JP2012200225A - Assisting tool for centrifugal separation of container for measuring cellular potential and method of centrifugal separation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an assisting tool for centrifugal separation, which can perform the centrifugal separation of a container for measuring cellular potential, without having the leakage of culturing liquid, etc., from the gap of a container body part and an electrode substrate, and to provide a method of centrifugal separation.SOLUTION: This assisting tool 2 for the centrifugal separation, which is used in performing the centrifugal separation of the container 1 for measuring the cellular potential, which includes the container body part 11 and the electrode substrate 12, is characterized by including a bottom surface 21 having the approximately same form as a bucket of the centrifugal separator, and a projecting part 22 which is arranged on an upper part of the bottom surface 21 and has the approximately same form as the electrode substrate 12 of the container 1 for measuring the cellular potential. On performing the centrifugal separation of the container 1 for measuring the cellular potential, the bottom surface of the electrode substrate 12 of the container 1 for measuring the cellular potential is supported by the projecting part 22 while the container 1 for measuring the cellular potential is loaded on the assisting tool 2 for the centrifugal separation.

Description

  The present invention relates to a centrifuge auxiliary tool for a cell potential measuring container mounted on an electric potential measuring device and measuring a cell potential, and a centrifuge method thereof.

  In modern new drug development, it is required to determine early on the toxicity caused by drugs. As one type of toxicity, drug-induced (acquired) QT prolongation syndrome that can cause serious arrhythmia is known.

  In drug-induced long QT syndrome, the QT interval on the electrocardiogram is prolonged after the drug is administered. From TdP (Torsades de pointes) It is a serious disease that causes movement, fainting, and sudden death. In fact, of the 25 drugs that were discontinued in the US market since 1980, 5 were suspected of causing drug-induced long QT syndrome.

  For this reason, in order to determine the toxicity caused by the drug, a measurement method for observing the influence of the drug on the activity of the ion channel from the change in the potential of the cell in the culture medium to which the drug is administered has been disclosed (Non-Patent Document). 1). A cell potential measuring container for more efficiently measuring by such a measuring method is disclosed (Patent Document 1), and this cell potential measuring container includes a plurality of wells that contain cells together with a culture solution, and The measurement electrode and the reference electrode are disposed on the bottom surface of each well. The cell potential is measured by mounting the cell potential measuring container on the potential measuring device.

  However, the above-mentioned cell potential measurement container needs to be provided with an electrode, and is provided with a substrate on which an electrode is provided separately from the container. Therefore, the cell potential measurement container is centrifuged with a centrifuge. In some cases, the center portion of the substrate is bent, so that a gap is formed between the container and the substrate, and the culture solution leaks.

URL: http: //www.brck.co.jp/MCS/qtscreencataloguejp1.pdf

International publication WO2011 / 010721 gazette

  The present invention has been made in view of such circumstances, and a centrifuge auxiliary tool and a centrifuge capable of centrifuging a cell potential measurement container without leakage of a culture solution or the like from between the container body and the electrode substrate. It is an object to provide a separation method.

  As a result of intensive studies by the present inventors in order to solve such problems, it is possible to prevent leakage of a culture solution or the like by supporting the electrode substrate using a centrifuge auxiliary tool during centrifugation of the cell potential measurement container. The method of suppressing was discovered and it came to this invention.

  A centrifugal auxiliary tool of the present invention includes a container body portion including a plurality of cylindrical bodies having upper and lower ends opened, and an electrode substrate including a substrate body and a plurality of electrodes disposed on the substrate body, The electrode substrate is joined to the lower end of the container body to form a plurality of wells, which are mounted on a potential measuring device and used for centrifuging a cell potential measuring container for measuring a cell potential. The centrifuge assisting tool includes a bottom surface having substantially the same shape as the bucket of the centrifuge, and a convex having substantially the same shape as the electrode substrate of the cell potential measuring container provided on the top of the bottom surface. And the electrode substrate of the cell potential measuring container by the convex part of the centrifuge assisting tool in a state where the cell potential measuring container is placed on the centrifuge assisting tool when the cell potential measuring container is centrifuged. The bottom of the The features.

  Further, the centrifugal separation method of the present invention includes a container body portion including a plurality of cylindrical bodies having upper and lower ends opened, and an electrode substrate including a substrate body and a plurality of electrodes disposed on the substrate body. A method for centrifuging a cell potential measuring container for measuring a cell potential mounted on a potential measuring device by joining the electrode substrate to the lower end of the container body and forming a plurality of wells. The centrifuge is provided with a centrifuge having a bottom surface having substantially the same shape as the centrifuge bucket and a convex portion having substantially the same shape as the electrode substrate of the cell potential measuring container provided on the top of the bottom surface. A step of placing the auxiliary tool with the bottom surface facing down, a step of placing the bottom surface of the electrode substrate of the cell potential measuring container and the convex portion of the centrifugal auxiliary tool on the centrifugation auxiliary tool, And above by centrifuge The cellular potential measuring container characterized in that it comprises a step of centrifugation.

  By using the centrifugation method of the present invention or performing centrifugation using the centrifugal auxiliary tool of the present invention, the cell potential measuring container can be used without leakage of the culture solution or the like from between the container body and the electrode substrate. Can be centrifuged.

It is a disassembled perspective view of the cell potential measuring container in the present invention. It is a perspective view of the centrifuge auxiliary tool of the present invention. It is a disassembled perspective view at the time of attaching the centrifuge auxiliary tool of this invention to a cell potential measuring container. It is a perspective view at the time of attaching the centrifugation auxiliary tool of this invention to the cell potential measurement container. FIG. 6 is a cross-sectional view (cross section taken along the line X-X ′ in FIG. 4) when the centrifugal auxiliary tool of the present invention is attached to the cell potential measuring container.

  Embodiments based on the present invention will be described below with reference to the drawings. In addition, this embodiment is only one embodiment of this invention, and can change an embodiment in the range which does not change the summary of this invention. In the present invention, the centrifuge refers to a bucket-type centrifuge that has a bucket inside, which is commonly used in the field of cell culture and the like, and a cell culture container or the like is placed in the bucket of the centrifuge. And centrifuge.

  As shown in FIG. 1, in the present invention, the cell potential measuring container 1 is mainly composed of a container body 11, an electrode substrate 12, and a double-sided tape 13. The resin container body 11 includes a plurality of cylindrical tubular bodies 111 having upper and lower ends opened. Each cylindrical body 111 constructs a sidewall of the well 14 (the well 14 represents the space shown in FIG. 5). The upper end of the container body 11 is open to accommodate cells and culture solution at the time of measurement, and the lower end is open because the electrode substrate 2 described later is used as the bottom surface. The shape of the cylindrical body 111 is not particularly limited in the present invention, but is preferably a cylindrical shape from the viewpoint of easy manufacturing.

  In the present invention, since the cell potential measuring container 1 is disposable, it is desirable that at least the container body 11 is made of resin in order to make the product inexpensive. The resin is not particularly limited as long as the container body 11 can be molded and is electrically insulating. If the specific example of resin is given, a polypropylene, a polystyrene, polyester, a polycarbonate, etc. will be mentioned, for example. In particular, from the viewpoint of low material cost, high transparency, and good appearance, the resin is preferably polystyrene. In addition, the manufacturing method of the container body 11 is not necessarily limited as long as it is easy to manufacture. However, in view of the complicated structure of the container body, it is usually desirable to manufacture by injection molding. .

  On the other hand, the electrode substrate 12 constructs the bottom surface of the well 14. The electrode substrate 12 includes a substrate body 121 made of an electrically insulating material, and a plurality of measurement electrodes 122 and reference electrodes 123 are disposed on the electrode substrate 121. The plurality of measurement electrodes 122 and the reference electrode 123 include at least copper. The measurement electrode 122 on the electrode substrate 12 in FIG. 1 is disposed substantially at the center of the C-shaped reference electrode 123. Then, all the measurement electrodes 122 and the reference electrodes 123 are led to the connectors 125 arranged in a line on one side of the electrode substrate by independent lead wires 124. The shape of the reference electrode 123 is C-shaped in order to secure a space for arranging the lead wire 124 in order to lead the measurement electrode 122 to the connector 125. Although not shown, the connector 125 is also disposed on the back surface of the electrode substrate 12 (the surface on which the measurement electrode 122 and the reference electrode 123 are not disposed). When the cell potential measuring container of the present invention is placed on a dedicated potential measuring device (not shown), the connector 125 on the back surface of the electrode substrate 12 is electrically connected to the dedicated potential measuring device, Allows measurement of cell potential.

  The substrate body 121 is made of a so-called electrically insulating material, and examples thereof include polypropylene, polystyrene, polyester, fluorine resin, polycarbonate, acrylic resin, paper phenol, paper epoxy, glass epoxy containing glass epoxy, and alumina. However, it is easy to construct the measurement electrode 122 and the reference electrode 123. From the viewpoint of high electrical conductivity, mechanical strength, and low cost, glass epoxy is generally selected. Many. However, the present invention is not limited to the material of the substrate body 121.

  The measurement electrode 122 is an electrode that contacts a cell and measures the activity of the ion channel of the cell as a potential, and the reference electrode 123 is a potential that becomes a so-called reference when contacting with a liquid (usually a culture solution) used at the time of measurement. It is an electrode for measuring. The measurement electrode 122 and the reference electrode 123 are disposed at such positions that one exists on the bottom surface of each well 14 when the electrode substrate 12 is joined to the container body 11 to form the well 14. Of course, the measurement electrode 122 and the reference electrode 123 are not electrically connected even at the bottom surface of each well 14. Examples of the material used for plating the measurement electrode 122 and the reference electrode 123 include gold, silver, carbon, platinum, ruthenium oxide, and palladium. These materials are plated on copper wiring formed on the substrate body.

  The container body 11 and the electrode substrate 12 are joined by a double-sided tape 13. Joining is performed so that the double-sided tape 13 does not exist at least at the lower end of the cylindrical body 111 of the container body 11. That is, the shape of the double-sided tape 13 is adjusted according to the shape of the joining margin of the container body. As a result, the electrode substrate 12 is exposed as the bottom surface of the well 14. The type of the double-sided tape 13 is not particularly limited as long as the bonding strength between the container body and the electrode substrate is sufficient and has electrical insulation, water resistance (water repellency) and chemical resistance. It is not a thing. For example, double-sided tapes such as Nichiban double-sided tape (manufactured by Nichiban Co., Ltd.) and structural bonding tapes (manufactured by Sumitomo 3M Limited) can be suitably used. In addition, after joining one side of the double-sided tape 13 to the joining margin of the container body, the other side of the double-sided tape 13 may be joined to the electrode substrate 12 or vice versa. Furthermore, in order to perform bonding accurately, for example, it is desirable to provide holes for positioning in the container body, the electrode substrate, and the double-sided tape, respectively, but the present invention is not necessarily limited to the presence or absence of the holes for positioning. Is not to be done.

  The cell potential measuring container 1 in the present invention is a disposable part used for a system that measures changes in cell potential with respect to a plurality of novel drug candidate compounds in each well 14. The cell potential measuring container of the present invention is used by being mounted on a dedicated potential measuring device (not shown). The configuration has a plurality of wells 14. The plurality of wells 14 are formed by bonding the electrode substrate 2 to the lower end of the container body 1 as described above. The formed well 14 is liquid-tight enough to accommodate a medium (usually a culture solution) used for measurement. A measuring electrode 22 and a reference electrode 23 are disposed on the bottom surface of each well 14. Thereby, the potential of the cell can be measured in each well 14. Here, the “cell” includes not only one cell but also a cell mass (spheroid) in which a plurality of cells are aggregated.

  If the culture solution does not reach the bottom surface of each well 14 at the time of the potential measurement, the potential cannot be measured accurately (for example, when air exists between the bottom surface of the well 14 and the culture solution). Therefore, an operation of centrifuging is performed so that the culture solution reaches the bottom surface of the well 14.

  Hereinafter, the centrifugal auxiliary tool 2 of the present invention will be described with reference to FIG. The centrifugal auxiliary tool 2 of the present invention includes a bottom surface 21 and a convex portion 22. The material of the centrifugal auxiliary tool 2 of the present invention is not particularly limited as long as it can be mounted on a bucket and holds the cell potential measuring container 1 with the convex portion 22. For example, polypropylene, polyacetal, poly Examples thereof include resin materials such as ether ether ketone and polytetrafluoroethylene, metal materials such as stainless steel and aluminum alloy, and elastic materials such as silicone rubber. Also, the manufacturing method is not particularly limited, but a method in which the bottom surface 21 and the convex portion 22 are integrally formed, and a method in which the bottom surface 21 and the convex portion 22 are separately molded and bonded are preferable.

  Since the bottom surface 21 is used by placing it on the bucket inside the centrifuge (not shown) with the bottom surface 21 facing downward, it may have any shape corresponding to the bucket, for example, a conventional square A square shape or a circular shape can be considered so as to correspond to a die or a round bucket. In addition, since the bottom surface 21 has substantially the same shape as the bucket, there are few gaps generated between the bucket and the centrifuge assisting tool 2 when the centrifuge assisting tool 2 is placed on the bucket, so that the operability is excellent. In the present embodiment, as shown in FIG. 2, a square bottom surface 21 having substantially the same shape as a square bucket is provided.

  The convex portion 22 is provided on the upper portion of the bottom surface 21. As shown in FIG. 3, the convex portion 22 is used by placing the above-described cell potential measuring container 1 from the top, so long as it has a shape corresponding to the bottom surface of the substrate body 121 of the electrode substrate 12 of the cell potential measuring container 1. For example, a square shape can be considered. Further, the convex portion 22 has substantially the same shape as the bottom surface of the substrate body 121, so that when the cell potential measuring container 1 is placed on the centrifuge assisting tool 2, a gap generated between the centrifuge assisting tool 2 and the cell potential measuring container 1. Therefore, it is possible to support the bottom surface of the substrate body 121 of the cell potential measuring container 1 with more surfaces, and to stably hold the substrate body 121 during centrifugation. In the present embodiment, as shown in FIG. 2, a square-shaped convex portion 22 having substantially the same shape as the substrate main body 121 of FIG. 1 is provided. The upward protruding length (height) of the convex portion 22 is not particularly limited as long as the cell potential measuring container 1 can be held. For example, the lower end of the container body 11 of the cell potential measuring container 1 and One that is larger than the height of the bottom surface of the substrate body 121 (substrate height h in FIG. 4) is conceivable, specifically 3 to 5 mm. By providing the convex portion 22 at such a height, the substrate body 121 of the electrode substrate 12 of the cell potential measurement container 1 is held from below even during centrifugation, so that a downward force is applied to the substrate body 121, It is possible to prevent the substrate body 121 from being bent.

Hereinafter, the centrifugation method according to the present embodiment will be described with reference to FIGS. The centrifugation method according to this embodiment is roughly divided into the following three steps.
(1) The first step of placing the centrifuge aid 2 on the centrifuge bucket.
(2) Second step of placing the cell potential measuring container 1 on the centrifuge auxiliary tool 2.
(3) Third step of centrifuging cell potential measuring container 1 with a centrifuge.

  In a 1st process, it mounts in the form which faces the bottom face of the centrifuge auxiliary tool 2 on the bucket of a centrifuge. At this time, since the bottom surface 21 of the centrifuge auxiliary tool 2 is provided in substantially the same shape as the bucket, it is possible to arrange the bucket and the bottom surface 21 with a small excess gap.

  In the subsequent second step, as shown in the exploded perspective view of FIG. 3, the bottom surface of the substrate body 121 of the cell potential measurement container 1 is in the order of the lid 3, the cell potential measurement container 1, and the centrifuge auxiliary tool 2 from the top. Arrange it so that it is at the bottom. The lid 3 is provided in a shape that covers the container body 11 so that foreign matter does not enter the cell potential measurement container 1 during centrifugation. When the cell potential measuring container 1 is placed on the convex portion of the centrifugation auxiliary tool 2, the convex portion 22 of the centrifugal auxiliary tool 2 is provided in substantially the same shape as the bottom surface of the substrate main body 121. It is possible to arrange the bottom surface and the convex portion 22 with little extra space. The state in which the cell potential measuring container 1 is placed on the convex portion of the centrifuge auxiliary tool 2 is as shown in FIG. 4, and the cross-sectional view thereof is as shown in FIG.

  Note that the order of the first step and the second step may be reversed. That is, the order of placing the bucket, the centrifugal auxiliary tool 2, and the cell potential measuring container 1 from the bottom in the centrifuge before the third step is not limited.

  Further, the third step is a step of closing the centrifuge lid and centrifuging the cell potential measuring container 1 arranged inside as described above.

  After passing through the above steps, the cell potential measuring container 1 is taken out from the centrifuge, so that the culture solution reaches the bottom surface of the well 14 of the cell measuring container 1, so that the cell potential measuring container 1 is mounted on the potential measuring apparatus and the cell Can be measured.

  The present invention can centrifuge the cell potential measurement container without leakage of a culture solution or the like from between the container body and the electrode substrate, thereby realizing rapid drug screening and assisting in the development of a new drug. Will.

DESCRIPTION OF SYMBOLS 1 Cell potential measurement container 11 Container trunk | drum 111 Cylindrical body 12 Electrode board | substrate 121 Substrate body 122 Measurement electrode 123 Reference electrode 124 Lead wire 125 Connector 13 Double-sided tape 14 Well 2 Centrifugal auxiliary tool 21 Bottom face 22 Convex part 3 Lid

Claims (2)

A container body portion including a plurality of cylindrical bodies having upper and lower ends opened;
An electrode substrate including a substrate body and a plurality of electrodes disposed on the substrate body, and the electrode substrate is joined to a lower end of the container body portion to form a plurality of wells. A centrifuge assisting tool used when centrifuging a cell potential measuring container for measuring the potential of a cell mounted thereon,
The centrifuge auxiliary tool includes a bottom surface having substantially the same shape as a bucket of a centrifuge, and a convex portion having substantially the same shape as the electrode substrate of the cell potential measuring container provided on the top of the bottom surface,
At the time of centrifugation of the cell potential measuring container, the bottom surface of the electrode substrate of the cell potential measuring container is supported by the convex portion of the assisting tool in a state where the cell potential measuring container is placed on the centrifugation assisting tool. A cell potential measuring container centrifugation aid. A container body portion including a plurality of cylindrical bodies having upper and lower ends opened;
An electrode substrate including a substrate body and a plurality of electrodes disposed on the substrate body, and the electrode substrate is joined to a lower end of the container body portion to form a plurality of wells. A method for centrifuging a cell potential measurement container for measuring the potential of a cell mounted thereon,
A centrifuge assisting tool comprising a centrifuge bucket having a bottom surface having substantially the same shape as that of the centrifuge bucket and a convex portion having substantially the same shape as the electrode substrate of the cell potential measuring container provided on the top of the bottom surface. Placing the bottom face down,
Placing the bottom of the electrode substrate of the cell potential measuring container on the centrifuge assisting tool and the convex part of the centrifuge assisting tool,
And a step of centrifuging the cell potential measuring container with the centrifuge.

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