Three-dimensional co-culture system for in vitro cultured embryos and its use

Abstract

The present invention provides a three-dimensional co-culture system, a method for using the three-dimensional co-culture system, and a kit including the three-dimensional co-culture system.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 378,929, filed October 10, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present invention relates to the field of biotechnology. Provided herein is a three-dimensional co-culture system for culturing embryos in vitro. The three-dimensional co-culture system includes: (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) a cell culture medium. [Background technology]

[0003] In vitro production of embryos offers several advantages over in vivo-derived embryo production, including but not limited to the introduction of genetic manipulations, allowing for efficient selection of superior genetic characteristics and rapid generation of animals with desired traits. Genetic engineering can provide a powerful tool to aid in understanding the fundamental mechanisms regulating physiology. The in vitro environment is still suboptimal for embryo growth and development, and therefore there is a need to develop superior in vitro embryo culture systems that mimic the in vivo environment to provide robust and viable embryos and achieve higher success rates for in vitro fertilization procedures, to develop efficient reproductive techniques for agriculturally important animals, and to develop efficient conservation of extinct and endangered species through reproductive techniques. Summary of the Invention

[0004] In one general aspect, the present invention relates to a three-dimensional co-culture system, which can include, for example, (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture medium. Also provided is a method for growing an embryo in a three-dimensional co-culture system, the method comprising: (a) culturing at least one somatic cell; (b) embedding the at least one somatic cell in a three-dimensional structure; (c) obtaining at least one embryo; and (d) embedding or placing the at least one embryo in or near the three-dimensional structure; wherein the at least one somatic cell and the at least one embryo are grown in or near the three-dimensional structure in a cell culture medium. Also provided is a kit comprising: (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture medium. In certain embodiments, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can include, for example, a gel-like substance or a structural scaffold. The gel-like substance can be selected from, for example, hydrogel, agarose, basement membrane extract, or extracellular matrix. In certain embodiments, the scaffold-based structure is produced by a 3D printer. In certain embodiments, the three-dimensional structure is a scaffold-free structure. The scaffold-free structure can, for example, comprise a cluster of cells that form the three-dimensional structure. In certain embodiments, the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells and / or cells of the reproductive tract. In certain embodiments, the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptilian embryo, a fish embryo, an amphibian embryo, and a marsupial embryo. The embryo can be, for example, a mammalian embryo. In certain embodiments, the cell culture medium is selected from at least one of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F-12), N2B27, M16, potassium-supplemented SOM (KSOM), tissue culture medium 199 (TCM-199), or a custom medium. The cell culture medium may contain, for example, sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg2+ ) may be included. In certain embodiments, the somatic cells are embedded in or placed near the three-dimensional structure. In certain embodiments, the embryos are embedded in or placed near the three-dimensional structure. In certain embodiments, the three-dimensional co-culture system further comprises a bioreactor.

[0005] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Brief explanation of the drawings]

[0006] [Figure 1A] Figure 1A is a representative image of a monolayer cell culture. [Figure 1B] Figure 1B is a representative image of three-dimensional (3D) cell culture. [Figure 2] Figure 2 shows representative images demonstrating the progression of 3D cell culture over an 8-day period. The somatic cells in the 3D culture proliferated and connected with each other to form 3D structures of cell clusters. Many structures were formed during the 8-day culture. [Figure 3] Figure 3 shows a representative image of a 3D co-culture system for culturing embryos in vitro. At least one embryo was embedded in a 3D structure of somatic cells, which provided favorable growth conditions for the embryo. The 3D structure also provided physical support and maintained its spatial and morphological shape during embryo growth and development. [Figure 4] FIG. 4 is an image demonstrating an E3.5 mouse embryo in contact with a three-dimensional system during in vitro culture. [Figure 5] FIG. 5 shows images demonstrating an E3.5 mouse embryo developed to the E5.5 stage in a three-dimensional in vitro culture system. [Figure 6] FIG. 6 is an image of a structure with expanded bodies attached to a 3D in vitro culture system with stem structures and placenta-like structures. [Figure 7] FIG. 7 is an image of a pre-implantation E4.5 mouse embryo. [Figure 8A] Figure 8A shows images of the development of an E4.5 mouse embryo in a 3D in vitro culture system. Figure 8A shows images from day 1. [Figure 8B] Figure 8B shows images of the developmental progression of an E4.5 mouse embryo in a 3D in vitro culture system, with images of day 2 shown in Figure 8B. [Figure 8C] Figure 8C shows images of the development of an E4.5 mouse embryo in a 3D in vitro culture system, with images at day 3. [Figure 8D] Figure 8D shows images of the developmental progression of an E4.5 mouse embryo in a 3D in vitro culture system, with Figure 8D being the fourth day. [Figure 8E] Figure 8E shows images of the development of an E4.5 mouse embryo in a 3D in vitro culture system, with images at day 5. DETAILED DESCRIPTION OF THE INVENTION

[0007] Various publications, articles, and patents are cited or described in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like which has been included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed.

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise specified, any numerical values, such as concentrations or concentration ranges, described herein can be understood to be modified in all instances by the term "about." Thus, numerical values ​​typically include ±10% of the recited value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (wt / vol) includes 0.9% (wt / vol) to 11% (wt / vol). As used herein, the use of numerical ranges expressly includes all possible subranges within such ranges, all individual numerical values, including integers and fractions of values ​​within such ranges, unless the context clearly indicates otherwise. Unless otherwise indicated, the term "at least" preceding a series of elements can be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0009] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or other variations thereof, are understood to mean the inclusion of the stated integer or groups of integers, but not the exclusion of other integers or groups of integers, and are intended to be inclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of components is not necessarily limited to only those components, but may include other components not expressly listed or included in the composition, mixture, process, method, article, or device. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present). As used herein, the conjunctive term "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are joined by "and / or," the first alternative refers to the applicability of the first element without the second element. The second alternative refers to the applicability of the second element without the first element. The third alternative refers to the applicability of the first and second elements together. Any one of these alternatives is understood to be within this meaning and therefore meets the requirements of the term "and / or" as used herein. Also, the simultaneous applicability of more than one alternative is understood to be within this meaning and therefore meets the requirements of the term "and / or."

[0010] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, indicates that any listed integer or group of integers is inclusive, but that additional integers or groups of integers cannot be added to a particular method, structure, or composition. As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, refers to the inclusion of any recited integer or group of integers, and any inclusion of any recited integer or group of integers that does not materially change the basic or novel properties of a particular method, structure, or composition. See MPEP § 2111.03. The words "right", "left", "bottom" and "top" designate directions on the drawings to which reference is made. It should also be understood that the terms "about," "approximately," "generally," "substantially," and similar terms used herein when referring to dimensions and characteristics of components in the preferred invention indicate that the described dimensions / characteristics are not rigid boundaries or parameters, and do not exclude minor variations that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters will include variations that do not alter the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0011] 3D co-culture system Provided herein are three-dimensional co-culture systems for culturing embryos in vitro. The three-dimensional co-culture systems provide an in vitro culture environment that more closely resembles the in vivo environment than flat monolayer culture systems. The three-dimensional co-culture systems also provide three-dimensional support for embryos during in vitro development. The three-dimensional co-culture systems and methods for using the three-dimensional co-culture systems provide an environment that results in superior embryo viability and development. Superior embryo viability and development can be manifested, for example, by high success rates in human in vitro fertilization (IVF), efficient reproductive techniques for agriculturally important animals, and the efficient preservation of extinct and endangered species through reproductive techniques. In one general aspect, the present invention relates to a three-dimensional co-culture system, which can include, for example, (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture medium. As used herein, the term "three-dimensional co-culture" is commonly understood by those skilled in the art and refers to a method of culturing at least one type of cell with at least one embryo, wherein the at least one type of cell and the at least one embryo are transplanted or seeded into or near an artificial structure (i.e., a three-dimensional structure) capable of supporting the three-dimensional co-culture of the at least one type of cell and the at least one embryo. The three-dimensional structure is essential for mimicking the in vivo environment, allowing the at least one cell and the at least one embryo to grow and / or develop in their own microenvironment. In certain embodiments, the three-dimensional co-culture system is suitable for use in in vitro cell culture.

[0012] In certain embodiments, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can include, for example, a gel-like substance or a structural scaffold. The gel-like substance can be selected from, for example, hydrogel, agarose, basement membrane extract, or extracellular matrix. The extracellular matrix can be, for example, synthetic or natural. Synthetic means that the extracellular matrix is ​​produced under laboratory conditions. Natural means that the extracellular matrix is ​​derived from and / or isolated from a particular organism. Gel-like substances are known in the art and are commercially available. See, for example, MyoGel (pharmasana.co.uk); OBAGEL® (Obatala; New Orleans, Louisiana), CETUREGEL™ (Jessen Biotechnology; Shanghai, China), JELLAGEL® (Jellagen; Wales), GROWDEX® (UPM Biomedicals; Helsinki, Finland), GELTREX™ (Thermo Fisher Scientific; Waltham, Massachusetts), and Matrigel® (Corning Life Sciences; Corning, New York). In certain embodiments, the scaffold-based structure can be made with a 3D printer. As used herein, the term "scaffold" refers to a structure comprising a biocompatible material. The material provides a suitable surface for the attachment and growth of at least one somatic cell and at least one embryo. The scaffold can provide mechanical stability and support. The scaffold can be a particular shape or form that influences or defines the three-dimensional shape or form assumed by a population of at least one somatic cell and at least one embryo. Such shapes or forms include, but are not limited to, a film (e.g., a shape having two dimensions substantially greater than the third), a ribbon, a cord, a sheet, a flat disk, a cylinder, a sphere, or a three-dimensional amorphous shape.

[0013] In certain embodiments, the three-dimensional structure is a scaffold-free structure. The scaffold-free structure can, for example, comprise a cluster of cells that form the three-dimensional structure. As used herein, "cell clumps" or "cell aggregation" refers to the formation of an aggregate by clustering and adhering of initially separate cells. Cell clumps are one of several major types of cellular organization and include cells that are loosely grouped together, not tightly bound, and therefore do not form tissue. Examples include the clustering of single-cell organisms or blood cells in suspension, and the aggregation of mesenchymal cells during cartilage formation. In certain embodiments, the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells, and / or cells of the reproductive tract (e.g., cells of the fallopian tube or cells of the uterus). The somatic cells may, for example, be of the same species as the embryo or of a different species. As used herein, "somatic cell" refers to any cell of an organism other than germ cells (ie, sperm and egg cells). In certain embodiments, the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptilian embryo, a fish embryo, an amphibian embryo, and a marsupial embryo. The embryo can be, for example, a mammalian embryo.

[0014] In certain embodiments, the cell culture medium is selected from at least one of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F-12), N2B27, M16, potassium-supplemented SOM (KSOM), tissue culture medium 199 (TCM-199), or a custom medium. The cell culture medium may contain, for example, sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ ) may be included. In certain embodiments, the somatic cells are embedded in or placed near the three-dimensional structure. In certain embodiments, the embryos are embedded in or placed near the three-dimensional structure. The somatic cells and / or embryos may be embedded in the three-dimensional structure or placed in holes / cavities created within the three-dimensional structure.

[0015] In certain embodiments, the 3D co-culture system further comprises a bioreactor. Bioreactors can enable precise and reproducible control over the environmental conditions of the embryo and somatic cell cultures. These environmental conditions can include, for example, temperature, pH, medium flow rate, oxygen, nutrient supply, and removal of unwanted metabolites. Furthermore, increasingly complex systems are being designed for simultaneous control of cell seeding onto the scaffold. The ability to maintain and monitor the environment during growth is common to these advanced systems. Several designs of bioreactors exist, including, but not limited to, rotating-wall vessels, direct perfusion systems, hollow fibers, spinner flasks, and mechanical force systems. Bioreactors are known in the art. See, e.g., Martin et al., "The role of bioreactors in tissue engineering," Trends Biotechnol. 22:80-86 (2004).

[0016] How to use In another general aspect, the present invention relates to a method for growing an embryo in a three-dimensional co-culture system, the method comprising: (a) culturing at least one somatic cell; (b) embedding the at least one somatic cell in a three-dimensional structure; (c) obtaining at least one embryo; and (d) embedding or placing the at least one embryo in or near the three-dimensional structure; wherein the at least one somatic cell and the at least one embryo grow in or near the three-dimensional structure in a cell culture medium. For example, the embryo can be embedded in a three-dimensional structure or placed in a hole / cavity created in the three-dimensional structure. The embryo can also be placed near the three-dimensional structure. After the embryo is placed in the three-dimensional co-culture system, the culture medium is changed or modified to maximize embryo development. As a non-limiting example, if the embryo has different requirements than somatic cells, the culture medium can be changed or modified to maximize embryo development. For example, bovine embryos can be cultured in a chemically defined culture medium, while somatic cells may prefer a medium supplemented with fetal bovine serum.

[0017] In certain embodiments of the methods of the present invention, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can include, for example, a gel-like substance or a structural scaffold. The gel-like substance can be selected from, for example, hydrogel, agarose, basement membrane extract, or extracellular matrix. Gel-like substances are known in the art and are commercially available. See, for example, MyoGel (pharmasana.co.uk); OBAGEL® (Obatala; New Orleans, Louisiana); CETUREGEL™ (Yesen Biotechnology; Shanghai, China); JELLAGEL® (Jellagen; Wales); GROWDEX® (UPM Biomedicals; Helsinki, Finland); GELTREX™ (Thermo Fisher Scientific; Waltham, Massachusetts); and Matrigel® (Corning Life Sciences; Corning, New York). In certain embodiments of the methods of the present invention, the scaffold-based structure is produced by a 3D printer. In certain embodiments of the methods of the present invention, the three-dimensional structure is a scaffold-free structure. The scaffold-free structure can, for example, comprise a cluster of cells forming the three-dimensional structure. In certain embodiments of the methods of the invention, the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells, and / or cells of the reproductive tract (e.g., cells of the fallopian tube or cells of the uterus). The somatic cells may, for example, be of the same species as the embryo or of a different species. In certain embodiments, the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptilian embryo, a fish embryo, an amphibian embryo, and a marsupial embryo. The embryo can be, for example, a mammalian embryo. In certain embodiments of the methods of the present invention, the cell culture medium is selected from at least one of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F-12), N2B27, M16, potassium-supplemented SOM (KSOM), tissue culture medium 199 (TCM-199), or custom media. The cell culture medium may contain, for example, sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ ) may be included. In certain embodiments of the methods of the present invention, the somatic cells are embedded in or placed near the three-dimensional structure, hi certain embodiments, the embryos are embedded in or placed near the three-dimensional structure. In certain embodiments of the methods of the present invention, the three-dimensional co-culture system further comprises a bioreactor.

[0018] kit In another general aspect, the present invention relates to a kit containing components necessary for a three-dimensional co-culture system, which may include, for example, (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture medium. In certain embodiments of the kit of the present invention, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can include, for example, a gel-like substance or a structural scaffold. The gel-like substance can be selected from, for example, hydrogel, agarose, basement membrane extract, or extracellular matrix. Gel-like substances are known in the art and are commercially available. See, for example, MyoGel (pharmasana.co.uk); OBAGEL® (Obatala; New Orleans, Louisiana); CETUREGEL™ (Yesen Biotechnology; Shanghai, China); JELLAGEL® (Jellagen; Wales); GROWDEX® (UPM Biomedicals; Helsinki, Finland); GELTREX™ (Thermo Fisher Scientific; Waltham, Massachusetts); and Matrigel® (Corning Life Sciences; Corning, New York). In certain embodiments of the kits of the present invention, the scaffold-based structure is produced by a 3D printer. In a specific embodiment of the kit of the present invention, the three-dimensional structure in the kit is a scaffold-free structure. The scaffold-free structure can, for example, comprise a cell cluster that forms a three-dimensional structure.

[0019] In certain embodiments of the kits of the invention, the somatic cells of the kit are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells, and / or cells of the reproductive tract (e.g., cells of the fallopian tube or cells of the uterus). The somatic cells may be, for example, of the same species as the embryo or of a different species. In certain embodiments, the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptilian embryo, a fish embryo, an amphibian embryo, and a marsupial embryo. The embryo can be, for example, a mammalian embryo. In certain embodiments of the kits of the invention, the cell culture medium is selected from at least one of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12 (DMEM / F-12), N2B27, M16, potassium-supplemented SOM (KSOM), tissue culture medium 199 (TCM-199), or custom media. The cell culture medium may contain, for example, sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ ) may be included. In certain embodiments of the kit of the present invention, the somatic cells of the kit are embedded in or placed near a three-dimensional structure, hi certain embodiments, the embryos of the kit are embedded in or placed near a three-dimensional structure. In certain embodiments, the kit further comprises a bioreactor.

[0020] Embodiment The present invention also provides the following non-limiting embodiments. Embodiment 1 is (a) Three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) Cell culture medium It is a three-dimensional co-culture system comprising: Embodiment 2 is the three-dimensional co-culture system according to embodiment 1, wherein the three-dimensional structure is a scaffold-based structure. Embodiment 3 is the three-dimensional co-culture system of embodiment 2, wherein the scaffold-based structure comprises a gel-like substance or a structural scaffold. Embodiment 4 is the three-dimensional co-culture system of embodiment 3, wherein the gel-like substance is selected from a hydrogel, agarose, a basement membrane extract, or an extracellular matrix. Embodiment 5 is a three-dimensional co-culture system according to embodiment 3, wherein the structural scaffold is made by a 3D printer. Embodiment 6 is the three-dimensional co-culture system according to embodiment 1, wherein the three-dimensional structure is a scaffold-free structure. Embodiment 7 is a three-dimensional co-culture system according to embodiment 6, wherein the scaffold-free structure comprises cell clusters that form a three-dimensional structure.

[0021] Embodiment 8 is a three-dimensional co-culture system according to any one of embodiments 1 to 7, wherein the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells and / or cells of the reproductive tract. Embodiment 9 is the three-dimensional co-culture system according to any one of embodiments 1 to 8, wherein the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptile embryo, a fish embryo, an amphibian embryo, and a marsupial embryo. Embodiment 10 is the three-dimensional co-culture system of embodiment 9, wherein the embryo is a mammalian embryo. Embodiment 11 is the three-dimensional co-culture system of any one of embodiments 1 to 10, wherein the cell culture medium is selected from at least one of MEM, DMEM, RPMI, DMEM / F-12, N2B27, M16, KSOM, TCM-199, or a custom medium. Embodiment 12 is a method for treating a cell culture medium containing sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ 12. The three-dimensional co-culture system according to any one of embodiments 1 to 11, comprising at least one of: Embodiment 13 is a three-dimensional co-culture system according to any one of embodiments 1 to 12, wherein the somatic cells are embedded in or placed near the three-dimensional structure. Embodiment 14 is a three-dimensional co-culture system according to any one of embodiments 1 to 13, wherein the embryo is embedded in or placed near the three-dimensional structure. Embodiment 15 is a three-dimensional co-culture system according to any one of embodiments 1 to 14, further comprising a bioreactor.

[0022] Embodiment 16 is (a) culturing at least one somatic cell; (b) embedding at least one somatic cell in the three-dimensional structure; (c) obtaining at least one embryo; (d) implanting or placing at least one embryo in or near the three-dimensional structure; A method of growing an embryo in a three-dimensional co-culture system, in which at least one somatic cell and at least one embryo are grown in or near a three-dimensional structure in cell culture medium. Embodiment 17 is the method of embodiment 16, wherein the three-dimensional structure is a scaffold-based structure. Embodiment 18 is the method of embodiment 17, wherein the scaffold-based structure comprises a gel-like material or a structural scaffold. Embodiment 19 is the method of embodiment 18, wherein the gel-like substance is selected from a hydrogel, agarose, a basement membrane extract, or an extracellular matrix. Embodiment 20 is the method of embodiment 18, wherein the structural scaffold is made by a 3D printer. Embodiment 21 is the method of embodiment 16, wherein the three-dimensional structure is a scaffold-free structure.

[0023] Embodiment 22 is the method of embodiment 21, wherein the scaffold-free structure comprises cell clusters that form a three-dimensional structure. Embodiment 23 is the method according to any one of embodiments 16 to 22, wherein the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells and / or cells of the reproductive tract. Embodiment 24 is the method of any one of embodiments 16 to 23, wherein the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptilian embryo, a fish embryo, an amphibian embryo, and a marsupial embryo. Embodiment 25 is the method of embodiment 24, wherein the embryo is a mammalian embryo. Embodiment 26 is the method of any one of embodiments 16 to 25, wherein the cell culture medium is selected from at least one of MEM, DMEM, RPMI, DMEM / F-12, N2B27, M16, KSOM, TCM-199, or custom medium. Embodiment 27 is a method for treating a cell culture medium containing sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ 27. The method according to any one of embodiments 16 to 26, comprising at least one of:

[0024] Embodiment 28 is (a) Three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) Cell culture medium The kit includes: Embodiment 29 is the kit according to embodiment 28, wherein the three-dimensional structure is a scaffold-based structure. Embodiment 30 is the kit of embodiment 29, wherein the scaffold-based structure comprises a gel-like substance or a structural scaffold. Embodiment 31 is the kit of embodiment 30, wherein the gel-like substance is selected from a hydrogel, agarose, a basement membrane extract, or an extracellular matrix. Embodiment 32 is a kit according to embodiment 30, wherein the structural scaffold is made by a 3D printer. Embodiment 33 is a kit according to embodiment 28, wherein the three-dimensional structure is a scaffold-free structure. Embodiment 34 is a kit according to embodiment 33, wherein the scaffold-free structure comprises cell clusters that form a three-dimensional structure. Embodiment 35 is a kit according to any one of embodiments 28 to 34, wherein the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells and / or cells of the reproductive tract.

[0025] Embodiment 36 is the kit of any one of embodiments 28 to 35, wherein the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptile embryo, a fish embryo, an amphibian embryo, and a marsupial embryo. Embodiment 37 is the kit of embodiment 36, wherein the embryo is a mammalian embryo. Embodiment 38 is a kit according to any one of embodiments 28 to 37, wherein the cell culture medium is selected from at least one of MEM, DMEM, RPMI, DMEM / F-12, N2B27, M16, KSOM, TCM-199, or custom medium. Embodiment 39 is directed to a method for preparing a cell culture medium containing sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ 39. The kit according to any one of embodiments 28 to 38, comprising at least one of: Embodiment 40 is a kit according to any one of embodiments 28 to 39, wherein the somatic cells are embedded in or placed near a three-dimensional structure. Embodiment 41 is a kit according to any one of embodiments 28 to 40, wherein the embryo is embedded in or placed near a three-dimensional structure. Embodiment 42 is a kit according to any one of embodiments 28 to 41, wherein the three-dimensional co-culture system further comprises a bioreactor. [Example]

[0026] Example 1: Three-dimensional co-culture system Preparation of three-dimensional structures (3D structures) GELTREX™ (Thermo Fisher Scientific; Waltham, MA) was thawed overnight on ice in a 4°C refrigerator. A 1000 μl tip rack, a 100 μl tip rack, and 10-1.5 ml tubes were also placed in the 4°C refrigerator overnight. After overnight incubation, the thawed GELTREX™ was removed from the refrigerator and placed on ice. Preparation of somatic cells for 3D co-cultureSomatic cells (e.g., cumulus cells) were cultured in cell culture medium. The cumulus cells were washed twice with PBS. Four drops of trypsin were added to a cell culture flask. The cells were incubated at 38.5°C for 5-10 minutes. The flask was gently tapped to separate the cells. The cells were divided into two 1.5 ml tubes. The cells were centrifuged at 500 g for 5 minutes. The supernatant was discarded after centrifugation. Plating somatic cells to create 3D structures 1 ml of standard DMEM medium was added to one of the tubes and the cells were mixed gently. 500 μl of cells were plated into two wells of a 4-well plate. The 4-well plate was placed in an incubator. This cell preparation grew as a monolayer and was used as a control. 200 μl of GELTREX™ was added to the other tube and gently mixed by pipetting the contents of the tube up and down three times. 50 μl of cells combined with GELTREX™ were plated into each well of a 4-well plate. The 4-well plate was placed in an incubator for 20-30 minutes. The 4-well plate was removed from the incubator and observed for solidification of the GELTREX™. Once the GELTREX™ had solidified, 450 μl of standard DMEM was added to each well of the 4-well plate. The 4-well plate was placed in an incubator and observed for the formation of a 3D cell culture. Continuing 3D culture The 3D cultures were inspected daily under an inverted microscope to observe cell growth and cell clump formation. When the medium color turned yellow, the cell culture medium was changed as needed, as indicated. The yellow color indicates a decrease in the pH of the cell culture medium. Photographs of the 3D cultures were taken using an EVOS microscope (Thermo Fisher Scientific; Waltham, MA) (Figure 2). On day 1 of plating, embryos were either embedded in the 3D co-culture or placed into holes / cavities within the 3D co-culture structure. After embryo placement, the 3D co-cultures were inspected daily for cell proliferation, structural integrity, and morphology in the 3D structures, and the culture medium was changed as needed to maximize embryo development. The culture medium was changed when the medium color turned yellow. The yellow color indicates a decrease in the pH of the medium. Photographs of the 3D co-cultures were taken to record the development of the embryos (Figure 3).

[0027] Example 2: Three-dimensional co-culture system for culturing embryos Preparation of 3D structures with bovine cumulus cells Thaw the GELTREX® solution overnight at 4°C. The next day, add the thawed GELTREX® solution to a pre-chilled 0.5 ml tube. The amount of GELTREX® can be varied based on the experiment and ranges from 200 to 600 μl. Ten microliters of a single-cell suspension of bovine cumulus cells was added to 100 μl of GELTREX® solution. The number of bovine cumulus cells per 100 μl of GELTREX® solution can vary from 1,000 to 10,000. The cells were mixed with the GELTREX® solution by gently pipetting up and down several times. The mixture of GELTREX® and cells was plated in a 48-well plate. 50 to 100 μl of the mixture was seeded into each well of the 48-well plate. The plate was incubated at 37°C for 10 minutes to allow the GELTREX® solution to solidify. At the end of the incubation period, fresh TCM-199 medium supplemented with 15% fetal bovine serum (FBS) was added to each well. The plate was then placed in an incubator at 37°C in a 5% CO2 atmosphere in air until use. Collection and culture of preimplantation mouse embryos Three-week-old CD-1 female mice (Charles River Laboratories, Wilmington, MA) were treated with 5 IU (international units) of PMSG (pregnant mare serum gonadotropin) (Biovendor R&D, Czech Republic), followed 48 hours later by 5 IU of hCG (human chorionic gonadotropin) (Millipore; Burlington, MA). The females were then mated overnight with breeding B6D2F1 or 129SV males (Jackson Laboratory; Bar Harbor, ME). The following morning, females were inspected for the presence of a mating plug, and only females with a plug were used for embryo collection. Preimplantation mouse embryos were collected from females with a plug. Mouse embryos were placed in the 3D GELTREX® and cell solution in a 48-well plate prepared as described above. Mouse embryos were cultured at 37°C in an atmosphere of 5% CO2 in air. Culture medium was changed every other day or as needed. Mouse embryo development was monitored daily.

[0028] result Development of preimplantation E3.5 mouse embryos cultured in a three-dimensional in vitro culture system Eleven E3.5 mouse embryos (CD-1 female × B6DF1 male) were placed in a 3D culture system using GELTREX® and bovine cumulus cells. The mouse embryos were cultured in TCM-199 medium supplemented with 15% FBS at 37°C in a 5% CO2 atmosphere in air. All E3.5 mouse embryos hatched on day 2 of culture, reaching the 3D system (Figure 4). The mouse embryos continued to develop and formed embryos comparable to E5.5 in the 3D culture system, which are equivalent to in vivo (Figure 5). Mouse embryos continued to develop in the 3D culture system, and six embryos developed structures with extension bodies, stem structures, and placenta-like structures attached to the 3D system (Figure 6). These structures were present until day 9 of culture. Development of rhythmic contractions in E4.5 mouse embryos cultured in a three-dimensional in vitro culture systemPreimplantation E4.5 mouse embryos (Figure 7) were collected from CD-1 females mated with B6DF1 males and placed in a 3D in vitro culture system. Mouse embryos were cultured in TCM-199XEP medium (Table 1) at 37°C in a 5% CO atmosphere. Figures 8A-8E illustrate the developmental progression of E4.5 mouse embryos. Embryos that reached the 3D system on day 1 of culture developed into E5.5 embryos on day 2 and continued to develop in the 3D culture on days 3, 4, and 5. [Table 1] On day 5 of culture, the embryos were treated with 0.5 μM retinoic acid. On day 8 of culture, the embryos exhibited rhythmic contractions similar to those of cardiomyocytes. The rhythmic contractions continued until day 18 of culture, when they slowed and eventually ceased, presumably due to nutrient depletion. Rhythmic contractions were observed in other embryos developed in the same culture system.

[0029] conclusion A static 3D in vitro embryo culture system has been developed and demonstrated to be capable of supporting the development of preimplantation mouse embryos beyond the postimplantation stage. The 3D system can also support organogenesis, as evidenced by rhythmic contractions of the developing embryo. This is the first static in vitro culture system capable of supporting postimplantation embryo development and organogenesis in vitro.

[0030] Those skilled in the art will recognize that changes can be made to the above-described embodiments without departing from these broad inventive concepts. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by this description.

Claims

1. (a) Three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture medium A three-dimensional co-culture system comprising:

2. The three-dimensional co-culture system according to claim 1 , wherein the three-dimensional structure is a scaffold-based structure.

3. The three-dimensional co-culture system of claim 2 , wherein the scaffold-based structure comprises a gel-like substance or a structural scaffold.

4. The three-dimensional co-culture system of claim 3 , wherein the gel-like substance is selected from a hydrogel, agarose, a basement membrane extract, or an extracellular matrix.

5. The three-dimensional co-culture system of claim 3, wherein the structural scaffold is produced by a 3D printer.

6. The three-dimensional co-culture system according to claim 1 , wherein the three-dimensional structure is a scaffold-free structure.

7. The three-dimensional co-culture system of claim 6 , wherein the scaffold-free structure comprises cell clusters that form a three-dimensional structure.

8. The three-dimensional co-culture system according to any one of claims 1 to 7, wherein the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells and / or cells of the reproductive tract.

9. The three-dimensional co-culture system according to any one of claims 1 to 8, wherein the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptilian embryo, a fish embryo, an amphibian embryo, and a marsupial embryo.

10. The three-dimensional co-culture system of claim 9, wherein the embryo is a mammalian embryo.

11. 11. The three-dimensional co-culture system of any one of claims 1 to 10, wherein the cell culture medium is selected from at least one of MEM, DMEM, RPMI, DMEM / F-12, N2B27, M16, KSOM, TCM-199 or a custom medium.

12. The cell culture medium contains sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ The three-dimensional co-culture system according to any one of claims 1 to 11, comprising at least one of the following:

13. The three-dimensional co-culture system according to any one of claims 1 to 12, wherein the somatic cells are embedded in the three-dimensional structure or placed in the vicinity of the three-dimensional structure.

14. The three-dimensional co-culture system according to any one of claims 1 to 13, wherein the embryo is embedded in or placed near the three-dimensional structure.

15. The three-dimensional co-culture system according to any one of claims 1 to 14, further comprising a bioreactor.

16. 1. A method for growing embryos in a three-dimensional co-culture system, comprising: (a) culturing at least one somatic cell; (b) embedding said at least one somatic cell in a three-dimensional structure; (c) obtaining at least one embryo; (d) implanting or placing the at least one embryo in or near the three-dimensional structure; The method, wherein said at least one somatic cell and at least one embryo are grown in a cell culture medium on or near said three-dimensional structure.

17. The method of claim 16, wherein the three-dimensional structure is a scaffold-based structure.

18. The method of claim 17 , wherein the scaffold-based structure comprises a gel-like substance or a structural scaffold.

19. 19. The method of claim 18, wherein the gel-like substance is selected from a hydrogel, agarose, a basement membrane extract, or an extracellular matrix.

20. 20. The method of claim 18, wherein the structural scaffold is made with a 3D printer.

21. The method of claim 16, wherein the three-dimensional structure is a scaffold-free structure.

22. 22. The method of claim 21, wherein the scaffold-free structure comprises cell clusters that form a three-dimensional structure.

23. 23. The method of any one of claims 16 to 22, wherein the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells and / or cells of the reproductive tract.

24. 24. The method of any one of claims 16 to 23, wherein the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptilian embryo, a fish embryo, an amphibian embryo, and a marsupial embryo.

25. 25. The method of claim 24, wherein the embryo is a mammalian embryo.

26. 26. The method of any one of claims 16 to 25, wherein the cell culture medium is selected from at least one of MEM, DMEM, RPMI, DMEM / F-12, N2B27, M16, KSOM, TCM-199, or custom medium.

27. The cell culture medium contains sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ 27. The method of any one of claims 16 to 26, comprising at least one of:

28. (a) Three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture medium Kit including:

29. The kit according to claim 28, wherein the three-dimensional structure is a scaffold-based structure.

30. 30. The kit of claim 29, wherein the scaffold-based structure comprises a gel-like substance or a structural scaffold.

31. 31. The kit of claim 30, wherein the gel-like substance is selected from a hydrogel, agarose, a basement membrane extract, or an extracellular matrix.

32. 31. The kit of claim 30, wherein the structural scaffold is made with a 3D printer.

33. 29. The kit of claim 28, wherein the three-dimensional structure is a scaffold-free structure.

34. 34. The kit of claim 33, wherein the scaffold-free structure comprises a cluster of cells that form a three-dimensional structure.

35. 35. The kit of any one of claims 28 to 34, wherein the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells and / or cells of the reproductive tract.

36. 36. The kit of any one of claims 28 to 35, wherein the embryo is selected from the group consisting of a mammalian embryo, an avian embryo, a reptilian embryo, a fish embryo, an amphibian embryo, and a marsupial embryo.

37. 37. The kit of claim 36, wherein the embryo is a mammalian embryo.

38. 38. The kit of any one of claims 28 to 37, wherein the cell culture medium is selected from at least one of MEM, DMEM, RPMI, DMEM / F-12, N2B27, M16, KSOM, TCM-199, or custom medium.

39. The cell culture medium contains sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ 39. The kit of any one of claims 28 to 38, comprising at least one of:

40. 40. The kit of any one of claims 28 to 39, wherein the somatic cells are embedded in or placed near the three-dimensional structure.

41. 41. The kit of any one of claims 28 to 40, wherein the embryo is embedded in or placed near the three-dimensional structure.

42. 42. The kit of any one of claims 28 to 41, wherein the three-dimensional co-culture system further comprises a bioreactor.

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