JPH11142392A - Method for evaluating toxicity with using cultured cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply evaluating toxicity or safety of objects to be evaluated, e.g. chemicals, cosmetics, detergents, etc., without carrying out a toxicity evaluation to the substances for many concentration points. SOLUTION: According to the toxicity evaluation method, an animal cultured cell is embedded, and a cultured collagen gel substance is processed for an effective time with a substance to be evaluated, whereby an intercellular enzyme emitted from the substance to be evaluated to the cultured substance is measured with time.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for evaluating the toxicity of a test substance using a collagen gel culture in which animal culture cells are embedded and cultured. The present invention relates to a method for evaluating the toxicity and safety of test substances such as cosmetics and detergents.

[0002]

2. Description of the Related Art Conventionally, toxicity or safety tests of test substances such as chemicals, cosmetics, detergents and the like have been carried out using laboratory animals, as represented by the Draize test method. However, the results show that, in addition to the problems of quantitativeness and objectivity, a large number of animals are required for one type of test substance, and this method is an alternative to animal experiments from an economic or animal welfare perspective. Is required.

[0003] As an alternative to animal experiments, a method using animal cells, particularly a method using a monolayer culture system, has been used. However, since the cells are present in the living body in a three-dimensional arrangement or in a state enclosed in a connective tissue such as collagen, the results in a monolayer culture system cannot always be extrapolated to the living body as it is. Absent. It is also known that a monolayer culture system loses its function in a living body.

[0004]

Thus, Bell et al. Reported that human fibroblasts were cultured in a collagen gel (US Pat. No. 4,485,096). The culture has the same morphology as fibroblasts in the dermal connective tissue, and a kit for evaluating toxicity using the culture is also commercially available. For toxicity evaluation using the culture, a method is mainly performed using the tetrazolium salt (MTT) and the survival rate at the end point of stimulation as an index. However, in this method, only one point of viability data is obtained from one culture, and in order to evaluate the toxicity of one test substance, the viability must be determined for several points at different concentrations. A plurality of cultures are required. Therefore, there is a problem that it is economically burdensome to evaluate the toxicity of many test substances.

[0005] In addition, as a method using a culture similar to the biological structure, a method for evaluating skin irritation is to use an effective amount of a human skin keratinocyte or fibroblast culture or a coculture of these cells. A method for measuring the stimulating potential of a composition by exposing to a test substance over a period of time and measuring cell viability by reduction of a tetrazolium salt to a formazan dye (Japanese Patent Application Laid-Open No. 6-180311)
Alternatively, a method of seeding animal cells on a collagen gel, periodically changing the medium, adding a test substance to the medium after a certain period of time, and measuring the function of the animal cells after a certain period of time (specifically) JP-A-8-163996) and the like are known. However, in each case, only one viability data can be obtained from one culture, which has the same problem as the method of Bell et al.

Further, as a toxicity evaluation method not using a tetrazolium salt, a safety evaluation test method is known in which cultured animal cells are treated with a test substance and lactate dehydrogenase released from the cells is measured. No. Hei 4-48695).
However, even when this measurement method is used, the toxicity evaluation has the same problem as described above.

[0007]

Means for Solving the Problems In order to reduce the number of required cultures, the present inventors have conducted intensive studies on methods for detecting and evaluating the toxicity of a test substance. The present inventors have found a toxicity evaluation method based on a change over time in the amount of lactate dehydrogenase released from a culture of a substance into a medium, and have completed the present invention.

That is, the present invention provides a method for treating a collagen gel culture in which animal culture cells are embedded and cultured with a test substance for an effective period of time so that intracellular cells released from the test substance into the culture can be treated. This is a toxicity evaluation method characterized by measuring an enzyme over time.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a collagen gel culture in which animal culture cells are embedded and cultured is used. The animal cultured cells to be used include fibroblasts, muscle cells, fat cells, vascular endothelial cells, and the like. Mesenchymal cells, particularly fibroblasts, are preferably used. Moreover, the cells may be established cells. As animal species, mammals such as human, rat, mouse and the like can be used.

[0010] Collagen gel can be prepared from rat tail or bovine tendon, but commercially available one can be suitably used.

[0011] The embedding culture means culturing a gelled mixture of animal cultured cells and collagen solution. Animal culture cells are embedded, and as a cultured collagen gel culture, for example, human cultured fibroblasts are embedded,
There are cultured collagen gel cultures, such as trade name "Matrex TM" (manufactured and sold by Toyobo).

As the test substance used in the present invention, chemicals, cosmetics, detergents and the like are widely used.

In the present invention, these test substances are dissolved in water, physiological saline, a medium, or the like, or added in a solid state to a cultured collagen gel culture in which animal culture cells are embedded and cultured. Process over

Here, the term "effective time" refers to a time sufficient for the test substance to act on animal cells in the collagen gel and exhibit the toxicity of the test substance, and is generally 30 minutes to 24 hours. Is desirable. While exposing the test substance to the collagen gel culture, the culture is performed while culturing in a medium so that the cell activity can be maintained. Examples of the medium to be used include a DMEM medium, an EMEM medium, and PBS, and it is particularly preferable to use a DMEM medium containing no serum. The culture is cultured in this medium at, for example, 37 ° C. for 30 minutes to 24 hours.

In particular, for a plate-like collagen gel, the upper surface is exposed to a test substance, and the other surface (lower surface) is exposed.
It is desirable to perform the culture while contacting the medium with the medium. In this case, as a culture device for collagen gel, there are Millicell (Millipore), cell culture insert (Falcon), and the like. For example, Transwell (Coaster) can be suitably used.

When animal cells in the collagen gel are killed by the test substance, the cell membrane is lysed by the cell death or the cell membrane is destroyed by the direct action of the test substance. Released during.

In the present invention, the above medium is collected over time during the treatment of the test substance, and the amount of intracellular enzymes contained therein is measured. Intracellular enzymes to be measured include lactate dehydrogenase, alkaline phosphatase, and the like.In particular, lactate dehydrogenase is present in cells for many cell types, and the amount of enzyme can be easily measured. It is convenient.

The amount of lactate dehydrogenase can be measured by a quantitative method using an antibody, etc., but it is generally convenient to measure the enzyme activity. In this case, a commercially available kit for measuring lactate dehydrogenase activity is preferably used.

The toxicity in the present invention is evaluated by observing the change over time in the amount of intracellular enzyme released into the medium. For example, using the length of time required to release a certain amount of enzyme as an index, it can be determined that the shorter the time, the stronger the toxicity. When comparing the toxicity between test substances with different concentrations, (concentration) x (time)
The product can be used as an index.

By using the method of the present invention, the toxicity can be evaluated at only one concentration point, and the required number of cultures can be reduced.

[0021]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to specific embodiments. Example 1 A collagen gel culture in which cultured human fibroblasts are embedded and cultured is a trade name of Matrex (manufactured and sold).
Toyobo) was used. The culture is produced by culturing a mixture of cultured human fibroblasts and collagen gel for a certain period of time.

The above collagen gel culture is placed in an assay plate (6-well plate), 5 ml of an assay medium (manufactured and sold by Toyobo) is added to the collagen gel culture, and the mixture is left at room temperature for 30 minutes. Washing was performed with assay medium. The assay medium in each well was aspirated and withdrawn, silicone was applied to the assay ring provided with the kit, and brought into close contact with the top surface of the collagen gel culture.

1.5 ml of an assay medium (manufactured and sold by Toyobo) was added to each well of the assay plate. 37
After placing the wells in an incubator at 95 ° C (humidity: 95% or more) for 1 hour to perform pre-culture, the assay medium in the wells was removed by suction, and 1.5 ml of assay medium was newly added to each well. Was. 80 μl of the six test substances shown in Table 1 dissolved in distilled water were added to the center of the assay ring attached to the upper surface of the culture. At this time, a collagen gel culture without any addition was used as a negative control. In a 37 ° C. incubator (having a humidity of 95% or more), the collagen gel culture to which the test substance was exposed was cultured, and 0.5, 1, 2, 4,
After 8 and 16 hours, 1 well is removed from the assay medium in the well.
00 μl of the medium was collected, and then a fresh amount of the assay medium was replenished, and the culture was continued.

The positive control sample contains 0.
A sample solution extracted from all tissues of the collagen gel culture with 1% Tween 20-assay medium was used.
Sampled assay medium (S), negative control (NC), positive control (PC),
For blank (fresh assay medium only, BL)
Lactate dehydrogenase activity was measured using a commercially available lactate dehydrogenase activity measurement kit (Wako Pure Chemical Industries, Ltd.). Assuming that the activity amount of the positive control was 100%, the damage rate of each sample was calculated by the following formula. Failure rate (T,%) = {(S) − (BL)} × 100 /
(PC). Here, the product of the exposure time (ET20) at which T = 20% and the concentration (C) of the test substance used was defined as a CT20 value.
Table 1 shows the test substances used and the results obtained.

[0025]

[Table 1]

As is evident from Table 1, the lower the CT20 value, the stronger the toxicity.

[0027]

According to the present invention, the toxicity can be evaluated only at one concentration point, and the required number of cultures can be reduced. Therefore, when screening for the toxicity of another type of test substance, the required cost can be reduced and the screening can be performed easily.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yoshihisa Kawamura 10-24 Toyocho, Tsuruga-shi, Fukui Toyobo Co., Ltd.

Claims (4)

[Claims] 1. A collagen gel culture in which animal culture cells are embedded and cultured, is treated with a test substance for an effective time, whereby the intracellular enzymes released from the test substance into the culture are treated with time. A method for evaluating toxicity, characterized in that the method is used to determine the toxicity. 2. The method according to claim 1, wherein the cultured animal cells are human fibroblasts. 3. The toxicity evaluation method according to claim 1, wherein the intracellular enzyme released from the test substance into the culture is lactate dehydrogenase. 4. A toxicity evaluation reagent comprising a collagen gel culture in which animal culture cells are embedded and cultured, a medium, and a reagent for measuring intracellular enzymes.