Design, engineering and utility of biotic games

Games are a significant and defining part of human culture, and their utility beyond pure entertainment has been demonstrated with so-called ‘serious games’. Biotechnology – despite its recent advancements – has had no impact on gaming yet. Here we propose the concept of ‘biotic games’, i.e., games that operate on biological processes. Utilizing a variety of biological processes we designed and tested a collection of games: ‘Enlightenment’, ‘Ciliaball’, ‘PAC-mecium’, ‘Microbash’, ‘Biotic Pinball’, ‘POND PONG’, ‘PolymerRace’, and ‘The Prisoner's Smellemma’. We found that biotic games exhibit unique features compared to existing game modalities, such as utilizing biological noise, providing a real-life experience rather than virtual reality, and integrating the chemical senses into play. Analogous to video games, biotic games could have significant conceptual and cost-reducing effects on biotechnology and eventually healthcare; enable volunteers to participate in crowd-sourcing to support medical research; and educate society at large to support personal medical decisions and the public discourse on bio-related issues

Longitudinal multiparameter assay of lymphocyte interactions from onset by microfluidic cell pairing and culture

Resolving how the early signaling events initiated by cell–cell interactions are transduced into diverse functional outcomes necessitates correlated measurements at various stages. Typical approaches that rely on bulk cocultures and population-wide correlations, however, only reveal these relationships broadly at the population level, not within each individual cell. Here, we present a microfluidics-based cell–cell interaction assay that enables longitudinal investigation of lymphocyte interactions at the single-cell level through microfluidic cell pairing, on-chip culture, and multiparameter assays, and allows recovery of desired cell pairs by micromanipulation for off-chip culture and analyses. Well-defined initiation of interactions enables probing cellular responses from the very onset, permitting single-cell correlation analyses between early signaling dynamics and later-stage functional outcomes within same cells. We demonstrate the utility of this microfluidic assay with natural killer cells interacting with tumor cells, and our findings suggest a possible role for the strength of early calcium signaling in selective coordination of subsequent cytotoxicity and IFN-gamma production. Collectively, our experiments demonstrate that this new approach is well-suited for resolving the relationships between complex immune responses within each individual cell.Singapore-MIT AllianceAmerican Association for Cancer Research. Pancreatic Cancer Action NetworkMassachusetts Institute of Technology. Department of Electrical Engineering and Computer Science (Frank Quick Faculty Research Innovation Fellowship

Ex vivo Dynamics of Human Glioblastoma Cells in a Microvasculature-on-a-Chip System Correlates with Tumor Heterogeneity and Subtypes

The perivascular niche (PVN) plays an essential role in brain tumor stem-like cell (BTSC) fate control, tumor invasion, and therapeutic resistance. Here, a microvasculature-on-a-chip system as a PVN model is used to evaluate the ex vivo dynamics of BTSCs from ten glioblastoma patients. BTSCs are found to preferentially localize in the perivascular zone, where they exhibit either the lowest motility, as in quiescent cells, or the highest motility, as in the invasive phenotype, with migration over long distance. These results indicate that PVN is a niche for BTSCs, while the microvascular tracks may serve as a path for tumor cell migration. The degree of colocalization between tumor cells and microvessels varies significantly across patients. To validate these results, single-cell transcriptome sequencing (10 patients and 21 750 single cells in total) is performed to identify tumor cell subtypes. The colocalization coefficient is found to positively correlate with proneural (stem-like) or mesenchymal (invasive) but not classical (proliferative) tumor cells. Furthermore, a gene signature profile including PDGFRA correlates strongly with the “homing” of tumor cells to the PVN. These findings demonstrate that the model can recapitulate in vivo tumor cell dynamics and heterogeneity, representing a new route to study patient-specific tumor cell functions

Spatiotemporally Controlled Cardiac Conduction Block Using High-Frequency Electrical Stimulation

Background: Methods for the electrical inhibition of cardiac excitation have long been sought to control excitability and conduction, but to date remain largely impractical. High-amplitude alternating current (AC) stimulation has been known to extend cardiac action potentials (APs), and has been recently exploited to terminate reentrant arrhythmias by producing reversible conduction blocks. Yet, low-amplitude currents at similar frequencies have been shown to entrain cardiac tissues by generation of repetitive APs, leading in some cases to ventricular fibrillation and hemodynamic collapse in vivo. Therefore, an inhibition method that does not lead to entrainment – irrespective of the stimulation amplitude (bound to fluctuate in an in vivo setting) – is highly desirable. Methodology/Principal Findings: We investigated the effects of broader amplitude and frequency ranges on the inhibitory effects of extracellular AC stimulation on HL-1 cardiomyocytes cultured on microelectrode arrays, using both sinusoidal and square waveforms. Our results indicate that, at sufficiently high frequencies, cardiac tissue exhibits a binary response to stimulus amplitude with either prolonged APs or no effect, thereby effectively avoiding the risks of entrainment by repetitive firing observed at lower frequencies. We further demonstrate the ability to precisely define reversible local conduction blocks in beating cultures without influencing the propagation activity in non-blocked areas. The conduction blocks were spatiotemporally controlled by electrode geometry and stimuli duration, respectively, and sustainable for long durations (300 s). Conclusion/Significance: Inhibition of cardiac excitation induced by high-frequency AC stimulation exhibits a binary response to amplitude above a threshold frequency, enabling the generation of reversible conduction blocks without the risks of entrainment. This inhibition method could yield novel approaches for arrhythmia modeling in vitro, as well as safer and more efficacious tools for in vivo cardiac mapping and radio-frequency ablation guidance applications

Microfluidic single-cell technologies for assaying lymphocyte interactions

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2015.Cataloged from PDF version of thesis.Includes bibliographical references (pages 130-144).Immune cells do not live in isolation but interact to coordinate their many actions. One of the chief routes they foster communication is through direct physical interactions that enables them to read and interpret signals mediated at membrane interfaces. Despite the critical importance of these direct interactions in determining crucial developmental and functional immunological responses, their dynamic nature together with vast heterogeneity and polyfunctionality of individual immune cells have presented technical challenges for their systematic investigation. In particular, only limited tools are available that can exert control over the individual cells and their microenvironments to be able to precisely define interactions and deeply profile their outcomes at the individual cell level to resolve emerging immune responses within each single-cell. To fill this critical void, this thesis presents the development and implementation of novel microfluidic technologies for single-cell analysis of direct cell-cell interactions in immunology. By combining carefully designed weir-based hydrodynamic traps with a multistep cell loading procedure, the microfluidic devices capture and controllably pair hundreds of cells in parallel. This approach provides requisite control over interactions with one-to-one interacting partners, well-defined and synchronous initiation of interactions, and enduring contacts. It also provides full control over the soluble microenvironment by solution exchange without losing cell registration. Accordingly, these features enable monitoring and assaying lymphocyte interactions longitudinally from the beginning with multiparametric single-cell measurements. These capabilities in turn allow probing into complete immune cell activation window from the very onset for direct correlation analyses within hundreds of individual cells in a single experiment. We apply these new 'microfluidic cell pairing' technologies to quantitative investigation of lymphocyte interactions to elucidate lymphocyte activation dynamics and their relation to diverse functional behaviors at the single-cell level. These studies help resolve qualitatively and quantitatively distinct calcium signaling patterns in single CD8 T cells based on varying T cell receptor affinities which correlate with differential cytokine output. Similar studies with natural killer (NK) cells identify a previously unreported inverse correlation between the strength of early calcium signaling and cytokine production, and further indicate a calcium-dependent mechanism for selective regulation of cytotoxicity and cytokine production in NK cells. Collectively, these findings provide essential insight into the regulation and evolution of immune responses within individual immune cells, and establish the potential of these new microfluidic technologies to address important questions on many aspects of cell-cell interactions across biology in general and in immunology in particular.by Burak Dura.Ph. D

Spatially and temporally controlled immune cell interactions using microscale tools

Many critical immunological responses are mediated by cell–cell interactions. Despite their capabilities, traditional techniques that rely on snapshot analysis or ensemble measurements can only provide a fragmentary picture of the complexity of these interactions. Emerging classes of new and versatile microscale tools hold great potential for enabling detailed investigation of these interactions with precise control in space and time, multiplexed measurement capability and high-throughput single-cell analysis. These features allow new ways of examining immune cell interactions that are not possible with traditional methods, improve the extent of information extracted from experiments, and reveal new findings. Here, we review recent developments in microscale tools that are paving the way for comprehensive analyses of cell–cell interactions in the immune system.Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science (Frank Quick Faculty Research Innovation Fellowship

Comparison of copper intrauterine device with levonorgestrel-bearing intrauterine system for post-abortion contraception

WOS: 000362674400018PubMed ID: 26180028Aim: The aim of this study was to compare the safety, bleeding pattern, effects, side-effects, complications and 6-month continuity rates of levonorgestrel-bearing intrauterine system (LNG-IUS) with conventional copper intrauterine device (Cu-IUD) inserted immediately after voluntary termination of pregnancy up to 10weeks of gestation. Methods: One hundred women who underwent voluntary pregnancy termination and preferred IUD insertion as a contraceptive method after counseling were enrolled. The patients were randomly allocated to Cu-IUD or LNG-IUS and followed up at 10 days, and at 1, 3 and 6 months. The expulsion rates, continuation rates, side-effects, and bleeding patterns were compared. Results: Fifty women in the Cu-IUD group and 44 women in the LNG-IUS group were followed up. The continuity and expulsion rate for Cu-IUD and LNG-IUS at the end of 6months was 74%, 12%, and 75%, 11.3%, respectively. In LNG-IUS users, the incidence of amenorrhea and the number of spotting days were higher and hemoglobin increased throughout the follow-up period. The side-effects related to both methods were not different from interval insertions. Conclusion: Immediate post-abortion intrauterine contraception with Cu-IUD or LNG-IUS is a safe, reliable method. The incidence of side-effects is similar, and there is only a slightly higher rate of expulsion but an acceptable rate of method continuation

Guidance of conduction path using high-frequency suprathreshold AC stimuli.

<p>A. Microelectrode array used in the experiment. Yellow arrows labeled 1 and 2 represent the conduction paths before and during the block, as derived from the isochrone maps in (C). SE, stimulation electrode used for pacing cells; BE, blocking electrode; RE, recording electrodes. B. Electrical recordings showing the change in conduction path during blocking by the increased time delay between stimulation pulse and LATs. Black bar represents the block duration. (* denotes one missed beat during the experiment). C. Isochrone maps revealing the direction change in conduction path.</p

Demonstration of spatiotemporally controlled electrical conduction block (50 µA_p-p, 5 kHz).

<p>Time-lapse of Ca²⁺ fluorescence showing propagation of electrical activity before, during and after application of the inhibitory stimulus. White dotted lines highlight the location and geometry of blocking electrode (electrode A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036217#pone-0036217-g001" target="_blank">Fig. 1A,D</a>). Red indicates high Ca²⁺ concentration, blue low.</p

Single-cell simulation results of suprathreshold AC inhibition compared to normal action potential.

<p>A–E. Prolonged action potential. Simulated responses to 1 kHz square wave applied between <i>t</i> = 10 ms and <i>t</i> = 510 ms. Prolonged action potential revealed by membrane potential (A, F) and intracellular Ca²⁺ levels (B, G). C. Inactivation of Na⁺ channels (during the block, fast and slow inactivation gating parameters – <i>h</i>₁ and <i>h</i>₂ – decreased to zero; activation gating variable <i>m</i> oscillated between 0 and 1 in comparison to resting values in H). D. Prolonged activation of inward Ca²⁺ channel (during the block, fast and slow inactivation gating parameters – <i>f</i>_L1 and <i>f</i>_L2 – remained lowered while activation gating variable <i>d</i>_L remained high compared to resting values in I). E. Prolonged activation of outward K⁺ channel (during the block, inactivation gating variable <i>s</i>_SUS lowered to 0.9 while activation gating variable <i>r</i>_SUS plateaued around 0.3 in comparison with resting values in J). F–J. Normal action potential. Simulated responses to a stimulus of 10 ms duration applied at <i>t</i> = 40 ms.</p