Three-dimensional co-culture system for culturing embryo in vitro and application thereof

Abstract

Provided herein are three-dimensional co-culture systems, methods of using the three-dimensional co-culture systems, and kits including the three-dimensional co-culture systems.

Description

[0001] Cross - reference to related applications

[0002] This application claims the priority of U.S. Provisional Application No. 63 / 378,929, filed on October 10, 2022, the disclosure of which is incorporated herein by reference in its entirety. Technical field

[0003] 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 comprises (a) a three - dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) a cell culture medium. Background art

[0004] The in vitro production of embryos has several advantages over the production of embryos from in - vivo sources, including but not limited to the effective selection of superior genes for transgenic modification to rapidly obtain animals with desired traits. Genetic engineering can provide powerful tools to help understand the basic mechanisms regulating physiology.

[0005] Since the in vitro environment remains sub - optimal for embryo growth and development, there is a need to develop better systems for culturing embryos in vitro to mimic the in - vivo environment, thereby providing robust and viable embryos, providing a higher success rate for the in vitro fertilization process, providing better efficiency in the reproductive technologies of agriculturally important animals, and providing better efficiency for the conservation of extinct and endangered species through reproductive technologies. Summary of the invention

[0006] In one aspect, the present invention relates to a three - dimensional co - culture system. The three - dimensional co - culture system can, for example, comprise (a) a three - dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) a cell culture medium.

[0007] Also provided is a method for culturing embryos in a three - dimensional co - culture system. The method comprises (a) culturing at least one somatic cell; (b) implanting at least one somatic cell into a three - dimensional structure; (c) obtaining at least one embryo; and (d) implanting at least one embryo into the three - dimensional structure or placing it near the three - dimensional structure; wherein at least one somatic cell and at least one embryo grow in or near the three - dimensional structure in a cell culture medium.

[0008] Also provided is a kit, which comprises (a) a three - dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) a cell culture medium.

[0009] In certain embodiments, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can, for example, include a gel-like material or a structural scaffold. The gel-like material can, for example, be selected from hydrogels, agarose, basement membrane extracts, or extracellular matrices. In certain embodiments, the scaffold-based structure is created by a three-dimensional printer.

[0010] In certain embodiments, the three-dimensional structure is a scaffold-free structure. The scaffold-free structure can, for example, include cell aggregates that form the three-dimensional structure.

[0011] 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 from the reproductive tract.

[0012] In certain embodiments, the embryo is selected from mammalian embryos, avian embryos, reptilian embryos, fish embryos, amphibian embryos, and marsupial embryos. The embryo can, for example, be a mammalian embryo.

[0013] In certain embodiments, the cell culture medium is selected from at least one of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12), N2B27, M16, Potassium-Supplemented Somatic Cell Medium (KSOM), Tissue Culture Medium - 199 (TCM-199), or a custom medium. The cell culture medium can, for example, contain at least one of sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ), or magnesium ions (Mg 2+ ).

[0014] In certain embodiments, the somatic cells are implanted into the three-dimensional structure or placed near the three-dimensional structure. In certain embodiments, the embryo is implanted into the three-dimensional structure or placed near the three-dimensional structure.

[0015] In certain embodiments, the three-dimensional co-culture system further includes a bioreactor. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The foregoing summary of the invention and the following detailed description of the preferred embodiments will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present application is not limited to the exact embodiments shown in the drawings.

[0017] Figure 1A and Figure 1B show representative images of a monolayer cell culture ( Figure 1A ) and a three-dimensional (3D) cell culture ( Figure 1B ).

[0018] Figure 2 Shows representative images demonstrating the progression of a three-dimensional cell culture over an 8-day period. Somatic cells within the three-dimensional culture proliferate and interconnect to form a three-dimensional structure of cell aggregates. During the 8-day culture, more structures are formed.

[0019] Figure 3 Shows representative images of embryos cultured in a three-dimensional co-culture system in vitro. At least one embryo is implanted into the three-dimensional structure of somatic cells, which provides favorable growth conditions for the embryo. The three-dimensional structure also provides physical support to maintain the spatial morphological shape of the embryo during its growth and development.

[0020] Figure 4 Shows images demonstrating the contact of E3.5 mouse embryos with the three-dimensional system during in vitro culture.

[0021] Figure 5 Shows images demonstrating the development of E3.5 mouse embryos to the E5.5 stage in a three-dimensional in vitro culture system.

[0022] Figure 6 Shows images of a structure having an expanse connected to a three-dimensional in vitro culture system via a stalk-like structure and a placenta-like structure.

[0023] Figure 7 Shows images of pre-implantation E4.5 mouse embryos.

[0024] Figures 8A to 8E Shows images of the developmental progression of E4.5 mouse embryos in a three-dimensional in vitro culture system. Figure 8A Shows an image of Day 1; Figure 8B Shows an image of Day 2; Figure 8C Shows an image of Day 3; Figure 8D Shows an image of Day 4; Figure 8E Shows an image of Day 5. Detailed Description

[0025] Various publications, articles, and patents are cited or described in the background art and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of the documents, acts, materials, devices, articles, etc. included in this specification is for the purpose of providing context for the present invention. Such discussion does not admit that any or all of these constitute a part of the prior art with respect to any invention disclosed or claimed herein.

[0026] 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. Otherwise, certain terms used herein have the meanings as set forth in this specification.

[0027] It should be noted that, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents.

[0028] Unless otherwise indicated, any numerical values, such as the concentrations or concentration ranges described herein, should be understood to be modified in all instances by the term "about". Thus, numerical values generally include ±10% of the recited value. For example, a concentration of 1 mg / mL includes concentrations from 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible sub-ranges, all individual numerical values within the range, including integers and fractional values within such ranges, unless the context otherwise clearly indicates.

[0029] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to ascertain using only routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed within the scope of the invention.

[0030] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", or "containing", or any other variants thereof will be understood to imply the inclusion of the stated integer or group of integers, but not the exclusion of any other integer or group of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a series of elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, mixture, process, method, article, or apparatus. Additionally, unless expressly stated to the contrary, "or" refers to an inclusive or rather than 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).

[0031] As used herein, the conjunctive term "and / or" between a plurality of recited elements shall be understood to include both individual and combined options. For example, in the case where two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within this meaning and thus meets the requirements of the term "and / or" as used herein. The concurrent applicability of more than one option is also understood to fall within this meaning and thus meets the requirements of the term "and / or".

[0032] As used herein, the term "consists of" or variations thereof, such as "consist of" or "consisting of", as used throughout the specification and claims, indicates the inclusion of any recited integer or group of integers, but does not allow the addition of additional integers or groups of integers to the specified method, structure, or composition.

[0033] As used herein, the term "consists essentially of" or variations thereof, such as "consist essentially of" or "consisting essentially of", as used throughout the specification and claims, indicates the inclusion of any recited integer or group of integers, and optionally includes any recited integer or group of integers that do not materially alter the basic or novel characteristics of the specified method, structure, or composition. See M.P.E.P. §2111.03.

[0034] The words "right", "left", "lower", and "upper" designate directions in the reference drawings.

[0035] It should also be understood that, as understood by one of ordinary skill in the art, the terms "about", "approximately", "generally", "substantially", and similar terms, as used herein when referring to the dimensions or characteristics of the components of the preferred invention, indicate that the described dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations that are functionally the same or similar. At a minimum, such references to numerical parameters will include variations that do not change the least significant digit using mathematical and industrial principles recognized in the art (e.g., rounding, measurement error, or other systematic errors, manufacturing tolerances, etc.).

[0036] Three-dimensional co-culture system

[0037] The present invention provides a three-dimensional co-culture system for culturing embryos in vitro. The three-dimensional co-culture system provides an in vitro culture environment that is closer to the in vivo environment than a planar monolayer culture system. The three-dimensional co-culture system also provides three-dimensional support for embryos during their in vitro development. The three-dimensional co-culture system and methods of using the three-dimensional co-culture system provide an environment that results in better embryo viability and development. Better embryo viability and development can be demonstrated, for example, by higher success rates of human in vitro fertilization (IVF), by better efficiency in reproductive techniques for agriculturally important animals, and by better efficiency in reproductive techniques for protecting extinct and endangered species.

[0038] In one general aspect, the present invention relates to a three-dimensional co-culture system. The three-dimensional co-culture system can, for example, include (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) a cell culture medium.

[0039] As used herein, the term "three-dimensional co-culture" is generally 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 implanted 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 critical for mimicking the in vivo environment to allow the at least one cell and the at least one embryo to grow and / or progress in their own microenvironment. In certain embodiments, the three-dimensional co-culture system is suitable for in vitro cell culture.

[0040] In certain embodiments, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can, for example, include a gel-like material or a structural scaffold. The gel-like material can, for example, be selected from hydrogels, agarose, basement membrane extract, or extracellular matrix. The extracellular matrix can, for example, be 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 materials are known in the art and are commercially available, see, for example, MyoGel (pharmasana.co.uk); (Obatala; New Orleans, LA), CETUREGEL TM (Yeasen Biotechnology; Shanghai, China), (Jellagen; Wales), (UPM Biomedicals; Helsinki, Finland), GELTREX TM(Thermo Fisher; Waltham, MA), and (Corning LifeSciences; Corning, NY).

[0041] In certain embodiments, the scaffold-based structure can be created by a 3D printer.

[0042] As used herein, the term "scaffold" refers to a structure comprising a biocompatible material that provides a surface suitable for adhesion and proliferation of at least one somatic cell and at least one embryo. The scaffold can provide mechanical stability and support. The scaffold can be of a particular shape or form so as to influence or define 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, membranes (e.g., having a two-dimensional form that is substantially greater than a third dimension), strips, cords, sheets, flat disks, cylinders, spheres, or three-dimensional amorphous shapes.

[0043] In certain embodiments, the three-dimensional structure is a scaffold-free structure. The scaffold-free structure can, for example, comprise cell aggregates that form the three-dimensional structure.

[0044] As used herein, "cell aggregate" or "cell aggregation" refers to initially isolated cells that aggregate together and adhere to form an aggregate. Cell aggregates are one of several major types of cellular organizational structures, which include cells that are loosely grouped together, not tightly connected, and thus do not form tissue. Examples can include the aggregation of single-celled organisms or blood cells in suspension and the condensation of mesenchymal cells during cartilage formation.

[0045] 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 from the reproductive tract (e.g., cells from the fallopian tube or cells from the uterus). The somatic cells can, for example, be from the same or a different species as the embryo.

[0046] As used herein, "somatic cell" refers to any cell of a living organism other than germ cells (i.e., sperm and egg cells).

[0047] In certain embodiments, the embryos are selected from mammalian embryos, avian embryos, reptilian embryos, fish embryos, amphibian embryos, and marsupial embryos. The embryo can, for example, be a mammalian embryo.

[0048] In certain embodiments, the cell culture medium is selected from at least one of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12), N2B27, M16, Potassium-Supplemented Somatic Cell Medium (KSOM), Tissue Culture Medium - 199 (TCM-199), or a custom medium. The cell culture medium can, for example, contain at least one of sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ), or magnesium ions (Mg 2+ ).

[0049] In certain embodiments, somatic cells are implanted into or placed near a three-dimensional structure. In certain embodiments, an embryo is implanted into or placed near a three-dimensional structure. The somatic cells and / or the embryo can be implanted into a pore / cavity generated within or placed within the three-dimensional structure.

[0050] In certain embodiments, the three-dimensional co-culture system further includes a bioreactor. The bioreactor is capable of precise and reproducible control of the environmental conditions for culturing the embryo and somatic cells. These environmental conditions can, for example, include temperature, pH, media flow, oxygen, nutrient supply, and waste metabolite removal. Additionally, increasingly complex systems are being designed to simultaneously control the seeding of cells into the scaffold. A common feature of these advanced systems is the ability to maintain and monitor the environment during growth.

[0051] There are several bioreactor designs, including but not limited to rotating wall vessels, direct perfusion systems, hollow fibers, stirred flasks, and mechanical force systems. Bioreactors are known in the art; see, for example, Martin et al., “The role of bioreactors in tissue engineering,” Trends Biotechnol. 22:80 - 86 (2004).

[0052] Method of Use

[0053] In another aspect, the present invention relates to a method of culturing an embryo in a three-dimensional co-culture system. The method includes (a) culturing at least one somatic cell; (b) implanting the at least one somatic cell into a three-dimensional structure; (c) obtaining at least one embryo; and (d) implanting the at least one embryo into the three-dimensional structure or placing it near the three-dimensional structure; wherein the at least one somatic cell and the at least one embryo grow within the three-dimensional structure of the cell culture medium or near the three-dimensional structure.

[0054] Embryos can, for example, be implanted into a three-dimensional structure or placed in a pore / cavity generated within a three-dimensional structure. Embryos can also be placed near a three-dimensional structure. After placing the embryos in a three-dimensional co-culture system, the culture medium is replaced or improved to enable optimal development of the embryos. As a non-limiting example, if the requirements of the embryos are different from those of somatic cells, the culture medium can be replaced or improved to enable optimal development of the embryos. For example, bovine embryos can be cultured in a chemically defined medium, while somatic cells can preferably be cultured in a medium supplemented with fetal bovine serum.

[0055] In certain embodiments of the method of the present invention, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can, for example, include a gel-like material or a structural scaffold. The gel-like material can, for example, be selected from hydrogels, agarose, basement membrane extract, or extracellular matrix. Gel-like materials are known in the art and are commercially available. See, for example, MyoGel (pharmasana.co.uk); (Obatala; New Orleans, LA), CETUREGEL TM (Yeasen Biotechnology; Shanghai, China), (Jellagen; Wales), (UPM Biomedicals; Helsinki, Finland), GELTREX TM (Thermo Fisher; Waltham, MA), and (Corning Life Sciences; Corning, NY).

[0056] In certain embodiments of the method of the present invention, the scaffold-based structure is created by a three-dimensional printer.

[0057] In certain embodiments of the method of the present invention, the three-dimensional structure is a scaffold-free structure. The scaffold-free structure can, for example, include cell aggregates that form a three-dimensional structure.

[0058] In certain embodiments of the method of the present invention, the somatic cells are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells, and / or cells from the reproductive tract (e.g., cells from the fallopian tube or cells from the uterus). The somatic cells can, for example, be from the same or a different species as the embryos.

[0059] In certain embodiments, the embryos are selected from mammalian embryos, avian embryos, reptilian embryos, fish embryos, amphibian embryos, and marsupial embryos. The embryos can, for example, be mammalian embryos.

[0060] In certain embodiments of the method 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 simplex optimized medium (KSOM), tissue culture medium-199 (TCM-199), or a custom medium. The cell culture medium can, for example, contain at least one of sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ), or magnesium ions (Mg 2+ ).

[0061] In certain embodiments of the method of the present invention, the somatic cells are implanted into or placed near a three-dimensional structure. In certain embodiments, the embryo is implanted into or placed near a three-dimensional structure.

[0062] In certain embodiments of the method of the present invention, the three-dimensional co-culture system further includes a bioreactor.

[0063] Kit

[0064] In another general aspect, the present invention relates to a kit comprising the components necessary for a three-dimensional co-culture system. The kit can, for example, include (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) a cell culture medium.

[0065] In certain embodiments of the kit of the present invention, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can, for example, include a gel-like material or a structural scaffold. The gel-like material can, for example, be selected from hydrogels, agarose, basement membrane extract, or extracellular matrix. Gel-like materials are known in the art and are commercially available, see, for example, MyoGel (pharmasana.co.uk); (Obatala; New Orleans, LA), CETUREGEL TM (Yeasen Biotechnology; Shanghai, China), (Jellagen; Wales), (UPM Biomedicals; Helsinki, Finland), GELTREX TM (Thermo Fisher; Waltham, MA), and (Corning Life Sciences; Corning, NY).

[0066] In certain embodiments of the kit of the present invention, the scaffold-based structure is created by a 3D printer.

[0067] In certain embodiments 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, include cell aggregates that form the three-dimensional structure.

[0068] In certain embodiments of the kit of the present invention, the somatic cells in the kit are selected from skin cells, bone cells, blood cells, connective tissue cells, cumulus cells, granulosa cells, and / or cells from the reproductive tract (e.g., cells from the fallopian tube or cells from the uterus). The somatic cells can, for example, be from the same or a different species as the embryo.

[0069] In certain embodiments, the embryo is selected from mammalian embryos, avian embryos, reptilian embryos, fish embryos, amphibian embryos, and marsupial embryos. The embryo can, for example, be a mammalian embryo.

[0070] In certain embodiments of the kit of the present invention, the cell culture medium is selected from at least one of Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Roswell Park Memorial Institute Medium (RPMI), Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F-12), N2B27, M16, Potassium-Supplemented Somatic Cell Medium (KSOM), Tissue Culture Medium-199 (TCM-199), or a custom medium. The cell culture medium can, for example, contain at least one of sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ), or magnesium ions (Mg 2+ ).

[0071] In certain embodiments of the kit of the present invention, the somatic cells in the kit are implanted into the three-dimensional structure or placed near the three-dimensional structure. In certain embodiments, the embryo in the kit is implanted into the three-dimensional structure or placed near the three-dimensional structure.

[0072] In certain embodiments, the kit further includes a bioreactor.

[0073] Embodiments

[0074] The present invention also provides the following non-limiting embodiments.

[0075] Embodiment 1 is a three-dimensional co-culture system, which includes:

[0076] (a) A three-dimensional structure;

[0077] (b) At least one somatic cell;

[0078] (c) At least one embryo; and

[0079] (d) A cell culture medium.

[0080] Embodiment 2 is a three-dimensional co-culture system according to Embodiment 1, wherein the three-dimensional structure is a scaffold-based structure.

[0081] Embodiment 3 is a three-dimensional co-culture system according to Embodiment 2, wherein the scaffold-based structure comprises a gel-like material or a structural scaffold.

[0082] Embodiment 4 is a three-dimensional co-culture system according to Embodiment 3, wherein the gel-like material is selected from hydrogels, agarose, basement membrane extracts or extracellular matrices.

[0083] Embodiment 5 is a three-dimensional co-culture system according to Embodiment 3, wherein the structural scaffold is created by a three-dimensional printer.

[0084] Embodiment 6 is a three-dimensional co-culture system according to Embodiment 1, wherein the three-dimensional structure is a scaffold-free structure.

[0085] Embodiment 7 is a three-dimensional co-culture system according to Embodiment 6, wherein the scaffold-free structure comprises cell aggregates that form a three-dimensional structure.

[0086] 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 from the reproductive tract.

[0087] Embodiment 9 is a three-dimensional co-culture system according to any one of Embodiments 1 to 8, wherein the embryos are selected from mammalian embryos, avian embryos, reptilian embryos, fish embryos, amphibian embryos and marsupial embryos.

[0088] Embodiment 10 is a three-dimensional co-culture system according to Embodiment 9, wherein the embryo is a mammalian embryo.

[0089] Embodiment 11 is a three-dimensional co-culture system according to 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.

[0090] Embodiment 12 is a three-dimensional co-culture system according to any one of Embodiments 1 to 11, wherein the cell culture medium contains sodium ions (Na + ) and potassium ions (K +) Calcium ion (Ca 2+ ) or magnesium ion (Mg 2+ ), or at least one of them.

[0091] Embodiment 13 is a three-dimensional co-culture system according to any one of Embodiments 1 to 12, wherein the somatic cells are implanted into the three-dimensional structure or placed near the three-dimensional structure.

[0092] Embodiment 14 is a three-dimensional co-culture system according to any one of Embodiments 1 to 13, wherein the embryo is implanted into the three-dimensional structure or placed near the three-dimensional structure.

[0093] Embodiment 15 is a three-dimensional co-culture system according to any one of Embodiments 1 to 14, wherein the three-dimensional co-culture system further includes a bioreactor.

[0094] Embodiment 16 is a method for culturing an embryo in a three-dimensional co-culture system, the method comprising:

[0095] (a) Culturing at least one somatic cell;

[0096] (b) Implanting at least one somatic cell into a three-dimensional structure;

[0097] (c) Obtaining at least one embryo; and

[0098] (d) Implanting at least one embryo into the three-dimensional structure or placing it near the three-dimensional structure;

[0099] 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.

[0100] Embodiment 17 is the method according to Embodiment 16, wherein the three-dimensional structure is a scaffold-based structure.

[0101] Embodiment 18 is the method according to Embodiment 17, wherein the scaffold-based structure includes a gel-like material or a structural scaffold.

[0102] Embodiment 19 is the method according to Embodiment 18, wherein the gel-like material is selected from hydrogels, agarose, basement membrane extracts, or extracellular matrix.

[0103] Embodiment 20 is the method according to Embodiment 18, wherein the structural scaffold is created by a three-dimensional printer.

[0104] Embodiment 21 is the method according to Embodiment 16, wherein the three-dimensional structure is a scaffold-free structure.

[0105] Embodiment 22 is the method according to Embodiment 21, wherein the scaffold-free structure comprises cell aggregates forming a three-dimensional structure.

[0106] 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 from the reproductive tract.

[0107] Embodiment 24 is the method according to any one of Embodiments 16 to 23, wherein the embryo is selected from mammalian embryos, avian embryos, reptilian embryos, fish embryos, amphibian embryos, and marsupial embryos.

[0108] Embodiment 25 is the method according to Embodiment 24, wherein the embryo is a mammalian embryo.

[0109] Embodiment 26 is the method according to 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 a custom medium.

[0110] Embodiment 27 is the method according to any one of Embodiments 16 to 26, wherein the cell culture medium contains at least one of sodium ions (Na + ), potassium ions (K + ), calcium ions (Ca 2+ ), or magnesium ions (Mg 2+ ).

[0111] Embodiment 28 is a kit, comprising:

[0112] (a) A three-dimensional structure;

[0113] (b) At least one somatic cell;

[0114] (c) At least one embryo; and

[0115] (d) A cell culture medium.

[0116] Embodiment 29 is the kit according to Embodiment 28, wherein the three-dimensional structure is a scaffold-based structure.

[0117] Embodiment 30 is the kit according to Embodiment 29, wherein the scaffold-based structure comprises a gel-like material or a structural scaffold.

[0118] Embodiment 31 is the kit according to Embodiment 30, wherein the gel-like material is selected from hydrogels, agarose, basement membrane extracts, or extracellular matrix.

[0119] Embodiment 32 is a kit according to Embodiment 30, wherein the structural scaffold is created by a 3D printer.

[0120] Embodiment 33 is a kit according to Embodiment 28, wherein the three-dimensional structure is a scaffold-free structure.

[0121] Embodiment 34 is a kit according to Embodiment 33, wherein the scaffold-free structure comprises cell aggregates forming a three-dimensional structure.

[0122] 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 from the reproductive tract.

[0123] Embodiment 36 is a kit according to any one of Embodiments 28 to 35, wherein the embryo is selected from mammalian embryos, avian embryos, reptilian embryos, fish embryos, amphibian embryos, and marsupial embryos.

[0124] Embodiment 37 is a kit according to Embodiment 36, wherein the embryo is a mammalian embryo.

[0125] 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 a custom medium.

[0126] Embodiment 39 is a kit according to any one of Embodiments 28 to 38, wherein the cell culture medium contains at least one of sodium ions (Na + )、potassium ions (K + )、calcium ions (Ca 2+ ) or magnesium ions (Mg 2+ ).

[0127] Embodiment 40 is a kit according to any one of Embodiments 28 to 39, wherein the somatic cells are implanted into the three-dimensional structure or placed near the three-dimensional structure.

[0128] Embodiment 41 is a kit according to any one of Embodiments 28 to 40, wherein the embryo is implanted into the three-dimensional structure or placed near the three-dimensional structure.

[0129] 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.

[0130] Examples

[0131] Example 1: Three-dimensional co-culture system

[0132] Preparation of three-dimensional structure (3D structure) : Thaw GELTREX TM (Thermo Fisher Scientific; Waltham, MA) overnight on ice in a 4°C refrigerator. Place 1000 μl pipette tips, 100 μl pipette tips, and 10 mL to 1.5 ml tubes in the 4°C refrigerator overnight as well. After overnight incubation, take out the thawed GELTREX TM from the refrigerator and place it on ice.

[0133] Preparation of somatic cells for three-dimensional co-culture : Culture somatic cells (e.g., cumulus cells) in cell culture medium. Wash the cumulus cells twice with PBS. Add 4 drops of trypsin to the cell culture flask. Incubate the cells at 38.5°C for 5 to 10 minutes. Gently tap the flask to detach the cells. Aliquot the cells into 2 ml to 1.5 ml tubes. Centrifuge the cells at 500 g for 5 minutes. Discard the supernatant after centrifugation.

[0134] Plating somatic cells and creating a three-dimensional structure : Add 1 ml of regular DMEM medium to one of the tubes and gently mix the cells. Plate 500 μl of the cells into 2 wells of a 4-well plate. Place the 4-well plate in the incubator. The cells prepared in this way serve as a monolayer culture and are used as a control.

[0135] Add 200 μl of GELTREX TM to another tube and gently mix the contents of the tube by pipetting up and down 3 times. Plate 50 μl of the cells with GELTREX TM into each well of the 4-well plate. Place the 4-well plate in the incubator for 20 to 30 minutes. Take out the 4-well plate from the incubator to observe the solidification of GELTREX TM . After the solidification of GELTREX TM , add 450 μl of regular DMEM to each well of the 4-well plate. Place the 4-well plate in the incubator to allow the formation of a three-dimensional cell culture.

[0136] Continuing three-dimensional culture : Check the three-dimensional culture under an inverted microscope every day to observe cell proliferation and the formation of cell aggregates. When the color of the culture medium turns yellow, indicating a decrease in the pH of the cell culture medium, replace the cell culture medium as needed. Take pictures of the three-dimensional culture with an EVOS microscope (Thermo Fisher Scientific; Waltham, MA) ( Figure 2 ).

[0137] On the first day of plating, embryos are implanted into the three-dimensional co-culture, or the embryos are placed in the wells / cavities within the three-dimensional co-culture structure. After placing the embryos, the cell proliferation, structural integrity, and morphology of the three-dimensional structure of the three-dimensional co-culture are examined daily, and the culture medium is replaced according to the optimal development needs of the embryos. When the color of the culture medium turns yellow, indicating a decrease in the pH of the culture medium, the culture medium is replaced. Photographs of the three-dimensional co-culture are taken and the embryonic development is recorded( Figure 3 )

[0138] Example 2: Three-dimensional co-culture system for culturing embryos

[0139] Preparing a three-dimensional structure with bovine cumulus cells : Thaw the solution overnight at 4°C. The next day, place the thawed solution in a pre-chilled 0.5 ml tube. The amount of

[0140] can vary based on the experiment and ranges from 200 μl to 600 μl. To each 100 μl of solution, add 10 μl of a single-cell suspension of bovine cumulus cells. The number of bovine cumulus cells in each 100 μl of solution can vary between 1,000 and 10,000. Mix the cells with the

[0141] solution by slowly pipetting up and down several times. and the cell mixture are plated onto a 48-well plate. In each well of the 48-well plate, inoculate 50 μl to 100 μl of the mixture.

[0142] Incubate the plate at 37°C for 10 minutes to allow the solution to solidify. At the end of the incubation, add fresh TCM-199 medium supplemented with 15% fetal bovine serum (FBS) to each well. Then place the plate in an incubator at 37°C in an environment with 5% CO2 in the air until use.

[0143] Collection and culture of pre-implantation mouse embryos

[0144] Three-week-old CD-1 female mice (BioVendor R&D, Czech Republic) were treated with 5 I.U (International Units) of PMSG (Pregnant Mare Serum Gonadotropin), and then 48 hours later, they were treated with 5 I.U of hCG (Human Chorionic Gonadotropin) (Millipore; Burlington, MA). The female mice were then mated overnight with male mice of the B6D2F1 or 129SV strains (Jackson Laboratory; Bar Harbor, ME). The female mice were checked for mating plugs the next morning, and only the female mice with plugs were used for embryo collection. Pre-implantation mouse embryos were collected from the plugged female mice.

[0145] The mouse embryos were placed in the three-dimensional and cell solution in a 48-well plate prepared as described above. The mouse embryos were cultured in an environment of 37 °C with 5% CO2 in the air.

[0146] The culture medium was changed every other day or as needed. The development of the mouse embryos was monitored daily.

[0147] Results

[0148] Development of pre-implantation E3.5 mouse embryos cultured in a three-dimensional in vitro culture system : Eleven (11) E3.5 mouse embryos (CD-1 female × B6DF1 male) were placed in the three-dimensional and bovine cumulus cell culture system. The mouse embryos were cultured in TCM-199 medium supplemented with 15% FBS in an environment of 37 °C with 5% CO2 in the air. All E3.5 mouse embryos hatched and reached the three-dimensional system on the second day during the culture period ( Figure 4 ).

[0149] The mouse embryos continued their development and formed embryos equivalent to their in vivo counterparts at E5.5 in the three-dimensional culture system ( Figure 5 ).

[0150] The mouse embryos continued their development in the three-dimensional culture system, and six (6) of the embryos developed into embryos with an expanded body, which were connected to the three-dimensional system through a stalk-like structure and a placenta-like structure ( Figure 6 ). The structure remained viable until day 9 of the culture.

[0151] Development of rhythmic contractions of E4.5 mouse embryos cultured in a three-dimensional in vitro culture system : Pre-implantation 4.5 mouse embryos were collected from plugged CD-1 female mice mated with B6DF1 male mice and placed in a three-dimensional in vitro culture system ( Figure 7 ). The mouse embryos were cultured in TCM-199XEP medium (Table 1) in an environment of 37 °C with 5% CO2 in the air. Figures 8A to 8EShows the progress of E4.5 mouse embryo development. The embryo touches the three-dimensional system on the first day of culture, develops into an E5.5 embryo on the second day, and continues to develop in three-dimensional culture on days 3, 4, and 5.

[0152] Table 1: TCM-199XEP medium

[0153] Components Volume TCM-199 + 15% FBS 9.8 mL IT-X stock solution, 100x 100 μL N-acetyl-L-cysteine, 100x 100 μL Estradiol, 1000x 10 μL Progesterone 1000x 10 μL In total 10ml

[0154] On the 5th day of culture, the embryos were treated with 0.5 μM retinoic acid. On the 8th day of culture, rhythmic contractions similar to cardiomyocytes were observed on the embryos. The rhythmic contractions continued until the 18th day of culture. Possibly due to nutrient depletion, the rhythmic contractions slowed down and eventually stopped. Rhythmic contractions were also observed in another embryo developed in the same culture system.

[0155] Conclusion

[0156] A static three-dimensional in vitro embryo culture system was developed and it was demonstrated that this three-dimensional in vitro culture system was able to support pre-implantation mouse embryo development to the post-implantation stage. The three-dimensional system was also able to support organogenesis, which was confirmed by the rhythmic contractions of the developing embryo. This is the first static in vitro culture system that can support post-implantation embryo development and organogenesis in vitro.

[0157] Those skilled in the art will understand that changes can be made to the embodiments described above without departing from their general inventive concept. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by this specification.

Claims

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

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 according to claim 2, wherein the scaffold-based structure comprises a gel-like material or a structural scaffold.

4. The three-dimensional co-culture system according to claim 3, wherein the gel-like material is selected from hydrogels, agarose, basement membrane extracts, or extracellular matrix.

5. The three-dimensional co-culture system according to claim 3, wherein the structural scaffold is created by a three-dimensional 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 according to claim 6, wherein the scaffold-free structure comprises cell aggregates 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 from 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 mammalian embryos, avian embryos, reptilian embryos, fish embryos, amphibian embryos, and marsupial embryos.

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

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

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

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

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

16. A method for culturing an embryo in a three-dimensional co-culture system, the method comprising: (a) Culturing at least one somatic cell; (b) Implanting the at least one somatic cell into the three-dimensional structure; (c) Obtaining at least one embryo; and (d) Implanting the at least one embryo into the three-dimensional structure or placing it near the three-dimensional structure; wherein the at least one somatic cell and the at least one embryo grow in the three-dimensional structure in the cell culture medium or grow near the three-dimensional structure.

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

18. The method according to claim 17, wherein the scaffold-based structure comprises a gel-like material or a structural scaffold.

19. The method according to claim 18, wherein the gel-like material is selected from hydrogels, agarose, basement membrane extracts, or extracellular matrix.

20. The method according to claim 18, wherein the structural scaffold is created by a three-dimensional printer.

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

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

23. The method according to 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 from the reproductive tract.

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

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

26. The method according to 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 a custom medium.

27. The method according to any one of claims 16 to 26, wherein the cell culture medium contains sodium ions (Na +) Potassium ion (K + ) Calcium ion (Ca 2+ ) or magnesium ion (Mg 2+ ), or at least one of them.

28. Kit, which comprises: (a) Three-dimensional structure; (b) At least one somatic cell; (c) At least one embryo; and (d) Cell culture medium.

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

30. The kit according to claim 29, wherein the scaffold-based structure comprises a gel-like material or a structural scaffold.

31. The kit according to claim 30, wherein the gel-like material is selected from hydrogels, agarose, basement membrane extracts or extracellular matrix.

32. The kit according to claim 30, wherein the structural scaffold is created by a 3D printer.

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

34. The kit according to claim 33, wherein the scaffold-free structure comprises cell aggregates forming a three-dimensional structure.

35. The kit according to 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 from the reproductive tract.

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

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

38. The kit according to 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 a custom medium.

39. The kit according to any one of claims 28 to 38, wherein the cell culture medium contains sodium ion (Na + ), potassium ion (K + ), calcium ion (Ca 2+ ) or magnesium ion (Mg 2+ ), or at least one of them.

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

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

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

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