CA2112897A1 - Bioassay method - Google Patents
Bioassay methodInfo
- Publication number
- CA2112897A1 CA2112897A1 CA 2112897 CA2112897A CA2112897A1 CA 2112897 A1 CA2112897 A1 CA 2112897A1 CA 2112897 CA2112897 CA 2112897 CA 2112897 A CA2112897 A CA 2112897A CA 2112897 A1 CA2112897 A1 CA 2112897A1
- Authority
- CA
- Canada
- Prior art keywords
- embryo
- test
- yolk sac
- test substance
- circulation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Diabetes (AREA)
- Endocrinology (AREA)
- Gastroenterology & Hepatology (AREA)
- Rheumatology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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.
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
`~/0'~3/01272 21 i 2 8 9 7 PCI/CB92/01210 ` ~-~ "j ~ s~ BIOASSAY METHOD
This invention relates to bioassays for potentially biologically harmful substances.
One method used in carr,ying out a bioassay is to extract rat embr,yos and incubate them floating in a nutrient medium containing the test substance.
Under normal circumstances it is dif f icult to administer directly substances to living post-implantation embryo such as a rat embryo as currently used, due to the presence of the embr,yonic visceral yolk sac. The yolk sac contains enzymes which may metabolise the test substances SQ 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 involYing ~,isolated cell populations grown in tissue culture or ~ ~ :
This invention relates to bioassays for potentially biologically harmful substances.
One method used in carr,ying out a bioassay is to extract rat embr,yos and incubate them floating in a nutrient medium containing the test substance.
Under normal circumstances it is dif f icult to administer directly substances to living post-implantation embryo such as a rat embryo as currently used, due to the presence of the embr,yonic visceral yolk sac. The yolk sac contains enzymes which may metabolise the test substances SQ 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 involYing ~,isolated cell populations grown in tissue culture or ~ ~ :
2;:12,~!~7 PCtl6B 9 2 / O ~ 2 ~
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 .~ethod 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 circulatïon of an embryo having a yolk sac wherein said test reagents bypass the visceral yolk sac of the embryo by cannulation of the vitelline circulation with microcannulae having an internal tip diameter of between 1 and 2 ~m, so that the reagents are not modified ~y visceral yolX sac metabolism, culturing the embryo in culture medium and assessing embryonic development.
United Kingdom Patent Office cl IDCTITI~'E SHEEr PCT Internatlonal Application .~vv~ ~ I v -, 2 1 1 2 8 9 7 r~ D ~ ~ J U 1 ! 1 ~ :
-2 JUNE !99 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.
During cannulation the embryo may be placed in a well on a microscope sta~e. 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 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.
` ~ ~ SU8STITUTE SHEET
211'~8!37 WO 93/01272 P~/GB92/0121~, A~ $~ A`S ~ - 4 ~
S ~ ~ r~S .~
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 ~itelline circulation or chlorioallantoic blood vessels, and the eg~ 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.
....
2~ 128~
~093/01272 PCT/GB92/01210 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 ~e an easier task than performing multiple micro-cannulations.
The effects of the test substances may be determined by auantitation means which may comprise immunological, biochemical and morphometric assays.
The biochemical assays may comp-ise protein, DNA, DNA synthesis, protein synthesis and enzyme synthesis and the morphometric assays may comprise crown-rump measurements, somite number measurements, yolk sac diameter measurements and vitelline circulation assessment. The morphometric assays may additionally comprise established morphometric scoring assays.
The method may be used to assess factors which are toxic specifically to the de~eloping embryo, or to assess angiogenic and anti-angiogenic factors. The visceral yolk sac may also be bypassed by cannulation o~
211~89;7 WO93/01272 PCTtGB92J0121~ ;
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 ~ascular 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 ~itro 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 comprisins a micro-injection pump, a micro-manipulator, a heating stage and dissection head for a microscope, together, if desired, with a mucroscope.
One typical method for a bioassay according to the invention will now be described.
The method comprises injecting test su~stances and appropriate control su~stances into a post-implantation Wistar rat embryo between 9 and 13 days old, b~passing the visceral yolk sac of the embryo so that the substances are not modified by ~isceral yolk ~1128~7 V~93/01272 PCT/GB9V01210 sac metabolism, culturing the injected embryo in culture medium and quantitating subsequent emhryonic development.
The yolk sac is bypassed by micro-cannulation of the vitelline circulation. The cannula is inserted so tha~ 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 ~m, and preferably of 1.2 ~m. 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 pum? 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 lS 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.
wo 93,0l2~2l ~ 2 8 3 7 - 8 - PCT/GB92/012:0 At l0.5 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 synthetlc 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 in~olved in toxification/detoxification reactions. One such .
system is the P450 Cytochrome system.
DNA content may be assayed with 4'-6-diamidino-2-phenylindole (DAPI).
~:' 2~2~)7 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 emb~yo heart. The embryo is then homogenised and an acid-insoluble fraction obtained and subjected to li~uid scintillation spectrophotometry to assess the entire mass response in ~itro to the test substance.
A test kit for carrying out the ~ioassay comprises microcan~ulae, sterile dyed black agar blocks for holding the embryos for the micro-cannulation procedure, cul'ure medium sachets, a work sta~ion comprising a micro-injection pump, a micro-manipulato-and a microscope comprisLng a dissecting head and a heated stagej the heating being desirable for maintaining ~iability 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.
WO 93/01~72 2 1 1 2 ~ n 7 1 o PCl /GB92/01210 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 ~isceral yolk sac metabolism.
Embryos may be used which are not surrounded by a ~isceral 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 ~he need for micro-cannuiation 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.
~' ~
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 .~ethod 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 circulatïon of an embryo having a yolk sac wherein said test reagents bypass the visceral yolk sac of the embryo by cannulation of the vitelline circulation with microcannulae having an internal tip diameter of between 1 and 2 ~m, so that the reagents are not modified ~y visceral yolX sac metabolism, culturing the embryo in culture medium and assessing embryonic development.
United Kingdom Patent Office cl IDCTITI~'E SHEEr PCT Internatlonal Application .~vv~ ~ I v -, 2 1 1 2 8 9 7 r~ D ~ ~ J U 1 ! 1 ~ :
-2 JUNE !99 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.
During cannulation the embryo may be placed in a well on a microscope sta~e. 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 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.
` ~ ~ SU8STITUTE SHEET
211'~8!37 WO 93/01272 P~/GB92/0121~, A~ $~ A`S ~ - 4 ~
S ~ ~ r~S .~
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 ~itelline circulation or chlorioallantoic blood vessels, and the eg~ 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.
....
2~ 128~
~093/01272 PCT/GB92/01210 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 ~e an easier task than performing multiple micro-cannulations.
The effects of the test substances may be determined by auantitation means which may comprise immunological, biochemical and morphometric assays.
The biochemical assays may comp-ise protein, DNA, DNA synthesis, protein synthesis and enzyme synthesis and the morphometric assays may comprise crown-rump measurements, somite number measurements, yolk sac diameter measurements and vitelline circulation assessment. The morphometric assays may additionally comprise established morphometric scoring assays.
The method may be used to assess factors which are toxic specifically to the de~eloping embryo, or to assess angiogenic and anti-angiogenic factors. The visceral yolk sac may also be bypassed by cannulation o~
211~89;7 WO93/01272 PCTtGB92J0121~ ;
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 ~ascular 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 ~itro 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 comprisins a micro-injection pump, a micro-manipulator, a heating stage and dissection head for a microscope, together, if desired, with a mucroscope.
One typical method for a bioassay according to the invention will now be described.
The method comprises injecting test su~stances and appropriate control su~stances into a post-implantation Wistar rat embryo between 9 and 13 days old, b~passing the visceral yolk sac of the embryo so that the substances are not modified by ~isceral yolk ~1128~7 V~93/01272 PCT/GB9V01210 sac metabolism, culturing the injected embryo in culture medium and quantitating subsequent emhryonic development.
The yolk sac is bypassed by micro-cannulation of the vitelline circulation. The cannula is inserted so tha~ 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 ~m, and preferably of 1.2 ~m. 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 pum? 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 lS 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.
wo 93,0l2~2l ~ 2 8 3 7 - 8 - PCT/GB92/012:0 At l0.5 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 synthetlc 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 in~olved in toxification/detoxification reactions. One such .
system is the P450 Cytochrome system.
DNA content may be assayed with 4'-6-diamidino-2-phenylindole (DAPI).
~:' 2~2~)7 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 emb~yo heart. The embryo is then homogenised and an acid-insoluble fraction obtained and subjected to li~uid scintillation spectrophotometry to assess the entire mass response in ~itro to the test substance.
A test kit for carrying out the ~ioassay comprises microcan~ulae, sterile dyed black agar blocks for holding the embryos for the micro-cannulation procedure, cul'ure medium sachets, a work sta~ion comprising a micro-injection pump, a micro-manipulato-and a microscope comprisLng a dissecting head and a heated stagej the heating being desirable for maintaining ~iability 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.
WO 93/01~72 2 1 1 2 ~ n 7 1 o PCl /GB92/01210 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 ~isceral yolk sac metabolism.
Embryos may be used which are not surrounded by a ~isceral 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 ~he need for micro-cannuiation 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 (27)
1. A method for bioassay comprising passing test reagents into the circulation of an embryo having a yolk sac wherein said test reagents bypass the visceral yolk sac of the embryo by cannulation of the vitelline circulation with microcannulae having an internal tip diameter of between 1 and 2 µm, so that the reagents are not modified by visceral yolk sac 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 claim 1, in which, during cannulation, the embryo is placed in a well on a microscope stage.
PCT/GB92/01121?
PCT/GB92/01121?
7. A method according to claim 6, in which the well is formed from agar.
8. A method according to claim 7, in which the well is dyed to provide contrast when viewing the blood vessels beneath the yolk sac surface.
9. A method according to any one of claims 6 to 8, in which the well is heated.
10. A method according to any one of claims 1 to 9, in which the vitelline circulation is cannulated with a microcannula.
11. A method according to any one of claims 1 to 10, in which the microcannula is coated with a siliconising agent.
12. A method according to any one of claims 1 to 11, in which the microcannula is connected to an injection pump.
13. A method according to any one of claims 1 to 12, in which the microcannula is used to inject a test substance containing a fluorescent dye.
14. A method according to any one of claims 1 to 13, comprising injecting a total volume of less than or equal to one microlitre.
15. A method according to any one of claims 1 to 14, in which a test substance is injected into the embryo over the course of four minutes.
16. A method according to any one of claims 1 to 15, in which, where the embryo is a rat embryo, it is cultured in heat-inactivated rat serum containing an antibiotic.
17. A method according to any one of claims 1 to 15, in which the embryo is cultured in synthetic or semi-synthetic medium.
18. A method according to claim 1, utilising embryos which are not surrounded by a visceral yolk sac.
19. A method according to claim 18, utilising chicken embryos in fertilised eggs.
20. A method according to claim 18 or claim 19, in which the test reagent is administered by micro-cannulation directly into the extra-embryonic circulation.
21. A method according to claim 19, in which the egg contents are released from the shell into a plastic container and incubated therein.
22. A method according to claim 19, in which a window is cut into the shed to access the embryo.
23. A method according to claim 18, in which the test reagent is introduced into the albumen.
24. A method according to any one of claims 1 to 23, in which the test substance is added with a radiolabel.
25. A method according to claim 2 or claim 18, in which the test substance is injected into the embryo heart.
26. A method according to claim 25, 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.
27. 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.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB919114638A GB9114638D0 (en) | 1991-07-06 | 1991-07-06 | Bioassay method |
| GB9114638.1 | 1991-07-06 | ||
| GB919114955A GB9114955D0 (en) | 1991-07-11 | 1991-07-11 | Bioassay method |
| GB9114955.9 | 1991-07-11 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2112897A1 true CA2112897A1 (en) | 1993-01-21 |
Family
ID=26299196
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2112897 Abandoned CA2112897A1 (en) | 1991-07-06 | 1992-07-03 | Bioassay method |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0594660A1 (en) |
| JP (1) | JPH06509469A (en) |
| AU (1) | AU2230992A (en) |
| CA (1) | CA2112897A1 (en) |
| IE (1) | IE922172A1 (en) |
| WO (1) | WO1993001272A2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0716690A1 (en) * | 1993-08-30 | 1996-06-19 | Northwestern University | Rat pluripotent embryonic stem cells and method of obtaining and using same |
| DE19606207A1 (en) * | 1996-02-21 | 1997-08-28 | Univ Duesseldorf H Heine | Method for determining the phototoxicity and / or photosensitivity of substances or mixtures of substances and their use |
| JP2006262863A (en) * | 2005-03-25 | 2006-10-05 | Gifu Univ | Method for evaluating external stimulation |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU461120A1 (en) * | 1972-10-30 | 1975-02-25 | Харьковский Научно-Исследовательский Институт Микробиовакцин И Сывороток | The method of determining the biological activity of substances |
| DE3530336A1 (en) * | 1985-08-24 | 1987-02-26 | Wittmann J Dr | Process for culturing the poultry embryo |
-
1992
- 1992-07-03 IE IE922172A patent/IE922172A1/en not_active Application Discontinuation
- 1992-07-03 JP JP5502083A patent/JPH06509469A/en active Pending
- 1992-07-03 AU AU22309/92A patent/AU2230992A/en not_active Abandoned
- 1992-07-03 CA CA 2112897 patent/CA2112897A1/en not_active Abandoned
- 1992-07-03 EP EP92914060A patent/EP0594660A1/en not_active Withdrawn
- 1992-07-03 WO PCT/GB1992/001210 patent/WO1993001272A2/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| WO1993001272A2 (en) | 1993-01-21 |
| IE922172A1 (en) | 1993-01-13 |
| WO1993001272A3 (en) | 1993-03-18 |
| AU2230992A (en) | 1993-02-11 |
| JPH06509469A (en) | 1994-10-27 |
| EP0594660A1 (en) | 1994-05-04 |
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