JPS6234391B2 - - Google Patents

Description

[Detailed description of the invention] Background of the invention Technical field The present invention relates to a cell culture device.

Prior Art and Problems Conventionally, cells have been cultured in large quantities to produce, for example, interferon, urokinase, vaccines, and the like. In this case, since cells can only be cultured in a monolayer, various efforts have been made to increase the culture area.

For example, Japanese Patent Application Laid-open No. 55-34671 describes a cell culture device in which two or more cell adhesion plates with at least a portion or all of the periphery having a raised edge are fixed in a container at appropriate intervals. There is. In the case of this device, when attaching seed cells to the cell attachment plate whose entire periphery has a high edge, a cell suspension is injected into the uppermost cell attachment plate, and this is poured into the cell attachment plate at the later stage. It overflows sequentially towards the board. In this case, the concentration of cells may change as the overflow progresses. Furthermore, in a device using a cell attachment plate with a partially raised edge, a somewhat complicated process is required for attaching cells to each cell attachment plate.

In any case, in the cell culture device described above, it is appropriate to attach the same type of cells to each cell attachment plate. However, in some cases, it may be desired to culture different types of cells in the same medium and obtain the desired product through the cooperation of these different types of cells cultured on mutually independent plates. However, it is difficult to achieve this objective with the conventional devices described above.

OBJECTIVES OF THE INVENTION Therefore, an object of the present invention is to provide a cell culture device that can easily attach cells at a constant concentration and can also culture different types of cells in the same environment.

According to the present invention, a plate-like body exhibiting hydrophilicity at least on the surface so that cells can attach and proliferate; There is provided a cell culture device comprising a formed protruding member, the protruding member having a through hole that penetrates the plate-like body in the thickness direction so that the rod-like member can be inserted therethrough. Ru.

DETAILED DESCRIPTION OF THE INVENTION The present invention will now be described in detail with reference to the accompanying drawings.

As shown in FIG. 1A (plan view) and FIG. 1B (cross-sectional view), the cell culture device according to the first aspect of the present invention includes a plate-like main body 10 and one protrusion 11 formed at the center thereof. Become. Plate body 10
At least its surface is hydrophilic so that cells can attach and proliferate. Therefore,
The plate-like main body 10 can be made of a non-cytotoxic hydrophilic material such as glass or a hydrophilic plastic such as hydroxyethyl methacrylate or a pullulan derivative, or it can be made of a hydrophobic material such as polystyrene and its surface is acid-treated. Alternatively, it may be made hydrophilic by applying a hydrophilic material or by other methods. The planar shape of the plate-like main body 10 may be circular as shown in the figure, or may be polygonal.

A protrusion 11 provided approximately at the center of one main surface of the plate-like body 10 has a through hole 12 that penetrates the plate-like body 10 in the thickness direction so that a rod (not shown) can be inserted therethrough. This through hole 12 is provided so that when a plurality of plate-like bodies 10 are stacked to culture cells, the rod-like body can be removably inserted into the through-hole so that the rod-like body can serve as a support. It is something. At this time, the protrusions 11 allow the plate-like bodies to be stacked on top of each other with a distance equal to the height of the protrusions.

The cell culture device according to the second embodiment shown in FIG. 2 includes a plate-like main body 20 similar to that shown in FIGS. ) have protrusions 21 having the same height. When the plate-like bodies 20 are stacked on top of each other, the protrusions 21 can separate the plate-like bodies from each other by an interval corresponding to the height thereof.

In each of the embodiments described above, it is preferable that the projections 11 or 21 do not significantly reduce the surface area of the plate-like body 10 or 21 to which cells adhere. These protrusions can be integrally molded at the same time as the plate-shaped main body, or they can be formed as a second part.
In the case of the embodiment shown in the figure, after forming the plate-like main body 20,
A protrusion 21 can be installed separately. Further, the protrusions must have at least a height that allows the culture solution to enter between the plate-like bodies 10 or 21 when they are stacked on top of each other, and to perform the desired culture sufficiently. Its minimum height can be determined by simple experiment.

Specific Effects of the Invention In order to culture cells using the cell culture device of the present invention described above, each culture device is housed in each chamber, a cell suspension is injected, and the cell culture device is incubated in a carbon dioxide atmosphere. Then, the cells are allowed to adhere to the surface of the plate-like body or are cultured to some extent. Next, the culture device is removed from the shear dish, and when the culture device of the first aspect is used, the third
As shown in the figure, a culture tank 30 is made of glass, plastic, stainless steel, or the like, and has a support rod 31 fixed upright in the center of its bottom surface. Each of the through-holes 22 is inserted through the support rods, and a desired number of plates are stacked one on top of the other. When the culture device of the second aspect is used, a support rod is not necessary, and it is sufficient to simply overlap the plate-like bodies. At this time, at least a portion of the peripheral edge of the plate-like main body of each culture device is spaced apart from the inner wall of the tank 30 so as not to come into contact with it. Next, a predetermined culture solution is injected with a pump or the like through the culture solution inlet 32 provided at the bottom of the tank 30, and is discharged from the outlet 33 as the case may be. Proliferate. Note that the inlet 32 and the outlet 33 may be connected via a pump or the like to circulate the culture solution. Further, a culture solution inlet may be provided above the tank 30. In addition,
When the plate-like body has a peripheral wall lower than the protrusion formed around the entire periphery, cells can be attached directly to the plate-like body without using a shear plate or the like.

Experimental examples using the cell culture device of the present invention will be described below.

Experimental Example 1 Twenty-two cell culture devices made of polystyrene whose surfaces were subjected to hydrophilic treatment as shown in Figures 1A and 1B were used. Each plate-like body has a diameter of 89 mm and a thickness of 1 mm, the protrusion has an outer diameter of 9 mm, a height of 3 mm, and an inner diameter of the through hole of 5 mm.
It was hot. The culture tank used was made of glass and had an inner diameter of 100 mm and a depth of 100 mm, with glass tubes at the top and bottom, and a glass rod with a length of 90 mm and a diameter of 4.9 mm was fixed upright in the center of the bottom.

First, subcultured monolayer HeLa cells were treated with trypsin, washed, and then the cell concentration was measured using a hemocytometer.

The cells were suspended in a culture solution containing Eagle's Mineral Essential Medium and 5% calf serum to prepare a suspension of 3×10 ⁴ cells/ml (medium).

Each cell culture device is housed in a sterilized shear dish with an inner diameter of 90 mm, and 10 ml of the above cell suspension is added to each cell culture device.
was injected, covered, and incubated in a carbon dioxide incubator for 10 to 24 hours to allow cells to adhere to the plate-like body.

Next, each cell culture device was taken out of the shear dish, and the glass rod of the culture tank was inserted through the through hole of each plate-like body. The culture solution was injected into the culture tank using a pump from the lower glass tube and discharged from the upper glass tube to circulate the culture solution. When cultured in this state for 6 days, HeLa cells were uniformly proliferating in a monolayer on each plate-like body.

Experimental Example 2 Sixty cell culture devices made of surface-hydrophilized polystyrene having the shape shown in FIG. 2 were used. Each plate-like body has a diameter
It is 500 mm long and 5 mm thick, and the protrusions are 30 mm in diameter and 6 mm in height, and are placed symmetrically at 250 mm intervals. The culture tank used was a stainless steel circular tank with an inner diameter of 600 mm and a depth of 700 mm, which had an inlet and an outlet for the culture medium at the upper and lower sides, respectively.

A culture solution and cell suspension were prepared in the same manner as in Experimental Example 1.

A cell culture device was housed in each container, which was a shell-like container with an inner diameter of 502 mm, a depth of 30 mm, and a wall thickness of 5 mm, and had a step on the upper end surface of the peripheral wall, and 400 ml of cell suspension was poured into each container. 20 sheets each were stacked using silicone packing, a cover was placed on the top layer, and the sheets were incubated in a constant temperature room at 37°C for 10 to 24 hours.

Next, the cell culture devices were taken out of the container and stacked in a culture tank so that the periphery did not touch the inner wall of the tank. This culture tank was placed in a constant temperature bath at 37°C, and a culture solution maintained at 37°C was poured into the culture tank, and cultured for 6 days. 5% _CO2 during this period
- A gas mixture of 95% air was introduced into the culture tank at a rate of 25 ml/min. As a result, HeLa cells were uniformly proliferating in a single layer on the surface of each cell culture device.

Specific Effects of the Invention According to the cell culture device of the present invention described above,
Cells can be attached separately to each plate-like body. Therefore, cells can be attached to the plate-like body at a constant rate, and it is easy to culture different types of cells in the same culture environment. Furthermore, since the stacked plate-like bodies can be taken out one by one, the growth state of cells on each plate-like body can be easily checked.

[Brief explanation of the drawing]

FIG. 1A is a plan view of a cell culture device according to the first embodiment of the present invention, and FIG. 1B is a line B-- in FIG. 1A.
2 is a plan view of the cell culture device according to the second embodiment of the present invention, and FIG. 3 is a sectional view taken along line B.
FIG. 2 is a perspective view showing a state in which the cell culture devices shown in FIG. A are stacked in a culture tank. 10, 20...Plate-shaped main body, 11, 21...Protrusion, 1
2... Through hole, 30... Culture tank, 31... Glass rod, 32... Inlet, 33... Outlet.

Claims (1)

[Claims] 1 A plate-like body exhibiting hydrophilic properties at least on the surface so that cells can attach and proliferate, and a protrusion formed in the center of the plate-like body so that the plate-like bodies can be stacked at intervals. 1. A cell culture device comprising: a member, the protruding member having a through hole extending through the plate-like main body in the thickness direction so that a rod-like member can be inserted therein.