Constitutive material modeling of cell: a micromechanics approach

The variations in mechanical properties of cells obtained from experimental and theoretical studies can be overcome only through the development of a sound mathematical framework correlating the derived mechanical property with the cellular structure. Such a formulation accounting for the inhomogeneity of the cytoplasm due to stress fibers and actin cortex is developed in this work. The proposed model is developed using the Mori-Tanaka method of homogenization by treating the cell as a fiber-reinforced composite medium satisfying the continuum hypothesis. The validation of the constitutive model using finite element analysis on atomic force microscopy (AFM) and magnetic twisting cytometry (MTC) has been carried out and is found to yield good correlation with reported experimental results. It is observed from the study that as the volume fraction of the stress fiber increases, the stiffness of the cell increases and it alters the force displacement behavior for the AFM and MTC experiments. Through this model, we have also been able to find the stress fiber as a likely cause of the differences in the derived mechanical property from the AFM and MTC experiments. The correlation of the mechanical behavior of the cell with the cell composition, as obtained through this study, is an important observation in cell mechanics.     

Statistical modeling of single target cell encapsulation

High throughput drop-on-demand systems for separation and encapsulation of individual target cells from heterogeneous mixtures of multiple cell types is an emerging method in biotechnology that has broad applications in tissue engineering and regenerative medicine, genomics, and cryobiology. However, cell encapsulation in droplets is a random process that is hard to control. Statistical models can provide an understanding of the underlying processes and estimation of the relevant parameters, and enable reliable and repeatable control over the encapsulation of cells in droplets during the isolation process with high confidence level. We have modeled and experimentally verified a microdroplet-based cell encapsulation process for various combinations of cell loading and target cell concentrations. Here, we explain theoretically and validate experimentally a model to isolate and pattern single target cells from heterogeneous mixtures without using complex peripheral systems.     

A tool for modeling strategic decisions in cell culture manufacturing

The development of a prototype tool for modeling manufacturing in a biopharmaceutical plant is discussed. A hierarchical approach to modeling a manufacturing process has been adopted to confer maximum user flexibility. The use of this framework for assessing the impact of manufacturing decisions on strategic technical and business indicators is demonstrated via a case study. In the case study, which takes the example of a mammalian cell culture process delivering a therapeutic for clinical trials, the dynamic modeling tool indicates how manufacturing options affect the demands on resources and the associated manufacturing costs. The example illustrates how the decision-support software can be used by biopharmaceutical companies to investigate the effects of working toward different strategic goals on the cost-effectiveness of the process, prior to committing to a particular option.     

An approximation method for estimating cell loss from a growing tumor

A simple self-consistent calculational scheme is developed for estimating cell loss for a growing tumor (or other population) when the growth fraction can be estimated at regular intervals. This is applied to published data for a particular much-studied Ehrlich ascites tumor. The loss rate is found to be substantially higher than that estimated by previous, less precise means.     

How dopamine transporter interacts with dopamine: insights from molecular modeling and simulation

By performing homology modeling, molecular docking, and molecular dynamics simulations, we have developed three-dimensional (3D) structural models of both dopamine transporter and dopamine transporter-dopamine complex in the environment of lipid bilayer and solvent water. According to the simulated structure of dopamine transporter-dopamine complex, dopamine was orientated in a hydrophobic pocket at the midpoint of the membrane. The modeled 3D structures provide some detailed structural and mechanistic insights concerning how dopamine transporter (DAT) interacts with dopamine at atomic level, extending our mechanistic understanding of the dopamine reuptake with the help of Na(+) ions. The general features of the modeled 3D structures are consistent with available experimental data. Based on the modeled structures, our calculated binding free energy (DeltaG(bind) = -6.4 kcal/mol) for dopamine binding with DAT is also reasonably close to the experimentally derived DeltaG(bind) value of -7.4 kcal/mol. Finally, a possible dopamine-entry pathway, which involves formation and breaking of the salt bridge between side chains of Arg(85) and Asp(476), is proposed based on the results obtained from the modeling and molecular dynamics simulation. The new structural and mechanistic insights obtained from this computational study are expected to stimulate future, further biochemical and pharmacological studies on the detailed structures and mechanisms of DAT and other homologous transporters.     

An approximation to the likelihood for a pedigree with loops

This paper presents a new approximation to the likelihood for a pedigree with loops, based on cutting all loops and extending the pedigree at the cuts. An opimum loop-cutting strategy and an iterative extension technique are presented. The likelihood for a pedigree with loops is then approximated by the conditional likelihood for the entire cut-extended pedigree given the extended part. The approximate likelihoods are compared with the exact likelihoods obtained using the program MENDEL for several small pedigrees with loops. The approximation is efficient for large pedigrees with complex loops in terms of computing speed and memory requirements.     

An efficient method of modeling material properties using a thermal diffusion analogy: an example based on craniofacial bone

The ability to incorporate detailed geometry into finite element models has allowed researchers to investigate the influence of morphology on performance aspects of skeletal components. This advance has also allowed researchers to explore the effect of different material models, ranging from simple (e.g., isotropic) to complex (e.g., orthotropic), on the response of bone. However, bone's complicated geometry makes it difficult to incorporate complex material models into finite element models of bone. This difficulty is due to variation in the spatial orientation of material properties throughout bone. Our analysis addresses this problem by taking full advantage of a finite element program's ability to solve thermal-structural problems. Using a linear relationship between temperature and modulus, we seeded specific nodes of the finite element model with temperatures. We then used thermal diffusion to propagate the modulus throughout the finite element model. Finally, we solved for the mechanical response of the finite element model to the applied loads and constraints. We found that using the thermal diffusion analogy to control the modulus of bone throughout its structure provides a simple and effective method of spatially varying modulus. Results compare favorably against both experimental data and results from an FE model that incorporated a complex (orthotropic) material model. This method presented will allow researchers the ability to easily incorporate more material property data into their finite element models in an effort to improve the model's accuracy.     

Three-dimensional molecular modeling with single molecule FRET

Single molecule fluorescence energy transfer experiments enable investigations of macromolecular conformation and folding by the introduction of fluorescent dyes at specific sites in the macromolecule. Multiple such experiments can be performed with different labeling site combinations in order to map complex conformational changes or interactions between multiple molecules. Distances that are derived from such experiments can be used for determination of the fluorophore positions by triangulation. When combined with a known structure of the macromolecule(s) to which the fluorophores are attached, a three-dimensional model of the system can be determined. However, care has to be taken to properly derive distance from fluorescence energy transfer efficiency and to recognize the systematic or random errors for this relationship. Here we review the experimental and computational methods used for three-dimensional modeling based on single molecule fluorescence resonance transfer, and describe recent progress in pushing the limits of this approach to macromolecular complexes.     

Noninvasive diagnosis of a single cell with a probe beam

It is important to distinguish a living/dead cell in cell culture, especially in the regenerate medicine field including cell therapy, since those cells are usually in short supply and consequently the ex vivo culture process should be operated strictly. Conventional methods for distinguishing a living from a dead cell usually require labeling with a dye, which spoils the culture of the cell. Here we show a simple noninvasive method for diagnosing a dead or alive cell with a probe beam. If a cell is alive, the active transport of materials across the cell membrane causes a change of concentration gradients, and this change further induces a change of deflection of a probe beam passing through a vicinity of the cell membrane. If a cell is dead, no or little change in deflection of the probe beam is induced because no or little active materials movement across the cell membrane exists. The deflection of the probe beam is monitored, and judgment on whether a cell is dead or alive from the deflection signal agreed with the conventional decision.     

Kinetics of single cells: observation and modeling of a stochastic process

The successive generation times for single cells of Escherichia coli K-12 were measured as described by A. Elfwing, Y. LeMarc, J. Baranyi, and A. Ballagi (Appl. Environ. Microbiol. 70:675-678, 2004), and the histograms they generated were used as empirical distributions to simulate growth of the population as the result of the multiplication of its single cells. This way, a stochastic birth model in which the underlying distributions were measured experimentally was simulated. To validate the model, analogous bacterial growth curves were generated by the use of different inoculum levels. The agreement with the simulation was very good, proving that the growth of the population can be predicted accurately if the distribution of the first few division times for the single cells within that population is known. Two questions were investigated by the simulation. (i) To what extent can we say that the distribution of the detection time, i.e., the time by which a single-cell-generated subpopulation reaches a detectable level, can be identified with that of the lag time of the original single cell? (ii) For low inocula, how does the inoculum size affect the lag time of the population?     

The cell as a material

To elucidate the dynamic and functional role of a cell within the tissue it belongs to, it is essential to understand its material properties. The cell is a viscoelastic material with highly unusual properties. Measurements of the mechanical behavior of cells are beginning to probe the contribution of constituent components to cell mechanics. Reconstituted cytoskeletal protein networks have been shown to mimic many aspects of the mechanical properties of cells, providing new insight into the origin of cellular behavior. These networks are highly nonlinear, with an elastic modulus that depends sensitively on applied stress. Theories can account for some of the measured properties, but a complete model remains elusive.     

Computational fluid dynamics modeling,a novel,and effective approach for developing scalable cell therapy manufacturing processes

Induced pluripotent stem cells (iPSCs) hold great potential to generate novel, curative cell therapy products. However, current methods to generate these novel therapies lack scalability, are labor-intensive, require a large footprint, and are not suited to meet clinical and commercial demands. Therefore, it is necessary to develop scalable manufacturing processes to accommodate the generation of high-quality iPSC derivatives under controlled conditions. The current scale-up methods used in cell therapy processes are based on empirical, geometry-dependent methods that do not accurately represent the hydrodynamics of 3D bioreactors. These methods require multiple iterations of scale-up studies, resulting in increased development cost and time. Here we show a novel approach using computational fluid dynamics modeling to effectively scale-up cell therapy manufacturing processes in 3D bioreactors. Using a GMP-compatible iPSC line, we translated and scaled-up a small-scale cardiomyocyte differentiation process to a 3-L computer-controlled bioreactor in an efficient manner, showing comparability in both systems.     

Production planning of a flexible manufacturing system in a material requirements planning environment

Early flexible manufacturing system (FMS) production planning models exhibited a variety of planning objectives; typically, these objectives were independent of the overall production environment. More recently, some researchers have proposed hierarchical production planning and scheduling models for FMS. In this article, we examine production planning of FMS in a material requirements planning (MRP) environment. We propose a hierarchical structure that integrates FMS production planning into a closed-loop MRP system. This structure gives rise to the FMS/MRP rough-cut capacity planning (FMRCP) problem, the FMS/MRP grouping and loading (FMGL) problem, and the FMS/MRP detailed scheduling problem. We examine the FMRCP and FMGL problems in detail and present mathematical programming models for each of these problems. In particular, the FMRCP problem is modeled as a generalized assignment problem (GAP), and a GAP-based heuristic procedure is defined for the problem. We define a two-phase heuristic for the FMGL problem and present computational experience with both heuristics. The FMRCP heuristic is shown to solve problems that exhibit a dependent-demand relation within the FMS and with FMS capacity utilization as high as 99 percent. The FMGL heuristic requires very little CPU time and obtains solutions to the test problems that are on average within 1.5 percent of a theoretical lower bound. This FMS/MRP production planning framework, together with the resulting models, constitutes an important step in the integration of FMS technology with MRP production planning. The hierarchical planning mechanism directly provides for system-level MRP planning priorities to induce appropriate production planning and control objectives on the FMS while simultaneously allowing for necessary feedback from the FMS. Moreover, by demonstrating the tractability of the FMRCP and FMGL problems, this research establishes the necessary groundwork upon which to explore systemwide issues pertaining to the coordination of the hierarchical structure.     

Growth of a single cell

A theory of growth of a cell which takes up nutrients by diffusion or active transport is discussed. The main conclusion is that the volume should grow at least as fast as the third power of the time. Existing experimental evidence is not a conclusive test of the theory, and further experiments to test it are proposed.     

Multiple proteins with single activities or a single protein with multiple activities: The conundrum of cell surface NADH oxidoreductases

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.     

Multiple proteins with single activities or a single protein with multiple activities: the conundrum of cell surface NADH oxidoreductases

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.     

1-Methyl-4-phenylpyridinium-induced down-regulation of dopamine transporter function correlates with a reduction in dopamine transporter cell surface expression

The mechanisms whereby 1-methyl-4-phenylpyridinium (MPP(+)) mediates cell death and Parkinsonism are still unclear. We have shown that dopamine transporter (DAT) is required for MPP(+)-mediated cytotoxicity in HEK-293 cells stably transfected with human DAT. Furthermore, MPP(+) produced a concentration- and time-dependent reduction in the uptake of [3H]dopamine. We observed a significant decrease in [3H]WIN 35428 binding in the intact cells with MPP(+). The saturation analysis of the [3H]WIN 35428 binding obtained from total membrane fractions revealed a decrease in the transporter density (B(max)) with an increase in the dissociation equilibrium constant (K(d)) after MPP(+) treatment. Furthermore, biotinylation assays confirmed that MPP(+) reduced both plasma membrane and intracellular DAT immunoreactivity. Taken together, these findings suggest that the reduction in cell surface DAT protein expression in response to MPP(+) may be a contributory factor in the down-regulation of DAT function while enhanced lysosomal degradation of DAT may signal events leading to cellular toxicity.