JPH06509469A - bioassay method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Shimono building permanent residence The present invention relates to bioassays for substances potentially harmful to living organisms.

One method used when performing bioassays is to extract rat embryos and The rat embryos are cultured while suspended in a nutrient medium containing the test substance. There is a law.

Under normal circumstances, the presence of an embryonic organ, the yolk sac, would It is difficult to directly administer a substance to an embryo after implantation with a living egg, such as a live embryo.

The yolk sac metabolizes the test substance and induces a seed substance that consists of more than the test substance itself. Contains enzymes that may accept conductors.

Furthermore, the test substance combines with metabolic products in the yolk sac to form the pure test substance. It is possible to generate adducts with different properties. Further problems test It is the subsequent metabolism of substances, metabolic products, and intermediate products. These and other reactions can cause either metabolic activation or metabolic deactivation of the test substance.

Extracts of independent cell populations or tissue homogenates, typically grown in tissue culture In vitro bioassays, including provide valuable information about the effects of potentially hazardous chemicals on systems biopsies for potentially mutagenic, carcinogenic, and angiogenic substances. The conventional method is preferably performed on multicellular tissue or whole animal tissue.

The present invention has advantages over existing test tube methods and provides Provides an in vitro bioassay that does not require the use of cell tissue or whole animal tissue.

However, such tissues can be used as backup or complete bioassays. It may also be desirable for the sake of law. Generally speaking, the method of the present invention No further testing is necessary if the substance is shown to be harmful.

The present invention allows reagents to be distributed throughout the embryonic circulation in such a way that they are not modified by any intermediate metabolism. This includes bioassays that pass through a tube, culture the embryo in a medium, and assess embryonic development. When using mammalian embryos, for example rat embryos, this method The reagent is injected into the embryo while bypassing the yellow groove, and the reagent is absorbed by organ yolk sac material metabolism. It may also be possible to prevent it from being modified.

The vitelline groove may be bypassed by cannulation of the vitelline circulation. cannula During the procedure, the embryo may be placed in a well on the microscope stage. This (bommi is cold) You can make it in the sky, or use contrast staining to observe the blood vessels under the yolk sulcus. You may give a strike. The cavity may be heated.

Cannulation of the yolk circulation can be performed with a microcannula (this microphone The locaniure can have a tip inner diameter of 1-2 μm.

The microcannula may be coated with a silicone treatment.

The microcannula may be connected to a microinjection pump, or may be injected with fluorescent dyes. For example, by injecting a test substance, which can consist of fluorescein or rhodamine, It can also be used to check whether the strike substance has been injected in a satisfactory manner. .

A total volume of test substance of 1 microliter or less may be injected into the embryo.

The embryo may be a 10-11 day old Wistar rat conceptus.

The test substance may be injected into the embryo over a period of 4 minutes.

After injection of the test substance, the rat embryos are treated with, for example, gentamicin, penicillin, Cultured in heat-inactivated rat serum containing antibiotics such as streptomycin. It's okay.

If the embryo is not a rat conceptus, it is incubated in heat-inactivated allogeneic serum containing antibiotics. Embryos may also be cultured.

Otherwise, the embryos, whether rat conceptus or not, should be placed in synthetic media or It may also be cultured in a synthetic medium.

The embryo used is a fertilized chicken egg that is not surrounded by the vitelline groove. You may. Reagents include embryonic circulation, possibly vitelline circulation or chlorinated allantoin. The acid can be conventionally administered, such as by microcannulation directly into the blood vessel.

You can also put the egg contents from the eggshell into a plastic container and culture it there.

Alternatively, the embryo may be accessed by opening a window in the eggshell.

However, by introducing reagents into the albumen in close proximity to the embryo, microcariculture It also avoids the need for

Processing methods other than direct cannulation ensure that the reagents are resistant to intrafetal transfer. It may be necessary to test sample embryos that have been protected from modification. , which is generally an easier task than performing multiple microcannulations. Dew.

The effectiveness of test substances is determined by immunological bioassays, biochemical bioassays, and cytostatic bioassays. can be determined by quantification means, which can include physical assays.

Biochemical bioassays include protein, DNA, DNA synthesis, protein synthesis and enzyme synthesis. The cytometric bioassay methods include crown lamp measurement, primordial number measurement, and yolk groove straightening. May include diameter measurements and vitelline circulation assessment. The cell estimation bioassay method additionally may include cytostochastic bioassays.

This method is particularly useful for assessing factors harmful to the developing embryo, angiogenesis, It may also be used to assess non-angiogenic factors. The organ vitelline groove is also an early developmental stage bypass by cannulation of the embryonic heart by a microcannula It's okay. The angiogenic activity of the test substance is assessed by morphometry of the developing vasculature. I can value it.

The test substance should be attached with a radiological label, e.g. tritiated thymidine and then May be injected into the embryonic heart. Next, the embryos were homogenized and the acid-insoluble components thus obtained is subjected to liquid scintillation spectrophotometry to determine the total mass response to the test material in a test tube. You may evaluate internally.

The invention also includes test kits for carrying out this method. This te The kit includes a microcannula, stained agar wells, culture medium, and microinjections. Heating stages and cutting heads for pumps, micromanipulators and microscopes A workstation including a microscope may optionally be included.

One representative bioassay method according to the present invention will be described below.

This method involves transferring the embryo's organ vitelline groove to the Wistar rat embryo after 9 to 13 days of egg implantation. Bypassing the test substance and appropriate Inject the antiseptic substance, culture the injected embryos in culture medium, and quantify subsequent embryonic development. It consists of becoming

Bypassing the vitelline groove is performed by microcannulation of the vitelline circulation. Kanyu The tube is inserted so that the flow of its contents is in the same direction as the blood flow. cannula The microcannula used in the The tip has an inner diameter of m. Typically, microcannulae are made of borosilicate. , water-repellent coating with trimethylchlorosilane to remove test substances and embryos. Tissue may be prevented from adhering to the walls of the cannula.

The microcannula is connected to a microinjection pump for a period of 1 second to 4 minutes. nanoliter or picolitre quantities. The test substance is fluorescein Contains fluorescent dyes such as in- or rhodamine, and the test substance is satisfactory. Check the embryo to see if it has been injected.

The test substance is injected into the embryo in a total volume of no more than 1 microliter. than this Significantly larger volumes may lead to artifacts in the method and embryo staining before the bioassay is complete. may cause

At 10.5 days, the rat conceptus has negligible vasculature in the vitelline groove; , which can be used to assess potential angiogenesis.

After cannulation, gentamicin, penicillin and streptomycin Culture the embryos in roller bottles of heat-inactivated rat serum containing antibiotics such as . However, it can be cultured in any suitable medium, such as synthetic or semi-synthetic media. It would be appropriate to do so.

The effectiveness of test substances is determined by immunological bioassays, biochemical bioassays and cell estimation. Determined by scientific bioassay. Biochemical bioassays, for example, and total DNA content as well as protein and DNA synthesis. The total mass response of the embryo can be evaluated. However, special enzymes or can bioassay proteins and detect biochemical organ systems associated with toxicization/detoxification reactions. It would be appropriate to carry out an evaluation done at the time of implementation. One such system is There is a P2S5 Cytochrome system.

The DNA content is 4'-6-diamino-2-phenylindole (DAPI) and biological Can be tested.

Cytometric bioassay methods include crown lamp measurement, protosegment number measurement, and yolk groove diameter measurement. and visual inspection of the vitelline circulation.

The test substance is labeled with a radioactive label, such as tritiated thymidine, and then Can be injected into the budding heart. Next, the embryos are homogenized and the resulting acid-insoluble components are transferred to a liquid system. The total mass response to the test substance is measured in vitro using anti-chillation spectrophotometry. evaluate.

The test kit for carrying out this bioassay method consists of a microcannula, a microphone Sterile stained black agar plates for holding embryos for locannulation work locks, medium sachets, microinjection pumps, micromanipulators and Includes a workstation consisting of an open head, a microscope that includes a heating stage, and do. Heating is desirable to maintain the viability of the embryo during the injection procedure, and is It is preferred to maintain the laboratory at a temperature sufficient for the present bioassay method.

One method used to perform the bioassay is to extract rat embryos and The test substance is cultured while floating on a nutrient medium containing the test substance.

When using mammalian embryos, especially rat embryos, this method uses the embryo's organ eggs. Bypass the yellow groove to prevent reagents from being modified by organ yolk sac material metabolism. Inject the reagent into the embryo.

The embryo used is a fertilized chicken egg that is not surrounded by the vitelline groove. You may. The reagents can be followed directly into the vitelline circulation, such as by microcannulation. Can be administered as usual. You can also remove the egg contents from the eggshell into a plastic container and place it inside. Alternatively, the embryo may be accessed by opening a window in the eggshell.

However, by introducing reagents into the albumen close to the embryo, It can also meet the need for renewal.

Processing methods other than direct cannulation ensure that the reagents are resistant to intrafetal transfer. It may be necessary to test sample embryos that have been protected from modification. , which is generally an easier task than performing multiple microcannulations. Dew.

Submission of a copy (translation) of the written amendment (Article 184-8 of the Patent Act) Deadline: January 6, 1994

Claims (30)

[Claims] 1. Pass the reagent through the embryonic circulation in such a way that it is not modified by any intermediate metabolism. A bioassay method characterized by culturing the embryo in a medium and evaluating the development of the embryo. Law. 2. The method according to claim 1, characterized in that the embryo is a mammalian embryo. How to do it. 3. In the method according to claim 2, the mammalian embryo is a rat embryo. A method characterized by: 4. In the method according to claim 3, the rat embryo is treated with a Winstar rat. A method characterized in that it is a fetal product. 5. In the method according to claim 3 or 4, the embryo is 10 to 11 days old. A method characterized by being of. 6. In the method according to any one of claims 1 to 5, The bud is the organ yolk sac, and the reagent bypasses the organ yolk sac of the embryo and the organ yolk sac material It is characterized by injecting the reagent into the embryo while preventing it from being modified by metabolism. How to do it. 7. The method according to claim 6, wherein the yolk sac is subjected to cannulation of the vitelline circulation. A method characterized by bypassing the 8. In the method according to claim 7, the embryo is subjected to microscopy during cannulation. A method characterized by placing the device in a recess above the stage. 9. The method according to claim 8, characterized in that the depression is made of agar. How to make it a sign. 10. In the method according to claim 9, the recesses are dyed to remove the subsurface of the yolk sac. A method characterized by providing contrast when observing blood vessels. 11. In the method according to any one of claims 8 to 10, , a method characterized by heating the recess. 12. In the method according to any one of claims 1 to 11, , a method characterized by cannulating the vitelline circulation with a microcannula . 13. The method according to claim 12, wherein the microcannula has a diameter of 1 to 2μ. A method characterized in that the tip has an inner diameter of m. 14. The method according to claim 12 or 13, in which the microcannula A method characterized in that the metal layer is coated with a silicone treatment agent. 15. In the method according to any one of claims 12 to 14, and connecting the microcannula to an infusion pump. 16. In the method according to any one of claims 12 to 15, The test substance containing the fluorescent dye is injected using a microcannula. A method characterized by: 17. In the method according to any one of claims 1 to 16, , a method characterized in that a total volume of less than or equal to 1 microliter is injected. 18. In the method according to any one of claims 1 to 17, , a method characterized in that the test substance is injected into the embryo over a period of 4 minutes. 19. In the method according to any one of claims 1 to 18, , in heat-inactivated rat serum containing antibiotics, if the embryo is a rat embryo. A method characterized by culturing an embryo. 20. In the method according to any one of claims 1 to 18, , a method characterized by culturing the embryo in a synthetic medium or a semi-synthetic medium. 21. The method of claim 1, wherein the organ is surrounded by a yolk sac. A method characterized by using a dead embryo. 22. In the method according to claim 21, the method uses embryos of fertilized chicken eggs. A method characterized by: 23. 22. The method according to claim 21 or 22, wherein the reagent is circulated outside the embryo. A method characterized in that the administration is performed by direct microcannulation. 24. A method according to claim 22, in which the egg contents are removed from the eggshell into plastic. A method characterized by transferring to a container and culturing therein. 25. In the method according to claim 22, the embryo is accessed by opening a window in the eggshell. A method characterized by: 26. The method according to claim 21, characterized in that the reagent is introduced into egg white. How to make it a sign. 27. In the method according to any one of claims 1 to 26, , a method characterized in that a radiological label is attached to a test substance. 28. The method of claim 28, wherein the test substance is injected into the embryonic heart. A method characterized by: 29. In the method according to claim 28, the embryo is evaluated for the effect of a test substance. Homogenize to obtain the acid-insoluble fraction for liquid scintillation spectrophotometry. The method is characterized by in vitro evaluation of the total mass response to the test substance. Method. 30. A test kit for performing bioassays and a microcannula , stained agar well, culture medium, microinjection pump, and micromanipure. a workstation consisting of a computer, a heating stage for the microscope, and a cutting head. A test kit comprising: