Micro-mechanical viscoelastic properties of crosslinked hydrogels using the nano-epsilon dot method

Engineering materials that recapitulate pathophysiological mechanical properties of native tissues in vitro is of interest for the development of biomimetic organ models. To date, the majority of studies have focused on designing hydrogels for cell cultures which mimic native tissue stiffness or quasi-static elastic moduli through a variety of crosslinking strategies, while their viscoelastic (time-dependent) behavior has been largely ignored. To provide a more complete description of the biomechanical environment felt by cells, we focused on characterizing the micro-mechanical viscoelastic properties of crosslinked hydrogels at typical cell length scales. In particular, gelatin hydrogels crosslinked with different glutaraldehyde (GTA) concentrations were analyzed via nano-indentation tests using the nano-epsilon dot method. The experimental data were fitted to a Maxwell Standard Linear Solid model, showing that increasing GTA concentration results in increased instantaneous and equilibrium elastic moduli and in a higher characteristic relaxation time. Therefore, not only do gelatin hydrogels become stiffer with increasing crosslinker concentration (as reported in the literature), but there is also a concomitant change in their viscoelastic behavior towards a more elastic one. As the degree of crosslinking alters both the elastic and viscous behavior of hydrogels, caution should be taken when attributing cell response merely to substrate stiffness, as the two effects cannot be decoupled

Engineering hydrogel viscoelasticity

The aim of this study was to identify a method for modifying the time-dependent viscoelastic properties of gels without altering the elastic component. To this end, two hydrogels commonly used in biomedical applications, agarose and acrylamide, were prepared in aqueous solutions of dextran with increasing concentrations (0, 2 and 5% w/v) and hence increasing viscosities. Commercial polyurethane sponges soaked in the same solutions were used as controls, since, unlike in hydrogels, the liquid in these sponge systems is poorly bound to the polymer network. Sample viscoelastic properties were characterised using the epsilon-dot method, based on compression tests at different constant strain-rates. Experimental data were fitted to a standard linear solid model. While increasing the liquid viscosity in the controls resulted in a significant increase of the characteristic relaxation time (), both the instantaneous () and the equilibrium ( ) elastic moduli remained almost constant. However, in the hydrogels a significant reduction of both and was observed. On the other hand, as expected, – an indicator of the equilibrium elastic behaviour after the occurrence of viscoelastic relaxation dynamics – was found to be independent of the liquid phase viscosity. Therefore, although the elastic and viscous components of hydrogels cannot be completely decoupled due to the interaction of the liquid and solid phases, we show that their viscoelastic behaviour can be modulated by varying the viscosity of the aqueous phase. This simple-yet-effective strategy could be useful in the field of mechanobiology, particularly for studying cell response to substrate viscoelasticity while keeping the elastic cue (i.e. equilibrium modulus, or quasi-static stiffness) constant

The automation of the \"Welding Machine\" for the DUs integration in the KM3NeT experiment

This note describes some technical aspects, in particular the control system, of one of the tools used during Process-1 of the integration of the Detection Units (DUs) in the KM3NeT experiment. In particular, the device is designed to seal the Break-out-box (BOB). The BOB is a box interface between a Digital Optical Module (DOM) and the electro-optical cable (VEOC) for power and optical connection of each optical module (DOM) to the DU. The original manual version of the tool developed by NIKHEF was then automatized by INFN-LNS in order to guarantee a replicable operation in the recursive process of the DU integration and two samples have been developed for the integration sites at LNS and Genova respectively. The general principle and performances of the automated welding machine will be presented shortly in a dedicated INFN report. Here we summarize the cycle of the operations performed by the machine and how they are managed by corresponding control program and related electronics boards

Environmental Control in Flow Bioreactors

The realization of physiologically-relevant advanced in vitro models is not just related to the reproduction of a three-dimensional multicellular architecture, but also to the maintenance of a cell culture environment in which parameters, such as temperature, pH, and hydrostatic pressure are finely controlled. Tunable and reproducible culture conditions are crucial for the study of environment-sensitive cells, and can also be used for mimicking pathophysiological conditions related with alterations of temperature, pressure and pH. Here, we present the SUITE (Supervising Unit for In Vitro Testing) system, a platform able to monitor and adjust local environmental variables in dynamic cell culture experiments. The physical core of the control system is a mixing chamber, which can be connected to different bioreactors and acts as a media reservoir equipped with a pH meter and pressure sensors. The chamber is heated by external resistive elements and the temperature is controlled using a thermistor. A purpose-built electronic control unit gathers all data from the sensors and controls the pH and hydrostatic pressure by regulating air and CO2 overpressure and flux. The system’s modularity and the possibility of imposing different pressure conditions were used to implement a model of portal hypertension with both endothelial and hepatic cells. The results show that the SUITE platform is able to control and maintain cell culture parameters at fixed values that represent either physiological or pathological conditions. Thus, it represents a fundamental tool for the design of biomimetic in vitro models, with applications in disease modelling or toxicity testin

Design and development of a dual-flow bioreactor mimicking intestinal peristalsis and permeability in epithelial tissue barriers for drug transport assessment.

we present a bioreactor system which combines a semi permeable membrane that simulates the osmotic nutrients interchange in the intestine circulation and rhythmic peristaltic movement. This custom-designed, pulsatile membrane bioreactor, with programmable manipulation, allows adjustment of luminal flow rate. In addition, this system is also capable of achieving the intestine-like drug distribution to the cells by the luminal perfusion of the hollow channel compartment. This dynamic platform can mimic the human intestine with increased accuracy to overcome many of the limitations with the previously described in vitro intestinal models, providing a more representative model of the human intestine

The new "Welding Machine" for the DUs integration in the KM3NeT experiment: Mechanical and Pneumatic parts

This note describes one of the tools used during Process-1 of the integration of the Detection Units (DUs) in the KM3NeT experiment. In particular, the device is designed to seal the Break-out-box (BOB). The BOB is a box interface between a Digital Optical Module (DOM) and the electro-optical cable (VEOC) for power and optical connection of each optical module (DOM) to the DU. The original manual version of the tool developed by NIKHEF was then automatized by INFN-LNS in order to guarantee a reproducible operation in the recursive process of the DU integration and two samples have been realized for the integration sites at LNS and Genova respectively

real time cellular impedance monitoring and imaging in a dual flow bioreactor

The generation of physiologically relevant in vitro models of biological barriers can play a key role in understanding human diseases and in the development of more predictive methods for assessing toxicity and drug or nutrient absorption. Here, we present an advanced cell culture system able to mimic the dynamic environment of biological barriers, while monitoring cell behaviour through real-time impedance measurements and imaging. Caco-2 cells were cultured in the Trans Epithelial Electric Impedance (TEEI) bioreactor under both flow and static conditions. The cells in dynamic conditions developed higher impedance values at low frequencies and showed a typical RC behaviour, while the controls showed minimal capacitive behaviour. These results highlighted the differences between flow and static conditions and the ability of the TEEI measurements to provide a more precise indication of monolayer formation

NEMO-SN1 Abyssal Cabled Observatory in the Western Ionian Sea

The NEutrinoMediterranean Observatory—Submarine Network 1 (NEMO-SN1) seafloor observatory is located in the central Mediterranean Sea, Western Ionian Sea, off Eastern Sicily (Southern Italy) at 2100-m water depth, 25 km from the harbor of the city of Catania. It is a prototype of a cabled deep-sea multiparameter observatory and the first one operating with real-time data transmission in Europe since 2005. NEMO-SN1 is also the first-established node of the European Multidisciplinary Seafloor Observatory (EMSO), one of the incoming European large-scale research infrastructures included in the Roadmap of the European Strategy Forum on Research Infrastructures (ESFRI) since 2006. EMSO will specifically address long-term monitoring of environmental processes related to marine ecosystems, marine mammals, climate change, and geohazards

Dependence of atmospheric muon flux on seawater depth measured with the first KM3NeT detection units: The KM3NeT Collaboration

KM3NeT is a research infrastructure located in the Mediterranean Sea, that will consist of two deep-sea Cherenkov neutrino detectors. With one detector (ARCA), the KM3NeT Collaboration aims at identifying and studying TeV–PeV astrophysical neutrino sources. With the other detector (ORCA), the neutrino mass ordering will be determined by studying GeV-scale atmospheric neutrino oscillations. The first KM3NeT detection units were deployed at the Italian and French sites between 2015 and 2017. In this paper, a description of the detector is presented, together with a summary of the procedures used to calibrate the detector in-situ. Finally, the measurement of the atmospheric muon flux between 2232–3386 m seawater depth is obtained