JP2007222037A - Cell-culturing and centrifuging tube - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell-culturing and centrifuging tube which does not have the possibility of contamination caused by the transfer of cultured cells and with which the cells can surely be collected. <P>SOLUTION: This cell-culturing and centrifuging tube is characterized by comprising a separation tube 2 and a stopper 1 for sealing the upper opening of the separation tube 2, disposing an opening in the stopper 1, disposing a culture gas-penetrable nonwoven fabric to cover the opening, disposing at least one baffle 5 on the inner wall surface of the separation tube 2, and further integrally forming the baffle 5 with the separation tube 2. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a cell culture centrifuge tube.

In general, cell culturing operations include cell suspension preparation, cell culturing in a culture gas atmosphere, medium exchange in the culture vessel, etc., and then passage to the culture vessel as needed. Repeat the operation.
Using the cells grown to the required number of cells by the above procedure, recovering the bioreactor that produces the cells, or using the expanded cells for medicine, and using living cells such as E. coli, yeast, and cultured cells It is used for the purpose of synthesizing proteins.

  In the above culturing operation, culture containers such as petri dishes, flasks, roller bottles, and the like, and centrifuge containers called centrifuge tubes are appropriately used. For example, a cell suspension having a predetermined concentration using a centrifuge container is used. After preparing the solution, the cell suspension is transferred to a culture vessel, and a culture operation is performed in a temperature and culture gas atmosphere suitable for culture.

In the subculture operation, the recovery of the bioreactor, and the recovery of the synthesized protein, the culturing operation is carried out by transferring from the culture vessel to the centrifuge vessel.
The essential performance of the culture vessel is mainly culture gas exchange, while the centrifuge tube requires a tube shape and centrifugal strength to perform centrifugation, so each vessel is used properly. Attention to the contamination that occurs when the container is transferred to the container is a great work effort (for example, Patent Document 1).

The transfer work is generally done manually with a pipette in a clean bench, but trained workers can contaminate bacteria and viruses and, depending on the purpose, endotoxin, keratin, and DNA degradation. It is necessary to pay close attention to contamination with enzymes, etc., and take time to work.
For this reason, there has been a problem in that the time taken for the transfer work causes the cells to be damaged and the number of cells that can be collected to decrease.
Even if the work was performed with extreme caution, contamination sometimes occurred and the subsequent operations could be affected.
JP 2000-157261 A

  This invention is made | formed in view of the said situation, and makes it a subject to provide the cell culture centrifuge tube which can extract | collect a cell reliably, without the possibility of the contamination by transfer.

  The cell culture centrifuge tube of the present invention includes a separation tube and a plug that seals the upper opening of the separation tube. The plug is provided with an opening, and is cultured so as to cover the opening. A non-woven fabric capable of gas permeation is provided.

  According to the present invention, since there is a plug body opening and a non-woven fabric is provided so as to cover the opening, the cell culture medium and the like are put in the cell culture centrifuge tube without being transferred to the culture vessel. In addition to being a centrifuge tube, it also functions as a culture tube. Further, since there is no transfer from the culture tube to the centrifuge tube, contamination of the culture solution can be prevented. Furthermore, after culturing, it can be used as a centrifuge tube for separating cells as it is.

  Further, the inner wall surface of the separation tube may be provided with at least one baffle plate. This enables efficient stirring when stirring the liquid. Furthermore, when the culture solution is shaken using a shaker, turbulent flow can be generated in the solution by the baffle plate, and efficient culture can be performed.

  According to the present invention, it is possible to provide a cell culture centrifuge tube that can be reliably collected without fear of contamination due to transfer.

Embodiments of the present invention will be described below with reference to the drawings. In all the drawings, common constituent elements are denoted by the same reference numerals, and detailed description thereof will be appropriately omitted in the following description.

  As shown in FIG. 1, the cell culture centrifuge tube according to this embodiment includes a separation tube (2) and a plug (1) that seals the upper opening of the separation tube (2) (FIG. 1). ), The plug (1) is provided with an opening (7) (FIG. 2 (a)), and a non-woven fabric (4) capable of permeable to culture gas is provided so as to cover the opening (7). (FIG. 2 (a)). 2A is a cross-sectional view taken along line AA ′ in FIG. 1, and FIG. 2B is a cross-sectional view taken along line BB ′ in FIG. 1.

  The purpose of the nonwoven fabric (4) provided on the plug (1) is to prevent contamination of viruses and bacteria while exchanging the culture gas. Therefore, the air permeability of the nonwoven fabric (4) is preferably 1 sec / 100 cc or more and 500 sec / 100 cc or less based on JISP8117. Moreover, when considering the quality maintenance after manufacture, it is preferable to use the nonwoven fabric (4) having a bacterial barrier property (ASTM F1608-95) of 3 years or more. The material of the nonwoven fabric (4) is not particularly limited, and can be selected from polyvinyl chloride, polypropylene, polycarbonate, polyethersulfone, nylon, polyethylene, polytetrafluoroethylene and the like in addition to the cellulosic material. Among these, a polyethylene nonwoven fabric (4) is particularly preferable in consideration of stability against radiation sterilization, water resistance, and chemical resistance.

The thickness of the non-woven fabric (4) is not particularly limited as long as the air permeability is satisfied. However, if it is in the range of 50 μm or more and 500 μm or less, the handleability is excellent and sufficient strength can be obtained. Therefore, it is preferable. The area of the nonwoven fabric (4) through which the culture gas can permeate may be any size as long as the gas that has substantially permeated through the nonwoven fabric (4) is supplied in a sufficient amount for cell culture. preferable because it is stable cultured as having 0.001 cm ² or more per 1ml, medium 1ml per 0.004 cm ² or more, as long as 0.07 cm ² or less, further sufficient gas permeability can be obtained a stable culture In addition, it is more preferable because the risk of the medium scattered on the plug (1) contacting the nonwoven fabric (4) during shaking during culture can be reduced.

The method for joining the plug (1) and the nonwoven fabric (4) needs to be joined with no gap and with a strength that does not come off during the culturing period, and pinching or welding such as heat, ultrasonic waves, and high frequencies is used. I can do things.
Among them, ultrasonic and high frequency welding means are preferable in terms of reliability and strength. The material of the plug (1) is not particularly limited, but is preferably made of a resin in terms of strength, moldability, handleability, price, radiation resistance (sterilization), and the like. Polyethylene resin, polypropylene resin, polystyrene resin, ABS resin, polycarbonate resin, etc. can be selected. When joining by welding by using a polyethylene resin for the plug (1) and using a non-woven fabric (4) made of polyethylene, it is particularly preferable because the reliability and strength of the joined portion are particularly excellent.

  In addition, it is preferable that the plug (1) and the separation tube (2) can be arbitrarily fitted or detached to add a cell suspension or change the medium, and the screw mechanism (6) is joined to the separation tube. This is particularly preferable in terms of certainty when the (2) and the plug (1) are tightened (FIG. 3).

It is preferable that at least one baffle plate (5) is provided on the inner wall surface of the cell culture centrifuge tube of the present embodiment (FIGS. 1 and 2B). In this case, when culturing using a cell culture centrifuge tube, the cell culture centrifuge tube of the present invention is placed in a shaker in an incubator such as an incubator and cultured while being shaken.
At that time, the culture solution inside the separation tube (2) is efficiently stirred by the baffle plate (5) provided inside the separation tube (2), so that the culture solution is passed through the nonwoven fabric (4). One of the important features of the present invention is that the components of the culture solution in the container can be made uniform at the same time that the gas exchange is performed.
Furthermore, when culturing cells in a suspended state such as suspension culture, the cells themselves are uniformly dispersed in the culture solution, so that stable culture is possible. The shape of the baffle plate (5) is not particularly limited, but it is preferably a shape that does not hinder pipetting operation or centrifugal cell recovery operation, and is along the separation tube (2) and the separation tube bottom (3). It can also be provided with a shape (FIGS. 3 and 4), or a twisted plate (8) (FIGS. 5A and 5B) in a protruding shape at the center of the separation tube bottom (3). Furthermore, it is possible to further increase the stirring efficiency by providing a plurality of baffle plates (5) radially from the separation tube bottom (3) (FIG. 7).
Moreover, it is possible to further increase the stirring efficiency by providing baffle plates (5) on the wall surfaces of the central portion of the separation tube (2) and the plurality of portions of the separation tube bottom (3) (FIG. 6).

The material of the separation tube (2) is not particularly limited, but in addition to the same reason as the plug (1), it is preferably made of a resin in terms of easy surface treatment for adherent cells, such as polyethylene resin, polypropylene resin, Polystyrene resin, ABS resin, polycarbonate resin, TPX resin, polyethylene terephthalate resin, etc. can be selected.
Considering transparency and centrifugal strength, polypropylene resin, polycarbonate resin, polyethylene terephthalate resin, and polystyrene resin are particularly preferable.
Further, by imparting hydrophilicity to the inner wall surface of the container by plasma treatment or corona treatment, it can be suitably used for adhesive cell culture.

The shape of the separation tube (2) is not particularly limited, but the tube shape is preferable in terms of centrifugal efficiency, and if the bottom surface is a conical (conical) or round (hemispheric) shape, it is more efficiently centrifuged. This is particularly preferred because the cells can be collected in one place in the container.
The volume of the separation tube (2) can be arbitrarily selected according to the culture scale, but it is preferably in the range of 10 ml or more and 250 ml or less because the handleability is particularly excellent.
The range of 10 ml or more and 100 ml or less is particularly preferable because it can be easily adapted to existing general centrifuges and shakers.

  Moreover, it is preferable that the baffle plate (5) is integrally formed with the separation tube (2). Thereby, since it becomes possible by the same process when forming a separation tube, it becomes possible to provide a cell culture centrifuge tube more efficiently and cheaply.

The cell culture centrifuge tube of the present invention is preferably sterilized. As a sterilization method, autoclave, dry heat sterilization, gas sterilization, radiation sterilization, or the like can be used.
Radiation sterilization is preferable in that there is no problem of residual gas affecting cells and mass processing is possible.

Cells that can be cultured using the present invention are not particularly limited. In addition to general cancerous cells, fungi such as Escherichia coli and yeast for the purpose of protein synthesis, or for regenerative medicine and antibody medicine It can be effectively used for living cells.
In particular, in the fields of regenerative medicine and antibody medicine using human cells, for which significant progress is expected in the future, prevention of contamination and efficient work are important issues, and the cell culture centrifuge tube of the present invention is effectively used. Can be used.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.

Example 1
A polypropylene resin (manufactured by Sumitomo Noblen Co., WP708) was used as a resin material to form a 50 ml capacity tube having a baffle plate (5) integrally with the separation tube (2) having the shape shown in FIG.
The tube had a height of 115 mm, an outer diameter of 30 mm, and a V-bottom with a capacity of 50 ml, and the baffle plate (5) had a length of 50 mm and a height of 5 mm.
Furthermore, the plug body (1) having the shape shown in FIG. 1 was formed by injection molding using a polyethylene resin (KM590L, manufactured by Nippon Polyolefin Co., Ltd.) as the resin material.
The shape of the plug (1) was 15 mm in height and 35 mm in outer diameter, and had an opening (7) having a diameter of 10 mm in the center.
A non-woven fabric (manufactured by Asahi DuPont, Tyvek 1059B, thickness 170 μm, air permeability 22 seconds / 100 cc) is joined to the opening (7) of the plug body (1) by ultrasonic welding, and culture gas can permeate. A plug (1) having an area of the nonwoven fabric (4) of 0.79 cm ² (0.026 cm ² per ml when 30 ml of medium was added) was obtained.
Example 1 was a product sterilized with 10 kGy of γ rays in a state where the tube and the plug (1) were combined.

(Example 2)
The same as Example 1 except that after forming the tube separation tube (2) by injection molding using polystyrene resin (HF Japan manufactured by PS Japan Co., Ltd.) as the resin material, the surface was given wettability by performing oxygen plasma treatment for 5 minutes. Example 2 was prepared under the conditions described above.

(Comparative Example 1)
A tube in which the baffle plate (5) was removed from the tube shown in Example 1 was molded by injection molding using polypropylene resin (manufactured by Sumitomo Noblen Co., WP708) as the resin material.
The shape of the tube is a shape having a height of 115 mm, an outer diameter of 30 mm, a V bottom of 50 ml capacity, and no baffle plate (5).
Furthermore, the plug body (1) in which the opening (7) is eliminated from the plug body (1) having the shape shown in Example 1 by injection molding using polyethylene resin (manufactured by Nippon Polyolefin Co., Ltd., KM590L) as the resin material. Was molded.
The shape of the plug (1) is 15 mm in height and 35 mm in outer diameter, and does not have an opening (7) in the center.
A combination of the above tube and the plug (1) sterilized with γ rays of 10 kGy was designated as Comparative Example 1.

(Comparative Example 2)
A comparative example of a tube sterilized with 10 kGy of γ rays in a state where a tube having the same baffle plate (5) as in Example 1 is combined with a plug body having no opening (7) as in Comparative Example 1 2.

Test Example Hepatoblastoma hepablastoma (HepG2) cells were adjusted to 1 × 10 ⁴ cells / ml with Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum. Example 1, Example 2, Comparative Example 1 Then, each 30 ml was dispensed into the centrifuge tubes manufactured in Comparative Example 2, and after closing each stopper (1), in a carbon dioxide incubator with a humidity of 37% at 37 ° C., a shaker (MULTI manufactured by Tokyo Science Machinery Co., Ltd.). The cells were cultured for 6 days with shaking at 170 rpm using a SHAKER MMS. The medium was changed twice during 6 days of culture.
After completion of the culture, each centrifuge tube taken out from the incubator was centrifuged at 1000 rpm for 5 minutes, and then all the cells in the centrifuge tube were collected and the number of cells was measured. The results are shown in Table 1.

As is clear from Table 1, the cell culture centrifuge tubes of the present invention produced in Example 1 and Example 2 showed good cell growth properties.
Further, in Example 1, the state of the cells after culturing grew in a state where several cell aggregates were dispersed in the medium, and in Example 2 in which the surface treatment for culture was performed, the aggregates in the medium In addition to the above, the cells were proliferating in a state where the cells were adhered and layered on the wall surface of the tube.
On the other hand, in the comparative example, most cells were killed, and in the comparative example 2 in which gas exchange was performed with the nonwoven fabric (4) of the plug (1), viable cells were confirmed, but gas exchange in the medium was performed. It was thought that it did not proliferate because it was not performed sufficiently.

  The present invention relates to a cell culture centrifuge tube that does not require transfer of a container during cell culture operation and centrifugation operation, and there is no risk of contamination, preparation of a cell suspension, medium in a culture container Since it is possible to safely and quickly perform operations such as exchange, passaging, and bioreactor recovery, fungi such as Escherichia coli and yeast for general cell culture research purposes and protein synthesis, or It can be effectively used in the field of regenerative medicine and antibody medicine for the purpose of regenerative medicine and antibody medicine, as well as regenerative medicine and antibody medicine using human cells.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Plug body 2 Separation pipe 3 Separation pipe bottom part 4 Nonwoven fabric 5 Baffle board 6 Screw 7 Opening part 8 Twist board

It is a perspective view of the cell culture centrifuge tube which shows the state at the time of culture | cultivation of this invention. It is sectional drawing which shows the opening part of a stopper, and the baffle plate of a separation pipe. It is a perspective view which shows the screw mechanism of a separation tube. It is a perspective view which shows other embodiment of a baffle plate. It is a perspective view which shows other embodiment of a baffle plate. It is a perspective view which shows other embodiment of a baffle plate. It is a perspective view which shows other embodiment of a baffle plate.

Claims (6)

Including a separation tube and a plug for sealing the upper opening of the separation tube,
An opening is provided in the plug, and a non-woven fabric capable of permeable to culture gas is provided so as to cover the opening.   The cell culture centrifuge tube according to claim 1, wherein at least one baffle plate is provided on an inner wall surface of the separation tube.   The cell culture centrifuge tube according to claim 1, wherein the baffle plate is integrally formed with the separation tube.   The cell culture centrifuge tube according to any one of claims 1 to 3, wherein an inner wall surface of the separation tube is plasma-treated.   The cell culture centrifuge tube according to any one of claims 1 to 4, wherein the stopper and the separation tube are detachable.   The cell culture centrifuge tube according to any one of claims 1 to 5, wherein at least one twist plate is provided at a bottom of the separation tube.

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