BIOASSAY METHOD

Abstract: A bioassay method comprises passing test reagents into the circulation of an embryo in such manner that the reagents are not modified by any intermediate metabolism. This can be effected in e.g. rat embryos by microcannulation directly into the vitelline circulation by passing the visceral yolk sac. In chick embryos the test reagent can be administered directly into the extra-embryonic circulation. The embryo is cultured and assessed as to its embryonic development.

Description: BIOASSAYMETHOD This invention relates to bioassays for potentially biologically harmful substances. One method used in carrying out a bioassay is to extract rat embryos and incubate them floating in a nutrient medium containing the test substance. Under normal circumstances it is difficult to administer directly substances to living post- implantation embryo such as a rat embryo as currently used, due to the presence of the embryonic visceral yolk sac. The yolk sac contains enzymes which may metabolise the test substances so that the embryo may receive derivatives of some substances rather than the test substances per se. Moreover, the test substances may combine with metabolites in the yolk sac, forming addition compounds with properties different to the pure test substances. A further complication is the subsequent metabolism of the test substance-metabolite intermediates. These and other reactions can lead to either metabolic activation or deactivation of the test substance. Although in vitro assays, generally involving isolated cell populations grown in tissue culture or extracts of tissue homogenates, can yield valuable information about the effects of potentially hazardous chemicals on various systems within such cell populations or homogenates, assays for potentially mutagenic, carcinogenic and angiogenic substances are preferably carried out in multicellular or whole animal systems. The present invention provides an in vitro method for bioassay which has advantages over existing in_ vitro methods and which avoids the need for the use of multi¬ cellular or whole animal systems as conventionally used; although such systems may also be desired as back-up or for a complete assay, in general, if the method of the present invention indicates that the substance is harmful, the requirement for further testing can in general be eliminated. The invention comprises a method for bioassay comprising passing test reagents into the circulation of an embryo in such manner that the reagents are not modified by any intermediate metabolism, culturing the embryo in culture medium and assessing embryonic development. When using mammalian, e.g. rat embryos, the method may comprise injecting test reagents into the embryo, bypassing the visceral yolk sac of the embryo so that the reagents are not modified by visceral yolk sac metabolism. The yolk sac may be bypassed by cannulation of the vitelline circulation. During cannulation the embryo may be placed in a-well on a microscope stage. The well may be formed from agar and may be dyed to provide contrast when viewing the blood vessels beneath the yolk sac surface. The well may be heated. The vitelline circulation may be cannulated by a microcannula, which may have an internal tip diameter of between 1 and 2 μ . The microcannula may be coated with a silicon- ising agent. The microcannula may be connected to a micro- injection pump and may be used to inject a test substance which may comprise a fluorescent dye, such as fluorescein or rhodamine, so that a check may be made as to whether the test substance has been satisfactorily injected. The test substance may be injected into the embryo in a total volume of less than or equal to one microlitre. The embryo may be a Wistar rat conceptus which may be between 10 and 11 days old. The test substance may be injected into the embryo over the course of four minutes. After injection of the test substances, the rat embryos may be cultured in heat-inactivated rat serum containing antibiotics such as gentamycin, penicillin and streptomycin, for example. Where the embryo is not a rat conceptus, the embryo may be cultured in heat-inactivated homologous serum which may contain antibiotics. Otherwise, the embryo, whether a rat conceptus or not, may be cultured in synthetic or semi-synthetic medium. Embryos may be used which are not surrounded by a visceral yolk sac, for example chicken embryos in fertilised eggs. The test reagent may be administered as before by micro-cannulation directly into the extra- embryonic circulation, possibly the vitelline circulation or chlorioallantoic blood vessels, and the egg contents may be released from the shell into a plastic container and incubated therein, or a window may be cut into the shell to access the embryo. However, it may be possible to avoid the nee for micro-cannulation by introducing the test reagen into the albumen close to the embryo. It may be necessary to test sample embryos whic have been treated otherwise than by direct cannulatio to ensure that the test reagent has survived th transfer into the embryo in unmodified form, but this would in general be an easier task than performin multiple micro-cannulations. The effects of the test substances may b determined by quantitation means which may compris immunological, biochemical and morphometric assays. The biochemical assays may comprise protein, DNA, DNA synthesis, protein synthesis and enzym synthesis and the morphometric assays may compris crown-rump measurements, somite number measurements, yolk sac diameter measurements and vitelline circulatio assessment. The morphometric assays may additionall comprise established morphometric scoring assays. The method may be used to assess factors whic are toxic specifically to the developing embryo, or t assess angiogenic and anti-angiogenic factors. Th visceral yolk sac may also be bypassed by cannulation o the embryo heart at an early stage of development with a microcannula. Angiogenic activity of the test substances may be assessed by morphometry of the developing blood vascular system. The test substance may be added with a radio- label, such as tritiated thymidine, and injected into the embryo heart. The embryo may then be homogenised and an acid-insoluble fraction obtained and subjected to liquid scintillation spectrophotometry to assess in vitro the entire mass response to the test substance. The invention also comprises test kits for carrying out the method. Such test kits may include microcannulae, dyed agar wells, culture media, a work station comprising a micro-injection pump, a micro- manipulator, a heating stage and dissection head for a microscope, together, if desired, with a microscope. One typical method for a bioassay according to the invention will now be described. The method comprises injecting test substances and appropriate control substances into a post- implantation Wiεtar rat embryo between 9 and 13 days old, bypassing the visceral yolk sac of the embryo so that the substances are not modified by visceral yolk sac metabolism, culturing the injected embryo in culture medium and quantitating subsequent embryonic development. The yolk sac is bypassed by micro-cannulation of the vitelline circulation. The cannula is inserted so that the flow of the cannula contents is with the direction of blood flow. The microcannula used in the cannulation has an internal tip diameter of between 1 and 2 urn, and preferably of 1.2 μm. Typically, the microcannula is made of borosilicate and may be given an aqueous-repellent coating with trimethylchlorosilane to prevent the test substances as well as the embryonic tissue adhering to the wall of the cannula. The microcannula is connected to a micro- injection pump set to deliver nano- or even pico-litre volumes over the course of one second to four minutes. The test substances comprise a fluorescent dye such as fluorescein or rhodamine and the embryo checked to see if the test substance has been satisfactorily injected: The test substance is injected into the embryo in a total volume of less than or equal to one microlitre. Volumes substantially greater than this lead to artefacts of the method and can lead to an embryo dying before the bioassay is complete. At 10.5 days the rat conceptus has a negligible blood vascular system in the yolk sac, which can be exploited in the assessment of potentially angiogenic substances. After cannulation, the embryo is cultured in roller bottles in heat-inactivated rat serum containing antibiotics such as gentamycin, penicillin and streptomycin. It will be appreciated, however, that the embryo may be cultured in any suitable medium such as a synthetic or semi-synthetic medium. The effects of the test substances are determined in immunological, biochemical and morphometric assays. The biochemical assays can for example be for total protein and total DNA content and for protein and DNA synthesis, thus assessing the entire mass response of the embryo to the test substances. It will be appreciated, however, that specific enzymes or proteins can be assayed and an assessment made of the induction, or otherwise, of biochemical systems which are involved in toxification/detoxification reactions. One such system is the P450 Cytochrome system. DNA content may be assayed with 4'-6-diamidino-2- phenylindole (DAPI). Morphometric assays comprise crown-rump measurements, somite number measurements, yolk sac diameter measurements and visual inspection of the vitelline circulation. The test substance can be added with a radio- label such as tritiated thymidine and injected into the embryo heart. The embryo is then homogenised and an acid-insoluble fraction obtained and subjected to liquid scintillation spectrophotometry to assess the entire mass response in. vitro to the test substance. A test kit for carrying out the bioassay comprises microcannulae, sterile dyed black agar blocks for holding the embryos for the micro-cannulation procedure, culture medium sachets, a work station comprising a micro-injection pump, a micro-manipulator and a microscope comprising a dissecting head and a heated stage, the heating being desirable for maintaining viability of the embryo during the injection procedure and being preferable to maintaining the laboratory at a sufficient temperature for this. One method used in carrying out a bioassay is to extract rat embryos and incubate them floating in a nutrient medium containing the test substance. When using mammalian, e.g. rat embryos, the method may comprise injecting test reagents into the embryo, bypassing the visceral yolk sac of the embryo so that the reagents are not modified by visceral yolk sac metabolism. Embryos may be used which are not surrounded by a visceral yolk sac, for example chicken embryos in fertilised eggs. The test reagent may be administered as before by micro-cannulation directly into the vitelline circulation, and the egg contents may be released from the shell into a plastic container and incubated therein, or a window may be cut into the shell to access the embryo. However, it may be possible to avoid the need for micro-cannulation by introducing the test reagent into the albumen close to the embryo. It may be necessary to test sample embryos which have been treated otherwise than by direct cannulation to ensure that the test reagent has survived the transfer into the embryo in unmodified form, but this would in general be an easier task than performing multiple micro-cannulations.

Claims: CLAIMS 1. A method for bioassay comprising passing test reagents into the circulation of an embryo in such manner that the reagents are not modified by any intermediate metabolism, culturing the embryo in culture medium and assessing embryonic development. 2. A method according to claim 1, in which the embryo is a mammalian embryo. 3. A method according to claim 2, in which the mammalian embryo is a rat embryo. 4. A method according to claim 3, in which the rat embryo is a Wistar rat conceptus. 5. A method according to claim 3 or claim 4, in which the embryo is between 10 and 11 days old. 6. A method according to any one of claims 1 to 5, in which the embryo has a visceral yolk sac and comprising injecting test reagents into the embryo, bypassing the visceral yolk sac of the embryo so that the reagents are not modified by visceral yolk sac metabolism. 7. A method according to claim 6, in which the yol sac is bypassed by cannulation of the vitellin circulation. 8. A method according to claim 7, in which, durin cannulation, the embryo is placed in a well on microscope stage. 9. A method according to claim 8, in which the wel is formed from agar. 10. A method according to claim 9, in which the wel is dyed to provide contrast when viewing the bloo vessels beneath the yolk sac surface. 11. A method according to any one of claims 8 to 10, in which the well is heated. 12. A method according to any one of claims 1 to 11, in which the vitelline circulation is cannulated with a microcannula. 13. A method according to claim 12, in which the microcannula has an internal tip diameter of between 1 and 2 urn. 14. A method according to claim 12 or claim 13, in which the microcannula is coated with a siliconising agent. 15. A method according to any one of claims 12 to 14, in which the microcannula is connected to an injection pump. 16. A method according to any one of claims 12 to 15, in which the microcannula is used to inject a test substance containing a fluorescent dye. 17. A method according to any one of claims 1 to 16, comprising injecting a total volume of less than or equal to one microlitre. 18. A method according to any one of claims 1 to 17, in which a test substance is injected into the embryo over the course of four minutes. 19. A method according to any one of claims 1 to 18, in which, where the embryo is a rat embryo, it is cultured in heat-inactivated rat serum containing an antibiotic. 20. A method according to any one of claims 1 to 18, in which the embryo is cultured in synthetic or semi- synthetic medium. 21. A method according to claim 1, utilising embryos which are not surrounded by a visceral yolk sac. 22. A method according to claim 21, utilising chicken embryos in fertilised eggs. 23. A method according to claim 21 or claim 22, in which the test reagent is administered by micro¬ cannulation directly into the extra-embryonic circulation. 24. A method according to claim 22, in which the egg contents are released from the shell into a plastic container and incubated therein. 25. A method according to claim 22, in which a window is cut into the shed to access the embryo. 26. A method according to claim 21, in which the test reagent is introduced into the albumen. 27. A method according to any one of claims 1 to 26, in which the test substance is added with a radiolabel. 28. A method according to claim 28, in which the test substance is injected into the embryo heart. 29. A method according to claim 28, in which the embryo is homogenised for assessment of the effects of the test substance, an acid-insoluble fraction obtained and subjected to liquid scintillation spectrophotometry to assess in-vitro the entire mass response to the test substance. 30. A test kit for carrying out a bioassay comprising microcannulae, dyed agar wells, culture media, a work station comprising a micro-injection pump, a micromanipulator, a heating stage and a dissection head for a microscope.

Inventors: CUMBERLAND PETER FREDERICK THO, PRATTEN MARGARET KATHRYN, CUMBERLAND, PETER, FREDERICK, THOMAS, PRATTEN, MARGARET, KATHRYN
Applicants: UNIV LEICESTER, UNIVERSITY OF LEICESTER
Application year: 1992
Main classification: C12N
IPC codes: A61K49, C12M1, C12Q1, G01N33