Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy.
Research Centre E. Piaggio, University of Pisa, Pisa, Italy.
PLoS One. 2020 Sep 11;15(9):e0238812. doi: 10.1371/journal.pone.0238812. eCollection 2020.
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 (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-regulation 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-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.
三维体外成熟（3D IVM）是一种有前途的方法，可以提高 IVM 效率，因为它可以防止卵丘-卵母细胞复合体（COC）扁平化并保持其结构和功能完整性。迄今为止报道的方法重复性低，验证研究有限。在这项研究中，优化了基于生物打印的生产工艺，用于生成含有 COC 的微珠（COC-微珠），并在大型动物模型（绵羊）中对其有效性进行了测试。生产了海藻酸盐微珠，并对其大小、形状和在培养条件下的稳定性进行了表征。COC 包封具有高效率和可重复性，并且卵丘完整性得以保留。COC-微珠进行了 IVM，COC 在标准 2D IVM 中培养作为对照。IVM 后，对卵母细胞进行核染色质构型、生物能量/氧化状态以及线粒体活性的基因生物标志物（TFAM、ATP6、ATP8）和卵母细胞发育能力（KHDC3、NLRP5、OOEP 和 TLE6）的转录活性分析。与 2D 对照相比，3D 系统更有效地支持卵母细胞核成熟（P<0.05）。卵质线粒体活性和活性氧（ROS）生成能力增加（P<0.05）。在 3D IVM 中观察到 TFAM、ATP6 和 ATP8 的上调以及 KHDC3、NLRP5 表达的下调。总之，用于生产 COC-微珠的新生物打印方法具有高重复性和高效率。此外，3D IVM 可提高卵母细胞核成熟和卵母细胞细胞质成熟的相关参数，可用于临床和毒理学应用。
