Cellular Colonies Research Articles

Photocytoxicity in vitro studies of ruthenium (II) complexes inducing cell death through targeting DNA

Photodynamic therapy (PDT) has been proven to be an important and effective therapy method with good spatial and temporal accuracy in clinical cancer treatment. Among many PDT candidates, Ru(II)-based compounds have received more attention due to their excellent photochemical and photophysical properties, high singlet oxygen (1O2) photogeneration, and DNA affinities. In this study, three ruthenium(II) complexes (RC1, RC2, RC3) have been designed by varying the aromatic plane area of the main ligand to adjust their 1O2 photogeneration, DNA affinities, DNA photodamage, and photocytoxicity. All complexes display no cytotoxicity in the dark against Hela, A549, HepG2, and BEL-7402 cells. RC1 displays no photocytotoxicity against all tumor cells. RC2 and RC3 showed high photocytotoxicity against BEL-7402 cells. The cellular uptake, cell migration, colonies assay, apoptosis analysis, and cell cycle analysis of RC2 and RC3 confirmed that the ruthenium(II) complexes might effectively enter the cancer cells, and lead to BEL-7402 cells apoptosis through DNA damage pathway. DNA photocleavage, DNA binding, and 1O2 measurement experiments demonstrated the DNA damage caused by RC2 and RC3 under light, compared to RC1, might be due to their high DNA affinities, large 1O2 quantum yields, and large lipophilicity by varying the aromatic plane area of the main ligand. Time dependent density functional theory (TDDFT) calculated results further show that two complexes exhibit the photocytotoxicity possibly via type II photoreaction mechanism because of the larger ΔS-T than O2 and strong SOC between Sn and Tm. Hence, rational design of ruthenium(II) complexes by increasing DNA affinities, 1O2 quantum yields and lipophilicity can be regarded as an effective approach for enhanced photodynamic therapy (PDT).

The population context is a driver of the heterogeneous response of epithelial cells to interferons

Isogenic cells respond in a heterogeneous manner to interferon. Using a micropatterning approach combined with high-content imaging and spatial analyses, we characterized how the population context (position of a cell with respect to neighboring cells) of epithelial cells affects their response to interferons. We identified that cells at the edge of cellular colonies are more responsive than cells embedded within colonies. We determined that this spatial heterogeneity in interferon response resulted from the polarized basolateral interferon receptor distribution, making cells located in the center of cellular colonies less responsive to ectopic interferon stimulation. This was conserved across cell lines and primary cells originating from epithelial tissues. Importantly, cells embedded within cellular colonies were not protected from viral infection by apical interferon treatment, demonstrating that the population context-driven heterogeneous response to interferon influences the outcome of viral infection. Our data highlights that the behavior of isolated cells does not directly translate to their behavior in a population, placing the population context as one important factor influencing heterogeneity during interferon response in epithelial cells.

P191 - Explants from high-grade bladder cancer demonstrate evident 3D-spheroids cellular colonies formation in vitro.

P191 - Explants from high-grade bladder cancer demonstrate evident 3D-spheroids cellular colonies formation in vitro.

Triplet-Net Classification of Contiguous Stem Cell Microscopy Images.

Cellular microscopy imaging is a common form of data acquisition for biological experimentation. Observation of gray-level morphological features allows for the inference of useful biological information such as cellular health and growth status. Cellular colonies can contain multiple cell types, making colony level classification very difficult. Additionally, cell types growing in a hierarchical, downstream fashion, can often look visually similar, although biologically distinct. In this paper, it is determined empirically that traditional deep Convolutional Neural Networks (CNN) and classical object recognition techniques are not sufficient to distinguish between these subtle visual differences, resulting in misclassifications. Instead, Triplet-net CNN learning is employed in a hierarchical classification scheme to improve the ability of the model to discern distinct, fine-grain features of two commonly confused morphological image-patch classes, namely Dense and Spread colonies. The Triplet-net method improves classification accuracy over a four-class deep neural network by ∼ 3 %, a value that was determined to be statistically significant, as well as existing state-of-the-art image patch classification approaches and standard template matching. These findings allow for the accurate classification of multi-class cell colonies with contiguous boundaries, and increased reliability and efficiency of automated, high-throughput experimental quantification using non-invasive microscopy.

High‐throughput formation of miniaturized cocultures of 2D cell monolayers and 3D cell spheroids using droplet microarray

AbstractMost of the biological processes, including cell signaling, cancer invasion, embryogenesis, or neural development, are dependent on and guided by the complex architecture and composition of cellular microenvironments. Mimicking such microenvironments in cell coculture models is crucial for fundamental and applied biology investigations. The ability to combine different cell types grown as both two‐dimensional (2D) monolayers and three‐dimensional (3D) spheroids in specific defined location inside a microculture environments is a key towards in vitro tissue modeling and towards mimicking complex in vivo cellular processes. In this study, we introduce and investigate a method to create in vitro models of 2D cell monolayers cocultured with 3D spheroids in defined preorganization. We demonstrate the possibility of creating such complex cellular microenvironments in a high‐throughput and automated manner by creating arrays of such droplets containing prearranged 2D and 3D cellular microcolonies. Furthermore, we demonstrate an application of this approach to study paracrine propagation of Wnt signaling between 2D and 3D cellular colonies. This method provides a general approach for the miniaturized, high‐throughput, and automated formation of complex coculture cellular microarchitectures that will be useful for mimicking various in vivo complex cellular structures and for studying complex biological processes in vitro.

Tracking the Travels

Tracking the Travels

Angiogenic Effect of a Nanoniosomal Deferoxamine-Loaded Poly(vinyl alcohol)-Egg White Film as a Promising Wound Dressing.

Owing to the noticeable increase in the number of patients with impaired wound healing capabilities, developing bioactive wound dressings with supportive physicomechanical and biological properties for clinical wound management has attracted much more attention nowadays. In this regard, engineered dressings with angiogenesis potential are vital for accelerated tissue regeneration. In the current study, nanoniosomal deferoxamine (DFO)-loaded transparent films of egg white-poly(vinyl alcohol) (PVA/EW/ND) were successfully fabricated at three different PVA/EW ratios (1:0, 1:1, and 1:1.5 wt/wt %) through the thin film hydration and solvent casting methods. The developed films' characterizations were carried out using scanning electron microscopy, Fourier transform infrared spectroscopy analysis, uniaxial tensile strength, water uptake, water vapor transmission rate, in vitro degradation, and drug release. The results demonstrated that the various weight ratios of PVA/EW have a significant effect on the microscopic morphology, equilibrium swelling, degradation, and mechanical properties of the films. The drug release profile exhibited a sustained release of DFO with controlled burst-lag phases resembling the Korsmeyer-Peppas pattern. The cytotoxicity and adhesion analysis using human dermal fibroblasts displays the biocompatibility of the developed PVA/EW/ND films and the formation of cellular colonies on the surface. The in vitro angiogenic capability of the developed films evaluated by the scratch wound assay and microbead-assisted tube formation study showed a significant increase in the rate of migration of human umbilical vein endothelial cells and in the number of tube-like structures. Therefore, the achieved results suggest that the presented PVA/EW/ND film has promising potential for effective wound healing applications.

Microstructural refinement and mechanical response of Al2O3–ZrO2 eutectics fabricated by a novel pulse discharge plasma assisted melting method

In the present work, microstructural refinement and mechanical response of Al2O3–ZrO2 eutectics fabricated by a pulse discharge plasma assisted melting (PDPAM) method were investigated. The solidified microstructure evolves from polygonal eutectic colonies into irregular cellular colonies with increasing the superheating temperature of the melt from 1820 °C to 1900 °C. The average eutectic spacing inside the colonies decreases from 1.80 ± 0.10 μm to 0.25 ± 0.06 μm, and the coarse inter-colonial structure is refined, which is attributed to the increase in undercooling temperature. High-temperature microstructural stability of Al2O3–ZrO2 eutectics is improved significantly as contrasted with the as-sintered ceramics. Besides, the load dependence of Vickers hardness for Al2O3–ZrO2 eutectics is investigated.

Self-shaping liquid crystal droplets by balancing bulk elasticity and interfacial tension

The shape diversity and controlled reconfigurability of closed surfaces and filamentous structures, universally found in cellular colonies and living tissues, are challenging to reproduce. Here, we demonstrate a method for the self-shaping of liquid crystal (LC) droplets into anisotropic and three-dimensional superstructures, such as LC fibers, LC helices, and differently shaped LC vesicles. The method is based on two surfactants: one dissolved in the LC dispersed phase and the other in the aqueous continuous phase. We use thermal stimuli to tune the bulk LC elasticity and interfacial energy, thereby transforming an emulsion of polydispersed, spherical nematic droplets into numerous, uniform-diameter fibers with multiple branches and vice versa. Furthermore, when the nematic LC is cooled to the smectic-A LC phase, we produce monodispersed microdroplets with a tunable diameter dictated by the cooling rate. Utilizing this temperature-controlled self-shaping of LCs, we demonstrate life-like smectic LC vesicle structures analogous to the biomembranes in living systems. Our experimental findings are supported by a theoretical model of equilibrium interface shapes. The shape transformation is induced by negative interfacial energy, which promotes a spontaneous increase of the interfacial area at a fixed LC volume. The method was successfully applied to many different LC materials and phases, demonstrating a universal mechanism for shape transformation in complex fluids.

Medical Plaster Enhancement by Coating with Cistus L. Extracts within a Chitosan Matrix: From Natural Complexity to Health Care Simplicity

Our investigation was focused on the preparation and characterization of novel plasters based on Carboxymethyl Chitosan derivative (CMC), to be used for the treatment of radiation dermatitis with Biologic Active Compounds (BACs) in a moist wound-healing environment. After performing the extraction and characterization of BACs from Cistus L., we optimized the BACs/CMC solution for subsequent plaster preparation. Then, plasters were prepared by dip-coating with a different number of layers, and we characterized them by Environmental Scanning Electron Microscopy (ESEM), Contact Angle (CA) and release tests in water for 24 h. Taking into account the flexibility of the plasters and the amount of released BACs after 24 h, the sample obtained after two dip-coating steps (2La) appeared promising in regard to comfortable mechanical properties and active principles administration. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test performed on keratinocytes cultured in standard medium shows that cells treated with released extract from 2La start to proliferate, extend cellular viability and form colonies typical for epidermal cells.

Inhibitory effects of cynaropicrin on human melanoma progression by targeting MAPK, NF-κB, and Nrf-2 signaling pathways in vitro.

Malignant melanoma is the deadliest skin cancer, due to its propensity to metastasize. MAPKs and NF-κB pathways are constitutively activated in melanoma and promote cell proliferation, cell invasion, metastasis formation, and resistance to therapeutic regimens. Thus, they represent potential targets for melanoma prevention and treatment. Phytochemicals are gaining considerable attention for the management of melanoma because of their several cellular and molecular targets. A screening of a small library of sesquiterpenes lactones selected cynaropicrin, isolated from the aerial parts of Centaurea drabifolia subsp. detonsa, for its potential anticancer effect against melanoma cells. Treatment of human melanoma cells A375 with cynaropicrin resulted in inhibition of cell proliferation and induction of caspase-3-dependent apoptosis. Furthermore, cynaropicrin reduced several cellular malignant features such migration, invasion, and colonies formation through the inhibition of ERK1/2 and NF-κB activity. Cynaropicrin was able to reduce intracellular reactive oxygen species generation, which are involved in all the stages of carcinogenesis. Indeed, cynaropicrin increased the expression of several antioxidant genes, such as glutamate-cysteine ligase and heme oxygenase-1, by promoting the activation of the transcription factor Nrf-2. In conclusion, our results individuate cynaropicrin as a potential adjuvant chemotherapeutic agent for melanoma by targeting several protumorigenic signaling pathways.

Selective Photodynamic Effects on Breast Cancer Cells Provided by p123 Pluronic®- Based Nanoparticles Modulating Hypericin Delivery.

Breast cancer is the most relevant type of cancer and the second cause of cancer- related deaths among women in general. Currently, there is no effective treatment for breast cancer although advances in its initial diagnosis and treatment are available. Therefore, the value of novel anti-tumor therapeutic modalities remains an immediate unmet need in clinical practice. Following our previous work regarding the properties of the Pluronics with different photosensitizers (PS) for photodynamic therapy (PDT), in this study we aimed to evaluate the efficacy of supersaturated hypericin (HYP) encapsulated on Pluronic® P123 (HYP/P123) against breast cancer cells (MCF-7) and non-tumorigenic breast cells (MCF-10A). Cell internalization and subcellular distribution of HYP/P123 was confirmed by fluorescence microscopy. The phototoxicity and citototoxicity of HYP/P123 was assessed by trypan blue exclusion assay in the presence and absence of light. Long-term cytotoxicity was performed by clonogenic assay. Cell migration was determined by the wound-healing assay. Apoptosis and necrosis assays were performed by annexin VFITC/ propidium Iodide (PI) by fluorescence microscopy. Our results showed that HYP/P123 micelles had high stability and high rates of binding to cells, which resulted in the selective internalization in MCF-7, indicating their potential to permeate the membrane of these cells. Moreover, HYP/P123 micelles accumulated in mitochondria and endoplasmic reticulum organelles, resulting in the photodynamic cell death by necrosis. Additionally, HYP/P123 micelles showed effective and selective time- and dose dependent phototoxic effects on MCF-7 cells but little damage to MCF-10A cells. HYP/P123 micelles inhibited the generation of cellular colonies, indicating a possible capability to prevent the recurrence of breast cancer. We also demonstrated that HYP/P123 micelles inhibit the migration of tumor cells, possibly by decreasing their ability to form metastases. Taken together, the results presented here indicate a potentially useful role of HYP/P123 micelles as a platform for HYP delivery to more specifically and effectively treat human breast cancers through photodynamic therapy, suggesting they are worthy for in vivo preclinical evaluations.

Multi-scale microstructural investigation of a laser 3D printed Ni-based superalloy

The heterogeneous microstructure of a laser 3D printed Ni-based superalloy was examined at multiple length scales. The sub-millimeter-sized columnar crystal grains are composed of micron-sized cellular colonies. The crystal grains grow in epitaxy with the substrate under the large temperature gradient and high cooling rate. The cell boundaries, decorated with γ/γ′ eutectics, μ-phase precipitates and high density of dislocations, show enrichment of γ′ forming elements and low-angle misorientations. Dislocations trapped in the intra-cellular regions are characterized as statistically stored dislocations with no detectable contribution to lattice curvature, and are the results of the interaction between dislocations and γ′ precipitates.

A Novel Methodology for Characterizing Cell Subpopulations in Automated Time-lapse Microscopy.

Time-lapse imaging of cell colonies in microfluidic chambers provides time series of bioimages, i.e., biomovies. They show the behavior of cells over time under controlled conditions. One of the main remaining bottlenecks in this area of research is the analysis of experimental data and the extraction of cell growth characteristics, such as lineage information. The extraction of the cell line by human observers is time-consuming and error-prone. Previously proposed methods often fail because of their reliance on the accurate detection of a single cell, which is not possible for high density, high diversity of cell shapes and numbers, and high-resolution images with high noise. Our task is to characterize subpopulations in biomovies. In order to shift the analysis of the data from individual cell level to cellular groups with similar fluorescence or even subpopulations, we propose to represent the cells by two new abstractions: the particle and the patch. We use a three-step framework: preprocessing, particle tracking, and construction of the patch lineage. First, preprocessing improves the signal-to-noise ratio and spatially aligns the biomovie frames. Second, cell sampling is performed by assuming particles, which represent a part of a cell, cell or group of contiguous cells in space. Particle analysis includes the following: particle tracking, trajectory linking, filtering, and color information, respectively. Particle tracking consists of following the spatiotemporal position of a particle and gives rise to coherent particle trajectories over time. Typical tracking problems may occur (e.g., appearance or disappearance of cells, spurious artifacts). They are effectively processed using trajectory linking and filtering. Third, the construction of the patch lineage consists in joining particle trajectories that share common attributes (i.e., proximity and fluorescence intensity) and feature common ancestry. This step is based on patch finding, patching trajectory propagation, patch splitting, and patch merging. The main idea is to group together the trajectories of particles in order to gain spatial coherence. The final result of CYCASP is the complete graph of the patch lineage. Finally, the graph encodes the temporal and spatial coherence of the development of cellular colonies. We present results showing a computation time of less than 5 min for biomovies and simulated films. The method, presented here, allowed for the separation of colonies into subpopulations and allowed us to interpret the growth of colonies in a timely manner.

Comparative in vitro study of photodynamic activity of hypericin and hypericinates in MCF-7 cells

In this work we present a comparative in vitro study of photodynamic activity between hypericin (HYP) and some hypericinates (hypericin ionic pair with lysine or N-methylglucamine) in human mammary adenocarcinoma cells (MCF-7). The toxicity and phototoxicity of hypericin and hypericinates were compared, as well as their cellular uptake and localization and mutagenic, genotoxic and clonogenic capacity. Our results demonstrate that different cationic moieties promote differences in the hypericinate solubility in a biological environment, and can influence the cellular localization and the phototoxicity of the photosensitizer. It was verified that hypericinates have better efficiency to generate singlet oxygen than HYP, and a lower aggregation in biological medium. In vitro assays have shown that HYP and the hypericinates are able to permeate the MCF-7 cell membrane and accumulated in organelles near the nucleus. The difference in location, however, was not determinant to the cell death mechanism, and a higher prevalence of apoptosis for all studied compounds occurred. The photodynamic studies indicated that hypericinates were more effective than HYP and were able to inhibit the formation of cellular colonies, suggesting a possible ability to prevent the recurrence of tumors. It also appears that all compounds have relative safety for mutagenicity and genotoxicity, which opens up a further safe route for application in in vivo studies.

Evaluation of Porous TiNi-based Alloy as a Scaffold for Liver Tissue Engineering

Complex analysis of development of cells of liver – hepatocytes in the biocompatible scaffold, fabricated of porous-permeable TiNi-based alloy has been performed. Specific stages of development of cellular population in pores of scaffold are displayed in the present investigation. The stepwise characteristic of developing of hepatocytes in porous-permeable structure from TiNi-based alloy is demonstrated. Nanoporous surface of pores in incubator permit cells to be reliably fixed and produce growth to cellular colonies, and porous-permeable structure of scaffold allows penetrating to nutrients from environment during all time of investigation in vivo. Examination on CCl4-induced hepatitis has displayed effective antitoxic activity of population of hepatocytes in scaffold from TiNi-based alloy and their prolonged effect.

Coarse-graining bacteria colonies for modelling critical solute distributions in picolitre bioreactors for bacterial studies on single-cell level.

SummaryMicrofluidic single‐cell bioreactors have found widespread application to investigate growth and gene expression of microbial model organisms, but yet there are few attempts to systematically characterize different design and cultivation concepts. Quantitative measurements of critical solute concentrations, e.g. limiting nutrients, are not yet feasible within the typical volumes in the range of picolitres. A way to gain new insights about the mass transport within those volumes is by simulation, but the complex geometry resulting from the multitude of cells within a colony leads to time and resource consuming computational challenges. In this work, six different concepts for the model representation of cellular microcolonies within microfluidic monolayer growth chamber devices are compared. The Gini coefficient is proposed as new measure for inhomogeneity within cellular colonies. An example cell colony is represented by a single point source, a cylindrical volume with homogeneous reaction rates with and without adjusted diffusion coefficient, as point sources for each single cell and as rod‐shaped, diffusion blocking, three‐dimensional cells with varying shapes. Simulated concentration profiles across the chambers depended strongly on the chosen cell representation. The representation with the lowest degree of abstraction, three‐dimensional cells, leads to complex geometries and high computational effort, but also gives a conservative and therefore preferable estimate for the cultivation conditions within a given cultivation chamber geometry. Interestingly, the cylindrical volume with adjusted diffusion coefficient gives similar results but requires far less computational effort. Therefore, it is proposed to use the three‐dimensional cells for detailed studies and to determine parameters for the cylindrical volume with adjusted diffusion coefficient, which can then be used for experimental design, screening of parameter spaces, and similar applications.

Effect of Co on Discontinuous Precipitation Transformation with TCP Phase in Ni-based Alloy Containing Re

The effect of Co on discontinuous precipitation (DP) transformation involving the formation of topologically close-packed (TCP) phase was investigated in three Ni-Cr-Re model alloys containing different levels of Co. One typical TCP phase, σ, was generated within DP cellular colonies along the migrating grain boundaries in experimental alloys during aging treatment. As a result of the increased solubility of Re in the γ matrix and enlarged interlamellar spacing of σ precipitates inside of growing DP colonies, Co addition suppressed the formation of σ phase and associated DP colonies. This study suggests that Co could potentially serve as a microstructural stabilizer in Re-containing Ni-base superalloys, which provides an alternative method for the composition optimization of superalloys.

Implementation of an Agent-Based Parallel Tissue Modelling Framework for the Intel MIC Architecture

Timothy is a novel large scale modelling framework that allows simulating of biological processes involving different cellular colonies growing and interacting with variable environment. Timothy was designed for execution on massively parallel High Performance Computing (HPC) systems. The high parallel scalability of the implementation allows for simulations of up to 109 individual cells (i.e., simulations at tissue spatial scales of up to 1 cm3 in size). With the recent advancements of the Timothy model, it has become critical to ensure appropriate performance level on emerging HPC architectures. For instance, the introduction of blood vessels supplying nutrients to the tissue is a very important step towards realistic simulations of complex biological processes, but it greatly increased the computational complexity of the model. In this paper, we describe the process of modernization of the application in order to achieve high computational performance on HPC hybrid systems based on modern Intel® MIC architecture. Experimental results on the Intel Xeon Phi™ coprocessor x100 and the Intel Xeon Phi processor x200 are presented.

Ageing characteristics of the metastable gamma phase in U–9 wt.% Mo alloy: experimental observations and thermodynamic validation

Ageing characteristics of the metastable bcc γ-phase in U–9 wt.% Mo alloy, a candidate for high uranium density nuclear fuel for research and test reactors, have been investigated in this study. Analyses of the aged microstructures, employing X-ray diffraction and various microscopy techniques, revealed the decomposition mechanism of the metastable γ-phase to the stable α-U and γ′ (U2Mo) phases. A discontinuous precipitation reaction, leading to the generation of partially transformed cellular colonies with lamellae comprising of either the α-phase or the γ′-phase in γ-phase matrix, was found to be operative. The in situ transformation of γ interlamellar regions to the γ′-phase was noticed occasionally within the γ + α lamellar colonies. Thermodynamic analysis of the U–Mo system, using free energy–composition diagrams, could associate the observed attributes of the decomposition of γ-phase to the extent of Mo segregation and the chemical driving force required for the nucleation of α- and γ′-phases in the γ-matrix.