Cat vitrified oocytes culture in 3D liquid marble microbioreactors

Liquid marbles, also known as pearl drops, are three-dimensional (3D) microbioreactors in which living cells can survive, proliferate and react to stimuli. The pillar on which this technology is established is the use of non-adhesive, hydrophobic molecules to create a casing that maintains the cells suspended in a small volume of inner liquid and has the right porosity to allow free exchange of gases (1). Microorganisms, tumor spheroids, red blood cells and embryonic stem cells have been cultured in liquid marbles, and their survival and growth were successfully obtained (1–3). This microbioreactor, that better resembles the in vivo conditions compared to two-dimensional (2D) cultures, might also be beneficial for female gametes, and especially for the low-competence cryopreserved oocytes. However, only one study with fresh sheep gametes (4) provided some information on the usefulness of this environment for in vitro maturation (IVM). In this study, the efficiency of liquid marbles as 3D microbioreactors for the IVM of vitrified domestic cat oocytes was assessed and compared with that of traditional microdrops of medium (2D). Material and methods: Fresh ovaries (n = 30) from domestic queens were obtained after surgery and cumulus oocytes complexes (COCs) were collected. Sixty-five COCs were vitrified by Cryotop method (5) and, after warming, morphologically intact (6) vitrified oocytes (VOs, n = 59) were cultured in 3D liquid marbles (n = 30) or in 2D microdrops of medium (n = 29). To create the 3D microbioreactor, a chemically inert hydrophobic powder (polytetrafluoroethylene, PTFE; Sigma-Aldrich, St. Louis, MO, USA) was used following a published protocol (4). Oocytes were matured for 24 h in a controlled atmosphere (38.5°C and 5% CO2 in air) in TCM199 supplemented with 10% FBS, 10 ng/mL EGF, 0.6 mM cysteine (Sigma-Aldrich) and 0.5 IU/mL FSH + 0.5 IU/mL LH (Pluset, Calier, Spain). Chromatin configurations were determined by bisbenzimide (Hoechst 33342; Sigma-Aldrich) staining, and data were analyzed by Chi-square test, with the level of significance set at p < 0.05. Results: Vitrified oocytes resumed meiosis at similar proportions in 3D and 2D culture conditions (3D: 50% vs. 2D: 55.2%; p = 0.69), and the same trend was observed for full maturation (3D: 13.3% vs. 2D: 13.8%; p = 0.96). Conclusions: Liquid marble technology, even if promising for different types of somatic cells, in these experimental conditions had the same effect as traditional 2D culture on cat immature vitrified oocytes IVM. To fully restore and boost the developmental competence of feline cryopreserved oocytes other systems remain to be investigated. References: 1) Arbatan et al., Adv Healthc Mater 2012;1:467–9. 2) Arbatan et al., Adv Healthc Mater 2012;1:80–3. 3) Sarvi et al., Adv Healthc Mater 2015;4:77–86. 4) Ledda et al., J Assist Reprod Genet 2016;33:513–8. 5) Kuwayama, Theriogenology 2007;67:73–80. 6) Apparicio et al., Reprod Dom Anim 2013;48:240–4.