Thomas A. Zangle

On the propagation of concentration polarization from microchannel-nanochannel interfaces. Part I: Analytical model and characteristic analysis.

We develop two models to describe ion transport in variable-height micro- and nanochannels. For the first model, we obtain a one-dimensional (unsteady) partial differential equation governing flow and charge transport through a shallow and wide electrokinetic channel. In this model, the effects of electric double layer (EDL) on axial transport are taken into account using exact solutions of the Poisson-Boltzmann equation. The second simpler model, which is approachable analytically, assumes that the EDLs are confined to near-wall regions. Using a characteristics analysis, we show that the latter model captures concentration polarization (CP) effects and provides useful insight into its dynamics. Two distinct CP regimes are identified: CP with propagation in which enrichment and depletion shocks propagate outward, and CP without propagation where polarization effects stay local to micro- nanochannel interfaces. The existence of each regime is found to depend on a nanochannel Dukhin number and mobility of the co-ion nondimensionalized by electroosmotic mobility. Interestingly, microchannel dimensions and axial diffusion are found to play an insignificant role in determining whether CP propagates. The steady state condition of propagating CP is shown to be controlled by channel heights, surface chemistry, and co-ion mobility instead of the reservoir condition. Both models are validated against experimental results in Part II of this two-paper series.

On the Propagation of Concentration Polarization from Microchannel-Nanochannel Interfaces Part II. Numerical and Experimental Study

We present results of a combined computational and experimental study of the propagation of concentration polarization (CP) zones in a microchannel-nanochannel system. Our computational model considers the combined effects of bulk flow, electromigration, and diffusion and accurately captures the dynamics of CP. Using wall charge inside the nanochannel as a single fitting parameter, we predict experimentally observed enrichment and depletion shock velocities. Our model can also be used to compute the existence of CP with propagating enrichment and depletion shocks on the basis of measured ion mobility and wall properties. We present experiments where the background electrolyte consists of only a fluorescent ion and its counterion. These results are used to validate the computational model and to confirm predicted trends from an analytical model presented in the first of this two-paper series. We show experimentally that the enrichment region concentration is effectively independent of the applied current, while the enrichment and depletion shock velocities increase in proportion to current density.

Live-cell mass profiling: an emerging approach in quantitative biophysics

Cell mass, volume and growth rate are tightly controlled biophysical parameters in cellular development and homeostasis, and pathological cell growth defines cancer in metazoans. The first measurements of cell mass were made in the 1950s, but only recently have advances in computer science and microfabrication spurred the rapid development of precision mass-quantifying approaches. Here we discuss available techniques for quantifying the mass of single live cells with an emphasis on relative features, capabilities and drawbacks for different applications.

Chemical dissection of the cell cycle: Probes for cell biology and anti-cancer drug development

Cancer cell proliferation relies on the ability of cancer cells to grow, transition through the cell cycle, and divide. To identify novel chemical probes for dissecting the mechanisms governing cell cycle progression and cell division, and for developing new anti-cancer therapeutics, we developed and performed a novel cancer cell-based high-throughput chemical screen for cell cycle modulators. This approach identified novel G1, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes. We further characterized the M-phase inhibitors and highlight the most potent M-phase inhibitor MI-181, which targets tubulin, inhibits tubulin polymerization, activates the spindle assembly checkpoint, arrests cells in mitosis, and triggers a fast apoptotic cell death. Importantly, MI-181 has broad anti-cancer activity, especially against BRAFV600E melanomas.

Effects of constant voltage on time evolution of propagating concentration polarization

We extend the analytical theory of propagating concentration polarization (CP) to describe and compare the effects of constant-voltage versus constant-current conditions on the transient development of CP enrichment and depletion zones. We support our analysis with computational and experimental results. We find that at constant voltage, enrichment and depletion regions spread as t(1/2) as opposed to the previously observed t(1) scaling for constant current conditions. At low, constant voltages, the growth and propagation of CP zones can easily be misinterpreted as nonpropagating behavior.

Quantifying Biomass Changes of Single CD8+ T Cells during Antigen Specific Cytotoxicity

Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.

Quantification of Biomass and Cell Motion in Human Pluripotent Stem Cell Colonies

Somatic cell reprogramming to pluripotency requires an immediate increase in cell proliferation and reduction in cell size. It is unknown whether proliferation and biomass controls are similarly coordinated with early events during the differentiation of pluripotent stem cells (PSCs). This impasse exists because PSCs grow in tight clusters or colonies, precluding most quantifying approaches. Here, we investigate live cell interferometry as an approach to quantify the biomass and growth of HSF1 human PSC colonies before and during retinoic acid-induced differentiation. We also provide an approach for measuring the rate and coordination of intracolony mass redistribution in HSF1 clusters using live cell interferometry images. We show that HSF1 cells grow at a consistent, exponential rate regardless of colony size and display coordinated intracolony movement that ceases with the onset of differentiation. By contrast, growth and proliferation rates show a decrease of only ∼15% decrease during early differentiation despite global changes in gene expression and previously reported changes in energy metabolism. Overall, these results suggest that cell biomass and proliferation are regulated independent of pluripotency during early differentiation, which is distinct from what occurs with successful reprogramming.

Rapidly quantifying drug sensitivity of dispersed and clumped breast cancer cells by mass profiling

Live cell mass profiling is a promising new approach for rapidly quantifying responses to therapeutic agents through picogram-scale changes in cell mass over time. A significant barrier in mass profiling is the inability of existing methods to handle pleomorphic cellular clusters and clumps, which are more commonly present in patient-derived samples or tissue cultures than are isolated single cells. Here we demonstrate automated Live Cell Interferometry (LCI) as a rapid and accurate quantifier of the sensitivity of single cell and colony-forming human breast cancer cell lines to the HER2-directed monoclonal antibody, trastuzumab (Herceptin). The relative sensitivities of small samples (<500 cells) of four breast cancer cell lines were determined tens-to-hundreds of times faster than is possible with traditional proliferation assays. These LCI advances in clustered sample assessment and speed open up the possibility for therapeutic response testing of patient-derived solid tumor samples, which are viable only for short periods ex vivo and likely to be in the form of cell aggregates and clusters.

Live cell interferometry quantifies dynamics of biomass partitioning during cytokinesis.

The equal partitioning of cell mass between daughters is the usual and expected outcome of cytokinesis for self-renewing cells. However, most studies of partitioning during cell division have focused on daughter cell shape symmetry or segregation of chromosomes. Here, we use live cell interferometry (LCI) to quantify the partitioning of daughter cell mass during and following cytokinesis. We use adherent and non-adherent mouse fibroblast and mouse and human lymphocyte cell lines as models and show that, on average, mass asymmetries present at the time of cleavage furrow formation persist through cytokinesis. The addition of multiple cytoskeleton-disrupting agents leads to increased asymmetry in mass partitioning which suggests the absence of active mass partitioning mechanisms after cleavage furrow positioning.

Nongenetic origins of cell-to-cell variability in B lymphocyte proliferation

Significance This study addresses why splenic B lymphocytes show differential cell proliferation and death decisions, and whether these may be predictable. Biology provides examples of both stochastic decision making and highly deterministic developmental programs. Prior studies of B lymphocytes suggested these cells make stochastic decisions, but the key experiment—long-term tracing of individual cell lineages—has not been done. Overcoming the technical challenges, we found that B cell fate decisions are largely nonstochastic. Using a mathematical model, we found that they are predictable, as long as the state of the molecular network in founder cells is known. That allowed us to identify the molecular determinants of proliferative fate decisions, which potentially constitute novel drug targets and biomarkers for B cell-mediated diseases. Rapid antibody production in response to invading pathogens requires the dramatic expansion of pathogen-derived antigen-specific B lymphocyte populations. Whether B cell population dynamics are based on stochastic competition between competing cell fates, as in the development of competence by the bacterium Bacillus subtilis, or on deterministic cell fate decisions that execute a predictable program, as during the development of the worm Caenorhabditis elegans, remains unclear. Here, we developed long-term live-cell microscopy of B cell population expansion and multiscale mechanistic computational modeling to characterize the role of molecular noise in determining phenotype heterogeneity. We show that the cell lineage trees underlying B cell population dynamics are mediated by a largely predictable decision-making process where the heterogeneity of cell proliferation and death decisions at any given timepoint largely derives from nongenetic heterogeneity in the founder cells. This means that contrary to previous models, only a minority of genetically identical founder cells contribute the majority to the population response. We computationally predict and experimentally confirm nongenetic molecular determinants that are predictive of founder cells’ proliferative capacity. While founder cell heterogeneity may arise from different exposure histories, we show that it may also be due to the gradual accumulation of small amounts of intrinsic noise during the lineage differentiation process of hematopoietic stem cells to mature B cells. Our finding of the largely deterministic nature of B lymphocyte responses may provide opportunities for diagnostic and therapeutic development.