Enzymatic cross-linking collagen-based bioink for 3D bioprinting of endometrial model with endometrial epithelial organoids

The human endometrium, a dynamic tissue responsive to sex hormones, plays a pivotal role in female reproduction and menstrual cycling, comprising diverse cell populations including epithelial and stromal cells. The development of in vitro endometrial models holds substantial potential for tissue repair, drug discovery, and disease modeling. However, the construction of biomimetic structural in vitro models capable of supporting long-term culture and preserving relevant biological functions has been hindered by limitations in both material properties and fabrication strategies. Here, an enzymatically cross-linked collagen-based bioink was developed, enabling extrusion bioprinting with favorable printability, biocompatibility, and bioactivity. The enzyme-catalyzed cross-linking process was optimized through room-temperature pre-crosslinking, and its evolution was described using a storage modulus ( G′ )-time kinetic model, enabling prediction of the printable window and reliable fabrication of three-dimensional (3D) constructs. Then, monoculture and co-culture systems of endometrial epithelial organoids (EEOs) and endometrial stromal cells (ESCs) embedded in collagen-based hydrogels were established. Utilizing the bioink and printing strategy, an in vitro endometrial model was 3D bioprinted, exhibiting sustained viability (≥11 days) and hormone-responsive functions comparable to or showing enhanced functional characteristics relative to commonly used Matrigel-based or collagen-based systems. This biomimetic platform provides a physiologically relevant in vitro model for investigating endometrial biology, elucidating pathological mechanisms, and facilitating drug screening applications.