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

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.

Bioengineering Approaches to Improve In Vitro Performance of Prepubertal Lamb Oocytes

“…Although bioengineering strategies are becoming widespread in the area of cell culture, including ARTs, to the best of our knowledge such approaches have never been applied in prepubertal oocytes in studies conducted to date, but only in adult oocytes [36,51,[58][59][60][61][62].…”

“…The aim of the present study was to mimic as much as possible the physiological follicular conditions, such as COC microencapsulation, for 3D-IVM. The first step was to provide prepubertal COCs with a 3D environment using a one-step automated bioprinting method [36]. As the results show, the applied technique was able to improve prepubertal oocyte nuclear maturation rates and to reduce the incidence of chromosomal abnormalities.…”

“…Alginate microbeads including COCs (COC-microbeads) were fabricated using the spherical hydrogel generator (SpHyGa) [36,39]. Briefly, the SpHyGa was loaded with a syringe filled with a 1% sodium alginate solution in IVM medium and fitted with a needle with a 508 µm internal diameter, adapted to ovine COC size [36]. COCs were added to the alginate solution by using a Gilson-type pipette with sterile tips.…”

“…Oocytes were evaluated in relation to their meiotic stage, and classified as germinal vesicles (GVs), metaphase to telophase I (MI to TI), and MII with the first polar body extruded [41]. Oocytes showing either multipolar meiotic spindle, irregular chromatin clumps, or absence of chromatin were considered to be abnormal [36]. Oocytes with the first polar body (PB) extruded and one pronucleus were classified as parthenogenetically activated [55].…”

Creating an Artificial 3-Dimensional Ovarian Follicle Culture System Using a Microfluidic System

“…Recently, second meiotic metaphase oocytes were obtained through multistep in human [ 12 ]. Meanwhile, 3-dimensional (3D) culture techniques have utilized a wide range of approaches such as embedding cells in extracellular matrixes [ 13 , 14 , 15 ], printing 3D scaffolds [ 16 ], 3D bioprinting of biocompatible materials and cells [ 17 ], decellularization/ recellularization of tissues [ 18 ] and also the use of more sophisticated microfluidics technology [ 19 , 20 ].…”