Functional mimicry of uterine tissue using endometrial organoids in rats

Endometrial organoids (EOs) have gained attention as a promising in vitro model for investigating uterine physiology, reproductive disorders, and embryo-maternal interactions, providing an alternative to in vivo studies while minimizing ethical concerns. Despite their increasing use across species, a well-characterized rat EO model is limited.

We established and validated a rat EO platform that recapitulates the structural and functional characteristics of the native endometrium.

We established and validated a rat EO platform that recapitulates the structural and functional characteristics of the native endometrium. Organoids were generated from epithelial-rich stem-cell populations isolated from adult female rats and cultured in 3D Matrigel. EO formation efficiency was assessed in relation to plasma progesterone concentration, and organoids were evaluated for long-term viability, cryopreservation tolerance, and morphological consistency over serial passages. Functional relevance was examined by real-time polymerase chain reaction and RNA sequencing of sex steroid hormone receptors (progesterone receptor and estrogen receptor α) and CD34. GFP (Green Fluorescent Protein)-labeled EOs were transplanted into the uterine lumen of wild-type rats to evaluate engraftment and persistence.

Rat EOs displayed morphological and molecular characteristics comparable to native uterine tissue, maintaining viability and integrity over multiple passages and after cryopreservation. Immunohistochemical analyses using epithelial (E-cadherin), stromal (Vimentin), and proliferative (Ki-67) markers confirmed the presence of multiple cell types resembling those in native uterine tissue. Formation efficiency positively correlated with circulating progesterone concentrations. Gene expression confirmed key endometrial markers, including hormone receptors and stromal-associated genes. GFP-expressing EOs successfully engrafted into wild-type uterine lumens and persisted long term, demonstrating functional and structural compatibility with the in vivo uterine environment.

The rat EO model developed here provides a physiologically relevant platform for studying endometrial biology, enabling research on reproductive mechanisms and disease modeling. Its ability to mimic and engraft in the uterine environment suggests applications in regenerative medicine and therapeutic transplantation.

This rat EO model provides a physiologically relevant platform for studying uterine biology and reproductive mechanisms without extensive animal use. Its ability to mimic and engraft in the uterine environment supports potential applications in disease modeling, drug testing, and regenerative medicine.