Decellularized scaffolds of porcine uterus reconstruct the endometrial microenvironment to promote uterine epithelial cell differentiation and organoid formation.

Endometrial epithelial organoids (EEOs) represent a valuable tool for investigating endometrial development and establishing in vitro models of related diseases. Traditional organoid cultures, which are dependent on mouse-derived Matrigel, fail to recapitulate species-specific microenvironments. This study optimized the fabrication of porcine uterine decellularized extracellular matrix (DECM) scaffolds using a freeze-thaw cycle combined with low-damage chemical decellularization, significantly enhancing the biomimetic properties. We preserved collagen, glycosaminoglycans (GAG), and key structural proteins while accurately reconstructing the native uterine fibrous-pore network. Gradient thickness scaffolds (20-100 µm) prepared via frozen sectioning exhibit tunable elastic moduli, accommodating diverse cellular behaviors. These DECM scaffolds not only supported the co-culture of porcine endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs) but also induced the self-organization of EECs into structures resembling the native endometrial luminal epithelium/glandular epithelium (CK-18+). Furthermore, in the absence of steroid hormones, the DECM scaffolds supported differentiation toward FoxA2+ glandular epithelium, whereas Matrigel formed only FoxA2- vesicular-like structures, highlighting the scaffolds' advantage in enhancing cellular diversity. This study provides an advanced tool for elucidating endometrial microenvironment regulatory mechanisms and offers critical insights for improving organoid culture techniques and developing endometrial-specific synthetic scaffolds for its cultivation.