JPH07203946A - Tool for removing culture medium - Google Patents

Abstract

PURPOSE:To provide a means for removing a culture medium capable of simply carrying out the removal of the old culture medium in exchanging the culture medium without damaging a cell or a tissual mass in culturing the cell or tissual mass in a culture vessel (plate well) of a small volume. CONSTITUTION:This tool for removing a culture medium is composed of a water absorbing material and provided by forming the material into a cylindrical member 1 having a circular or a polygonal cross section and a hollow part 2 or a columnar member 3 having a recessed part 4 at the tip thereof.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is used in the fields of tissue culture, cell culture, etc., and is used for removing old medium at the time of medium exchange in three-dimensional culture for forming tissue and cell clusters. The present invention relates to a medium removing member and a medium removing device.

[0002]

2. Description of the Related Art Recently, in the field of cell culture, it has become popular to form cell clusters for the purpose of maintaining the function of cells and perform the culture for a long period of time. During the culture, waste products and the like due to the metabolism of cells will be discharged into the medium, and it is necessary to periodically exchange the medium in the long-term culture.

To replace the medium, the old medium is first removed and then a new medium is added. To remove old medium, conventionally, the medium is sucked from the incubator with a suction device or a pipette. In the case of three-dimensional culture, the adhesion between the cells and the culture surface is not strong, and there is a problem that the tissue mass or cell mass in the culture is sucked. Particularly, in the case of a 96-well plate, since the diameter of the well is small and the volume is small, even if the medium is weakly sucked, the cell mass is sucked.

Although it is not intended to replace the medium for recovering the liquid in the well of the 96-well plate,
There is a system for collecting and collecting a supernatant containing radioactivity released from cells for a target cytotoxicity experiment in a water-feeding filter. This is one in which 96 filters (a porous material obtained by compressing fibers into a non-woven fabric) are arranged and fixed at positions corresponding to each well of a flat 96-well plate, and the supernatant in 96 wells is Can be collected every time. The purpose of collecting the supernatant with this system is to measure the radioactivity in the supernatant, and in order to improve the measurement accuracy of the radioactivity, make sure that the cells to which the radioactivity has been applied are not sucked into the filter. At the tip of the is attached a removable membrane with holes smaller than the cells.

However, when this system is used for removing the old medium at the time of replacing the medium when culturing the cell mass, if the old medium is to be completely recovered, the tip of the filter is placed in the well of the plate. It is necessary to bring it to the bottom, and at that time, there is a risk of pressing the cell mass or tissue mass in culture. Also, on the membrane attached to the filter tip,
Cells and tissues will attach. If the filter is shallowly placed in the well to prevent such a situation, the old medium cannot be sufficiently removed.

[0006]

SUMMARY OF THE INVENTION The present invention is primarily directed to plates with small volume wells such as 96-well plates.
The purpose is to solve such problems in medium exchange when culturing cell or tissue mass, and the purpose is to replace the old medium in the well of the plate with the cell or tissue mass in culture. To provide a medium removing member or a medium removing device that can be removed without damaging or accidentally aspirating and can be applied to a part of wells of a multi-plate having a large number of wells or a row unit of wells. Especially.

[0007]

[Means for Solving the Problems] As a result of earnest research to achieve such an object, the present inventors have found that by forming a recess in the tip of a water-absorbent filter, the cell mass or tissue mass in culture can be removed. The inventors have found that the medium in the wells of the plate can be almost completely removed without aspiration or damage, and have completed the present invention.

That is, the present invention is a medium removing member which is made of a water-absorbing material and is formed into a hollow cylindrical shape having a circular or polygonal cross section or a columnar shape having a recess at the tip, Furthermore, this member is made of a plastic molded product and is fitted and fixed to the tip of a carrier having a hollow portion or a recess at the rear end, and the medium removing member is fixed on the base. The medium removing device is characterized in that the medium removing device is fitted and fixed in a concave portion or a protruding portion which is regularly arranged in one or more rows.

The present invention will be described in detail below with reference to the drawings. FIG. 1 is an overall view of a medium removing member according to an embodiment of the present invention, FIG. 2 is an enlarged view of the tip of the medium removing member, and FIG. 3 is a sectional view of the tip of the medium removing member.

As shown in FIG. 1, the medium removing member according to the present invention has a tubular shape (1) or a columnar shape (3), and the cross-sectional shape thereof is usually circular, but a culture container to be used at the time of use. Depending on the shape of the wells of the plate
There is no problem with making a polygon such as a quadrangle or a hexagon. Further, the columnar member (3) is provided with a recess (4) at its tip, but for convenience in use, the recess (5) may be provided at the rear end as well, as shown in FIG. 1 (C).

The material constituting the medium removing member of the present invention is
There is no particular designation as long as it is water resistant and structurally water absorbent. Examples of such a material include a bundle of fibers such as acetate, a plastic foam body, a sintered body or the like formed into a hollow cylindrical shape or a rod shape. Since only the liquid medium is recovered by the member (1, 3) of the present invention, the size of the fine pores of the material is not particularly limited, and the liquid medium can be absorbed promptly and reliably. Anything can be done.

In the case of the tubular member (1) having the hollow portion (2), the outer diameter is preferably set to be substantially equal to the inner diameter of the well of the target plate in order to secure a large amount of water absorption per unit length. . The cross-sectional shape of the hollow part (2) is not particularly limited, but it is desirable that the hollow part (2) has a circular shape because the cell mass formed by three-dimensional culture is close to a spherical shape. 1 of the inner diameter of the well of the target plate
It is preferably about / 3 to 2/3. If it is larger than this, the amount of water absorption per unit length becomes small, the length of the tubular member (1) needs to be made large, and the strength of the member becomes weak and handling during use becomes difficult. On the other hand, if it is smaller than this, the tip of the tubular member (1) may come into contact with a tissue mass or a cell mass, which is not preferable.

In the case of the columnar member (3) having the recess (4) at its tip, its outer diameter is equal to or slightly smaller than the inner diameter of the well of the plate to be examined, as in the case of making it cylindrical as described above. Is desirable. There is a distance of about 1.5 mm between the bottom (6) of the recess (4) and the tip (7),
It suffices that the inner diameter at the projecting end portion is secured to be about 1/3 to 2/3 of the inner diameter of the well. Although there is no particular regulation of the shape, since the shape of the target cell mass is close to a sphere, the vertical cross-section taken along the central axis is trapezoidal or semicircular as shown in FIGS. 3 (b) and (c). Is desirable.

Next, a method of using the medium removing member according to the present invention will be described. When the target culture vessel (plate well) is one and the medium removal member is used alone, the rear end is grasped with tweezers for operation, but the rear end is water-insoluble. It is desirable to impart non-water absorption and prevent the tweezers from being contaminated by a method of impregnating and hardening the resin. It should be noted that, even when the dispenser (8) or the base (10) is used as described below, it is desirable that the rear end of the medium removing member be subjected to a non-water-absorption treatment in order to prevent contamination of the dispenser (8) and the substrate (10). Of course.

Usually, a plurality of wells of a multi-well plate (for example, 96 wells) are often used in units of rows or columns. In such a case, as shown in FIG. 4,
If a dispenser (8) used for preparative dispensing of a sample is used, it can be operated with a feeling similar to that of a tip for a dispenser, and after use, a medium removing member (9) attached to the tip of the dispenser (8) Can also be ejected by the ejector of the dispenser (8),
It is convenient. At the time of use, a recess (5) provided on the rear end side of the hollow portion (2) of the tubular member (1) or the rear end side of the columnar member (3) is fitted to each tip of the dispenser (8), It suffices to fix them, and the medium in a plurality of wells can be simultaneously removed by using a dispenser such as 8 or 12 depending on the situation. Also, the mounting operation to the tip of the dispenser (8) is similar to the method of mounting the tip for the dispenser, and the hollow portion (2) on the rear end side of the culture medium removing member is used.
Alternatively, it can be easily mounted by aligning the recess (5) on the rack and inserting the tip of the dispenser (8), and aseptic operation is ensured.

Here, one problem is that the medium removing member (9) is porous and not so strong that it is strongly fitted when it is attached to the tip of the dispenser (8).
It is easy to break. In order to solve such a problem, as shown in FIG. 5, a medium removing member (9) is previously attached to the tip of a carrier (10) made of a plastic molded product, and the carrier (10) is attached. ) It is preferable to fit the rear end side of the dispenser (8) to the tip end of the dispenser (8). Carrier (1
As for the shape of 0), the tip side can be fitted in the hollow portion (2) or the recess (5) at the rear end of the medium removing member (9) as shown in FIG. ), The medium removing member (9) has a hollow portion or a recess so that the rear end portion thereof can be fitted, and the rear end side has a hollow portion or a recess portion that can be fitted to the tip end of the dispenser (8) or the like. Just do it. In this way, the non-water absorption treatment of the rear end of the medium removing member (9) is not always necessary.

Further, as shown in FIG. 6, the rear end of the medium removing member (9) according to the present invention is provided in a concave portion or a protruding portion which is regularly arranged in one or a plurality of rows on a flat plate-shaped substrate (11). The medium remover with the side fitted and fixed may be prepared in advance and used. The number of rows of recesses or protrusions provided on one substrate (11) and the number of rows in one row may be determined according to the needs at the site of use and are not particularly limited. The shape of the base (11) does not necessarily have to be a flat plate shape, and may be a block or box shape, or a flat plate provided with a recess or a protrusion on the side surface.

The fixing of the medium removing member to the base (11) is
Although it may be directly bonded as shown in FIG.
As shown in (b), the base (11) is provided with a recess (12), but as shown in FIG. 7 (c), an annular rib (13) is provided,
The rear end of the medium removing member (9) may be inserted and fixed therein. In this method, the tubular member (1) and the columnar member (3)
Can be applied to any of Further, as shown in FIGS. 7D and 7E, the protrusion (14) is provided to form the hollow portion (2) on the rear end side of the tubular member (1) and the recess (() on the rear end side of the columnar member (3). 5) may be fitted.

The material of the substrate (11) is required to be water resistant and non-water absorbent in order to prevent the hands from being contaminated by the medium during use. Although it is not specified as long as it has these properties, a plastic molded product is preferable in view of mass productivity, and polyethylene, polypropylene, ABS resin, etc. are selected from the viewpoint of moldability, impact resistance, molding material cost, etc. It is good to be

By adsorbing and coating poly-HEMA (poly-hydroxyethylmethacrylate) or the like on the culture surface of the culture container, the cells of the adhesive system form a cell mass without adhering to the culture surface, and have a long function in hepatocytes and the like. Can be maintained for a period. In the case of a small culture area such as a 96-well plate, when the above-mentioned poly-HEMA is coated and cells are seeded at a certain concentration and cultured, several cell clusters are formed and the cell clusters are collected in the center of the well of the plate. Come on. Especially if the bottom of the well is hemispherical,
Form two cell masses.

To exchange the culture, the medium removing member of the present invention is inserted from the tip to the bottom of the well of the plate. Not only the hollow part (2) of the tubular member (1) but also the recess (4) of the columnar member (3) does not contact the center of the bottom surface of the well, so that the formed cell mass is broken or the cell mass is destroyed. Does not stick to the medium removing member. Due to the surface tension of the medium, the old medium moves to a portion of the medium removing member that is in contact with the well, and is completely sucked to complete the removal of the old medium.

[0022]

EFFECTS OF THE INVENTION By using the medium removing member or medium removing device according to the present invention, the removal of the old medium in the medium exchange during the three-dimensional culture in a well with a small volume such as a 96-well plate can be performed. As a medium removing means for three-dimensional culture, it can be completed completely without sucking the cell clumps and without destroying the cell clumps, and it can respond to the situation without waste even in the case of small-scale culture experiments. It is suitable.

[Brief description of drawings]

FIG. 1 is an overall view showing a medium removing member according to an embodiment of the present invention.

FIG. 2 is an enlarged view showing an example of the tip shape of the medium removing member of the present invention.

FIG. 3 is a cross-sectional view showing an example of the tip shape of the medium removing member of the present invention.

FIG. 4 is a view showing a usage example in which the medium removing member of the present invention is attached to the tip of a dispenser.

FIG. 5 is a view showing another embodiment of the medium removing member of the present invention.

FIG. 6 is a view showing a usage example (medium removal device) in which the medium removal member of the present invention is fitted and fixed to a base.

FIG. 7 is a view for explaining a method of fixing the medium removing member of the present invention to the base.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Cylindrical member 2 Hollow part 3 Columnar member 4, 5, 12 Recess 9 Medium removal member 10 Carrier 11 Base 13 Rib 14 Projection

Claims (8)

[Claims] 1. A medium removing member, which is made of a water-absorbent material and is formed into a hollow cylindrical shape having a circular or polygonal cross section. 2. A culture medium removing member, which is made of a water-absorbing material, has a circular or polygonal cross-sectional shape, and is formed in a columnar shape having a recess at the tip. 3. The medium removing member according to claim 2, wherein the columnar member has a recess at the rear end. 4. The medium removing member according to any one of claims (1) to (3), wherein the rear end portion of the member is subjected to non-water absorption treatment. 5. The rear end of the member according to claim 1 or 2 at the front end of a carrier made of a plastic molded product and having a hollow portion or a recess at the front end and the rear end. A medium removing member characterized in that parts are fitted and fixed. 6. The rear end of the member according to claim 1 or 3, wherein the front end of the carrier is a plastic molded product and has a hollow portion or a recess at the front end and the rear end. A medium removing member which is fitted and fixed to a portion. 7. The rear end portion of the member according to claim 1 or 2 is fitted and fixed in a recess provided on the base in a regular arrangement in one or a plurality of rows. And a medium remover. 8. A hollow portion or a recess at the rear end of the member according to claim 1 or 3 is fitted to a protrusion provided on the base in a regular arrangement in one or a plurality of rows. , A fixed medium removing device.