JP2003052361A - Base plate for forming aggregate with controlled outline of adhesion-depending cell and method for cell culture - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a base plate for forming an aggregate with a controlled outline for adhesion-depending cells, with which base plate culture cells can grow for a long time by mutually giving a foothold by adhering between cells even when the cells flow on a culture liquid. SOLUTION: The base plate 11 is formed of a non-cell adhesive material to which culturing cells do not adhere, and it has dent parts 13 for controlling the outlines of cell aggregates formed by adhering between cells seeded.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a substrate for forming an outline control aggregate of adhesion-dependent cells and a cell culture method.

[0002]

2. Description of the Related Art When developing a new drug, a cost of several tens of billions of yen is invested to develop one commercial product (new drug). Most of the cost is used for toxicity evaluation tests of drug candidates, mainly in animal experiments. In the first place, the development of a new drug is the work of chemically modifying one chemical substance that has been found to have a drug effect, and discovering the molecular structure with the maximum drug effect and the minimum side effect. At this time, in the early stages of development, each new drug candidate substance that has been subjected to a wide variety of chemical modifications is subjected to toxicity tests, etc. by animal experiments to screen for highly toxic drugs. Due to the large number of animals, an extremely large number of experimental animals will be killed. Since these animals are very expensive, the cost of animal experiments for toxicity evaluation in the early stages of drug discovery becomes enormous. In addition, from the viewpoint of animal welfare, international criticism of animal experiments is increasing, and there is an urgent need to develop drug discovery tools that can replace or greatly reduce animal experiments. In recent years, what has attracted attention as a drug discovery tool and has been partially put into practical use is a cultured cell obtained by culturing and growing the cell in vitro.
This is used to test the physiological activity and toxicity of new drugs. However, the skin cells that are practically used are mainly skin cells that are easy to culture and proliferate, and cells that are responsible for drug metabolism in the body that are truly desired as drug discovery tools (eg, hepatocytes, kidney cells). The cultured cells using Escherichia coli have not yet been put to practical use because there is no culture system that exerts their functions sufficiently outside the body. At present, the following methods are mainly used to obtain the above-mentioned cultured cells. The most common method is to uniformly coat a culture dish with a cell-adhesive protein such as collagen as a carrier,
This is a method in which a culture solution is supplied, cells are seeded, the cells are allowed to adhere to a carrier, and growth is performed. Alternatively, by arranging cells on the substrate by using a substrate formed with a surface pattern in which cell adhesion is different, culturing cells on the surface of the substrate, and adhering the cells only to sites where they can easily adhere. Techniques for making it happen have also been developed. Furthermore, in the case of specific cells, when the cells are seeded on a cell non-adhesive substrate, they may adhere to each other to form a cell aggregate called a spherical spheroid. In this case, it is known that the cells maintain their survival for a long time by supplying scaffolds to each other without adhering to the base.

[0003]

The main reason why cells involved in drug metabolism in vivo cannot exert their original functions when cultured in vitro is that the three-dimensional arrangement of cells is reproduced in vitro. It is believed that this is because it is not possible. In in vitro cell culture, a method of seeding cells on a flat surface is mainly used, but it must be said that this state is far from the state of cells in a living body. Furthermore, when cells are cultured using the above-mentioned general cell culture method and the cells adhere to the culture substrate, it is very difficult to transplant them to another site without damaging the cells. There is also. When forming spheroids, it is relatively easy to transplant to another site because it is not adhered to the substrate, but it is difficult to control the shape and size, and if the size becomes too large, the spheroids will grow. It has been revealed that the cells existing in the central part of the plant do not function well in excretion of waste products, uptake of nutrients and oxygen, and the aggregate dies from the central part. Therefore, in order to solve the above problems, it is a major problem to establish a culture system capable of controlling the positions of cells and the shape of cells when they are aggregated.

As a result of earnest studies in view of the above-mentioned problems, the present inventor has surprisingly used an agarose gel substrate having a concave portion as a culture substrate so that cell aggregates can be obtained while cells are suspended. The present invention has been completed based on the finding that it is possible to form a film and control its shape.

[0005]

A substrate for forming an outer shape control aggregate of adhesion-dependent cells according to the present invention is made of a non-cell-adhesive material to which cultured cells do not adhere, and seeded cells are It is characterized by having a concave portion that regulates the outer shape of the cell aggregate formed by adhesion. Agarose gel can be used as the non-adhesive material. Agarose is a polysaccharide that has many hydroxyl groups and is hydrophilic. It is well known that almost no cells adhere to agarose gel. Since this agarose gel has a shape-retaining property after gelling, it can be easily molded into a required shape using a molding die. The cell non-adhesive material is not limited to agarose gel, and may be any material as long as it has moldability and does not have cell adhesiveness.

Further, in the cell culture method according to the present invention, the above-mentioned substrate for forming the outer shape control aggregate of adhesion-dependent cells is housed in a culture container, a culture solution is supplied onto the substrate, and a large number of the substrate are placed in the recesses of the substrate. Cells are seeded and maintained under the required conditions, the cells precipitated at the bottom of the base recess are adhered to each other, and the outer shape of the cell aggregate to which a large number of cells have adhered is substantially aligned with the shape of the recess on the recess wall surface. It is characterized in that it is allowed to grow while being regulated.

The seeded cells are granular and independent of each other in the initial stage of culture, but eventually adjacent cells come into contact with each other to form cell aggregates. It was confirmed that the contact sites adhere to each other, and the adhered sites serve as a scaffold, and even cells having an adhesion dependency can survive for a long period of time. Furthermore, it was revealed that, surprisingly, the outer shape (contour) of the aggregate was restricted to the shape of the recess, and a cell aggregate having a shape different from the conventional spherical shape was obtained. The shape of the concave portion is arbitrary depending on the shape of the shaping mold, and thus, the cell aggregate having any shape can be used as long as the shaping mold is a processable shape. For example, when culturing hepatic parenchymal cells, it mimics the fact that hepatic parenchymal cells in a living body are arranged in parallel in a rod shape, and by forming recesses in parallel groove shapes, it is very similar to that in the living body. It can be a linear hepatocyte aggregate. The culture medium and the culture and growth conditions do not need to be particularly special, and the culture medium and the usual conditions can be used.

In addition to hepatocytes, any type of cells that adhere to each other, such as muscle cells, nerve cells, vascular endothelial cells, endocrine cells, skin / mucosal cells, etc., can be used. Can be obtained in the shape of. Since the cell aggregates are suspended in the culture medium and not attached to the substrate, they can be easily aspirated and collected with a pipette (a kind of dropper), etc., and can be transplanted to other sites without destroying the cells. it can. That is, it can be diverted to various uses while being shaped into an arbitrary shape.

For example, the positional relationship between cells in a living body and the shape of an aggregate can be directly restored by transplanting various cultured cells cultured in an in vitro culture system as described above to restore them. Information can be transmitted by contact, and functions caused by shape (such as muscle contraction function) can be reproduced. Further, by reseeding the aggregated hepatocytes on a substrate using a material capable of inducing polarity, it can be expected that the polar structure that the cells had in vivo will be restored. Hepatocytes in the liver are linear aggregates surrounded by various non-parenchymal cells, and the linear hepatocyte aggregates that can be created as described above. By using, it can be expected to be applied to an artificial liver.

[0010]

EXAMPLE FIG. 1 shows a substrate 11 made of agarose gel. As shown in the figure, multiple parallel grooves (recesses,
Valley 13 is formed. The substrate 11 may be formed of agarose gel alone, but may be supported on a supporting substrate (not shown) such as glass. The width, the depth, and the width of the crests of the groove 11 on the substrate 11 were all 0.1 mm, and the side where the groove was formed was a square of 1 cm. The agarose gel is made by mixing 6 wt% of agarose powder with water, boiling it once to dissolve it, and then cooling it into a gel, which is then molded into the shape shown in the figure using a molding die (not shown). Then, the substrate 11 was formed. The substrate 11 was housed in a culture dish (culture vessel, not shown), and a culture solution was supplied into the culture dish so as to be immersed in the groove 13. The culture medium is PSN (Peni
cillin, Streptomycin, Neomycin: All are antibiotics)
Williams Medium E (Williams
^, medium E) 90 vol% to 10 vol% FBS (Fetal b)
(Ovine serum: fetal bovine serum) was used as a general culture medium. On the substrate 11, mature mouse hepatocytes were seeded at 80,000 cells / cm ² . The culture dish was housed in an incubator (not shown), and cultured for 24 hours in an air atmosphere at a temperature of about 37 ° C. and a carbon dioxide concentration of about 5%.
An enlarged photograph of the growth of the cells after growing is shown in FIG. The cells seeded on the substrate 11 fall into the groove 13 by gravity. In the initial stage of culture, hepatocytes were in the form of particles, but as the culture proceeded, the individual cells began to come into contact with each other on the culture medium to form aggregates. And 2
After the lapse of 4 hours, as shown in FIG. 2, the aggregate grew so as to fill the groove 13 of the substrate 11 almost completely. This cell aggregate was not adhered to the substrate 11 and could be easily transplanted to another site by aspirating with a pipette. It was confirmed that the individual cells of the cell aggregate were adhered and survived for 2 days.

[0011]

As described above, according to the substrate for controlling the outer shape control aggregate of adhesion-dependent cells and the method for culturing cells according to the present invention, the cell aggregate having the outer shape in which the grown cells have a substantially concave shape. Was formed. Moreover, since this cell aggregate is not adhered to the substrate, it can be easily transplanted to other sites without damaging the cells, and the shape was three-dimensionally controlled in addition to toxicity evaluation. It has an effect that it can be applied to various uses requiring a cell aggregate.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a substrate.

FIG. 2 is a photograph showing cell aggregates after 24 hours of culture and growth.

[Explanation of symbols]

11 board 13 groove (recess)

Claims (2)

[Claims] 1. A non-cell-adhesive material that does not adhere to cultured cells, and has a recessed portion that regulates the outer shape of a cell aggregate formed by adhering seeded cells to each other. A substrate for forming an outer shape control aggregate of adhesion-dependent cells. 2. A substrate for forming an outer shape control aggregate of adhesion-dependent cells according to claim 1 is housed in a culture container, a culture solution is supplied onto the substrate, and a large number of cells are seeded in the recesses of the substrate. ,
Maintaining the required conditions, cells that have settled to the bottom of the base recess are allowed to adhere to each other, and the cell aggregate to which a large number of cells have adhered is grown while the outer shape of the cell aggregate is regulated by the recess wall surface so as to follow the shape of the recess. A method for culturing cells, which comprises: