One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM effi- ciency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its struc- tural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for gener- ating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regula- tion of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbe- ads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear mat- uration and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications