Bioengineering Approaches to Improve In Vitro Performance of Prepubertal Lamb Oocytes

Abstract

Juvenile in vitro embryo technology (JIVET) provides exciting opportunities in animal reproduction by reducing the generation intervals. Prepubertal oocytes are also relevant models for studies on oncofertility. However, current JIVET efficiency is still unpredictable, and further improvements are needed in order for it to be used on a large-scale level. This study applied bioengineering approaches to recreate: (1) the three-dimensional (3D) structure of the cumulus–oocyte complex (COC), by constructing—via bioprinting technologies—alginate-based microbeads (COC-microbeads) for 3D in vitro maturation (3D-IVM); (2) dynamic IVM conditions, by culturing the COC in a millifluidic bioreactor; and (3) an artificial follicular wall with basal membrane, by adding granulosa cells (GCs) and type I collagen (CI) during bioprinting. The results show that oocyte nuclear and cytoplasmic maturation, as well as blastocyst quality, were improved after 3D-IVM compared to 2D controls. The dynamic 3D-IVM did not enhance oocyte maturation, but it improved oocyte bioenergetics compared with static 3D-IVM. The computational model showed higher oxygen levels in the bioreactor with respect to the static well. Microbead enrichment with GCs and CI improved oocyte maturation and bioenergetics. In conclusion, this study demonstrated that bioengineering approaches that mimic the physiological follicle structure could be valuable tools to improve IVM and JIVET.

3D Spheroid Human Dermal Papilla Cell as an Effective Model for the Screening of Hair Growth Promoting Compounds: Examples of Minoxidil and 3,4,5-Tri-O-caffeoylquinic acid (TCQA)

“…Another alternative that was proposed to mimic the ECM environment is animal-derived polymers, but ethical and variability concerns were raised in this case [49]. For this purpose, hydrogels from a non-animal origin such as alginate are gaining enormous consideration due to their applicability for tissue regeneration such as improving the in vitro performance of prepubertal lamb oocytes [50,51]. Thus, in order to improve the efficacy of our 3D model, we have proposed using compounds and biomolecules that can activate hair growth-and ECM-related pathways in 3D spheroid systems to further enhance DPC inductive properties [52,53].…”

Investigating and Modelling an Engineered Millifluidic In Vitro Oocyte Maturation System Reproducing the Physiological Ovary Environment in the Sheep Model

“…Follicular contents were released in sterile Petri dishes containing phosphate buffered saline (PBS) and observed under a Nikon SMZ18 stereomicroscope equipped with a transparent heating stage set up at 37 °C (Okolab S.r.l., Napoli, Italy). Only COCs with at least three intact cumulus cell layers and homogenous cytoplasm were selected for culture [ 56 ].…”

“…In a previous study, we found that millifluidic IVM (mIVM) improved oocyte mitochondrial membrane potential, intracellular ROS levels, and mitochondria/ROS colocalization, markers of healthy oocyte cytoplasmic maturation. In that study, a previously developed 3D mIVM system with COC-including bioprinted alginate microbeads was used [ 55 ] and inserted into the millifluidic device [ 56 ].…”

Biomaterials and advanced technologies for the evaluation and treatment of ovarian aging

“… Christiani A Amorim [ 268 ] 2009 alginate matrix Small human preantral follicles Survived in vitro culture in alginate matrix for 7 days. Antonella Mastrorocco[ 269 ] 2021 Alginate microspheres Lamb cumulus oocyte complexes Increased the nuclear maturation rate of preadolescent oocytes and reduced the incidence of chromosome abnormality. Parisa Jamalzaei [ 270 ] 2020 ALG hydrogel Mouse preantral follicles Survival rate of 0.5%ALG cultured follicles was significantly higher than 0.75% and 1%ALG cultured follicles.…”

“…In addition, alginate culture systems can be used for IVM. Recently, Mastrorocco et al developed a 3D IVM protocol for lamb COC encapsulated in the core of alginate microspheres [ 269 ]. This technique could increase the nuclear maturation rate of preadolescent oocytes and reduce the incidence of chromosome abnormalities, thus improving the in vitro performance of preadolescent lamb oocytes.…”