudes radially outward from the outer peripheral surface of the holder 2, and a hanging portion that extends from the tip end of the flange portion 4a toward the one end surface along the axial direction. It consists of part 4b. And the 1st nail | claw part 4c which protrudes to radial direction inward is formed in the front-end | tip of this drooping part 4b. The first claw portion 4c is a second claw portion that is formed on the outer peripheral surface of the ring-shaped fixing portion 5 erected on the periphery of the opening of the recess 11 of the holder arrangement portion 10 and projects radially outward. It engages with 5b and plays the role which fixes the holder 2 to the holder arrangement | positioning part 10 so that attachment or detachment is possible.

  As shown in FIG. 5, the filter 3 has a two-layer structure of a membrane filter 3a on which cells are placed and a mesh filter 3b for supporting the membrane filter 3a. Since the membrane filter 3a is a field of reaction that directly contacts cells and reagents, it is preferably made of a material that has appropriate chemical resistance and does not easily adhere to cells. For example, hydrophilic polytetrafluoroethylene, It can be made of hydrophilic nylon, hydrophilic polyvinylidene fluoride, hydrophilic polycarbonate, hydrophilic polypropylene, hydrophilic polysulfone, hydrophilic polyethersulfone, or the like. The pore diameter may be selected according to the size of the cell to be treated, and is not particularly limited in the present invention, but is usually 1 to 5 μm. On the other hand, the mesh filter 3b is provided in order to suppress deformation of the membrane filter 3a at the time of liquid dispensing or the like, and is made of a material having lower elasticity and higher strength than the membrane filter 3a, such as nylon. Made in mesh with resin. The mesh size is not particularly limited, but can be, for example, 20 to 500 μm. By making the filter 3 have a two-layer structure, it is possible to prevent the filter 3 from being damaged in the cell processing or recovery process on the filter 3 and to reduce deformation. Thereby, the function of the filter 3 can fully be exhibited.

  Such a filter 3 can be integrated with the holder 2 by placing a membrane filter 3a and a mesh filter 3b on a mold and then performing outsert molding using a synthetic resin constituting the holder 2. . Thereby, the holder 2 with the filter 3 can be produced with few processes. In addition, the holder 2 and the filter 3 can be produced separately, and the filter 3 can be fixed to the holder 2 with an adhesive or the like.

  The sample dispensing unit 20 can be rotated about the Z axis (vertical axis) and can move up and down along the Z axis, and the tip of the rotating arm 21 (on the opposite side to the rotation axis). A pipette for sample dispensing attached to the end of the sample. The arm 21 is configured to be vertically movable and rotatable by a stepping motor 20a. Further, the sample dispensing unit 20 includes a pump unit (not shown) for aspirating and dispensing a sample with a sample dispensing pipette. A cell suspension containing cells, which is accommodated in a vial 72 transferred by a vial transfer unit 71 comprising a belt conveyor, is sucked by the pipette for sample dispensing and dispensed onto the filter 3 of the holder 2. . The vial transfer unit 71 includes a drive motor 71a for transferring the belt conveyor.

  As shown in FIG. 6, the reagent dispensing unit 30 includes a support column 32 erected on a base plate 31 on which the cell processing apparatus 1 is disposed, and an arm 33 protruding in the horizontal direction from the upper end of the support column 32. And a reagent dispensing pipette 34 held vertically by the arm 33. A reagent injection tube 73 is connected to the base side end of the reagent dispensing pipette 34, and the other end of the reagent injection tube 73 is connected to a reagent container 74 (see FIG. 1) via pump means (not shown). ) Is connected to the reagent bottle 75 arranged in (1).

  The reagent dispensing unit 30 is arranged along the outer periphery of the holder arrangement unit 10 in order to dispense the reagent to a plurality of holders arranged in the holder arrangement unit 10. When the holder 2 is rotationally moved to the reagent dispensing position, the pump means is driven to dispense a predetermined amount of reagent onto the filter 3 of the holder 2 from the tip of the reagent dispensing pipette 34.

  Further, a liquid level sensor 76 for detecting the liquid on the filter 3 is attached to the arm 33, and the liquid level of the liquid on the filter 3 is detected by the liquid level sensor 76. It is configured to detect the distance to the surface. Then, in accordance with a signal from the liquid level sensor 76, as will be described later, suction means for making negative pressure on the back side of the filter 2 of the holder 2 (the side opposite to the side where the cell suspension or reagent is dispensed). The drive is stopped.

  Similar to the sample dispensing unit 20, the recovery liquid supply unit 40 includes an arm 41 that can rotate around the Z axis and can move up and down along the Z axis. The arm 41 is configured to be vertically movable and rotatable by a stepping motor 40a. A cylindrical body 41 (see FIGS. 7 to 8) for collecting the cells on the filter 3 treated with the reagent is connected near the tip of the arm 41. A recovery liquid supply tube (not shown) is connected to the base side of the cylindrical body 41, and the recovery liquid accommodated in the container is connected to the recovery liquid supply tube and the cylindrical body 41 by pump means (not shown). It is supplied to the filter 3 via.

  As shown in FIGS. 7 to 8, a recovery liquid supply member 42 that supplies a constant flow of recovery liquid to the surface of the filter 3 is provided at the tip of the cylindrical body 41. The recovery liquid supply member 42 includes a facing surface 43 that is disposed to face the cell existing region on the filter 3, and a spacer portion that is provided on the facing surface 43 and defines a distance between the filter 3 and the facing surface 43. 44 and a supply port 45 for supplying the recovered liquid.

  In the recovery liquid supply member 42, the recovery liquid is supplied from the supply port 45 to the spiral flow path 46 formed by the cell existence region on the filter 3, the facing surface 43 and the spacer portion 44. In this case, the recovery liquid supply member 42 is disposed in a state in which the spacer portion 44 is in contact with the cell existing region on the filter 3 in the step of supplying the recovery liquid for releasing the cells. By providing the recovery liquid supply member 42 in a state where the spacer portion 44 is in contact with the cell existing area on the filter 3, the recovery liquid is supplied to the flow path parallel to the filter 3 with respect to the cells on the filter 3. As a result, a flow of the recovered liquid parallel to the surface of the filter 3 is created. This makes it possible to supply the recovery liquid so that a uniform and strong shearing force is applied to the cell existing area on the filter surface, and to release the cells from the filtration membrane in a clean form without damaging the cells. Thus, it can be efficiently recovered in the recovery liquid while being dispersed in the recovery liquid. Furthermore, since the flow path is spiral, a uniform and powerful flow recovery liquid can be supplied to the cell existence region of the filter 3 in the cell existence region on the circular filter surface. The shearing force for releasing the cells from the filter 3 can be adjusted by the flow rate of the collected liquid supplied per unit time, the height of the spacer portion 44, and the width of the flow path formed by the spacer portion 44. It can be set as appropriate according to the state and amount of cells to be treated.

  The recovery liquid supplied to the flow path flows through the cell existence region in a spiral shape, and then discharges the recovery liquid containing cells released from the filter 3 provided near the end of the spiral flow path. From the outlet 47, it is discharged | emitted by the storage part 48 which stores a collection liquid. The storage portion 48 is formed of a recess, and is formed on the surface of the recovered liquid supply member 42 opposite to the facing surface 43.

  Similar to the sample dispensing unit 20, the sample collection unit 50 can be rotated about the Z axis and can be moved up and down along the Z axis, and the distal end portion (the rotation of the rotation arm 51). A specimen collection pipette (not shown) attached to the end opposite to the moving shaft is provided. The arm 51 is configured to be vertically movable and rotatable by a stepping motor 50a. Further, the sample collection unit 50 includes pump means (not shown) for aspirating and dispensing the sample with a sample collection pipette. The specimen recovery unit 50 recovers the recovery liquid (including cells released from the filter 3) in the storage portion 48 of the recovery liquid supply member 42. The mouthpiece of the sample collection pipette is arranged to be offset by about 3 to 20 degrees from the axis of the sample collection pipette. By moving the arm 51 up and down, the mouthpiece is It arrange | positions in the collection | recovery liquid in the storage part 48, Then, the pump means which is not shown in figure is driven and collection | recovery liquid is aspirated.

  After the collected liquid is aspirated, the arm 51 rotates by a predetermined angle, and then the sample collecting pipette discharges the collected liquid into the tube 49 transferred by the tube transfer unit 130 formed of a belt conveyor. The tube transfer unit 130 includes a drive motor 130a for transferring the belt conveyor.

  The recovered liquid supply member 42 from which the recovered liquid in the reservoir 48 has been sucked is cleaned by the cleaning spitz 80. As shown in FIG. 9, the cleaning spitz 80 includes a bottomed cylindrical body 81 having an open top, and a cleaning liquid inlet port 83 and an outlet port 84 are provided on a side wall 82 of the bottomed cylindrical body 81. Is formed. A tube 85 for supplying cleaning liquid and a tube 86 for discharging are connected to the inlet port 83 and the outlet port 84, respectively. The inlet port 83 and the outlet port 84 are connected to pump means (not shown). The cleaning liquid is supplied into the cleaning spitz 80 from the cleaning liquid bottle 76 through the inlet port 83 by the pump means. The cleaning liquid supplied into the cleaning spitz 80 is sucked and discharged through the outlet port 84 by the pump means. Further, a drainage port 88 is formed at the bottom 87 of the bottomed cylindrical body 81, and the cleaning liquid in the bottomed cylindrical body 81 is sucked by suction means (not shown). In the bottomed cylindrical body 81, the recovered liquid supply member 42 is moved up and down a plurality of times, and the recovered liquid supply member 42 is cleaned by the cleaning liquid supplied from the inlet port 83 during that time.

  The holder attaching / detaching unit 60 has an arm 62 having a gripping portion 61 that can be opened and closed at the tip, and this arm 62 is configured to be movable up and down and back and forth. The arm 62 in the retracted position is moved forward, the gripping portion 61 is then closed, the mouth of the holder 2 is gripped, and the arm 62 is raised in this state, and the holder 2 is taken out from the recess 11. Then, by moving the arm 62 forward and opening the gripping portion 61, the gripped holder 2 is dropped onto the holder discarding portion 63. The used holder 2 accumulated in the holder disposal unit 63 is appropriately collected and discarded by the user.

  In FIG. 1, reference numeral 90 denotes a light-shielding cover, which prevents the fluorescent dye used for staining cells from fading by light and ceases to emit light when irradiated with an excitation laser beam used in a flow cytometer. Is provided.

Next, the structure of the suction unit 120, the mechanism for sucking the liquid on the filter 3, and the mechanism for applying pressure to the back side of the filter 3 will be described with reference to FIG.
The suction unit 120 includes a holder placement unit 10, a first valve 14, a second valve 15, a third valve 19, a negative pressure source 16, and a positive pressure source 17. One end of a pipe 13 is connected to the first port 12 formed on the back side of the holder 2, and the other end side of the pipe 13 passes through the first valve 14 and the second valve 15. It is connected to a negative pressure source 16 that is a suction unit and a positive pressure source 17 that is an air pressure source. One end of the atmosphere release pipe 8 is connected to the second port 18 formed on the side surface of the holder arrangement portion 10, and the other end of the atmosphere release pipe 8 is opened to the atmosphere via the third valve 19. Has been. The first valve 14 and the third valve 19 are opening / closing valves having an opening / closing function, while the second valve 15 is a switching valve for switching a flow path.

  First, when a part of the liquid dispensed on the filter 3 remains on the filter 3 and the remainder is sucked through the filter 3, the first valve 14 is opened and the third valve 19 is closed. To do. Next, after the second valve 15 is switched to the negative pressure source 16 side, the negative pressure source 16 is driven to make the back side of the filter 3 have a negative pressure. Thereby, the liquid on the filter 3 can be sucked through the filter 3. At that time, the distance to the liquid level of the liquid on the filter 3 is detected by the liquid level sensor 76, and the first valve 14 is closed and the third valve 19 is opened according to the signal from the liquid level sensor 76. Then, the back side of the filter 3 is opened to atmospheric pressure. This makes it possible to suck unnecessary liquid while reliably leaving a predetermined amount of liquid on the filter 3.

  In addition, when cells that have been subjected to predetermined processing such as reagent processing and washing are supplied with a recovery liquid and recovered in the recovery liquid, the third valve 19 is opened, the first valve is opened, and the second valve is opened. After switching to the negative pressure side, the liquid inside the holder placement unit 10 and the tube transfer unit 13 was discharged and replaced with air, the third valve 19 was closed, and then the second valve 15 was switched to the positive pressure source 17 side. Later, the positive pressure source 17 is driven to make the back side of the filter 3 a positive pressure. When the recovery liquid is supplied onto the filter 3 in order to release the cells on the filter 3, the recovery liquid supplied onto the filter 3 by the recovery liquid becomes a leakage flow that passes through the filter 3 by the supply pressure. The flow parallel to the filter surface cannot be produced effectively. Further, it is conceivable that the filter 3 bends so as to protrude downward, and thereby the flow of the recovery liquid is disturbed and the cells cannot be recovered efficiently. However, deformation of the filter 3 and the leakage flow can be prevented by making the back side of the filter 3 positive with air. As a result, the collected liquid can be efficiently and smoothly supplied to the cell existence region of the filter 3, and the cells on the filter 3 can be efficiently collected.

  The first valve 14 is preferably closed after the back side of the filter 3 is set to a predetermined positive pressure. After a predetermined pressure is applied to the back side of the cell-existing surface of the filter 3, the communication between the positive pressure source 17 and the back side of the filter 3 is interrupted, so that the space on the back side of the filter 3 is sealed. Therefore, the deformation of the filter 3 when the recovered liquid is supplied onto the filter 3 can be further reduced.

  The predetermined pressure (positive pressure) is preferably set to be equal to or higher than the liquid pressure of the recovered liquid supplied onto the filter 3 and below the bubble point of the filter 3 so that the filter 3 does not allow air to pass through. When the recovery liquid is supplied onto the filter 3 in such a state, the deformation and leakage flow of the filter 3 are prevented, and the flow of the recovery liquid can be efficiently formed.

  Next, the rotation drive unit 110 shown in FIG. 10 will be described. The rotation drive unit 110 includes a rotation drive source (not shown) that rotates the holder installation unit 10 at a predetermined pitch. Then, the holder installation unit 10 rotates by an amount corresponding to the number of pulses of the drive pulse signal supplied from the control unit 101 to the rotation drive unit 110 and stops.

  The control unit 101 determines the amount of rotational movement of the holder installation unit 10 from the origin position of the holder installation unit 10 by counting the number of pulses of the supplied drive pulse signal, and the rotational movement of the holder installation unit 10 is determined. It is possible to control. Further, the control unit 101 controls the rotation driving unit 110 so as to rotate the holder installation unit 10 counterclockwise by 1 pitch every 3 minutes.

As shown in FIG. 11, the control unit 101 mainly includes a CPU 101a, a ROM 101b, a RAM 101c, and a communication interface 101d.
The CPU 101a can execute the computer program stored in the ROM 101b and the computer program read out to the RAM 101c. The ROM 101b stores a computer program to be executed by the CPU 101a, data used for executing the computer program, and the like. The RAM 101c is used for reading a computer program stored in the ROM 101b. Further, when these computer programs are executed, they are used as a work area of the CPU 101a.

  The communication interface 101d includes a sample dispensing unit 20, a reagent dispensing unit 30, a collection liquid supply unit 40, a sample collection unit, a rotation drive unit 110, a suction unit 120, a holder detachment unit 60, a cleaning spitz 80, a tube transfer unit 130, and It has a function for transmitting a command from the CPU 501a for driving each part of the vial transfer unit 71.

  Next, an example of a cell processing method using the cell processing apparatus according to the present embodiment will be described. In this method, the cells in the cell suspension are, for example, cells collected from a patient, and are used for obtaining information that supports diagnosis by examining the cells. For example, for the diagnosis of cervical cancer, a cell suspension in which cervical cells are suspended in a solvent is used.

  As shown in FIG. 1, the holder installation unit 10 can install 51 holders 2 and is rotated counterclockwise by 1 pitch every 3 minutes by the rotation drive unit 110, and each process is performed at each predetermined position. That is, each process is performed at 51 predetermined positions. Corresponding to each predetermined position, position numbers 1P to 51P are given. Hereinafter, the processing steps in the holder 2 installed in the holder installation unit 10 will be described in correspondence with the position numbers using the flowcharts shown in FIGS. In addition, about the control of the rotation drive part 110 by the control part 101, since the holder installation part 10 is only rotated counterclockwise by 1 pitch for 3 minutes, it abbreviate | omits about the description.

<Position numbers 1P to 12P>
First, the user installs the holder 2 on 1P to 12P of the holder installation unit 10.

<Position number 13P>
Next, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid onto the filter 3 of the 13P holder 2 (step S1). Next, the control unit 101 performs a suction process by controlling the suction unit 120 (step S2).
Here, the suction processing in step S2 will be specifically described with reference to the flowchart shown in FIG. The control unit 101 controls the suction unit 120 to apply a weak negative pressure from the back side of the filter 3 and starts suction of the liquid on the filter 3 (step S101). The liquid level on the filter 3 is detected by the liquid level sensor 76, and it is determined whether or not the liquid level is at a predetermined level (step S102). If it is not at the predetermined liquid level, the process of step S102 is repeated until the predetermined liquid level is reached. When the liquid level is at a predetermined level, the control unit 101 closes the first valve 14, opens the third valve 19, opens the back side of the filter 3 to atmospheric pressure, and closes it immediately thereafter. As a result, suction by negative pressure is stopped (step 103). Thus, the liquid level on the filter 3 is detected, so that the filter 3 is always wet with the liquid.
Note that the subsequent suction processing is the same as that in step S2, and a description thereof will be omitted.

<Position number 14P>
Next, the control unit 101 contains a vial 72 containing a cell suspension (about 1 × 10 5 cells having a diameter of about 3 to 200 μm contained in 1 mL of fixative) prepared from a cervical scraping specimen. Is transferred to the suction position by the vial transfer unit 71 (step S3), and the cell suspension is sucked from the transferred vial 72 by the sample pipetting pipette of the sample dispensing arm 20 and distributed onto the filter 3 of the holder 2. Note (step S4). The filter 3 used was a two-layer structure in which a hydrophilic PTFE flat membrane filter (pore diameter 0.4-5 μm) as a solid-liquid separation membrane was laminated on a net-like sheet of about 60 μm.

<Position numbers 15P to 16P>
Next, at 15P, the control unit 101 performs a suction process by controlling the suction unit 120 (step S5). And the control part 101 dispenses dilution / washing liquid on the filter 3 of the holder 2 by controlling the reagent dispensing unit 30 (step S6).
Next, in 16P, the control unit 101 performs similar processing (steps S7 and S8).

<Position number 17P>
Next, at 17P, the control unit 101 performs the suction process by controlling the suction unit 120 (step S9). Then, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid from the reagent bottle 75 onto the filter 3 of the holder 2 (Step S10). Further, the control unit 101 performs the suction process by controlling the suction unit 120 (step S11).

<Position number 18P>
Next, in 18P, the control unit 101 dispenses the activator from the reagent bottle 75 by controlling the reagent dispensing unit 30 (step S12).

<Position numbers 19P to 20P>
Next, in 19P to 20P, the control unit 101 does not perform processing (time is provided for reacting the cells with the activator).

<Position numbers 21P to 22P>
Next, at 21P, the control unit 101 performs the suction process by controlling the suction unit 120 (step S13). Then, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid from the reagent bottle 75 onto the filter 3 of the holder 2 (Step S14).
Next, in 22P, the control unit 101 performs similar processing (steps S15 and S16).

<Position number 23P>
Next, at 23P, the control unit 101 performs a suction process by controlling the suction unit 120 (step S17). Then, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid from the reagent bottle 75 onto the filter 3 of the holder 2 (step S18). Further, the control unit 101 performs the suction process by controlling the suction unit 120 (step S19).

<Position number 24P>
Next, in 24P, the control unit 101 dispenses the activation reaction terminator from the reagent bottle 75 by controlling the reagent dispensing unit 30 (step S20).

<Position numbers 25P to 28P>
Next, in 25P-28P, the control part 101 does not perform a process (time for reacting an activation agent and an activation reaction terminator is provided).

<Position number 29P>
Next, at 29P, the control unit 101 performs a suction process by controlling the suction unit 120 (step S21). Then, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid from the reagent bottle 75 onto the filter 3 of the holder 2 (step S22). Furthermore, the control unit 101 performs the suction process by controlling the suction unit 120 (step S23).

<Position number 30P>
Next, at 30P, the control unit 101 dispenses the blocking agent from the reagent bottle 75 onto the filter 3 by controlling the reagent dispensing unit 30 (step S24).

<Position numbers 31P to 34P>
Next, in 25P-28P, the control part 101 does not perform a process (time for reacting a cell and a blocking agent is provided).

<Position number 35P>
Next, in 35P, the control unit 101 performs the suction process by controlling the suction unit 120 (step S25). And the control part 101 dispenses the antibody from the reagent bottle 75 on the filter 3 of the holder 2 by controlling the reagent dispensing unit 30 (step S26).

<Position numbers 36P to 39P>
Next, in 36P-39P, the control part 101 does not perform a process (time for reacting a cell and an antibody is provided).

<Position numbers 40P to 41P>
Next, at 40P, the control unit 101 controls the suction unit 120 to perform a suction process (step S27). Then, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid from the reagent bottle 75 onto the filter 3 of the holder 2 (step S28).
Next, in 41P, the control unit 101 performs the same process (steps S29 and S30).

<Position number 42P>
Next, in 42P, the control unit 101 controls the suction unit 120 to perform a suction process (step S31). Then, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid from the reagent bottle 75 onto the filter 3 of the holder 2 (step S32). Further, the control unit 101 performs a suction process by controlling the suction unit 120 (step S33).

<Position number 43P>
Next, in 43P, the control unit 101 dispenses the staining solution from the reagent bottle 75 by controlling the reagent dispensing unit 30 (step S34).

<Position numbers 44P to 45P>
Next, in 44P to 45P, the control unit 101 does not perform processing (time is provided for reacting the antibody with the staining).

<Position numbers 46P-48P>
Next, at 46P, the control unit 101 controls the suction unit 120 to perform a suction process (step S35). Then, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid from the reagent bottle 75 onto the filter 3 of the holder 2 (step S36).
Next, in 47P to 48P, the control unit 101 performs similar processing (steps S37 to S40).

<Position number 49P>
Next, at 49P, the control unit 101 performs the suction process by controlling the suction unit 120 (step S41). Then, the control unit 101 controls the reagent dispensing unit 30 to dispense the dilution / washing liquid from the reagent bottle 75 onto the filter 3 of the holder 2 (step S42). Further, the control unit 101 performs the suction process by controlling the suction unit 120 (step S41).
Next, the third valve is opened, the first valve is opened, the liquid inside the holder placement unit 10 and the tube transfer unit 13 is discharged and replaced with air, the third valve 19 is closed, and the first valve 14 is opened. After applying a positive pressure to the back side of the filter 3 that is equal to or lower than the bubble point of the filter 3 and higher than the liquid pressure of the recovered liquid supplied onto the filter 3 when the recovered liquid is supplied, the first valve 14 Is closed to close the back side of the filter 3 (step S44).

<Position number 50P>
Next, at 50P, the control unit 101 controls the tube transfer unit 130 to move the tube 49 to the collection position (step S45).
The arm 41 of the recovery liquid supply unit 40 is rotated and lowered, and the spacer portion 44 of the recovery liquid supply member 42 shown in FIGS. 7 to 8 is arranged in contact with the cell existing region on the filter 3 (step). S46). Next, the recovery liquid is supplied from the supply port 45 located at the center of the recovery liquid supply member 42 to the spiral flow path 46 formed by the cell existence region on the filter 3, the facing surface 43 and the spacer portion 44. (Step 47). As a result, a uniform flow parallel to the surface of the filter 3 is formed, and the cells on the filter 3 are released by this flow and collected in the collected liquid. At this time, the recovered liquid supply member 42 may be reciprocated several times up and down to promote the release of cells firmly attached to the filter 3 and the dispersion of cells.
Next, the control unit 101 controls the sample recovery unit 50 to store the recovery liquid containing the cells released from the filter 3 contained in the storage part 48 of the recovery liquid supply member 42 into the tube 49. It collect | recovers and encloses (step S48). Further, the control unit 101 controls the recovered liquid supply unit 40 and the cleaning spitz 80 to clean the recovered liquid supply member 42 with the cleaning liquid (step S49). By setting the tube 49 at a predetermined position of the flow cytometer, the sample can be measured and analyzed.

<Position number 51P>
Next, the control unit 101 controls the holder detaching unit 60 to remove the holder 2 on which the holder installation unit 10 is installed and discard it to the holder discarding unit 63 (step 50).

  In the embodiment, the user installs the holder 2 in the holder installation unit 10, but a supply unit that automatically supplies the holder 2 to the holder installation unit 10 may be provided.

It is a plane explanatory view of one embodiment of the cell treatment device of the present invention. It is an air circuit diagram of the holder arrangement | positioning part in the cell processing apparatus shown by FIG. It is a section explanatory view of a holder which has a filter, and a holder arrangement part. It is sectional explanatory drawing of the holder arrangement | positioning part with which the holder was mounted | worn. It is sectional explanatory drawing of the edge part vicinity of a holder. It is sectional explanatory drawing of a reagent dispensing unit and a holder arrangement | positioning part. It is explanatory drawing which looked at the collection liquid supply member in a collection liquid supply unit from the lower part. It is explanatory drawing which looked at the collection liquid supply member in a collection liquid supply unit from the upper part. It is a section explanatory view of cleaning Spitz. It is explanatory drawing of the unit which comprises a cell processing apparatus. It is explanatory drawing of the control part which controls a cell processing apparatus. It is a flowchart which shows the example of the cell processing using the cell processing apparatus of this invention. It is a flowchart which shows the example of the cell processing using the cell processing apparatus of this invention. It is a flowchart which shows the example of the cell processing using the cell processing apparatus of this invention. It is a flowchart which shows the example of the cell processing using the cell processing apparatus of this invention. It is a flowchart which shows the example of the cell processing using the cell processing apparatus of this invention. It is a flowchart of the attraction | suction process in this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Cell processing apparatus 2 Holder 3 Filter 4 Locking part 5 Fixing part 10 Holder arrangement | positioning part 11 Recess 12 First port 13 Pipe 14 First valve 15 Second valve 16 Negative pressure source 17 Positive pressure source 18 Second port 19 First 3 valve 20 specimen dispensing unit 21 arm 30 reagent dispensing unit 32 support 33 arm 40 collection liquid supply unit 42 collection liquid supply member 43 facing surface 44 spacer section 45 supply port 46 channel 47 discharge port 48 storage section 50 sample collection unit 60 Holder Desorption Unit 71 Vial Transfer Unit 72 Vial 73 Reagent Injection Tube 74 Reagent Storage Unit 75 Reagent Bottle 76 Liquid Level Sensor 80 Washing Spitz 90 Shading Cover

Claims (11)

A cell processing apparatus for processing cells on a filtration membrane,
An arrangement part for arranging a holder having the filtration membrane;
A first dispensing unit that dispenses a cell suspension containing cells into the holder disposed in the placement unit;
A second dispensing unit that dispenses a reagent to the holder disposed in the placement unit;
A liquid supply section for supplying a liquid for releasing cells on the filtration membrane;
An air pressure source that applies pressure to the back side of the cell-existing surface of the filtration membrane,
A cell suspension is dispensed onto the filtration membrane of the holder by the first dispensing unit, a reagent is dispensed to cells on the filtration membrane by the second dispensing unit, and the filtration is performed by the liquid supply unit. A cell processing apparatus, wherein a liquid is supplied so as to collect reagent-treated cells from a membrane.   The cell treatment device according to claim 1, further comprising a suction unit that sucks the liquid dispensed in the holder disposed in the placement unit through a filtration membrane.   The cell treatment apparatus according to claim 2, wherein the suction unit sucks so that a predetermined liquid remains on the filtration membrane.   The cell processing apparatus according to claim 3, further comprising a liquid level sensor that detects a liquid on the filtration membrane.   The liquid supply member is provided on a facing surface arranged to face a cell existing region on the filtration membrane, a spacer portion provided on the facing surface and defining an interval between the filtration membrane and the facing surface, and supplies the liquid The cell processing apparatus according to claim 1, further comprising a supply port. The cell processing apparatus according to claim 5 , wherein the flow path formed by the cell existence region, the opposing surface, and the spacer portion is spiral. The cell processing apparatus according to claim 1 , wherein the pneumatic pressure source includes valve means for blocking a communication state with a back side of a cell existing surface of the filtration membrane.   The cell processing apparatus according to any one of claims 1 to 7, wherein the placement unit is configured to be able to place the holder in an annular shape and to be rotatable.   The cell treatment device according to any one of claims 1 to 8, wherein the filtration membrane has a two-layer structure of a membrane filter on which cells are placed and a mesh filter for supporting the membrane filter. A cell processing apparatus for processing cells on a filtration membrane,
A holder arrangement part capable of arranging a plurality of holders having the filtration membrane;
A first dispensing unit for dispensing a cell suspension containing cells into a holder disposed in the holder placement unit;
A second dispensing unit for dispensing a reagent to the holder disposed in the holder arranging unit;
A suction part capable of sucking the liquid dispensed into the holder through a filtration membrane;
A control unit for controlling the suction operation of the suction unit;
A recovery liquid supply unit that supplies a recovery liquid for releasing the cells on the filtration membrane treated with the reagent, and in the process of processing the cells on the filtration membrane, a predetermined amount of liquid is applied to the filtration membrane. A cell processing apparatus configured to control the suction of the liquid by the suction unit so as to remain. A cell processing apparatus for processing cells on a filtration membrane,
An arrangement part for arranging a holder having the filtration membrane;
A first dispensing unit that dispenses a cell suspension containing cells into the holder disposed in the placement unit;
A second dispensing unit that dispenses a reagent to the holder disposed in the placement unit;
A cell on the filtration membrane having a facing surface disposed facing the filtration membrane, a spacer portion provided on the facing surface, defining a distance between the filtration membrane and the facing surface, and a supply port for supplying the liquid And a liquid supply unit for supplying a liquid for releasing the cell.

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