JP2003250517A - Substrate for cell culture and method for cell culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substrate for cell culture capable of carrying out three- dimensional coculture of cells in a state laminated in the thickness direction of the substrate in order to maximize effects of intercellular contact, and a method for cell culture. <P>SOLUTION: The substrate 10 for the cell culture is composed of a cell nonadhesive material without making the cell to be cultured adhere. The substrate 10 has slitlike through-holes 12 for culturing the different cells in the laminated state in the thickness direction of the substrate 10 in the interior thereof. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a cell culture substrate and a cell culture method.

[0002]

2. Description of the Related Art When developing a new drug, a toxicity test is conducted by an animal experiment to screen a drug having a low toxicity, but an extremely large number of animals (mouse) are killed. Further, since these animals are very expensive, it is estimated that the cost for the animal experiment for the toxicity evaluation at the early stage of drug discovery is several hundred billion yen, which is a factor of enormous cost. Therefore, in recent years, as a substitute for animals, cultured cells in which cells are cultured and grown in vitro are being developed, and the physiological activity and toxicity of drugs are tested using these. For example, as an alternative method, some cell cultures such as cultured skin cells are being used as alternatives to animal experiments, but it is important to test with cells that have the role of metabolizing drugs in the body. Therefore, it has not reached the level of a perfect substitute.

Based on the above situation, if liver cells that metabolize drugs in vivo are cultivated in vitro and the same ability as when they exist in vivo can be expressed, they will be sacrificed in animal experiments. It can be an alternative to animal experiments that can significantly reduce the number of animals. To achieve the above, liver cells,
In particular, it is important to in vitro culture hepatic parenchymal cells, which are directly responsible for the function of metabolic drugs, in good condition. In a living body, a plurality of types of cells are often present in contact with each other in a three-dimensional array. Conventionally, it is known that a substance to which cells are difficult to adhere or a substance to which cells are easily adhered is applied to a substrate, and a plurality of types of cells are seeded to arrange the cells two-dimensionally.

[0004]

Such restriction of cell adhesion sites is often used as a method for co-culturing. However, in the two-dimensional co-culture system, the contact between different kinds of cells is limited only to the boundary between different kinds of cells, and the effect of the contact between cells cannot be fully utilized.

Therefore, the present invention provides a substrate for cell culture and a cell culture method capable of three-dimensional co-culturing in which cells are stacked in the thickness direction of the substrate in order to maximize the effect of cell-cell contact. .

[0006]

[Means for Solving the Problems] That is, the cell culture substrate according to the present invention is a cell culture substrate made of a non-cell adhesive material to which cells to be cultivated do not adhere, and different cells are internally formed. Has a slit-shaped through hole that is cultured in a laminated state in the thickness direction of the substrate.
Silicone rubber or agarose gel can be used as the non-adhesive material. Cells do not adhere to silicone rubber. Agarose is a polysaccharide and has a hydroxyl group and has hydrophilicity, but it is considered that cells are not adhered because the hydrophilicity is too strong. When using this agarose gel, the agarose content is preferably 6 wt% or more. When the agarose content is 6 wt% or more, the agarose gel has a shape-retaining property, so that it can be easily molded into a required shape using a molding die. The non-adhesive material is not limited to silicone rubber or agarose gel.

The cell culture device according to the present invention is characterized in that the cell culture substrate is brought into close contact with a culture dish made of a cell adhesive material. Further, in the cell culture method according to the present invention, a culture solution is supplied to the cell culture device, a large number of first cells are seeded on the substrate, and the seeded cells are maintained at a required temperature condition. It is characterized by comprising a step of culturing in a state of being adhered to the culture dish in the through hole, and a step of culturing a second cell on the first cell cultured in the through hole.

[0008]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows a cell culture substrate 10. The substrate 10 is made of silicon rubber and has a disk shape with a thickness of about 1 mm. In this substrate 10, slit-shaped through holes 12 were formed in parallel. The width of the through hole 12 was set to 0.5 mm. Since the size of liver parenchymal cells is about 50 μm, about 10 cells are arranged in the through holes 12. The length and width of the through hole 12 are not particularly limited.

The through hole 12 can be formed by laser processing or the like. The substrate 10 is not limited to the one made of silicon rubber, and may be made of a material such as agarose gel that does not adhere to cells. As described above, when the agarose content is 6 wt% or more, the agarose gel has a shape-retaining property, and thus the agarose gel can be easily molded into a required shape using a molding die.

When culturing cells, as shown in FIG. 2, the substrate 10 is provided with TC12 wells having cell adhesiveness.
The cell culture device 14 is brought into close contact with the culture dish 14 to prepare a cell culture device.
By pressing the substrate 10 onto the surface of the culture dish 14, the substrate 1
0 adheres to the culture dish 14, and the cells are placed on the substrate 10 and the culture dish 14.
There is no entry between and.

The culture solution is supplied to the extent that the substrate 10 is immersed in the culture dish 14, and the first cells are seeded in the culture solution. The first cells gradually settle in the slit-shaped through holes 12 and grow in a state of being adhered to the culture dish 14. The cell has a through hole 12
It spreads in a single layer inside. Then, the second cell is seeded in the culture medium. The second cells also settle and spread in the through holes 12,
Propagate using the first cell as a scaffold. The second cell is also cultured in a monolayer state.

FIG. 3 is a schematic diagram showing a state after the culture is completed. Layered first cell 16 and second cell 1
Since it has a three-dimensional structure in which 8 and layers are in contact with each other, a cell array almost equivalent to that in vivo can be expected, and the function expressed in vivo can be exhibited despite the culture in vitro.
It can be suitably used as a hepatotoxicity evaluation chip or the like.

The culture medium and the culture and growth conditions are not particularly limited, and the usual culture medium and conditions can be used. Alternatively, instead of singly culturing, for example, hepatocytes and other cells may be co-cultured to highly stabilize the function of hepatocytes and to obtain hepatocytes in culture close to biological tissues. In addition to hepatocytes, cells of a type in which cells adhere to each other, for example, muscle cells, nerve cells, vascular endothelial cells, endocrine cells, skin / mucosal cells and the like can be cultured.

[0014]

Example The cell culture apparatus shown in FIG. 2 was used. PSN (Penicillin, Streptomycin,
Neomycin: 10 vol% FB in 90 vol% of WE medium (Williams' Medium E) containing antibiotics)
A medium (culture solution) supplemented with S (Fetal bovine serum) was supplied in such an amount that the substrate 10 was immersed, and hepatocytes isolated from the mouse were suspended (sown) in the medium. Furthermore, EGF 10ng / ml (Epidermal growth
factor: epidermal growth factor), insulin 1μ
g / ml was added to promote the proliferation of hepatocytes. 1 cell
Using 1 ml of medium per well, 150,000 cells were seeded. After seeding, RNA in the medium and cells was periodically collected, and albumin producing ability and cytochrome P450 mRNA expression were measured. Fig. 4 shows albumin production ability. In the figure, TC plane culture refers to a comparative example in which cells are uniformly seeded on the front surface of a culture dish without using a substrate, and TC patterning culture is the case of this example. As is clear from the figure, also in the case of the example, it can be seen that the albumin producing ability is maintained for about 100 hours as in the comparative example. That is, even when the cell culture is performed in the slit-shaped through-hole, the albumin production ability equivalent to that in the flat culture is maintained. FIG. 5 shows data of mRNA expression test using cytochrome P450. The measurement is RT-
By PCR method. As is clear from the figure, also in the case of Example, mRNA was expressed for about 48 hours as in Comparative Example. As is clear from the above, the function of liver parenchymal cells is not impaired even if the cell adhesion site is restricted to the through hole. Then, as described above, after culturing and proliferating the first layer (first cells) in the through-holes, culturing the cells in the second layer (second cells) and subsequent layers in a three-dimensional laminated state Thus, it has become possible to manufacture a cell chip that retains almost the same cell function as that in vivo, even in vitro.

[0015]

As described above, according to the cell culture substrate and cell culture method of the present invention, heterogeneous cells can be cultured and propagated in a laminated state having a three-dimensional structure, so that they can be used in vitro. It has become possible to manufacture a cell chip that retains almost the same cell function as in vivo.

[Brief description of drawings]

FIG. 1 is an explanatory diagram of a cell culture substrate.

FIG. 2 is an explanatory diagram of a cell culture device.

FIG. 3 is a schematic diagram showing a state where the culture is completed.

FIG. 4 is a graph showing albumin production ability.

FIG. 5 is a graph showing data of an mRNA expression test using cytochrome P450.

[Explanation of symbols]

10 substrates 12 through holes 14 culture dish 16 First cell 18 second cell

Claims (4)

[Claims] 1. A cell culture substrate made of a non-cell-adhesive material to which cells to be cultured do not adhere, wherein:
A cell culture substrate having slit-shaped through holes for culturing different cells in a stacked state in the thickness direction of the substrate. 2. The cell culture substrate according to claim 1, wherein the non-adhesive material is silicon rubber or agarose gel. 3. An apparatus for cell culture, wherein the cell culture substrate according to claim 1 or 2 is in close contact with a culture dish made of a cell adhesive material. 4. A culture solution is supplied to the cell culture device according to claim 3, a large number of first cells are seeded on the substrate, and the seeded cells are penetrated by maintaining the temperature conditions as required. A method of culturing cells, comprising a step of culturing while adhering to the culture dish in the holes, and a step of culturing second cells on the first cells cultured in the through holes. .