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KR-20260062147-A - Method for preparation of artificial endometrium model using 3D printing and electrospining

KR20260062147AKR 20260062147 AKR20260062147 AKR 20260062147AKR-20260062147-A

Abstract

The present invention relates to a method for manufacturing an artificial endometrial model using 3D printing and electrospinning, and to the use of the artificial endometrial manufactured according to this method.

Inventors

  • 박석희
  • 이경아
  • 김경화
  • 김영서
  • 전용필

Assignees

  • 부산대학교 산학협력단
  • 성신여자대학교 연구 산학협력단
  • 차의과학대학교 산학협력단

Dates

Publication Date
20260507
Application Date
20241025

Claims (20)

  1. i) a step of preparing a cage by 3D printing a biocompatible first polymer to have a bottom surface and a side surface, wherein the top surface is open and the side surface has a plurality of windows formed with an area of (0.7 to 2.5 mm) × (0.7 to 2.5 mm); ii) a step of preparing a uniaxially aligned nanofiber array by electrospinning a biocompatible second polymer; iii) A step of transferring the uniaxially aligned nanofiber array prepared as above onto a cage; iv) a step of injecting endometrial stromal cells (EnSC), fibrinogen, and thrombin through a side window into the cage into which the nanofiber array has been transferred to form a fibrin gel containing endometrial stromal cells; and v) a step of transferring and culturing endometrial epithelial cells (EnEC) onto a nanofiber array of the cage; a method for manufacturing an artificial endometrial model.
  2. In paragraph 1, A manufacturing method in which step i) above is performed by a material extrusion-based 3D printing technique or a photopolymerization-based 3D printing technique.
  3. In paragraph 1, A manufacturing method in which step ii) above is performed by electrospinning using a gap-based fiber recovery technique.
  4. In paragraph 1, A method of manufacturing in which the nanofiber array prepared in step ii) above consists of strands with an average fiber diameter of 0.4 to 1 μm.
  5. In paragraph 1, A manufacturing method in which the above step iii) is performed 10 to 25 times while rotating at a predetermined angle for each transfer.
  6. In paragraph 1, The above-mentioned biocompatible first polymer and biocompatible second polymer are each independently polycaprolactone (PCL), poly(glycolic acid; PGA), poly(lactic acid; PLA), poly(D,L-lactic-co-glycolic acid) (PLGA), poly(L-lactide-co-D,L-lactide)), polyhydroxybutyrate (PHB), polyhydroxyvalerate, polyvalerolactone (PVL), polydioxanone (PDO, PDS), polyethylene glycol diacrylate (PEGDA), and polycaprolactone diacrylate (PCLDA). A method of manufacturing a polymer selected from the group consisting of poly(β-aminoester) diacrylates, polyurethane acrylate (PUA), N-vinyl pyrrolidone (NVP), polymethyl methacrylate (PMMA), copolymers thereof, and mixtures thereof, wherein the polymers are the same or different from one another.
  7. In paragraph 1, A manufacturing method further comprising the step of forming a barrier of a predetermined height by 3D printing a biocompatible first' polymer along the upper perimeter of the cage to which the nanofiber array has been transferred after step iii) above.
  8. In paragraph 1, A method of preparation in which the above endometrial epithelial cells include soluble cells, insoluble cells, or both.
  9. An artificial endometrium comprising: a bottom surface and a side surface formed of a biocompatible first polymer, the side surface having a plurality of windows formed with an area of (0.7 to 2.5 mm) × (0.7 to 2.5 mm); a nanofiber mat located at the open top of the cage, wherein a uniaxially aligned nanofiber array composed of strands formed of the biocompatible first polymer with an average fiber diameter of 0.4 to 1 μm thickness is transferred 10 to 25 times while undergoing orthogonal rotation; a barrier formed of the biocompatible first polymer extending along the periphery of the side surface of the cage; a fibrin gel comprising endometrial stromal cells (EcSCs) injected into the interior of the cage; and endometrial epithelial cells (EnECs) located on the nanofiber mat.
  10. In Paragraph 9, An artificial endometrium manufactured by the method of any one of paragraphs 1 through 8.
  11. In Paragraph 9, The artificial endometrium described above is an artificial endometrium that mimics the microenvironment of the human endometrium.
  12. In Paragraph 9, The artificial endometrium described above is an in vitro model for uterine disease-related drug screening, factor function studies, and implantation rate enhancement studies.
  13. An implantation-related factor screening system comprising the artificial endometrium and trophoblast cell-mimicking spheroids of claim 9.
  14. A screening method for implantation-related factors comprising the step of placing trophoblast cell-mimicking spheroids on a nanofiber mat and co-culturing them with endometrial epithelial cells in the implantation-related factor screening system of claim 13 in the presence or absence of implantation-related factor candidate substances.
  15. In Paragraph 14, A screening method in which the above-mentioned implantation-related factor is applied inside a cage containing fibrin gel or on a nanofiber mat.
  16. In Paragraph 14, A screening method that observes the morphology of co-cultured spheroids to determine the degree of attachment, adhesion, or penetration into the cage on a nanofiber mat, and determines whether it is an implantation-promoting or inhibiting factor.
  17. An implantation-related gene analysis system comprising the artificial endometrium and trophoblast cell-mimicking spheroids of claim 9.
  18. A method for analyzing implantation-related genes, comprising the step of placing trophoblast cell-mimicking spheroids on a nanofiber mat and co-culturing them with endometrial epithelial cells in the implantation-related factor screening system of claim 17.
  19. In Paragraph 18, A gene analysis method that obtains information on genes whose expression is upregulated or downregulated by more than twofold by comparing and analyzing genes isolated from spheroids, endometrial epithelial cells, or endometrial stromal cells before and after co-culture.
  20. In Paragraph 18, A genetic analysis method that provides genetic information about trophoblast-endometrial interactions.

Description

Method for preparation of artificial endometrium model using 3D printing and electrospining The present invention relates to a method for manufacturing an artificial endometrial model using 3D printing and electrospinning, and to the use of the artificial endometrial manufactured according to this method. Embryo implantation is a sophisticated process involving coordinated interactions between a developing embryo and the endometrium. The endometrium is a complex tissue composed of two types of cells—endometrial epithelial cells (EnEC) and endometrial stromal cells (EnSC)—and must reach a receptive state in preparation for embryo implantation. To prepare for embryo implantation, the endometrium must reach a receptive state through morphological and functional changes within a short period of the mid-secretory phase of the female menstrual cycle, known as the window of implantation (WOI); endometrial receptivity is regulated by ovarian steroid hormones such as estrogen and progesterone. The process of embryo implantation can be classified into three main stages: apposition, attachment/adhesion, and invasion. In humans, the blastocyst is properly oriented when the internal cell mass (ICM) faces the endometrium. Subsequently, the trophectoderm (TE) contacts the implantation site of the endometrial epithelium, adheres to the epithelial surface, and then penetrates the endometrial epithelial layer. Finally, the blastocyst invades the endometrial stroma and comes into contact with the mother's blood circulation along with the decidua. Since in vivo research in humans is ethically impractical, animal in vivo models are used for studies on endometrial function and embryo implantation. While animal models are highly useful, significant discrepancies still exist when compared to humans, particularly regarding fertility, which is lower in humans compared to other mammalian species. Consequently, the successful establishment of implantation requires a better understanding of the complex regulatory mechanisms of embryo implantation based on in vitro models of the human endometrium. Meanwhile, two-dimensional (2D) in vitro models are the simplest culture systems for research. In particular, features related to the endometrial microenvironment, including failure of ovarian steroid hormone response, autocrine and/or paracrine interactions, and secretion, are insufficient in these models. Furthermore, the aforementioned 2D models present technical challenges such as long-term culture and the loss of various phenotypes in cultured cells. Therefore, three-dimensional (3D) in vitro models of the human endometrium are being developed as an important alternative for embryo implantation research. Endometrial cells are known to be cultured in various 3D hydrogel matrices such as Matrigel, collagen, fibrin-agarose, gelatin, and alginate. Additionally, the aforementioned 3D hydrogel models provide cell-cell communication and cell-matrix interactions while maintaining cell characteristics and phenotypes. However, the above 3D hydrogel model still has incomplete endometrial dynamics and function caused by a lack of access to culture media (nutritives, parasecretory and growth factors). Therefore, as a good tool for understanding embryo implantation, a more physically and physiologically relevant research model similar to the human endometrial microenvironment is required. Figure 1 illustrates the overall process for fabricating a 3D biomimetic artificial endometrial model. (a) shows the fabrication of a 3D ECM-mimicking scaffold through a combined process of electrospinning and 3D printing, in which a nanofiber membrane based on the basement membrane concept was constructed to facilitate the attachment of endometrial epithelial cells to stromal cells within a fibrin gel. (b) shows a schematic diagram illustrating the construction of an artificial endometrial culture system containing JAR spheroids, in which human endometrial stromal cells, fibrinogen, and thrombin are assembled within the wells of the artificial endometrium to form a fibrin gel. Once formed, the fibrin gel is allowed to form within one hour of culture prior to the addition of the medium. Subsequently, human endometrial epithelial cells were dispensed onto the nanofiber membrane of the artificial endometrium to form a monolayer, thereby constructing the artificial endometrial system. It took one day to form a tight monolayer. After developing GFP-expressing JAR spheroids using a spheroid culture technique, they were transferred to the upper part of an artificial endometrial system and attachment experiments were initiated. Figure 2 illustrates the exploration of solution concentration, printing temperature, and conditions for suitable mesh formation. (a) shows SEM images and measured diameters of electrospun fibers prepared with three different concentrations of solution, and (b) shows the 3D printing deposition of a polymer melt on an electrospun mat with different noz