JP2016036274A - Cell peeling method, cell peeling device, and cell culture system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cell peeling method suitable for industrial applications aiming at mass culture of cells.SOLUTION: The invention comprises a peeling bath 2; a culture medium supply line 3 which supplies a culture medium 14 comprising a carrier in which cells are attached to the peeling bath; a peeling liquid supply line 5 which supplies a peeling liquid to the peeling bath; a filter 9 which is provided in a lower part of the peeling bath and has meshes larger than the cell and smaller than the carrier; a waste-liquid line 6 connected to the peeling bath below the filter; an agitating device 8 agitating the peeling liquid in the peeling bath. The peeling liquid is supplied from a peeling liquid supply line into the peeling bath releasing the medium through the waste-liquid line to peel the cells from the carrier by the agitating device.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a cell detachment method, a cell detachment apparatus, and a cell culture system for detaching adherent cells from a cell adhesion base after cell suspension culture.

  There are two types of cells such as animal cells and plant cells, which are adherent cells that require a scaffold for growth during cultivation, and floating cells that do not require a scaffold for growth. Cultured cells are widely used for production of useful substances.

  Examples of cell culture methods include stationary culture and suspension culture. For example, when adherent cells are cultured by the stationary culture method, the culture solution is introduced into a petri dish or a T-flask or a 6-24 hole plate, and the cells are allowed to adhere to the bottom surface, or Cultivates cells by solidifying the culture solution with agar or the like and attaching the cells to the solidified culture solution.

In the case of the stationary culture method in which the cells are attached to the bottom surface or the solidified culture solution, the culture is performed on a flat surface, so that only 10 ⁴ cells can be cultured per 1 cm ² , and 10 ⁸ cells are cultured. In order to do so, an area of about 2000 cm ² is required.

  On the other hand, when adherent cells are cultured by the floating culture method, the cells are attached to a microcarrier (carrier) having a diameter of about 180 μm, and the carrier is suspended in a culture solution introduced into a container such as a bottle. Cells are cultured.

When cells are cultured by the suspension culture method, 10 ⁶ cells can be cultured per mL, and even when 10 ⁸ cells are cultured, a culture solution of about 100 mL is sufficient.

  In both the static culture method and the floating culture method, when the cultured cells are peeled off from the adhesion target, an operator can add a peeling solution such as EDTA solution (ethylenediaminetetraacetic acid) to the cells to make the cells easy to peel off. By repeating suction and discharge with a pipette, a shear stress was applied between the carrier and the cells, and the cells were detached from the carrier and collected.

  In Patent Document 1, the lid of the dispensing container is opened, the cell suspension is sucked from the container into the chip, and the process of discharging the cell suspension into the well of the dispensing container is automatically performed. A liquid feeding method, a liquid feeding unit, and an automatic culture system that prevent dripping from the chip by forming an air layer at the tip of the chip when the cell suspension is sucked are disclosed. Yes.

  Patent Document 2 discloses a work table in which various operations for cell culture are performed inside a work space, and a robot that automatically performs predetermined work such as planting predetermined cells in a container brought in through a pass box. There is disclosed an automatic cell culture apparatus that is provided with an arm and that can reliably sterilize the work space and can render the sterilization gas harmless in a closed space.

JP 2013-102723 A JP 2011-167405 A

  However, as in the past, simply removing the cells from the carrier while aspirating and discharging the carrier to which the cells are attached by a pipette requires a lot of time and effort to collect the cells, and mass culture of the cells. Could not be applied. Accordingly, the present invention aims to provide a cell detachment method, a cell detachment apparatus, and a cell culture system that are suitable for industrial applications aimed at mass culture of cells by solving such problems. It is.

  A first invention for achieving the object includes a first step of supplying a culture medium containing a carrier to which cells are attached to a peeling tank, a second step of separating the carrier from the culture medium, and the second step. A third step of applying a stripping solution to the carrier separated in the step of step 4, a fourth step of stirring the stripping solution containing the carrier in the stripping tank, and a stripping from the carrier by the fourth step. And a fifth step of separating the produced cells from the carrier.

  Furthermore, the second invention includes a peeling tank, a medium supply mechanism for supplying a medium containing a carrier with cells attached to the peeling tank, and a discharge mechanism for separating the medium from the carrier and discharging the medium outside the peeling tank. A stripping solution supply mechanism for supplying a stripping solution for stripping the cells from the carrier into the stripping tank, a stirring device for stirring the carrier and the stripping solution in the stripping tank, and the stirring device And a delivery mechanism for separating the cells detached from the carrier from the carrier and feeding them out of the separation tank.

  According to the 1st and 2nd invention, since the peeling liquid containing the support | carrier with which the cell was attached can be stirred directly, a cell can be reliably peeled from a support | carrier.

  Further, the third invention comprises a cell introduction device, a cell culture device, the cell peeling device described above, and a cell recovery device, wherein the cell introduction device provides a carrier on which cells are attached to the cell culture device. And the cell culture device suspends the carrier in the medium and cultures the cells on the carrier, and the cell peeling device peels the cells from the carrier contained in the medium supplied from the cell culture device. The cell collection device is a cell culture system that collects the detached cells. According to this invention, according to the first and second inventions, the cell recovery efficiency can be improved in order to recover the cells detached from the carrier.

  According to the present invention, it is possible to provide a cell detachment method, a cell detachment apparatus, and a cell culture system that are also suitable for industrial applications aimed at mass culture of cells.

1 is a system diagram of cell culture according to an embodiment of the present invention. It is a lineblock diagram showing a 1st embodiment of a cell exfoliation device concerning the present invention. It is process drawing explaining the cell peeling process using this cell peeling apparatus. It is a block diagram which shows 2nd Embodiment of the cell peeling apparatus which concerns on this invention. It is a perspective view of the stirring apparatus (fan unit) of this cell peeling apparatus. It is a front view which shows the modification of a fan unit. It is a block diagram which shows 3rd Embodiment of the cell peeling apparatus which concerns on this invention. It is a block diagram which shows 4th Embodiment of the cell peeling apparatus which concerns on this invention.

  Next, embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a block diagram showing an example of an industrial system (culture system) 26 intended for mass culture of cells. The cell culture system 26 includes a cell introduction device 27, a cell culture device 12, a cell detachment device 1, and a cell recovery device 28, and performs a series of processes from cell introduction to culture, detachment, and recovery. A solid line arrow indicates a state in which a plurality of blocks connected by a solid line are always connected to each other, and a broken line arrow indicates a state in which a plurality of blocks connected by a broken line are connected to each other as necessary. ing.

  The cell introduction device 27 is mainly composed of a medium tank 37 that is refrigerated and stored by a chemical solution tank 33, a carrier supply container 34, a cell supply unit 35, a refrigerator 36, and the like. The carrier supply container 34, the cell supply unit 35, and the medium tank 37 store the carrier 38, cells, and medium 14, respectively. The cell introduction device 27 is stored in the culture tank 39 of the cell culture device 12 together with the medium 14 and the carrier 38. Supply.

  The cell culture apparatus 12 includes a culture tank 39 in which a constant temperature state is maintained by a constant temperature tank 41 or the like, a medium circulation pump 42, and a waste liquid tank 43. The cell culture device 12 suspends the carrier 38 supplied from the cell introduction device 27 in the culture medium 14 and circulates the culture medium 14 by the culture medium circulation pump 42, thereby culturing the cells while floating in the culture medium.

  The cell detachment apparatus 1 includes a detachment tank 2 and a chemical solution tank 44 (a buffer solution supply source 15 and a detachment solution supply source 16 described later) in which various chemical solutions are stored, and is supplied from the chemical solution supplied from the chemical solution tank 44 and a stirring device. The cells are peeled from the carrier 38 in the peeling tank 2 by the shearing stress. Furthermore, the cell collection device 28 collects the cells detached by the cell separation device 1.

  FIG. 2 shows the cell peeling apparatus 1 that performs a peeling process for peeling cells from the carrier 38. The peeling tank 2 is a substantially cylindrical container, and has a conical shape whose lower end portion is reduced in diameter toward the lower side. A culture medium supply line 3, a buffer solution supply line 4, and a stripping solution supply line 5 are connected to the upper end of the stripping tank 2, and a waste liquid line 6 and a cell recovery line 7 are connected to the lower end of the stripping tank 2, respectively. Yes.

  A stirring device 8 for stirring the stripping solution in the stripping tank 2 is provided below the stripping tank 2. The stirring device 8 includes a swinging (shaking) mechanism of the peeling tank 2 and applies shear stress to the peeling liquid by stirring the peeling liquid by swinging of the peeling tank 2. A mesh-like filter 9 is horizontally provided in the lower part in the peeling tank 2. For swinging, the peeling tank 2 is reciprocated in the horizontal and vertical directions according to the required amplitude and frequency, the reciprocating circle movement of the peeling tank 2 at a predetermined speed, and the predetermined fulcrum is set according to the required amplitude and frequency. The operation | movement etc. which reciprocately tilt the peeling tank 2 in the center are included. For example, the peeling tank 2 is swung in the range of 10 rpm to 200 rpm.

  The filter 9 has a mesh of a size that allows cells to pass but does not allow the carrier 38 to pass. Here, the carrier 38 is a microcarrier made of sepharose, polystyrene, ceramic or the like, and has a diameter of about 180 μm. A fluidized bed 11 of the carrier 38 is formed on the filter 9.

  The culture medium supply line 3 is connected upstream with a cell culture device 12 for culturing cells, and a culture medium supply pump 13 is provided in the middle. The cell culture device 12 supplies the culture medium 14 including the carrier 38 with the cells attached thereto from the culture medium supply line 3 into the peeling tank 2 via the culture medium supply pump 13.

  The buffer solution supply line 4 is connected to the buffer solution supply source 15 on the upstream side, and supplies a buffer solution such as PBS (phosphate buffered saline) into the peeling tank 2 through the buffer solution supply line 4. The stripping solution supply line 5 is connected to the stripping solution supply source 16 on the upstream side, and supplies a stripping solution such as EDTA solution (ethylenediaminetetraacetic acid) into the stripping tank 2 through the stripping solution supply line 5.

  The waste liquid line 6 is connected to a waste liquid tank 43 (see FIG. 1) for discharging the excess medium 14 and the like on the downstream side. A waste liquid pump 17 is provided in the middle of the waste liquid line 6, and the liquid in the peeling tank 2 is discharged to the waste liquid tank 43 by driving the waste liquid pump 17.

  The cell collection line 7 is connected to the cell introduction line 19 on the downstream side through a sterile connector 18. A collection pump 21 is provided in the middle of the cell collection line 7. By driving the collection pump 21, cells in the separation tank 2 are collected in the cell collection container 22 through the cell introduction line 19. The cell collection container 22 constitutes a part of the cell collection device 28.

  The cell collection container 22 is, for example, a centrifuge tube, and the medium 14 is previously introduced therein and is closed by a lid 23. The cell introduction line 19 penetrates through the lid 23, and the cell introduction line 19 extends into the cell collection container 22 and opens near the bottom of the cell collection container 22. An air supply / discharge line 24 is connected to the lid body 23. The air supply / discharge line 24 opens above the liquid level of the culture medium 14, and an air filter 25 is provided in the middle.

  Next, the cell detachment process using the cell detachment apparatus 1 will be described using the process diagram of FIG.

STEP: 01
When the cell detachment process is started, the medium supply pump 13 is first driven, and the medium 14 containing the cells cultured in the cell culture device 12 is supplied into the detachment tank 2 via the medium supply line 3. At this time, the flow rate of the culture medium 14 supplied into the peeling tank 2 is determined by the number of cells to be collected.

STEP: 02
When the culture medium 14 having a desired flow rate is supplied into the peeling tank 2, the culture medium supply pump 13 is stopped and the waste liquid pump 17 is driven so that the culture medium 14 in the peeling tank 2 passes through the waste liquid line 6. To 43. At this time, since the mesh size of the filter 9 is smaller than the diameter of the carrier 38, the medium 14 is filtered, and the carrier 38 contained in the medium 14 is separated on the filter 9.

STEP: 03
When the culture medium 14 is discharged, next, PBS is supplied into the peeling tank 2 through the buffer solution supply line 4, and the culture medium 14 attached to the cell-attached carrier 38 is washed. The flow rate of PBS supplied into the peeling tank 2 is set to a flow rate sufficient for washing the carrier 38, for example, the same flow rate as that of the culture medium 14.

STEP: 04
After the PBS is kept in the peeling tank 2 for a predetermined time, the waste liquid pump 17 is driven, and the PBS is discharged from the peeling tank 2 through the waste liquid line 6. In addition, it is desirable to repeat two steps of STEP: 03 and STEP: 04 a plurality of times so that the culture medium 14 attached to the carrier 38 can be sufficiently washed.

STEP: 05
When the cleaning of the carrier 38 is completed, the EDTA liquid is then supplied into the peeling tank 2 through the peeling liquid supply line 5. When the EDTA solution acts on the carrier 38, the state of the interface between the cells and the carrier 38 becomes a state where the cells are easily detached from the carrier 38. The flow rate of the EDTA liquid supplied into the peeling tank 2 is set to a flow rate suitable for the carrier 38 deposited on the filter 9 to become the slurry fluidized bed 11. For example, when the liquid level of the EDTA liquid is about 5 mm above the filter 9 and the concentration of the carrier 38 in the EDTA liquid is about 5 g / L to 20 g / L, the fluidized bed 11 is suitable for stirring. It becomes the slurry form which has.

STEP: 06
After supplying the EDTA solution, the stirring device 8 is driven to swing the peeling tank 2. For example, the peeling tank 2 is reciprocated in the horizontal direction at a rotation speed of 10 rpm to 200 rpm. The fluidized bed 11 is agitated by the reciprocating motion of the separation tank 2, and shearing stress due to agitation and impact due to collision between the carriers 38 can be exerted between the carrier 38 and the cells, so that the cells are detached from the carrier 38. . The time for reciprocating the peeling tank 2 is desirably set to about 5 minutes, for example, so that the EDTA solution does not adversely affect the cells.

STEP: 07
After detaching the cells from the carrier 38, the recovery pump 21 is driven, and the cells are introduced into the medium 14 in the cell recovery container 22 together with the EDTA solution via the cell recovery line 7 and the cell introduction line 19, and the cells are detached. End the process. After the cell detachment process is completed, the cell recovery line 7 can be detached from the aseptic connector 18, and the cell recovery container 22 can be used for various applications.

  At this time, since the mesh size of the filter 9 is larger than that of the cells and smaller than that of the carrier 38, only the carrier 38 remains on the filter 9 and only the cells are collected in the cell collection container 22. In the cell collection container 22, about 10 times the medium 14 of the EDTA solution is introduced in advance, and the EDTA solution is diluted to a concentration that does not adversely affect the cells.

  A syringe may be connected to the air supply / discharge line 24 to collect cells using the syringe. When collecting cells by a syringe, the collection pump 21 can be omitted.

  In the above-described embodiment, the carrier 38 is deposited on the filter 9 in the peeling tank 2, and the EDTA liquid, which is a peeling liquid, is supplied to form a slurry fluidized bed 11, and the fluidized bed 11 is stirred to support the cells. Since the cells are separated from the cells 38 and the cells are collected, a large amount of cells can be collected easily and at once without the need for a pipette, and can be applied to industrial applications.

  Since the embodiment described above does not require a robot or the like for separating and collecting cells from the carrier 38, the apparatus configuration can be simplified and miniaturized, and the manufacturing cost can be reduced. .

  Furthermore, since the flow rate of the supplied EDTA solution is set to a minimum flow rate so that the carrier 38 becomes a slurry when the cells are peeled off, the EDTA solution can be diluted with the medium 14 in the cell recovery container 22, and the EDTA solution can be diluted. The influence of cells on the cells can be suppressed.

  Furthermore, by reducing the size of the cell peeling device 1, the entire cell culture system 26 including the cell introduction device 27, the cell culture device 12, and the cell recovery device 28 can be reduced in size.

  Next, a second embodiment of the cell peeling device 1 will be described. FIG. 4 shows a cell peeling apparatus 1 according to the second embodiment. This cell culture device is different from the cell peeling device (FIG. 1) of the first embodiment in that the stirring device 8 is changed. In the cell peeling apparatus of the first embodiment, the stirring device 8 is stirring the peeling tank 2 from the outside by a swing mechanism, whereas in the cell peeling apparatus of the second embodiment, the stirring tank 8 is placed in the peeling tank 2. The stirrer 8 is placed so that the stripping solution can be directly stirred. By doing so, the shear stress for peeling the cells from the carrier can be effectively applied to the carrier, and the cell recovery rate is improved.

  As shown in FIG. 4, the stirring device 8 includes a fan unit 62 having a plurality of rotating blades having a shape adapted to the inner wall (shape) of the lower part of the peeling tank 2, a rotating shaft 60 of the rotating blades, and an integrated rotating shaft. A passive pulley 56 that rotates, a drive shaft 55 having a drive pulley 54, a motor 52 that rotates the drive shaft, and a belt 58 wound around the passive pulley 56 and the drive pulley 54. . When the motor 52 rotates, the drive pulley 54 rotates, and the rotating shaft 60 rotates through the belt 58 and the passive pulley 56.

  FIG. 5 is a perspective view of the fan unit 62, and the fan unit 62 has six rotating blades 62A at equal intervals. The fan unit 62 rotates around a hollow rotating shaft 63. The filter 9 described above is attached to the lower end 62 </ b> B of the rotating shaft 63. Further, the upper end 62 </ b> C of the rotating shaft 63 is joined to the rotating shaft 60 in a watertight manner. The rotating shaft 60 extends to the outside of the peeling tank while extending in the vertical direction in the peeling tank 2, and the tip 60 </ b> A is joined to the extended end of the cell recovery line 7 in a watertight manner. The rotary shaft 60 can also pass a stripping solution containing cells inside.

  When the stripping solution is supplied into the stripping tank 2, the fan unit 62 is rotated by the rotation of the motor 52. Since part or all of the fan unit 62 is immersed in the stripping solution, when the fan unit 62 rotates, the stripping solution containing the carrier to which the cells are attached is directly stirred. When the stripping solution is directly stirred by the fan unit 62, shear stress is effectively applied to the carrier and the cells, and the cells are sufficiently detached from the carrier. As a mode of rotation of the fan unit 62, in addition to constant speed rotation, a mode in which the rotation speed is changed regularly or irregularly, a mode in which normal rotation and reverse rotation are repeated, a mode in which rotation and stop are repeated, and the like are used. Various aspects for enlarging can be employed. It should be noted that the stripping solution may be such that the carrier is immersed in the bottom of the stripping tank 2.

  When the fan unit 62 is rotated and the cells are sufficiently detached from the carrier, the fan unit 62 is stopped and the recovery pump 21 is driven. A filter 9 is attached to the tip of the fan unit 62, and the filter 9 is connected to the cell recovery line 7 via the cavity of the fan unit 62 and the cavity of the rotating shaft 60, so that the recovery pump 21 is driven. Then, the peeling solution containing the cells peeled from the carrier is collected in the cell collection container 22.

  FIG. 6 is a modification of the fan unit 62. The diameter of the filter 9 is enlarged. The tip of the fan unit 62 corresponding to this is also enlarged. By increasing the diameter of the filter 9, it is easy to prevent clogging of the filter.

  A third embodiment of the peeling apparatus 1 is shown in FIG. The peeling apparatus according to this embodiment is a fan unit having a fluid bed above the filter 9 having a diameter that is slightly smaller than the inner diameter of the peeling tank and that is within the height range of the fluidized bed 11. 62. According to this embodiment, the shear stress is directly applied to the fluidized bed by the rotation of the fan unit 62.

  A fourth embodiment of the peeling apparatus 1 is shown in FIG. In the stripping apparatus according to this embodiment, the recovery line 7 extends from the top of the stripping tank 2 to the bottom of the stripping tank, and the fan unit 62 of the stirring device 8 is provided at the bottom of the stripping tank 2 separately from the recovery line. The stripping solution is directly stirred. A filter 9 is provided at the bottom end of the separation tank 2 of the recovery line 7. According to this embodiment, since the fan unit 62 and the collection line 7 are separate, the complexity of the structure of the fan unit 62 can be avoided.

DESCRIPTION OF SYMBOLS 1 Cell peeling apparatus, 2 peeling tank, 3 culture medium supply line, 4 buffer supply line, 5 peeling liquid supply line, 6 waste liquid line, 7 cell collection line, 8 stirring apparatus, 9 filter, 14 culture medium, 38 support | carrier

Claims (8)

A first step of feeding a culture medium containing a carrier to which cells are attached to a peeling tank;
A second step of separating the carrier from the medium;
A third step of applying a stripping solution to the carrier separated in the second step;
A fourth step of stirring the stripping solution containing the carrier in the stripping tank;
A fifth step of separating the cells detached from the carrier by the fourth step from the carrier;
A cell detachment method comprising: A peeling tank;
A medium supply mechanism for supplying a medium containing a carrier with cells attached to the peeling tank;
A discharge mechanism for separating the medium from the carrier and discharging the medium out of the peeling tank;
A stripping solution supply mechanism for supplying a stripping solution for stripping the cells from the carrier into the stripping tank;
A stirring device for stirring the carrier and the stripping solution in the stripping tank;
A delivery mechanism that separates the cells detached from the carrier after the stirring by the stirring device from the carrier and sends them out of the peeling tank;
A cell peeling apparatus comprising:   The cell detachment apparatus according to claim 2, wherein the agitation apparatus agitates the carrier and the detachment liquid in the detachment tank from outside the detachment tank.   The cell detaching device according to claim 3, wherein the stirring device swings the stirring layer.   The cell detachment apparatus according to claim 2, wherein the agitator directly agitates the carrier and the detachment liquid in the detachment tank in the detachment tank.   The cell aggregating apparatus according to claim 5, wherein the agitation device has a rotating blade that rotates at a lower portion of the exfoliating apparatus. The discharge mechanism is provided in a lower part of the peeling tank, and the carrier is separated from the medium by a filter having a mesh larger than the cells and smaller than the carrier,
The cell peeling apparatus according to claim 2, wherein the recovery mechanism separates the peeling solution containing the cells from the carrier by the filter. A cell introduction device;
A cell culture device;
The cell detaching device according to any one of claims 2 to 7,
A cell recovery device;
With
The cell introduction device introduces a carrier with cells attached to the cell culture device,
The cell culture device suspends the carrier in a medium and cultures the cells on the carrier;
The cell detaching device detaches the cells from the carrier contained in the medium supplied from the cell culture device;
The cell collection device collects the detached cells;
Cell culture system.

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