<i>In Silico</i> Models for Dynamic Connected Cell Cultures Mimicking Hepatocyte-Endothelial Cell-Adipocyte Interaction Circle

<div><p>The biochemistry of a system made up of three kinds of cell is virtually impossible to work out without the use of <i>in silico</i> 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 <i>in vitro</i> 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 <i>in silico</i> model was then validated against experimental datasets coming from literature about the cited <i>in vitro</i> 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.</p></div

Block diagram showing an overall view of the metabolic pathways with respective interconnections implemented for hepatic cell.

<p>High-energy molecules (ATP, NADH, etc.) metabolism and energy function are not included for clarity, because they influence every subsystem.</p

Block diagram showing an overall view of the metabolic pathways with respective interconnections implemented for adipose cell.

<p>Block diagram showing an overall view of the metabolic pathways with respective interconnections implemented for adipose cell.</p

Measured [23] and simulated glucose and fatty acid trends in the culture medium for hepatic monocultures.

<p>Upper figures refer to static conditions, the other ones describe dynamic conditions. Solid lines represent the simulated data, while circles (for the static case) and squares (for the dynamic case) represent the corresponding experimental data. Measured values are expressed as means ± standard deviation for experiments run at least in triplicate: numerical values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111946#pone.0111946-Vinci1" target="_blank">[23]</a> and error bars represent the standard deviation. (A) Glucose trend in static conditions. (B) Fatty acid trend in static conditions. (C) Glucose trend in dynamic conditions. (D) Fatty acid trend in dynamic conditions.</p

Fatty Acid Translocase kinetic parameters for hepatic (CD36_EP), endothelial (CD36_ET) and adipose (CD36_AD) cells in a saturable process.

<p>Fatty Acid Translocase kinetic parameters for hepatic (CD36_EP), endothelial (CD36_ET) and adipose (CD36_AD) cells in a saturable process.</p

The main interface of the complete (3-way connected) model developed in this work.

<p>There are three principal blocks, represented by cell images: from top to bottom, they correspond to networks of reactions describing endothelial, adipose and hepatic metabolism. On the right side, it is possible to see blocks simulating extracellular fatty acid and glycerol concentrations.</p

Automatic Photo-Cross-Linking System for Robotic-Based In Situ Bioprinting

This work reports the design and validation of an innovative automatic photo-cross-linking device for robotic-based in situ bioprinting. Photo-cross-linking is the most promising polymerization technique when considering biomaterial deposition directly inside a physiological environment, typical of the in situ bioprinting approach. The photo-cross-linking device was designed for the IMAGObot platform, a 5-degree-of-freedom robot re-engineered for in situ bioprinting applications. The system consists of a syringe pump extrusion module equipped with eight light-emitting diodes (LEDs) with a 405 nm wavelength. The hardware and software of the robot were purposely designed to manage the LEDs switching on and off during printing. To minimize the light exposure of the needle, thus avoiding its clogging, only the LEDs opposite the printing direction are switched on to irradiate the newly deposited filament. Moreover, the LED system can be adjusted in height to modulate substrate exposure. Different scaffolds were bioprinted using a GelMA-based hydrogel, varying the printing speed and light distance from the bed, and were characterized in terms of swelling and mechanical properties, proving the robustness of the photo-cross-linking system in various configurations. The system was finally validated onto anthropomorphic phantoms (i.e., a human humerus head and a human hand with defects) featuring complex nonplanar surfaces. The designed system was successfully used to fill these anatomical defects, thus resulting in a promising solution for in situ bioprinting applications

Measured [23] and simulated glucose and fatty acid trends in the culture medium for endothelial monocultures.

<p>Upper figures refer to static conditions, the other ones describe dynamic conditions. Solid lines represent the simulated data, while circles (for the static case) and squares (for the dynamic case) represent the corresponding experimental data. Measured values are expressed as means ± standard deviation for experiments run at least in triplicate: numerical values are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111946#pone.0111946-Vinci1" target="_blank">[23]</a> and error bars represent the standard deviation. (A) Glucose trend in static conditions. (B) Fatty acid trend in static conditions. (C) Glucose trend in dynamic conditions. (D) Fatty acid trend in dynamic conditions.</p

Enzymatic parameters ( for direct and indirect reactions, , kinetic rate expressions) relating to the new enzymes introduced in the model for lipid metabolism.

<p>Enzymatic parameters ( for direct and indirect reactions, , kinetic rate expressions) relating to the new enzymes introduced in the model for lipid metabolism.</p

Measured [24] and simulated metabolite trends in the culture medium for the 3-way connected system (dynamic conditions).

<p>Solid lines represent the simulated data, while squares represent the corresponding experimental data. Measured values are expressed as means ± standard deviation: numerical values were extracted from plots reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111946#pone.0111946-Vinci2" target="_blank">[24]</a> and error bars represent the standard deviation. From three to six replicates were run for each experiment. (A) Glucose trend in culture medium. (B) Fatty acid trend in culture medium. (C) Glycerol trend in culture medium.</p