Gianni Orsi

Mapping the Pharyngeal and Intestinal pH of Caenorhabditis elegans and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors

Extended dynamic range pH-sensitive ratiometric nanosensors, capable of accurately mapping the full physiological pH range, have been developed and used to characterize the pH of the pharyngeal and intestinal lumen of Caenorhabditis elegans in real-time. Nanosensors, 40 nm in diameter, were prepared by conjugating pH-sensitive fluorophores, carboxyfluorescein (FAM) and Oregon Green (OG) in a 1:1 ratio, and a reference fluorophore, 5-(and-6)-carboxytetramethylrhodamine (TAMRA) to an inert polyacrylamide matrix. Accurate ratiometric pH measurements were calculated through determination of the fluorescence ratio between the pH-sensitive and reference fluorophores. Nanosensors were calibrated with an automated image analysis system and validated to demonstrate a pH measurement resolution of ±0.17 pH units. The motility of C. elegans populations, as an indicator for viability, showed nematodes treated with nanosensors, for concentrations ranging from 50.00 to 3.13 mg/mL, were not statistically different to nematodes not challenged with nanosensors up to a period of 4 days (p < 0.05). The nanosensors were also found to remain in the C. elegans lumen >24 h after nanosensor challenge was removed. The pH of viable C. elegans lumen was found to range from 5.96 ± 0.31 in the anterior pharynx to 3.59 ± 0.09 in the posterior intestine. The pharyngeal pumping rate, which dictates the transfer of ingested material from the pharynx to the intestine, was found to be temperature dependent. Imaging C. elegans at 4 °C reduced the pharyngeal pumping rate to 7 contractions/min and enabled the reconstruction of rhythmic pH oscillations in the intestinal lumen in real-time with fluorescence microscopy.

Mixing of Two Miscible Liquids in T-shaped Microdevices

Numerical simulations were performed to study the flow fields and mixing characteristics of liquid flows converging in a T-shaped micromixer, when the two inlet fluids are both water or water and ethanol. We showed that at smaller Reynolds number, Re < 100, mixing is controlled by transverse diffusion, and therefore by the residence times of each fluids. Accordingly, mixing ethanol and water is slightly easier than mixing water with water, due to the fact that, as ethanol is slightly more viscous than water and therefore it is slower, the residence time of water-ethanol mixtures is larger than that of the water-water case. On the other hand, at larger Reynolds number, mixing water and ethanol may take considerably longer, as the onset of engulfment is retarded and occurs at larger Reynolds number, namely increasing from Re ≅ 140 in the water-water case to Re ≅ 230 in the water-ethanol case. This is due to the fact that a water-ethanol mixture has a viscosity that is up to almost three times larger than that of water; therefore, at the confluence of the T-mixer, the water and the ethanol streams are separated by a quite viscous layer of a water-ethanol mixture, that hampers any vortex formation, thus retarding mixing.

A new 3D concentration gradient maker and its application in building hydrogels with a 3D stiffness gradient.

For a deeper knowledge of phenomena at cell and tissue level, for understanding the role on bimolecular signalling and for the development of new drugs it is important to recreate in vitro environments that mimic the physiological one. Spatial gradients of soluble species guide the cells’ morphogenesis, and they range in a three‐dimensional (3D) environment. Gradients of mechanical properties, which have a 3D pattern, could lead cell migration and differentiation. In this work, a new 3D Concentration Gradient Maker able to generate 3D concentration gradients of soluble species was developed, which could be used for differential perfusion of scaffolds. The same device can be applied to build hydrogel matrixes with a 3D gradient of mechanical properties. Computational dynamic fluid analysis was used to develop the gradient generator; the validation of the 3D gradient of stiffness was carried out using finite elements analysis and experimental studies. The device and its application could bring improvements in studying phenomena related to cell chemotaxis and mechanotaxis, but also to differentiation in the simultaneous presence of gradients in both soluble chemical species and substrate stiffness. Copyright

Combining Inkjet Printing and Sol-Gel Chemistry for Making pH-Sensitive Surfaces

Today biomedical sciences are experiencing the importance of imaging biological parameters with luminescence methods. Studying 2D pH distribution with those methods allows building knowledge about complex cellular processes. Immobilizing pH sensitive nanoparticles inside hydrogel matrixes, in order to guarantee a proper SNR, could easily make stable and biocompatible 2D sensors. Inkjet printing is also well known as tool for printing images onto porous surfaces. Recently it has been used as a free-form fabrication method for building three-dimensional parts, and now is being explored as a way of printing electrical and optical devices. Inkjet printing was used either as a rapid prototyping method for custom biosensors. Sol-gel method is naturally bound with inkjet, because the picoliter-sized ink droplets evaporate quickly, thus allowing quick sol-gel transitions on the printed surface. In this work will be shown how to merge those technologies, in order to make a nanoparticles doped printable hydrogel, which could be used for making 2D/3D smart scaffolds able to monitor cell activities. An automated image analysis system was developed in order to quickly have the pH measurements from pH nanosensors fluorescence images.

HEMETβ: improvement of hepatocyte metabolism mathematical model

This article describes hepatocyte metabolism mathematical model (HEMETβ), which is an improved version of HEMET, an effective and versatile virtual cell model based on hepatic cell metabolism. HEMET is based on a set of non-linear differential equations, implemented in Simulink®, which describes the biochemical reactions and energetic cell state, and completely mimics the principal metabolic pathways in hepatic cells. The cell energy function and modular structure are the core of this model. HEMETβ as HEMET model describes hepatic cellular metabolism in standard conditions (cell culture in a plastic multi-well placed in an incubator at 37°C with 5% of CO2) and with excess substrates concentration. The main improvements in HEMETβ are the introductions of Michaelis–Menten models for reversible reactions and enzymatic inhibition. In addition, we eliminated hard non-linearities and modelled cell proliferation and every single aminoacid degradation pathway. All these innovations, combined with a user-friendly aspect, allow researchers to create new cell types and validate new experimental protocols just varying ‘peripheral’ pathways or model inputs.

The influence of mesh topology in the abdominal wall repair process

The tissue integration and the formation of adhesions in the repair of abdominal wall defects are principally led to the topology and the mechanical properties of implanted prosthesis. In this study we analyzed the influence of the topology of the meshes for abdominal wall repair, made of polypropylene (PP), evaluating its ability to prevent and to minimize the formation of adhesions, and to promote tissue ingrowth. Two series of in vivo studies were performed. In the first, two types of PP meshes, a lightweight macroporous mesh (LWM) and a heavyweight microporous mesh (HWM) were compared with determine the optimal porosity for tissue integration. In the second, a composite mesh, Clear Mesh Composite (CMC), made of a LWM sewn on a PP planar smooth film, was compared with a PP planar film, to demonstrate how two different topologies of same material are able to induce different tissue integration with the abdominal wall and different adhesion with internal organs. In both studies, the prostheses were implanted in Wistar rats and histological analysis and mechanical characterization of tissue coupled with the implants were performed. LWM showed better host tissue ingrowth in comparison to HWM. CMC prosthesis showed no adhesions to the viscera and no strong foreign body reaction, moreover its elasticity and anisotropy index were more similar to that of natural tissue. These results demonstrated that the surface morphology of PP surgical meshes allowed to modulate their repair ability.

ADMET: ADipocyte METabolism mathematical model

White fat cells have an important physiological role in maintaining triglyceride and free fatty acid levels due to their fundamental storage property, as well as determining insulin resistance. ADipocyte METabolism is a mathematical model that mimics the main metabolic pathways of human white fat cell, connecting inputs (composition of culture medium) to outputs (glycerol and free fatty acid release). It is based on a set of nonlinear differential equations, implemented in Simulink® and controlled by cellular energetic state. The validation of this model is based on a comparison between the simulation results and a set of experimental data collected from the literature.

In silico models for dynamic connected cell cultures mimicking hepatocyte-endothelial cell-adipocyte interaction circle

The biochemistry of a system made up of three kinds of cell is virtually impossible to work out without the use of in silico models. Here, we deal with homeostatic balance phenomena from a metabolic point of view and we present a new computational model merging three single-cell models, already available from our research group: the first model reproduced the metabolic behaviour of a hepatocyte, the second one represented an endothelial cell, and the third one described an adipocyte. Multiple interconnections were created among these three models in order to mimic the main physiological interactions that are known for the examined cell phenotypes. The ultimate aim was to recreate the accomplishment of the homeostatic balance as it was observed for an in vitro connected three-culture system concerning glucose and lipid metabolism in the presence of the medium flow. The whole model was based on a modular approach and on a set of nonlinear differential equations implemented in Simulink, applying Michaelis-Menten kinetic laws and some energy balance considerations to the studied metabolic pathways. Our in silico model was then validated against experimental datasets coming from literature about the cited in vitro model. The agreement between simulated and experimental results was good and the behaviour of the connected culture system was reproduced through an adequate parameter evaluation. The developed model may help other researchers to investigate further about integrated metabolism and the regulation mechanisms underlying the physiological homeostasis.

Volume of Mixing Effect on Fluid Counter-Diffusion

The counter-current diffusion-driven mixing process of two miscible fluids is studied in the absence of gravity, assuming that the mixture is non-regular, that is its volume is smaller than the sum of the initial volumes of the two components. Two competing effects are present in the mixing region: on one hand, the mass flow rate of each species increases, due to the larger density of the fluid; on the other hand, though, the volumetric flux is retarded by the inward convection due to volume disappearance, which opposes the outward velocity field due to diffusion. This intuition is confirmed by the analytical result of a 1D non-ideal mixing process, showing that, in the presence of the convection induced by a volume decrease: (a) the process is self-similar; (b) the mass flux of each species at the interface increases by approximately 0.8e, where e is the maximum relative volume decrease; and (c) the volume flux of each species decreases by approximately a 0.2e amount. This result is further confirmed by ...

LTI Models for 3-Iodothyronamine Time Dynamics: A Multiscale View

3-Iodothyronamine (Tι AM) is a novel relative of thyroid hormone that plays a role in critical body regulatory processes such as glucose metabolism, thermal regulation, and heart beating. This paper was aimed at characterizing time dynamics of T1AM and its catabolite 3-iodothyroacetic acid (TA1) in different biological scales with linear time-invariant models. Culture medium samples coming from culture of H9c2 murine cells and perfusion liquid samples from perfused rat heart were collected after the injection of a T1AM bolus. T1AM and TA1 concentrations in the samples were assayed with high-performance liquid chromatography coupled to tandem mass spectrometry. Kinetic constants relative to T1AM transport and conversion were estimated with weighted least-squares method. We found that these constants can be related with an allometric power law depending on mass, with a negative exponent of -0.27 ± 0.19, implying that the velocity of conversion and internalization of Ti AM decreases with increasing of system mass.