JPH08308562A - Animal cell incubator and method for measuring drug metabolic activity using the same - Google Patents

Abstract

(57) [Abstract] [Purpose] By increasing the function of cultured animal cells and maintaining an environment similar to that in vivo, economically, efficiently and efficiently Provided is an incubator that can be used for evaluation of metabolism of a drug. [Structure] A culture surface is coated with a collagen gel containing telopeptides removed or treated with a reducing agent, the collagen gel containing type I collagen as a main component, and animal cells are sown on the culture surface to culture the drug metabolizing enzyme. The activity is maintained for a long time.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention enhances the function of cultured animal cells to maintain an environment similar to that in vivo,
Animal cell incubator for economically, efficiently and reproducibly performing metabolism, metabolic toxicity, and drug efficacy test by metabolism of new chemical substances and pharmaceuticals conventionally used in animals, and a drug using the same The present invention relates to a method for measuring metabolic activity.

[0002]

2. Description of the Related Art Conventionally, tests of metabolic efficacy and metabolic toxicity of new chemical substances and pharmaceuticals have been conducted using individual animals, but it is difficult to quantify test results and to determine causal relationships. In addition to the drawbacks such as the above, a large number of individual animals are required for one kind of substance, and therefore there is a demand for an alternative method to animal experiments from the viewpoint of animal welfare. The alternative method for animal experiments reduces the number of experimental animals used, reduces the economic burden of raising animals, and develops methods and materials that can be used for rational and convenient evaluation of metabolic drug efficacy and metabolic toxicity. The purpose is to do.

As one of the alternative methods for animal experiments, a method of culturing animal cells taken out of the body and examining the drug sensitivity and drug metabolizing ability thereof has been considered, but a commonly used tissue culture (tissue With the monolayer culture method using a (culture) petri dish, in vivo functions are immediately lost, and the cells themselves die immediately, making it difficult to study drug metabolism stably with good reproducibility. Is not a sufficient alternative.

On the other hand, although attempts have been made to use established (immortalized) cells, established cells have lost most of the original functions possessed by normal cells in vivo, and their use range is limited. To be done. Also, mainly for primary culture hepatocytes,
As a three-dimensional culture method for maintaining an environment similar to that in the living body,
Spheroid culture method (5th Annual Meeting of the Japanese Society for Alternatives to Animal Experiments p30-33 ('91)) and Matrigel culture method (5th Annual Meeting of the Japanese Society for Alternatives to Animal Experiments p38-)
41 ('91)) has been used as an alternative method. However, in the case of the spheroid culture method, 2-3
There is a problem with cell necrosis in the central part of spheroids due to long-term culture for more than a week and permeability of substances to cells inside. Further, in the case of the Matrigel culture method, the composition of Matrigel has not been completely clarified, and there is a part that is not clear whether the added test substance alone is effective.

[0005]

Therefore, in order to solve the above problems of the prior art, the present inventors have
A previously proposed method for culturing animal cells, that is, a method in which telopeptide-free type I collagen is used as a substrate material, and a specific substance is added to the medium (JP-A-3-4780), and a reducing agent Type I processed in
By using a method using collagen (Japanese Patent Laid-Open No. 6-292565) to maintain cultured animal cells in an environment similar to that in the living body, it is a complicated reaction in the cells and is most likely to be lost due to deterioration of the culture environment. It was found that the drug metabolizing ability, which is one of the functions, can be maintained for a long period of time, and the present invention has been completed.

That is, the object of the present invention is to use a collagen gel obtained by gelling type I collagen from which telopeptides have been removed or type I collagen treated with a reducing agent as a substrate material for culture, and to evaluate drug metabolizing ability. The object of the present invention is to provide an animal cell incubator that can be maintained for a long period of time, and further to use it to investigate the state of drug metabolism of animal cells.

[0007]

The inventors of the present invention have completed the present invention as a result of extensive studies to achieve the above object. That is, the present invention is to coat a culture surface with a collagen gel obtained by gelling type I collagen, in particular telopeptide-free type I collagen or type I collagen treated with a reducing agent, as a substrate material for culture, An animal cell incubator characterized by maintaining the activity of a drug-metabolizing enzyme stably for a long period of time by inoculating and culturing animal cells on the above, and a method for measuring drug-metabolizing activity using the same. is there.

The present invention will be described in detail below. As the type I collagen used as the main component of the substrate material for animal cell culture used in the present invention, there are calf dermis, porcine skin, rat tail tendon, etc. as its origin. However, the purified type I collagen can be used as appropriate.

As a method of removing the telopeptide, there is a method of using pepsin which is an enzyme of biological origin. The reducing agent that can be used in the reducing agent treatment is one that can be used in an aqueous solution of sodium borohydride,
No reduction of ester or amide (peptide bond) occurs,
Any substance that causes reduction of the aldehyde group can be used, and as long as it has such an action, an appropriate substance can be used regardless of the type.

The substrate material for animal cell culture used in the present invention contains, as a main component, the above-mentioned telopeptide-free type I collagen or collagen treated with a reducing agent. It is possible to use either the one to be used or the one in which an appropriate other substrate material is allowed to coexist, and collagen, which is the main component thereof, is gelled and coated on the surface of the incubator, The method is characterized by culturing animal cells in. The collagen component ratio may be 70% or more, preferably 90% or more.

The method of gel formation is as follows. Prepare a collagen solution containing telopeptide-removed type I collagen or type I collagen treated with a reducing agent as a main component at a desired concentration, and add the solution to a phosphate buffer solution. , Hepes
(N- [2 hydroxyethyl] piperazine-N '-[2
Ethanesulfonic acid]) Neutralize using sodium hydroxide aqueous solution, etc., dispense this solution into the well of a Petri dish or plate, and let it stand at 20 ° C or higher, preferably 37 ° C, and it will gel in several hours. The culture surface is coated. The gel portion obtained is thoroughly washed with a phosphate buffer or a culture solution and used as an animal cell incubator. When it is stored for a long time, it can be stored for a long time by storing it at about 4 ° C.

The method for culturing animal cells using the animal cell incubator of the present invention is only required to seed the cells on the collagen gel, and the cells adhere to the upper surface of the collagen gel within a few hours to 1 day after the seeding. To do. The animal cells can be applied to hepatocytes, mammary gland cells, kidney cells, and other appropriate animal cells, and the type thereof is not particularly limited.

These animal cells are repeatedly exchanged with a medium on a collagen gel for 3 to 14 days, preferably 7 to 14 days, and kept in an environment similar to that in the living body. Various toxicity tests can be evaluated by adding a test substance such as to measure the function of animal cells. Every 1 to 4 days as the medium replacement interval,
The shorter the length, the better, but it is usually performed every 2 to 3 days from the viewpoint of the amount of work. Further, the period of culturing is not particularly limited, and it can be used for about 3 days, but the drug metabolizing ability of animal cells is stable for 1 to 2 weeks depending on the medium to be used. Depending on the situation, metabolic ability can be maintained for 4 weeks or longer.

The effect of the test substance on metabolism can be evaluated by adding the test substance to the medium and measuring the metabolites in the animal cells and the medium after a certain period of time. Depending on the characteristics (type) of cell function, a change may appear in about 1 hour after the addition of the test substance, but it is usually preferable to measure after 1 to 4 days have elapsed. In addition, the evaluation of chronic toxicity due to the metabolites of the test substance is carried out for a period of 1 to 2 months or longer by using a medium to which the test substance is added at a relatively low concentration and continuing the culture while repeating the medium exchange. This is possible by measuring the time-dependent change in the function of animal cells over a period of time. The period for continuing the measurement is not particularly limited, but is limited to the period in which the function of the animal cell is maintained at a required level.

In addition, for example, the evaluation of carcinogenicity by metabolites is carried out by continuously adding a low concentration of the test substance to the medium as in the case of the evaluation of chronic toxicity, and then intracellularly expressing the carcinogenic marker enzyme or the like. It becomes possible by checking the presence or absence of expression. Furthermore, since the substance ingested in vivo is first metabolized in the liver, the metabolic function of hepatocytes is applied using the incubator of the present invention, and various substances after metabolism of the test substance by hepatocytes are used. It is also possible to evaluate the effect on animal cells. Specifically, it can be examined by co-culturing hepatocytes and target cells, or by causing the hepatocytes to secrete a metabolite using the incubator of the present invention and bringing the culture supernatant into contact with the target cells. .

[0016]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the types of cells used and the metabolic activity to be measured are not limited to these examples. [Example 1] Of the components eluted from the calf dermis into the hydrochloric acid aqueous solution of PH3, the components gelled in the neutral (PH7-8) physiological saline were recovered to obtain untreated type I collagen. . The obtained type I collagen was dissolved in a hydrochloric acid solution of PH3 at a concentration of 0.3%, and 10 times concentration of phosphate buffered saline and reconstitution buffer (2.2% NaHC
O ₃ , 4.77% Hepes, 0.05% NaOH solution) was mixed at a ratio of 8: 1: 1, and spread on a culture surface of a plastic petri dish to a thickness of 1 to 2 mm.
Gelation was performed in an incubator at 0 ° C. to prepare an animal cell incubator.

[Example 2] A 0.3% solution (PH3) of type I collagen (untreated collagen) having telopeptides eluted from the dermis of calves with hydrochloric acid of PH3 and a 3% solution of pepsin (PH3) were used. Mix at a ratio of 1: 1 and 4 ℃
The mixture was stirred for about 1 day. This reaction solution was mixed with 10 times concentration of phosphate buffered saline and reconstitution buffer (2.2% Na).
HCO ₃ , 4.77% Hepes, 0.05% NaOH
Solution) was mixed at a ratio of 8: 1: 1, heated to 37 ° C. and allowed to stand for about 1 day to gel the collagen, and then the collagen gel alone was separated by centrifugation and washed to give 1 mM. It was dissolved in (mmol) hydrochloric acid to obtain a pepsin-treated collagen solution.

0.3 of the obtained pepsin-treated collagen
% Solution (PH3), 10-fold concentrated phosphate buffered saline and reconstitution buffer (2.2% NaHCO ₃ , 4.77).
% Hepes, 0.05% NaOH solution) 8: 1:
Mix in a ratio of 1 and add 1 to the culture surface of the plastic petri dish.
After laying it to a thickness of 2 mm to 2 mm, it was gelled in an incubator at 37 ° C. to prepare an animal cell incubator.

[Example 3] In the same manner as in Example 1, a 0.3% solution (PH3) of type I collagen having telopeptides eluted from the calf dermis with PH3 hydrochloric acid was used, and 10
Double concentration phosphate buffered saline and 1% sodium borohydride aqueous solution were mixed at a ratio of 10: 1.2: 1, and 4
Stirring was carried out at ℃ for about 1 day. Then, the mixture was heated to 37 ° C. and allowed to stand for about 1 day to gel the collagen. Then, only the collagen gel was separated by centrifugation, washed, and dissolved in 1 mM hydrochloric acid to obtain a reduction-treated collagen solution. This was processed like Example 1 and the animal cell culture device was produced.

The purity of the purified collagen of Examples 1 to 3 is
The result of protein staining of the electrophoretic gel and measurement by densitometry was 80 to 95%. Comparative Example 1 The culture surface of a petri dish was coated with type I collagen at a rate of 1 μg / cm ² .

[Cultivation test of animal cells in medium containing serum] Hepatocytes were separated from rats by the collagenase perfusion method and placed in the cell incubators of Examples 1, 2, 3 and Comparative Example 1.
The seeds were seeded at a concentration of 5 × 10 ⁵ / cm ² and cultured at 37 ° C. in a 5% carbon dioxide gas incubator. The culture solution L-15 medium, 10% fetal bovine serum, insulin 10- ⁷ M, dexamethasone ^{10- 7 M, EGF (Epidemal Growth} Facto
r) Using 10 ng / ml, proline 30 μg / ml, and dimethyl sulfoxide 2% added, a culture solution was added to the upper surface of the gel layer to a height of 1 to 2 mm or more,
Culture was continued while changing the medium every 2-3 days.

Then, while culturing, the maintenance status of the activity of P-450, which is one of the metabolic enzymes in hepatocytes, was evaluated. That is, 1 μg / ml of 3-methylcholanthrene was added as a P-450 activity inducer to the medium of each sample on the 3rd, 7th, 14th, 21st, and 28th day of culturing,
After further culturing for 24 hours, 7-ethoxycoumarin was added thereto, and 7-ethoxycoumarin was metabolized and produced by the action of P-450.
By measuring the amount of hydroxycoumarin, P-4
The maintenance status of 50 activity (monooxygenase activity) was measured. At the same time, P-450 activity when 3-methylcholanthrene was not added was also measured.

[Animal cell culture test using serum-free medium]
Hepatocytes were separated from rats by the collagenase perfusion method and placed in the cell incubator of Examples 1, 2, 3 and Comparative Example 1 at 5 × 1.
The cells were seeded at a concentration of 0 ⁵ / cm ² and cultured at 37 ° C in a 5% carbon dioxide gas incubator. The culture solution L-15 medium, insulin 10- ⁷ M, dexamethasone 10- ⁷ M, EGF
Using 10 ng / ml and 30 μg / ml of proline, a culture solution was added to the upper surface of the gel layer to a height of 1 to 2 mm or more, and the culture was continued while exchanging the medium every 2-3 days. Then, while culturing, the maintenance status of the P-450 activity was measured in the same manner as above with and without the addition of the P-450 activity inducer 3-methylcholanthrene.

The results are shown in FIG. 1 (when 3-methylcholanthrene is added) and FIG. 2 (when 3-methylcholanthrene is not added). When comparing FIG. 1 and FIG. Hepatocytes in gel culture, especially hepatocytes in collagen gel culture that has been subjected to pepsin treatment and reduction treatment,
By adding a P-450 activity inducer, P-450
It can be seen that the metabolic ability of 450 is activated and that the hepatocytes metabolize the artificial compound in the same way as in the living body. Further, from FIG. 1, in culture on pepsin-treated and reduction-treated collagen gel, P-450 activity was observed even after 3 weeks of culture, whereas collagen-coated incubator which is a conventional incubator. Then, the P-450 activity was at a low level immediately after the start of the culture, and the activity almost disappeared after the 7th day,
It can be seen that drug metabolism activity cannot be maintained.

As described above, the type I collagen-coated cell incubator has a low drug-metabolizing activity and therefore is not useful for evaluation tests of drug-metabolism toxicity and drug efficacy, whereas pepsin-treated and reduced-treated collagen is used. Primary hepatocytes cultured in an incubator in which a culture surface is coated with gel can maintain a drug metabolic activity represented by P-450, which is one of the most easily lost functions, for a long period of time. It was confirmed that it could be applied to the drug metabolism toxicity as an alternative to the experiment and the evaluation test of the drug efficacy of drug metabolites.

[0026]

EFFECTS OF THE INVENTION According to the present invention, metabolism tests of chemical substances and pharmaceuticals can be carried out without sacrificing a large number of experimental animals, and the economic burden for breeding a large number of experimental animals can be reduced. In addition, it is possible to provide a rational and convenient method for evaluating a metabolite.

[Brief description of drawings]

FIG. 1 shows the time course of P-450 activity in each incubator when 3-methylcholanthrene was added, (a) using serum-containing medium and (b) using serum-free medium. It's time.

FIG. 2 shows the case where 3-methylcholanthrene is not added,
It is a figure which shows the time-dependent change of P-450 activity in each incubator, (a) is a serum containing culture medium use, (b) is a serum-free culture medium use.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Hiroto Nakajima, 1st Taya-cho, Sakae-ku, Yokohama-shi, Kanagawa Sumitomo Electric Industries, Ltd. Yokohama Works (72) Naoki Niihara 1st, Taya-cho, Sakae-ku, Yokohama-shi, Kanagawa Sumitomo Electric Ki Industry Co., Ltd. Yokohama Works

Claims (6)

[Claims] 1. The activity of a drug-metabolizing enzyme is obtained by coating a culture surface with a collagen gel (culture substrate) containing gelled type I collagen as a main component, and inoculating animal cells onto the collagen gel and culturing the cells. An animal cell incubator characterized by being maintained. 2. The animal cell culture device according to claim 1, wherein the type I collagen is type I collagen from which telopeptides have been removed or type I collagen treated with a reducing agent. 3. The animal cell incubator according to claim 1, wherein the primary culture hepatocytes are the subject of culture. 4. Animal cells are cultured using the animal cell incubator according to any one of claims 1 to 3, and a drug is further added to the culture solution to obtain animal cells. A method for measuring drug-metabolizing activity, which comprises examining the state of drug metabolism of the drug. 5. The animal cell incubator according to any one of claims (1) to (3) is seeded with animal cells, and the cells are cultured for 3 to 14 days while repeating the medium exchange, A method for measuring drug-metabolizing activity, which comprises adding a test substance such as a drug to a culture medium, and measuring a metabolite in the culture medium or cells after a certain period of time has elapsed. 6. The method for measuring drug metabolic activity according to claim 5, wherein the animal cell is a primary cultured hepatocyte and the metabolite is a substance metabolized by the metabolic enzyme P-450.