WO2014148508A1

WO2014148508A1 - Cell culture vessel

Info

Publication number: WO2014148508A1
Application number: PCT/JP2014/057390
Authority: WO
Inventors: 聖真田中; 健一森井; 裕美河原; 弓削　類
Original assignee: 株式会社ジェイ・エム・エス; 株式会社スペース・バイオ・ラボラトリーズ
Priority date: 2013-03-22
Filing date: 2014-03-18
Publication date: 2014-09-25
Also published as: JP6153357B2; JP2014183753A

Abstract

Provided is a cell culture vessel capable of culturing cells more efficiently in a microgravity environment. A cell culture vessel (1) provided with: a vessel main body (10) having an opening (11); a cap (20) removably mounted on the opening (11); a gas-permeable region (22) for permitting the passage of gas, having liquid tightness and being provided in the cap (20); and a film (30) which allows cultured cells to adhere thereto, disposed in the interior of the vessel main body (10). The film (30) is preferably housed in the vessel main body (10) in a folded state folded by a plurality of folding lines (31) extending parallel to each other at a predetermined distance.

Description

Cell culture vessel

The present invention relates to a cell culture vessel capable of efficiently culturing cells such as somatic stem cells, embryonic stem cells (ES cells), and induced pluripotent stem cells (iPS cells).

Conventionally, when culturing cells such as somatic stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), sterilized resin cell culture containers are used (for example, patents) Reference 1). More specifically, the cell culture is performed by filling a liquid culture medium together with the cells to be cultured in the cell culture container, and allowing the cell culture container filled with the cells and the medium to stand in a predetermined environment.

In recent years, there has also been proposed a technique for growing cells while suppressing stem cell differentiation by culturing stem cells in a microgravity environment (for example, 10 ⁻³ G) using a gravity-dispersed simulated microgravity device. (For example, refer to Patent Document 2).

Japanese Patent Laid-Open No. 10-179137 Japanese Patent Laid-Open No. 2003-9852

By the way, in many cases, cells grow by adhering to the inner surface of the cell culture container. When cells are cultured using a cell culture container as proposed in Patent Document 1 in a normal gravity environment, the cells grow while adhering to the bottom surface of the cell culture container.
On the other hand, when cells are cultured using a similar cell culture container in a microgravity environment, the cells grow while adhering to the entire inner surface of the cell culture container. Therefore, in a microgravity environment, cell differentiation can be suppressed and cell culture efficiency can be improved.
In the present situation where regenerative medical technology using stem cells is developed, a cell culture container capable of culturing cells more efficiently is required.

Therefore, an object of the present invention is to provide a cell culture vessel capable of culturing cells more efficiently in a microgravity environment.

The present invention includes a container body having an opening, a cap that is detachably attached to the opening, a gas-permeable region that is provided in the cap, has liquid-tightness, and allows gas flow, It is related with a cell culture container provided with the film body which can be arranged inside the container main body and to which a cultured cell can adhere.

Further, it is preferable that the film body is accommodated in the container body in a state of being folded in a pleated shape by a plurality of fold lines extending in parallel with each other at a predetermined interval.

Further, the container body has a bottom wall part, a side wall part rising from the periphery of the bottom wall part, and a main body part having an upper wall part arranged in parallel with the bottom wall part at the standing end of the side wall part, A cylindrical neck part having one end side connected to the side wall part and the opening part formed on the other end side, and the length between the two folding lines arranged adjacent to each other in the film body is The diameter is preferably equal to or smaller than the diameter of the opening.

Further, the container body includes a cylindrical portion in which the opening is formed on one end side, and a reduced diameter portion that is disposed on the other end side of the cylindrical portion and decreases in diameter from the proximal end side toward the distal end side. It is preferable to provide.

Further, it is preferable that the film body is folded in a pleated shape by a plurality of fold lines extending in parallel with each other at a predetermined interval, and the plurality of fold lines are arranged along the longitudinal direction of the container body.

Further, it is preferable that the plurality of broken lines are parallel to a direction in which gravity is applied when the cell culture container is centrifuged.

Further, it is preferable that the film body is accommodated in the container body in a state of being rolled into a cylindrical shape.

Moreover, it is preferable that the length between the two broken lines arranged adjacent to each other is equal to or less than the radius of the cylindrical portion.

The film body is preferably composed of a polyethylene terephthalate film.

The surface of the film body is preferably subjected to charge treatment.

The cell of the present invention can be cultured more efficiently in a microgravity environment.

It is a figure which shows an example of the apparatus used in order to culture the cell culture container of this invention in a microgravity environment. It is a perspective view which shows the cell culture container which concerns on 1st Embodiment of this invention. It is a disassembled perspective view which shows the cell culture container of 1st Embodiment. It is a top view which shows the cell culture container of 1st Embodiment. FIG. 5 is a sectional view taken along line AA in FIG. 4. FIG. 5 is a sectional view taken along line BB in FIG. 4. It is a perspective view which shows the cell culture container which concerns on 2nd Embodiment of this invention. It is a front view which shows the cell culture container of 2nd Embodiment. It is a disassembled perspective view which shows the cell culture container of 2nd Embodiment. It is CC sectional view taken on the line of FIG. FIG. 9 is a sectional view taken along line DD of FIG. It is a disassembled perspective view which shows the modification of the cell culture container of 2nd Embodiment. It is sectional drawing which shows the other modification of the cell culture container of 2nd Embodiment, and is a figure corresponding to FIG. It is a perspective view which shows the other modification of the cell culture container of 2nd Embodiment.

Hereinafter, preferred embodiments of the cell culture container of the present invention will be described with reference to the drawings.
The cell culture container 1 of the present invention is suitably used in a microgravity environment. The microgravity environment, refers to a very gravity smaller environment than on the ground, for example, and the environment ^{(10 -3} G) in the space station, gravity distributed simulated microgravity device (e.g., JP 2003-9852 An environment that can be realized using a gazette). Alternatively, from the viewpoint of reducing the influence of gravity, the above-mentioned cell culture vessel can be suitably used for a Rotary Cell Culture System (RCCS; Synthecon), which is a three-dimensional culture system.
In a microgravity environment or an environment that reduces the effect of gravity that can be achieved with a single-axis rotating culture vessel such as RCCS, cells can reduce the effect of gravity. Therefore, the cells can grow by adhering not only to the upper surface of the horizontal plane but also to all surfaces inside the cell culture container 1.

As shown in FIG. 1, the gravity-dispersed simulated microgravity device 100 includes a container accommodating portion 110 that can accommodate a plurality of or a single cell culture container 1, and the container accommodating portion 110 that has a first shaft 125. A first support portion 120 that rotatably supports a rotation shaft, a second support portion 130 that rotatably supports a container housing portion 110 with a second shaft 135 orthogonal to the first shaft 125 as a rotation shaft, and a container A control unit (not shown) that controls the rotation of the housing unit 110. Then, the simulated microgravity device 100 disperses the gravity applied to the container housing portion 110 by rotating the container housing portion 110 about two

orthogonal axes

125 and 135, thereby simulating a microgravity environment. Realize.

First, the cell culture container 1 according to the first embodiment of the present invention will be described with reference to FIGS.
As shown in FIGS. 2 and 3, the cell culture container 1 of the first embodiment includes a container body 10 having an opening 11, a cap 20 that is detachably attached to the container body 10, and the interior of the container body 10. And a film body 30 accommodated in the container.

The container main body 10 includes a box-shaped main body portion 12 and a neck portion 13.
As shown in FIGS. 4 to 6, the main body 12 includes a bottom wall 121 having a hexagonal shape in plan view, a side wall 122 rising from each of six sides that are peripheral edges of the bottom wall 121, and a side wall An upper wall portion 123 disposed parallel to the bottom wall portion 121 is provided at the standing end of 122. An opening is formed in one of the six side walls 122.

The neck 13 is configured in a cylindrical shape. One end side of the neck portion 13 is connected to a portion where the opening of the side wall portion 122 where the opening is formed is formed. The other end side of the neck 13 constitutes the opening 11 of the container body 10. A thread 131 is formed on the outer peripheral surface of the neck 13 near the end on the opening 11 side.
In the first embodiment, as shown in FIGS. 2 and 6, the neck 13 is slightly inclined so that the end on the opening 11 side is located on the upper wall 123 side than the end on the side wall 122 side. It extends. Further, the diameter of the neck portion 13 is substantially equal to the height of the side wall portion 122.

The container body 10 described above is made of a synthetic resin or glass having transparency. The material of the container body 10 is generally composed of polystyrene from the viewpoints of the above-described transparency and cell adhesion. However, PP (polypropylene) or PET (polyethylene terephthalate) may be used to improve the strength during centrifugation.
The inner surface of the container body 10 is preferably subjected to charge treatment such as plasma discharge in order to improve cell adhesion.

The cap 20 includes a cap main body 21, a gas permeable region 22 provided in the cap main body 21, and an O-ring 23 (see FIG. 3).
The cap main body 21 includes a cylindrical fitting portion 211 that is fitted on the outer peripheral surface of the container main body 10 (neck portion 13), and an end surface portion 212 that closes one end side of the fitting portion 211. A thread groove 213 having a shape corresponding to the thread 131 formed in the container body 10 (neck portion 13) is formed on the inner surface of the fitted portion 211.

The gas permeable region 22 is a region having liquid-tightness and allowing a gas flow. The gas permeable region 22 is provided on the end surface portion 212 of the cap body 21. The gas permeable region 22 includes a plurality of through holes 221 formed in the end surface portion 212 and a gas permeable film 222 disposed on the inner surface side of the end surface portion 212 in the cap body 21.
The gas permeable membrane 222 allows a gas such as carbon dioxide or oxygen to flow without passing a liquid. As the gas permeable membrane 222, a film of polytetrafluoroethylene (PTFE), polyethylene, silicone resin, poly-4-methylpentene-1, polyisoprene, polybutadiene, ethylene vinyl acetate copolymer, polystyrene, and the like is about 100 μm thick. The film-like structure is mentioned.

The O-ring 23 is disposed on the inner surface side of the cap body 21. The O-ring 23 maintains the liquid tightness between the cap 20 and the container body 10 when the cap 20 is attached to the container body 10.

The film body 30 is composed of a polyethylene terephthalate film (hereinafter also referred to as a PET film). In the first embodiment, as shown in FIGS. 4 and 5, the film body 30 is formed by alternately folding rectangular PET films at a plurality of fold lines 31 formed in parallel with each other at a predetermined interval. It is composed of folds.
A length L <b> 1 (see FIG. 5) between two folding lines 31 arranged adjacent to each other in the film body 30 is equal to or smaller than the diameter of the opening 11. The length L1 between the two folding lines 31 is preferably substantially equal to the diameter of the opening 11 from the viewpoint of increasing the surface area of the film body 30 accommodated in the container body 10.

The surface (both sides) of the film body 30 is preferably subjected to charge treatment such as plasma discharge in order to improve cell adhesion. The charge treatment on the surface of the film body 30 is performed on the PET film in a state before the folding line 31 is formed. Thereby, the charge process to the film body 30 can be performed easily and uniformly.
The thickness of the film body 30 is preferably 30 μm to 150 μm, more preferably 50 μm to 100 μm, from the viewpoint of maintaining good foldability.
Further, the contact angle of water on the surface of the film body 30 subjected to the charge treatment is preferably 50 ° to 70 ° at room temperature (25 ° C.) from the viewpoint of improving cell adhesion.

In the cell culture container 1 described above, the film body 30 is accommodated in the container body 10 in the following procedure. First, the film body 30 is contracted so that the pleats of the film body 30 folded in a pleat shape are close to each other. Next, the contracted film body 30 is pushed into the container body 10 from the opening 11. Then, the film body 30 that passes through the neck portion 13 and reaches the main body portion 12 is released from the contracted state and spreads inside the main body portion 12. Thereby, the cell culture container 1 by which the film body 30 folded in the shape of a pleat is arrange | positioned inside the container main body 10 is manufactured.

The above cell culture container 1 is used as follows.
First, the cap 20 of the sterilized cell culture container 1 is removed, the container body 10 in which the film body 30 is accommodated is filled with a liquid medium, and then the cells are seeded. Thereafter, the cap 20 is attached to the container body 10 filled with the liquid medium and seeded with the cells. Here, the liquid medium is preferably filled and filled so that no air remains in the container body 10. Thus, when cells are cultured in a microgravity environment, the cells can be prevented from coming into contact with air, so that the cells can be adhered to the entire inner surface of the container body 10 and the entire surface of the film body 30.
Next, the cell culture vessel 1 is placed in a microgravity environment, for example, by mounting it on a simulated microgravity device 100 as shown in FIG.

The cells cultured in the cell culture vessel 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, hepatic stem cells, bone cells, chondrocytes, muscle cells, cardiomyocytes, nerve cells, tendon cells, fat cells, pancreatic cells, hepatocytes, kidney cells, Examples thereof include differentiated cells such as hair matrix cells and blood cells, or precursor cells thereof.

As the liquid medium, those usually used for cell culture can be used without particular limitation. Specific examples include alpha α-MEM medium, RPMI-1640 medium, and MEM basic medium.

In addition to chemical components such as sodium, potassium, calcium, magnesium, phosphorus, chlorine, amino acids, vitamins, hormones, antibiotics, fatty acids, sugars, etc., these liquid media contain serum and A biological component such as a cell growth factor (cytokine) may be contained. However, by culturing cells in a microgravity environment, stem cells can be accelerated while maintaining an undifferentiated state without using biological components such as serum and cell growth factors (cytokines).

Next, a procedure for recovering cells grown in culture will be described.
When recovering the cultured cells, first, the liquid medium filled in the cell culture container 1 is removed, and then a cell peeling agent such as trypsin is added to the container body 10, so that the container body 10 and the film body 30 are added. Peel off the cells attached to the.
Next, the detached cells are transferred to a centrifuge tube using a pipette or the like, and centrifuged, for example, under conditions of 1000 rpm, 4 ° C., and 5 minutes.
Thereby, the cells cultured using the cell culture container 1 are collected.

According to the cell culture container 1 of 1st Embodiment demonstrated above, there exist the following effects.

(1) The cell culture container 1 was configured to include a film body 30 disposed inside the container body 10. Thereby, when cells are cultured in a microgravity environment, the cells can be adhered on the inner surface of the container body 10 and the surface of the film body 30, so that the area of the region to which the cells can adhere can be increased. Moreover, in the cell culture container 1, as a technique for increasing a region to which cells can adhere, that is, a technique for increasing the surface area of a cell adhesion target, a porous material, hollow fiber, or microbead is accommodated in the container body. A method can be considered. However, when these materials are used as cell adhesion targets, sterilization of these materials and surface treatment such as charge treatment become difficult. Moreover, it is difficult to peel off the proliferated cells, and the cell recovery rate is lowered. Furthermore, the cell growth is poor. Therefore, the film body 30 was used as a cell adhesion target. As a result, sterilization and surface treatment can be easily performed, and the proliferated cells can be easily detached. Furthermore, since the adhesiveness of the cells is excellent, the proliferation of the cells can be improved.

(2) The film body 30 was folded in a pleat shape and accommodated in the container body 10. Thereby, since the area of the area | region which a cell can adhere | attach can be increased more, the efficiency of cell culture can be improved more.

(3) The container body 10 is configured to include the body portion 12 and the neck portion 13, and the length L1 between the fold lines 31 of the film body 30 is configured to be equal to or smaller than the diameter of the opening portion 11. Thereby, since the film body 30 folded in a pleat shape can be easily accommodated in the container body 10, the cell culture container 1 can be easily manufactured.

(4) The cell culture container 1 is mainly manufactured from polystyrene from the viewpoints of cell adhesion, ease of processing, and the like. However, when the film body is made of polystyrene, the film body is easily broken when the film body is folded, and it is difficult to process the film body into a pleated shape. Then, the film body 30 was comprised with the polyethylene terephthalate film. Thereby, generation | occurrence | production of the crack of the film body 30 at the time of folding the film body 30 can be prevented. Therefore, since the ease of processing of the film body 30 can be improved, the film body 30 having a shape suitable for cell culture can be easily manufactured.

(5) The surface of the film body 30 was subjected to charge treatment. Thereby, a functional group can be provided to the surface of the film body 30, and the hydrophilicity of the film body 30 can be enhanced. Therefore, the adhesiveness of the cells to the surface of the film body 30 can be further improved.

Next, a second embodiment of the cell culture container of the present invention will be described with reference to FIGS.
The cell culture container 1A of the second embodiment is different from the first embodiment in the shape of the container body 10A and the arrangement of the film body 30 inside the container body 10A. In the description after the second embodiment, the same constituent elements are denoted by the same reference numerals, and the description thereof is omitted or simplified.

In the cell culture container 1A of the second embodiment, the container body 10A is formed in a cylindrical shape in which an opening 11A is formed on one end side and the other end side is closed, as shown in FIG. The container body 10 </ b> A includes a cylindrical portion 14 and a reduced diameter portion 15.
The cylindrical portion 14 is disposed on one end side of the container body 10A, that is, on the side where the opening portion 11A is formed. A screw thread 141 is formed on the outer peripheral surface of the cylindrical portion 14 near the end on the opening 11A side.
The reduced diameter portion 15 is disposed at the end of the cylindrical portion 14 opposite to the side where the opening 11A is formed. The reduced diameter portion 15 is formed in a conical shape having a reduced diameter from the proximal end side toward the distal end side.

The container body 10A is formed in the same shape and size as a centrifuge tube that can be used in a centrifuge (for example, the same shape and size as a centrifuge tube (conical tube) having a capacity of 50 ml).

The cap 20 includes a cap body 21, a gas permeable region 22 provided in the cap body 21, and an O-ring 23.
The cap main body 21 includes a cylindrical fitting portion 211 that is fitted on the outer peripheral surface of the container main body 10, and an end surface portion 212 that closes one end side of the fitting portion 211. A thread groove 213 having a shape corresponding to the thread 121 formed on the container body 10 (cylindrical portion 14) is formed on the inner surface of the fitted portion 211.

In 2nd Embodiment, as shown in FIG.9 and FIG.11, the film body 30 folded in the shape of a pleat is accommodated in the inside of 10 A of container main bodies so that the fold line 31 may follow the longitudinal direction of 10 A of container main bodies. That is, the surface of the film body 30 is disposed along the longitudinal direction of the container body 10A.
More specifically, the film body 30 is rolled into a cylindrical shape and accommodated in the container body 10 so that the extending direction of the fold line 31 is the height direction in a state of being folded in a pleat shape.
In the second embodiment, the height H of the folds, that is, the length between the two folding lines 31 arranged adjacent to each other is equal to or less than the radius r of the cylindrical portion 14, and more preferably, the radius r of the cylindrical portion 14. 70% to 90% (see FIG. 11).

According to the cell culture container 1 of 2nd Embodiment demonstrated above, there exist the following effects other than the effect of (1), (2), (4), (5) mentioned above.

(6) The container body 10A was configured in a cylindrical shape, and the container body 10A was configured to include the cylindrical portion 14 and the reduced diameter portion 15. Thereby, when recovering the cultured cells, the cells cultured on the surface of the container body 10A and the film body 30 are peeled off, and then the cell culture container 1A is placed in a centrifuge and centrifuged. The cultured cells are easily collected at the bottom of the cylindrical container body 10A. Further, when the cell culture container 1A after culturing the cells is centrifuged, the cells are collected in the reduced diameter portion 15 while preventing the film body 30 from moving to the tip side of the container body 10A. Therefore, the efficiency of cell culture using the cell culture vessel 1A can be further improved. In addition, since the cultured cells can be collected without being transferred to another container, it is difficult to cause contamination during cell collection.

(7) The film body 30 was folded in a pleated shape at a plurality of fold lines 31, and accommodated in the container body 10A so that the fold line was along the longitudinal direction of the container body 10A. Thereby, since the surface area of the film body 30 accommodated in 10 A of container main bodies can be increased, the area of the area | region which a cell can adhere | attach can be increased more. In addition, since the folding line 31 is arranged along the longitudinal direction of the container main body 10A, the surface of the film body 30 can be aligned in the separation direction (the direction in which gravity is applied) by centrifugation when performing centrifugation. Therefore, the cultured cells are easily separated and collected at the bottom of the cylindrical container body 10A.

(8) The film body 30 was accommodated in the container body 10A in a state of being rolled into a cylindrical shape. Thereby, the film body 30 folded in a pleat shape can be accommodated in the container main body 10A with a larger surface area. Further, since the film body 30 can be evenly arranged inside the container body 10A, it is difficult to cause a bias in the fluidity of the medium filled in the cell culture container 1A. Therefore, the efficiency of cell culture can be further improved.

The preferred embodiments of the cell culture container of the present invention have been described above. However, the present invention is not limited to the above-described embodiments, and can be appropriately changed.

For example, the arrangement of the film body 30 inside the container body 10 is not limited to the arrangement of the first embodiment and the second embodiment. That is, as shown in FIG. 12, a plate-like PET film 32 smaller than the inner diameter of the container main body 10A is arranged in a plurality of layers at predetermined intervals in the thickness direction on the container main body 10A of the second embodiment. In addition, the film body 30 may be configured by integrating the plurality of PET films 32 with a disk-shaped support plate 33 that is substantially equal to the inner diameter of the container body 10. Moreover, as shown in FIG. 13, you may accommodate the PET film in the container main body 10 in the state rolled round, and may comprise the film body 30. As shown in FIG. Moreover, as shown in FIG. 14, you may accommodate the film body 30 folded in the shape of a pleat in 10 A of container main bodies, without rounding into a cylinder shape.

In the first embodiment and the second embodiment, the film body 30 is made of a PET film, but the present invention is not limited to this. That is, you may comprise a film body with other synthetic resin films, such as a polystyrene film and a polypropylene film.

Further, the shape of the container body 10 is not limited to the shape of the first embodiment and the second embodiment. That is, the container body may be formed in a rectangular parallelepiped shape or a cubic shape.

DESCRIPTION OF

SYMBOLS

1,1A

Cell culture container

10,10A Container

main body

11,11A Opening part 12 Main body part 13 Neck part 14 Cylindrical part 15 Reduced diameter part 20 Cap 22 Gas permeation area 30 Film body 31 Folding line

Claims

A container body having an opening;
A cap detachably attached to the opening;
A gas permeable region provided in the cap, having liquid tightness and allowing a gas flow;
A cell culture container comprising: a film body disposed inside the container main body and capable of adhering cultured cells.
The cell culture container according to claim 1, wherein the film body is accommodated in the container body in a state of being folded in a pleated shape by a plurality of fold lines extending in parallel with each other at a predetermined interval.
The container body is
A main body portion having a bottom wall portion, a side wall portion standing up from a peripheral edge of the bottom wall portion, and an upper wall portion arranged in parallel to the bottom wall portion at an upright end of the side wall portion;
A cylindrical neck having one end connected to the side wall and the opening formed on the other end;
The cell culture container according to claim 2, wherein a length between two fold lines arranged adjacent to each other in the film body is equal to or less than a diameter of the opening.
The container body is
A cylindrical portion in which the opening is formed on one end side;
The cell culture container according to claim 1, further comprising a reduced diameter portion disposed on the other end side of the cylindrical portion and having a diameter reduced from the proximal end side toward the distal end side.
The cell culture according to claim 4, wherein the film body is folded in a pleated shape by a plurality of fold lines extending in parallel with each other at a predetermined interval, and the plurality of fold lines are arranged along a longitudinal direction of the container body. container.
The cell culture container according to claim 5, wherein the plurality of broken lines are parallel to a direction in which gravity is applied when the cell culture container is centrifuged.
The cell culture container according to claim 5 or 6, wherein the film body is accommodated inside the container body in a state of being rolled into a cylindrical shape.
The cell culture container according to claim 7, wherein a length between two fold lines arranged adjacent to each other is equal to or less than a radius of the cylindrical portion.
The cell culture container according to any one of claims 1 to 8, wherein the film body is composed of a polyethylene terephthalate film.
10. The cell culture container according to claim 1, wherein the surface of the film body is subjected to charge treatment.