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 (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.
Highlights
The cumulus-oocyte complex (COC) is a multicellular structure characterized by three-dimensional (3D) architecture and bi-directional crosstalk between the oocyte and its surrounding cumulus cells
Bioengineering approaches have been employed to develop 3D culture methods in different cell systems and, over the last decade, there has been growing interest in cell microencapsulation, a procedure involving the enveloping of cells within a semipermeable polymeric matrix able to mimic the native tissue architecture and physiology. 3D culture methods for in vitro oocyte maturation (IVM) after COC microencapsulation have become a goal for researchers in the field of Assisted Reproductive Technologies (ARTs) [15] as microencapsulation may prevent COCs flattening on the bottom of the plate, typical of a traditional 2D culture system, which causes reduction of about 50% of the cell surface exposed to medium [16]
Microbead stability and oocyte maturation rate were analyzed in 1% alginate and by using the 508 μm needle. 3D microbeads produced with 1% alginate and the 508 μm needle remained stable in shape and size when incubated over 24 hours at 38.5 ̊C under 5% CO2 in air (Table 4)
Summary
The cumulus-oocyte complex (COC) is a multicellular structure characterized by three-dimensional (3D) architecture and bi-directional crosstalk between the oocyte and its surrounding cumulus cells. Paracrine interactions among these cells govern various processes of oogenesis culminating in oocyte meiosis resumption, maturation and ovulation [1,2,3,4]. The majority of ART studies have been conducted in conventional 2D culture systems. This may have led to limitations due to loss of cell native phenotype [14]. Bioengineering approaches have been employed to develop 3D culture methods in different cell systems and, over the last decade, there has been growing interest in cell microencapsulation, a procedure involving the enveloping of cells within a semipermeable polymeric matrix able to mimic the native tissue architecture and physiology. 3D culture methods for IVM after COC microencapsulation have become a goal for researchers in the field of ARTs [15] as microencapsulation may prevent COCs flattening on the bottom of the plate, typical of a traditional 2D culture system, which causes reduction of about 50% of the cell surface exposed to medium [16]
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