JPH07108233B2 - Toxicity test method using cultured cells - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a toxicity test method using cultured cells, and more particularly to an in vitro toxicity test method useful as an alternative method to the Draize eye mucous membrane irritation test.

[0002]

2. Description of the Related Art In the fields of medicine, cosmetics, agricultural chemicals, medical materials and the like, toxicity tests using animals have been widely conducted so far in order to ensure safety. However, this toxicity test is not only economically because it requires a lot of labor, time and cost, but also from the viewpoint of animal welfare and the objective reliability of test results. The development of toxicity test methods that can be substituted for various animal experiments has been demanded.

For example, a test substance was dropped on the eyes of a rabbit,
In the case of the Draize test, which visually evaluates the degree of changes in ocular tissues such as the cornea, iris, and conjunctiva over time, and comprehensively evaluates the irritancy of the test substance, objective reliance is placed on the irritation evaluation. It is particularly cruel, not only because it is non-specific and requires a high degree of skill in the determination, but also because the test site is the eye, the development of alternatives is urgently needed (e.g., toxicology). Forum, Vol. 10, No. 2, pp. 137-143 (1987)).

Therefore, as a method that may be an alternative to the Draize method, a toxicity test method for examining the effect of a test substance on the colony-forming ability of corneal cells of rabbit eyes has already been proposed (see above). References). However,
In the case of this test method, pretreatment of cultured cells is complicated,
A relatively long culture time is required to form the colonies necessary for the determination, and the number of colonies formed must be counted each time, which is inefficient.

[0005]

SUMMARY OF THE INVENTION The present invention further improves the toxicity test method using such cultured cells to obtain objectively reliable determination results with good reproducibility in a relatively short time. , An in vitro toxicity test method useful as an alternative to the Draize method, which can be easily obtained.

[0006]

That is, the present invention provides (i) calcium chloride <0.1 mM and methionine 8.0-1.
Rabbit corneal epithelial cells were cultured in a serum-free medium characterized by containing 4.0 mg / l, and (ii) the concentration of calcium chloride in the medium was 0.1 mM or more, preferably 0.
After increasing to 1 to 10 mM, culturing the cells is continued, (iii)
Furthermore, the cells are continuously cultured in the presence of the test substance,
(iv) Next, the present invention relates to a toxicity test method using cultured cells, which comprises adding neutral red to a culture system and measuring the amount of neutral red taken up by living cells.

As the serum-free medium used in step (i), the serum-free medium for animal cell culture according to the previous application by the present applicant may be used (see Japanese Patent Application No. 3-34150). In this case, if the concentrations of calcium chloride and methionine are out of the above ranges, it becomes difficult to obtain an effective effect of proliferating rabbit corneal epithelial cells. Examples of suitable serum-free medium include the following mediums I to III.

Medium I (units are mg / l unless otherwise stated) Component Content Alanine 8.91 Arginine 210.67 Asparagine monohydrate 15.01 Aspartic acid 8.52 Cysteine hydrochloride monohydrate 38.64 Glutamic acid 14.70 Glutamine 511.5 Glycine 7.51 Histidine hydrochloride monohydrate 44.02 Isoleucine 51.16 Leucine 39.35 Lysine hydrochloride 23.74 Methionine 11.19 Phenylalanine 12.39 Proline 23. 03 Serine 36.78 Threonine 7.74 Tryptophan 6.54 Tyrosine 9.06 Valine 19.92 Biotin 0.020 Folic acid 1.06 Lipoic acid 0.21 Nicotinic acid amide 0.330 Calcium pantothenate 0.620 Pyridoxine hydrochloride 0 .140 Lvov Rabin 0.207 Thiamine hydrochloride 0.675 Vitamin B ₁₂ 0.922 Adenine 12.16 Choline chloride 6.98 Glucose 1082 Inositol 10.81 Hypoxanthine 2.04 Ptericin dihydrochloride 0.0805 Sodium pyruvate 55.0 Thymidine 0.727 Calcium chloride dihydrate 0.03 mM Potassium chloride 201.29 Magnesium chloride hexahydrate 124.0 Sodium chloride 7597.2 Sodium dihydrogen phosphate heptahydrate 294.8 Potassium dihydrogen phosphate 42.19 Sulfuric acid Copper pentahydrate 0.0025 Iron sulfate heptahydrate 1.1 1 Sodium selenium 0.001934 Manganese sulfate pentahydrate 0.000065 Sodium silicate nonahydrate 0.0711 Ammonium molybdate tetrahydrate 0.1 000618 Ammonia Vanaji Mu 0.000293 Nickel chloride hexahydrate 0.000059 Tin chloride dihydrate 0.000056 Zinc chloride heptahydrate 0.2157 Hepes 3336.2 Sodium hydrogen carbonate 1176 Phenol red sodium salt 1.242 Insulin 5 μg / ml Hydrocortisone 0.5 μg / ml EGF 10 ng / ml BPE 0.4% (w / w)

Medium II (units are mg / l unless otherwise stated) Component content Alanine 7.13 Arginine 189.5 Asparagine monohydrate 12.10 Aspartic acid 7.51 Cysteine hydrochloride monohydrate 35.13 Glutamic acid 14.70 Glutamine 400.0 Glycine 6.06 Histidine hydrochloride monohydrate 11.93 Isoleucine 51.16 Leucine 39.35 Lysine hydrochloride 20.09 Methionine 9.00 Phenylalanine 9.81 Proline 23. 03 Serine 33.00 Threonine 5.96 Tryptophan 6.13 Tyrosine 7.25 Valine 19.92 Biotin 0.020 Folic acid 1.06 Lipoic acid 0.21 Nicotinic acid amide 0.330 Calcium pantothenate 0.620 Pyridoxine hydrochloride 0 .140 Riboflavi 0.207 Thiamine hydrochloride 0.675 Vitamin B ₁₂ 0.922 Adenine 12.16 Choline chloride 6.98 Glucose 1082 Inositol 10.81 Hypoxanthine 2.04 Ptericin dihydrochloride 0.0805 Sodium pyruvate 55.0 Thymidine 0.727 Calcium chloride dihydrate 0.03 mM Potassium chloride 201.29 Magnesium chloride hexahydrate 124.0 Sodium chloride 7597.2 Sodium dihydrogen phosphate heptahydrate 294.8 Potassium dihydrogen phosphate 42.19 Sulfuric acid Copper pentahydrate 0.0025 Iron sulfate heptahydrate 1.1 1 Sodium selenium 0.001934 Manganese sulfate pentahydrate 0.000065 Sodium silicate nonahydrate 0.0711 Ammonium molybdate tetrahydrate 0.1 000618 Ammonia vanadium 000293 Nickel chloride hexahydrate 0.000059 Tin chloride dihydrate 0.000056 Zinc chloride heptahydrate 0.2157 Hepes 3336.2 Sodium hydrogen carbonate 1176 Phenol red sodium salt 1.242 Insulin 5 μg / ml Hydrocortisone 0.5 μg / ml EGF 10 ng / ml BPE 0.4% (w / w)

Medium III (unless otherwise indicated units are mg / l) Component Content Alanine 4.46 Arginine 168.54 Asparagine monohydrate 7.51 Aspartic acid 8.52 Cysteine hydrochloride monohydrate 36.88 Glutamic acid 14.70 Glutamine 550.0 Glycine 3.75 Histidine hydrochloride monohydrate 54.50 Isoleucine 77.40 Leucine 59.03 Lysine hydrochloride 23.74 Methionine 13.00 Phenylalanine 14.00 Proline 17. 27 Serine 31.53 Threonine 10.00 Tryptophan 11.23 Tyrosine 11.00 Valine 41.00 Biotin 0.0075 Folic acid 0.88 Lipoic acid 0.10 Nicotinic acid amide 0.513 Calcium pantothenate 0.762 Pyridoxine hydrochloride 0 .5 4 Riboflavin 0.075 Thiamine hydrochloride 0.675 Vitamin B ₁₂ 0.205 adenine 12.16 choline chloride 6.98 glucose 1045 inositol 21.62 Puterishin dihydrochloride 0.08 thymidine 0.36 Calcium chloride dihydrate 0 0.03 mM potassium chloride 260.9 magnesium chloride hexahydrate 61.0 sodium chloride 7597.2 sodium dihydrogen phosphate heptahydrate 141.96 copper sulfate pentahydrate 0.0025 iron sulfate heptahydrate 0.70 Sodium selenium 0.001934 Manganese sulphate pentahydrate 0.000121 Sodium silicate nonahydrate 0.0711 Ammonium molybdate tetrahydrate 0.000618 Ammonia vanadium 0.000293 Nickel chloride hexahydrate 0.000059 Tin chloride Dihydrate 0.0 00056 Zinc chloride heptahydrate 0.072 Hepes 3336.2 Sodium hydrogen carbonate 1176 Phenol red sodium salt 1.13 Insulin 5 μg / ml Hydrocortisone 0.5 μg / ml EGF 10 ng / ml BPE 0.4% (w / w)

Rabbit corneal epithelial cells are obtained by separating and collecting rabbit eyeballs as they are, or by using an appropriate treatment solution,
For example, the culture may be performed using the media I to III and then used. In the present invention, the number of rabbits to be sacrificed is significantly reduced because objective test results can be reproducibly obtained using a small amount of cell sample. For example, according to the present invention, three rabbits are required to judge and evaluate the toxicity of one concentration for one test substance by the Draize method. By using corneal epithelial cells harvested from rabbits,
It is possible to examine the toxicity of four test substances for four test substances.

The culture conditions for the step (i) of rabbit corneal epithelial cells in the above serum-free medium are not particularly limited, but are usually 35 to 38 ° C., preferably 37 ° C., and the number of cells implanted is 4000 / cm ² is 4 to 7 days, and the culture atmosphere is carbon dioxide gas of 3 to 7
%, Preferably 5% air is generally used.

In step (ii), calcium chloride is added to the above serum-free medium and the concentration thereof is 0.1 mM or more,
After raising to preferably 0.1 to 10 mM, the rabbit corneal epithelial cells are further subjected to 35 to 38 ° C., preferably 37 ° C.,
When the number of cells implanted is 4000 / cm ² , the cells are cultured for 4 to 7 days. The culture atmosphere is the same as in (i). The concentration of calcium chloride in this culture step (ii) was 0.1
If it is lower than mM, as shown in FIG. 1, a sufficient amount of neutral red is not taken up by cells, so that the measurement accuracy is lowered and the toxicity test becomes unreliable. Note that FIG.
In the figure, the horizontal axis is the concentration of calcium chloride in serum-free medium.
(mM), and the vertical axis represents the absorbance of neutral red at 540 nm (A ₅₄₀ × 10 ⁵ ).

In step (iii), the rabbit corneal epithelial cells cultured as described above are continuously cultured in the presence of the test substance. The culture conditions are usually 35 to 38 ° C., preferably 37 ° C., and the number of cells implanted is 4000 cells / cm ^2.
In the case of, the period is 4 to 7 days, and the culture atmosphere is the same as in the case of (i). Examples of the test substance include various active ingredients, auxiliary ingredients, and the like, which are added to medicines, agricultural chemicals, cosmetics and the like. These test substances are usually dissolved in a suitable solvent, for example, 10 mM phosphate buffer (pH 7.0), ethanol, a 1: 1 mixture of the phosphate buffer and ethanol,
After subjecting the solution to filter sterilization treatment, the medium used in step (ii) is appropriately diluted to a desired concentration, and then added in an appropriate amount to the culture cell-containing medium obtained in step (ii).

In step (iv), neutral red is further added, and the amount of neutral red taken up by living cells is measured. In this case, usually, neutral red is added in an amount of 0 to 200 mg / medium using the medium used in step (ii).
After diluting to l, it is added to the culture medium, and then the culture system is kept under the above-mentioned culture conditions for 2 to 3 hours, and then the uptake amount of neutral red is measured. In order to measure the uptake, the culture system is usually subjected to the following post-treatment. That is, after removing the culture supernatant, the calcium chloride-containing formalin solution
(For example, 1% formalin solution containing 1% calcium chloride)
To fix the cultured cells and remove the supernatant to remove neutral red that is not taken up by the cells, and then add an acetic acid-containing ethanol solution (eg, 1% acetic acid-containing 50% ethanol solution). By doing so, the neutral red incorporated into the living cells is extracted. The amount of neutral red contained in the extract may be measured by a conventional method, for example, a colorimetric method or a spectrophotometric method, and the absorbance using a spectrophotometer, for example,
The method of measuring the absorbance at 540 nm is convenient.

In the above toxicity test method, the stronger the toxicity of the test substance, the smaller the amount of neutral red taken up. Therefore, the toxicity of the test substance can be determined by measuring the amount of neutral red taken up. Becomes As illustrated in the following examples, the determination result of the toxicity test method according to the present invention shows a good correlation with the determination result of the conventionally used Draize eye mucous membrane irritation test method.

[0017]

EXAMPLES The present invention will be described below with reference to examples. Example 1 1. Corneal epithelial cells isolated from the eyeballs of rabbits were cultured in the above medium I at 37 ° C. in a 5% CO ₂ incubator for 5 minutes.
Incubate for a day. 2. Next, calcium chloride was added to medium I to give 0.15
Plate cells in 96 mM culture medium I '
Inoculate (0.1 ml per well (cell number 2,500
Cells) and cultured at 37 ° C. for 3 days. 3. 0.1 ml of the evaluation drug is added to each well, and the culture is continued for 2 days. As the evaluation drug, the surfactant 1 shown in Table 1 was used.
One kind and one kind of preservative were dissolved in each solvent, sterilized by filtration, and then diluted with the medium I ′ for use. 4. Add medium containing 0.1 ml of Neutral Red 15 μg and place in 37 ° C., 5% CO ₂ incubator for 3 hours. After removing the neutral red that had not been taken up by the cells, 0.2 ml of a 1% formalin solution containing 1% calcium chloride was added to fix the cells to the plate. 5. After removing the supernatant, 0.1 ml of a 50% ethanol solution containing 1% ethanol is added to extract neutral red into which live cells are incorporated. After 20 minutes, the absorbance at 540 nm was measured with a microplate reader. 6. The percentage of the neutral red uptake of cells treated with the evaluation drug to the amount of neutral red uptake of cells without the evaluation drug was calculated. Neutral red uptake is 50% from each evaluated drug concentration and its percentage value
The evaluation drug concentration NR ₅₀ (w / w% × 10 ⁵ ) was calculated.
The results are shown in Table 2.

[0018]

[Table 1]

[0019]

[Table 2]

Comparative Example 1 An aqueous solution of a test sample was applied to the left eye of a healthy Japanese white rabbit weighing approximately 2 kg.
(1%, 10%, 50% w / w concentration) was added dropwise by 100 μl, and the animals were kept as they were without washing. After that, over time (1,
The degree of eye damage was evaluated at 3, 6, 24 hours and 4 and 7 days). From the results, a drug concentration DS ₂₀ (w / w%) giving a Draize evaluation point of 20 was determined. The results are shown in Table 2.

Using the results shown in Table 2, in order to examine the correlation between the Draize eye mucous membrane irritation test and the toxicity test using rabbit corneal epithelial cells, the DS of Draize eye mucous membrane irritation of each of the evaluated drugs was plotted on the X axis. Toxicity test using rabbit corneal epithelial cells for each drug evaluated on the Y-axis, the logarithmic value of ₂₀ (w / w%) N
It was plotted so as to have a logarithmic value of R ₅₀ (w / w% × 10 ⁵ ) (see FIG. 2). As a result, the regression equation Y = 1.4 52X +
The correlation was 0.765 and the correlation coefficient was 0.711, indicating good correlation.

Example 2 Corneal epithelial cells isolated from the eyeball of rabbit were treated with the above-mentioned medium I.
Was cultured for 5 days at 37 ° C. Cell culture plate
(96 wells) 0.1 ml per well (2,50 cells)
(0) inoculate. The inoculated cells were further cultured for 5 days in a medium in which the concentration of calcium chloride in medium I was increased to 0.15 mM. As a result, when the cells are counted on the hemocytometer, 2
It was 2,500. On the other hand, when this measurement was carried out by the amount of neutral red uptake of live cells,
An absorbance of 0 nm gave 0.80.

Example 3 Corneal epithelial cells were cultured at 37 ° C. using the medium I as in Example 2.
The cells were cultured for 5 days. The cells are inoculated into a culture plate (96 wells) in an amount of 0.1 ml per well (2,500 cells). The inoculated cells were further adjusted to a calcium chloride concentration of 0.5.
It was cultured for 5 days in a medium adjusted to mM. As a result, the number of cells was 25,000 when counted on a hemocytometer. On the other hand, when this measurement was carried out with the amount of neutral red taken up by living cells, the absorbance at 540 nm was 0.85.

Comparative Example 2 Corneal epithelial cells were cultured at 37 ° C. in the same manner as in Example 2 except that the medium I was used.
The cells were cultured for 5 days. The cells are inoculated into a culture plate (96 wells) in an amount of 0.1 ml per well (2,500 cells). The inoculated cells were further cultured in medium I for 5 days. As a result, when the cells were counted on a hemocytometer, the number was 17,400. On the other hand, when this measurement was carried out with the amount of neutral red uptake of living cells, 0.21 was obtained at an absorbance of 540 nm.

[0025]

INDUSTRIAL APPLICABILITY The toxicity test method using the cultured cells according to the present invention provides an objectively reliable toxicity determination result with good reproducibility in a relatively short time using a small amount of rabbit corneal epithelial cells. , Because it can be easily obtained,
It can be performed without requiring time and cost, and it can drastically reduce the number of sacrificed rabbits as compared with the Draize method, and has a good correlation with the toxicity determination result by the Draize method. Therefore, it is particularly useful as an alternative method to the Draize method. Therefore, the toxicity test method according to the present invention includes, for example, pesticides, surfactants for cosmetic raw materials, preservatives, ophthalmic solutions such as eye drops and wetting solutions for contact lenses, or ophthalmic solutions such as contact lenses, intraocular lenses or artificial corneas. It is particularly useful as a toxicity test method for medical materials and the like.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between the concentration of calcium chloride in a serum-free medium used in the present invention and the amount of neutral red uptake per cell.

FIG. 2 is a graph showing the correlation between the result of the toxicity test according to the present invention (Example 1) and the result of the Draize eye mucous membrane irritation test.

[Explanation of symbols]

 1 POE-LE 2 POE-ST 3 CFAD 4 PEG-MO 5 SLGL 6 SGCS 7 SLS 8 ST 9 BC 10 DMAC 11 STAC 12 SB

Claims (3)

[Claims] 1. (i) Rabbit corneal epithelial cells are cultured in a serum-free medium characterized by containing calcium chloride <0.1 mM and methionine 8.0-14.0 mg / l, (ii) After increasing the concentration of calcium chloride in the medium to 0.1 mM or more, preferably 0.1 to 10 mM, culturing the cells is continued, and (iii) further culturing the cells in the presence of the test substance. (Iv) Next, neutral red is added to the culture system, and the uptake amount of neutral red by living cells is measured, and a toxicity test method using cultured cells. 2. The toxicity test method according to claim 1, wherein the test substance is dissolved in a phosphate buffer and / or ethanol and added to the culture system. 3. The method of claim 1, wherein the amount of neutral red uptake is determined by measuring the absorbance at 540 nm using a spectrophotometer.