JPS5878583A - Cultivation apparatus of cell - Google Patents

Abstract

PURPOSE:To obtain a cultivation apparatus for cells, capable of easily applying the cells in a given concentration to a protruding member, by forming the protruding member on at least one main surface of a platy body exhibiting the hydrophilic nature. CONSTITUTION:A cultivation apparatus for cells consisting of a platy body 10 having at least the surface consisting of a hydrophilic material, e.g. glass or a hydrophilic plastic, to apply the cells thereto and a protruding part 11 formed in the central part thereof. Preferably, the protruding part 11 has a hole 12 passing through the platy body 10 in the thickness direction to insert a rod-like body, e.g. a supporting rod 31, therethrough. In use, the supporting rod 31 fixed upright at the center of the bottom of a cultivation tank 30 is inserted in each hole 12 of the platy body 10, and a desired number of the platy bodies 10 are placed on top of each other.

Description

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

Prior art and melting point Conventionally, cells have been cultured in large quantities to produce, for example, interferon, urokinase, vaccines, etc. In this case, cells can only be cultured in a monolayer, so it is important to consider how the culture area can be used. C: Should it be made larger? (:
@h's ingenuity has been put into place.

For example, Japanese Patent Application Laid-Open No. 55-34671 describes a cell culture device in which two or more cell adhesion plates having at least a part or all of the periphery with a raised edge are fixed in a container at additional intervals. There is.

In the case of this device, the cell adhesion plate 6 has a raised edge around the entire periphery.
: When attaching cells to become seeds, a turbidity-resistant solution is injected into the uppermost cell attachment plate (=-), and this is sequentially overflowed toward the subsequent cell attachment plate. In this case,
If over 70-<1=, the concentration of cells may change. In addition, in a device using a cell adhesion plate with a part of the riverside edge height, each cell attachment plate is 51IiII! ζ: Adding cells to the layer is a somewhat complicated process.

In any case, in the above cell culture device, each cell with N1
It is appropriate for the tli' knee to be layered with similar cells. However, in some cases, different types of cells may be
Cooperation of these cells of different polarity, cultured in a culture medium and on mutually independent plates, may be desired to obtain the desired product, but the above-mentioned conventional devices cannot achieve this purpose. difficult to achieve.

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

According to this invention 6: 4 = so that cells can attach and proliferate.
A plate-like body exhibiting water-producing properties at least on its surface, and a protruding member formed on at least a part of the principal surface of the plate-like body so as to be able to accommodate nine cough plate-like bodies at intervals from each other. A cell culture device consisting of the following is provided.

In a preferred embodiment of the present invention, the projecting member is a projection provided at the center of the plate-like body, and has a through-hole extending through the plate-like body in the thickness direction so that the rod-like body can be inserted therein. It consists of what you have. Alternatively, the protruding member may consist of a plurality of protrusions provided on the entire surface of the plate-like main body.

■Detailed explanation of the invention The present invention will be described in detail below with reference to the accompanying drawings.

First aspect 11g of the present invention = The cell culture device according to No. 1A
As shown in Figure (top view) and Figure 1B (sectional view),
It consists of a plate-like main body 10 and one protrusion 11 formed at the center thereof. At least the surface of the plate-like main body 10 exhibits hydrophilicity so that cells can attach and proliferate.

Therefore, the plate-like 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.

The surface of the plate-like main body 10 may be made hydrophilic by acid treatment, coating with a hydrophilic material, or other methods. It may also be polygonal.

Approximately at the center of the life box of the plate-shaped main body 10 (=the protrusion 11 provided has a through hole 12 that penetrates the two-dimensional plate-shaped main body 10 in the thickness direction so that a rod-shaped body (not shown) can pass through it. When culturing cells by stacking a plurality of plate-like bodies 1#, a rod-like body should be removably inserted into this through-hole 12. At this time, each class-like body can be aligned with each other with an interval equal to the height of the projection 114'.

s2 Second state shown in Figure 4 @S: The cell culture device according to 1
! A plate-like main body 20 similar to that shown in Figures J&lA and sIB and a plurality of pieces (in the figure, 4
The protrusions 21 have the same height. When the plate-like bodies 20 are fitted together, the protrusions 21 can separate the plate-like bodies 6 from each other by an interval corresponding to the height thereof.

In each of the embodiments described above, the protrusions 11 or 21 greatly reduce the surface area of the plate-like body 10 or 21 on which the cells are layered.
It is preferable that these protrusions do not cause a decrease in C. These protrusions can be integrally molded simultaneously with the molding of the plate-like body 20, or, in the case of the embodiment shown in FIG. , Anki 21 can be installed separately. Further, the protrusions need to have at least a size such that when the plate-like main bodies 1o or 21 are stacked one on top of the other, the culture solution can enter between them and the desired culture can be carried out sufficiently. Its minimum height can be determined by simple experiment.

■Specific effect of the invention To culture cells using the cell culture device of the present invention described above, each culture device is housed in each petri dish, cell suspension is injected, and a carbon dioxide atmosphere is placed. Cells are allowed to adhere to the plate-like body surface C: or to be cultured to some extent. Then 1. Remove the culture device from the petri dish.

When the culture device of the first aspect is used, 1
As shown in Figure 83 (= bottom center part support rod 31
The supporting rods C of the culture tank 30 made of additional glass, plastic, and stainless steel rings are inserted through each through hole 22 and the desired number of rods are combined. When the second aspect # is used as the culture device, a support rod is not required and it is sufficient to simply overlap the two single oak plate-like bodies. At least a part of the tank 30 is spaced apart from the inner wall of the tank 30 without contacting it.

Next, a predetermined culture solution is injected with a pump or the like from the culture solution inlet 32 provided at the bottom of the tank 30, and then discharged from the outlet 33, and the culture tank 5 is maintained at a predetermined temperature. Proliferate cells while Note: The inlet 32 and the outlet 33 may be connected via a pump or the like to circulate the culture solution, and a culture solution inlet may be provided above the tank 30. Note that if the plate-like body has a wall lower than the protrusion formed all around it, cells can be attached directly to the plate-like body C2 without using a petri dish. .

Experimental examples using the cell culture 1N device of the present invention will be described below.

Experimental Example 1 Twenty-two cell culture devices made of polystyrene with a water-repellent surface treatment and shaped as shown in Fig. 1 and IB were used. The outer diameter was 9u, the height was 3U, and the circular diameter of the through hole was 5cm.The culture tank used was made of glass, had glass tubes at the top and bottom, had an inner diameter of 1100t, a depth of 100u, and had a curved bottom. Nakao was given a glass rod with a length of 90 m and a diameter of 4.9 fins to stand upright.

First, subcultured monolayer HeLa (He1m) cells were treated with trypsin, washed, and then the cell concentration was measured using a hemocytometer.

2. Suspend the above cells in culture solution C, which is made by adding 5% calf serum to Eagle's Mineral Essentials Medium.3. X
A suspension of 10' (cells)/m/(medium) was prepared.

Each cell culture device was housed in a sterilized Petri dish with an inner diameter of 90 μ, injected with 10 m/each of the above cell suspension, covered, and incubated in a carbon dioxide incubator for 10 to 24 hours. Cells were attached to the plate-like body y11'8.

Next, each cell culture device was taken out from the petri dish, and the glass rod of the culture tank was inserted through the through hole of each plate-like body.

The culture 1& was injected into the culture tank through the glass tube at the bottom of the culture tank, and the culture solution was circulated by discharging it through the glass tube at the top. When cultured in this state for 6 days, a single layer of HeLa cells (1 monolayer) was found to proliferate evenly on each oak-like body.

Experimental Example 2 Sixty cell culture devices made of surface and hydrated polystyrene having the shape shown in FIG. 2 were used. Each plate-shaped body is @500uI in diameter
It is 5M thick, and the protrusion has a diameter of 3011 mm and a height of 86 mm.
These are installed in symmetrical positions at intervals of 250U. The culture tank used was a stainless-steel type tank with a circular diameter of 600 U and a depth of 7001 lJI, which had an inlet and an outlet for the culture solution at the upper and lower sides, respectively.

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

Each cell culture device is housed in a petri dish-like container with an inner diameter of 502 gJ+1, a depth of 5 oiu, and a wall thickness of 5 U, and a two-tiered upper end surface. The containers were stacked with 20 sheets each through a silicone backing, a cover was placed on the top layer, and the containers were inked in a constant temperature room at 37.degree. C. for 24 hours from 1.theta.

Next, take out the cell culture device from the container, and the culture tank circle ζ = the area around it is the tank gate! i! (=Contact culture solution was injected and cultured for 6 days.During this period, 5% 00.-9
A gas mixture of 5% air was pumped into the S culture tank at a rate of 25 m//min. As a result, on the surface of each cell culture device, HeLa cells were proliferating evenly in a single layer.

■Specific effects of the invention Cell culture II of this invention as described above! According to the apparatus, cells can be separately attached to each machine body. Therefore, it is possible to layer the plate-like bodies (cells) at a constant ratio, and it is easy to culture cells of different types under the same culture environment. Since the cells can be taken out one by one, the growth state of the cells on each plate-like body can be easily checked.

[Brief explanation of drawings]

WILM diagram is a plan view of the cell culture device according to the first embodiment of the present invention, FIGS. 1 and 8 are cross-sectional views taken along line IB-IB in FIG. 1A, and FIG. 2 is a plan view of the cell culture device according to the first embodiment of the present invention. FIG. 3 is a perspective view showing the state in which the cell culture devices shown in FIG.・Protrusion, 1
2... Through hole, 30... Culture tank, 31... Glass rod, 32... Inlet, 33... Outlet. Hiko Dozuo Shiki, the representative of the deceased

Claims (3)

[Claims] (1) A plate-like body exhibiting hydrophilicity at least on the surface so that cells can attach and proliferate, and at least a portion of the plate-like body so that the plate-like bodies can overlap each other with two gaps. A cell culture device consisting of a protruding member formed on a surface. (2) The protruding member is one protrusion provided in the central part of the plate-shaped body (2) and has a through hole that penetrates the plate-shaped body in the thickness direction so that the rod-shaped body can be inserted. A cell culture device according to claim 1. (3) The cell culture device according to claim 1, wherein the projecting member comprises a plurality of protrusions provided on one main surface of the plate-like body.