JP6732245B2 - Cell culture device - Google Patents

Description

The present invention relates to a cell culture device capable of efficiently culturing a large amount of adherent cells or cell lines, particularly somatic stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and the like. ..

Generally, cells are cultivated by filling a liquid medium together with the cells to be cultivated in a culturing vessel of a culturing apparatus, and leaving the culturing vessel filled with the cells and the medium to stand in a predetermined environment.

Recently, there is a strong demand for culturing a large number of cells in order to actually use the cultivated cells in a living body. As an example of mass production, it is known that, as in Patent Document 1, a zigzag-folded film body in a cylinder or a radially-folded film body is put in one culture container and cultured in a large amount. ing. However, in Patent Document 1, observing the adhered state of cells adhered to the bent film body is not disclosed and is not considered.

A configuration as disclosed in Patent Document 2 is known as an example of a culture device that mass-produces cells and observes cells in a container of the culture device. In Patent Document 2, the state of cells in the stacked containers of the culture device is observed by stacking the containers of the culture device in multiple stages and observing from an oblique direction.

JP, 2014-183753, A JP, 2004-344016, A

However, in the culture device of Patent Document 2, since the culture vessels are stacked, a large number of culture vessels cannot be stacked if the height of the stacked culture vessels is too high or the height is limited. Further, since the cells in the stacked culture vessels are observed obliquely, there is a drawback that the observation is limited and cannot be observed sufficiently.

Therefore, it is an object of the present invention to provide a cell culture device capable of effectively culturing a large amount of cells and observing the culture state more accurately.

According to the present invention, substrates for culturing cells are provided in parallel in a multi-tiered manner in one culture container, and observation windows are provided in these multi-tiered substrates to show the state of cells attached to the superposed substrates. Provided is a cell culture device capable of observing cells.

Specifically, the first aspect of the present invention is such that a tubular container body, an opening provided at one end of the container body, and an opening provided at the other end of the container body are integrally provided and contract toward the tip. With a diameter pool
A lid member attached to the opening, a gas-permeable portion that is provided on the lid member, has liquid-tightness, and allows gas to flow, and is disposed inside the container body, and cultured cells on the surface. Is provided with a plurality of substrates that can be bonded, each of the substrates is made of a plate material, the surfaces of the substrates are arranged along the longitudinal direction of the container body, and are stacked in parallel at predetermined intervals in multiple stages, When viewed from the surface wall of the container body facing the surfaces of the plurality of substrates, the surface condition of the second substrate positioned inside the first substrate is set on the first substrate positioned on the surface wall side. It is characterized in that an observation window on the surface wall side is provided so that it can be observed from the side.

A second invention is characterized in that, in the first invention, the observation window is a through hole, and a state of a substrate positioned inside a substrate provided with the through hole can be observed. ..

In a third aspect based on the second aspect, the plurality of substrates are provided in at least three or more steps having a third substrate inside the second substrate, and the through hole is provided in the first substrate. A second substrate through hole for observing the surface condition of the second substrate; a third substrate through hole provided in the first substrate for observing the surface condition of the third substrate; and the second substrate. And a third substrate through hole at a position overlapping with the third substrate through hole provided in the first substrate.

In a fourth aspect based on the third aspect, the substrate is provided in N steps, and is located inside the (N)th substrate located on the back wall opposite to the front wall of the container body. to (M) th (M) substrate through-hole for can observe the surface state of the substrate is, from the first substrate second - is provided (M 1) through successively to the substrate, ( M-1) th to the surface condition of the substrate can microscopy (M - 1) through holes for the substrate, wherein the first substrate second (M - 2) provided through in succession to the substrate cage, sequentially, is - - ((X -2) M) substrate through hole (M (X -2)) th (X is 2 to M a natural number) for the surface state of the substrate can microscopy first , from the first substrate first, characterized in that it (M - 1) - (X ) provided through sequentially to the substrate.

5th invention is located in a ( N ) th board|substrate located in the back surface wall on the opposite side to the said surface wall of the said container main body in 4th invention, and is located inside the said (N)th board|substrate. An observation window on the back wall side is provided so that the surface state of the (N-1)th substrate can be observed from the back wall side.

In a sixth aspect based on the fifth aspect, the surface state of the (N-2)th substrate is set on the (N)th substrate and the (N-1)th substrate from the back wall side. It is characterized in that a through hole for the (N−2)th substrate on the back wall side for observation is continuously provided.

A seventh invention is characterized in that, in the invention according to any one of the first to sixth inventions, the lid member is provided with an access port capable of inserting and withdrawing an injection needle while keeping airtightness. To do.

An eighth invention is characterized in that, in any one of the first to seventh inventions, the lid member is assembled in the opening portion so as to be openable and closable.

A ninth invention is the invention of any one of the first to seventh inventions, wherein the plurality of substrates are arranged in parallel at equal intervals and parallel to a direction in which gravity is applied when centrifuging the cell culture device. It is arranged in.

According to the first aspect of the present invention, the substrates can be cultivated by stacking the substrates in parallel in multiple stages in a single container, so that a large number of cells can be cultured on a plurality of substrates at one time and the mass productivity is excellent. In addition, it is possible to observe the cell attachment state or proliferation state of the second substrate inside the first substrate through the observation window on the surface wall side.

According to the second invention, the state of the substrate positioned inside the substrate having the through hole can be observed through the through hole without being disturbed by the substrate having the through hole, and the cell attachment state of the substrate can be observed. And the growth state can be observed.

According to the third invention, the cell attachment state and culture state of the second substrate and the third substrate inside the first substrate can be reliably and accurately observed through the through hole.

According to the fourth invention, the through hole for the (M) substrate for observing the surface state of the (M)th substrate continuously penetrates from the first substrate to the (M - 1) substrate. and it is provided with, the surface state of the (M-1) th substrate, first to be observed (M - 1) substrate through-hole, the first substrate second - to (M 2) substrate, continuous and provided so as to penetrate successively, (M - (X -2)) th (X is a natural number 2 or more M or less) to the surface state of the substrate, can be observed the (M - (X - 2)) substrate through-hole, from the first substrate the (M - (X - to 1)) substrate, since provided through continuously, it is provided to overlap the multistage substrate, inner The culture state of the substrate located at can be observed.

According to the fifth invention, the (N)-th substrate located on the back wall opposite to the front wall of the tubular container body is located inside the (N)-th substrate (N-1). Since a rear surface wall side observation window is provided for observing the surface condition of the second substrate from the rear surface wall side, it is possible to observe from both sides, and more accurate observation can be performed.

According to the sixth aspect, the surface state of the (N)th substrate, the (N-1)th substrate, and the (N-2)th substrate can be observed from the back surface side from the back surface side. Therefore, the multi-stage substrate can be observed from both sides, and the cell attachment state can be observed more accurately.

According to the seventh invention, the needle is stabbed Komu and withdrawing things, exchange seeding or medium of the cells, or to investigate the component change of the medium in the middle culture by extraction analysis of medium in the container, the culture medium It is possible to adjust the concentration and the like while maintaining airtightness.

According to the eighth aspect, it is possible to easily open the lid member, set the substrate, and collect the cultured cells that have accumulated in the reservoir after centrifugation.

According to the ninth invention, since the plurality of substrates are arranged in parallel with the direction in which gravity is applied when centrifuging the cell culture device, when centrifuging, the substrates are made to follow the separation direction by centrifugation. The cultured cells can be easily separated and collected in the reservoir.

FIG. 1 is a perspective view showing an outline of the cell culture device of the first embodiment. FIG. 2 is a perspective view of the cell culture device of FIG. 1 in a disassembled state. FIG. 3 is a sectional view taken along line III-III of the lid member of FIG. FIG. 4 is a sectional view taken along line IV-IV of the lid member of FIG. FIG. 5 is a perspective view for explaining an image of the substrate in the cell culture device of the first embodiment. FIG. 6 is a front view for explaining the outline of the centrifuge. FIG. 7 is a perspective view showing an outline of the cell culture device of the second embodiment. FIG. 8 is a cross-sectional view of the cell culture device of FIG. 7 taken along the line VIII-VIII. FIG. 9 is a sectional view taken along line IX-IX of the cell culture device of FIG. 7. FIG. 10 is a perspective view showing the outline of the cell culture device of the third embodiment. 11 is a sectional view taken along line XI-XI of the cell culture device of FIG. FIG. 12 is a cross-sectional view of the cell culture device of FIG. 10 taken along the line XII-XII. 13 is a sectional view taken along line XIII-XIII of the cell culture device of FIG. FIG. 14 is a schematic view of a part of the cell culture device of the fourth embodiment.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that the following description of the preferred embodiments is merely an example in essence, and is not intended to limit the present invention, its application, or its use.
(Embodiment 1)
The cell culture device of Embodiment 1 will be described with reference to FIGS. 1 to 5. As shown in FIGS. 1 and 2, the cell culture device 1 according to the first embodiment includes a container body 10 having a rectangular cross section, a lid member 20 detachably attached to the container body 10, and a container body 10. And a substrate 30 housed inside. The cell culture device of the present invention is suitable for an airtight cell culture device, and an embodiment will be described with reference to an airtight cell culture device.

The container body 10 is, for example, a rectangular parallelepiped shape having a length of 17 mm, a width of 17 mm, and a length of 110 mm, and is made of transparent synthetic resin or glass. From the viewpoint of the above-mentioned transparency and good adhesion of cells, the material of the container body 10 is generally made of polystyrene, but PP (polypropylene) is used to improve the strength during centrifugation. ), or PET (polyethylene terephthalate).

An opening 15 having a rectangular cross section is provided on one end side of the container body 10, and a conical reservoir 17 having a diameter reduced from the base end side to the tip side is integrally provided on the other end side of the container body 10. Has been. Note that the shape of the reservoir 17 is not limited to the shape of this embodiment. For example, a quadrangular pyramid shape may be used because it is easy to manufacture the quadrangular pyramid shape in accordance with the shape of the container body having an outer shape as well. When the cells are collected by centrifugation in the reservoir 17, it is preferable that the cross section is circular, so that at least the inner shape is preferably elliptical or circular. For example, the outer shape may be a quadrangular pyramid shape, and the inside may be a conical shape or an elliptical shape.

The opening 15 is formed by an upper end surface 10a that is hermetically pressed against the lid member 20, and an inclined surface 10b that extends from the upper end surface 10a in the longitudinal direction of the container body 10 and that has a reduced diameter.

The lid member 20 is made of polystyrene and is attached to the opening 15 so as to be openable and closable. In the present invention, opening and closing means that the lid member 20 is movably attached to the container body 10 by a hinge or the like to open and close the opening 15, or the lid member 20 is detachably attached to the container body 10. The opening 15 is opened by removing the opening 15 and the opening 15 is hermetically held by attaching the opening 15.

The lid member 20 has a rectangular shape, the outer peripheral lower surface 20a of the lid member 20 is pressed against the upper end surface 10a of the container body 10 forming the opening 15, and the inclined surface 20b of the lid member 20 becomes the inclined surface 10b. It is pressed and attached. At the time of mounting, the lid member 20 is pushed into the container body 10 from the outside, and the inclined surface 20b of the lid member 20 is brought into direct contact with the inclined surface 10b of the container body 10 to be pressure-welded, and the lower end surface 20a of the lid member 20 and the container body 10 are It is pressed so as to approach the upper end surface 10 a of the device 10. A rubber seal ring 21 is interposed between the lower end surface 20a of the lid member 20 and the upper end surface 10a of the container body 10 to keep the liquid tightness higher.

The lid member 20 is fitted with a gas-permeable portion 25 that is liquid-tight and allows the flow of gas. The gas permeable portion 25 is made of a cylindrical rubber member, and an engaging portion (not shown) is swelled in the middle thereof. The engaging portion engages with the engaging concave portion (not shown) of the lid member 20 so as to come into close contact therewith. Although the engaging portion and the engaging recess are not shown in the first embodiment, they have the same structure as the engaging portion 27a and the engaging recess 20c.

Like the gas permeable region disclosed in Japanese Unexamined Patent Publication No. 2014-183752, the gas permeable portion 25 includes a plurality of through holes and a gas permeable film arranged on the inner surface side of the lid member 20. May be. This gas permeable membrane allows the passage of gases such as carbon dioxide and oxygen without passing a liquid. As the gas permeable film, a film of polytetrafluoroethylene (PTFE), polyethylene, silicone resin, poly-4-methylpentene-1, polyisoprene, polybutadiene, ethylene vinyl acetate copolymer, polystyrene or the like having a thickness of about 100 μm is used. An example is a film-shaped one.

Further, the lid member 20 is provided with an access port 27 capable of inserting and withdrawing an injection needle (not shown) while keeping airtightness. The access port 27 is pierced with an injection needle to inoculate cells or exchange the medium, extract the medium in the container and analyze it to investigate the change in the components of the medium during the culture, and adjust the concentration of the medium airtightly. It can be done while maintaining sex. The access port 27 is made of, for example, an elastic synthetic rubber member. In this embodiment, the access port 27 is made of a cylindrical rubber member, and the engaging portion 27a is bulged in the middle. The engaging portion 27a is adapted to engage with the engaging concave portion 20d of the lid member 20 to be in close contact therewith. Note that the material and structure of the access port described above are merely examples, and the present invention is not limited to this.

Inside the container body 10, a plurality of substrates 30 to which cultured cells can adhere are arranged on the surface. The substrate 30 is made of a polystyrene plate material (plate thickness: 0.5 mm), and is stacked in parallel with the rectangular polystyrene columns 31 provided at both ends at regular intervals in five steps to perform welding or the like. It is fixed by fixing means. As shown in FIGS. 1 and 2, the substrate 30 is housed inside the container body 10 along the longitudinal direction of the container body 10. That is, the surface of the substrate 30 is arranged along the longitudinal direction of the container body 10.

The support column 31 is provided only on the side close to the lid member 20 when it is inserted into the container body 10, and is not provided on the reservoir portion 17 side. As will be described later, this is to prevent the column 31 from obstructing the flow of cells when the cultured cells attached to the surface of the substrate 30 are collected in the pool 17 by centrifugation. If the support of the substrate 30 is prioritized, the support columns 31 may be provided on the reservoir 17 side.

The plurality of substrates 30 have the same size and shape, and as shown in FIGS. 1 and 5, the first substrate 301, the second substrate 302, and the third substrate 303 are sequentially viewed from the surface wall 11 of the container body 10. , The fourth substrate 304, and the fifth substrate 305 are arranged side by side in parallel at an integral interval. The substrate 30 is housed inside the container body 10 along the longitudinal direction of the container body 10. That is, the surface of the substrate 30 is arranged along the longitudinal direction of the container body 10.

As shown in FIG. 5, the second substrate 302, the third substrate 303, the fourth substrate 304, and the fifth substrate 305 located inside the surface wall 11 of the container body 10 are obstructed by the first substrate 301 and the like. Therefore, in the first embodiment, the surface states of the second substrate 302, the third substrate 303, the fourth substrate 304, and the fifth substrate 305 can be observed with a microscope from the surface wall 11 side. An observation window 40 on the front surface wall side and an observation window 50 on the rear surface wall side for allowing microscopic observation from the rear surface wall 12 side are provided.

As shown in FIG. 5, the observation window 40 on the surface wall side is formed of a through hole having a circular cross section, and the first substrate 301 includes a second substrate 302, a third substrate 303, a fourth substrate 304, and a fifth substrate 305. Through holes for observing the surface state of the second substrate, that is, the through holes 412 for the second substrate, the through holes 413 for the third substrate, the through holes 414 for the fourth substrate, and the through holes 415 for the fifth substrate are linearly arranged at regular intervals. It is provided above. Then, in the second substrate 302, through holes for observing the surface states of the third substrate 303, the fourth substrate 304, and the fifth substrate 305, that is, the through holes 423 for the third substrate and the through holes 424 for the fourth substrate. The through holes 425 for the fifth substrate are sequentially provided immediately below the through holes of the first substrate 301. In the third substrate 303, through holes for observing the surface states of the fourth substrate 304 and the fifth substrate 305, that is, the through holes 434 for the fourth substrate and the through holes 435 for the fifth substrate are sequentially arranged on the first substrate. It is provided directly below the through hole of 301. Through holes for observing the surface state of the fifth substrate 305, that is, through holes 445 for the fifth substrate, are sequentially provided directly below the through holes of the first substrate 301 in the fourth substrate 304.

The observation window 50 on the back wall side is formed of a through hole having a circular cross section, and the fifth substrate 305 is used to observe the surface states of the first substrate 301, the second substrate 302, the third substrate 303, and the fourth substrate 304. Through holes for the first substrate, the through holes 551 for the second substrate, the through holes 553 for the third substrate, and the through holes 554 for the fourth substrate are sequentially provided on a straight line at regular intervals. There is. Then, in the fourth substrate 304, through holes for observing the surface states of the first substrate 301, the second substrate 302, and the third substrate 303, that is, the first substrate through hole 541, the second substrate through hole 542. The through holes 543 for the third substrate are sequentially provided immediately below the through holes of the fifth substrate 305. In the third substrate 303, through holes for observing the surface state of the first substrate 301 and the second substrate 302, that is, the through holes 531 for the first substrate and the through holes 532 for the second substrate are sequentially arranged in the fifth substrate. It is provided directly below the through hole of 305. Through holes for observing the surface state of the first substrate 301, that is, through holes 521 for the first substrate, are sequentially provided in the second substrate 302 immediately below the through holes of the fifth substrate 305.

Each through hole has a diameter of about 2 mm, but it is preferable that the diameter is as small as possible if the cells can be observed under a microscope.

Since the culture solution can flow through these through-holes, it is possible to prevent the concentration and stagnation of the culture solution, and it is possible to more evenly culture the cells and further promote the culture, as compared with those without the through-holes. ..
The substrate 30 and the support column 31 are not fixed to the container body 10 and are arranged with a slight gap therebetween so that they can be inserted into or taken out from the container body 10. In addition, in order to prevent the substrate 30 from rattling in the container body 10, the support columns 31 are engaged with the engaging recesses 20e of the lid member 30. This engaging means is only an example, and other engaging means may be used. For example, a recess or the like for engaging the support column 31 may be provided inside the container body 10 so that the engaging and disengaging can be performed. It is also possible that the support pillar can be attached to or removed from the container body 10 directly or through the elastic member without rattling, without any recess.

An example of the centrifugal separator 2 will be described based on FIG.

For example, a centrifuge as disclosed in JP 2010-115175 A can be used. The centrifuge 2 accommodates the culture vessel 10, the centrifuge vessel 6 whose bottom is rotated outward in the radial direction, the rotation drive mechanism 3 which rotates the centrifuge vessel 6, and the rotation by the rotation drive mechanism 3. And a control unit 4 for periodically varying the speed. The centrifuge container 6 has a cylindrical shape, and the bottom portion has a smoothly reduced diameter. The culture container 10 after culturing the cells is inserted and attached from the top to the centrifugation container 6 with the tip side facing down, with the lid member 20 kept sealed. The centrifugal separator 2 is an example, and the present invention is not limited to the centrifugal separator having this structure.
The above cell culture device 1 is used as follows.

First, the lid member 20 of the sterilized cell culture device 1 is removed, the substrate 30 is housed in the container body 10, the liquid medium is filled, and the lid member 20 is attached. Next, the cell culture device 1 is accommodated in an incubator with the container body 10 lying down so that the surface of the substrate 30 is substantially along the horizontal direction, and the cells are cultured.

As a result, the cells adhere to the substrate 30 in the container body 10 in the laid state and proliferate. In order to uniformly grow the cells on the front and back surfaces of the substrate 30, the container body 10 may be turned upside down, automatically inverted, automatically horizontally rotated, or three-dimensionally rotated. You can

The cells cultured by the cell culture device 1 include somatic stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (EG cells), mesenchymal stem cells, In addition to stem cells such as neural stem cells, vascular endothelial stem cells, hematopoietic stem cells, and hepatic stem cells, bone cells, chondrocytes, muscle cells, cardiomyocytes, nerve cells, tendon cells, adipocytes, pancreatic cells, hepatocytes, kidney cells, Examples include differentiated cells such as hair matrix cells and blood cells, or their precursor cells.

In addition, as the liquid medium, those usually used for cell culture can be used without particular limitation. Specific examples thereof include α-MEM medium, RPMI-1640 medium, MEM basic medium and the like.

In addition to these chemical components such as sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, hormones, antibiotics, fatty acids, and sugars, these liquid media contain serum and serum in order to enhance the cell growth effect. A biological component such as a cell growth factor (cytokine) may be contained. However, addition of biological components such as serum and cell growth factor may cause infection with BSE and the like and cancer of cells, and therefore it is preferable not to use these biological components.

The surface (both sides) of the substrate 30 is preferably coated with an adhesion factor such as laminin or fibronectin, or subjected to electric charge treatment such as plasma discharge in order to improve cell adhesiveness. .. The electric charge processing on the surface of the substrate 30 may be performed by plasma processing for each sheet after stacking in five steps and supporting the pillars 31 at predetermined intervals, and then stacking in five steps and supporting the pillars 31. .. The thickness of the substrate 30 is preferably 0.5 to 3 mm, particularly 1.0 to 3 mm in order to maintain rigidity and flatness. The distance between the substrates 30 may be several μm as long as cells can be cultured, but a small distance is disadvantageous in mass production, and is 1.0 to 5.0 mm in consideration of mass productivity. It is preferable.

Further, the contact angle of water on the surface of the substrate 30 which has been subjected to the electric charge treatment is preferably 50 degrees to 70 degrees at room temperature (25° C.) from the viewpoint of improving cell adhesiveness.

Next, the procedure for observing the state of culturing and proliferating under a microscope will be described.

When cells are cultured using the cell culture device 1 of the first embodiment, the cells grow by adhering to the front surface or the back surface of the substrate 30 in the cell culture device. In order to observe the cell proliferation state and cell adhesion state at this time, a microscope is set, for example, on the surface wall side of the container body 10 and the microscope (not shown) is focused on the first substrate 301. The surface state of the first substrate 301 is observed. Next, the container body 10 is slid, the microscope is focused on the second substrate 302, the second substrate through hole 412 provided in the first substrate 301 is passed through, and the surface state of the second substrate 302 is observed. To do. The surface condition of the third substrate 303, the surface condition of the fourth substrate 304, and the surface condition of the fifth substrate 305 are sequentially observed. That is, the surface state of the third substrate 303 is observed through the third substrate through hole 413 provided in the first substrate 301 and the third substrate through hole 423 provided in the second substrate 302. The fourth substrate through hole 414 provided in the first substrate 301, the fourth substrate through hole 424 provided in the second substrate 302, and the fourth substrate through hole 434 provided in the third substrate 303 are transparent to 4 The surface condition of the substrate 304 is observed. The fifth substrate through hole 415 provided in the first substrate 301, the fifth substrate through hole 425 provided in the second substrate 302, the fifth substrate through hole 435 provided in the third substrate 303, and the fourth substrate 304 are provided. The surface state of the fifth substrate 305 is observed through the provided fifth substrate through hole 445.

Similarly, the surface states of the fifth substrate 305, the fourth substrate 304, the third substrate 303, the second substrate 302, and the first substrate 301 are also observed from the rear surface wall 12. The observation procedure is the same as the observation from the surface wall 11, and detailed description thereof will be omitted here.

In the above observation procedure, the first substrate 301, the second substrate 302, the third substrate 303, the fourth substrate 304, and the fifth substrate 305 are described in order, but the order is not limited to this order. , Other order is acceptable. Alternatively, the present invention may be applied to the case of observing only a part of the substrates instead of observing all the substrates. The observation may be performed only from the front surface wall 11 side or the rear surface wall 12 side.

Since the substrate 30 has a rectangular cross section, the front surface wall 11 and the rear surface wall 12 can be arranged in parallel with the respective substrates 30, and since the front surface wall 11 and the rear surface wall 12 have a constant thickness, the observed portion is not distorted. The substrate to be observed can be observed more accurately directly from the front of the observation windows 40 and 50 (without passing through another substrate).

In this way, by observing, it is possible to monitor the growth state and adhesion state of each cell, and perform necessary treatments, such as addition and change of medium, change of culture conditions, change of position and orientation of culture vessel, etc. be able to.

Next, a procedure for recovering cells that have been cultured and proliferated will be described.

When the cultured cells are collected, first, the liquid medium filled in the cell culture device 1 is removed, and then a cell release agent such as trypsin is added to the container body 10 to remove the cells adhered to the substrate 30. It is made to be peeled off or easily peeled off. Then, the cell culture device 1 is directly installed in the centrifuge 2 and centrifuged (for example, 500 rpm, room temperature, 5 min).

As a result, the separated cells are collected in the reservoir 17 of the container body 10. Since the surface of the substrate 30 is arranged along the longitudinal direction of the container body 10, when the centrifugation is performed, the surface of the substrate 30 can be aligned with the separation direction by the centrifugation, so that the cultured cells can be easily transferred. They are separated and collected in the reservoir 17 of the cylindrical container body 10. Further, the substrate 30 is restricted from moving downward by the reservoir 17. As a result, cells can be collected in the reservoir 17 while preventing the substrate 30 from moving to the tip side of the container body 10.

The cell culture device 1 of the first embodiment described above has the following effects.

The container body 10 was formed in a tubular shape, and a plurality of substrates 30 were arranged in parallel inside the container body 10. Accordingly, a large number of substrates 30 can be arranged in parallel and set in the culture container 10, so that a large amount of cells can be cultured. In particular, since the substrate 10 has the observation window 30, it is possible to observe even during the culture or even the state of the cells attached to the substrate 30 stacked inside, and the culture environment can be appropriately controlled. As a result, a large amount of cells can be cultured, the environment for adhering to the substrate can be maintained, and this leads to large-scale production of cells.
In addition, since it is possible to culture cells by stacking substrates made of rectangular plate materials in multiple stages, it is possible to culture a large number of cells on a plurality of substrates at one time, which is excellent in mass productivity. It is a container with a rectangular outer shape, and many containers can be stacked and placed in the culture space in the incubator, and the culture space can be used efficiently.

Also, after culturing cells, they can be set in a centrifuge and centrifuged without removing the cultured cells in the container, so there is no need to replace the cultured cells, and the cultured cells may be contaminated with foreign substances such as bacteria. And has excellent workability. When collecting the cultured cells, the cultured cells are peeled off on the surface of the substrate 30 and then the cell culture device 1 is centrifuged. Therefore, by disposing the substrate 30 inside the container body 10 such that the surface of the substrate 30 is along the longitudinal direction of the container body 10, the surface of the substrate 30 is separated by centrifugation when centrifugation is performed. Since the cells are arranged in the separation direction (the direction in which gravity is applied), the cultured cells can be easily separated and collected at the bottom of the cylindrical container body 10. Therefore, the area of the region where cells can adhere can be increased, and the cultured cells can be easily collected, so that the efficiency of cell culture can be further improved.

Since the lid member 20 is detachably provided in the opening 15 of the container body 10, the lid member 20 is opened at the time of collecting the cultured cells, and the substrate is taken out from the opening 15 first to make the inside of the culture container 10 hollow. After that, since the cultured cells in the reservoir 17 can be taken out, the workability is easy and the cells can be promptly collected without omission.

After the cell culture, the culture cells in the container can be set in the centrifuge 2 and centrifuged without removing the culture cells in the container, so that the culture cells do not have to be replaced, and the culture cells may be contaminated with various bacteria. And has excellent workability. In addition, since cells can be cultured by stacking substrates in multiple stages, a large number of substrates can be cultured at one time, which is excellent in mass productivity.
(Embodiment 2)
The second embodiment will be described based on FIGS. 7 to 9. In the description of the second embodiment, the same components will be denoted by the same reference symbols, and the description thereof will be omitted or simplified. The second embodiment differs from the first embodiment in the shapes of the culture container 101 and the opening 151.

The culture container 101 has a substantially cylindrical shape, and the upper surface 101a and the lower surface 101b of the culture container 101 are partly flat. That is, when observing the observation windows 40 and 50 of the substrate 30 from the outside of the culture container 101, it is preferable that the culture container 101 has a constant thickness and is parallel to the substrate 30. The upper surface 101a and the lower surface 101b are flat surfaces.

The lid member 201 includes a disc-shaped bottom member 201a that closes the opening 151 of the container body 101, and a cylindrical screw portion 201b that is continuous with the outer periphery of the bottom member 201a. The threaded portion 201b is screwed with the threaded portion 101b formed on the outer periphery of the container body 101 to hermetically close the opening 151.

As shown in FIG. 8, according to the shape of the culture vessel 101, the width of the substrate 30 is widest in the third substrate 303a, and sequentially narrows in the order of the substrates 302a and 304a and the substrates 301a and 305a. The observation windows 40 and 50 are provided at the center in the width direction. The lid member 201 is a screw-in type and is removable.

With this structure, the shape of the centrifuge vessel 6 of the centrifuge 2 can be matched with the cylindrical shape of the centrifuge vessel 6, so that the vessel body 101 can be fitted into the centrifuge vessel 6 for use, and the existing centrifuge apparatus can be effectively used. Available.

In the second embodiment, the lid member 201 is screwed to the container body 101 with a screw to be airtightly attached, but a seal member may be interposed to further improve airtightness.

Further, the container body 101 is formed in the same shape and size as the centrifugal separation tube 6 usable in the centrifugal separation device 2 (for example, the same shape and size as a centrifugal separation tube (conical tube) having a capacity of 50 ml), Instead of the centrifuge tube 6, it is possible to directly assemble and centrifuge.
(Embodiment 3)
The third embodiment will be described based on FIGS. 10 to 13. In the description of the third embodiment, the same constituents will be given the same reference numeral, and the description thereof will be omitted or simplified. The third embodiment is different from the first embodiment in the opening 152 and the lid member 202.

The opening 152 of the container body 102 according to the third embodiment includes a flat wall 102b extending from the upper end 101a of the container body 102 toward the inside of the container body 102 in the radial direction, and a pool portion along the container body 102 from the flat wall 102b. The inner wall 102c extending in the direction of 17 is provided. An opening 152 is formed on the inner side of the inner wall 102c in the radial direction.

The lid member 202 is made of a medical rubber member such as medical precision rubber (clean rubber (Sumitomo Rubber Industries, Ltd.)), and has a rectangular shape elastically pressed against the rectangular inner peripheral surface 102 a of the container body 102. The outer peripheral surface 202a of the container main body 102 is provided with a projecting portion 232 that is sandwiched between the outer peripheral wall 102a and the inner wall 102c of the container body 102.

When fitting the lid member 202, the lid member 202 is elastically forcibly reduced to a diameter equal to or smaller than the inner diameter of the inner wall 102c from the outside of the opening portion 152, and then once inside the container basic body 102 (the inner wall 102c. The lid member 202 is pushed inward. Then, after the outer periphery of the lid member 202 returns to the original outer diameter that comes into contact with the outer peripheral wall 102a of the container body 102, the lid member 20 is moved in the direction in which the protrusion 252 of the lid member 202 is in the space. Make it fit.
In addition, when removing, it is sufficient to perform the reverse operation.
Similar to the first embodiment, the gas permeable portion 25a and the puncture portion 27a are fitted into the lid member 202.

In the third embodiment, since the lid member 202 made of a rubber material for medical use is elastically pushed and fitted into the opening 152, sufficient airtightness can be ensured. Further, the gas permeable portion 25a and the access port 27a can also be fitted and assembled in the lid member 202, and the mounting work is easy.

(Embodiment 4)
The fourth embodiment will be described based on FIG. In the description of the fourth embodiment, the same constituents will be given the same reference numeral, and the description thereof will be omitted or simplified. The fourth embodiment is different from the first embodiment in the shape of the lid member 203.

As shown in FIG. 14, a lid member 203 is rotatably attached by a hinge. Specifically, a non-engaging portion 103a is provided so as to project on the outer periphery of the upper end portion of the container body 103. The engaging member 6 is rotatably supported by the lid member 203. The engaging member 6 is composed of a U-shaped frame member, is provided with an engaging portion 6a that engages with the non-engaging portion 103a at the tip portion, and a bracket 203a provided on the outer periphery of the lid member 203 at the rear end portion. A shaft supporting portion 6b is rotatably supported. Sealing rings 213a and 213b are provided between the engaging member 6 and the container body 103.
(Other embodiments)
It is possible to combine the respective embodiments. For example, the structure of the fourth embodiment is used for the second embodiment.

As a means for observing the cell attachment state and the culture state on the substrate, a microscope observation is generally used, but the observation window of the present invention is not limited to this microscope observation, and when it is used visually or under a microscope. Other than the above, for example, using a digital camera to save the observation data and display it on the monitor screen of the personal computer, use a video camera to save the observation data and to display the moving image and still image on the personal computer monitor surface. It also includes the case of using a loupe to display.

The shape of the through hole is not limited to the circular shape, and various shapes such as an elliptical shape, a polygonal shape, and a slit shape can be applied. Further, the size of the through holes may be changed such that the size of the through holes is gradually increased or gradually decreased without making them all the same size.

In addition, by making the observation window a through hole, it is excellent in that the surface state of the inner substrate at a position overlapping the substrate having the through hole can be observed without being disturbed by the substrate having the through hole. However, the observation window may be used without providing a through hole, by providing a thin film, or by subjecting a portion corresponding to the observation window to a treatment that makes it difficult for cells to adhere.

For example, the number of the substrates 30 inside the container body 10 has been described as an example, but the number is not limited to five and may be another number such as ten.

The support column 31 is not limited to the structure of the above-described embodiment, and may be any one as long as it can hold the plurality of substrates 30 at regular intervals in the container body 10. For example, a plate material may be used, and a lattice structure may be used. But good. In addition, the convex portion may be provided on one substrate 30 and the concave portion may be provided on the other substrate 30 facing each other so that the convex portion and the concave portion are fitted to each other, without forming the substrate 30 as a separate member.

Further, the positional relationship between the container body 10 and the substrate 30 may be such that when the substrate 30 is placed sideways in the incubator without being moved, the substrate 30 may be slightly floating from the inner surface of the container body 10. The inner surface of the main body 10 is provided with a partial convex portion, or the substrate is provided with a partial convex portion, or a space member is arranged between the inner surface of the container main body 10 and the substrate 30. It's just good.

As shown in FIG. 6, since the centrifuge container 6 swings and revolves around, it often has a cylindrical shape. Therefore, as disclosed in Japanese Patent Laid-Open No. 2010-115143, when the existing centrifuge is adopted, it is preferable that the culture vessel 10 also has a circular cross section. However, even if the cross-sectional shape of the first embodiment is rectangular, the cross-sectional shape of the culture container does not have to be circular as long as the rectangular-shaped culture container 10 can be stored in the centrifugation container 6 and centrifugation can be performed. For example, the cross section may be polygonal, semicircular, or elliptical.

The front surface wall 11 and the rear surface wall 12 of the container body 10 are transparent or semi-transparent, and the surface condition of the first substrate 301 and the fifth substrate 305 can be observed by a microscope without doing anything. Alternatively, the inner surface of the back wall 12 may be treated so that cultured cells do not adhere to it so that the transparency is maintained. Even in this case, a treatment for preventing the above-mentioned cultured cells from adhering may be applied only to the observation window portion.

INDUSTRIAL APPLICABILITY The present invention can be used for a cell culture device capable of culturing cells such as cell culture, adherent cells or cell lines, particularly somatic stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. It has a high quality.

DESCRIPTION OF SYMBOLS 1 Cell culture device 2 Centrifuge 10 Container body 17 Reservoir 20 Lid member 30 Substrate 31 Strut 40 Observation wall 50 on the front wall side Observation window 50 on the rear wall side

Claims (9)

A tubular container body,
An opening provided on one end side of the container body,
A reservoir portion that is integrally provided on the other end side of the container body and has a diameter reduced toward the tip,
A lid member attached to the opening,
A gas permeable portion that is provided on the lid member, has liquid tightness, and allows the passage of gas,
A plurality of substrates arranged inside the container body and capable of adhering cultured cells to the surface,
Equipped with
Each of the substrates is made of a plate material, the surface of the substrate is arranged along the longitudinal direction of the container body, and stacked in parallel at predetermined intervals in multiple stages,
When viewed from the surface wall of the container body facing the surfaces of the plurality of substrates, the surface condition of the second substrate positioned inside the first substrate on the first substrate positioned on the surface wall side is the surface state. A cell culture device characterized in that an observation window on the surface wall side is provided so that the cell can be observed from the wall side. 2. The cell culture device according to claim 1, wherein the observation window is a through hole, and the state of the substrate located inside the substrate provided with the through hole can be observed. In claim 2,
The plurality of substrates are provided in at least three or more stages having a third substrate inside the second substrate,
The through hole is
A second substrate through hole provided in the first substrate for observing a surface state of the second substrate;
A third substrate through hole provided in the first substrate for observing a surface state of the third substrate;
A cell culture device having a through hole for a third substrate, which is provided on the second substrate and overlaps with a through hole for a third substrate provided on the first substrate. In claim 3,
The substrate is provided in N stages,
For the (M)th substrate for observing the surface condition of the (M)th substrate located inside the (N)th substrate located on the back wall opposite to the front wall of the container body A through hole is continuously provided from the first substrate to the (M - 1)th substrate,
The for can observe the surface state of the (M-1) th substrate (M - 1) substrate through-hole, from the first substrate second - to (M 2) substrates, provided through continuously Has been
Sequentially, (M - (X -2)) th (X is a natural number 2 or more M or less) to be observed the surface condition of the substrate of the (M - (X -2)) substrate through-hole, the first 1 substrate first to (M - - (X 1) ) substrate, a cell culture apparatus which is characterized in that provided through continuously. In claim 4,
The surface state of the (N-1)th substrate located inside the ( N ) th substrate on the ( N ) th substrate located on the back wall opposite to the front wall of the container body. The cell culture device is provided with an observation window on the back wall side for observing from the back wall side. In claim 5,
On the (N)-th substrate and the (N-1)-th substrate, the surface state of the (N-2)-th substrate can be observed from the back-wall side, and the (N-) 2) A cell culture device characterized in that through holes for a substrate are continuously provided. In any one of Claim 1 thru|or 6,
The cell culture device, wherein the lid member is provided with an access port capable of inserting and withdrawing an injection needle while maintaining airtightness. In any one of Claim 1 thru|or 7,
The cell culture device, wherein the lid member is openably and closably attached to the opening. In any one of Claim 1 thru|or 8,
A cell culture device, wherein the plurality of substrates are arranged in parallel at equal intervals and parallel to a direction in which gravity is applied when the cell culture device is centrifuged.

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