Method for culturing bird embryos outside the eggshell

Abstract

To provide a method for culturing avian embryos outside the eggshell that can reliably achieve high embryo survival rates and low embryonic developmental abnormality rates, and that is relatively easy to perform, as well as a method for evaluating the toxicity of test substances using such a culture method. [Solution] The embryo and yolk are removed from the fertilized bird egg and transferred together with avian aqueous oocyte white into a roughly cylindrical well in a culture vessel. The opening of the well, which is filled with avian aqueous oocyte white, is then covered with a porous membrane, and the avian embryo is cultured by turning it over. Preferably, the cultured avian embryo is further cultured in a roughly cylindrical well in which a porous membrane is provided along the inner circumference, and the amount of avian aqueous oocyte white is adjusted so that there is a space between the well and the avian yolk below the opening.

Description

Technical Field

[0001] The present invention relates to a method for culturing avian embryos outside the eggshell and a method for evaluating the toxicity of a test substance using such a culturing method.

Background Art

[0002] Attempts to remove avian embryos from the eggshell and allow them to develop have been made for a long time. For example, a method of culturing embryos outside the eggshell by using a surrogate eggshell for the entire period from immediately after fertilization of a chicken embryo to hatching has been reported (Non-Patent Document 1). This surrogate eggshell culturing method divides the development from the 1-cell stage to hatching into three stages. Specifically, System I is used to culture embryos in the body of a female bird, System II is used to culture embryos for about three days from the blastoderm stage after egg laying to the embryonic formation stage, and System III is used to culture embryos from the embryonic development stage to hatching. Different systems are used for each stage, and the chicken embryos are transferred according to the progress of embryonic development. In addition, as a culturing method for System II, a method has been reported in which avian egg yolk containing an embryo is transferred to an artificial container of an egg type slightly larger than the volume of the avian egg yolk, filled with avian watery egg white, sealed with a hydrophobic fluororesin film, and cultured by rotation (Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The object of the present invention is to provide a method for culturing bird embryos outside the eggshell that can reproducibly achieve a high embryo survival rate and a low rate of embryonic developmental abnormalities, and that is relatively easy to perform, as well as a method for evaluating the toxicity of a test substance using such a culture method. [Means for solving the problem]

[0006] The inventors of this invention have been diligently conducting research to solve the above problems. In the process, they discovered that when the embryo and yolk are extracted from a fertilized bird egg, transferred together with avian aqueous oocyte white in a roughly cylindrical well in a culture vessel, and the opening of the well filled with avian aqueous oocyte white is covered with a porous membrane, and the avian embryo is cultured by turning the egg, the embryo survival rate is high and the rate of embryonic developmental abnormalities is low. Furthermore, they confirmed that when the avian embryo cultured by turning the egg is further cultured in a roughly cylindrical well with a porous membrane along the inner circumference and the amount of avian aqueous oocyte white adjusted so that there is a space between it and the avian yolk below the opening, the survival rate of the avian embryo can be effectively increased for at least the first two-thirds of the period from egg-laying to hatching (12 days after culture outside the eggshell in the case of quail embryos). This invention was completed based on these findings.

[0007] In other words, the present invention is as follows: [1] A method for culturing a bird embryo outside the eggshell, comprising the following steps (a) and (b). (a) A step of transferring the embryo and yolk of a bird fertilized egg and the aqueous oval white of a bird into one or more wells of a roughly cylindrical shape in a culture vessel; (b) A step of covering the opening of the well filled with avian aqueous egg white with a porous membrane and culturing the avian embryo by turning the egg; [2] The method according to [1] above, further comprising step (c) below. (c) A step of further culturing the avian embryo cultured by turning eggs in step (b) in one or more substantially cylindrical wells in a culture vessel in the presence of avian aqueous egg white, wherein the well has a porous membrane along its inner circumferential surface, there is a space between the opening of the well and the avian egg yolk, and the total culture period of steps (b) and (c) is 12 days or less; [3] The method according to [2] above, wherein the culture period in step (b) is 72 hours or less. [4] The method according to [2] or [3] above, wherein the total incubation period of steps (b) and (c) is 5 to 12 days. [5] A method for evaluating the toxicity of a test substance, comprising the following steps (a), (b), and (p), wherein in step (b), the test substance is exposed to a bird embryo before or during egg turning culture. (a) A step of transferring the embryo and yolk of a bird fertilized egg and the aqueous oval white of a bird into one or more wells of a roughly cylindrical shape in a culture vessel; (b) A step of covering the opening of the well filled with avian aqueous egg white with a porous membrane and culturing the avian embryo by turning it over; (p) A step of evaluating the toxicity of the test substance using abnormalities in bird embryos exposed to the test substance as an indicator; [6] A method for evaluating the toxicity of a test substance, comprising the following steps (a), (b), (c), and (p), wherein in step (b), the test substance is exposed to a bird embryo before or during egg turning culture, or in step (c), the test substance is exposed to a bird embryo before or during culture. (a) A step of transferring the embryo and yolk of a bird fertilized egg and the aqueous oval white of a bird into one or more wells of a roughly cylindrical shape in a culture vessel; (b) A step of covering the opening of the well filled with avian aqueous egg white with a porous membrane and culturing the avian embryo by turning it over; (c) A step of further culturing the avian embryo cultured by turning eggs in step (b) in one or more substantially cylindrical wells in a culture vessel in the presence of avian aqueous egg white, wherein the well has a porous membrane along its inner circumferential surface, there is a space between the opening of the well and the avian egg yolk, and the total culture period of steps (b) and (c) is 12 days or less; (p) A step of evaluating the toxicity of the test substance using abnormalities in bird embryos exposed to the test substance as an indicator; [7] The method according to [5] or [6] above, wherein the incubation period in step (b) is 72 hours or less. [8] The method according to [6] or [7] above, wherein the toxicity of the test substance is evaluated using abnormalities in bird embryos exposed to the test substance as an indicator 5 to 12 days after culturing in steps (b) and (c). [9] The method according to any one of [1] to [8] above, wherein the culture vessel has at least 12 wells.

[10] The method according to any of [1] to [9] above, wherein the bird is a quail. [Effects of the Invention]

[0008] The present invention provides a method for culturing avian embryos outside the eggshell, which can reproducibly achieve high embryo survival rates and low embryonic developmental abnormalities, and can be performed with relatively simple procedures. Therefore, this culture method allows for relatively simple and accurate evaluation of whether avian embryos have genetic diseases, using embryonic developmental abnormalities as an indicator. Furthermore, this culture method also allows for relatively simple and accurate evaluation of the toxicity of a test substance by exposing avian embryos to the test substance and using embryonic abnormalities as an indicator. Thus, it is useful as an alternative to animal testing for evaluating the toxicity of candidate active ingredients in pharmaceuticals, quasi-drugs, functional foods, food additives, pesticides, chemicals, etc. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1A is a schematic diagram of one embodiment of step (b) described above. Figure 1B is a schematic diagram of one embodiment of step (c) described above. [Figure 2]Figure 2A shows microscopic images of quail embryos cultured outside the eggshell in 12 wells on days 2.5, 3.0, 3.5, 4.0, and 4.5. Figure 2B shows the results of analyzing the survival rate of the quail embryos cultured outside the eggshell in 12 wells on days 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0. [Figure 3] This figure shows the results of analyzing the incidence of morphological abnormalities in the somites (Figure 3A), brain (Figure 3B), and heart (Figure 3C) of quail embryos 40 hours after extrashell culture, following the application of sodium valproate (VPA) at various concentrations (0 [mol / L], 0.1 [mol / L], 0.2 [mol / L], or 0.3 [mol / L]) to the surface of the embryos. The asterisk (*) in the figure indicates a statistically significant difference (p<0.05) compared to the result when VPA was 0 (mol / L). [Figure 4] This figure shows the results of analyzing the body length (Figure 4A) and interocular distance (Figure 4B) of quail embryos 40 hours after being cultured outside the eggshell with VPA at various concentrations (0 [mol / L], 0.1 [mol / L], 0.2 [mol / L], or 0.3 [mol / L]) dropped onto the surface of the embryos in 12 wells. The "**" in the figure indicates a statistically significant difference (p<0.01) compared to the result when the VPA concentration was 0 (mol / L). [Figure 5] This figure shows the results of analyzing the incidence of morphological abnormalities in the brain (Figure 5A) and heart (Figure 5B) of quail embryos after egg-turning culture in 12 wells, with various concentrations of VPA (0 [mol / L], 0.1 [mol / L], 0.2 [mol / L], or 0.3 [mol / L]) added dropwise at 24 hours, followed by further egg-turning culture, and analysis at 40 hours. The asterisk (*) in the figure indicates a statistically significant difference (p<0.05) compared to the result when VPA was 0 (mol / L). [Figure 6]After culturing quail embryos outside the eggshell in a 12-well plate and then performing subculture, at the 24th hour, VPA at various concentrations (0 [mol / L], 0.1 [mol / L], 0.2 [mol / L], or 0.3 [mol / L]) was dropped, followed by further subculture. The figure shows the results of analyzing the body length (Figure 6A) and interocular distance (Figure 6B) of the embryos at the 40th hour. In the figure, "**" indicates that there is a statistically significant difference (p < 0.01) compared to the results when VPA is 0 (mol / L). [Figure 7] At 48 - 55 hours after culturing quail embryos outside the eggshell in a 12-well plate, the embryos were transferred to a 12-well plate with a porous membrane provided along the inner circumferential surface and further cultured. The microscopic images at the 5th day (Figure 7A) and the microscopic images at the 8th day (Figure 7B - D) are shown.

Embodiments for Carrying out the Invention

[0010] <The Present Culture Method> As a method for culturing avian embryos outside the eggshell according to the present invention, a step (a) of transferring an avian embryo and avian egg yolk in a fertilized avian egg and avian albuminous egg white into one or more substantially cylindrical wells in a culture container; and a step (b) of covering the opening of the well filled with avian albuminous egg white with a porous membrane and performing subculture of the avian embryo. Without particular limitation as long as it is a method for culturing the avian embryo outside the eggshell (more specifically, in a state without an eggshell), and further culturing the avian embryo subcultured in step (b) in one or more substantially cylindrical wells in a culture container in the presence of avian albuminous egg white, wherein the well is provided with a porous membrane along the inner circumferential surface of the well, there is a space (air layer) between the opening of the well and the avian egg yolk, and the total of the culture periods of step (b) and step (c) is within 12 days. It is preferable to further include the step (c). The culture in such step (c) may be a culture in a stationary state or a culture in a slightly shaken state (shaking culture).

[0011] <The Present Evaluation Method 1> As one aspect of the method for evaluating the toxicity of a test substance of the present invention, it sequentially includes the above step (a); the above step (b); and a step (p) of evaluating the toxicity of the test substance using the abnormalities of avian embryos exposed to the test substance. In the above step (b), there is no particular limitation as long as the test substance is exposed to avian embryos before (preferably, immediately before) or during transfer egg culture. (In this specification, it may be referred to as "this evaluation method 1").

[0012] <This evaluation method 2> As another aspect of the method for evaluating the toxicity of a test substance of the present invention, it sequentially includes the above step (a); the above step (b); the above (c); and the above step (p). In the above step (b), the test substance is exposed to avian embryos before or during transfer egg culture, or in the above step (c), the test substance is exposed to avian embryos before (preferably, immediately before) or during culture. There is no particular limitation as long as it is a method for evaluating the toxicity of the test substance (in this specification, it may be referred to as "this evaluation method 2"). In addition, this evaluation method 1 and this evaluation method 2 may be collectively referred to as "this evaluation method" in this specification, and this culture method, this evaluation method 1, and this evaluation method 2 may be collectively referred to as "this method" in this specification.

[0013] <Explanation of terms> As the "avian fertilized egg" in the above step (a), it is preferably an avian fertilized egg taken out from the eggshell of an egg immediately after laying (for example, within 0 to 12 hours, 0 to 6 hours, 0 to 3 hours, 0 to 1 hour, 0 to 40 minutes, 0 to 20 minutes, 0 to 10 minutes, 0 to 6 minutes, 0 to 3 minutes, etc. after laying) by a bird, or an avian fertilized egg taken out from the eggshell of an egg immediately after laying that has been stored at a temperature at which embryonic development hardly progresses (for example, within the range of 10 to 14°C).

[0014] In this specification, "birds" are not particularly limited, and examples include chickens, quails, turkeys, ducks, etc. Since the effects are demonstrated in the following examples, quails can be preferably exemplified.

[0015] In this method, "watery egg white" refers to the internal and / or external watery egg white contained in the egg white, and is different from thick egg white. Avian watery egg white can be collected, for example, from unfertilized or fertilized eggs of birds according to standard methods. The avian watery egg white may be derived from a different species of bird than the fertilized avian egg used in this method, but it is preferable to use watery egg white derived from the same species of bird as the fertilized avian egg used in this method.

[0016] The "culture vessel" used in this method may have one or more wells that are roughly cylindrical (not egg-shaped [elliptical]), and it is preferable that it be equipped with a lid to cover the top of the culture vessel to prevent contamination and evaporation of moisture from the wells. From the viewpoint of culturing multiple bird fertilized eggs simultaneously on the same culture vessel, it is preferable that the culture vessel has multiple (two or more) wells. Examples of lower limits for the number of wells include 2 or more, 4 or more, 6 or more, 8 or more, 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, etc. Examples of upper limits for the number of wells include 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, etc. These lower and upper limits can be arbitrarily combined. The culture vessel described above is preferably one that has 12 or more wells (at least 12 wells).

[0017] In this specification, "egg turning culture" means culturing a fertilized egg while it is rotating in a circular motion (rotation) at a constant angle (egg turning angle) in both forward and reverse directions.

[0018] In this specification, the "Hamburger-Hamilton developmental stages" (sometimes referred to as "HH stages") were originally a classification of chicken embryo developmental stages based on the morphological characteristics of the embryo (see "Journal of Morphology, Vol. 88, 49-92, (1951)"). However, since the development and morphogenesis of avian embryos other than chickens progress similarly to that of chicken embryos, HH stages have also been defined for the development of avian embryos other than chickens (e.g., quail) based on the morphology of the embryo at each stage (e.g., number of somites, head, limb buds, etc.) (see, for example, "Journal of Anatomy, Vol. 216, 3-15, (2010)").

[0019] In this method, the temperature used when culturing avian fertilized eggs is, for example, in the range of 37 to 40°C (preferably 37.5 to 38.5°C), and the humidity used when culturing avian fertilized eggs is, for example, 50% or more (preferably 80% or more).

[0020] In step (b) above, "the well filled with avian aqueous egg white" means a state in which the liquid level of avian aqueous egg white in the well is at a height such that the porous membrane and the avian aqueous egg white come into contact when the opening of the well is covered with the porous membrane.

[0021] In this specification, "porous membrane" means a membrane that has multiple pores on its surface (top and bottom surface) that enable gas exchange, and a porous membrane may further have functions such as low friction, non-stick properties, and drug resistance.

[0022] The average pore diameter on the surface of the porous film described above is not particularly limited. Examples of lower limits include 0.001 μm or more, 0.003 μm or more, 0.006 μm or more, 0.01 μm or more, 0.03 μm or more, 0.06 μm or more, 0.1 μm or more, 0.3 μm or more, 0.45 μm or more, 1 μm or more, etc. Examples of upper limits include 1000 μm or less, 600 μm or less, 300 μm or less, 200 μm or less, 100 μm or less, 60 μm or less, 30 μm or less, 10 μm or less, 6 μm or less, 3 μm or less, 2 μm or less, 1 μm or less, etc. These lower and upper limits can be combined in any way.

[0023] The substrate for the porous membrane described above is not particularly limited and can include, for example, polytetrafluoroethylene (PTFE), polydimethylsiloxane (PDMS), polycarbonate (PC), polyester (PET), polystyrene (PS), TAC (triacetylcellulose), polyketone (PK), nylon (Ny), low-density polyethylene (LDPE), medium-density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone (PES), polyethylene naphthalate, polypropylene, acrylic, and the like.

[0024] In step (c) above, "a porous membrane is provided along the inner circumferential surface of the well" means a state in which a porous membrane is provided along the inner circumferential surface of a substantially cylindrical well, and the porous membrane may be in contact with part or all of the inner circumferential surface of the well, or it may not be in contact with the inner circumferential surface of the well. Furthermore, it is preferable that the height of the porous membrane is about the same as or greater than the height of the upper tip of the bird egg yolk when placed in the well, and about the same as or less than the height of the well.

[0025] The total culture period for steps (b) and (c) above should be within 12 days. The lower limit can be, for example, 1 day, 2 days, 3 days, 4 days, or 5 days, and the upper limit can be, for example, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, or 6 days. These lower and upper limits can be combined in any way. The total culture period for steps (b) and (c) above is preferably within the range of 5 to 12 days.

[0026] The above-mentioned test substances can be any substance that can be subject to toxicity evaluation, and examples include candidate active ingredients in pharmaceuticals, quasi-drugs, functional foods, food additives, pesticides, chemicals, etc. (e.g., peptides, polypeptides, antibodies, nucleotides, polynucleotides, sugars, lipids, organic compounds, inorganic compounds, etc.), pathogens, radioactive materials, etc.

[0027] In step (p) above, "abnormalities in avian embryos exposed to the test substance" refers to any abnormalities observed compared to normally developing avian embryos (specifically, avian embryos not exposed to the test substance). Examples include: decreased survival rate compared to normally developing avian embryos (specifically, avian embryos not exposed to the test substance); delayed progression of developmental stages; increased incidence of morphological abnormalities in tissues or organs such as somites, brain, and heart of the embryo; shortening of body length and / or interorbital distance; etc.

[0028] <Process (a)> In step (a) above, the contents of the avian fertilized egg to be transferred into the well only need to include at least the embryo and yolk. Other contents of the avian fertilized egg (e.g., egg white [thick egg white and / or watery egg white]) may be transferred into the well together with the embryo and yolk, or they may be removed without being transferred into the well. Since the embryo in an avian fertilized egg is located on the surface of the yolk, the embryo can be transferred into the well together with the yolk by transferring the yolk as is.

[0029] <Process (b)> In step (b) above, egg turning culture of bird embryos can be performed using an incubator equipped with an automatic egg turning device (for example, the P-008 model manufactured by Showa Frankie Co., Ltd.) at any egg turning interval (for example, 0 minutes [continuous turning], within the range of 0 to 5 minutes, within the range of 0 to 15 minutes, within the range of 15 to 30 minutes, within the range of 30 to 60 minutes, etc.) and any egg turning angle (for example, within the range of 30 to 60 degrees, within the range of 60 to 90 degrees, within the range of 80 to 100 degrees, 90 degrees, etc.).

[0030] In step (b) above, the culture period for egg turning culture of avian embryos may be any period after transferring the avian embryos to the wells. Examples of lower limits include 0.5 days (12 hours) or more, 1 day (24 hours) or more, 1.5 days (36 hours) or more, 2 days (48 hours) or more, etc. Examples of upper limits include 4 days (96 hours) or less (up to HH stage 24 if the bird is a quail), 3.5 days (84 hours) or less (up to HH stage 22 if the bird is a quail), 3.0 days (72 hours) or less (up to HH stage 21 if the bird is a quail), etc. These lower and upper limits can be combined in any way. In step (b) above, a culture period of 3.0 days (72 hours) or less is preferred for egg turning culture of avian embryos.

[0031] In step (b) above, the egg-turning culture of avian embryos may be performed with a lid covering the culture container, or without a lid. Figure 1A schematically shows one embodiment in which the egg-turning culture in step (b) above is performed with a lid covering the culture container, from day 0 to day 3 after extra-shell culture.

[0032] <Process (c)> In step (c) above, the method for further culturing the avian embryo cultured by turning the eggs in step (b) is to culture the avian embryo cultured by turning the eggs in step (b) together with the yolk in a substantially cylindrical well in which a porous membrane is provided along the inner circumference and the amount of avian aqueous oocyte white is adjusted so that there is a space between the avian yolk below the opening. For example, a porous membrane is provided along the inner circumference of the well after turning the eggs, and a portion of the avian aqueous oocyte white present in the well is removed so that there is a space between the opening of the well and the avian yolk, and the avian embryo is cultured in the same well; a porous membrane is provided along the inner circumference, A method may be described as follows: a culture vessel having one or more wells of a roughly cylindrical shape is newly prepared, the bird embryos and yolks cultured by turning eggs in step (b) are transferred into the wells, and if necessary, bird aqueous egg whites are transferred, and the amount of bird aqueous egg whites is adjusted so that a space is created between the opening of the well and the bird egg yolks, and then the bird embryos are cultured; or, in step (b), the bird embryos are cultured by turning eggs in a roughly cylindrical well with a porous membrane pre-prepared along the inner circumference, and then a portion of the bird aqueous egg whites in the well is removed so that a space is created between the opening of the well and the bird egg yolks, and the bird embryos are cultured in the same well.

[0033] In step (c) above, the culture period for the bird embryo should be such that the sum of the culture period in step (b) and the culture period in step (c) is within 12 days (288 hours) (if the bird is a quail, up to HH stage 42 [within the period not applicable to animal experiments]). For example, if the culture period in step (b) is within 3.0 days (72 hours), the culture period in step (c) should be within 9 days (216 hours); if the culture period in step (b) is within 3.5 days (84 hours), the culture period in step (c) should be within 8.5 days (204 hours); and if the culture period in step (b) is within 4 days (96 hours), the culture period in step (c) should be within 8 days (192 hours).

[0034] In step (c) above, it is preferable to cover the top of the wells with a porous membrane or a lid of the culture vessel to prevent contamination and evaporation of water from the wells. Figure 1B schematically shows one embodiment in which the culture in step (c) above is carried out from day 2 to day 12 after extra-eggshell culture with the culture vessel covered with a lid.

[0035] In step (b) of the evaluation method described above, a method for exposing bird embryos to the test substance before or during egg turning culture, and in step (c) of evaluation method 2 described above, a method for exposing bird embryos to the test substance before or during culture, include, for example, dropping the test substance onto the surface of the bird embryo to directly expose the bird embryo to the test substance; or indirectly exposing the bird embryo present in the egg yolk to the test substance by injecting it into the egg yolk or by dissolving the test substance in the bird aqueous egg white in the well; and so on.

[0036] In step (b) of the evaluation method described above, the timing of exposure of the avian embryos during egg-turning culture to the test substance can be any time after the start of egg-turning culture. Examples of lower limits include 0.5 days (12 hours) or later, 1 day (24 hours) or later, 1.5 days (36 hours) or later, 2 days (48 hours) or later, 2.5 days (60 hours) or later, etc. Examples of upper limits include within 3.0 days (72 hours) after the start of egg-turning culture, within 2.5 days (60 hours) or later, within 2 days (48 hours) or within 1.5 days (36 hours), etc. These lower and upper limits can be combined in any way.

[0037] In step (c) of evaluation method 2, the timing of exposure of the cultured bird embryo to the test substance can be any time after the start of culture, and the lower limit is, for example, 0.5 days (12 hours) or later, 1 day (24 hours) or later, 1.5 days (36 hours) or later, 2 days (48 hours) or later, 2.5 days (60 hours) or later, 3.0 days (72 hours) or later, 3.5 days (84 hours) or later, 4.0 days (96 hours) or later, 4.5 days (108 hours) or later, 5.0 days (120 hours) or later, 5.5 days (132 hours) or later, 6.0 days (144 hours) or later, 6.5 days (156 hours) or later, and 7.0 days (168 hours) or later. The upper limits can be set as follows: for example, within 8.0 days (192 hours) after the start of culture, within 7.5 days (180 hours), within 7.0 days (168 hours), within 6.5 days (156 hours), within 6.0 days (144 hours), within 5.5 days (132 hours), within 5.0 days (120 hours), within 4.5 days (108 hours), within 4.0 days (96 hours), within 3.5 days (84 hours), within 3.0 days (72 hours), within 2.5 days (60 hours), within 2 days (48 hours), within 1.5 days (36 hours), etc. These lower and upper limits can be combined arbitrarily.

[0038] <Process (p)> In step (p) described above, a method for evaluating the toxicity of the test substance using abnormalities in bird embryos exposed to the test substance as an indicator is as follows: if the bird embryos exposed to the test substance show abnormalities, the test substance can be evaluated as toxic; and if the bird embryos exposed to the test substance do not show abnormalities, the test substance can be evaluated as not toxic.

[0039] The timing for performing step (p) in evaluation method 1 can be any time after exposure of the test substance to the bird embryo. Examples of lower limits include 0.5 days (12 hours) or later, 1 day (24 hours) or later, 1.5 days (36 hours) or later, 2 days (48 hours) or later, 2.5 days (60 hours) or later, etc. Examples of upper limits include within 3.0 days (72 hours), within 2.5 days (60 hours), within 2 days (48 hours), within 1.5 days (36 hours), etc. These lower and upper limits can be combined in any way.

[0040] The timing for performing step (p) in evaluation method 2 can be any time after exposure of the test substance to the bird embryo, and the lower limit can be, for example, 0.5 days (12 hours) or later, 1 day (24 hours) or later, 1.5 days (36 hours) or later, 2 days (48 hours) or later, 2.5 days (60 hours) or later, 3.0 days (72 hours) or later, 3.5 days (84 hours) or later, 4.0 days (96 hours) or later, 4.5 days (108 hours) or later, 5.0 days (120 hours) or later, 5.5 days (132 hours) or later, 6.0 days (144 hours) or later, 6.5 days (156 hours) or later, 7.0 days (168 hours) or later, etc. The upper limits can be listed as follows: for example, within 8.0 days (192 hours), within 7.5 days (180 hours), within 7.0 days (168 hours), within 6.5 days (156 hours), within 6.0 days (144 hours), within 5.5 days (132 hours), within 5.0 days (120 hours), within 4.5 days (108 hours), within 4.0 days (96 hours), within 3.5 days (84 hours), within 3.0 days (72 hours), within 2.5 days (60 hours), within 2 days (48 hours), within 1.5 days (36 hours), etc. These lower and upper limits can be combined arbitrarily.

[0041] The present invention will be described more specifically below with reference to examples, but the technical scope of the present invention is not limited to these examples. In the following examples, 8.0 to 9.0 g fertilized quail eggs laid immediately after spawning were used as fertilized quail eggs. Watery egg white was collected from fertilized quail eggs by a standard method and stored under refrigeration. [Examples]

[0042] [Example 1] Examination of culture conditions for quail embryos up to day 3 after extrashell culture. The culture conditions for quail embryos up to day 3 (72 hours) after extra-shell culture were investigated. In the case of egg-turning culture of quail embryos using an incubator, unless otherwise specified, continuous turning was performed with no turning interval and a turning angle of 90 degrees. Unless otherwise specified, a 12-well multi-well plate was used for the culture vessel. Unless otherwise specified, a PTFE membrane with a pore size of 0.45 μm was used.

[0043] 1.Material 1-1 Culture container As culture vessels, 12-well multiwell plates (cylindrical wells with a diameter of 21.4-22.0 mm and a height of 17 mm; VTC-P12; manufactured by Bioremo) or 6-well multiwell plates (cylindrical wells with a diameter of 34.8-35.5 mm and a height of 17.5 mm; VTC-P6; manufactured by Bioremo) were used.

[0044] 1-2 Porous membrane As porous membranes, we used polytetrafluoroethylene (PTFE) membranes with four different pore sizes (0.1 μm, 0.2 μm, 0.45 μm, or 1.0 μm) (WP-010, WP-020, WP-045, and WP-100 Poreflon membranes, respectively) (manufactured by Sumitomo Electric Fine Polymer Co., Ltd.), or polydimethylsiloxane (PDMS) sheets with two different pore sizes (100 μm or 200 μm) (manufactured by CScrie Co., Ltd.).

[0045] 2. Method Quail embryos were cultured outside the eggshell according to the following procedure [1] to [4]. [1] The eggshells of fertilized quail eggs, which had been disinfected with Osban disinfectant, were cracked open, and their contents were transferred to a petri dish (6 cm dish), after which the thick egg white was removed. [2] After adding watery oocyte white to each well of the culture vessel to a height of about 1 / 3, the yolk containing the embryo, from which the thick oocyte white had been removed, was transferred to each well. [3] After filling the wells with watery egg white up to the openings, the porous membrane was placed over all the wells and then sealed. [4] Using an incubator (P-008 model, manufactured by Showa Franki Co., Ltd.), the eggs were cultured for 3 days (72 hours) with egg turning at a temperature of 37.8 degrees Celsius, humidity of 81-89%, no interval between egg turning (continuous turning), and an egg turning angle of 90 degrees. The survival rate (embryonic survival rate) and embryonic developmental abnormality rate of quail embryos were calculated on the 3rd day after culture outside the eggshell.

[0046] 3.Results Regarding the egg turning interval during quail embryo culture, the conditions shown in Table 1 were investigated. The results showed that when embryos were cultured under conditions of static incubation without egg turning, the embryo survival rate was significantly lower compared to the case with no egg turning interval (continuous turning), and the rate of embryonic developmental abnormalities was significantly higher compared to the case with no egg turning interval (continuous turning) (see Table 1). On the other hand, with three different egg turning intervals (15 minutes, 30 minutes, or 60 minutes), the embryo survival rate was similar to the case with no egg turning interval (continuous turning), and although there was a slight increase in the rate of embryonic developmental abnormalities as the egg turning interval lengthened, there was no significant difference compared to the case with no egg turning interval (continuous turning) (see Table 1).

[0047] Furthermore, regarding the egg turning angle during quail embryo culture, the conditions shown in Table 2 were examined. The results showed that when cultured under conditions of an egg turning angle of 30 degrees or 60 degrees, the embryo development rate decreased slightly compared to when the egg turning angle was 90 degrees, and the embryo development abnormality rate increased slightly compared to when the egg turning angle was 90 degrees, but no significant difference was observed (see Table 2). These results indicate that quail embryos can be cultured with a high survival rate and low rate of embryonic developmental abnormalities for at least 3 days (72 hours) by rotating them at any desired angle. At 72 hours after extrashell culture, the quail embryos were in HH stage 20-21 (approximately 10% were in HH stage 20 and approximately 90% were in HH stage 21).

[0048] [Table 1] In the table, "Embryo Development Abnormalities" refers to embryos in which abnormalities or death were observed (the same applies to the following tables). In the table, "*" indicates a statistically significant difference compared to "Continuous Ovulation Turning" (p<0.05).

[0049] [Table 2]

[0050] Next, when culturing quail embryos by turning the eggs, the conditions for the type of porous membrane covering the entire well were investigated. The results showed that regardless of whether PTFE membranes with four different pore sizes (0.1 μm, 0.2 μm, 0.45 μm, or 1.0 μm) or PDMS sheets with two different pore sizes (100 μm or 200 μm) were used, the embryo survival rate was high and the rate of embryonic developmental abnormalities was low, so no significant difference was observed depending on the type of porous membrane (see Tables 3 and 4). These results indicate that the type of porous membrane covering the entire well during quail embryo culture does not adversely affect embryo survival rate or the incidence of embryonic abnormalities.

[0051] [Table 3]

[0052] [Table 4]

[0053] Next, we changed the culture vessel (12-well multiwell plate) to a 6-well multiwell plate and investigated the conditions under which the thickened egg white was not removed from the quail embryos. As a result, when quail embryos were cultured using a 6-well multiwell plate, and when quail embryos were cultured without removing the thickened egg white, the embryo survival rate and the rate of embryonic developmental abnormalities were similarly maintained (see Table 5).

[0054] These results indicate that, regardless of whether or not the thick egg white is removed, culturing quail embryos in cylindrical wells with a diameter of at least 21.4–35.5 mm and a height of approximately 17–17.5 mm can achieve high embryo survival rates and low rates of embryonic developmental abnormalities.

[0055] [Table 5]

[0056] Furthermore, we investigated whether quail embryos could be cultured beyond the third day after extrashell culture. As a result, while quail embryos maintained a high survival rate until days 3.5 to 4.0, the survival rate rapidly declined thereafter, and 0% (0 / 24) of embryos were found to be alive on day 5 (see Figure 2). These results indicate that high embryo survival rates can be achieved for quail embryos up to 3.5 to 4.0 days after egg-turning culture outside the eggshell. The reason for the rapid decline in quail embryo survival rates after 4.0 days of culture outside the eggshell may be that, as the vascular system develops, it comes into contact not only with the porous membrane but also with the plastic of the well, potentially increasing the proportion of quail embryos that die.

[0057] [Example 2] Toxicity evaluation of a test substance using a culture method up to day 3 after extrashell culture of quail embryos. The toxicity of the test substance was evaluated using a culture method up to day 3 (72 hours) after extrashell culture of quail embryos. 1.Material 1-1 Culture container A 12-well multiwell plate (cylindrical wells with a diameter of 21.4-22.0 mm and a height of 17 mm; VTC-P12; manufactured by Bioremo) was used as the culture vessel.

[0058] 1-2 Porous membrane As the porous membrane, a PTFE membrane with a pore size of 0.45 μm (WP-045-80 Poreflon membrane) (manufactured by Sumitomo Electric Fine Polymer Co., Ltd.) was used.

[0059] 2. Method The toxicity of the test substance (chemical substance) was evaluated using a method for culturing quail embryos outside the eggshell, following the procedures [1] to [4] below. [1] The eggshells of fertilized quail eggs, which had been disinfected with Osban disinfectant, were cracked open, and their contents were transferred to a petri dish (6 cm dish), after which the thick egg white was removed. [2] After adding watery oocyte white to each well of the culture vessel to a height of about 1 / 3, the yolk containing the embryo, from which the thick oocyte white had been removed, was transferred to each well. [3] After filling the wells with aqueous egg white up to the opening, VPA (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) of various concentrations (0 [mol / L], 0.1 [mol / L], 0.2 [mol / L], 0.3 [mol / L], 0.4 [mol / L], or 0.5 [mol / L]) was dropped onto the surface of the quail embryos, and porous membranes were placed over all the wells and sealed. [4] Using an incubator (P-008 model, manufactured by Showa Franki Co., Ltd.), embryos were cultured by turning at a temperature of 37.8 degrees Celsius, a humidity of 81-89%, with no interval between turning (continuous turning), and a turning angle of 90 degrees. Embryo growth at 32 hours after extra-shell culture (see Table 6) and at 72 hours after extra-shell culture (see Table 7) was observed. At 40 hours after extra-shell culture, the incidence of morphological abnormalities in the somites, brain, and heart of embryos that had reached the standard developmental stage was calculated (see Figure 3), and body length and interocular distance were measured (see Figure 4).

[0060] 3.Results When quail embryos were cultured in the presence of VPA, the rate of normal development decreased, the rate of developmental failure and death increased in a VPA concentration-dependent manner compared to quail embryos cultured in the absence of VPA (see Tables 6 and 7), the incidence of morphological abnormalities in the somites, brain, and heart of the embryo increased (see Figure 3), and the body length and interorbital distance decreased (see Figure 4).

[0061] In addition, experiments were similarly conducted in which VPA at various concentrations (0 [mol / L], 0.1 [mol / L], 0.2 [mol / L], or 0.3 [mol / L]) was dropped onto the surface of quail embryos 24 hours after eggshell culture, instead of at 0 hours after eggshell culture, and the embryos were cultured for 40 hours. At 40 hours after eggshell culture, embryonic growth was observed (see Table 8), the incidence of morphological abnormalities in the brain and heart of embryos that had reached the standard developmental stage was calculated (see Figure 5), and body length and interocular distance were measured (see Figure 6).

[0062] As a result, when VPA was added to quail embryos 24 hours after extrashell culture, the rate of normal development decreased, the rate of developmental failure and death increased in a VPA concentration-dependent manner compared to quail embryos that were not added with VPA (see Table 8), the incidence of morphological abnormalities in the brain and heart of the embryos increased (see Figure 5), and body length and interocular distance decreased (see Figure 6).

[0063] These results indicate that when the test substance is dropped onto quail embryos outside the eggshell and the culture method described in this study is performed, the toxicity of the test substance can be evaluated using embryonic abnormalities such as a decrease in the rate of normal embryonic development, an increase in the rate of developmental failure, an increase in the rate of death, an increase in the incidence of morphological abnormalities in somites, brain, and heart, and shortening of body length and interorbital distance as indicators. At 32 hours after culture outside the eggshell, the quail embryos were in HH stage 7-9 (14% were in HH stage 7, 45% in HH stage 8, and 41% in HH stage 9), and at 72 hours after culture outside the eggshell, the quail embryos were in HH stage 19-21 (9% were in HH stage 19, 13% in HH stage 20, and 78% in HH stage 21).

[0064] [Table 6] In the table, "standard development" means that the quail embryo developed within the range of developmental stages (standard developmental stages) when cultured outside the eggshell in the absence of VPA (with PBS added) (the same applies to the following tables). In the table, "developmental delay" means that a morphological abnormality was observed that was delayed or severe compared to the standard developmental stage (the same applies in the following tables).

[0065] [Table 7]

[0066] [Table 8]

[0067] [Example 3] Investigation of a method for culturing quail embryos outside the eggshell for a longer period of time. Based on the results of Example 1, we investigated methods for culturing quail embryos outside the eggshell for a longer period of time. 1.Material 1-1 Culture container A 12-well multiwell plate (cylindrical wells with a diameter of 21.4-22.0 mm and a height of 17 mm; VTC-P12; manufactured by Bioremo) was used as the culture vessel.

[0068] 1-2 Porous membrane As the porous membrane, a PTFE membrane with a pore size of 0.45 μm (WP-045-80 Poreflon membrane) (manufactured by Sumitomo Electric Fine Polymer Co., Ltd.) was used.

[0069] 2. Method Quail embryos were cultured outside the eggshell according to the following procedure [1] to [6]. [1] The eggshells of fertilized quail eggs, which had been disinfected with Osban disinfectant, were cracked open, and their contents were transferred to a petri dish (6 cm dish), after which the thick egg white was removed. [2] After adding watery oocyte white to each well of the culture vessel to a height of about 1 / 3, the yolk containing the embryo, from which the thick oocyte white had been removed, was transferred to each well. [3] After filling the wells with watery egg white up to the openings, the porous membrane was placed over all the wells and then sealed. [4] Using an incubator (P-008 model, manufactured by Showa Franki Co., Ltd.), the eggs were cultured by turning them at a temperature of 37.8 degrees Celsius, a humidity of 81-89%, with no interval between turning (continuous turning), and a turning angle of 90 degrees. After 48-55 hours, a culture vessel was prepared with wells having a porous membrane along the inner circumference, and the yolk containing the embryo and the watery oocyte white were transferred to each well. [5] After adjusting the amount of watery egg white so that there is space below the opening of the well, the lid was put on. [6] Using an incubator (P-008 model, manufactured by Showa Franki Co., Ltd.), the quail embryos were cultured at a temperature of 37.8 degrees Celsius and a humidity of 81-89% under static conditions for 12 days, and the survival rate of the quail embryos was analyzed.

[0070] 3.Results When quail embryos were cultured outside the eggshell after being turned, and then transferred to wells with a porous membrane along the inner circumference at 48-55 hours for further culture, 75% (18 / 24) of the embryos survived on day 5 after culture outside the eggshell (corresponding to HH stages 26-27) (see Figure 7A), 66.7% (16 / 24) survived on day 8 after culture outside the eggshell (corresponding to HH stages 35-36) (see Figure 7B), and 42% (10 / 24) survived on day 12 after culture outside the eggshell (corresponding to HH stage 42). Furthermore, no embryonic developmental abnormalities were observed in these surviving embryos.

[0071] These results indicate that culturing quail embryos that have been turned and cultured to a predetermined developmental stage in a roughly cylindrical well with a porous membrane along the inner circumference and an adjusted amount of quail watery egg white to create a space between the quail yolk below the opening effectively increases the survival rate of quail embryos for at least 12 days after extrashell culture. Furthermore, it is shown that contact of the porous membrane with the developing vascular system is important for the survival of quail embryos. Separation of quail fingers is observed on day 8 after extrashell culture (see Figures 7C and 7D), suggesting that toxicity evaluation of the test substance can be performed using the presence or absence of limb bud and finger morphological abnormalities as indicators by culturing extrashell culture for up to day 8. [Industrial applicability]

[0072] This invention contributes to an alternative to animal testing for evaluating the toxicity of candidate active ingredients in pharmaceuticals, quasi-drugs, functional foods, food additives, pesticides, chemicals, and the like.

Claims

1. A method for culturing bird embryos outside the eggshell, comprising the following steps (a) and (b). (a) A step of transferring the embryo and yolk of a bird fertilized egg and the aqueous oval white of a bird into one or more wells of a roughly cylindrical shape in a culture vessel; (b) A step of covering the opening of the well filled with avian aqueous egg white with a porous membrane and culturing the avian embryo by turning the egg;

2. The method according to claim 1, further comprising step (c) below. (c) A step of further culturing the avian embryo cultured by turning eggs in step (b) in one or more substantially cylindrical wells in a culture vessel in the presence of avian aqueous egg white, wherein the well has a porous membrane along its inner circumferential surface, there is a space between the opening of the well and the avian egg yolk, and the total culture period of steps (b) and (c) is 12 days or less;

3. The method according to claim 2, wherein the culture period in step (b) is 72 hours or less.

4. The method according to claim 2, wherein the total incubation period of steps (b) and (c) is 5 to 12 days.

5. A method for evaluating the toxicity of a test substance, comprising the following steps (a), (b), and (p), wherein step (b) involves exposing a bird embryo to the test substance before or during egg-turning culture. (a) A step of transferring the embryo and yolk of a bird fertilized egg and the aqueous oval white of a bird into one or more wells of a roughly cylindrical shape in a culture vessel; (b) A step of covering the opening of the well filled with avian aqueous egg white with a porous membrane and culturing the avian embryo by turning the egg; (p) A step of evaluating the toxicity of the test substance using abnormalities in bird embryos exposed to the test substance as an indicator;

6. A method for evaluating the toxicity of a test substance, comprising the following steps (a), (b), (c), and (p), wherein in step (b), the test substance is exposed to a bird embryo before or during egg-turning culture, or in step (c), the test substance is exposed to a bird embryo before or during culture. (a) A step of transferring the embryo and yolk of a bird fertilized egg and the aqueous oval white of a bird into one or more wells of a roughly cylindrical shape in a culture vessel; (b) A step of covering the opening of the well filled with avian aqueous egg white with a porous membrane and culturing the avian embryo by turning the egg; (c) A step of further culturing the avian embryo cultured by turning eggs in step (b) in one or more substantially cylindrical wells in a culture vessel in the presence of avian aqueous egg white, wherein the well has a porous membrane along its inner circumferential surface, there is a space between the opening of the well and the avian egg yolk, and the total culture period of steps (b) and (c) is 12 days or less; (p) A step of evaluating the toxicity of the test substance using abnormalities in bird embryos exposed to the test substance as an indicator;

7. The method according to claim 6, wherein the culture period in step (b) is 72 hours or less.

8. The method according to claim 6, wherein the toxicity of the test substance is evaluated using abnormalities in bird embryos exposed to the test substance as an indicator 5 to 12 days after culturing in steps (b) and (c).

9. The method according to any one of claims 1 to 8, wherein the culture vessel has at least 12 wells.

10. The method according to any one of claims 1 to 8, wherein the bird is a quail.

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