JPWO2014157204A1 - Incubator - Google Patents

Abstract

The culture apparatus includes a container that forms a closed space for storing a medium for culturing cells, a blade 52 that is housed in the closed space, and scrapes off deposits on the bottom surface 41a of the container, and the blade 52. A scraper 5 having a support part 51 for supporting the blade, a levitating mechanism for levitating and fixing the scraper 5 to the container so that the blade 52 is separated from the bottom surface 41a of the container, and a blade disposed outside the closed space. And a drive unit for moving the scraper in a state where 52 is in contact with the bottom surface 41a of the container, the scraper 5 has a magnetic force generator 53, and the drive unit sandwiches the magnetic force generator 53 in the moving direction of the scraper 5. The magnetic force generators 38 and 39 are disposed on the scraper 5, and power is transmitted to the scraper 5 through the magnetic force generators 53, 38 and 39.

Description

  The present disclosure relates to an apparatus for culturing cells.

  A culture method is widely used in which cells are cultured in a container such as a petri dish, and the cells adhering to the bottom surface of the container are physically or chemically detached and recovered after the culture. For example, in fields such as regenerative medicine where it is assumed that cultured cells are returned to the body, the physical method of scraping cells with a scraper is better than the chemical method of peeling cells with chemicals. Is suitable.

  Patent Document 1 discloses an apparatus used for detaching cells by a physical method. The apparatus includes a blade that is inserted into the container, a holder that presses the blade against the bottom surface of the container, and a rotating unit that rotates the blade together with the holder. Scrap the upper cells.

  Patent Documents 2 and 3 disclose techniques for driving a scraper by magnetic force.

  Patent Document 4 discloses a wiper device that separates a blade rubber from a surface to be wiped by a magnetic force.

  U.S. Patent No. 6,099,077 discloses a culture assembly in which a scraper is placed in a sealed annular dish.

  Patent Document 6 discloses a multistage culture system in which a plurality of circular plates for culturing cells are stacked in multiple stages.

  Patent Document 7 discloses a magnetic support mechanism that can float a floating part with respect to a fixed part by a magnetic force and maintain the floating state. The fixed portion has two permanent magnets spaced apart from each other, and the floating portion has one permanent magnet disposed between the two permanent magnets.

JP 2006-325517 A US Pat. No. 6,790,654 U.S. Pat. No. 5,930,344 JP-A-10-167014 Special table 2008-545427 gazette Japanese Patent Publication No. 60-36277 JP 2001-284438 A

  An object of the present disclosure is to provide a culture apparatus and a culture method useful for culturing and collecting cells in a closed system.

  A culture apparatus according to the present disclosure includes a container that forms a closed space for storing a medium for culturing cells, a blade that is stored in the closed space, and scrapes off deposits on the bottom surface of the container, and A scraper having a support portion for supporting the blade, a levitating mechanism for levitating and fixing the scraper to the container so that the blade is separated from the bottom surface of the container, and the blade is disposed outside the closed space. And a drive unit that moves the scraper in contact with the scraper, the scraper having a first magnetic force generator, and the drive unit is disposed so as to sandwich the first magnetic force generator in the moving direction of the scraper. It has a 2nd and 3rd magnetic force generation body, and transmits motive power to a scraper via the 1st, 2nd and 3rd magnetic force generation body.

  According to this culture apparatus, the scraper can be separated from the bottom surface of the container during the culture so as not to inhibit the cell culture. Further, since the scraper is fixed to the container in a floating state, the first magnetic force generator can be easily positioned when the container is installed on the drive unit. For this reason, the arrangement of the first, second, and third magnetic force generators can be easily adjusted so that the second and third magnetic force generators sandwich the first magnetic force generator in the moving direction of the scraper. By adjusting the arrangement in this way, a sufficient force can be transmitted from the second and third magnetic force generators to the first magnetic force generator, and the scraper can be moved firmly. Therefore, the cells can be efficiently cultured and the cultured cells can be scraped off firmly.

  The levitation mechanism may further include a fourth magnetic force generator disposed outside the container, and the scraper may be lifted by the magnetic force between the first and fourth magnetic force generators.

  The support portion may have a rotation shaft hole, and the container may have a substantially circular bottom surface and a rotation shaft for insertion into the rotation shaft hole of the scraper. The rotating shaft may have a flange portion whose outer diameter is larger than the inner diameter of the rotating shaft hole on the upper end side, and the upper peripheral edge portion and the flange portion of the rotating shaft hole may each have a recessed portion or a protruding portion to be fitted. The rotation shaft and the rotation shaft hole may guide the vertical movement of the scraper in the container. The diameter of the bottom surface of the container may be 30 to 400 mm.

  The levitation mechanism may further include a plate to which the fourth magnetic force generator is fixed and a plate guide for guiding the slide of the plate. The scraper can be lifted by sliding the plate, and if it is necessary to lift the scraper again, adjust the position of the scraper and slide the plate again. Can surface.

  The scraper has two first magnetic force generators, the support portion has a rod shape, the two first magnetic force generators are arranged in the longitudinal direction of the support portion, and the levitation mechanism has 2 Each of the fourth magnetic force generators has a shape extending in one direction, the two fourth magnetic force generators are fixed to be aligned in the longitudinal direction of the plate, and the plate guide is long The plate may be guided to slide along the direction. The plate and the plate guide are disposed above the container, and the scraper may float by the attractive force between the first and fourth magnetic force generators. The plate and the plate guide are disposed below the container, and the scraper may float by repulsive force between the first and fourth magnetic force generators.

  The flatness of the bottom surface of the container may be 40 to 800 μm. The material of the blade may be silicone rubber. The container may have a liquid feeding port and a vent port on the side wall.

  The support portion is provided so as to be rotatable about a single axis intersecting the bottom surface of the container, and the drive unit has a drive rotating body provided so as to be rotatable about the single axis. The second and third magnetic force generators are provided in the drive rotor, and the first, second, and third magnetic force generators each have a rectangular shape in plan view, and the first magnetic force generator, the second magnetic force generator, The third magnetic force generator is provided so as to be inclined with respect to each other in a plan view when the phase shift of the support portion with respect to the drive rotator is not generated, and the tilt becomes small when the phase shift occurs. It may be set as follows.

  The support portion is provided so as to be rotatable about a single axis intersecting the bottom surface of the container, and the drive unit has a drive rotating body provided so as to be rotatable about the single axis. The second and third magnetic force generators are provided in the drive rotator, and the first, second, and third magnetic force generators each have a rectangular shape in plan view, and the first, second, and third magnetic force generators. Each of the generators has a shape extending in one direction in plan view, and the first magnetic force generator and the second and third magnetic force generators do not have a phase shift of the driven rotor relative to the drive rotor. Sometimes, they are provided so as to be inclined with respect to each other in a plan view, and the inclinations may be set to be small when a phase shift occurs.

  If the magnetic force generators are configured not to tilt when no phase shift occurs, the tilt between the magnetic force generators increases when the phase shift occurs. Compared to such a configuration, since the inclination between the magnetic force generators becomes smaller during the rotation of the drive rotator and the support portion, the power transmitted from the second and third magnetic force generators to the first magnetic force generator is increased. . Therefore, the cells can be scraped more firmly.

  The first, second and third magnetic force generators are permanent magnets, the poles of the first permanent magnet are arranged along the moving direction, and the poles of the second magnetic force generator are the same as those of the first magnetic force generator. The poles are arranged vertically so as to attract the poles on the second magnetic force generator side of the poles, and the poles of the third magnetic force generator attract the poles on the third magnetic force generator side among the poles of the first magnetic force generator. You may line up and down. In this case, the first, second, and third magnetic force generators form a magnetic force line loop, and more sufficient power can be transmitted from the drive unit to the scraper. Therefore, the cells can be scraped more firmly.

The scraper may have two or more blades or one blade having a notch in the middle. The container has a substantially circular bottom surface and a rotation shaft, and the support portion includes a rotation shaft hole into which the rotation shaft is inserted, and one or more blades extending in the radial direction from the rotation shaft hole. And a first magnetic force generator may be disposed on the arm. The scraper has two blades and two first magnetic force generators, and the support portion has two arms extending in different radial directions from the rotation shaft hole, and the two blades are 2 The two first magnetic force generators may be respectively disposed on the two arms. The blade supported by one arm extends from the vicinity of the rotation shaft hole to the position of the radius r1 of the container, and the blade supported by the other arm extends from the position of the radius r2 of the container to the tip side of the arm. The radius r1 and the radius r2 may satisfy the condition of the formula (1).
r1 ≧ r2 (1)

  The levitation mechanism can switch between a floating state of the scraper and a released state of the scraper in both directions. In the levitation state, the levitation mechanism raises the scraper when the first magnetic force generator is in the levitation position. And the drive unit starts the movement of the scraper with the second and third magnetic force generators sandwiching the flying position in the moving direction, and the second and third magnetic force generators move. The scraper may be configured to stop moving with the levitation position sandwiched in the direction. In this case, it is possible to adjust the arrangement of the first, second and third magnetic force generators so that the second and third magnetic force generators sandwich the first magnetic force generator in the moving direction of the scraper. Can be executed.

  According to the present disclosure, it is possible to provide a culture apparatus and a culture method that are useful for culturing and collecting cells in a closed system.

It is sectional drawing which shows schematic structure of the culture apparatus which concerns on this embodiment. It is a perspective view of an incubator. It is a perspective view which shows the state which removed the crown-shaped member in FIG. It is sectional drawing which follows the IV-IV line | wire in FIG. It is sectional drawing which shows the state which inserted the plate in FIG. It is sectional drawing which follows the VI-VI line in FIG. It is a top view which shows the positional relationship between magnetic force generation bodies. In FIG. 7, it is a top view which shows the state which the phase shift of the driven rotary body with respect to the drive rotary body produced. It is a top view which shows typically other embodiment of the incubator which concerns on this invention. It is a top view which shows the other example of a scraper. It is a top view which shows the other example of a scraper. It is a top view which shows the other example of a scraper. It is a top view which shows the other example of a scraper.

  Hereinafter, embodiments will be described in detail with reference to the drawings. In the description, elements having the same or the same function are denoted by the same reference numerals, and redundant description is omitted.

<Culture equipment>
A culture apparatus 1 shown in FIG. 1 is an apparatus that can culture cells in an incubator and then scrape adherent cells on the bottom of the incubator by remote control. The culture apparatus 1 includes an incubator 2 and a drive unit 3. Examples of cells to be cultured include adherent cells (dendritic cells, mesenchymal stem cells and fibroblasts, microglia cells, osteoblasts, cardiomyocytes, embryonic stem cells, induced pluripotent stem cells, etc.). For example, it can be differentiated into dendritic cells by culturing monocytes in leukocytes.

  The incubator 2 includes a container 4, a scraper 5, and a buoyancy generation unit (levitation mechanism) 6. The container 4 contains a medium for culturing cells. The scraper 5 is accommodated in the container 4 together with the culture medium, and rotates in the container 4 after cell culture to scratch the bottom surface 41a of the container 4. The buoyancy generating unit 6 is disposed on the top of the container 4 and separates the scraper 5 from the bottom surface 41a. The drive unit 3 is disposed outside the container 4 and rotates the scraper 5 after cell culture.

(container)
The container 4 is made of, for example, a transparent resin material, and includes a circular bottom plate 41 and side walls 42 provided along the periphery of the bottom plate 41 as shown in FIGS. At the center of the inner surface 41a (bottom surface 41a) of the bottom plate 41, a convex portion 43 for attaching the scraper 5 is formed. The upper part of the container 4 is closed with a transparent film (not shown) as a lid. The peripheral edge of the film is joined to the upper end of the side wall 42 by, for example, welding. Thereby, the closed space S <b> 1 is formed in the container 4.

  The bottom surface 41a of the container 4 is substantially circular. The diameter of the bottom surface 41a is preferably 30 to 400 mm, more preferably 50 to 250 mm, and still more preferably 110 to 250 mm. If the diameter is 30 mm or more, a sufficient amount of cells can be cultured in one container 4. On the other hand, if it is 400 mm or less, a sufficiently high flatness container can be produced by injection molding using a resin material. . The flatness of the bottom surface 41a of the container 4 is preferably 40 to 800 μm, more preferably 40 to 500 μm, and still more preferably 40 to 150 m. If the container has a flatness of 40 μm or more, a container satisfying this condition can be produced even if a resin material is used. On the other hand, if the container has a flatness of 800 μm or less, uneven seeding is less likely to occur.

  The side wall 42 has a liquid feeding port 44 and a ventilation port 45. The liquid feeding port 44 is a port for allowing the medium and cells to flow in or out, and communicates with the closed space S1. The liquid supply port 44 is formed integrally with the side wall 42 and has a cylindrical shape protruding obliquely upward from the vicinity of the bottom plate 41. A liquid feeding tube (not shown) is detachably connected to the liquid feeding port 44. The aeration port 45 is a port for allowing the gas in the closed space S1 to flow in or out along with the inflow and outflow of the culture medium and the cells, and enters the closed space S1 via a filter (not shown) for preventing foreign matter from entering. Communicate. The ventilation port 45 is formed integrally with the side wall 42 and has a cylindrical shape protruding sideways. The position of the ventilation port 45 is different from the position of the liquid feeding port 44 in the circumferential direction of the side wall 42. The position of the base end portion of the ventilation port 45 is higher than the position of the base end portion of the liquid feeding port 44. With such an arrangement, leakage of the culture medium and cells from the vent port 45 is prevented.

(Scraper)
As shown in FIGS. 3 and 4, the scraper 5 accommodated in the closed space S <b> 1 includes a driven rotating body (supporting portion) 51, two blades 52, and two permanent magnets (first magnetic force generation). Body) 53. The two permanent magnets 53 constitute a levitation mechanism of the scraper 5 together with the buoyancy generating unit 6.

  The driven rotating body (supporting portion) 51 is made of, for example, the same resin material as the material of the container 4, and includes two cylindrical arms 54 and two driven arms 55 extending from the peripheral surface of the cylindrical portion 54 to opposite sides. , 56.

  The cylindrical portion 54 stands upright with respect to the bottom surface 41 a and surrounds the convex portion 43. A pin (rotating shaft) 21 is inserted through the hole (rotating shaft hole) of the cylindrical portion 54 from above, and the lower end portion of the pin 21 is attached to the convex portion 43. A flange portion 21 a is formed at the upper end portion of the pin 21. The flange portion 21a regulates the rise of the cylindrical portion 54. The driven rotator 51 is attached to the bottom plate 41 by a pin 21 and is rotatable about an axis CL1 orthogonal to the bottom surface 41a. Depending on the material of the lid covering the container 4, the pin 21 may be provided on the lid.

  The driven rotor 51 can float with the pin 21 as a guide, and the flying height is limited by the flange portion 21a. A rotation restricting convex portion 21b is formed under the flange portion 21a, and a concave portion 54a that can be fitted to the convex portion 21b is formed at the upper end portion of the cylindrical portion 54. For this reason, when the driven rotating body 51 rises to the upper limit, the driven rotating body 51 becomes non-rotatable due to the fitting of the convex portion 21b and the concave portion 54a.

  The lengths of the driven arms 55 and 56 are different from each other, and the driven arm 55 is shorter than the driven arm 56. The distal end of the driven arm 55 is located between the cylindrical portion 54 and the side wall 42, and the distal end of the driven arm 55 is located in the vicinity of the side wall 42. The angle formed by the two driven arms 55 and 56 is not necessarily 180 °, but is preferably in the range of 160 ° to 200 °. If this angle is 160 ° to 200 °, the scraper 5 can be lifted or lowered in a well-balanced manner by using the magnetic force of the permanent magnets 53 arranged on different sides across the rotation axis.

  The two blades 52 are detachably attached under the driven arms 55 and 56, respectively. The blade 52 is made of, for example, an elastic material such as silicone rubber, and extends along the extending direction of the driven arms 55 and 56 and protrudes downward from the driven arms 55 and 56. A thin blade edge 52a is formed at the lower edge of the blade 52 (see FIG. 6). The blade edge portion 52 a contacts the bottom surface 41 a and scratches the bottom surface 41 a as the driven rotating body 51 rotates. Further, the blade edge portion 52 a is separated from the bottom surface 41 a as the driven rotating body 51 floats.

The blade 52 attached to the shorter driven arm 55 is located near the center of the bottom surface 41a and extends from the vicinity of the cylindrical portion 54 to the position of the radius r1 of the bottom surface 41a. The blade 52 attached to the longer driven arm 56 is located outside the bottom surface 41a and extends from the position of the radius r2 of the bottom surface 41a to the tip side. Here, the radius r1 and the radius r2 satisfy the condition of the formula (1).
r1 ≧ r2 (1)

  The above formula (1) means that a part of the range where the blade 52 of the driven arm 55 is scratched and the range where the blade 52 of the driven arm 56 is scratched overlap. As a result, the bottom surface 41a can be scratched with a small gap only by rotating the scraper 5 once.

  The two permanent magnets 53 are disposed on the driven arms 55 and 56, respectively. The permanent magnet 53 is separated from the rotation axis CL <b> 1 and is located on the blade 52. The permanent magnet 53 is a rectangular parallelepiped extending along the protruding direction of the driven arms 55 and 56. That is, the permanent magnet 53 has a rectangular shape (rectangular shape) extending in one direction in plan view. The N pole 53N and the S pole 53S of the permanent magnet 53 are aligned in the width direction of the driven arms 55 and 56 (see FIG. 6). The permanent magnet 53 is accommodated in a magnet accommodating portion 57 formed on each of the driven arms 55 and 56, and the magnet accommodating portion 57 is sealed by a lid member 58.

(Buoyancy generating unit)
As shown in FIG. 2, the buoyancy generating unit 6 includes a plate 61 and a crown-shaped member 62 that holds the plate 61 on the container 4. The plate 61 has a thin plate shape extending in one direction, and is disposed horizontally along the radial direction of the container 4. The plate 61 has two iron plates (fourth magnetic force generators) 63 arranged in the longitudinal direction. The two iron plates 63 attract the two permanent magnets 53 to generate buoyancy in the scraper 5. As described above, the iron plate 63 also corresponds to a magnetic force generator because it generates a magnetic force in cooperation with the permanent magnet 53.

  The crown-shaped member 62 includes an annular frame 64 attached to the upper end portion of the side wall 42, and a plate guide 65 that spans the annular frame 64 along the radial direction of the container 4. The plate guide 65 extends so as not to pass through the positions of the liquid feeding port 44 and the ventilation port 45. Both ends of the plate guide 65 are open so that the plate 61 can be taken in and out.

  The plate 61 and the plate guide 65 are disposed above the container 4 and float the scraper 5 by the attractive force between the permanent magnet 53 of the scraper 5 and the iron plate 63 of the plate 61. In the state where the plate 61 is removed from the plate guide 65, no buoyancy is generated in the scraper 5, so that the blade 52 contacts the bottom surface 41a as shown in FIG. On the other hand, when the plate 61 is inserted into the plate guide 65 and the scraper 5 is along the plate 61, buoyancy is generated in the scraper 5, so that the scraper 5 is lifted as shown in FIG. Separate from. At this time, the convex portion 21b of the pin 21 and the concave portion 54a of the cylindrical portion 54 are fitted, and the scraper 5 cannot rotate. Thereby, the scraper 5 is firmly fixed to the container. Since the scraper is fixed to the container also when the permanent magnet 53 is attracted to the iron plate 63, the convex portion 21b and the concave portion 54a are not essential. 4 and 5, illustration of the plate guide 65 is omitted.

  Since the pair of the iron plate 63 and the permanent magnet 53 is disposed at two positions sandwiching the pin 21, the buoyancy of the scraper 5 is generated at two positions sandwiching the pin 21. For this reason, the scraper 5 can be kept in a floating state more firmly. The floating state of the scraper 5 can be released by sliding the plate 61 and shifting the positions of the permanent magnet 53 and the iron plate 63. Thereafter, to raise the scraper 5 again, the scraper 5 can be lifted by magnetic force by first adjusting the angle of the scraper 5 to the position of the plate guide 65 and then inserting the plate 61 into the plate guide 65. Thus, the levitation mechanism can switch between the floating state of the scraper 5 and the released state of the scraper 5 in both directions. In the floating state, the permanent magnet 53 is positioned below the plate guide 65 (for levitation). The scraper 5 is levitated and fixed to the container.

Since the plate guide 65 extends so as not to pass through the liquid feeding port 44 and the ventilation port 45, the plate 61 and the scraper 5 are held without passing through the liquid feeding port 44 and the ventilation port 45. For this reason, liquid supply via the liquid supply port 44 or ventilation through the ventilation port 45 is not hindered by the scraper 5. The state in which liquid feeding or air feeding is hindered is, for example, a state in which a part of the liquid feeding port 44 or the ventilation port 45 is blocked by the end of the driven arm 55.
(Drive unit)

  As shown in FIG. 1, the drive unit 3 includes a housing 31, a power source 32, a drive rotator 33, and two attractive force generators 34. The housing 31 supports the container 4 horizontally. Hereinafter, the arrangement of the power source 32, the drive rotator 33, and the attractive force generator 34 will be described with reference to the container 4 supported by the casing 31.

  The power source 32 is provided in the housing 31 and is located below the container 4. The power source 32 has a rotating shaft 32a protruding upward, and incorporates a motor and a speed reducer (not shown) that rotate the rotating shaft 32a. The central axis CL2 of the rotation shaft 32a coincides with the rotation axis CL1 of the scraper 5.

  The drive rotator 33 is fixed to the upper end portion of the rotary shaft 32a, and has two drive arms 35 and 36 extending from the peripheral surface of the rotary shaft 32a to opposite sides. The length of the drive arm 35 corresponds to the length of the driven arm 55, and the length of the drive arm 36 corresponds to the length of the driven arm 56.

  Two attractive force generators 34 are provided on the drive arms 35 and 36, respectively. When the drive arms 35 and 36 are positioned below the driven arms 55 and 56, the attractive force generator 34 is positioned below the permanent magnet 53 of the driven arms 55 and 56.

  As shown in FIG. 6, the attractive force generator 34 includes a rectangular iron plate 37 and permanent magnets 38 and 39 (second and third magnetic force generators). The iron plate 37 is fixed horizontally on the drive arms 35 and 36, and the permanent magnets 38 and 39 are fixed on the iron plate 37. The permanent magnets 38 and 39 are rectangular parallelepipeds extending in one direction, and are arranged such that the extending directions are along two parallel sides of the iron plate 37. For this reason, the permanent magnets 38 and 39 have a rectangular shape in plan view.

  The attractive force generator 34 is arranged such that the permanent magnets 38 and 39 are sandwiched between the permanent magnets 53 in the moving direction of the permanent magnets 53 as the driven rotating body 51 rotates. The permanent magnet 38 disposed on the N pole 53N side of the permanent magnet 53 is disposed such that the S pole 38S is on the top and the N pole 38N is on the bottom. That is, the poles of the permanent magnets 38 are lined up and down so as to attract the poles of the permanent magnets 53 on the permanent magnet 38 side. The permanent magnet 39 disposed on the S pole 53S side of the permanent magnet 53 is disposed such that the N pole 39N is on the top and the S pole 39S is on the bottom. That is, the poles of the permanent magnets 39 are lined up and down so as to attract the poles of the permanent magnets 53 on the permanent magnet 39 side.

  By arranging the three permanent magnets 53, 38, 39 in this manner, a magnetic field line loop ML connecting these magnets is formed, and a strong attractive force is generated between the attractive force generator 34 and the permanent magnet 53. The iron plate 37 functions as a path of magnetic lines connecting the permanent magnets 38 and 39 and contributes to the enhancement of the attractive force.

  The drive rotator 33 shown in FIG. 1 is rotated by the power source 32 with the attractive force generator 34 positioned below the permanent magnet 53. Prior to this, the plate 61 is removed from the plate guide 65, the scraper 5 is lowered, and the blade 52 contacts the bottom surface 41a. When the drive rotator 33 rotates, power is transmitted to the driven rotator 51 via the attractive force generator 34 and the permanent magnet 53. The scraper 5 is rotated by this power. That is, the power source 32 rotates the drive rotator 33 and rotates the scraper 5 by the power transmitted through the attractive force generator 34 and the permanent magnet 53.

  Since the pair of the attractive force generating part 34 and the permanent magnet 53 is disposed at two positions sandwiching the pin 21, a couple force centered on the axis CL <b> 1 of the pin 21 is generated in the driven rotating body 51. By generating this couple, the driven rotor 51 can be rotated more smoothly around the axis CL1.

  Here, the positional relationship between the attractive force generator 34 and the permanent magnet 53 in plan view will be described. During the rotation of the drive rotator 33 and the driven rotator 51, a shift between the position of the drive rotator 33 and the position of the driven rotator 51 occurs due to the delay of the driven rotator 51. Hereinafter, this shift is referred to as “phase shift”. FIG. 7 is a plan view showing the attractive force generator 34 and the permanent magnet 53 in a state where no phase shift occurs. The state in which no phase shift occurs is a state in which the attractive force generating unit 34 is located below the permanent magnet 53 and the attractive force between the attractive force generating unit 34 and the permanent magnet 53 does not act in the rotation direction of the driven rotor 51. means.

  As shown in FIG. 7, the permanent magnet 53 is along the extending direction of the driven rotor 51. The attractive force generator 34 is inclined with respect to the extending direction of the driven rotor 51. A direction D1 in which the attractive force generator 34 tilts with respect to the extending direction of the driven rotator 51 is set on the opposite side of the rotational direction D2 of the drive rotator 33. That is, when there is no phase shift, the attractive force generator 34 is inclined in the direction D1 with respect to the permanent magnet 53. For this reason, the inclination of the attractive force generator 34 with respect to the permanent magnet 53 becomes smaller when a phase shift occurs as shown in FIG. The angle at which the attractive force generating section 34 tilts with respect to the permanent magnet 53 (hereinafter simply referred to as “the tilt angle of the attractive force generating section 34”) is set to be substantially zero when a phase shift occurs. Specifically, the angle of phase shift is measured in advance, and the inclination angle of the attractive force generator 34 is set so as to coincide with the measured value. The angle of phase shift can be measured, for example, by actually rotating the scraper 5 by the drive unit 3 in a state where the inclination angle of the attractive force generator 34 is temporarily set.

  The inclination of the attractive force generator 34 with respect to the permanent magnet 53 is the inclination of the sides of the permanent magnets 38 and 39 with respect to the side of the permanent magnet 53. The inclination of the attractive force generator 34 with respect to the permanent magnet 53 is also the inclination of the extending direction of the permanent magnets 38 and 39 with respect to the extending direction of the permanent magnet 53.

<Culture method>
A culture method using the culture apparatus 1 will be described. First, the incubator 2 is prepared. At this time, as shown in FIG. 5, it is preferable that the scraper 5 floats, that is, the plate 61 is put in the plate guide 65.

(Seeding process)
Next, a liquid feeding tube is connected to the liquid feeding port 44, and the culture solution is injected into the container 4 through the liquid feeding port 44 (step (A)). The culture solution is a turbid liquid in which cells are dispersed in a liquid medium. Cell seeding may be performed by supplying the turbid solution into the container 4, or previously storing the medium or cells in the container 4 and then supplying the cells or medium to the incubator in the container 4. You may go by.

(Culture process)
The incubator 2 is placed horizontally in an incubator and left for a predetermined period to culture the cells in the container 4 (step (B)). In the culturing process, the cells adhere to the bottom surface 41a. Since the scraper 5 is levitated by the buoyancy generating unit 6, the entire area of the bottom surface 41a can be used for cell culture. In addition, you may exchange a culture medium in the middle as needed. The medium can be exchanged via the liquid feeding port 44.

(Scraping process)
When the culture process is completed, the incubator 2 is taken out from the incubator and installed on the housing 31 of the drive unit 3. At this time, the positions of the drive rotator 33 and the driven rotator 51 are aligned. Next, the scraper 5 is lowered by removing the plate 61 from the plate guide 65, and the blade 52 is brought into contact with the bottom surface 41a. Next, the drive rotator 33 is rotated. Then, power is transmitted to the driven rotor 51 via the attractive force generator 34 and the permanent magnet 53, and the scraper 5 rotates. With this rotation, the blade 52 scratches the bottom surface 41a, so that the cells are peeled from the bottom surface 41a. Specifically, the scraper 5 is rotated with the magnetic force as a driving force by causing the attractive force generator 34 positioned below the incubator 2 to circularly move along the bottom plate 41 of the incubator 2 around the position corresponding to the pin 21. The blade 52 scrapes off deposits on the bottom surface 41a (step (C)).

  Since the scraper 5 is stored in the container 4 in advance, it is not necessary to insert the scraper 5 into the container 4. Further, the power for rotating the scraper 5 is transmitted from the outside of the container 4 to the inside of the container 4 through the attractive force generator 34 and the permanent magnet 53. Therefore, the cells can be detached without opening the closed space S1 of the container 4.

  Here, since the drive rotator 33 and the driven rotator 51 are not directly connected, when the scraper 5 is rotated, the phase shift occurs due to the phase delay of the scraper 5. As described above, the inclination of the attractive force generator 34 with respect to the permanent magnet 53 becomes smaller when a phase shift occurs. If the attractive force generation unit 34 and the permanent magnet 53 are configured not to tilt with respect to each other when no phase shift occurs, the tilt of the attractive force generation unit 34 with respect to the permanent magnet 53 causes a phase shift. Sometimes grows. Compared to such a configuration, since the inclination of the attractive force generating unit 34 with respect to the permanent magnet 53 becomes smaller during the rotation of the drive rotating body and the driven rotating body, the attractive force between the permanent magnet 53 and the attractive force generating unit 34 is large. Become. Furthermore, the angle at which the attractive force generating unit 34 tilts with respect to the permanent magnet 53 becomes substantially zero when a phase shift occurs, so the attractive force between the attractive force generating unit 34 and the permanent magnet 53 becomes larger. By increasing the attractive force between the attractive force generating section 34 and the permanent magnet 53, the blade 52 is firmly attached to the bottom surface 41a, and sufficient power is transmitted from the drive rotating body 33 to the driven rotating body 51. Therefore, the cells can be sufficiently detached.

  Further, in the form in which the poles of the permanent magnets 38, 39, 53 are arranged as shown in FIG. 6, if the inclination of the attractive force generating section 34 with respect to the permanent magnet 53 becomes large, both ends of the permanent magnet 53 In the part, the attracting poles move away and the repulsive poles approach each other. For example, in FIG. 6, the S pole 38S and the N pole 53N are moved away from each other, and the N pole 39N and the N pole 53N approach each other. By reducing the inclination of the attractive force generating unit 34 with respect to the permanent magnet 53, such a phenomenon is also prevented, so that the attractive force between the attractive force generating unit 34 and the permanent magnet 53 is further increased.

  The blade 52 is sandwiched between the permanent magnet 53 and the attractive force generator 34. For this reason, in addition to the weight of the scraper 5, an attractive force between the attractive force generator 34 and the permanent magnet 53 acts on the blade 52. As a result, the contact pressure between the blade 52 and the bottom surface 41a is increased, so that the cells can be more sufficiently detached.

(Recovery process)
When the scraping step is completed, the detached cells are collected from the liquid feeding port 44 together with the medium (step (D)). It is preferable that the container 4 is detached from the drive unit 3 and the plate 61 is inserted into the plate guide 65 to float the scraper 5. By floating the scraper 5, the medium and cells can be collected without being interrupted by the scraper 5.

  By using the culturing device 1, without opening the closed space S <b> 1 of the container 4, that is, in a state where the upper opening of the container 4 is covered with a lid material from the start of the seeding process to the end of the recovery process, Recovery can be performed. That is, according to this embodiment, cell culture and recovery can be performed in a closed system.

  By using the culturing device 1, without opening the closed space S <b> 1 of the container 4, that is, in a state where the upper opening of the container 4 is covered with a lid material from the start of the seeding process to the end of the recovery process, Recovery can be performed. That is, according to this embodiment, cell culture and recovery can be performed in a closed system.

  According to the culture apparatus 1, the scraper 5 can be separated from the bottom surface 41 a of the container 4 at the time of culture so as not to inhibit cell culture. Further, since the scraper 5 is fixed to the container 4 in a floating state, the first magnetic force generator 53 can be easily positioned when the container 4 is installed on the drive unit 3. For this reason, the first, second, and third magnetic force generators 53, 38, 38 are arranged so that the second and third magnetic force generators 38, 39 sandwich the first magnetic force generator 53 in the moving direction of the scraper 5. The arrangement of 39 can be easily adjusted. By adjusting the arrangement in this manner, a sufficient force can be transmitted from the second and third magnetic force generators 38 and 39 to the first magnetic force generator 53, and the scraper can be moved firmly. Therefore, the cells can be efficiently cultured and the cultured cells can be scraped off firmly.

  The levitating mechanism can switch between the levitating state of the scraper 5 and the levitating release state of the scraper 5, and in the levitating state, when the first magnetic force generator 53 is in the levitating position, the scraper 5 is levitated and fixed to the container 4, and the drive unit 3 starts the movement of the scraper 5 with the second and third magnetic force generators 38 and 39 sandwiching the levitating position in the moving direction. The second and third magnetic force generators 38 and 39 may be configured to stop the movement of the scraper 5 with the floating position sandwiched in the moving direction. In this case, the first, second, and third magnetic force generators 53, 38, 38 are arranged so that the second and third magnetic force generators 38, 39 sandwich the first magnetic force generator 53 in the moving direction of the scraper 5. The arrangement of 39 can be adjusted without omission.

  The incubator 2 exemplified by the present embodiment includes a container for storing a medium for culturing cells, a blade that is stored in the container, and scrapes off deposits on the bottom surface of the container and the blade. A scraper having a supporting portion for supporting, and a levitating mechanism for levitating the scraper in the container such that the blade is separated from the bottom surface of the container.

  According to the above incubator, when the cells are cultured, the scraper is raised above the surface of the medium, so that the cells can be prevented from adhering to the scraper, and chemical substances from the blade can be prevented from eluting into the medium. it can. The material of the blade is not particularly limited as long as it has a certain degree of elasticity and can ensure cytotoxicity and other safety. Specific examples include fluorine rubber, butyl rubber, isobrene rubber, butadiene rubber, ethylene propylene rubber, thermoplastic elastomer, polyethylene, polypropylene, and silicone rubber. Silicone rubber is most preferable from the viewpoint of elasticity and slipperiness.

  The levitation mechanism can levitate the scraper by magnetic force. For example, the scraper may have a first magnetic force generator, and the first magnetic force generator may constitute a levitation mechanism together with a fourth magnetic force generator disposed outside the container. Alternatively, the levitation mechanism may further include a fourth magnetic force generator disposed outside the container, and the scraper may be levitated by the magnetic force between the first and fourth magnetic force generators. The term “magnetic force generator” as used herein refers to a member (iron plate, cobalt plate) that generates an attractive force between a magnet (permanent magnet and electromagnet) that is a magnetic field generation source and a magnet that is not a magnetic field generation source. And a nickel plate).

  In order to stably maintain the state in which the scraper has floated, the first and fourth magnetic force generators can be configured as follows. That is, the first magnetic force generator may be formed of a pair of magnetic force generators and the support portion may be formed of a rod-shaped member, and the pair of magnetic force generators may be provided on the rod-shaped member so as to be aligned in the longitudinal direction of the rod-shaped member. . The fourth magnetic force generator is composed of a pair of magnetic force generators, and the levitation mechanism is configured so that the pair of magnetic force generators are fixed at positions separated from each other, and the plate slides along the longitudinal direction of the scraper. You may have a plate guide part to guide. By sliding a plate on which a pair of magnetic force generators are arranged, the scraper can be lifted, and if it is necessary to lift the scraper again, the position of the scraper is adjusted and The scraper can be lifted again by sliding the plate.

  When the plate and the plate guide portion are arranged above the container, the scraper is lifted by the attractive force between the first and fourth magnetic force generators. On the other hand, when a plate and a plate guide part are arrange | positioned under a container, a scraper floats by the repulsive force between the 1st and 4th magnetic force generation body.

  The first magnetic force generator disposed in the scraper can be used not only for floating the scraper but also for remotely operating the scraper with magnetic force. In this case, the rod-shaped member has a rotation shaft hole provided between a pair of magnetic force generators as the first magnetic force generator, and the container is inserted into the substantially circular bottom surface and the rotation shaft hole of the scraper. A configuration having a rotation shaft is preferable. By adopting such a configuration, it becomes possible to scrape cells on the bottom surface by rotating the scraper. Further, the rotation shaft and the rotation shaft hole can play a role of guiding the vertical movement of the scraper in the container.

  The rotating shaft of the container has a flange portion whose outer diameter is larger than the inner diameter of the rotating shaft hole on the upper end side, and the upper peripheral edge portion and the flange portion of the rotating shaft hole each have a recessed portion or a protruding portion to be fitted. Good. By adopting such a configuration, the floating scraper can be made non-rotatable. Thus, the scraper can be kept in a floating state even under a slight impact, which is useful when, for example, it is desired to ship the incubator in a floating state.

  The diameter of the bottom surface of the container can be 30 to 400 mm. The flatness of the bottom surface of the container is preferably 40 to 800 μm. The flatness referred to here is a value obtained on the basis of measurement using a CNC coordinate measuring machine Futcio Apex 910 manufactured by Mitutoyo Corporation. That is, measure eight equidistant points on a concentric circle every approximately 10 mm from the center of the container, and assume two parallel planes passing through the highest and lowest points of the bottom of the container, and the distance between these planes Is flatness.

  The incubator may be used to culture cells in a closed system. In this case, the incubator may further include a lid member disposed so as to close the upper opening of the container. Moreover, it is preferable that a container has a liquid port and a ventilation | gas_flowing port in a side wall.

  The culture apparatus 1 exemplified by the present embodiment is in a state where the blade is in contact with the bottom surface so as to scrape off the incubator and the deposits (cells, cell sheets, secretions during culture, etc.) on the bottom surface of the culture device. And a drive unit for moving the scraper. As described above, the levitating mechanism of the incubator floats the scraper by magnetic force, the scraper has the first magnetic force generator, and the first magnetic force generator is disposed outside the container. The levitation mechanism is configured together with the magnetic force generator. The drive unit has second and third magnetic force generators arranged below the incubator and movable in a direction along the bottom surface of the incubator, and the scraper has the first magnetic force generator, the second and second magnetic force generators. The blade moves in a state in which the blade is in contact with the bottom surface by the magnetic force between the three magnetic force generators.

  According to the above culture apparatus, when the cells are cultured, the scraper is lifted to a position higher than the surface of the medium, thereby preventing the cells from adhering to the scraper and preventing chemical substances from eluting from the blade into the medium. it can. In addition, the drive unit is useful for culturing in a closed system because it enables the scraper to be operated by magnetic force from the outside of the container.

  When the container of the incubator has a substantially circular bottom surface and a rotating shaft as described above, the culture apparatus 1 can be configured as follows. That is, the culture apparatus 1 includes the incubator and a drive unit that moves the scraper while the blade is in contact with the bottom surface so as to scrape off deposits on the bottom surface of the incubator. The drive unit includes second and third magnetic force generators that are arranged below the incubator and circularly move along the bottom surface of the incubator around a position corresponding to the rotation axis, and the scraper includes the first The blade rotates with the magnetic force between the magnetic force generator and the second and third magnetic force generators in contact with the bottom surface. By adopting such a configuration, it becomes possible to scrape cells on the bottom surface by rotating the scraper.

  The culture method exemplified by this embodiment includes (A) a step of supplying cells or a medium to a culture vessel having a scraper in the container, (B) a step of culturing the cells in the container, and (C) the end of the culture. Then, the scraper is levitated to a position higher than the medium in the container, including a step of scraping off the deposits on the bottom surface of the container with a blade of the scraper and (D) discharging the cultured cells from the container. (B) process is implemented in a state.

  According to the above culture method, in the step (B), by raising the scraper to a position higher than the medium surface, it is possible to prevent the cells from adhering to the scraper and to prevent chemical substances from eluting from the blade into the medium. it can. The step (A) may be performed in a state where the scraper is floated at a position higher than the medium in the container, and the step (D) may be performed in this state.

  The culture method may be performed in a closed system. In this case, it is preferable that the cover material covers the upper opening of the container at least from the start of the step (A) to the end of the step (D).

  In the step (C), the first and second magnetic force generators and the second and third magnetic forces are moved by moving the second and third magnetic force generators disposed below the incubator in a direction along the bottom surface of the incubator. The scraper may be moved using the magnetic force between the generator and the generator as a driving force, and the attached matter on the bottom surface may be scraped off with a blade. When the container of the incubator has a substantially circular bottom surface and a rotation axis as described above, in the step (C), the second and third magnetic force generators arranged below the incubator correspond to the rotation axis. The scraper is rotated by using the magnetic force between the first magnetic force generator and the second and third magnetic force generators as a driving force and circularly moved on the bottom surface by a blade. The deposits may be scraped off.

  The culture apparatus 1 disclosed by the present embodiment includes a container that stores a medium for culturing cells, a scraper that is housed in the container together with the medium, scrapes the bottom surface of the container, and a drive that drives the scraper. A scraper, a driven rotating body provided to be rotatable about a single axis intersecting the bottom surface, a blade projecting from the driven rotating body and contacting the bottom surface, and spaced apart from the single axis. A driven-side magnetic force generator provided on the driven rotator, and the drive unit is provided so as to be rotatable about one axis, and the drive rotator is separated from the one axis. The drive side magnetic force generator that draws the driven side magnetic force generator and the drive rotator are rotated, and the scraper is rotated by the power transmitted through the drive side and driven side magnetic force generators. The drive-side and driven-side magnetic force generators each have a rectangular shape in plan view, and when the driven rotor does not have a phase shift with respect to the drive rotor, they are inclined to each other in plan view. The inclination is set to be small when a phase shift occurs.

  The driven-side and drive-side magnetic force generators may each have a shape that extends in one direction in plan view.

  In this culture apparatus, since the scraper is accommodated in the container in advance, it is not necessary to insert the scraper into the container. The power for rotating the scraper is transmitted from the outside of the container to the inside of the container through the magnetic force generator. Therefore, the cells can be detached without opening the container.

  Here, the driven-side and driving-side magnetic force generators are provided so as to be inclined with respect to each other in a plan view when the driven rotating body is not phase-shifted with respect to the driving rotating body, and the tilt is shifted in phase. It is set to be small when it occurs. If the magnetic force generators are configured not to tilt when no phase shift occurs, the tilt between the magnetic force generators increases when the phase shift occurs. Compared to such a configuration, since the inclination between the magnetic force generators becomes smaller during the rotation of the drive rotator and the driven rotator, the attractive force between the magnetic force generators increases. As a result, the blade firmly adheres to the bottom surface, and sufficient power is transmitted from the drive rotating body to the driven rotating body. Therefore, the cells can be sufficiently detached.

  The driven rotator is provided with a first magnet as a driven-side magnetic force generator, and the drive rotator is provided with second and third magnets as a drive-side magnetic force generator. The poles are arranged along the moving direction accompanying the rotation of the driven rotor, the second and third magnets are arranged so as to sandwich the first magnet in the moving direction, and the poles of the second magnet are the first magnets The poles of the third magnet are arranged vertically so as to attract the poles on the second magnet side, and the poles of the third magnet are arranged vertically so as to attract the poles of the first magnet on the third magnet side. May be. In this case, the first to third magnets can form a magnetic force line loop to generate a stronger attractive force. Therefore, the cells can be more sufficiently detached.

  The culture method exemplified by this embodiment includes a culture process in which the culture apparatus 1 is used to store the medium in a container and the cells are cultured, and a scraping process in which the scraper is rotated by the drive unit to scrape the bottom surface. In the scraping step, the drive rotator is rotated so that the inclination of the magnetic force generator on the drive side and the driven side is reduced due to the phase shift of the driven rotator with respect to the drive rotator.

  The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above-described embodiments, and various modifications can be made without departing from the scope of the present invention. For example, the shape of the bottom surface 41a of the container 4 is not limited to a substantially circular shape, and may be another shape. A container 4A shown in FIG. 9 has a substantially rectangular bottom surface 4a. The scraper 5A is configured to float by a magnetic force. Further, the scraper 5A is configured to move from the one side 4b side of the container 4A to the opposite side 4c side, and the cells on the substantially rectangular bottom surface 4a can be scraped off by the two blades 52A.

  When one long blade is adopted without dividing the blade into two, the followability of the blade to the unevenness and the bending of the bottom surface tends to be insufficient. Therefore, by limiting the length of the blade to be used so that the entire bottom surface can be covered with a plurality of blades, the followability to the unevenness and bending of the bottom surface is improved, and the adherent cells are scraped more efficiently. Can be taken. The length of the blade is preferably 20 to 50% of the radius when the culture dish is circular (container 4), and 10 to 50% of the width of the culture dish when the culture dish is rectangular (container 4A). Preferably there is. Preferably, a plurality of blades are used and the total length of the blades is longer than the radius or width of the culture dish.

  10 to 13 show other examples of the scraper that can be mounted on the container 4. As shown in FIG. 10, the angle formed by the driven arms 55 and 56 may be about 90 ° or 90 ° or less. When this angle is within 90 °, the influence of the vertical movement between the two driven arms 55 and 56 is small, and the flatness of the bottom surface of the container can be easily handled. The lower limit of this angle is about 30 °. Because of the lever principle, it is easy to set the driving force of the driven arm 56 (long arm) and the driven arm 55 (short arm) equally, so that the blade arranged in the long arm is shorter than the blade arranged in the short arm. Is preferred. Although it is assumed here that the two driven arms 55 and 56 are integrally formed and rotate at the same time, they may be configured to be able to rotate independently.

  As shown in FIG. 11, the lengths of the two driven arms 55 and 56 may be equal to each other. Further, the driven rotating body 51 does not necessarily have to have the two driven arms 55 and 56, and may have only one driven arm 59 as shown in FIG. 11 and 12, one blade 52 may extend from the cylindrical portion 54 to the side wall 42, but if a long blade is used, the tendency to follow the unevenness and deflection of the bottom surface tends to be insufficient. It is in. Therefore, it is preferable to employ a plurality of short blades as shown in FIG. Or as shown in FIG. 12, you may improve the said followable | trackability by making the notch 52b in the middle of a braid | blade. In the scraper 5B shown in FIG. 13, three blades 52 are arranged on one wide driven arm 59.

  As shown in FIGS. 3 and 10-13, when the culture dish is circular, from the viewpoint of carrying out an efficient scraping operation, the blade is 95% or more of the bottom area of the culture dish by rotating the scraper once. It is preferable to cover. On the other hand, as shown in FIG. 9, when the culture dish is rectangular, from the same viewpoint, the scraper moves from one of the culture dishes to the other (one-way movement), so that the blade is 95% or more of the bottom area of the culture dish. It is preferable to cover the area. That is, it is preferable that the blade scrapes off deposits in an area of 95% or more of the bottom area of the culture dish by one rotation or one-way movement of the scraper (unit operation of the scraper).

  In the above embodiment, the case where the plate 61 and the plate guide 65 are disposed above the container 4 is illustrated, but the plate 61 and the plate guide 65 are disposed below the container 4, and the permanent magnet 53 and the plate 61 of the scraper 5 are disposed. The scraper 5 may be levitated by repulsive force between the magnet and the magnet (fourth magnetic force generator) disposed on the surface.

  Instead of inclining the attractive force generator 34 with respect to the extending direction of the driven rotor 51, the permanent magnet 53 may be inclined with reference to the extending direction of the driven rotor 51. Further, both the attractive force generator 34 and the permanent magnet 53 may be inclined with reference to the extending direction of the driven rotor 51. In either case, the inclination of the attractive force generating unit 34 and the permanent magnet 53 is set so that the direction D1 in which the attractive force generating unit 34 is inclined with respect to the permanent magnet 53 is opposite to the rotational direction D2 of the drive rotating body 33. To do.

  When the permanent magnets 53, 38, and 39 have a rectangular shape in a plan view, the permanent magnets 53, 38, and 39 do not necessarily have a shape that extends in one direction in a plan view, and may have a square shape, for example. Further, when the permanent magnets 53, 38, and 39 have a shape extending in one direction in a plan view, the permanent magnets 53, 38, and 39 do not necessarily have a rectangular shape in a plan view, and may have an elliptical shape, for example.

  The magnetic force generators of the scraper 5 and the drive unit 3 are not limited to permanent magnets, and the magnetic force generators of the buoyancy generating unit 6 are not limited to iron plates. As long as the magnetic force is generated between the plate 61 and the driven rotator 51 and the magnetic force is generated between the drive rotator 33 and the driven rotator 51, any magnetic force generator is adopted for each part. Good. For example, the magnetic force generator of the drive rotator 33 may be an iron plate. The magnetic force generator of the driven rotor 51 may be an iron plate, and the magnetic generators of the drive rotor 33 and the plate 61 may be permanent magnets. Furthermore, it is possible to employ an electromagnet as a magnetic force generator.

  The lengths of the two driven arms 55 and 56 may be equal to each other. The driven rotating body 51 does not necessarily have the two driven arms 55 and 56, and may have only one driven arm 55. In these cases, one blade 52 may extend from the cylindrical portion 54 to the side wall 42.

  The levitation mechanism is not limited to the one that causes the scraper 5 to levitate by magnetic force. For example, the scraper 5 may be levitated by an elastic member built in the container 4.

  The drive unit 3 starts the movement of the scraper 5 with the permanent magnets 38 and 39 sandwiching the floating position (position below the plate guide 65) in the movement direction, and the permanent magnets 38 and 39 are moved in the movement direction. You may be comprised so that the movement of the scraper 5 may be stopped in the state which pinched | interposed the said position for levitation | floating. A specific example of this is as follows. When the incubator 2 is installed on the housing 31, a positioning portion such as a protrusion is provided on the housing 31 so that the position of the plate guide 65 is constant. The rotation of the drive rotating body 33 is started with the permanent magnets 38 and 39 sandwiching the floating position in the moving direction, and the drive rotation is performed with the permanent magnets 38 and 39 sandwiching the floating position in the moving direction. The power source 32 is controlled so as to stop the rotation of the body 33.

  Hereinafter, examples will be described, but the present invention is not limited to these examples.

<Comparison evaluation 1>
The following Examples 1 to 3 were compared with Comparative Example 1.
Example 1

An apparatus having the same configuration as that of the culture apparatus 1 (the incubator 2 and the drive unit 3) according to the above embodiment was produced. The arrangement of the poles of the permanent magnets 38, 39, 53 is as shown in FIG. The diameter of the bottom surface of the container 4 was 230 mm. RAW264.7 cells were cultured and collected using this apparatus. The scraper was lifted when the cells were cultured, lowered when scraped, and lifted again when the cell suspension was recovered after scraping. Detailed culture and recovery procedures were as follows.
1. First, the scraper in the culture dish was lifted upward by a magnetic force so that the blade provided in the scraper was not immersed in the medium. Thereafter, the cells were seeded in the culture dish by suspending RAW264.7 cells (cell number: 3 × 10 ⁶ ) in 50 mL of medium. This culture dish was placed in a CO ₂ incubator and cultured at 37 ° C. for 2 days. A D-MEM medium containing 10% fetal bovine serum (Dulbecco's Modified Eagle's Medium (w / NaHCO ₃ w / o Glutamine) manufactured by DS Pharma Biomedical Co., Ltd.) was used as the medium.
2. After the culture for 2 days, the medium was changed, and the culture was further performed in a CO ₂ incubator at 37 ° C. for 2 days. Here, the same medium as described above was used.
3. After culturing for about four days in total, the surface of the culture dish was observed with a microscope, and the cells were uniformly adhered to the entire culture dish.
4). Cells adhering to the bottom of the culture dish were scraped with a scraper and collected. Specifically, first, the scraper that had surfaced was lowered. Thereafter, the scraper was rotated by a magnetic force, and the cells were scraped with a blade provided in the scraper. Thereafter, the culture dish was tilted while the scraper was lifted again, and the suspension was recovered.

(Example 2)
RAW264.7 cells (as in Example 1 except that the culture for about 4 days and medium change during the period were performed with the scraper lowered, that is, with the blade in contact with the bottom of the culture dish. The number of cells: 3 × 10 ⁶ ) was cultured and collected. When the surface of the culture dish after culturing was observed with a microscope, a portion having an extremely low cell density was observed compared with the surrounding area. Cells adhered to the bottom of the culture dish were collected with a scraper.

(Example 3)
RAW264.7 cells (cell number: 3 × 10 ⁶ ) in the same manner as in Example 1 except that the suspension was collected with the scraper lowered, that is, with the blade in contact with the bottom surface of the culture dish. Was cultured and recovered. The cells adhering to the bottom of the culture dish were scraped with a scraper and the suspension was collected, and then the surface of the culture dish was observed with a microscope. As a result, the remaining suspension and many cells suspended therein were observed.

(Comparative Example 1)
Culture and collection of RAW264.7 cells (cell number: 3 × 10 ⁶ ) in the same manner as in Example 1 except that only one permanent magnet was placed on the iron plate 37 instead of the permanent magnets 38 and 39. Went. The cells adhered to the bottom surface of the culture dish were scraped with a scraper, and the suspension was collected, and then the surface of the culture dish was observed with a microscope. As a result, many adhered cells were observed.

(Comparison result)
The number of cells collected according to Example 1 was 2.2 × 10 ⁸ cells. The number of cells collected according to Example 2 was 1.7 × 10 ⁸ cells. The number of cells collected according to Example 3 was 1.8 × 10 ⁸ cells. The number of cells collected in Comparative Example 1 was 1.6 × 10 ⁸ cells.

  The main reason that the number of cells collected in Example 2 is smaller than that in Example 1 was that the blade was always in contact with the bottom of the culture dish in Example 2, so that the medium was exchanged or the culture dish was moved. It is inferred that the cells were scraped off by the blade at an unfavorable timing due to the movement of the scraper and discarded together with the medium.

  The main reason that the number of cells collected in Example 3 is smaller than that in Example 1 is presumed that in Example 3, a part of the suspension was blocked by the blade and remained in the container at the time of suspension collection. Is done.

  The main reason that the number of cells collected in Comparative Example 1 is smaller than that in Example 1 is presumed that in Comparative Example 1, the transmission of force to the scraper was insufficient, so that cells were left behind.

<Comparison evaluation 2>
The following Examples 4 to 6 and Comparative Example 2 were compared.

Example 4
An apparatus having the same configuration as that of the culture apparatus 1 (the incubator 2 and the drive unit 3) according to the above embodiment was produced. The diameter of the bottom surface of the container 4 is 230 mm. The length of the blade 52 of the driven arms 55 and 56 is 60 mm.

  The positional relationship between the attractive force generator 34 and the permanent magnet 53 in plan view was set as follows. In a state where no phase shift occurs, the permanent magnet 53 is along the extending direction of the driven rotor 51. The attractive force generator 34 is inclined by 5 ° in the direction D1 with reference to the extending direction of the driven rotor 51.

Hela cells were cultured in the container 4 by the culture method according to the above embodiment. After completion of the culture, the container 4 was placed on the drive unit 3 and the driven rotating body 51 was rotated about 2 times by rotating the driving rotating body 33 twice at 1 rpm. The phase shift angle was measured during rotation, and the cell detachment rate was measured after rotation.
The cell detachment rate is a value calculated by the following equation.
Cell detachment rate = (number of cells after culture−number of remaining cells after scraping process) / number of cells after culture

(Example 5)
Except for the positional relationship between the attractive force generator 34 and the permanent magnet 53 in the culture apparatus 1, the Hela cells were cultured, the phase shift angle was measured, and the cell detachment rate was measured in the same manner as in Example 4. The positional relationship between the attractive force generator 34 and the permanent magnet 53 in plan view was set as follows. In a state where no phase shift occurs, the attractive force generator 34 follows the extending direction of the driven rotor 51. The permanent magnet 53 is inclined by 5 ° in the direction D2 with reference to the extending direction of the driven rotor 51.

(Example 6)
Except for the positional relationship between the attractive force generator 34 and the permanent magnet 53 in the culture apparatus 1, the Hela cells were cultured, the phase shift angle was measured, and the cell detachment rate was measured in the same manner as in Example 4. The positional relationship between the attractive force generator 34 and the permanent magnet 53 in plan view was set as follows. In a state where there is no phase shift, the attractive force generator 34 is inclined 2.5 ° in the direction D1 with respect to the extending direction of the driven rotor 51. The permanent magnet 53 is inclined by 2.5 ° in the direction D2 with reference to the extending direction of the driven rotor 51.

(Comparative Example 2)
Except for the positional relationship between the attractive force generator 34 and the permanent magnet 53 in the culture apparatus 1, the Hela cells were cultured, the phase shift angle was measured, and the cell detachment rate was measured in the same manner as in Example 4. The positional relationship between the attractive force generator 34 and the permanent magnet 53 in plan view was set as follows. In a state where no phase shift occurs, the attractive force generator 34 and the permanent magnet 53 are along the extending direction of the driven rotor 51.

(Comparison result)
The angle of phase shift in Example 4 was about 5.1 °. The cell detachment rate according to Example 4 was 98.3%. The angle of phase shift in Example 5 was about 5.4 °. The cell detachment rate according to Example 5 was 94.7%. The angle of phase shift in Example 6 was about 5.5 °. The cell detachment rate according to Example 6 was 95.8%.

  In Comparative Example 2, a vibration phenomenon that repeats a delay and a sudden follow-up occurred in the driven rotor 51 during rotation. Upon sudden follow-up of the driven rotating body 51, slight floating of the blade 52 was confirmed. Due to fluctuations associated with the vibration phenomenon, the angle of phase shift could not be measured. The cell detachment rate in Comparative Example 2 was 65%.

  From the above results, it was confirmed that by adjusting the positional relationship between the attractive force generator 34 and the permanent magnet 53, the scraper can be rotated more reliably and the cells can be sufficiently detached.

  The present disclosure can be used for an apparatus and a method for culturing cells.

  DESCRIPTION OF SYMBOLS 1 ... Culture device, 2 ... Incubator, 3 ... Drive unit, 4, 4A ... Container, 5, 5A, 5B ... Scraper, 6 ... Buoyancy generating unit (levitation mechanism), 21 ... Pin (rotating shaft), 21a ... Flange Part, 21b ... convex part, 38, 39 ... permanent magnet (second or third magnetic force generator), 41a ... bottom face of container, 42 ... side wall, 44 ... liquid feeding port, 45 ... vent port, 51 ... driven rotation Body (supporting part), 52 ... blade, 52b ... notch, 53 ... permanent magnet (first magnetic force generator), 54 ... cylindrical part, 54a ... concave, 61 ... plate, 63 ... iron plate (fourth magnetic force generation) Body), 65 ... plate guide, S1 ... closed space.

Claims (21)

A container constituting a closed space for accommodating a medium for culturing cells;
A scraper that is housed in the enclosed space and has a blade for scraping off deposits on the bottom surface of the container and a support portion that supports the blade;
A levitating mechanism for levitating and fixing the scraper to the container so that the blade is separated from the bottom surface of the container;
A drive unit that is disposed outside the enclosed space and moves the scraper in a state where the blade is in contact with the bottom surface of the container; and
The scraper has a first magnetic force generator,
The drive unit includes second and third magnetic force generators disposed so as to sandwich the first magnetic force generator in the moving direction of the scraper, and generates the first, second, and third magnetic force generators. A culture apparatus that transmits power to the scraper through the body.   The culture according to claim 1, wherein the levitation mechanism further includes a fourth magnetic force generator disposed outside the container, and the scraper is levitated by the magnetic force between the first and fourth magnetic force generators. apparatus. The support portion has a rotation shaft hole,
The culture apparatus according to claim 2, wherein the container has a substantially circular bottom surface and a rotation shaft for insertion into the rotation shaft hole of the scraper.   The rotating shaft has a flange portion whose outer diameter is larger than the inner diameter of the rotating shaft hole on the upper end side, and the upper peripheral edge portion of the rotating shaft hole and the flange portion each have a recessed portion or a protruding portion to be fitted. Item 4. The culture apparatus according to Item 3.   The culture apparatus according to claim 3 or 4, wherein the rotation shaft and the rotation shaft hole guide the vertical movement of the scraper in the container.   The culture apparatus according to any one of claims 3 to 5, wherein a diameter of a bottom surface of the container is 30 to 400 mm.   The culture apparatus according to any one of claims 2 to 6, wherein the levitation mechanism further includes a plate on which the fourth magnetic force generator is fixed, and a plate guide that guides the slide of the plate. The scraper has two of the first magnetic force generators,
The support portion has a rod shape, and the two first magnetic force generators are provided so as to be aligned in the longitudinal direction of the support portion,
The levitation mechanism has two of the fourth magnetic force generators,
The plate has a shape extending in one direction, and the two fourth magnetic force generators are fixed so as to be aligned in the longitudinal direction of the plate,
The culture apparatus according to claim 7, wherein the plate guide guides the plate so as to slide along a longitudinal direction.   The culture apparatus according to claim 7 or 8, wherein the plate and the plate guide are disposed above the container, and the scraper floats by an attractive force between the first and fourth magnetic force generators.   The culture apparatus according to claim 7 or 8, wherein the plate and the plate guide are disposed below the container, and the scraper is floated by a repulsive force between the first and fourth magnetic force generators.   The culture apparatus according to any one of claims 1 to 10, wherein the flatness of the bottom surface of the container is 40 to 800 µm.   The culture apparatus according to any one of claims 1 to 11, wherein a material of the blade is silicone rubber.   The culture apparatus according to any one of claims 1 to 12, wherein the container has a liquid feeding port and a ventilation port on a side wall. The support portion is provided so as to be rotatable around a single axis intersecting the bottom surface of the container,
The drive unit includes a drive rotator provided to be rotatable about the one axis, and the second and third magnetic force generators are provided to the drive rotator.
The first, second and third magnetic force generators each have a rectangular shape in plan view,
The first magnetic force generator and the second and third magnetic force generators are provided so as to be inclined with respect to each other in plan view when no phase shift of the support portion with respect to the drive rotating body occurs. The culture apparatus according to claim 1, wherein the inclination is set to be small when the phase shift occurs. The support portion is provided so as to be rotatable around a single axis intersecting the bottom surface of the container,
The drive unit includes a drive rotator provided to be rotatable about the one axis, and the second and third magnetic force generators are provided to the drive rotator.
The first, second and third magnetic force generators each have a rectangular shape in plan view,
The first, second and third magnetic force generators each have a shape extending in one direction in plan view,
The first magnetic force generator and the second and third magnetic force generators are provided so as to be inclined with respect to each other in plan view when no phase shift of the support portion with respect to the drive rotating body occurs. The culture apparatus according to claim 1, wherein the inclination is set to be small when the phase shift occurs. The first, second and third magnetic force generators are permanent magnets;
The poles of the first permanent magnet are arranged along the moving direction,
The poles of the second magnetic force generator are arranged vertically so as to attract the poles on the second magnetic force generator side among the poles of the first magnetic force generator,
The pole of the third magnetic force generator is arranged vertically so as to attract the pole on the third magnetic force generator side among the poles of the first magnetic force generator. Culture equipment.   The culture apparatus according to claim 1, wherein the scraper has two or more blades or one blade having a notch in the middle. The container has a substantially circular bottom surface and a rotation axis,
The support portion includes a rotation shaft hole into which the rotation shaft is inserted, and an arm that extends in the radial direction from the rotation shaft hole and supports one or more blades.
The culture apparatus according to claim 17, wherein the first magnetic force generator is disposed on the arm. The scraper has two blades and two first magnetic force generators,
The support portion includes two arms extending in different radial directions from the rotation shaft hole,
The two blades are respectively arranged on the two arms,
The culture device according to claim 18, wherein the two first magnetic force generators are also arranged on the two arms, respectively. The blade supported by one of the arms extends from the vicinity of the rotation shaft hole to the position of the radius r1 of the container, and the blade supported by the other arm of the blade from the position of the radius r2 of the container It extends to the tip side of the arm,
The culture apparatus according to claim 19, wherein the radius r1 and the radius r2 satisfy the condition of the formula (1).
r1 ≧ r2 (1) The levitation mechanism can switch between a floating state of the scraper and a released state of the scraper in both directions, and when the first magnetic force generator is in a levitation position in the floating state. , Levitating and fixing the scraper to the container,
The drive unit starts the movement of the scraper with the second and third magnetic force generators sandwiching the levitation position in the moving direction, and the second and third magnetic force generators are The culture apparatus according to any one of claims 1 to 20, wherein the culture apparatus is configured to stop the movement of the scraper while sandwiching the position for levitation in the movement direction.

Images