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Microfluidic droplet arrays potentially allow to carry out assays involving complex workflows at the single-cell level. In recent developments (e.g., [H.-H. Jeong et al., Lab Chip, 2016, 16, 1698), each droplet of the array can be addressed individually thanks to onchip pneumatic valves. In this experimental and theoretical work, we investigate the multiple interaction regimes between one or several droplets and a pneumatic valve. In particular, two main trapping modes are characterized, with very distinct flow features. We quantify the potentially huge increase of hydrodynamic resistance induced by the droplet/valve interaction. Finally, we rationalize the main transitions between trapped and non-trapped regimes through a generic theoretical model of the balance between capillary, hydrodynamic and elastic forces.

Background:In vitro maturation of immature testicular tissue (ITT) cryopreserved for fertility preservation is a promising fertility restoration strategy. Organotypic tissue culture proved successful in mice, leading to live births. In larger mammals, including humans, efficiently reproducing spermatogenesis ex vivo remains challenging. With advances in biomaterials technology, culture systems are becoming more complex to better mimic in vivo conditions. Along with improving culture media components, optimizing physical culture conditions (e.g., tissue perfusion, oxygen diffusion) also needs to be considered. Recent studies in mice showed that by using silicone-based hybrid culture systems, the efficiency of spermatogenesis can be improved. Such systems have not been reported for ITT of large mammals.Methods: Four different organotypic tissue culture systems were compared: static i.e., polytetrafluoroethylene membrane inserts (OT), agarose gel (AG) and agarose gel with polydimethylsiloxane chamber (AGPC), and dynamic i.e., microfluidic (MF). OT served as control. Porcine ITT fragments were cultured over a 30-day period using a single culture medium. Analyses were performed at days (d) 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity, diameters, and tissue core integrity were evaluated on histology. Immunohistochemistry was used to identify germ cells (PGP9.5, VASA, SYCP3, CREM), somatic cells (SOX9, INSL3) and proliferating cells (Ki67), and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured in supernatants using ELISA.Results: ITT fragments survived and grew in all systems. ST diameters, and Sertoli cell (SOX9) numbers increased, meiotic (SYCP3) and post-meiotic (CREM) germ cells were generated, and testosterone was secreted. When compared to control (OT), significantly larger STs (d10 through d30), better tissue core integrity (d5 through d20), higher numbers of undifferentiated spermatogonia (d30), meiotic and post-meiotic germ cells (SYCP3: d20 and 30, CREM: d20) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cell (Ki67/VASA) numbers, Leydig cell (INSL3) numbers and testosterone levels were not significantly different between systems.Conclusions: Using a modified culture system (AGPC), germ cell survival and the efficiency of porcine germ cell differentiation were moderately improved ex vivo. We assume that further optimization can be obtained with concomitant modifications in culture media components.

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Study question What is the impact of different static and dynamic organotypic culture systems on the outcome of prepubertal porcine immature testicular tissue (ITT) culture? Summary answer Using a hybrid silicone-agarose static culture system improves germ cell survival and differentiation in ITT ex-vivo. What is known already In mice, organotypic culture of ITT led to the birth of healthy fertile pups. In other larger mammals, including humans, it remains challenging to efficiently reproduce spermatogenesis ex-vivo. With the advent of tissue engineering, culture systems are becoming more complex to better mimic the in vivo environment by modifying and/or improving physical culture conditions (i.e. tissue perfusion, oxygen diffusion, direct/indirect contact with medium). Recent studies in rodents showed that by using hybrid static and dynamic silicone-based organotypic culture systems, the efficiency of spermatogenesis can be significantly improved. Such culture systems have not been reported in large mammals yet. Study design, size, duration Testes were collected from three 5-7 days old piglets as a by-product of castration for a Belgian farm. The fresh ITT fragments were immediately used for culture experiments. Fragments were cultured for a 30 days duration in the different static i.e. polytetrafluoroethylene membrane inserts (CT), agarose gel (AG) and agarose gel with polydimethylsiloxane cover (AGPC) and dynamic i.e. microfluidic (MF) systems using a single medium. The CT system served as control. Participants/materials, setting, methods Cultured ITT fragments were monitored every 4-5 days by serial imaging. Analyses were performed at days 0, 5, 10, 20 and 30. Seminiferous tubule (ST) integrity and diameters were evaluated on Periodic Acid Schiff-stained slides. Immunohistochemistry was used to identify: germ (PGP9.5, VASA, SYCP3, CREM), somatic (SOX9, INSL3) and proliferating (Ki67) cells and to assess oxidative stress (MDA) and apoptosis (C-Caspase3). Testosterone was measured using ELISA. A two-way ANOVA was used for statistical analyses. Main results and the role of chance All systems supported the survival, growth and maturation of ITT fragments in vitro. Between days 0 and 30, ST diameters increased in all systems (p < 0.000) (day 0: 61.2±6.5μm; day 30: CT: 75.3±18.1μm, AG: 77.7±15.7μm, AGPC: 84.9±13.6μm, 66.9±12.9μm). Sertoli cell (SOX9) numbers also increased while spermatogonia (PGP9) numbers per ST decreased (day 0: 6.94±0.31, day 30: CT: 1.84±0.64, AG: 2.65±0.88, AGPC: 3.47±0.7, MF: 2.11±0.84). Meiotic germ cells (SYCP3, CREM) were absent at day 0 and were generated starting day 5 (SYCP3) and 10 (CREM) in the different systems and maintained till day 30 (SYCP3 per ST; CT: 0.31±0.21, AG: 0.5±0.31, AGPC: 0.69±0.20, MF: 0.38±0.20 and CREM per ST: CT: 0.18±0.18, AG: 0.25±0.13, AGPC: 0.34±0.32, MF: 0.14±0.14). When compared to control (CT), significantly larger STs (days 10 to 30), a higher number of spermatogonia (PGP9) (day 30; AGPC: 3.72±0.70, CT: 1.84±0.64, p = 0.04), meiotic SYCP3 + (day 20, AGPC: 0.95±0.49, CT: 0.46±0.39, p = 0.01) and CREM+ cells (day 20, AGPC: 0.49±0.21, CT: 0.23±0.64, p = 0.02) were observed in the AGPC system. Apoptosis, lipid peroxidation (MDA), ST integrity, proliferating germ cells (Ki67/VASA), Leydig cells (INSL3) and testosterone levels were not significantly different between the different systems over the culture period. Limitations, reasons for caution The small sample size and inter-tissue variability could have limited the results of this study. Also it is not excluded that further differences, in terms of tissue growth, germ cell survival and differentiation, could be observed between the different systems if a more complex culture medium is used. Wider implications of the findings In contrast to rodents, modifying physical culture conditions minimally impacts the outcome of porcine ITT maturation ex-vivo. Due to anatomo-physiological similarities between pigs and humans, it is likely for similar results to be observed if applied to human ITT. Studies aiming at determining optimal culture medium components are rather needed. Trial registration number not applicable