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

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Published on: November 2, 2020

Antonella Mastrorocco1¤a, Ludovica Cacopardo2, Nicola Antonio Martino1¤b, Diana Fanelli3, Francesco Camillo3, Elena Ciani1, Bernard A. J. Roelen4, Arti Ahluwalia2,5☯, Maria Elena Dell’Aquila1☯

1 Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy; 2 Research Centre E. Piaggio, University of Pisa, Pisa, Italy; 3 Department of Veterinary Sciences, University of Pisa, Pisa, Italy; 4 Department of Clinical Sciences, Embryology, Anatomy and Physiology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands; 5 Department of Information Engineering, University of Pisa, Pisa, Italy

☯ These authors contributed equally to this work.
¤a Current address: Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
¤b Current address: Department of Veterinary Sciences, University of Torino, Torino, Italy

Abstract

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating 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 (TFAMATP6ATP8) and oocyte developmental competence (KHDC3NLRP5OOEP 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-regulation of TFAMATP6 and ATP8 and down-regulation of KHDC3NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.

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