Organoids are artificially assembled cell masses developed to reproduce organ functionality as the source tissue [1] and recently proposed in assisted reproduction technologies [2]. The aim of this study was to establish a 3D in vitro maturation (IVM) culture system, in the juvenile sheep model, by using the bioprinting technology, in view of modeling ovarian follicle organoids. Three experiments were conducted for: 1) defining a time interval allowing COC scheduling for encapsulation and IVM by using the Earle’ s/Hank’ s M199 (EH medium) holding procedure; 2) establishing the dataset to produce tailored alginate microbeads allowing inclusion of intact COCs and 3) comparing the effects of 3D versus 2D IVM culture on oocyte nuclear maturation rate. In experiment 1 (Exp. 1), COCs recovered from the ovaries of slaughtered juvenile sheep underwent 24h or 48h EH holding [3] and IVM [4]. COCs cultured immediately after retrieval were used as controls. After IVM, oocytes found at the metaphase II stage were analyzed for bioenergetic status expressed as mitochondria (mt) distribution pattern [4]. In Exp. 2, the spherical hydrogel generator (Sphyga) [5] was used to generate COC-including microbeads and variations of needle diameter and alginate concentration were analyzed. In Exp. 3, the meiotic stage of oocytes cultured in the 3D vs 2D system were compared. Data were analysed by the Chi-square test (significance at P<0.05). In Exp. 1, 318 COCs were analyzed (4 replicates). No deleterious effects on nuclear maturation rate were observed after 24h EH holding (54/108, 50% vs 55/101, 54.5% for treated and controls, respectively; P>0.05) whereas 48h significantly affected maturation rate (33/109, 30.3% vs 55/101, 54.5% for treated and controls, respectively, P<0.001). No significant effect of EH holding on mt pattern was found on either 24 or 48h EH holding (32/55, 58.2% vs. 25/54, 46.3% vs. 15/33, 45.5% for control, 24hEH and 48hEH, respectively; P>0.05). In Exp. 2, working parameters of Sphyga to get reproducible COC-including microbeads mimicking their 3D morphology were set up. In Exp. 3, 132 oocytes were analysed (3 replicates). No differences were observed in oocyte maturation rates in 3D vs 2D system (48/67, 71.6% vs 55/65, 84.6% for 3D and 2D, respectively; NS). In conclusions: 1) 24h EH holding allows to preserve ovine COC viability and competence; 2) microbead size and viscoelastic properties are sensitive parameters to obtain efficient COC encapsulation; 3) the established 3D IVM system allowed to obtain oocyte maturation at comparable rates than conventional 2D system.
ENCAPSULATION OF OVINE CUMULUS-OOCYTE COMPLEXES IN TAILORED ALGINATE MICROBEADS – TOWARDS MODELING OF OVARIAN FOLLICLE ORGANOIDS
Mastrorocco A;
2018-01-01
Abstract
Organoids are artificially assembled cell masses developed to reproduce organ functionality as the source tissue [1] and recently proposed in assisted reproduction technologies [2]. The aim of this study was to establish a 3D in vitro maturation (IVM) culture system, in the juvenile sheep model, by using the bioprinting technology, in view of modeling ovarian follicle organoids. Three experiments were conducted for: 1) defining a time interval allowing COC scheduling for encapsulation and IVM by using the Earle’ s/Hank’ s M199 (EH medium) holding procedure; 2) establishing the dataset to produce tailored alginate microbeads allowing inclusion of intact COCs and 3) comparing the effects of 3D versus 2D IVM culture on oocyte nuclear maturation rate. In experiment 1 (Exp. 1), COCs recovered from the ovaries of slaughtered juvenile sheep underwent 24h or 48h EH holding [3] and IVM [4]. COCs cultured immediately after retrieval were used as controls. After IVM, oocytes found at the metaphase II stage were analyzed for bioenergetic status expressed as mitochondria (mt) distribution pattern [4]. In Exp. 2, the spherical hydrogel generator (Sphyga) [5] was used to generate COC-including microbeads and variations of needle diameter and alginate concentration were analyzed. In Exp. 3, the meiotic stage of oocytes cultured in the 3D vs 2D system were compared. Data were analysed by the Chi-square test (significance at P<0.05). In Exp. 1, 318 COCs were analyzed (4 replicates). No deleterious effects on nuclear maturation rate were observed after 24h EH holding (54/108, 50% vs 55/101, 54.5% for treated and controls, respectively; P>0.05) whereas 48h significantly affected maturation rate (33/109, 30.3% vs 55/101, 54.5% for treated and controls, respectively, P<0.001). No significant effect of EH holding on mt pattern was found on either 24 or 48h EH holding (32/55, 58.2% vs. 25/54, 46.3% vs. 15/33, 45.5% for control, 24hEH and 48hEH, respectively; P>0.05). In Exp. 2, working parameters of Sphyga to get reproducible COC-including microbeads mimicking their 3D morphology were set up. In Exp. 3, 132 oocytes were analysed (3 replicates). No differences were observed in oocyte maturation rates in 3D vs 2D system (48/67, 71.6% vs 55/65, 84.6% for 3D and 2D, respectively; NS). In conclusions: 1) 24h EH holding allows to preserve ovine COC viability and competence; 2) microbead size and viscoelastic properties are sensitive parameters to obtain efficient COC encapsulation; 3) the established 3D IVM system allowed to obtain oocyte maturation at comparable rates than conventional 2D system.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
