Development of a three-dimensional culture model of endometrium to study embryo-uterine interactions

Three-dimensional (3D) cultured cells exhibit morphological and physiological properties more similar to in vivo cells than conventional monolayer cultured cells. They provide more physiologically relevant architecture, enabling more accurate simulation of tissue structure and function [ 1 ]. One of the main advantages of 3D cell culture is its ability to replicate the complex interactions between cell-cell and cell-extracellular matrix (ECM) that occur in living tissues. It enhances cellular functions such as proliferation, differentiation, and gene expression in ways that are more representative of in vivo conditions [ 2 ]. Cells in 3D culture tend to maintain a more natural morphology and demonstrate improved tissue-specific functions. For instance, hepatocytes in 3D culture exhibit enhanced albumin production and cytochrome P450 activity compared to two-dimensional conditions [ 3 ]. In recent years, 3D cultures have attracted attention for their unique characteristics and are now applied not only in basic research on cell physiology but also in drug discovery, regenerative medicine, and alternative methods to animal testing. Broadly, 3D culture systems can be categorized into scaffold-based and scaffold-free systems. Scaffold-based methods employ natural ECM components (e.g., collagen, Matrigel), hydrogels, or synthetic polymers to support cell adhesion, proliferation, and differentiation [ 4 , 5 ]. Scaffold-free strategies, including hanging-drop cultures, low-adhesion plates, rotary cell culture systems, and microfluidic platforms, promote cellular self-assembly into spheroids or organoids without an exogenous matrix [ 6 , 7 , 8 ]. Despite the many advantages of 3D culture, it is technically more difficult than monolayer culture and requires culture methods specific to each tissue and cell type. The endometrium is a dynamic tissue that undergoes cyclical remodeling, proliferation, differentiation, and shedding in response to hormonal cues. Conventional two-dimensional (2D) culture systems have provided important insights into endometrial cell biology; however, they fail to recapitulate the complex architecture, ECM interactions, and multicellular organization present in vivo . By bridging the gap between in vitro experimentation and in vivo physiology, 3D endometrial cell culture systems represent a promising platform for advancing reproductive biology research. The present review provides an overview of 3D culture of endometrial cells, including in-gel culture and spheroids, with a review of in vitro implantation models using embryonic cells.

In vitro models have been widely employed to investigate the dynamic interactions between embryos and the maternal endometrium, which are critical for implantation and pregnancy establishment. Early studies utilized co-culture systems of embryos with endometrial epithelial or stromal cells, demonstrating improved embryonic development and providing insight into paracrine communication [ 39 , 40 ]. These approaches have greatly contributed to understanding the mechanisms of embryo attachment and maternal receptivity, offering novel platforms for reproductive research and potential clinical applications in assisted reproduction. Endometrial epithelial cells from rodents are difficult to passage in culture, making it challenging to utilize the cells for three-dimensional culture or spheroid formation. To address this issue, we cultured finely dissected rat endometrial tissue pieces directly on non-adherent plates to create endometrial hetero spheroid-like structures ( Fig. 3A and B Fig. 3. Co-culture of cultured explants and hatched blastocyst in rat. (A) Rat uterine explant immediately after separation from 1.5 day’s post coitus rat uterine horn. (B) The rat uterine explant after 3 days in culture. (C) The hatched embryo attached to the uterine explant after 48 h in co-culture. (D) Enlarged view of Fig. 3C . (E) Attachment rate of embryos to the explants after steroid hormone treatment. The results are expressed as the percentage of attached embryos of each condition. n, number of embryos used for each experimental condition. Part of this figure is reproduced from Islam et al ., Cell Tissue Res., 2017 with permission from the publisher [ 41 ]. ), rather than using isolated endometrial cells [ 41 ]. Treatment with steroid hormones regulated the expression of Muc1 (mucin 1), Pr (progesterone receptor), Areg (Amphiregulin) and Igfbp1 (insulin-like growth factor-binding protein 1) in the cultured rat explants, demonstrating its responsiveness to the steroid hormones similar to that observed in vivo . In addition, the expression of decidualization marker genes, Prl8a2 (prolactin family 8, subfamily a, member 2) and Bmp2 (bone morphogenetic protein 2), was up-regulated by medroxyprogesterone acetate (MPA) and dibutyryl-cAMP treatment, indicating that the cultured explants have the potential for decidualization. When the cultured explants were co-cultured with hatched blastocysts, stable attachment was confirmed within 48 h ( Figs. 3 C and D). Furthermore, the P4-treated group showed a significantly higher attachment rate compared to the control group, while no attachment was observed in the estradiol (E2)-treated group ( Fig. 3E ). Despite the necessity of comprehensive investigation, the results suggest that the cultured rat uterine explants can be a useful in vitro model to study uterine functions and early implantation. Co-culture of cultured explants and hatched blastocyst in rat. (A) Rat uterine explant immediately after separation from 1.5 day’s post coitus rat uterine horn. (B) The rat uterine explant after 3 days in culture. (C) The hatched embryo attached to the uterine explant after 48 h in co-culture. (D) Enlarged view of Fig. 3C . (E) Attachment rate of embryos to the explants after steroid hormone treatment. The results are expressed as the percentage of attached embryos of each condition. n, number of embryos used for each experimental condition. Part of this figure is reproduced from Islam et al ., Cell Tissue Res., 2017 with permission from the publisher [ 41 ].

Three-dimensional culture systems of endometrial cells represent a promising platform for advancing the understanding of uterine biology and pathology. Recent advances have enabled the generation of endometrial organoids—self-organizing, stem cell–derived structures that recapitulate essential features of the endometrium, including epithelial differentiation, hormone responsiveness, and tissue remodeling. The organoid-based and microfluidic “endometrium-on-a-chip” systems further enhance physiological relevance by enabling long-term culture, hormonal responsiveness, and spatial architecture resembling the uterine lining [ 16 , 42 ]. The integration of endometrial organoids with other tissue or organ-on-a-chip systems could facilitate the study of complex physiological processes, such as the crosstalk between the endometrium, embryo, and immune system. Advances in bioengineering, including scaffold design, extracellular matrix optimization, and co-culture with stromal or immune cells, are expected to further enhance the physiological relevance of these models. In conclusion, 3D cultures of endometrial cells are paving the way toward next-generation organoid platforms, which will enable detailed analyses of endometrial function and embryo–endometrium interactions. Such models hold promise not only for improving livestock production but also for contributing to progress in infertility treatment.

The authors declare that they have no conflict of interest.