Microfluidic and Static Organotypic Culture Systems to Support Ex Vivo Spermatogenesis From Prepubertal Porcine Testicular Tissue: A Comparative Study.

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.

Predictability in a hydrodynamic pilot-wave system: Resolution of walker tunneling.

A walker is a macroscopic coupling of a droplet and a capillary wave field that exhibits several quantumlike properties. In 2009, Eddi et al. [Phys. Rev. Lett. 102, 240401 (2009)PRLTAO0031-900710.1103/PhysRevLett.102.240401] showed that walkers may cross a submerged barrier in an unpredictable manner and named this behavior "unpredictable walker tunneling." In quantum mechanics, tunneling is one of the simplest arrangements where similar unpredictability occurs. In this paper, we investigate how unpredictability can be unveiled for walkers through an experimental study of walker tunneling with precision. We refine both time and position measurements to take into account the fast bouncing dynamics of the system. Tunneling is shown to be unpredictable until a distance of 2.6 mm from the barrier center, where we observe the separation of reflected and transmitted trajectories in the position-velocity phase-space. The unpredictability is unlikely to be attributable to either uncertainty in the initial conditions or to the noise in the experiment. It is more likely due to changes in the drop's vertical dynamics arising when it interacts with the barrier. We compare this macroscopic system to a tunneling quantum particle that is subjected to repeated measurements of its position and momentum. We show that, despite the different theoretical treatments of these two disparate systems, similar patterns emerge in the position-velocity phase space.

Liquid dispensing in the adhesive hairy pads of dock beetles.

Many insects can climb on smooth inverted substrates using adhesive hairy pads on their legs. The hair-surface contact is often mediated by minute volumes of liquid, which form capillary bridges in the contact zones and aid in adhesion. The liquid transport to the contact zones is poorly understood. We investigated the dynamics of liquid secretion in the dock beetle Gastrophysa viridula by quantifying the volume of the deposited liquid footprints during simulated walking experiments. The footprint volume increased with pad-surface contact time and was independent of the non-contact time. Furthermore, the footprint volume decreased to zero after reaching a threshold cumulative volume (approx. 30 fl) in successive steps. This suggests a limited reservoir with low liquid influx. We modelled our results as a fluidic resistive system and estimated the hydraulic resistance of a single attachment hair of the order of MPa · s/fl. The liquid secretion in beetle hairy pads is dominated by passive suction of the liquid during the contact phase. The high calculated resistance of the secretion pathway may originate from the nanosized channels in the hair cuticle. Such nanochannels presumably mediate the transport of cuticular lipids, which are chemically similar to the adhesive liquid.

Pick up and release of micro-objects: a motion-free method to change the conformity of a capillary contact.

We propose a new 3D-printed capillary gripper equipped with a textured surface for motion-free release. The gripper classically picks up micro-objects thanks to the capillary forces induced by a liquid bridge. Micro-objects are released by decreasing the volume of this bridge through evaporation. The latter can be either natural or speeded up by a heating source (an IR laser or the Joule effect). The volume reduction changes the conformity of the contact between the gripper and the object. We analyze the gripper performance and the capillary force generated, and then we rationalize the release mechanism by defining the concept of contact conformity in the context of capillary forces.

Rupture of a Liquid Bridge between a Cone and a Plane.

In this work, a systematic experimental study of the rupture of an axially symmetric liquid bridge between a cone and a plane was performed, with focus on the volume distribution after break up. A model based on the Young-Laplace equation is presented, and its solutions are compared to experimental data. Cones and conical cavities with different aperture angles were used in our experiments. We found that this aperture influences the potential pinning of the contact line, the meniscus shape, and therefore the liquid transfer. For half aperture angles α < 70°, where no pinning was observed, the liquid bridge slips off from the cone and almost no transfer to the cone is observed. However, at α > 70°, contact line pinning on the cone induces a net liquid transfer to the cone at rupture. In the case of conical cavities, a maximum of liquid transfer is observed for at α = 110°. The distance at which the rupture of the liquid bridge occurs is also discussed. The model can fairly predict the transfer ratio and the rupture height of the liquid bridge.

Adhesive elastocapillary force on a cantilever beam.

This paper reports an experimental and theoretical investigation of a cantilever beam in contact with an underlying substrate, in the presence of an intervening liquid bridge. The beam is deflected in response to the adhesive capillary forces generated by the liquid. Three main regimes of contact are observed, similarly to other elastocapillary systems already reported in the literature. We measured both the position of the liquid meniscus and the force at the beam clamp in the direction normal to the substrate, as functions of the distance between the beam clamp and the substrate. The resulting force-displacement curve is not monotonic and it exhibits hysteresis in the second regime that we could attribute to solid-solid friction at the beam tip. In the third regime, the adhesive force measured at the clamp strongly increases as the beam approaches the substrate. A 2-dimensional beam model is proposed to rationalize these measurements. This model involves several non-linearities due to geometrical constraints, and its solution with a minimum of iterations is not trivial. The model correctly reproduces the force-displacement curve under two conditions: friction is considered in the second regime, and the reaction force applied by the substrate on the beam is distributed in the third regime. These results are discussed in the context of the adhesion of setal tips involved in the terrestrial locomotion of beetles.

Liquid secretion and setal compliance: the beetle's winning combination for a robust and reversible adhesion.

This paper is a brief review and discussion of the recent literature on the hairy adhesive pads of beetles, with the focus on two features of these pads, firstly, compliant setal tips and secondly, a liquid secretion, that together guarantee robust cycles of attachment/detachment on smooth and rough substrates. The compliance is required to ensure sufficient contact between the setal tips and the substrate with a minimum of elastically stored energy at the contact interface. The secretion fills potential gaps between both surfaces, generates capillary adhesive forces, and enhances self-cleaning of these microstructures. Furthermore, the secretion might prevent setal dehydration and subsequently maintain setal tip compliancy. The paper also pinpoints a series of open questions on the physical mechanisms at play to passively regulate the contact forces developed by these hairy pads during locomotion.

Introduction to focus issue on hydrodynamic quantum analogs.

Hydrodynamic quantum analogs is a nascent field initiated in 2005 by the discovery of a hydrodynamic pilot-wave system [Y. Couder, S. Protière, E. Fort, and A. Boudaoud, Nature 437, 208 (2005)]. The system consists of a millimetric droplet self-propeling along the surface of a vibrating bath through a resonant interaction with its own wave field [J. W. M. Bush, Annu. Rev. Fluid Mech. 47, 269-292 (2015)]. There are three critical ingredients for the quantum like-behavior. The first is "path memory" [A. Eddi, E. Sultan, J. Moukhtar, E. Fort, M. Rossi, and Y. Couder, J. Fluid Mech. 675, 433-463 (2011)], which renders the system non-Markovian: the instantaneous wave force acting on the droplet depends explicitly on its past. The second is the resonance condition between droplet and wave that ensures a highly structured monochromatic pilot wave field that imposes an effective potential on the walking droplet, resulting in preferred, quantized states. The third ingredient is chaos, which in several systems is characterized by unpredictable switching between unstable periodic orbits. This focus issue is devoted to recent studies of and relating to pilot-wave hydrodynamics, a field that attempts to answer the following simple but provocative question: Might deterministic chaotic pilot-wave dynamics underlie quantum statistics?

Interaction of two walkers: Perturbed vertical dynamics as a source of chaos.

Walkers are dual objects comprising a bouncing droplet dynamically coupled to an underlying Faraday wave at the surface of a vibrated bath. In this paper, we study the wave-mediated interaction of two walkers launched at one another, both experimentally and theoretically. Different outcomes are observed in which either the walkers scatter or they bind to each other in orbits or promenade-like motions. The outcome is highly sensitive to initial conditions, which is a signature of chaos, though the time during which perturbations are amplified is finite. The vertical bouncing dynamics, periodic for a single walker, is also strongly perturbed during the interaction, owing to the superposition of the wave contributions of each droplet. Thanks to a model based on inelastic balls coupled to the Faraday waves, we show that this perturbed vertical dynamics is the source of horizontal chaos in such a system.

Multi-scale tarsal adhesion kinematics of freely-walking dock beetles.

In this experimental study, living dock beetles are observed during their free upside-down walk on a smooth horizontal substrate. Their weight is balanced by the adhesion of hairy structures present on their tarsomeres. The motions involved in the attachment and detachment of these structures were characterized by simultaneously imaging the beetle from the side at the body scale, and from the top at the scale of a single tarsal chain. The observed multi-scale three-dimensional kinematics of the tarsi is qualitatively described, then quantified by image processing and physically modelled. A strong asymmetry is systematically observed between attachment and detachment kinematics, in terms of both timing and directionality.

Two-frequency forcing of droplet rebounds on a liquid bath.

Droplets can bounce indefinitely on a liquid bath vertically vibrated in a sinusoidal fashion. We here present experimental results that extend this observation to forcing signals composed of a combination of two commensurable frequencies. The Faraday and Goodridge thresholds are characterized. Then a number of vertical bouncing modes are reported, including walkers. The vertical motion can become chaotic, in which case the horizontal motion is an alternation of walk and stop.

Elasto-capillarity in insect fibrillar adhesion.

The manipulation of microscopic objects is challenging because of high adhesion forces, which render macroscopic gripping strategies unsuitable. Adhesive footpads of climbing insects could reveal principles relevant for micro-grippers, as they are able to attach and detach rapidly during locomotion. However, the underlying mechanisms are still not fully understood. In this work, we characterize the geometry and contact formation of the adhesive setae of dock beetles (Gastrophysa viridula) by interference reflection microscopy. We compare our experimental results to the model of an elastic beam loaded with capillary forces. Fitting the model to experimental data yielded not only estimates for seta adhesion and compliance in agreement with previous direct measurements, but also previously unknown parameters such as the volume of the fluid meniscus and the bending stiffness of the tip. In addition to confirming the primary role of surface tension for insect adhesion, our investigation reveals marked differences in geometry and compliance between the three main kinds of seta tips in leaf beetles.

Quantumlike statistics of deterministic wave-particle interactions in a circular cavity.

A deterministic low-dimensional iterated map is proposed here to describe the interaction between a bouncing droplet and Faraday waves confined to a circular cavity. Its solutions are investigated theoretically and numerically. The horizontal trajectory of the droplet can be chaotic: it then corresponds to a random walk of average step size equal to half the Faraday wavelength. An analogy is made between the diffusion coefficient of this random walk and the action per unit mass ℏ/m of a quantum particle. The statistics of droplet position and speed are shaped by the cavity eigenmodes, in remarkable agreement with the solution of Schrödinger equation for a quantum particle in a similar potential well.

Dynamics and statistics of wave-particle interactions in a confined geometry.

A walker is a droplet bouncing on a liquid surface and propelled by the waves that it generates. This macroscopic wave-particle association exhibits behaviors reminiscent of quantum particles. This article presents a toy model of the coupling between a particle and a confined standing wave. The resulting two-dimensional iterated map captures many features of the walker dynamics observed in different configurations of confinement. These features include the time decomposition of the chaotic trajectory in quantized eigenstates and the particle statistics being shaped by the wave. It shows that deterministic wave-particle coupling expressed in its simplest form can account for some quantumlike behaviors.

Rain-induced ejection of pathogens from leaves: revisiting the hypothesis of splash-on-film using high-speed visualization.

Plant diseases are a major cause of losses of crops worldwide. Although rainfalls and foliar disease outbreaks are correlated, the detailed mechanism explaining their link remains poorly understood. The common assumption from phytopathology for such link is that a splash is generated upon impact of raindrops on contaminated liquid films coating sick leaves. We examine this assumption using direct high-speed visualizations of the interactions of raindrops and leaves over a range of plants. We show that films are seldom found on the surface of common leaves. We quantify the leaf-surface's wetting properties, showing that sessile droplets instead of films are predominant on the surfaces of leaves. We find that the presence of sessile drops rather than that of films has important implications when coupled with the compliance of a leaf: it leads to a new physical picture consisting of two dominant rain-induced mechanisms of ejection of pathogens. The first involves a direct interaction between the fluids of the raindrop and the sessile drops via an off-centered splash. The second involves the indirect action of the raindrop that leads to the inertial detachment of the sessile drop via the leaf's motion imparted by the impact of the raindrop. Both mechanisms are distinct from the commonly assumed scenario of splash-on-film in terms of outcome: they result in different fragmentation processes induced by surface tension, and, thus, different size-distributions of droplets ejected. This is the first time that modern direct high-speed visualizations of impacts on leaves are used to examine rain-induced ejection of pathogens at the level of a leaf and identify the inertial detachment and off-center splash ejections as alternatives to the classically assumed splash-on-film ejections of foliar pathogens.

Optimal concentrations in nectar feeding.

Nectar drinkers must feed quickly and efficiently due to the threat of predation. While the sweetest nectar offers the greatest energetic rewards, the sharp increase of viscosity with sugar concentration makes it the most difficult to transport. We here demonstrate that the sugar concentration that optimizes energy transport depends exclusively on the drinking technique employed. We identify three nectar drinking techniques: active suction, capillary suction, and viscous dipping. For each, we deduce the dependence of the volume intake rate on the nectar viscosity and thus infer an optimal sugar concentration consistent with laboratory measurements. Our results provide the first rationale for why suction feeders typically pollinate flowers with lower sugar concentration nectar than their counterparts that use viscous dipping.