Osmotic injury and cytotoxicity for hMSCs in contact with Me2SO: The effect of cell size distribution.

The paper discusses the impact of cell size on cytotoxicity and expansion lysis during the osmotic excursions resulting from the contact of hMSCs from UCB with Me2SO. It builds upon the mathematical model recently presented by the authors, which pertains to a population of cells with uniform size. The objective is to enhance the model's relevance by incorporating the more realistic scenario of cell size distribution, utilizing a Population Balance Equations approach. The study compares the capability of the multiple-sized model to the single-sized one to describe system behavior experimentally measured through cytofluorimetry and Coulter counter when, first, suspending hMSCs in hypertonic solutions of Me2SO (at varying osmolality, system temperature, and contact times), and then (at room temperature) pelleting by centrifugation before suspending the cells back to isotonic conditions. Simulations demonstrate that expansion lysis and cytotoxic effect are not affected by cell size for the specific system hMSCs/Me2SO, thus confirming what was found so far by the authors through a single-size model. On the other hand, simulations show that, when varying the adjustable parameters of the model that are expected to change from cell to cell lineages, expansion lysis is sensitive to cell size, while cytotoxicity is not, being mainly influenced by external CPA concentration and contact duration. More specifically, it is found that smaller cells suffer expansion lysis more than larger ones. The findings suggest that different cells from hMSCs may require a multiple-sized model to assess cell damage during osmotic excursions in cryopreservation.

hMSCs in contact with DMSO for cryopreservation: Experiments and modeling of osmotic injury and cytotoxic effect.

In this study a combined analysis of osmotic injury and cytotoxic effect useful for the optimization of the cryopreservation process of a cell suspension is carried out. The case of human Mesenchymal Stem Cells (hMSCs) from Umbilical Cord Blood (UCB) in contact with dimethyl sulfoxide (DMSO) acting as Cryo-Protectant Agent (CPA) is investigated from the experimental as well as the theoretical perspective. The experimental runs are conducted by suspending the cells in hypertonic solutions of DMSO at varying osmolality, system temperature, and contact times; then, at room temperature, cells are pelleted by centrifugation and suspended back to isotonic conditions. Eventually, cell count and viability are measured by means of a Coulter counter and flow-cytometer, respectively. Overall, a decrease in cell count and viability results when DMSO concentration, temperature, and contact time increase. A novel mathematical model is developed and proposed to interpret measured data by dividing the cell population between viable and nonviable cells. The decrease of cell count is ascribed exclusively to the osmotic injury caused by expansion lysis: excessive swelling causes the burst of both viable as well as nonviable cells. On the other hand, the reduction of cell viability is ascribed only to cytotoxicity which gradually transforms viable cells into nonviable ones. A chemical reaction engineering approach is adopted to describe the dynamics of both phenomena: by following the kinetics of two chemical reactions during cell osmosis inside a closed system it is shown that the simultaneous reduction of cell count and viability may be successfully interpreted. The use of the Surface Area Regulation (SAR) model recently proposed by the authors allows one to avoid the setting in advance of fixed cell Osmotic Tolerance Limits (OTLs), as traditionally done in cryopreservation literature to circumvent the mathematical simulation of osmotic injury. Comparisons between experimental data and theoretical simulations are provided: first, a nonlinear regression analysis is performed to evaluate unknown model parameters through a best-fitting procedure carried out in a sequential fashion; then, the proposed model is validated by full predictions of system behavior measured at operating conditions different from those used during the best-fit procedure.

Coupling of mechanical deformation and reaction in mechanochemical transformations.

Driven by the promise of alternative synthetic routes to fine chemicals and pharmaceuticals, mechanochemistry is going through a period of intense growth. Mechanical forces are successfully utilized to activate chemical reactions involving an ever-growing variety of inorganic and organic substances with the aim of developing solvent-less processes to be used in the greener chemical industry of tomorrow. Down this path, the proper understanding of the relationships between processing variables, macroscopic transformation kinetics and microscopic chemistry represents one of the fundamental challenges to face. In this work, we develop a kinetic model that, taking into account the intrinsic statistical nature of the mechanical processing of powders by ball milling, combines a phenomenological description of the rheological behaviour of molecular solids with the chemistry of interface reactions. Specifically, we use discrete deformation maps to account for the co-deformation of molecular solids and the consequent increase of the interface area between initially segregated reactants. We assume that the chemical reaction only occurs, with a certain probability, when reactants come into contact due to relocations induced by shearing. No diffusion is allowed. The systematic variation of the amount of powder involved in individual impacts, the composition of powder mixtures and the reaction probability at the interface provide us with a complete overview of the kinetic scenario. In particular, we present the different kinetic curves that can be originated from interface reaction, pointing out how statistical, mixing and chemical factors affect the mechanochemical kinetics. Eventually, we suggest how experimental findings can be used to gain information on the underlying mechanochemistry based on the outcomes of our kinetic modeling.

Insights into the model of non-perfect osmometer cells for cryopreservation: A parametric sweep analysis.

Recently, a mathematical model able to describe the non-perfect osmotic behavior of cells during cryopreservation was proposed. The model improves the two-parameter formalism typically adopted in cryopreservation literature by allowing the transmembrane permeation of ions/salt, through the temporary opening of mechanosensitive channels whenever membrane stretching occurs: cells can reach an equilibrium volume different from the initial one, when isotonic conditions are re-established after contacting with impermeant or permeant solutes, such as sucrose or a cryoprotectant agent like dimethyl sulfoxide, respectively. Although the model was conceived as a conservative development of the two-parameter formalism to avoid over-parameterization, a complex picture of the system emerges. To describe this, first an appropriate non-dimensional version of the model equations is derived. Then, a parametric sweep analysis is performed and discussed to highlight the features of the novel model in comparison with the two-parameter formalism: the conditions by which the first reduces to the second are identified. Only equilibrium equations with impermeant sucrose may be analytically derived from the model: their validity is here extended much more than originally assumed. When permeant dimethyl sulfoxide comes into play, the temporary opening of mechanosensitive channels is difficult to predict and prevents the derivation of the equilibrium equations: in this case, a numerical integration of system dynamics up to steady state is required to determine the cell volume at equilibrium. In conclusion, cell volume at equilibrium depends on the position of the temporal window of mechanosensitive channels opening, which, in general, is a complex function of model parameters and operating conditions.

A Theoretical Study on Trehalose + Water Mixtures for Dry Preservation Purposes.

The properties of trehalose + water mixtures are studied as a function of mixture composition and temperature using molecular dynamics simulations. As trehalose disaccharide has been proposed for dry preservation purposes, the objective of this work is to analyse the nanoscopic properties of the considered mixtures, in terms of aggregation, clustering, interactions energies, and local dynamics, and their relationships with hydrogen bonding. The reported results allow a detailed characterization of hydrogen bonding and its evolution with mixture composition and thus inferring the effects of trehalose on water structuring providing results to justify the mechanisms of trehalose acting as preservation agent.

Optimization of Brilliant Blue R photocatalytic degradation by silver nanoparticles synthesized using Chlorella vulgaris.

Synthesis of silver nanoparticles (Ag NPs) using microalgae is gaining recognition for its environmentally friendly and cost-effective nature while maintaining high activity of NPs. In the present study, Ag NPs were synthesized using a methanolic extract of Chlorella vulgaris and subjected to calcination. The X-ray diffraction (XRD) analysis showed a crystalline nature of the products with Ag2O and Ag phases with an average crystalline size of 16.07 nm before calcination and an Ag phase with 24.61 nm crystalline size after calcination. Fourier transform infrared spectroscopy (FTIR) revealed the capping functional groups on Ag NPs, while scanning electron microscopy (SEM) displayed their irregular morphology and agglomeration after calcination. The organic coating was examined by energy-dispersive X-ray spectroscopy (EDX) and thermogravimetric (TGA) analyses, confirming the involvement of the metabolites. The UV-Vis analysis showed a difference in optical properties due to calcination. Synthesized Ag NPs were applied for the photodegradation of hazardous dye Brilliant Blue R in visible light. Different values of light intensity, catalyst dose, initial dye concentration, and pH were tested to identify the optimal set of operating conditions. The highest degradation efficiency of 90.6% with an apparent rate constant of 0.04402 min-1 was achieved after 90 min of irradiation in the highest tested catalyst dosage.

A novel population balance model to investigate the kinetics of in vitro cell proliferation: part I. Model development.

In biotechnology and biomedicine reliable models of cell proliferation kinetics need to capture the relevant phenomena taking place during the mitotic cycle. To this aim, a novel mathematical model helpful to investigate the intrinsic kinetics of in vitro culture of adherent cells up to confluence is proposed in this work. Specifically, the attention is focused on the simulation of proliferation (increase of cell number) and maturation (increase of cell size and DNA content) till contact inhibition eventually takes place inside a Petri dish. Accordingly, the proposed model is based on a population balance (PB) approach that allows one to quantitatively describe cell cycle progression through the different phases the cells of the entire population experienced during their own life. In particular, the proposed model has been developed as a 2D, multi-staged, and unstructured PB, by considering a different sub-population of cells for any single phase of the cell cycle. These sub-populations are discriminated through cellular volume and DNA content, that both increase during the mitotic cycle. The adopted mathematical expressions of the transition rates between two subsequent phases and the temporal increase of cell volume and DNA content are thoroughly analyzed and discussed with respect to those ones available in the literature. Specifically, the corresponding uncertainties and pitfalls are pointed out, by also taking into account the difficulties and the limitations involved in the quantitative measurements currently practicable for these biological systems. A novel mathematical expression for contact inhibition in line with the PB model developed is also formulated, along with a proper comparison between modeled and measurable DNA distributions. The strategy for a reliable, independent tuning of the adjustable parameters involved in the proposed model along with its numerical solution is outlined in Part II of this work, where it is also shown how it can be profitably used to gain a deeper insight into the phenomena involved during cell cultivation under microgravity conditions.

A novel population balance model to investigate the kinetics of in vitro cell proliferation: part II. Numerical solution, parameters' determination, and model outcomes.

Based on the general theoretical model developed in Part I of this work, a series of numerical simulations related to the in vitro proliferation kinetics of adherent cells is here presented. First the complex task of assigning a specific value to all the parameters of the proposed population balance (PB) model is addressed, by also highlighting the difficulties arising when performing proper comparisons with experimental data. Then, a parametric sensitivity analysis is performed, thus identifying the more relevant parameters from a kinetics perspective. The proposed PB model can be adapted to describe cell growth under various conditions, by properly changing the value of the adjustable parameters. For this reason, model parameters able to mimic cell culture behavior under microgravity conditions are identified by means of a suitable parametric sensitivity analysis. Specifically, it is found that, as the volume growth parameter is reduced, proliferation slows down while cells arrest in G0/G1 or G2/M depending on the initial distribution of cell population. On the basis of this result, model capabilities have been tested by means of a proper comparison with literature experimental data related to the behavior of synchronized and not-synchronized cells under micro- and standard gravity levels.

Structural Characterisation and Assessment of the Novel Bacillus amyloliquefaciens RK3 Exopolysaccharide on the Improvement of Cognitive Function in Alzheimer's Disease Mice.

In this study Bacillus amyloliquefaciens RK3 was isolated from a sugar mill effluent-contaminated soil and utilised to generate a potential polysaccharide with anti-Alzheimer's activity. Traditional and molecular methods were used to validate the strain. The polysaccharide produced by B. amyloliquefaciens RK3 was purified, and the yield was estimated to be 10.35 gL-1. Following purification, the polysaccharide was structurally and chemically analysed. The structural analysis revealed the polysaccharide consists of α-d-mannopyranose (α-d-Manp) and ß-d-galactopyranose (ß-d-Galp) monosaccharide units connected through glycosidic linkages (i.e., ß-d-Galp(1→6)ß-d-Galp (1→6)ß-d-Galp(1→2)ß-d-Galp(1→2)[ß-d-Galp(1→6)]ß-d-Galp(1→2)α-d-Manp(1→6)α-d-Manp (1→6)α-d-Manp(1→6)α-d-Manp(1→6)α-d-Manp). The scanning electron microscopy and energy-dispersive X-ray spectroscopy imaging of polysaccharides emphasise their compactness and branching in the usual tubular heteropolysaccharide structure. The purified exopolysaccharide significantly impacted the plaques formed by the amyloid proteins during Alzheimer's disease. Further, the results also highlighted the potential applicability of exopolysaccharide in various industrial and pharmaceutical applications.

A methodology to investigate the intrinsic effect of the pulsed electric current during the spark plasma sintering of electrically conductive powders.

A novel methodology is proposed for investigating the effect of the pulsed electric current during the spark plasma sintering (SPS) of electrically conductive powders without potential misinterpretation of experimental results. First, ensemble configurations (geometry, size and material of the powder sample, die, plunger and spacers) are identified where the electric current is forced to flow only through either the sample or the die, so that the sample is heated either through the Joule effect or by thermal conduction, respectively. These ensemble configurations are selected using a recently proposed mathematical model of an SPS apparatus, which, once suitably modified, makes it possible to carry out detailed electrical and thermal analysis. Next, SPS experiments are conducted using the ensemble configurations theoretically identified. Using aluminum powders as a case study, we find that the temporal profiles of sample shrinkage, which indicate densification behavior, as well as the final density of the sample are clearly different when the electric current flows only through the sample or through the die containing it, whereas the temperature cycle and mechanical load are the same in both cases.

A simulation model for stem cells differentiation into specialized cells of non-connective tissues.

A novel mathematical model to simulate stem cells differentiation into specialized cells of non-connective tissues is proposed. The model is based upon material balances for growth factors coupled with a mass-structured population balance describing cell growth, proliferation and differentiation. The proposed model is written in a general form and it may be used to simulate a generic cell differentiation pathway during in vitro cultivation when specific growth factors are used. Literature experimental data concerning the differentiation of central nervous stem cells into astrocytes are successfully compared with model results, thus demonstrating the validity of the proposed model as well as its predictive capability. Finally, sensitivity analysis of model parameters is also performed in order to clarify what mechanisms most strongly influence differentiation and cell types distribution.

Insights on Cryopreserved Sheep Fibroblasts by Cryomicroscopy and Gene Expression Analysis.

Cryopreservation includes a set of techniques aimed at storing biological samples and preserving their biochemical and functional features without any significant alterations. This study set out to investigate the effects induced by cryopreservation on cultured sheepskin fibroblasts (CSSF) through cryomicroscopy and gene expression analysis after subsequent in vitro culture. CSSF cells were cryopreserved in a cryomicroscope (CM) or in a straw programmable freezer (SPF) using a similar thermal profile (cooling rate -5°C/min to -120°C, then -150°C/min to -196°C). CSSF volume and intracellular ice formation (IIF) were monitored by a CM, while gene expression levels were investigated by real-time polymerase chain reaction in SPF-cryopreserved cells immediately after thawing (T0) and after 24 or 48 hours (T24, T48) of post-thaw in vitro culture. No significant difference in cell viability was observed at T0 between CM and SPF samples, while both CM and SPF groups showed lower viability (p < 0.05) compared to the untreated control group. Gene expression analysis of cryopreserved CSSF 24 and 48 hours post-thawing showed a significant upregulation of the genes involved in protein folding and antioxidant mechanisms (HPS90b and SOD1), while a transient increase (p < 0.05) in the expression levels of OCT4, BCL2, and GAPDH was detected 24 hours post-thawing. Overall, our data suggest that cryostored CSSF need at least 24 hours to activate specific networks to promote cell readaptation.

Osmotic behaviour of human mesenchymal stem cells: Implications for cryopreservation.

Aimed at providing a contribution to the optimization of cryopreservation processes, the present work focuses on the osmotic behavior of human mesenchymal stem cells (hMSCs). Once isolated from the umbilical cord blood (UCB) of three different donors, hMSCs were characterized in terms of size distribution and their osmotic properties suitably evaluated through the exposure to hypertonic and isotonic aqueous solutions at three different temperatures. More specifically, inactive cell volume and cell permeability to water and di-methyl sulfoxide (DMSO) were measured, being cell size determined using impedance measurements under both equilibrium and dynamic conditions. Experimental findings indicate that positive cell volume excursions are limited by the apparent increase of inactive volume, which occurs during both the shrink-swell process following DMSO addition and the subsequent restoration of isotonic conditions in the presence of hypertonic solutions of impermeant or permeant solutes. Based on this evidence, hMSCs must be regarded as imperfect osmometers, and their osmotic behavior described within a scenario no longer compatible with the simple two-parameter model usually utilized in the literature. In this respect, the activation of mechano-sensitive ion-channels seemingly represents a reasonable hypothesis for rationalizing the observed osmotic behavior of hMSCs from UCB.

Development of processes for environmental protection based on self-propagating reactions.

The possibility of exploiting self-propagating reactions for environmental protection is discussed in this paper. In particular, results obtained at the laboratory scale and related to the fixation and consolidation of high level radioactive wastes, the recycling of silicon sludge and aluminum dross produced by semiconductor industries and aluminum foundries, the treating and recycling of a highly toxic solid waste from electrolytic zinc plants, and the degradation of chlorinated aromatics, are examined with particular emphasis on the latter case. Specifically, the self-propagating destruction of hexachlorobenzene and 2-(2-4-dichlorophenoxy)-propanoic acid with calcium hydride as reductive substrate is demonstrated. In fact, the heat liberated by the reactions involved is large enough to guarantee the self-sustaining character of the process within a wide range of reactants compositions. Moreover, no residual chlorinated organic compounds were found in the final solid product. Some reactor engineering aspects, as well as other significant future scientific and technological issues, are also addressed in view of large-scale applicability of processes based on self-propagating reactions. To date, the batch reactor technology seems to be more easily applicable, although the use of continuous reactors is not excluded in the next future.

Adsorption of organic compounds onto activated carbons from recycled vegetables biomass.

The removal of organic species from aqueous solution by activated carbons is investigated. The latter ones are prepared from olive husks and almond shells. A wide range of surface area values are obtained varying temperature and duration of both carbonization and activation steps. The adsorption isotherm of phenol, catechol and 2,6-dichlorophenol involving the activated carbons prepared are obtained at 25 degrees C. The corresponding behavior is quantitatively correlated using classical isotherm, whose parameters are estimated by fitting the equilibrium data. A two component isotherm (phenol/2,6-dichlorophenol) is determined in order to test activated carbon behavior during competitive adsorption.