Advancing bovine in vitro fertilization through 3D printing: the effect of the 3D printed materials.

Nowadays there is an increasing demand for assisted reproductive technologies due to the growth of infertility problems. Naturally, fertilization occurs in the oviduct, where the oviductal epithelial cells (OECs) secrete many molecules that affect the embryo's metabolism and protect it from oxidative stress. When the OECs are grown in 3D culture systems, they maintain a great part of their functional characteristics, making them an excellent model for in vitro fertilization (IVF) studies. In this work, we aimed to evaluate the suitability of different 3D-printing processes in conjunction with the corresponding set of commercially available biomaterials: extrusion-based processing using polylactic acid (PLA) and polycaprolactone (PCL) and stereolithography or digital-light processing using polyethylene-glycol-diacrylate (PEGDA) with different stiffness (PEGDA500, PEGDA200, PEGDA PhotoInk). All the 3D-printed scaffolds were used to support IVF process in a bovine embryo assay. Following fertilization, embryo development and quality were assessed in terms of cleavage, blastocyst rate at days 7 and 8, total cell number (TCN), inner cell mass/trophectoderm ratio (ICN/TE), and apoptotic cell ratio (ACR). We found a detrimental effect on cleavage and blastocyst rates when the IVF was performed on any medium conditioned by most of the materials available for digital-light processing (PEGDA200, PEGDA500). The observed negative effect could be possibly due to some leaked compound used to print and stabilize the scaffolds, which was not so evident however with PEGDA PhotoInk. On the other hand, all the extrusion-based processable materials did not cause any detrimental effect on cleavage or blastocyst rates. The principal component analysis reveals that embryos produced in presence of 3D-printed scaffolds produced via extrusion exhibit the highest similarity with the control embryos considering cleavage, blastocyst rates, TCN, ICN/TE and ACR per embryo. Conversely, all the photo-cross linkable materials or medium conditioned by PLA, lead to the highest dissimilarities. Since the use of PCL scaffolds, as well as its conditioned medium, bring to embryos that are more similar to the control group. Our results suggest that extrusion-based 3D printing of PCL could be the best option to be used for new IVF devices, possibly including the support of OECs, to enhance bovine embryo development.

A computational model of cell viability and proliferation of extrusion-based 3D-bioprinted constructs during tissue maturation process.

3D bioprinting is a novel promising solution for living tissue fabrication, with several potential advantages in many different applicative sectors. However, the implementation of complex vascular networks remains as one of the limiting factors for the production of complex tissues and for bioprinting scale-up. In this work, a physics-based computational model is presented to describe nutrients diffusion and consumption phenomena in bioprinted constructs. The model-a system of partial differential equations that is approximated by means of the finite element method- allows for the description of cell viability and proliferation, and it can be easily adapted to different cell types, densities, biomaterials, and 3D-printed geometries, thus allowing a preassessment of cell viability within the bioprinted construct. The experimental validation is performed on bioprinted specimens to assess the ability of the model to predict changes in cell viability. The proposed model constitutes a proof of concept of digital twinning of biofabricated constructs that can be suitably included in the basic toolkit for tissue bioprinting.