Embryo culture method

Abstract

The invention provides an embryo culture method. Specifically, the invention comprises the step of culturing an embryo, in particular before implantation, on a culture dish provided with an extracellular matrix layer having a sufficient thickness on the surface thereof. The present invention makes it possible, for example, to develop an embryo into an implanted embryo.

Description

Technical Field

[0001] The present invention relates to a method for culturing embryos. Background Art

[0002] In order to study embryonic development and infertility, an in vitro embryo culture method has been developed. Non-patent document 1 discloses a method for culturing mouse blastocysts to the egg cylinder embryo stage. In non-patent document 1, it is not shown that the cultured embryos form gastrulation. In non-patent document 1, it is disclosed that embryonic development is more efficiently carried out to the egg cylinder embryo stage by removing a part of the embryo of E4.5, but the undamaged embryo cannot be normally developed to the later stage.

[0003] Non-Patent Document 2 discloses a method for culturing embryos after implantation. Specifically, Non-Patent Document 2 discloses that embryos at E5.5 or E6.5 were successfully developed to E11.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent literature 1: Bedzhov et al., Nature Protocols 9, 2732-2739, 2014

[0007] Non-patent literature 2: Aguilera-Castrejon et al., Nature 593, 119-124, 2021 Summary of the invention

[0008] The present invention provides a method for culturing embryos.

[0009] Non-patent literature 1 is equivalent to the culture method of the embryo from pre-implantation to the egg cylinder embryonic stage, and non-patent literature 2 is equivalent to the culture method of the embryo after implantation. The method for reproducing the embryonic development from the pre-implantation development stage to the post-implantation development stage (such as gastrulation stage) in vitro is still unknown. Therefore, the present invention provides the conditions and methods for culturing the embryo from the pre-implantation development stage to the gastrulation stage in vitro. In the present invention, a method for developing it without destroying the embryo is provided.

[0010] According to the present invention, the following inventions are provided.

[0011] (1) A method for culturing embryos in vitro, comprising:

[0012] Providing a culture container having an inner bottom surface covered with an extracellular matrix layer, wherein the embryo is placed on the extracellular matrix layer and contained in a sufficient amount of culture medium suitable for culturing the embryo; and

[0013] Then, the embryo placed on the extracellular matrix layer is cultured under conditions suitable for its culture.

[0014] Here, the thickness between the inner bottom surface of the extracellular matrix layer and the upper surface of the extracellular matrix in contact with the embryo is greater than or equal to a predetermined thickness.

[0015] The prescribed thickness is a thickness sufficient to prevent the embryo from adhering to the inner bottom surface during culture or to prevent the embryo from adhering to the inner bottom surface during culture and to increase the adhesion area on the inner bottom surface, and

[0016] The embryo is an embryo at any first developmental stage between the blastocyst stage and the gastrula stage.

[0017] Thus, the embryo in the blastocyst stage can be developed to any second developmental stage between the blastocyst stage and the gastrula stage, and the second developmental stage is the same developmental stage as the first developmental stage or a later developmental stage.

[0018] (2) The method according to (1) above, wherein the culture medium is a serum-free medium.

[0019] (3) The method according to (1) or (2) above, wherein the culture medium is a chemically defined medium.

[0020] (4) The method according to any one of (1) to (3) above, wherein the site where the embryo is placed is inside a hole formed on the upper surface of the extracellular matrix layer.

[0021] (5) The method according to (4) above, wherein the embryo is placed in contact with the inner wall of the hole.

[0022] (6) The method according to any one of (1) to (5) above, wherein the embryo is placed on the extracellular matrix layer with the parietal trophectoderm facing upward or downward.

[0023] (7) The method according to any one of (1) to (6) above, wherein the first developmental stage is a late blastocyst and the second developmental stage is a gastrula.

[0024] (8) The method according to any one of (1) to (7) above, wherein:

[0025] The above culture medium contains the test compound,

[0026] The method also includes a step of confirming whether there is a difference in the growth of the embryo after culture in the presence of the test compound compared to the growth of the embryo after culture in the absence of the test compound, if there is a difference, it means that the test compound affects the development of the embryo from the first developmental stage to the second developmental stage, and / or if there is no difference, it means that the test compound does not affect the development of the embryo from the first developmental stage to the second developmental stage.

[0027] (9) A method according to any one of (1) to (7) above, further comprising the step of culturing the test embryo at the first developmental stage under the condition that a normal embryo at the first developmental stage develops from the first developmental stage to the second developmental stage; if the test embryo does not develop normally from the first developmental stage to the second developmental stage, it indicates that there is an abnormality in the development of the test embryo from the first developmental stage to the second developmental stage; and / or if the test embryo develops normally from the first developmental stage to the second developmental stage, it indicates that the test embryo has the ability to develop from the first developmental stage to the second developmental stage.

[0028] (10) A culture system for culturing embryos, wherein:

[0029] including a culture container for culturing embryos,

[0030] The culture container is a culture container having an inner bottom surface covered with an extracellular matrix layer.

[0031] Here, the thickness of the inner bottom surface in the extracellular matrix layer and the upper surface of the extracellular matrix in contact with the embryo is greater than a predetermined thickness, and the predetermined thickness is a thickness sufficient to prevent the embryo from adhering to the inner bottom surface or adhering to and expanding the adhesion area on the inner bottom surface during culture.

[0032] The embryo is an embryo at any first developmental stage between the blastocyst stage and the gastrula stage.

[0033] Thus, the embryo in the blastocyst stage can be developed to any second developmental stage between the blastocyst stage and the gastrula stage, and the second developmental stage is the same developmental stage as the first developmental stage or a later developmental stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1A A petri dish is shown with a multiwell plate arranged inside it.

[0035] Figure 1B A cross-sectional view showing a multi-well plate and the holes in the multi-well plate.

[0036] Figure 1C A petri dish is shown with a multiwell plate arranged inside it.

[0037] Figure 1D A cross-sectional view of a hole without a coating on the inner bottom surface is shown.

[0038] Figure 1E Shown is a cross-sectional view of a well with an extracellular matrix membrane on the inner bottom surface.

[0039] Figure 1F A cross-sectional view of a well having an extracellular matrix having a thickness on the inner bottom surface used in 3D on top culture is shown.

[0040] Figure 1G The cross-sectional view of a well having an extracellular matrix with depressions on the inner bottom surface used in 3D in-hole culture is shown.

[0041] Figure 2A The optical microscope images and tissue immunostaining results of late blastocyst stage embryos (E4.5 embryos) cultured for one day on wells without a coating layer on the inner bottom surface are shown. Figure 2A In the experiment, it was observed that the embryo adhered to the inner bottom surface and its adhesion area expanded toward the outside of the embryo. Figure 2A It was also observed that the trophoblast layer extended outward from the embryo on the culture surface.

[0042] Figure 2B The optical microscope images and tissue immunostaining results of late blastocyst stage embryos cultured for 1 day on wells with an extracellular matrix membrane on the inner bottom surface are shown. Figure 2B In the study, it was observed that the embryo adhered to the inner bottom surface with extracellular matrix membrane, and the adhesion area expanded toward the outside of the embryo. Figure 2B It was also observed that the trophoblast layer stretched outwards towards the embryonic side of the culture medium.

[0043] Figure 2C The optical microscope images and tissue immunostaining results of late blastocyst stage embryos cultured for 1 day in 3D top culture are shown. Figure 2C In the experiment, the embryos maintained their shape and no adhesion to the inner bottom surface was observed.

[0044] Figure 2D Figure 2 shows optical microscope images and tissue immunostaining results of late blastocyst-stage embryos cultured for one day in 3D wells. Figure 2D In the experiment, the embryos maintained their shape and no adhesion to the inner bottom surface was observed.

[0045] Figure 3 The diagram shows the results of tissue immunostaining of late blastocyst-stage embryos cultured for 2 days in 3D well culture.

[0046] Figure 4AThe diagram shows the results of tissue immunostaining of late blastocyst-stage embryos cultured for 3 days in 3D well culture.

[0047] Figure 4B The diagram shows the results of tissue immunostaining of late blastocyst-stage embryos cultured for 3 days in 3D well culture in IVCM containing human serum.

[0048] Figure 4C The diagram shows the results of tissue immunostaining of late blastocyst-stage embryos cultured for 4 days in 3D well culture in IVCM containing human serum.

[0049] Figure 5 The results of tissue immunostaining of blastocysts (E3.5 embryos) cultured for one day and embryos cultured for three days in 3D top culture are shown.

[0050] Fig. 6A A cross section of a culture vessel having an extracellular matrix layer used in 3D top layer culture is shown.

[0051] Figure 6B A cross section of a culture container having an extracellular matrix layer used in 3D in-well culture is shown.

[0052] Figure 6C The structure of the blastocyst is shown.

[0053] Figure 7 The configuration of the culture system is shown.

[0054] Figure 8 The results are from 72 hours of 3D culture of E4.5 embryos in wells containing hydrogels modified with laminin-derived peptides. The culture was performed in a xeno-free environment.

[0055] Fig.9A The results are shown in Figure 1. E4.5 embryos were cultured in 3D wells containing gellan gum (Gelrite) containing basement membrane extracellular matrix (BME) for 24 hours.

[0056] Fig. 9B The results are shown for 24 hours of 3D culture of E4.5 embryos in wells containing gellan gum containing recombinant human vitronectin (VTN-N). Culture on gellan gum containing VTN-N coating was performed in a xeno-free environment.

[0057] Fig. 10A The figure shows embryos obtained by culturing primate (common marmoset) blastocysts for 3 days, hatching from the hyaloid, then culturing for 5 days in hIVCM containing 4-hydroxyestradiol, and then culturing for 15 days in hIVCM. The culture was continued for 20 days.

[0058] Fig. 10B These are immunostaining images of embryos obtained after 20 days of culture. DETAILED DESCRIPTION

[0059] In this specification, "embryo" may be a mammalian embryo, such as a primate such as a human, a gorilla, a chimpanzee, an orangutan, a bonobo, a marmoset, a rhesus monkey, and a cynomolgus monkey; a rodent such as a mouse, a rat, and a hamster; a carnivore such as a dog and a cat; a perissodactyl such as a horse, a donkey, and a mule; an embryo of an even-toed ungulate such as a cow, a pig, a llama, an alpaca, a goat, and a sheep. In one embodiment, the embryo is not a marsupial embryo.

[0060] In the present specification, "in vitro" means performed outside the body, especially in an artificially prepared culture medium. In contrast, "in vivo" means in vivo, and "in utero" means in utero.

[0061] In this specification, "extracellular matrix" refers to a substance that is present outside the cell, fills the extracellular space and acts as a physical support of the cell. Extracellular matrix is ​​usually insoluble. As the extracellular matrix of vertebrates such as people, collagen, proteoglycan, fibronectin, vitronectin and laminin can be listed. As the extracellular matrix of connective tissue (stroma), a large amount of type I collagen, proteoglycan (versican, decorin etc.) and fibronectin are included. In addition, as the extracellular matrix of basement membrane, type IV collagen, heparan sulfate proteoglycan, laminin and nidogen can be listed. In one embodiment, the extracellular matrix of basement membrane includes laminin. Basement membrane is present between epithelium and connective tissue.

[0062] In this specification, "culture" refers to maintaining cells, cell masses and / or embryos, growing them or developing them to a later developmental stage under conditions suitable for culture. Cultivation is usually carried out in a culture medium suitable for culture. The culture medium contains nutrients required for culture, and can maintain cells, cell masses and / or embryos, grow them or develop them to a later developmental stage. As the culture medium loses nutrients or accumulates waste in the culture medium, it is replaced with a new culture medium after a certain period of time. As culture medium, serum-containing culture medium, serum-free culture medium and chemically defined culture medium can be listed. Serum-free culture medium is a culture medium that does not contain serum. In addition, chemically defined culture medium is a culture medium with clear types and amounts of its constituent components. Serum-free culture medium and chemically defined culture medium can be albumin-free culture medium. Albumin-free culture medium is a culture medium that does not contain albumin. As examples of other culture mediums, xeno-free culture medium and cytokine-free culture medium can be listed. Xeno-free culture medium refers to a culture medium that does not contain xenogeneic components, particularly components of xenogeneic animals.

[0063] According to the present invention, a method for culturing embryos in vitro is provided. According to the present invention, a method for growing embryos in vitro and a method for developing embryos in vitro to a more advanced developmental stage are also provided. The embryo develops from the blastocyst stage through the late blastocyst stage and the egg cylinder embryo stage to the gastrula stage. In one way, the embryo can be an embryo in the late blastocyst stage. In one way, the embryo is a naturally produced embryo. In one way, the embryo is an artificially made embryo. In one way, the embryo can be a blastocyst (embryo-like structure). In one way, the embryo can be an ETX embryo. Blastocysts, for example, can be embryos obtained by co-culturing ES cells, trophoblast stem cells (TS cells) and extraembryonic endoderm cells (XEN cells) (corresponding to embryos in the developmental period of the late blastocyst stage, embryos in the developmental period of the egg cylinder embryo stage, or embryos in the developmental period of the gastrula stage) (for example, with reference to Sozen B., et al., Nature Cell Biology, 20:979-989, 2018). It is known that the embryo proposed in Sozen, 2018 is not a complete embryo. The method of the present invention enables pre-implantation embryos (such as late blastocyst stage embryos) and embryos at subsequent developmental stages to grow or develop into post-implantation embryos (such as gastrula stage embryos) in vitro.

[0064] The method of the present invention may include culturing embryos in vitro. The method of the present invention may preferably include developing embryos to a later developmental stage in vitro. Furthermore, the method of the present invention may include growing or developing preimplantation embryos (e.g., embryos in the late blastocyst stage) and embryos in the developmental stage thereafter into postimplantation embryos (e.g., embryos in the gastrula stage) in vitro.

[0065] The embryo of blastocyst stage develops to the embryo of gastrula stage through late blastocyst stage and egg cylinder embryo stage. In the method of the present invention, the embryo can be an embryo of any first developmental stage between blastocyst stage (particularly late blastocyst) and gastrula stage. In a preferred embodiment, the embryo of the first developmental stage can be an embryo of blastocyst stage (that is, blastocyst, preferably late blastocyst). The blastocyst is equivalent to an embryo of the germinal vesicle stage with a cleavage cavity induced by repeated cleavage of a fertilized egg. The blastocyst has a spherical appearance, contains a blastocyst cavity (cleavage cavity) and an internal cell mass inside, and is covered with a trophectoderm. In vivo, the blastocyst is implanted by being surrounded by endometrial epithelium. The gastrula is an embryo formed by the epiblast from the internal cell mass being trapped inside at the primitive streak. By forming a gastrula, a gastrula with three cell layers of ectoderm, mesoderm and endoderm is generated. Therefore, the presence of ectoderm, mesoderm or endoderm in the embryo indicates that the embryo is at least a developmental stage after the gastrula. In a preferred embodiment, the first developmental stage is a pre-implantation developmental stage, and the second developmental stage is a post-implantation developmental stage.

[0066] In the method for the present invention, embryo growth can be made to the embryo of any second developmental stage from the blastocyst stage (particularly the late blastocyst stage) to the gastrula stage. Here, the second developmental stage is the developmental stage identical with the first developmental stage or the developmental stage in a later period. The second developmental stage is preferably the developmental stage later than the first developmental stage, and can be the developmental stage later than more than 1 day, more than 2 days, more than 3 days or more than 4 days. For example, in a preferred embodiment, the embryo of the second developmental stage is a gastrula.

[0067] In the research so far, there is no report that embryos in the blastocyst stage can be cultured in vitro to develop into gastrula embryos with primitive streaks. It is clarified in the present invention that by culturing embryos under specific conditions, embryos in the blastocyst stage can be developed into gastrula embryos in vitro. In the method of the present invention, embryos can be cultured in a complete state (that is, an undamaged state) in vitro. The method of the present invention preferably does not include damaging embryos or partially or completely destroying embryos. In the present invention, the parietal endoderm and ventral endoderm are formed by the primitive endoderm of the cultured embryo, and the parietal endoderm can form Reichert's membrane.

[0068] In the method of the present invention, the embryo can be cultured in a culture container having an inner bottom surface covered with an extracellular matrix layer. The culture container has a shape that can store a liquid culture medium inside and culture the embryo therein. The culture container generally has a bottom surface and a side wall extending upward from the outer periphery of the bottom surface, and has an opening at the top, which can store a liquid such as a culture solution inside (for example, see Figure 1B ). Then, the embryo is cultured in a culture solution stored in a culture container. The size of the culture container is not particularly limited as long as it can be used for embryo culture. For example, a container with an opening diameter of 3 mm to 10 mm, such as 3 mm to 5 mm, can be preferably used. During culture, the opening can be covered with a lid covering the opening. In one embodiment, the culture container can be a hole of a multi-well plate. The multi-well plate has a plurality of holes arranged on a plate-like substrate.

[0069] When cultivating blastocyst on the inner bottom surface without extracellular matrix layer, blastocyst adheres to the above-mentioned inner bottom surface in cultivation, expands the adhesion area and stretches around. In addition, even with extracellular matrix layer, when the thickness of layer is thin, blastocyst also adheres to the above-mentioned inner bottom surface in cultivation equally, expands the adhesion area and stretches around. Like this, embryo can not normally develop into gastrula. Although unconstrained in theory, at this moment, embryo cell may destroy embryo, hinder the growth of normal embryo to the attachment on the inner bottom surface. Extracellular matrix layer can directly coat the bottom surface of container, but support layer can also be sandwiched between extracellular matrix layer and the bottom surface of container. Support layer is the support (such as solid support) that can stably support extracellular matrix layer, as long as there is no particular restriction on unacceptable cytotoxicity, for example, can comprise the support with biocompatibility, for example, can comprise the support in the group consisting of hydrogels such as agar, gellan gum. Extracellular matrix can be stacked on the above-mentioned support layer or solid phased on the above-mentioned support layer surface. In the case where the support has a hole (with depth and opening) for introducing the embryo, the inner surface of the hole is also preferably covered with an extracellular matrix layer, and at least the part contacted by the embryo is an extracellular matrix layer. The embryo can contact the bottom and / or side of the hole during cultivation. In the case where the hole is roughly cylindrical, the embryo can contact the bottom and side. In the case where the hole is roughly conical, the embryo can contact the side. In the case where the hole is roughly spherical, the embryo can contact one or more points or more than one region of the curved surface of the hole. The opening or inner periphery of the hole is not particularly limited, for example, it can be about 50 μm to about 200 μm, for example, about 70 μm to about 150 μm. The extracellular matrix layer can also contain a biocompatible diluent in addition to containing the extracellular matrix. As a biocompatible diluent, for example, an aqueous solution, a buffer solution, a physiological saline solution and a culture medium can be cited. The extracellular matrix layer can also be a mixture (gel composition) of an effective amount of the extracellular matrix and a support (preferably cell non-adhesive, for example, a hydrogel). On the extracellular matrix layer comprising an effective amount of the extracellular matrix, the embryo (eg, until the gastrula stage, preferably the late gastrula stage) can develop normally.

[0070] In the method of the present invention, blastocysts are cultured in a culture container having an inner bottom surface coated with an extracellular matrix layer with sufficient thickness. That is, in the present invention, the thickness of the upper surface of the extracellular matrix contacted by the inner bottom surface in the extracellular matrix layer is more than the specified thickness. And, the thickness specified is sufficient or necessary for preventing the embryo from adhering to the inner bottom surface or adhering to and expanding the adhesion area on the bottom surface during cultivation. This thickness is the thickness that can carry out so-called three-dimensional culture (3D culture). Three-dimensional culture can list the culture using a support and the culture not using a support. In the present invention, a support based on an extracellular matrix is ​​utilized in the culture of the embryo. As an extracellular matrix, animal extracellular matrix extraction hydrogel, protein hydrogel, peptide hydrogel, polymer hydrogel, etc. can be listed. As an extracellular matrix, a basement membrane extraction hydrogel can also be used. In a preferred embodiment, the extracellular matrix includes more than one, more preferably more than two, more preferably more than three, and more preferably all, selected from the group consisting of type IV collagen, heparan sulfate proteoglycan, laminin, and nestin. In the present invention, it is preferred to use an animal extracellular matrix extraction hydrogel as the extracellular matrix, and it is more preferred to use a basement membrane extraction hydrogel as the extracellular matrix. These extracellular matrices may be xeno-free. Xeno-free means that it does not contain xenogeneic components, especially components of other animal species. In a preferred embodiment, as the extracellular matrix, Matrigel TM and Cultrex TM UltiMatrixReduced Growth Factor, BME001, can be preferably used in the culture of the present disclosure. Matrigel TM and Cultrex TMUltiMatrix Reduced Growth Factor is a solubilized basement membrane extracellular matrix secreted by Engel breth-Holm-Swarm mouse sarcoma cells. In a preferred embodiment, the extracellular matrix may contain laminin, collagen IV, entactin and heparan sulfate proteoglycans. In a preferred embodiment, the extracellular matrix contains 35-40% (e.g., about 39%) laminin, 35-50% (e.g., about 45%) collagen IV and 10-15% (e.g., about 13%) entactin, and may also contain heparan sulfate proteoglycans. Entrytin strengthens the hydrogel structure based on laminin and collagen IV, so by containing entactin, the extracellular matrix can have high tensile strength, which is conducive to 3D in-hole culture and 3D top layer culture. In the present invention, the extracellular matrix layer may contain an effective amount of extracellular matrix or its components or partial peptides. In the present invention, the extracellular matrix layer may contain an effective amount of extracellular matrix or its components or partial peptides and a cell non-adhesive support (e.g., hydrogel). The extracellular matrix may be a laminin-derived peptide or a laminin α-1 chain-derived peptide, for example, a peptide consisting of an amino acid sequence at positions 2116 to 2120 of a sequence registered as GenBank Accession No. KAI4045526.1 or a portion of laminin containing the amino acid sequence. Alternatively, it may be a peptide consisting of an amino acid sequence at positions 950 to 954 of a sequence registered as GenBank Accession No. EAL24388.1 or a portion of laminin containing the amino acid sequence. The extracellular matrix layer may contain vitronectin (for example, human vitronectin, for example, an amino acid sequence having GenBank Accession No. EAW51082.1) or a portion thereof (VTN-N).

[0071] The thickness of the upper surface of the extracellular matrix that the inner bottom surface in the extracellular matrix layer contacts with the embryo can be appropriately set by a person skilled in the art based on the above. For example, the thickness can be, for example, more than 50 μm, more than 100 μm, more than 200 μm, more than 300 μm, more than 400 μm, more than 500 μm, more than 600 μm, more than 700 μm, more than 800 μm, more than 900 μm, or more than 1000 μm. Thus, it is possible to prevent the cells contained in the germinal vesicle from directly contacting the bottom surface.

[0072] Reference Fig. 6A A culture container 10 having an inner bottom surface covered with an extracellular matrix layer used in the method of the present invention will be described. Fig. 6AThe cross section of the culture vessel when culturing embryo is shown. The culture vessel 10 has a bottom surface 15 and a sidewall 11 extending upward from the periphery of the bottom surface 15. The culture vessel 10 has an extracellular matrix layer 13 on the inner bottom surface 15a. That is, the culture vessel 10 has an inner bottom surface 15a coated with an extracellular matrix layer 13. The extracellular matrix layer has sufficient thickness as described above, and the embryo 14 is cultivated under the state that the embryo 14 is carried on the extracellular matrix layer. In the method of the present invention, it is believed that it is not necessary to realize the complete blocking of the contact of the cultivated embryo 14 to the inner bottom surface 15a by the extracellular matrix layer, but by operating as described above, the complete blocking of the contact of the cultivated embryo 14 to the inner bottom surface 15a can be realized by the extracellular matrix layer.

[0073] In the method of the present invention, as long as the extracellular matrix layer can completely block the contact of the cultured embryo 14 with the inner bottom surface 15a, the shape of the extracellular matrix layer is free. Figure 6B The culture container 20 shown. For example, Figure 6B As shown, the extracellular matrix layer 13 can also have a hole 16 of sufficient size for embryo 14 being imported into its upper surface. The hole 16 can be, for example, a roughly cylindrical hole. In this case, the thickness between the upper surface 16a of the extracellular matrix contacted by the above-mentioned bottom surface 15a in the above-mentioned extracellular matrix layer and the embryo can be set as described above. In the method of the present invention, it is believed that it is not necessary to realize the complete blocking of the contact of the inner bottom surface 15a of the cultivated embryo 14 by the extracellular matrix layer, but by the extracellular matrix layer, it is possible to realize the complete blocking of the contact of the inner bottom surface 15a of the cultivated embryo 14. The hole can have a depth greater than the height of the embryo. The hole can have an opening of sufficient size for the embryo to enter. The hole can have a shape of sufficient size for the embryo to enter. The embryo can contact with the bottom surface and / or the side in the hole in cultivation.

[0074] In the method of the present invention, as long as the embryo develops normally, it can be placed on the extracellular matrix layer in any direction. In one embodiment of the present invention, the wall trophectoderm can be placed on the extracellular matrix layer in a manner such that the wall trophectoderm is downward, or the wall trophectoderm membrane is upward and the internal cell mass is downward. For example, in one embodiment, the embryo is an embryo of a rodent (e.g., a mouse or rat), and the wall trophectoderm can be placed on the extracellular matrix layer in a manner such that the wall trophectoderm is downward (e.g., in the embodiment of the present invention). Figure 6C In a preferred embodiment of the present invention, the embryo is a primate (e.g., human) embryo, and can be placed on the extracellular matrix layer with the wall trophectoderm membrane facing upward and the inner cell mass facing downward. Figure 6CAs shown, the blastocyst 30 is composed of the trophectoderm 31 surrounding the internal cell mass 32 and the blastocyst cavity 33. The wall trophectoderm is the trophectoderm 31 located on the blastocyst cavity 33 side rather than on the internal cell mass 32 side. In other words, it can be placed so that the internal cell mass 32 is upward and the blastocyst cavity 33 is downward.

[0075] In a preferred embodiment of the present invention, the embryo can be cultured in a manner that does not contact a solid support (particularly an artificial solid support, preferably a cell-adhesive solid support) other than the extracellular matrix layer. In a preferred embodiment of the present invention, the embryo can be cultured, for example, without a feeder layer.

[0076] In the method of the present invention, conditions suitable for culture can be appropriately determined by those skilled in the art. For example, conditions suitable for culture can be 35°C to 39°C (e.g., about 37°C) and about 3 to 7% CO2 (e.g., about 5% CO2). In one embodiment, for example, conditions suitable for culture can be 35°C to 39°C (e.g., about 37°C), about 3 to 7% CO2 (e.g., about 5% CO2), and about 15 to 25% O2 (e.g., about 20% O2). In one embodiment, conditions suitable for culture can be about 37°C, about 5% CO2, and hypoxic conditions. Hypoxia refers to conditions with an oxygen partial pressure significantly lower than the oxygen partial pressure in the atmosphere (about 20%). Hypoxic conditions can be, for example, conditions with an oxygen partial pressure of 5% to 15%.

[0077] For the culture medium used in the method of the present invention, a serum-containing culture medium, a serum-free culture medium or a chemically defined culture medium can be used. The culture medium can be used appropriately as long as it is a culture medium known as a culture medium suitable for embryo culture. The culture medium, for example, can contain KnockOut® in addition to a basal culture medium (basal culture medium for embryo culture such as DMEM, DMEM / F12, or modified DMEM / F12). TMSerum substitutes such as serum substitutes, L-glutamine (e.g., L-alanine-L-glutamine dipeptide), antibiotics (e.g., penicillin, streptomycin), insulin, transferrin, selenium, ethanolamine, reducing agents (e.g., monothioglycerol), and L-cysteine ​​(e.g., N-acetyl-L-cysteine) and other additives. In a preferred embodiment, the culture medium used in the method of the present invention can be a serum-free medium containing modified DMEM / F12. In one embodiment, the culture medium contains serum. In one embodiment, the culture medium contains human serum or at least one of its components. In one embodiment, the culture medium can be a mixture of human serum or at least one of its components and a chemically defined medium. In one embodiment, the culture medium is a xeno-free medium containing serum. In one embodiment, the culture medium is a xeno-free medium containing at least one of its components. In one embodiment, the xeno-free medium can be a mixture of human serum or at least one of its components and a chemically defined medium.

[0078] The present invention can be used to investigate the effect of the test compound on the implantation and / or development of the embryo. For example, when embryos such as normal embryos and abnormal embryos are cultured in the presence of the test compound and compared with the absence of the test compound, when the development of the embryo is different, it means that the test compound has a physiological activity that affects the development of the embryo. Thus, the effect of the test compound on the development of normal embryos and the effect of the test compound on abnormal embryos can be investigated. For embryos with poor implantation and abnormal embryos, compounds that normalize their development can become infertility treatment drugs. Therefore, the method of the present invention can also be used to screen infertility treatment drugs. Here, the test compound refers to a compound to be examined, for example, a compound that confirms its effect on the implantation and / or development of the embryo. The test compound can be a compound that has an effect on the implantation and / or development of the embryo, a compound that is suspected of having an effect on the implantation and / or development of the embryo, or a compound that has no effect on the implantation and / or development of the embryo. This is because, even if the compound itself has no effect, the effect on the embryo is unclear depending on the form of the compound, such as the content of the composition containing the compound.

[0079] The present invention can also be used to test the developmental capacity of the examined embryo. For example, according to the method of the present invention, the examined embryo in the first developmental stage is cultivated under the condition that the normal embryo in the first developmental stage develops from the first developmental stage to the second developmental stage. If the above-mentioned examined embryo does not develop normally from the first developmental stage to the second developmental stage, it means that the development of the embryo in the first developmental stage to the second developmental stage is abnormal, and / or if the above-mentioned examined embryo develops normally from the first developmental stage to the second developmental stage, it means that the above-mentioned examined embryo has the ability to develop from the first developmental stage to the second developmental stage. In this way, it is possible to test whether there is developmental abnormality in the embryo. Thus, for example, it is possible to check the adaptability to infertility treatment. Here, the examined embryo refers to the embryo to be checked, for example, the embryo to confirm implantation and / or developmental capacity.

[0080] According to the present invention, by combining other methods of developing blastocyst from fertilized egg and the method of developing to an earlier developmental stage from gastrula, embryo can be developed to an earlier developmental stage of gastrula from fertilized egg. The method of developing blastocyst from fertilized egg is developed with mouse (Manipulating the Mouse Embryo: a Laboratory Manual 4th ed. (Cold Spring Harbor Laboratory Press, 2014), which is then clinically applied to the in vitro fertilization of people (Steptoe et al., Lancet, 1978). For example, those skilled in the art can prepare blastocyst by fertilized egg based on the above content, by making fertilized egg differentiate under the condition that is suitable for being differentiated into blastocyst. As the method of developing to early developmental stage (hind limb formation period: E11) from gastrula, for example, the rotary culture method (Aguilera-Castrejon et al., Nature, 2021). For example, those skilled in the art can prepare embryos at the third developmental stage from gastrula embryos by allowing the gastrula embryos to differentiate under conditions suitable for differentiation to the third developmental stage from the gastrula embryos to the hind limb formation stage based on the above content. In this way, according to the present invention, it is expected that a method can be established to reproduce all steps from fertilized eggs to hind limb formation stage embryos in vitro without destroying the embryos. Therefore, according to the present invention, an in vitro method is provided that can obtain hind limb formation stage embryos from fertilized eggs.

[0081] According to the present invention, there is provided a culture system, which is a culture system (culture device) 50 for culturing embryos, wherein:

[0082] comprising a culture container 52,

[0083] The culture container 52 is a culture container having an inner bottom surface covered with an extracellular matrix layer.

[0084] Here, the thickness of the bottom surface of the extracellular matrix layer and the upper surface of the extracellular matrix in contact with the embryo is greater than a predetermined thickness, and the predetermined thickness is a thickness sufficient to prevent the embryo from adhering to the bottom surface or adhering to and expanding the adhesion area on the bottom surface during culture.

[0085] The embryo is an embryo at any first developmental stage between the blastocyst stage and the gastrula stage.

[0086] Thus, the embryo in the blastocyst stage can be developed to any second developmental stage between the blastocyst stage and the gastrula stage, and the second developmental stage is the same developmental stage as the first developmental stage or a later developmental stage.

[0087] The culture system can be used to develop the embryo from the first development stage to the second development stage. The details of the first development stage and the second development stage are as described above.

[0088] As an example, see Figure 7 The composition of the culture system is described. Figure 7 As shown, the culture system has a storage vault 51 for accommodating a culture container 52, and also has a thermometer 53 for measuring the temperature in the storage vault 51, a hygrometer 54 for measuring the humidity in the storage vault, and a thermostat 55 for controlling the temperature of the storage vault. In a preferred embodiment, the interior of the culture container 52 is controlled under sufficient humidity conditions. The control of humidity can be achieved, for example, by arranging a moisture retaining device (such as a moisture retaining sponge or paper towel, etc.) and the culture container 52 in the same space. In one embodiment, the culture container 52 is a multi-well plate, which is arranged inside another open system container (such as an open system container with an openable and closable lid, such as a Petri dish) (see Figure 1A and 1C ). In addition, the moisture retaining device is arranged in another open system container, and in particular, it is capable of maintaining the humidity in an open system container (such as a petri dish) with a closed lid. In a preferred embodiment, the culture system 50 further comprises a gas concentration sensor 56 in the storage chamber 51, and the gas concentration sensor 56 is capable of measuring the carbon dioxide concentration and / or the oxygen concentration. In a preferred embodiment, the culture system may further comprise an oxygen concentration controller 57 for controlling the oxygen concentration based on the measured oxygen concentration. The oxygen concentration controller 57 may supply nitrogen gas into the storage chamber in order to make the storage chamber hypoxic.

[0089] According to the present invention, provided are embryos from the late blastocyst stage to the egg cylinder embryo stage and from the egg cylinder embryo stage to the gastrula stage obtained by the method of the present invention, and a culture obtained by the method of the present invention containing the embryos.

[0090] Example

[0091] Materials and methods

[0092] Mouse embryos

[0093] Females were used for natural mating at 3±1 months after birth. Noon on the day when vaginal plugs were detected was set as E0.5. This study was approved by the Animal Experiment Committee of Kyushu University.

[0094] For mouse embryos, on embryonic day E3.5 or E4.5 (12:00), ICR female mice mated with ICR males, B6C3 female mice mated with C57BL / 6N and C3H / HeN hybrid mice (B6C3) males, B6C3-Otx2 female mice mated with B6C3-Otx2 em1(tdTomato)Utr After mating with a male, the uterus of B6C3 female mice was returned and recovered using M2 medium (Sigma-Aldrich M7167) and cultured in culture medium (in vitro culture medium: IVCM modified from IVC2 medium (Bedzhov et al., 2014)). The composition of IVCM is as follows: modified DMEM / F12 (Thermo Fisher Scientific, 12634010), 30% KSR (Thermo Fisher Scientific, 10828010), 1×GlutaMAXI (Thermo Fisher Scientific, 35050061), 1×Penicillin-Streptomycin (Thermo Fisher Scientific, 15140122), 1×ITS-X (Thermo Fisher Scientific, 51500056), 1×Monothioglycerol (FUJIFILM, 195-15791), 200 ng / ml progesterone (Sigma-Aldrich, P8783) and 25 μM N-acetyl-L-cysteine ​​(Sigma-Aldrich, A7250). This step was performed at 37°C using a stereomicroscope (manufactured by Leica) equipped with ThermoPlate (manufactured by Tokai Hito Co., Ltd., TPiD-TLDX).

[0095] Culture of mouse embryos

[0096] For embryo culture, use 8-well μ-slides (Ibidi, 80826) or angiogenesis μ-slides (Ibidi, 81506) and place them in a 10 cm Petri dish with a moistened paper towel ( Figure 1A -C). In the culture of E3.5 embryos, the zona pellucida was removed using acidic Tyrode's solution (Sigma-Aldrich, T1788) as previously described (Bedzhov et al.,). The zona pellucida-free blastocysts were cultured on 8-well μ-slides at 37°C and 5% CO2 for 24 hours using IVCM supplemented with 8 nM β-estradiol (Sigma-Aldrich, E8875) and 10 ng / ml osteopontin (Biolegend, 763602). E4.5 embryos or pre-cultured E3.5 embryos were cultured with IVCM on angiogenic μ-slides at 37°C and 5% CO2. In 2D culture, the cells were cultured on 2D slides coated with basement membrane extract (BME) (R&D systems, Cultrex TM UltiMatrix Reduced Growth Factor, BME001)( Figure 1E ) or uncoated ( Figure 1D Embryos were cultured on angiogenesis μ-slides. BME coating was performed on a cooling module (CorningCoolBox XT, 432022). BME was placed on the angiogenesis μ-slide and immediately removed, and the μ-slide was then incubated at 37°C for 10 minutes to form a gel ( Figure 1E In 3D top layer culture or 3D in-well culture, 10 μl (for 3D top layer experiment) or 15 μl (for 3D in-well experiment) of BME was placed in the inner well of the angiogenesis μ-slide and incubated at 37°C for 10 minutes ( Figure 1F , 1G In 3D well culture, a micropipette tip is used to create a hole with a diameter of 150 to 200 μm in the gel ( Figure 1G ). The embryos were cultured in the wells with the wall trophectoderm facing downward. After the initial 30 hours of culture, the medium was replaced with pre-heated IVCM and cultured at 37°C, 5% CO2. The medium was then replaced at 12:00 every day. Images were acquired using a stereomicroscope (Leica) or a BZ-X700 microscope (Keyence). All embryos were cultured using the chemically defined medium described above. No serum, such as fetal bovine serum, was added to the chemically defined medium.

[0097] Immunostaining

[0098] Embryos were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Fixed embryos were cleared using the CUBIC protocol (Tainaka et al., 2018) with some modifications as shown below. Fixed embryos were treated overnight at 37°C in an incubator with a shaker (hybridization incubator) using CUBIC-L (Tokyo Chemical Industry, T3740). To stop the clearing treatment, wash twice for 30 minutes each with PBS while gently shaking at room temperature. For the primary antibody incubation, the cells were incubated overnight at 4°C using a rocker shaker, and then incubated for 2 hours at room temperature using a fluorescently labeled AlexaFluor secondary antibody (Thermo Fisher Scientific). Both the primary and secondary antibodies were diluted with 1% BSA, 0.1% Tween-PBS. The cells were immersed in CUBIC-R+ (Tokyo Chemical Industry, T3741) and incubated overnight at 37°C in a hybridization incubator. Images were acquired using an inverted SP8 confocal microscope (Leica) and analyzed using LAS X software (Leica) or Imaris software (Oxford instruments).

[0099] result

[0100] In embryonic development, methods for culturing preimplantation embryos (Bedzhov et al., Nature Protocols 9, 2732-2739, 2014, etc.) and postimplantation embryos (Aguilera-Castrejon et al., Nature 593, 119-124,, 2021, etc.) have been developed. Preimplantation embryos are embryos from fertilization to E4.5 in mice, and postimplantation embryos are embryos after E5.0. In Bedzhov et al., 2014, a method for developing blastocysts from E3.5 to E4.5 in vitro beyond the implantation period was disclosed, and in Aguilera-Castrejon et al., 2021, a method for reproducing normal development from E5.5 to E11 in vitro was disclosed. However, it has not yet been achieved to efficiently develop preimplantation embryos to the gastrula stage in vitro. E4.5 embryos are blastocyst stage embryos, and E5.5 embryos correspond to egg cylinder embryos.

[0101] like Figure 1A As shown, a moistened paper towel was placed on the side of a 10 cm petri dish, and an 8-well μ-slide was placed in the center of the petri dish. Figure 1B As shown, the angiogenesis μ-slide has holes, and embryos can be introduced into each hole. Figure 1CAs shown, moistened paper towels were placed on the side of a 10 cm petri dish, and an angiogenesis μ-slide was placed in the center of the petri dish.

[0102] Apply different coatings to each well. Figure 1D The wells shown have bottom surfaces without any coating (non-coating), as shown Figure 1E The bottom surface of the membrane having a basement membrane extract (BME) as an extracellular matrix formed on the surface thereof (2D BME), as shown in FIG. Figure 1F The wells into which the thick blocks of basement membrane matrix were introduced (3D top layer) and Figure 1G The IVCM culture medium is introduced into the hole (in the 3D hole) with a small hole of the size of the embryo entering. The embryo (E4.5 embryo) of the mouse blastocyst stage is introduced into each hole. The embryo comes from an ICR female mouse. In the 3D hole culture, the embryo is buried in the above-mentioned small hole. Then, it is cultured at 37°C for 1 day. The embryos cultured for 1 day are observed using an optical microscope and a fluorescent microscope.

[0103] The results are as follows Figure 2A As shown in ~D. Figure 2A and 2B As shown, the embryos cultured in the holes with the bottom surface not coated adhered to the plate surface, became flat, and observed that the trophoblast spread on the plate. In contrast, the embryos cultured in the 3D top layer and the 3D holes developed normally. In the embryos cultured in the 3D top layer and the 3D holes, the situation of adhering to the plate surface and expanding was not observed. The ratio of the embryos (equivalent to the embryos of E5.0~E5.3) of normal development is shown in Table 1.

[0104] [Table 1]

[0105]

[0106] As shown in Table 1, it can be seen that the ratio of normal development of embryos cultured in 3D wells was significantly higher than that of embryos cultured under other coating conditions.

[0107] The late blastocysts (E4.5) were cultured in IVCM medium for 2 days by 3D well culture. When observed under an optical microscope, the cultured embryos had a cylindrical (oval cylinder) morphology. In addition, the cultured embryos were subjected to tissue immunostaining. In tissue immunostaining, the expression of Cdx2, Oct4, Gata6, and Eomes was detected using primary antibodies that specifically bind to each other and labeled secondary antibodies. The results are shown in Figure 2. Figure 3 As shown. Figure 3As shown, embryos cultured in 3D wells for 2 days formed Gata6-positive parietal endoderm. The parietal endoderm formed Reichert's membrane. In contrast, the formation of Reichert's membrane was not found in previous reports (Bedzhov et al., Nature Protocols 9, 2732-2739, 2014).

[0108] In addition, late blastocysts (E4.5) were cultured in IVCM medium for 3 days by 3D in-well culture. The embryos were from ICR female mice. The cultured embryos were provided for tissue immunostaining. In tissue immunostaining, the expression of Cdx2, Oct4, T, and Eomes was detected using primary antibodies and labeled secondary antibodies that specifically bind to each other. T is a marker for primitive streak and mesoderm, and Eomes is a marker for primitive streak and extraembryonic ectoderm. The resulting embryos grew to embryos in the early primitive streak stage. The results of tissue immunostaining are shown in Figure 2. Figure 4A As shown. Figure 4A As shown, the expression of T and Eomes was observed in the embryos cultured for 3 days. This shows that the embryos have formed the primitive streak. In addition, it is clear that the embryo develops to the gastrula stage. The formation of the primitive streak was not shown in previous reports (Bedzhovet al., Nature Protocols 9, 2732-2739, 2014). Under the above conditions, the development efficiency of the embryos equivalent to the developmental stage of E6.5 is 1 / 5. Therefore, in the hIVCM culture medium made by adding human serum instead of KSR of IVCM culture medium, culture is carried out under the same conditions as above. The composition of hIVCM culture medium is as follows: modified DMEM / F12 medium (Thermo Fisher Scientific), 30% human serum (Gaojin Biotechnology), 1×GlutaMax I (Thermo Fisher Scientific), 1×Penicillin-Streptomycin (Thermo Fisher Scientific), 1×ITS-X (Thermo Fisher Scientific), 200ng / mL progesterone (Sigma-Aldrich, P8783) and 10μM Mito-TEMPO (Selleck, S9733). As a result, embryos from E4.5 embryos to the developmental stage equivalent to E6.5 (refer to Figure 4B ) increased to 3 / 5. The 3D well culture was continued for 4 days. Figure 4C As shown in FIG. 1 , embryos at a developmental stage equivalent to E7.5 (late gastrulation embryos) were obtained. Figure 4CThe characteristics of the embryo obtained in the experiment are shown in the area surrounded by the white dotted line. The expression of Otx2, a marker of ectoderm, was confirmed ventrally, suggesting embryonic development to the late gastrulation stage embryo.

[0109] The blastocysts (E3.5) were cultured for 4 days. The embryos were from ICR female mice. Specifically, the first day was cultured in IVCM medium supplemented with β-estradiol and osteopontin, and then in 3D top layer for 3 days. The obtained embryos grew to the embryos of the early primitive streak stage. The results of tissue immunostaining are shown in Figure 5 As shown. Figure 5 As shown in the figure, the expression of T and Eomes was observed in embryos cultured for 3 days. This indicates that the embryos have formed a primitive streak. In addition, it was confirmed that the embryos developed to the gastrula stage.

[0110] According to the present example, embryonic development stages including pre- and post-implantation stages from E4.5 (late blastocyst) to E7.5 (late gastrulation embryo) were successfully reproduced in vitro.

[0111] In order to analyze the effects of basement membrane components on embryonic development, VitroGel (TWG007: IKVAV, TWG008: YIGSR, TheWell Bioscience Inc.) can be used instead of basement membrane components to implement the same 3D well culture of embryos as above. VitroGel was mixed with culture medium (3:1), and the resulting mixture was transferred to the wells of the inner well of the angiogenesis μ-slide to gel. Incubate at room temperature for 20 minutes, and then add culture medium to the gel. In the 3D well culture, a micropipette tip was used to open holes with a diameter of 150 to 200 μm in the gel. E4.5 embryos were cultured in IVCM for 72 hours. The embryos were from B6C3 female mice mated with C57BL / 6N and C3H / HeN hybrid mice (B6C3) males. The results are as shown in the figure. Figure 8 As shown. Figure 8 As shown, the hydrogel containing the peptide as a partial peptide derived from laminin supports embryonic development. It is thus clear that the hydrogel containing the laminin-derived peptide can function as an extracellular matrix layer (or support).

[0112] Gelrite (gellan gum; Fujifilm Wako Pure Chemical Industries, Ltd.) was used in the culture in the 3D top layer and 3D wells. Gelrite is a non-adhesive support for cells. Gelrite powder was dissolved in water (0.8%) and then boiled in a microwave oven. To form a gel of gellan gum, 12 μl of a mixture of 0.8% gellan gum / water was transferred to the wells of the inner wells of the angiogenesis μ-slide. As an extracellular matrix, BME001 or recombinant human vitronectin (VTN-N) was used. Recombinant human vitronectin (VTN-N) is derived from the region of positions 62 to 478 of human vitronectin. The solution was incubated at room temperature for 20 minutes, and then 1% BME001 / culture solution or 5 μg / mL VTN-N was added to the gel as an extracellular matrix and incubated overnight to allow the extracellular matrix to fully and deeply penetrate into the gellan gum. In order to perform 3D well culture, a micropipette tip was used to open a hole with a diameter of 70 to 100 μm in the gel infiltrated with the extracellular matrix. Under this condition, the embryo was in contact with the gellan gum containing the extracellular matrix, but not with the bottom surface of the well. In hIVCM, E4.5 embryos (with tdTomato operably connected downstream of the Otx2 gene) were cultured in 3D wells for 24 hours. The results are shown in Fig.9A and B. Embryos from B6C3-Otx2 em1(tdTomato)Utr B6C3 female mice mated with males. Fig.9A and B, when BME001 was used as the extracellular matrix ( Fig.9A ) and when using VTN-N ( Fig. 9B ) under the same conditions, the embryos developed normally.

[0113] Otx2 is a marker of post-implantation embryos expressed in the epiblast, and the expression of tdTomato, which was genetically introduced downstream of the Otx2 gene, suggests that the embryo has developed to the post-implantation stage. Fig.9A As shown in A and B, one day after the start of 3D well culture, all embryos expressed tdTomato driven by the Otx2 promoter, suggesting that the embryos developed to post-implantation embryos. This suggests that basement membrane extracts support embryonic development. It is clear that the above hydrogels containing BME001 or VTN-N (basement membrane components) play the role of an extracellular matrix layer (or support).

[0114] The above is an experiment using mouse embryos. Next, primate embryos were cultured using the method of the present invention.

[0115] Common marmoset (Callithrix jacchus) embryos (blastocysts) were kindly provided by Dr. Erika Sasaki (Central Research Institute for Laboratory Animals). To thaw the embryos in the frozen vials, 900 μL of SPB2 (pre-warmed for more than 1 hour at 38°C, 5% CO2, 5% O2) was added. The embryos were washed three times with PB1. The embryos were transferred to a drop of ORIGIO Sequential Blast containing 20% ​​FBS and 2 mM L-glutamine medium (pre-warmed for more than 1 hour at 38°C, 5% CO2, 5% O2) and cultured at 38°C, 5% CO2, 5% O2 until the embryos hatched. Then, the embryos were cultured in hIVCM medium by the 3D top layer method. Half of the medium was replaced with hIVCM medium containing 10 nM 4-hydroxyestradiol and cultured at 38°C, 5% CO2, 5% O2 for 5 days. Then, half of the culture medium was further replaced with hIVCM culture medium and cultured at 37°C and 5% CO2. Half of the culture medium was replaced at 12:00 every day. The embryos obtained were photographed using a stereomicroscope (Leica). Fig. 10A As shown in Figure 2, embryos were successfully cultured after 20 days. When the embryos were immunostained for the primitive streak marker T, the results were as follows: Fig. 10B As shown in the figure, it is clear that the embryo has reached the gastrulation stage. This shows that primates can also develop from pre-implantation embryos to post-implantation embryos in vitro.

[0116] Bedzhov, I., Leung, CY, Bialecka, M., and Zernicka-Goetz, M. (2014). In vitro culture of mouse blastocysts beyond the implantation stages. Nat Protoc 9, 2732-2739.

[0117] Tainaka, K., Murakami, TC, Susaki, EA, Shimizu, C., Saito, R.,Takahashi, K., Hayashi-Takagi, A., Sekiya, H., Arima, Y., Nojima, S., et al., (2018). Chemical

[0118] Landscape for Tissue Clearing Based on Hydrophilic Reagents. CellReports 24, 2196-2210.e2199.

Claims

1. A method for culturing embryos in vitro, wherein include: Providing a culture container having an inner bottom surface covered with an extracellular matrix layer, wherein the embryo is placed on the extracellular matrix layer and contained in a sufficient amount of culture medium suitable for culturing the embryo; and Then, the embryos placed on the extracellular matrix layer are cultured under conditions suitable for their culture. Here, the thickness of the inner bottom surface of the extracellular matrix layer and the upper surface of the extracellular matrix in contact with the embryo is greater than or equal to a predetermined thickness, The prescribed thickness is a thickness sufficient to prevent the embryo from adhering to the inner bottom surface during culture or to prevent the embryo from adhering to the inner bottom surface during culture and to expand the adhering area on the inner bottom surface, and The embryo is an embryo at any first developmental stage between the blastocyst stage and the gastrula stage, Thus, the embryo in the blastocyst stage can be developed to any second developmental stage between the blastocyst stage and the gastrula stage, and the second developmental stage is the same developmental stage as the first developmental stage or a later developmental stage.

2. The method according to claim 1, in, The culture medium was serum-free.

3. The method according to claim 1 or 2, in, The culture medium was a chemically defined medium.

4. The method according to any one of claims 1 to 3, in, The embryo is placed inside the hole formed on the upper surface of the extracellular matrix layer.

5. The method according to claim 4, in, The embryo is placed in contact with the inner wall of the hole.

6. The method according to any one of claims 1 to 5, in, The embryo is placed on the extracellular matrix layer with the mural trophectoderm either facing up or facing down.

7. The method according to any one of claims 1 to 6, in, The first developmental stage is the late blastocyst and the second developmental stage is the gastrula.

8. The method according to any one of claims 1 to 7, in, The culture medium contains a test compound, The method further comprises the step of confirming whether there is a difference in the growth of the embryo after culture in the presence of the test compound compared to the growth of the embryo after culture in the absence of the test compound, If there is a difference, it means that the tested compound affects the development of the embryo from the first development stage to the second development stage, and / or If there is no difference, it means that the tested compound does not affect the development of the embryo from the first development stage to the second development stage.

9. The method according to any one of claims 1 to 7, in, The method further comprises the step of culturing the embryo under test at the first development stage under the condition that the normal embryo at the first development stage develops from the first development stage to the second development stage. If the examined embryo does not develop normally from the first development stage to the second development stage, it indicates that the development of the examined embryo from the first development stage to the second development stage is abnormal, and / or If the examined embryo develops normally from the first development stage to the second development stage, it means that the examined embryo has the ability to develop from the first development stage to the second development stage.

10. A culture system, which is a culture system for culturing embryos, in, including a culture container for culturing embryos, The culture container is a culture container having an inner bottom surface covered with an extracellular matrix layer. Here, the thickness of the bottom surface of the extracellular matrix layer and the upper surface of the extracellular matrix in contact with the embryo is greater than a predetermined thickness, and the predetermined thickness is a thickness sufficient to prevent the embryo from adhering to the bottom surface or adhering to and expanding the adhesion area on the bottom surface during culture, The embryo is an embryo at any first developmental stage between the blastocyst stage and the gastrula stage, Thus, the embryo in the blastocyst stage can be developed to any second developmental stage between the blastocyst stage and the gastrula stage, and the second developmental stage is the same developmental stage as the first developmental stage or a later developmental stage.

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