JP6724649B2 - Cell culture device - Google Patents

Description

The present invention relates to a cell culture device that cultures cells using a culture bag.

In recent years, in the fields of medicine and biochemistry such as drug production, gene therapy, regenerative medicine, and immunotherapy, cells (including tissues, microorganisms, viruses, etc.) can be efficiently cultured in large quantities in an artificial environment. It has been demanded.

In order to meet such demands, the applicant has constructed a closed environment using a bag-shaped culture bag made of a flexible material to efficiently culture cells while reducing the risk of contamination. We have been repeatedly studying the technology related to cell culture that can be performed.

For example, in the patent document 1, the present applicant connects a plurality of culture bags via a transfer tube, transfers cells cultured in a culture bag having a small volume to a culture bag having a larger volume, and thereby the density of cells is increased. We have proposed a cell culture method for efficiently proliferating cells while maintaining the appropriateness.

Further, in Patent Document 1, a culture bag arranged at a relatively high position is tilted so that a transfer port provided in the culture bag is on a lower side, and a content liquid (cultured cells It is also proposed to transfer the suspended cell suspension) to a culture bag placed at a relatively low position by sending it from a transfer port. And, as an example of the device for tilting the culture bag, Patent Document 1 shows an example in which the base on which the culture bag is placed is pivoted with one end thereof pivoted to tilt the culture bag. ..

JP, 2005-188392, A

However, according to subsequent studies by the applicant, when the content liquid is sent from the transfer port provided in the culture bag to collect or transfer the cultured cells, the vicinity of the transfer port in the culture bag is considered. It has been found that the cells tend to be retained in the culture bag, and the retained cells remain in the culture bag and cannot be recovered.

This is considered to be due to the following reasons. That is, the transfer port is usually formed of a tubular member having a certain thickness, and as shown in FIG. 4, when the culture bag 10 is placed on the base 2p, the transfer port and the inner bottom surface of the culture bag 10 are transferred. A step corresponding to the thickness of the transfer port 11 is formed between the port 11 and the flow hole 12, and in the periphery of the transfer port 11, the inner bottom surface of the culture bag 10 is bent by the flexible material. Wrinkles are likely to occur. Therefore, it is considered that the cells C that could not reach the flow-feeding hole 12 of the transfer port 11 because of being blocked by such steps or wrinkles stay in front of the transfer port 11.
4 is an explanatory view showing the conventional technique, and shows a cross section of the culture bag 10 placed on the base 2p on the transfer port 11 side.

The present invention has been made on the basis of such findings, and when recovering or transferring cells cultured using a culture bag, the cells easily reach the flow port of the transfer port provided in the culture bag. By doing so, it is an object of the present invention to provide a cell culture device capable of collecting or transferring more cells.

The cell culture device according to the present invention is a cell culture device that performs cell culture using a culture bag made of a flexible material, and is provided in a base on which the culture bag is placed and the culture bag. A holder for holding a transfer port, wherein the base is provided with a notch at a position where the transfer port faces when the culture bag is placed, and the holder is The lowermost part of the flow port of the transfer port is configured to hold the transfer port in a state of being positioned below a reference surface including a mounting surface of the base on which the culture bag is mounted. ..

According to the present invention, when collecting or transferring cells cultured using a culture bag, it is possible to reduce the amount of cells remaining in the culture bag due to the cells remaining in the vicinity of the transfer port, and to increase the number of cells. It can be collected or transferred.

It is explanatory drawing which shows the outline of the cell culture apparatus which concerns on embodiment of this invention. It is explanatory drawing which shows the outline of the cell culture apparatus which concerns on embodiment of this invention. FIG. 3 is a sectional view taken along line AA of FIG. 2. It is explanatory drawing which shows a prior art.

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

An outline of the embodiment of the cell culture device according to the present invention is shown in simplified form in FIGS. 1 and 2. The cell culture device 1 shown in these figures is a device for carrying out cell culture using a culture bag 10 made of a flexible material.
1 shows a state before the culture bag 10 is attached to the cell culture device 1, and FIG. 2 shows a state where the culture bag 10 is attached to the cell culture device 1.

The culture bag 10 preferably has gas permeability (oxygen permeability, carbon dioxide permeability) necessary for cell culture so that cell culture can be performed in a closed system. Furthermore, in order to achieve high cell growth efficiency, it is preferable that the cells have low cytotoxicity, low elution properties, and radiation sterilization suitability. It is preferable that it has such transparency that it can be seen through.

As the flexible material forming the culture bag 10, for example, polyethylene, polypropylene, ethylene-α-olefin copolymer, ethylene-vinyl acetate copolymer, ethylene and acrylic acid or methacrylic acid copolymer and metal ions are used. Examples of the ionomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, polystyrene-based thermoplastic elastomer, and resin film made of a thermoplastic resin such as tetrafluoroethylene-hexafluoropropylene copolymer (FEP) are used. These resins may be used as a single layer or may be used by laminating same or different kinds of resins.

The culture bag 10 is formed in a bag shape using such a flexible material. For example, the peripheral portion may be sealed by heat fusion and formed into a desired shape such as a rectangular shape, but it was prepared for culturing such cells together with cells to be cultured. The specific form is not limited as long as the medium can be accommodated in the culture without hindrance.

Further, the culture bag 10 is provided with a transfer port 11 to which a transfer tube can be connected. By providing the transfer port 11 as described above, for example, the medium is supplied from the medium bag in which the medium is stored to the culture bag 10, or the cultured cells are collected from the culture bag 10 after the completion of the culture and used for collection. Operations such as transfer to a bag or another culture bag can be performed while maintaining a closed system via a transfer tube connected to the transfer port 11.

The transfer port 11 is made of a tubular member through which a culture medium, cells, etc. can flow, and includes, for example, polyethylene, polypropylene, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polybutadiene resin, silicone rubber, polyurethane-based thermoplastic elastomer. A thermoplastic resin such as a thermoplastic polyester elastomer, a thermoplastic silicone elastomer, a vinyl chloride, a thermoplastic polystyrene elastomer, or a thermoplastic resin such as FEP is used to form a flow hole 12 penetrating in the axial direction by injection molding or extrusion molding. It can be molded into a predetermined shape.

Although not particularly shown, in order to prevent the top surface side and the bottom surface side of the culture bag 10 from sticking to the transfer port 11 and clogging the flow-feeding hole 12, the culture bag is started from its proximal end. A port blockage prevention piece that projects into 10 may be provided.

In addition, for example, when the culture bag 10 is manufactured using a resin film, the transfer port 11 is provided between the resin film forming the top surface side of the culture bag 10 and the resin film forming the bottom surface side. Can be provided by sandwiching the above member and heat-sealing it.

The cell culture device 1 includes a base 2 on which such a culture bag 10 is placed, and a holder 3 that holds a transfer port 11 provided on the culture bag 10.

The base 2 is a base having a mounting surface on which the culture bag 10 is mounted. In the illustrated example, the fixtures 21 provided upright at the four corners of the base 2 are provided at the four corners of the culture bag 10. The culture bag 10 can be fixed on the mounting surface by locking the formed holes 13.

Although not particularly shown, the base 2 may be pivotally supported by one end side of which is pivotally supported. By doing so, the culture bag 10 placed on the base 2 is tilted so that the transfer port 11 is on the lower side, and the liquid content of the culture bag 10 is sent from the transfer port 11 by Although it can be transferred to a collection bag or another culture bag, the means for causing the content liquid of the culture bag 10 to flow from the transfer port 11 is not limited to this. For example, a peristaltic pump may be attached to a transfer tube connected to the transfer port 11 so that the liquid content of the culture bag 10 is sent from the transfer port 11 by its operation.

In addition, the base 2 is provided with a cutout 22 at a portion facing the transfer port 11 provided in the culture bag 10 when the culture bag 10 is placed. The notch 22 is located on the base 2 so that the transfer port 11 can be pushed down against the mounting surface of the base 2 without the transfer port 11 interfering with the base 2. It can be formed along the contour of the transfer port 11 (see FIG. 2).
Then, as shown in FIG. 3, the pushed transfer port 11 has a holder with the lowermost part of the flow hole 12 positioned below a reference surface including the mounting surface of the base 2. It is held at 3.
Note that FIG. 3 is a cross-sectional view taken along the line AA of FIG. 2, and the reference plane is indicated by a chain line.

In the illustrated example, as an example of the holder 3 that holds the transfer port 11, a receiving portion 3 a provided with an insertion portion through which the transfer port 11 is inserted and an upper portion of the transfer port 11 inserted through the insertion portion are provided. The holding tool 3 including a pressing plate 3b that presses is illustrated in a simplified manner. A specific configuration of the holder 3 as long as the transfer port 11 can be held in a state in which the lowermost part of the flow hole 12 of the transfer port 11 is located below the reference surface including the mounting surface of the base 2. Although not limited to this, it is preferable that the transfer port 11 can be held so that the central axis of the transfer port 11 is parallel to the mounting surface of the base 2.

As described above, a step is formed between the inner bottom surface of the culture bag 10 placed on the base 2 and the flow port 12 of the transfer port 11, or the culture bag around the transfer port 11 is formed. When wrinkles are generated on the inner bottom surface of 10, when the content liquid is sent from the transfer port 11 to collect or transfer the cultured cells, the transfer port 11 is blocked by the step or wrinkle. The cells C, which could not reach the delivery hole 12 of the above, stay in front of the transfer port 11 (see FIG. 4).
On the other hand, in the present embodiment, the transfer port 11 is held so that the lowermost part of the flow hole 12 of the transfer port 11 is located below the reference surface including the mounting surface of the base 2. The feed hole 12 of the transfer port 11 is located on an extension of the inner bottom surface of the culture bag 10 held on the base 3 and placed on the base 2. As a result, no step is formed between the inner bottom surface of the culture bag 10 and the flow port 12 of the transfer port 11, and the wrinkles on the inner bottom surface of the culture bag 10 pulled by the pushed transfer port 11 are not formed. Since the generation of cells is suppressed, the cells C in the content liquid easily reach the flow-feeding hole 12 of the transfer port 11 (see FIG. 3 ). As a result, according to the present embodiment, it is possible to effectively avoid the cells from staying in front of the transfer port 11 and collect or transfer more cells.

As described above, in the present embodiment, the lowermost portion of the flow-feeding hole 12 of the transfer port 11 is positioned below the reference surface including the mounting surface of the base 2 so that the cells in the content liquid are It is made easy to reach the flow hole 12 of the transfer port 11. In order to achieve such an effect more favorably, when the transfer port 11 is held by the holder 3, the center of the flow port 12 of the transfer port 11 is placed on the base 2. It is preferable to be located on or below the reference plane including the surface, and it is preferable that the uppermost part of the flow-feeding hole 12 of the transfer port 11 is located above the reference plane. ..

Although the present invention has been described with reference to the preferred embodiments, it is needless to say that the present invention is not limited to the above-described embodiments and various modifications can be made within the scope of the present invention.

For example, in the above-described embodiment, a simplified flat plate shape is illustrated and described as an example of the base 2, but the specific form of the base 2 is not limited to this. As proposed by the applicant in Japanese Patent Application No. 2005-017823, the base 2 has a partition that can project from the mounting surface at a predetermined height when the partition is accommodated in the base. The upper end surface of the piece may be embedded so as to be flush with the mounting surface. Furthermore, as proposed by the present applicant in Japanese Patent Application No. 2005-017824, the base 2 is provided with a pressing plate for pressing the culture bag 10 placed on the base 2, and the amount of the liquid content of the culture bag 10 is It can also be attached so that it can be moved up and down according to.

In short, the present invention comprises a base 2 on which the culture bag 10 is placed, and a holder 3 for holding the transfer port 11 provided on the culture bag 10, and the base 2 is provided with the culture bag 10. A cutout portion 22 is formed at a position where the transfer port 11 faces when placed, the holder 3 is placed, and the culture bag 10 is placed at the lowest part of the flow hole 12 of the transfer port 11. As long as the transfer port 11 is held in a state of being positioned below the reference surface including the mounting surface of the base 2, the detailed configuration other than this is limited to the above-described embodiment. It can be changed as appropriate without any need. Further, the detailed configurations described in the above-described embodiments can be appropriately selected and combined.

Hereinafter, the present invention will be described in more detail with reference to specific examples.

[Example 1]
Using a polyethylene resin film, the peripheral portion was sealed by heat fusion to prepare a culture bag 10 formed into a rectangular shape with a length of 320 mm and a width of 90 mm. 230 mL of the sample culture solution (including cultured cells and medium) was accommodated in the culture bag 10 and attached to the cell culture device 1 as shown in FIG. At that time, the transfer port 11 located on the notch 22 formed in the base 2 is pushed down so that the lowermost part of the flow-feeding hole 12 of the transfer port 11 is a base on which the culture bag 10 is placed. The transfer port 11 is held by the holder 3 in a state in which the transfer port 11 is located below the reference surface including the mounting surface.

Next, one end side of the base 2 is pivotally supported and rotated, and the culture bag 10 placed on the base 2 is tilted so that the transfer port 11 is on the lower side. The liquid was flown from the transfer port 11 by a drop, and allowed to stand until the flow from the transfer port 11 was not observed.
In addition, a pressure plate for pressing the upper surface side of the culture bag 10 was installed on the base 2 so that uneven distribution of the content liquid when the culture bag 10 was tilted was less likely to occur. Further, the inclination angle of the base 2 was set to about 10°.

Then, the culture bag 10 was removed from the cell culture device 1, and a syringe containing 5 mL of physiological saline and 7 mL of air was connected to the transfer port 11 and injected into the culture bag 10. Then, the inside of the culture bag 10 is washed with injecting physiological saline, and the mixed solution of the culture solution and the physiological saline remaining in the culture bag 10 is recovered, and the recovered solution is put into a 15 mL centrifuge tube and is rotated at 1200 rpm for 3 minutes. Centrifuge for minutes. The recovered liquid after centrifugation is mixed again with a pipette, the amount of recovered liquid is measured, diluted 10 times, and the number of cells is measured by a hemocytometer according to a standard method to calculate the number of remaining cells in the recovered liquid. did. The results are shown in Table 1.

[Comparative Example 1]
The content liquid of the culture bag 10 was sent from the transfer port 11 by a drop under the same conditions as in Example 1 except that the base 2p (see FIG. 4) in which the cutout portion 22 was not formed was used. After that, the inside of the culture bag 10 was washed with physiological saline, and the number of remaining cells in the recovered solution was calculated. Based on this, the reduction rate of the number of remaining cells with respect to Comparative Example 1 of Example 1 was also calculated. The results are shown in Table 1.

[Example 2 and Comparative Example 2]
Under the same conditions as in Example 1 and Comparative Example 1, respectively, except that the cell concentration of the sample culture solution was different, after the content liquid of the culture bag 10 was flowed by a drop from the transfer port 11, the inside of the culture bag 10 was changed. After washing with physiological saline, the number of residual cells in the recovered solution was calculated. Based on this, the reduction rate of the number of remaining cells with respect to Comparative Example 2 of Example 2 was also calculated. The results are shown in Table 1.

From these results, the number of remaining cells in the culture bag 10 is adjusted by arranging that the lowermost part of the flow hole 12 of the transfer port 11 is located below the reference surface including the mounting surface of the base 2. It was confirmed that the number of cells was reduced and more cells could be collected.

INDUSTRIAL APPLICABILITY The present invention can be suitably used in regenerative medicine, immunotherapy, antibody drug production, etc., in which a large amount of cells are cultured in a closed system.

DESCRIPTION OF SYMBOLS 1 Cell culture device 2 Base 22 Notch 3 Holding device 10 Culture bag 11 Transfer port 12 Flow hole

Claims (3)

A cell culture device for culturing cells using a culture bag made of a flexible material,
A base on which the culture bag is placed,
A holder for holding the transfer port provided in the culture bag,
In the base, a cutout portion is formed at a portion where the transfer port faces when the culture bag is placed,
The transfer port is such that the lowermost part of the flow-feeding hole of the transfer port is located below a reference surface including a mounting surface of the base on which the culture bag is mounted. A cell culture device for holding a cell culture medium. The cell culture device according to claim 1, wherein the holder holds the transfer port in a state in which a center of a flow hole of the transfer port is located on the reference surface or below the reference surface. .. The cell culture device according to claim 1 or 2, wherein the holder holds the transfer port in a state in which the uppermost part of the flow-feeding hole of the transfer port is located above the reference surface.

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