Behavior Of Mammalian Cells Research Articles

Collective behaviors of mammalian cells on amine-coated silicon nanowires

Intensive studies with vertical nanowire (NW) arrays have illustrated broad implications for manipulating mammalian cells in vitro, but how cellular responses are influenced by the presence of NWs has not been thoroughly investigated. Here, we address collective cellular behaviors, including surface area of cells, membrane trafficking, focal adhesion distribution and dynamics, and cytoskeletal protein distribution on amine-coated silicon (Si) NWs with different physical properties. The degree of HeLa cell spreading was inversely proportional to the surface area occupied by the NWs, which was not affected by manipulation of membrane trafficking dynamics. In the presence of a diffusive focal complex around the NWs, strong, well organized focal adhesion was hardly visible on the NWs, implying that the cells were interacting weakly with the NW-embedded surface. Furthermore, we found that actin filament formation of the cells on long NWs was not favorable, and this could explain our observation of reduced cell spreading, as well as the decreased number of focal adhesion complexes. Taken together, our results suggest that cells can survive on silicon NWs by adjusting their morphology and adhesion behavior through actively organizing these molecules.

High-throughput characterisation of osteogenic differentiation of human mesenchymal stem cells using pore size gradients on porous alumina.

The ability to control the cellular response is of critical importance when designing advanced biomaterials for applications in tissue engineering and regenerative medicine. An important aspect of biointerfacial interactions is surface topography at the nanoscale and therefore this needs to be taken into consideration. Here, a pore size gradient in porous alumina (pAl) was fabricated with pore sizes ranging from 50 nm to 3 μm. The attachment behaviour and osteogenesis of human mesenchymal stem cells (hMSCs) was investigated along this topography gradient for up to 2 weeks. Generally, cell attachment density and spreading area decreased with increasing pore size. Pore wall width and solid surface fraction also played a key role in cell adhesion. After 2 weeks, osteogenesis of hMSCs was enhanced by porous topography with a pore size of 120-230 nm in diameter and 10 nm pore wall width, compared with other topographies of the gradient. The results demonstrate that the gradient format allows in-depth high-throughput screening of surface parameters that are important for the control of mammalian cell behaviour, thereby advancing the development of new and improved biomaterials for e.g. orthopaedic and tissue engineering applications.

Genetically programmed superparamagnetic behavior of mammalian cells

Although magnetic fields and paramagnetic inorganic materials were abundant on planet earth during the entire evolution of living species the interaction of organisms with these physical forces remains a little-understood phenomenon. Interestingly, rather than being genetically encoded, organisms seem to accumulate and take advantage of inorganic nanoparticles to sense or react to magnetic fields. Using a synthetic biology-inspired approach we have genetically programmed mammalian cells to show superparamagnetic behavior. The combination of ectopic production of the human ferritin heavy chain 1 (hFTH1), engineering the cells for expression of an iron importer, the divalent metal ion transferase 1 (DMT1) and the design of an iron-loading culture medium to maximize cellular iron uptake enabled efficient iron mineralization in intracellular ferritin particles and conferred superparamagnetic behavior to the entire cell. When captured by a magnetic field the superparamagnetic cells reached attraction velocities of up to 30μm/s and could be efficiently separated from complex cell mixtures using standard magnetic cell separation equipment. Technology that enables magnetic separation of genetically programmed superparamagnetic cells in the absence of inorganic particles could foster novel opportunities in diagnostics and cell-based therapies.

Synthetic two-way communication between mammalian cells

The design of synthetic biology-inspired control devices enabling entire mammalian cells to receive, process and transfer metabolic information and so communicate with each other via synthetic multichannel networks may provide new insight into the organization of multicellular organisms and future clinical interventions. Here we describe communication networks that orchestrate behavior in individual mammalian cells in response to cell-to-cell metabolic signals. We engineered sender, processor and receiver cells that interact with each other in ways that resemble natural intercellular communication networks such as multistep information processing cascades, feed-forward-based signaling loops, and two-way communication. The engineered two-way communication devices mimicking natural control systems in the development of vertebrate extremities and vasculature was used to program temporal permeability in vascular endothelial cell layers. These synthetic multicellular communication systems may inspire future therapies or tissue engineering strategies.

An approach for assessing hydrogel hydrophobicity

Hydrogel hydrophobicity is an important modulator of mammalian cell behavior, drug payload release, and medical device fouling. Contact angle and protein adsorption measures are two common methods for evaluating hydrogel hydrophobicity. However, protein adsorption is a complex phenomenon which is challenging to interpret in terms of gel hydrophobicity alone. In addition, the permeability of hydrogels can be problematic for contact angle assessment, as this method can only be strictly applied to smooth, solid, and nonpermeable surfaces. Therefore, the development of a technique for measuring hydrogel hydrophobicity which is simple, sensitive, and independent of variations in gel permeability would significantly advance the ability to finely tune this variable. The present technical note develops a method for quantifying the hydrophobicity of hydrogels by exploiting their capacity to swell differentially in solvents of distinct polarities. To validate this technique, hydrogels of varying hydrophobicities were prepared by combining hydrophilic poly(ethylene glycol) diacrylate (PEGDA) with either hydrophobic 3-(trimethoxysilyl) propyl methacrylate (TMSPM) or hydrophilic 2-hydroxyethyl methacrylate (HEMA). The ratio of hydrogel swelling in 70% isopropanol to that in water was termed the hydrophobicity index (H-index) and was determined for each gel type. The measured H-indices reflected known differences in the hydrophobicities of HEMA, TMSPM, and PEGDA and, in contrast to contact angle assessments, appeared to be independent of variations in hydrogel permeability. In addition, the trend in H-indices agreed well with the trend in protein adsorption across hydrogel formulations, although the H-indices appeared to be able to resolve more subtle differences in gel hydrophobicity than protein adsorption measures.

Quantifying collective behavior in mammalian cells

Collective social behaviors generate one emergent response from many individual actors without central coordination: bees swarm, birds flock, and fish school. Single-celled bacteria and amoebae collectively swarm, detecting chemical signals emitted by close neighbors to synchronize their motion. Within organisms, collective behaviors drive multicellular processes during migration and wound healing. Similar to unicellular organisms, mammalian cells coordinate their responses during these complex processes by detecting changes in concentration of signaling molecules. However, the signaling pathways used by mammalian cells are typically more interdependent than those in unicellular organisms, and gene expression varies with the time-dependent concentration of signaling molecules. Understanding the role of intercellular communications for collective behavior in mammalian cells thus demands sensitive measurements of responses to chemical signals. In PNAS, Sun et al. (1) quantify the spatiotemporal evolution of calcium response to ATP stimulation in fibroblasts. They find that the response of dense colonies of cells to ATP stimulation is faster and more synchronized than that of isolated cells. By quantitatively correlating responses between neighboring pairs of cells, they identify pacemaker cells; by physically separating cells in a hydrogel matrix, they confirm that cells communicate via gap junctions to synchronize their responses. These experiments show that both temporal and spatial dynamics of collective response in mammalian cells depend on intercellular signaling.

Screening Mesenchymal Stem Cell Attachment and Differentiation on Porous Silicon Gradients

AbstractThe profound effects that nanoscale surface topography exerts on cell behavior are highly relevant to the development of advanced biomaterials and to advances in tissue engineering and regenerative medicine. Here, an asymmetric anodization procedure is used to produce n‐type porous silicon (pSi) gradients with pore sizes ranging from tens to hundreds of nanometers in diameter and changes in the ridge nanoroughness from a few to tens nanometers. Rat mesenchymal stem cells (rMSCs) adhere poorly at the regions with small pore size but high ridge roughness. Cell adhesion is increased gradually towards the large pore size but low ridge roughness end of the pSi gradients. Surface topography influences cell differentiation, but not cell proliferation. Osteogenesis of rMSCs is enhanced by porous topography with a ridge roughness lower than 10 nm, while adipogenesis of rMSCs is enhanced on the entire pSi gradient compared with flat Si substrates. The results demonstrate that the gradient format allows in‐depth screening of surface parameters that are important for the control of mammalian cell behavior, thereby advancing the development of new and improved biomaterials for orthopaedic and tissue engineering applications.

Realizing Antithetic Integral Feedback Control in Mammalian Cells.

Genetic circuit engineering has emerged as a powerful methodology to program the behavior of mammalian cells to respond to internal and external cues. This approach is now used to develop new therapeutics and improve production processes. However, genetic interaction networks are complex and hard to engineer rationally. Moreover, a design may fail, and it may not be possible to identify the root cause of its breakdown. Introducing designated regulatory circuitry in the form of integral feedback can introduce performance guarantees by ensuring robust and precise operation.

Modulation of Longevity and Tissue Homeostasis by the Drosophila PGC-1 Homolog

In mammals, the PGC-1 transcriptional coactivators are key regulators of energy metabolism, including mitochondrial biogenesis and respiration, which have been implicated in numerous pathogenic conditions, including neurodegeneration and cardiomyopathy. Here, we show that overexpression of the Drosophila PGC-1 homolog (dPGC-1/spargel) is sufficient to increase mitochondrial activity. Moreover, tissue-specific overexpression of dPGC-1 in stem and progenitor cells within the digestive tract extends life span. Long-lived flies overexpressing dPGC-1 display a delay in the onset of aging-related changes in the intestine, leading to improved tissue homeostasis in old flies. Together, these results demonstrate that dPGC-1 can slow aging both at the level of cellular changes in an individual tissue and also at the organismal level by extending life span. Our findings point to the possibility that alterations in PGC-1 activity in high-turnover tissues, such as the intestine, may be an important determinant of longevity in mammals.

CHO Proteome Alterations Induced by Reverse Transformation

To the Editor, Much attention has been focused on the attempt to undertake global analysis of protein expression and in particular to analyze the changes in protein expression associated with several processes such as differentiation and cell cycle progression. In general, cell differentiation— or proliferation—requires de novo protein synthesis and/or post-translational modification, in particular phosphorylation of proteins such as transcription factors. Consequently, there is a need to identify the proteins that are being modified, as well as to know when and where in the cell modifications occur. We focused our studies on the Chinese hamster ovary (CHO) mammalian tumor cell line, a good model to study the cell differentiation. It is known that cyclic 30,50monophosphate adenosine (cAMP) transforms CHO cells, in vitro: they lose characteristics associated with malignancy and adopt characteristics of normal fibroblasts (‘‘reverse transformation’’) [3, 7, 9]. Puck et al. [8–10] found that cAMP regulates the structure of the cell cytoskeleton, which in turn strongly influences many differentiation properties including the assumption of normal or cancerous behavior in mammalian cells. In a previous study, we analyzed how CHO-K1 cells display different nuclear protein contents when undergoing differentiation by cAMP [14]; our Mass Spectrometry (MS) investigation revealed a change in the composition of nuclear proteins associated to an inhibition of the protein expression with possible implications for the control of cell reverse transformation [14]. The mechanism of the transformation, however, is as yet obscure and need further exploration in all compartments of cell proteome which we have carried out with this study. Fundamental changes in properties of the cell membrane have indeed been reported but the molecular mechanisms generating these structural changes are largely unknown. The changes in the concentration of the different metabolites and the different protein composition associated with morphological changes observed are also largely unknown [8]. This study wants to explore more deeply the effect of cAMP differentiation on the proteome of CHO cells, considering that the changes in the levels of nuclear envelopeassociated proteins and chromatin-condensing proteins [14] constitutes only the first step toward developing an understanding of cellular mechanisms for controlling nuclear shape. Due to the complexity of eukaryotic proteomes, and the divergence of protein properties, it is beneficial to prepare standardized partial proteomes in order to maximize the coverage of the proteome and to increase the chance to visualize low abundance proteins and make them accessible for subsequent analysis [1]. The strategies used to prepare such a complex proteomic samples for MS analysis involve many steps. For protein extraction we chose to perform a subcellular fractionation to separate the whole cellular proteome in four fractions. In literature, there are several works in which this approach to proteome analysis is adopted [1, 6, 12, 13], but always to analyze the behavior of a restrict number of proteins, for example to investigate changes in subcellular localization of regulatory proteins impacted by experimental or disease parameters. R. Spera C. Nicolini Nanoworld Institute, Fondazione El.B.A. Nicolini, Largo Redaelli 7, Pradalunga, Bergamo, Italy e-mail: rspera@ibf.unige.it

Tissue Morphing Control on Dynamic Gradient Surfaces

In this report, we develop smart surfaces for the spatial and temporal control of mammalian cell behavior. We integrate a bioactive surface strategy with a photo-electroactive surface strategy to generate dynamic ligand surface gradients for controlling cell adhesion, tissue shape morphing, and cell tissue migration.

Immortalization and functional characterization of rat arachnoid cell lines

Modeling the behavior of mammalian arachnoid cells is critical to understand hydrocephalus and other brain disorders involving abnormal flow of cerebrospinal fluid, yet relatively little is known about the physiology of arachnoid cells due to lack of a robust three-dimensional model system. Explanted primary cultures have been the only option to study transport across arachnoid cell membranes, but practical limitations of primary culture include slow growth, early senescence, and poor reproducibility. The purpose of this study was to create immortalized rat arachnoid cell lines to permit in vitro study of arachnoid granulations and properties of cerebrospinal fluid (CSF) flow. We established and partially characterized two immortalized cell lines generated from primary rat arachnoid cells, using retroviral gene transfer of SV40 large T antigen (SV40 LTAg) either with or without human telomerase (hTERT). The established cell lines stably express either SV40 LTAg alone, or SV40 LTAg and hTERT, and demonstrate high proliferative rate, contact inhibition at confluence, and stable expression of protein markers characteristic of native arachnoid cells over more than 160 passages.

Behaviors of NIH-3T3 Fibroblasts on Graphene/Carbon Nanotubes: Proliferation, Focal Adhesion, and Gene Transfection Studies

Carbon-based materials, including graphene and carbon nanotubes, have been considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for stem cell differentiation, and components of implant devices. Despite the potential biomedical applications of these materials, only limited information is available regarding the cellular events, including cell viability, adhesion, and spreading, that occur when mammalian cells interface with carbon-based nanomaterials. Here, we report behaviors of mammalian cells, specifically NIH-3T3 fibroblast cells, grown on supported thin films of graphene and carbon nanotubes to investigate biocompatibility of the artificial surface. Proliferation assay, cell shape analysis, focal adhesion study, and quantitative measurements of cell adhesion-related gene expression levels by RT-PCR reveal that the fibroblast cells grow well, with different numbers and sizes of focal adhesions, on graphene- and carbon nanotube-coated substrates. Interestingly, the gene transfection efficiency of cells grown on the substrates was improved up to 250% that of cells grown on a cover glass. The present study suggests that these nanomaterials hold high potential for bioapplications showing high biocompatibility, especially as surface coating materials for implants, without inducing notable deleterious effects while enhancing some cellular functions (i.e., gene transfection and expression).

Role of calcium signalling and phosphorylations in disruption of the epithelial junctions by Pseudomonas aeruginosa quorum sensing molecule

In Pseudomonas aeruginosa, cell–cell communication based on acyl-homoserine lactone (HSL) quorum sensing molecules is known to coordinate the production of virulence factors and biofilms by the bacterium. Incidentally, these bacterial signals can also modulate mammalian cell behaviour. We demonstrate here that 3O-C 12-HSL can induce changes in calcium signalling through influx and release of calcium from thapsigargin-sensitive stores and delocalization of inositol 1,4,5-trisphosphate receptors (IP 3R), but not of ryanodine receptors (RyR). In parallel, P. aeruginosa 3O-C 12-HSL disrupts junctions in human Caco-2 cells as evidenced by a reduction of the expression and distribution of ZO-3 and JAM-A. Using co-immunoprecipitation we also found an alteration in the binding of ZO-3 to JAM-A in protein complexes. Moreover, 3O-C 12-HSL-treatment resulted in tyrosine hyperphosphorylation of ZO-3 and JAM-A. On the contrary, serine and threonine residues of ZO-1 and JAM-A became less phosphorylated after exposition of 3O-C 12-HSL. The 3O-C 12-HSL-induced intracellular calcium signalling and alteration in the phosphorylation status of junction proteins furthermore correlated with changes in the association between JAM-A–ZO-3. The calcium inhibitors thapsigargin, xestospongin C, and dantrolene partly prevented the 3O-C 12-HSL-induced decreases in TER and increases in the paracellular flux of 10 kDa dextran. These findings clearly suggest that P. aeruginosa 3O-C 12-HSL can cause the loss of epithelial barrier function via calcium signalling and further alteration in the phosphorylation status of junction proteins; and that bacterial quorum sensing signals represent inter-kingdom signalling.

Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase-deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

An Off-Lattice Hybrid Discrete-Continuum Model of Tumor Growth and Invasion

We have developed an off-lattice hybrid discrete-continuum (OLHDC) model of tumor growth and invasion. The continuum part of the OLHDC model describes microenvironmental components such as matrix-degrading enzymes, nutrients or oxygen, and extracellular matrix (ECM) concentrations, whereas the discrete portion represents individual cell behavior such as cell cycle, cell-cell, and cell-ECM interactions and cell motility by the often-used persistent random walk, which can be depicted by the Langevin equation. Using this framework of the OLHDC model, we develop a phenomenologically realistic and bio/physically relevant model that encompasses the experimentally observed superdiffusive behavior (at short times) of mammalian cells. When systemic simulations based on the OLHDC model are performed, tumor growth and its morphology are found to be strongly affected by cell-cell adhesion and haptotaxis. There is a combination of the strength of cell-cell adhesion and haptotaxis in which fingerlike shapes, characteristic of invasive tumor, are observed.

Imaging Viral Behavior in Mammalian Cells with Self‐Assembled Capsid–Quantum‐Dot Hybrid Particles

Unique spectral properties of quantum dots (QDs) enable ultrasensitive and long-term biolabeling. Aiming to trace the infection, movement, and localization of viruses in living cells, QD-containing virus-like particles (VLPs) of simian virus 40 (SV40), termed SVLP-QDs, are constructed by in vitro self-assembly of the major capsid protein of SV40. SVLP-QDs show homogeneity in size ( approximately 24 nm), similarity in spectral properties to unencapsidated QDs, and considerable stability. When incubated with living cells, SVLP-QDs are shown to enter the cells by caveolar endocytosis, travel along the microtubules, and accumulate in the endoplasmic reticulum. This process mimics the early infection steps of SV40. This is the first paradigm of imaging viral behaviors with encapsidated QDs in living cells. The method may provide a new alternative for various purposes, such as tracing viruses or viral components, targeted nanoparticle delivery, and probing of drug delivery.

Monomeric fluorescent timers that change color from blue to red report on cellular trafficking.

Based on the mechanism for chromophore formation in red fluorescent proteins, we developed three mCherry-derived monomeric variants, called fluorescent timers (FTs), that change their fluorescence from the blue to red over time. These variants exhibit distinctive fast, medium and slow blue-to-red chromophore maturation rates that depend on the temperature. At 37 degrees C, the maxima of the blue fluorescence are observed at 0.25, 1.2 and 9.8 h for the purified fast-FT, medium-FT and slow-FT, respectively. The half-maxima of the red fluorescence are reached at 7.1, 3.9 and 28 h, respectively. The FTs show similar timing behavior in bacteria, insect and mammalian cells. Medium-FT allowed for tracking of the intracellular dynamics of the lysosome-associated membrane protein type 2A (LAMP-2A) and determination of its age in the targeted compartments. The results indicate that LAMP-2A transport through the plasma membrane and early or recycling endosomes to lysosomes is a major pathway for LAMP-2A trafficking.

A Mathematical Model of Liver Cell Aggregation In Vitro

The behavior of mammalian cells within three-dimensional structures is an area of intense biological research and underpins the efforts of tissue engineers to regenerate human tissues for clinical applications. In the particular case of hepatocytes (liver cells), the formation of spheroidal multicellular aggregates has been shown to improve cell viability and functionality compared to traditional monolayer culture techniques. We propose a simple mathematical model for the early stages of this aggregation process, when cell clusters form on the surface of the extracellular matrix (ECM) layer on which they are seeded. We focus on interactions between the cells and the viscoelastic ECM substrate. Governing equations for the cells, culture medium, and ECM are derived using the principles of mass and momentum balance. The model is then reduced to a system of four partial differential equations, which are investigated analytically and numerically. The model predicts that provided cells are seeded at a suitable density, aggregates with clearly defined boundaries and a spatially uniform cell density on the interior will form. While the mechanical properties of the ECM do not appear to have a significant effect, strong cell-ECM interactions can inhibit, or possibly prevent, the formation of aggregates. The paper concludes with a discussion of our key findings and suggestions for future work.

MCAK-Independent Functions of ch-Tog/XMAP215 in Microtubule Plus-End Dynamics

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.