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US-20260125639-A1 - THREE-DIMENSIONAL CO-CULTURE SYSTEM FOR EMBRYOS CULTURED IN VITRO AND USES THEREOF

US20260125639A1US 20260125639 A1US20260125639 A1US 20260125639A1US-20260125639-A1

Abstract

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

Inventors

  • Leyi Li
  • Eriona Hysolli
  • Cassdiy BEARD
  • Jie Ouyang

Assignees

  • COLOSSAL BIOSCIENCES INC.

Dates

Publication Date
20260507
Application Date
20231010

Claims (20)

  1. 1 . A three-dimensional co-culture system comprising: (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture media.
  2. 2 . The three-dimensional co-culture system of claim 1 , wherein the three-dimensional structure is a scaffold-based structure or a scaffold-free structure.
  3. 3 . The three-dimensional co-culture system of claim 2 , wherein the scaffold-based structure comprises a gel-like material or structural scaffold.
  4. 4 . The three-dimensional co-culture system of claim 3 , wherein: the gel-like material is selected from a hydrogel, an agarose, a basement membrane extract, or an extracellular matrix; or the structural scaffold is created by a three-dimensional printer.
  5. 5 - 6 . (canceled)
  6. 7 . The three-dimensional co-culture system of claim 2 , wherein the scaffold-free structure comprises cell aggregates forming a three-dimensional structure.
  7. 8 . The three-dimensional co-culture system of claim 1 , wherein: the somatic cell is selected from a skin cell, a bone cell, a blood cell, a connective tissue cell, a cumulus cell, a granulosa cell, and/or a cell from the reproductive tract; and/or 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.
  8. 9 . (canceled)
  9. 10 . The three-dimensional co-culture system of claim 8 , wherein the embryo is a mammalian embryo.
  10. 11 . The three-dimensional co-culture system of claim 1 , wherein the cell culture media: is selected from at least one of MEM, DMEM, RPMI, DMEM/F-12, N2B27, M16, KSOM, TCM-199, or a custom-made medium; and/or comprises at least one of a sodium ion (Na + ), a potassium ion (K + ), a calcium ion (Ca 2+ ), or a magnesium ion (Mg 2+ ).
  11. 12 . (canceled)
  12. 13 . The three-dimensional co-culture system of claim 1 , wherein the somatic cell and/or the embryo is embedded in or placed near the three-dimensional structure.
  13. 14 . (canceled)
  14. 15 . The three-dimensional co-culture system of claim 1 , wherein the three-dimensional co-culture system further comprises a bioreactor.
  15. 16 . A method of 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 at least one embryo are grown in or near the three-dimensional structure in cell culture media.
  16. 17 . The method of claim 16 , wherein the three-dimensional structure is a scaffold-based structure or a scaffold-free structure.
  17. 18 . The method of claim 17 , wherein the scaffold-based structure comprises a gel-like material or structural scaffold.
  18. 19 . The method of claim 18 , wherein: the gel-like material is selected from a hydrogel, an agarose, a basement membrane extract, or an extracellular matrix; and/or the structural scaffold is created by a three-dimensional printer.
  19. 20 - 21 . (canceled)
  20. 22 . The method of claim 16 , wherein the scaffold-free structure comprises cell aggregates forming a three-dimensional structure.

Description

CROSS REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application No. 63/378,929, filed on Oct. 10, 2022, the disclosure of which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION This invention relates to the field of biotechnology. Provided herein are three-dimensional co-culture systems for culturing embryos in vitro. The three-dimensional co-culture systems comprise (a) a three-dimensional structure, (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture media. BACKGROUND OF THE INVENTION In vitro production of embryos has several advantages over in vivo-derived embryo production, including, but not limited to the efficient selection of superior genetics for the transfer of genetic modification to rapidly obtain animals with desirable traits. Genetic engineering can provide a powerful tool to aid in the understanding of basic mechanisms regulating physiology. As the in vitro environment is still suboptimal for embryo growth and development, there is a need to develop a better system for culturing embryos in vitro to mimic the in vivo environment to provide robust and viable embryos for a higher success rate for in vitro fertilization procedures, better efficiency in reproductive technologies in agriculturally important animals, and better efficiency for the conservation of extinct and endangered species through reproductive technologies. BRIEF SUMMARY OF THE INVENTION In one general aspect, the 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) cell culture media. Also provided are methods of growing an embryo in a three-dimensional co-culture system. The methods comprise (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 at least one embryo are grown in or near the three-dimensional structure in cell culture media. Also provided are kits comprising (a) a three-dimensional structure; (b) at least one somatic cell; (c) at least one embryo; and (d) cell culture media. In certain embodiments, the three-dimensional structure is a scaffold-based structure. The scaffold-based structure can, for example, comprise a gel-like material or a structural scaffold. The gel-like material can, for example, be selected from a hydrogel, an agarose, a basement membrane extract, or an extracellular matrix. In certain embodiments, the scaffold-based structure is created by a three-dimensional printer. In certain embodiments, the three-dimensional structure is a scaffold-free structure. The scaffold-free structure can, for example, comprise cell aggregates forming a three-dimensional structure. In certain embodiments, the somatic cell is selected from a skin cell, a bone cell, a blood cell, a connective tissue cell, a cumulus cell, a granulosa cell, and/or a cell from 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, for example, be a mammalian embryo. In certain embodiments, the cell culture media is selected from at least one of minimum essential media (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 SOM (KSOM), tissue culture medium-199 (TCM-199), or a custom-made medium. The cell culture media can, for example, comprise at least one of a sodium ion (Na+), a potassium ion (K+), a calcium ion (Ca2+), or a magnesium ion (Mg2+). In certain embodiments, the somatic cell is embedded in or placed near the three-dimensional structure. In certain embodiments, the embryo is embedded in or placed near the three-dimensional structure. In certain embodiments, the three-dimensional co-culture system further comprises a bioreactor. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings. FIGS. 1A and 1B show representative images of a monolayer cell culture (FIG. 1A) and a three-dimensional (3D) cell culture (FIG. 1B). FIG. 2 shows representative images demonstrating the progress of a three-dimensional cell culture over the course of 8 days. The somatic cells within the three-dimensional culture proliferated and connected to