Patent ID: 12227725

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, a preferred embodiment of the present invention will be described with reference to drawings.

(Cell Culture Bag)

First, a cell culture bag used in the present embodiment will be described with reference toFIG.1prior to description of a cell culturing device according to the present embodiment. A cell culture bag1shown inFIG.1(a)toFIG.1(c)is provided with: a bag body2configured from an upper surface film21and a lower surface film22which are stacked to heat seal a periphery20; and a port3attached to this bag body2. A size of the bag body2is not particularly limited, and is preferably adjusted to 50 to 500 mm in length and 50 to 500 mm in width, for example.

It should be noted that, in each drawing includingFIG.1, illustration of a flow path of a tube connected to the port3or the like, and illustration of a closing-off means which closes up the flow path, such as a pinch, a valve and the like are omitted.

As shown inFIG.1(c), a plurality of recesses4each serving as a cell culture portion are formed in the lower surface film22. An opening diameter (diameter) D of in the recess4is preferably 0.3 to 10 mm, and a depth d is preferably 0.1 mm or more, in order to suppress cell movement in the bag body2so that the cells under culture may retain in one recess4. The recesses4may be formed into the same opening diameter in all of the recesses4, or may include two or more kinds of recesses having a different opening diameter. For example, the lower surface film22may be divided into a plurality of regions, and the opening diameter of the recess4may be differed for each region.

Moreover, in the cell culture bag1, in order to facilitate gathering of the cells in a bottom of the recess4, as shown inFIG.1(b), the recess4has a spherical crown shape. It should be noted that the shape of the recess4is not limited thereto. For example, the recess4may have a shape dented in an earthenware mortar shape (conical shape). In order to facilitate gathering of the cells in the bottom of the recess4, a ratio d/D of a depth d to a diameter D of the recess4is preferably adjusted to 0.05 to 1.

Moreover, the recesses4are preferably aligned in a staggered shape as shown inFIG.1(c)so that an occupied area of the recess4in the lower surface film22may be as large as possible, but the recesses4may be aligned in a lattice shape, when necessary.

As shown inFIG.1(b), the upper surface film21has a swelling shape which is swollen into a plateau shape configured from: a substantially flat top surface part21acovering an upper part of a whole of the plurality of recesses4; and an inclined part21bformed on a circumference of the top surface part21a. Thus, in comparison with a flat pouch-shaped container prepared only by stacking two plastic films and sealing a periphery, even if the bag body2is filled with the culture medium, deformation in which the periphery of the lower surface film22is lifted up is suppressed.

The upper surface film21and the lower surface film22which form the bag body2are formed of a plastic film having gas permeability. The gas permeability of this plastic film is preferably 5,000 mL/(m2·day·atm) or more in an oxygen transmission rate measured at a test temperature of 37° C. in accordance with determination of gas-permeability rate in JIS K 7126.

Specific examples of a material to be used in the plastic film which forms the bag body2include a thermoplastic resin such as polyethylene, polypropylene, an ethylene-vinyl acetate copolymer, polyester, a silicone-based elastomer, a polystyrene-based elastomer, a tetrafluoroethylene-hexafluoropropylene copolymer (FEP) or the like. The materials may be used in a single layer or by laminating the same kind or different kinds of materials, but if heat sealability upon heat sealing the periphery is taken into consideration, the material preferably has a layer which functions as a sealant layer.

Moreover, a thickness of the plastic film which forms the bag body2is preferably 30 to 200 μm at which the film has moderate shape retainability so that the upper surface film21may keep the swelling shape while having flexibility, and the lower surface film22may keep a shape of the recess4.

The port3is formed of a tubular member through which the medium, the cells or the like can flow, and can be formed of a thermoplastic resin such as polyethylene, polypropylene, vinyl chloride, a polystyrene-based elastomer, FEP or the like, for example.

First Embodiment

Next, a cell culturing method and device according to an embodiment of the present invention will be described with reference toFIGS.2and3.FIG.2shows a schematic cross-sectional view of a cell culturing device100according to a first embodiment, in whichFIG.2(a)shows a state in which a pressing member is raised, andFIG.2(b)shows a state in which the pressing member falls.

The cell culturing device100according to the present embodiment is provided with: a stand5on which a cell culture bag1is placed; a pressing member6having a bottom surface6awhich presses a portion of a top surface part21aof an upper surface film21; and a support mechanism7which supports the pressing member6.

InFIG.2, illustration of a pump as a liquid feed means for injecting and discharging a culture medium through a port3of the cell culture bag1, and a releasing means for releasing spheroids from recesses4of the cell culture bag1is omitted. Specific examples of the pump include a peristaltic pump connected to the port3through a tube.

The liquid feed means is not limited to the pump, and may be a syringe, for example.

The stand5has a placement surface5aon which the cell culture bag1is horizontally placed. On this placement surface5a, an opening5bis formed as a shape for receiving each of a plurality of recesses4formed in a lower surface film22of the cell culture bag1. Thus, the placement surface5asupports the lower surface film22in non-contact with the recesses4, and therefore flattening and deformation of the recesses4are avoided, whereby outflow of the cells in the recesses4is prevented, and further gas permeability from the lower surface film22is improved.

The shape for receiving each of the plurality of recesses4is not limited to the opening, and may be a recess or a wire net shape, for example.

The pressing member6is a plate-shaped member having a substantially rectangular planar shape according to the top surface part21aof the cell culture bag1, and has a flat bottom surface6a.

The support mechanism7is configured from: a frame71provided on the stand5; guide pins72which are extended upward from four corners of an upper surface of the pressing member6to penetrate the frame71so as to be able to vertically move; and a biasing means73which biases the pressing member6downward. The biasing means73are configured from magnets73which are arranged on the upper surface of the pressing member6, and the frame71with the same pole sides facing each other. According to repulsive force applied between the permanent magnets73, the pressing member6vertically moves according to a height of the upper surface film21.

The biasing means73is not limited to the permanent magnet. Moreover, the biasing means73may be omitted, and according to self-weight of the pressing member6, the pressing member6may vertically move according to the height of the upper surface film21.

Confine

Next, the cell culturing method according to the present embodiment will be described with reference toFIGS.3and4. Illustration of the support mechanism7of the cell culturing device shown inFIG.2is omitted inFIGS.3and4.

As shown inFIG.3(a), a spheroid C formed during cell culture is contained at a uniform cell concentration in each recess4.

As shown inFIG.3(b), in a closing-off step, all of the plurality of recesses4are closed off using the upper surface film21by discharging a culture medium S contained in a bag body2through the port3by means of a pump (not shown). The upper surface film21is pressed by the flat bottom surface6aof the pressing member6, and therefore the upper surface film21is flatted, whereby each of the plurality of recesses4is securely closed off using the upper surface film21. Thus, the spheroid C is confined in each recess4, and therefore in the next releasing step, outflow of the spheroid C from each recess4is prevented.

When closing off the plurality of recesses4using the upper surface film21, each recess4may be tightly closed or need not be tightly closed. When the plurality of recesses4are not tightly closed, the plurality of recesses4only need to be closed to such an extent that overflow of the cells from the recesses4can be prevented.

Next, as shown inFIG.4, in the releasing step, the spheroids C are released, in some or all of the plurality of closed-off recesses4, from inner surfaces of the recesses4.

In the releasing step, some or all of the plurality of recesses4should be provided with vibration or oscillation. In that case, the cell culturing device100may be wholly provided with vibration or oscillation, or physical force of vibration or oscillation may be applied to the lower surface film22which forms the recesses4. Vibration or oscillation applied to the recesses4is also transmitted to the spheroids C adhering to the inner surfaces of the recesses4, whereby the spheroids C are released from the inner surfaces of the recesses4.

It should be noted that, as a means for providing vibration or oscillation, a vibrator8attached to the stand5of the cell culture device as shown inFIG.4may be used, or an oscillation device may be used. Moreover, vibration may be directly applied to the recesses4by directly bringing the vibrator into contact with the recesses4. Moreover, vibration or oscillation may be selectively applied to a part of the recesses4.

Moreover, vibration or oscillation may be reciprocating motion, or may be rotating motion by a test tube mixer, for example.

Here,FIG.5is a photograph showing an inside of one recess4to which the cells are inseminated in the present embodiment. A culture medium (StemFit (registered trademark)) manufactured by Ajinomoto Co., Inc. was used herein, and 3×104undifferentiated iPS cells (cell strain 1231A3) were inseminated per recess (well).

FIG.5(a)shows the recess after 48 hours from insemination. As shown in the figure, the cells inseminated adhere not only to a bottom center of the recess but also to every place of the inner surface of the recess to form the spheroids. It should be noted that, in the figure, the spheroids adhering to a circumferential inner surface of the recess are away from a focal plane of the photograph, and therefore do not appear clearly.

FIG.5(b)shows the recess after the closing-off step. The spheroids still adhere to every place of the inner surface only by closing off the recess4.

In the closing-off step, the peristaltic pump was used as the liquid feed means, and the culture medium was discharged through the port3until the upper surface film21closes off each recess4at a liquid feed rate of 1.0 mL/min.

FIG.5(c)shows the recess immediately after the releasing step. The stand5was vibrated using the vibrator8. As shown in the figure, a great number of small spheroids released from the circumferential inner surface of the recess4gather in the bottom center of the recess.

In the releasing step, the test tube mixer8being the vibrator8was used as the releasing means. Then, as shown inFIG.4, each recess4of the cell culture bag1was provided with a physical stimulus from outside by pressing the test tube mixer8to a lower surface of the stand5of the cell culturing device100for one minute. As the test tube mixer8, horizontal vibration type PresentMixer 6076881 having a vibration rate of about 2800 revolutions per minute, manufactured by TIETECH Co., Ltd., was used.

FIG.5(d)shows the recess after 15 hours from the releasing step. After the releasing step, the culture medium was injected into the bag body2and culture was resumed. As shown in the figure, one spheroid is formed in the bottom center of the recess4.

Thus, according to the present embodiment, the spheroids are released from the inner surface and gathered in one place while preventing movement of spheroids between the recesses. Thus, proliferation/differentiation induction efficiency of spheroids can be improved by efficiently forming one spheroid in one recess4.

Further, even if one spheroid is formed in one recess4, when the spheroids adhere to the inner surfaces of the recesses4, the proliferation/differentiation induction efficiency of spheroids can be improved by closing off the recesses4again and providing the recesses4with the physical stimulus, and releasing the spheroids from the inner surfaces to change adhesion sites of the spheroids while preventing movement of the spheroids between the recesses4.

Second Embodiment

Next, a second embodiment of the present invention will be described with reference toFIG.6. In the figure, illustration of the same components as in the first embodiment is partly omitted.

In the present embodiment, after the closing-off step described in the first embodiment, in a releasing step, as shown inFIG.6(a), a cell culture bag1is vertically inverted together with the cell culturing device100shown inFIG.1while closing off each recess4, in place of providing the recesses4of the cell culture bag with the physical stimulus from outside.

Moreover, as shown inFIG.6(b), in the releasing step, the cell culture bag1may be inclined together with the cell culturing device100shown inFIG.1while closing off each recess4in place of providing the recesses of the cell culture bag with the physical stimulus from outside. An inclination angle of the cell culture bag1relative to a horizontal line is not particularly limited, and the cell culture bag1may be inclined up to 90 degrees, for example.

Further, as shown inFIG.6(c), in the releasing step, the cell culture bag1may be vertically inverted and inclined together with the cell culturing device100shown inFIG.1while closing off each recess4, in place of providing the recesses with the physical stimulus from outside.

Thus, in the present embodiment, spheroids C adhering to the inner surfaces of the recesses4are released due to gravity by a simple operation of vertically inverting and/or inclining the culturing bag1. Moreover, each recess4is closed off using an upper surface film21, and therefore movement of released cells or spheroids C to other recesses4is avoided.

Moreover, the present embodiment is preferable when the cells and the aggregates adhering to the inner surfaces of the recesses4of the cell culture bag1are released only by vertically inverting and/or inclining the cell culture bag1.

The cell culture bag1may be vertically inverted and/or inclined manually, or may be vertically inverted and/or inclined by a mechanism of a robot arm and the like.

Moreover, in the releasing step of the first embodiment described above, the cell culture bag1may be further vertically inverted and/or inclined as in the present embodiment. Moreover, also in the releasing step in third and fourth embodiments described later, the cell culture bag1may be further vertically inverted and/or inclined as in the present embodiment.

Third Embodiment

Next, a third embodiment of the present invention will be described with reference toFIG.7. In the figure, illustration of the same components as in the first embodiment is partly omitted.

In the present embodiment, after the closing-off step described in the first embodiment, in a releasing step, each recess4is provided with a physical stimulus by thrusting up each recess from below by means of a thrust-up member9arranged below each of a plurality of recesses4, in place of providing the recesses4of the cell culture bag with vibration or oscillation.

The thrust-up member9vertically moves in an opening5bformed on a placement surface5aof a stand5by means of an actuator (not shown). A bottom of a crown spherical recess4curved in a protrusion shape downward is thrusted up by means of the thrust-up member9, whereby swelling is inverted so as to be curved in the protrusion shape upward. The spheroids C adhering to the inner surfaces of the recesses4are released by thrusting up the recesses4.

When the recess4remains in an inverted state upward, an original crown spherical shape curved in the protrusion shape downward can be recovered, for example, by injecting a culture medium into a bag body2next time, and then pressing the bag body2by means of a pressing member6or the like to increase internal pressure of the bag body2. Moreover, the shape of the recess4can be recovered also by sucking the recess4inverted upward from below, for example.

An example in which the thrust-up members9simultaneously thrust up all of the recesses4is shown inFIG.7, but the thrust-up members may selectively thrust up a part of the recesses4.

Moreover, one thrust-up member9may sequentially thrust up the recesses4one by one. In that case, the actuator (not shown) should be configured in such a manner that the actuator not only vertically moves the thrust-up member9, but also moves the thrust-up member in a horizontal direction to sequentially position the thrust-up members below desired recesses4.

Moreover, the recesses4may be provided with the physical stimulus by thrusting up the recesses4by means of the thrust-up members9and simultaneously applying vibration or oscillation to a cell culture bag1to release the spheroids C adhering to the inner surfaces of the recesses4.

Thus, even in cell culture using the cell culture bag1, adhesion positions of the spheroids C can be changed in the recesses4while preventing movement of the spheroids C between the recesses4.

Thus, culture/differentiation induction efficiency of cells can be improved.

Fourth Embodiment

Next, a cell culturing method and device according to a fourth embodiment of the present invention will be described with reference toFIG.8.

A structure of the cell culturing device according to the present embodiment can be formed in the same manner as in the second embodiment. In the present embodiment, after the closing-off step described in the first embodiment, unnecessary spheroids C1are selectively released and eliminated.

As shown inFIG.8(a), in a releasing step, spheroids C1in a part of a plurality of closed-off recesses4are selectively released. Also in the present embodiment, the same releasing means as in the first embodiment is used.

Next, as shown inFIG.8(b), in a clearing step, closing-off of the recesses4using an upper surface film21is cleared by injecting a culture medium into a bag body2through a port3by means of a pump. As a result of clearing closing-off of the recesses4, released spheroids C1are formed into a state of allowing outflow to an outside of the recesses4.

Next, as shown inFIG.8(c), in a discharging step, the selectively released spheroids C1are discharged together with the culture medium S by discharging the culture medium in the bag body2through the port3by means of the pump. Thus, unnecessary spheroids C1can be selectively released and removed.

While the unnecessary spheroids C1are selectively released and removed, the spheroids C which are not selected in other recesses4adhere to the inner surface of each recess4, and therefore are not discharged through the port3.

It should be noted that, in the present embodiment, the unnecessary spheroids C1are selected, but the spheroids to be selected are not limited to the unnecessary spheroids, and may be selected as an extraction sample, for example. Moreover, the spheroids in two or more recesses4can also be selectively released.

Fifth Embodiment

Next, a fifth embodiment of the present invention will be described with reference toFIG.9.

A structure of a cell culturing device according to the present embodiment can be formed in the same manner as in the fourth embodiment.

In the present embodiment, in the discharging step described in the fourth embodiment, selectively released spheroids C1are discharged together with a culture medium S through a port3to an outside of a bag body2in a state in which a cell culture bag1is vertically inverted and inclined so that the port3may be located below a plurality of recesses4.

The released spheroids C1may be discharged together with the culture medium S through the port3to the outside of the bag body2in a state in which the cell culture bag1is only vertically inverted without being inclined.

Moreover, the present embodiment is preferable when the cells and the aggregates adhering to inner sides of the recesses4of the cell culture bag1are not released only by vertically inverting the cell culture bag1.

Moreover, an operation of vertically inverting and/or inclining the cell culture bag1may be performed in the discharging step, or may be performed in a stage previous to the discharging step.

Thus, the selectively released spheroids C1in the releasing step move to the outside of the recesses4by vertically inverting the cell culture bag1, and easily discharged through the port3. Further, if the cell culture bag1is inclined so that the port3may be located below the plurality of recesses4, the released spheroids C1can be further easily discharged through the port3.

As described above, the present invention is described by showing preferred embodiments, but the present invention is not limited to the embodiments described above, and various modifications can be obviously made within the scope of the present invention. In the above-described embodiment, a rod-like thrust-up member is used, but a shape of the thrust-up member is not limited thereto. Moreover, in the above-described embodiment, an example of pressing the upper surface film by means of the pressing member is described, but the pressing member may be omitted in the present invention.

Moreover, in the above-described embodiment, an example of releasing the spheroids adhering to the inner surface of the cell culture bag is described, but the present invention can be applied also to a case where the cells not forming the spheroids are released from the inner surfaces of the recesses of the cell culture bag.

INDUSTRIAL APPLICABILITY

The present invention can be used as a technology on efficiently culturing various cells.

The entire contents of the documents described in the description concerning the present application and the description of the Japanese application serving as a basis of claiming the priority concerning the present application to the Paris Convention are incorporated by reference herein.

EXPLANATION OF NUMERICAL SYMBOLS

1Cell culture bag2Bag body20Periphery21Upper surface film21aTop surface part21bInclined part22Lower surface film3Port4Recess (well)5Stand5aPlacement surface5bOpening6Pressing member6aBottom surface7Support mechanism71Frame72Guide pin73Biasing means (magnet)8Vibrator (test tube mixer)9Thrust-up member100Cell culturing deviceC, C, C1Cells or cell aggregates (spheroids)S Culture medium