JPH02203778A - Multiple-type culture device for cell or the like - Google Patents

Abstract

PURPOSE:To obtain a multiple-type culture device capable of sufficiently culturing cells, etc., consisting of an upper culture medium, lower culture medium and a plate to fix these culture mediums, wherein a porous membrane is attached to the bottom of each culture medium. CONSTITUTION:The bottom of a cylindrical lower culture medium 1 (preferably comprising colorless transparent resin) is equipped with a flange 2 and a leg 3 and dented parts 4 are installed at 120 deg. intervals on the upper periphery of the culture medium 1. A porous membrane is attached to the bottom of the culture medium 1. Blades 6 are placed protrusively in three directions at 120 deg. intervals on the upper periphery of a cylindrical upper medium 5 (preferably comprising colorless transparent resin) and the blades 6 are engaged with the dented parts 4 of the lower culture medium 1. A porous membrane is also attached to the bottom of the culture medium 5. The upper culture medium 5 and the lower culture medium 1 are allowed to stand in a superimposed state in each hole of a plate 7.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to an apparatus for efficiently cultivating cells, etc. (prior art and its problems); Breed improvement using genetic manipulation or cell fusion; Techniques for producing new useful substances have recently made remarkable progress.

Attempts are also being made to use the metabolic activity of cells, particularly microorganisms, to decompose environmental pollutants and difficult-to-decompose substances into harmless substances.

There are many types of cells, especially microorganisms, and their living patterns vary widely.Furthermore, their growth patterns and metabolites often change when the environment changes, and growth conditions are easy to set and easy to handle. In addition, it grows and multiplies extremely rapidly, withstands harsh environments well, preserves seeds well even under extreme conditions, and does not become extinct. It has characteristics such as being extremely large in amount, adapting quickly to the environment, and quickly acquiring the ability to assimilate new substances.
Utilizing these characteristics for research and production activities has been practiced for a long time. Compared to microorganisms, animal cells are difficult to propagate and the choice of growth conditions is narrower, but active efforts are being made to impart useful characteristics through breeding.

In order to promote these research and production activities, efficient cell culture is required, but traditionally,
It is not always easy to separate and recover cells and metabolic products, and the process for separation and recovery is required, making the entire culture process inefficient.

In order to deal with these problems, for example, the patent application
No. 293049 is an invention in which a membrane filter is fixed to the bottom of a synthetic resin cylinder, and cells, etc. are cultured within the cylinder while allowing medium, drugs, ions, and secreted metabolites to freely pass through the membrane filter, and further The inventors of the present application have completed a high-density culture device in which almost no cells leak out by sealing everything except the cell injection port using a filter with an absolute pore size of 0.1 to 5 μm. )9
A patent application (unpublished) was filed as No.

All of these devices were excellent cell culture devices, but because they were limited to only one cell culture section, they were used for cultivating many types of cells and studying their interactions. It was an inconvenience.

(Problems to be Solved by the Invention) As mentioned above, efficient cell culture is not necessarily achieved using conventional culture vessels, and it is also easy to separate the cells and recover metabolic products. It wasn't. Furthermore, while culturing different types of cells without direct contact,
It was also not suitable for studying that interaction. The present invention solves these problems and provides a multiplex culture device that can efficiently culture cells and the like.

(Means for Solving the Problems) The present invention is an invention of a multiple culture device for cells, etc., consisting of a lower incubator, a lower incubator, and a culture plate, each part of which is isolated by a porous membrane.

There are no particular limitations on the material, shape, and dimensions of the culture plate used in the present invention. Efficient culture can be performed by using a multi-layer plate (porous culture plate).

The culture plate serves as a storage tank for culture fluid, chemicals, ions, hormones, etc., and supplies these to the cells in the upper culture vessel and the lower culture vessel, and metabolic products such as cells are also leached into the culture plate.

The upper incubator and the lower incubator are preferably molded from highly transparent synthetic resin.

In terms of culture efficiency, it is desirable that the incubator and the culture plate as well as the incubators be detachable from each other. A porous membrane is fixed to the lower part of each culture vessel, and in order to ensure that a sufficient amount of culture fluid, etc. is supplied from the lower part of the culture vessel, it is necessary to provide an appropriate amount of space at the lower part of the porous membrane. As a means for securing such a gap, an appropriate method such as providing legs at the bottom of each incubator can be adopted. Alternatively, the lower space can be secured by protruding multi-directional wings from the upper part of the incubator and locking them to the peripheral wall of the lower incubator or the culture plate.

The pore size of the porous membrane must be such that cultured cells, etc. do not pass through or leak out, and culture fluid, chemicals, ions, hormones, etc. can freely pass through. Therefore, it is generally suitable to have an absolute pore diameter in the range of 0°1 to 5 μm, although it varies depending on the type of cells.

It is desirable that the porous membrane be made of a material that can be sterilized in an autoclave and is stable against acids, alkalis, hydrocarbons, and the like. Suitable materials include cellulose ester, fluororesin, hydrophilic vinylidene fluoride, etc.
Of course, it is not limited to these.

The porous membrane fixed to the bottom of the upper culture vessel and the porous membrane fixed to the bottom of the lower culture vessel may be of the same material and absolute pore diameter, or may be different from each other. good.

Methods for fixing the porous membrane to the culture vessel include, for example, fixing by heat sealing (for example, at 270°C or higher), fixing using various solvents such as ketones and chlorine-based organic solvents, and other methods. It is also possible to fix by an appropriate method. It is important to maintain the flatness of the porous membrane for the purpose of using it in culture experiments, but this can be done, for example, by soaking the porous membrane in a mixture of ethanol and water in advance, keeping it at a moist temperature, and then fixing it. There is a way.

In the present invention, cells in the culture vessel do not leak out from inside the vessel. On the other hand, culture fluids, chemicals, ions, hormones, etc. can freely pass through the porous membrane, thereby making it possible to supply nutrients, etc. to cells. In addition, metabolic products such as cells also pass through the porous membrane and are discharged into the culture solution.

Therefore, it is extremely easy to separate cells and metabolic products during and after culturing.

Conventionally, in conventional plate culture, it has not been possible to react or infect adherent cells with drugs or viruses from below. On the other hand, when culturing adherent cells in the culture device of the present invention, the cells adhere to the porous membrane at the bottom of the culture vessel and proliferate, so the drug is reacted from both the upper and lower parts of the porous membrane. It is also suitable for studying the polarity of cells, such as round epithelial cells, because it is possible to infect cells only from the upper part or only from the lower part. In this way, cells can be cultured in a state close to in vivo, although in vitro.

As described above, since the culture device of the present invention is a multiple cell culture device consisting of a lower incubator, a lower incubator, and a culture plate, it is possible to simultaneously culture many types of cells in a non-contact manner. In other words, when culturing floating cells, two or three different types of cells (when also cultured in culture plates) are cultured without direct contact, while interactions due to the effects of their metabolic products etc. In addition, when culturing adherent cells, it is possible to study interactions due to growth patterns, effects of metabolic products, etc. while culturing two different types of cells without direct contact. can.

Even when floating cells and adherent cells are cultured simultaneously, it is possible to study their interaction in the same manner as above.

(Effect) By using the multiplex culture device of the present invention, In
Although cells are cultured in vitro, it is possible to study the interactions of cultured cells and cell polarity, and to efficiently conduct research on drug reactions, virus infections, etc. During or after culturing, each culture vessel can be separated and the growth state of cells etc. can be observed under a microscope, and metabolic products such as cells can be easily separated and collected from cells and analyzed.

(Examples) Examples of the present invention will be described below, but the present invention is not limited to the following examples.

Example 1 Made of colorless and transparent polystyrene resin, height 13
A cylindrical lower incubator (1) with a diameter of 301)I1)1 and an inner diameter of 27 mm was molded. A flange (2) and legs (3) were molded at the bottom of the culture mold. Concave portions (4) were carved at every 120° around the upper circumference of the incubator. A porous membrane of cellulose ester (absolute pore diameter: 0.45 μm) consisting of a mixture of cellulose acetate and cellulose nitrate was pre-soaked in a mixture of ethanol and water and kept at a moist temperature, and then fixed to the bottom of the culture vessel using methyl ethyl ketone as a solvent.

Next, an upper incubator (5) was formed into a cylindrical shape with a height of 12.5 mm, a diameter of 13 mm, and an inner diameter of 10 mm using a colorless and transparent polystyrene resin material. 1 in the upper cylinder of the incubator
A wing (6) having a length of 10 mm and a width of 2 mm was integrally molded and protruded in three directions for every 20@. The wings (6) of the upper incubator engage with the recesses (4) carved on the cylinder of the lower incubator, allowing the upper incubator to remain stably stationary, and the porous membrane located on the bottom surface of the upper incubator A gap necessary for supplying the culture solution etc. was ensured between the two without contacting the porous membrane of the incubator.

On the bottom of the upper incubator, a porous material made of cellulose ester similar to that used in the lower incubator (pore diameter 0.45 μm) was fixed using methyl ethyl ketone as a solvent.

On the other hand, the culture plate (7) was a multiwell plate made of polystyrene resin, 127 mm long, 86 mm wide, 17 mm high, and having 6 holes (inner diameter 36 mm, height 13 mm for each person).

Place the upper and lower incubators on each person's culture plate. All of these are removable and can be attached to each culture during or after culturing. It would be possible to separate the cells and observe the state of the cells under a microscope, or to collect and analyze the metabolic products excreted into the culture plate.

As mentioned above, the lower incubator is equipped with flanges and legs, so in the lower incubator, as in the case of the upper incubator, the lower part of the porous membrane is used for supplying culture solution, chemicals, ions, etc. Necessary and sufficient air gaps are ensured.

Example 2 A cylindrical lower culture vessel having a height of 13 mm, a diameter of 30 mm, and an inner diameter of 27 mm was molded from fluororesin. A flange and legs were molded into the bottom of the incubator.

Recesses were carved at every 120° on the upper circumference of the culture vessel. Porous membrane made of hydrophilic fluororesin (absolute pore diameter 3.0 μm)
was fixed to the bottom of the incubator by heat sealing (heating at 380°C through a thin aluminum layer).

Next, using fluororesin as the material, the height is 12.5 inches,
A cylindrical upper incubator with a diameter of 13 mm and an inner diameter of 10 mm was molded. The upper cylinder of the incubator has a length of 10 for every 120.
The blades are integrally molded to protrude in three directions. The wings engage with the recesses carved into the cylinder of the lower culture vessel, and the porous membrane on the bottom of the upper culture vessel does not come into contact with the porous membrane of the lower culture vessel, allowing the supply of culture fluid etc. between the two. The necessary and sufficient air space was ensured.

The bottom of the upper incubator is covered with a porous membrane (absolute pore diameter 3.0 μm) made of the same fluororesin as that used in the lower incubator.
was fixed by heat sealing (heating at 380°C through a thin aluminum layer).

On the other hand, the culture plate is made of polystyrene resin and [
1) 27m+a, width 86+nm, height 17m,
A multiwell plate with 6 wells (inner diameter 36 mm, height 13 mm for each person) was used.

Example 3 Made of polycarbonate resin, height 13mm.

A cylindrical lower incubator with a diameter of 30 mm and an inner diameter of 27 mm was molded. A flange and legs were molded at the bottom of the incubator.

Concave portions were carved at every 120° around the upper circumference of the incubator.

A porous membrane made of hydrophilic vinylidene fluoride (PVDF) (trade name rDuraporeJ, absolute pore diameter 0°22 μm) was fixed to the bottom of the culture vessel by heat sealing (270° C.).

Next, we made a polycarbonate resin material with a height of 12 cm.
．． An upper incubator with a diameter of 5 mm, a diameter of 13 mm, and an inner diameter of 10 mm was molded. The upper cylinder of the incubator has a length of 10 for every 120.
Meanwhile, wings with a width of 2 mm were integrally molded to protrude in three directions. The wings engage with the recesses carved into the cylinder of the lower culture vessel, and the porous membrane on the bottom of the upper culture vessel does not come into contact with the porous membrane of the lower culture vessel, allowing the supply of culture fluid etc. between the two. The necessary appropriate air gap was ensured.

The bottom of the upper culture vessel is made of wood-loving vinylidene fluoride (DVDF), which is the same as that used in the lower culture vessel! l! ! porous membrane (trade name rDuraporeJ, absolute pore size 0.22 μm)
) were fixed by heat sealing.

On the other hand, the culture plate is made of polystyrene resin and
41) 27 mm, j7II486 mm, height 17
mm, with 6 holes (inner diameter of each hole 36 mm, height 13 m)
A multi-sea urchin, Leprae, with m) was used.

[Brief explanation of the drawing]

The drawing shows an embodiment of the multiplex cell culture device of the present invention. FIG. 1 is a plan view. Fig. 2 is a sectional view taken along line A-A', Fig. 3 is a plan view of the upper incubator, Fig. 4 is a plan view of the lower incubator,
FIG. 5 is a plan view of the culture plate. Patent applicant: Japan E IJ Boa Kogyo Co., Ltd. 10 Procedural amendment (method/directive) May 16, 1989

Claims (4)

[Claims] (1) A multiple cell culturing device comprising an upper incubator, a lower incubator, and a culture plate, each of which is isolated by a porous membrane. (2) The multiple cell culturing device according to claim 1, wherein the upper incubator, the lower incubator, and the culture plate are detachable. (3) The cell etc. multiplex culturing device according to claim 1, wherein the porous membrane has an absolute pore diameter of 0.1 to 5 μm. (4) The cell, etc. multiplex culture device according to claim 1, wherein the porous membrane is made of cellulose ester or hydrophilic fluororesin.