3D Collagen Vascular Tumor-on-a-Chip Mimetics for Dynamic Combinatorial Drug Screening.

Disease models, including in vitro cell culture and animal models, have contributed significantly to developing diagnostics and treatments over the past several decades. The successes of traditional drug screening methods were generally hampered by not adequately mimicking critical in vivo features, such as a 3D microenvironment and dynamic drug diffusion through the extracellular matrix (ECM). To address these issues, we developed a 3D dynamic drug delivery system for cancer drug screening that mimicks drug dissemination through the tumor vasculature and the ECM by creating collagen-embedded microfluidic channels. Using this novel 3D ECM microsystem, we compared viability of tumor pieces with traditionally used 2D methods in response to three different drug combinations. Drug diffusion profiles were evaluated by simulation methods and tested in the 3D ECM microsystem and a 2D 96-well setup. Compared with the 2D control, the 3D ECM microsystem produced reliable data on viability, drug ratios, and combination indeces. This novel approach enables higher throughput and sets the stage for future applications utilizing drug sensitivity predicting algorithms based on dynamic diffusion profiles requiring only minimal patient tissue. Our findings moved drug sensitivity screening closer to clinical implications with a focus on testing combinatorial drug effects, an option often limited by the amount of available patient tissues.

Probing coordinated co-culture cancer related motility through differential micro-compartmentalized elastic substrates.

Cell development and behavior are driven by internal genetic programming, but the external microenvironment is increasingly recognized as a significant factor in cell differentiation, migration, and in the case of cancer, metastatic progression. Yet it remains unclear how the microenvironment influences cell processes, especially when examining cell motility. One factor that affects cell motility is cell mechanics, which is known to be related to substrate stiffness. Examining how cells interact with each other in response to mechanically differential substrates would allow an increased understanding of their coordinated cell motility. In order to probe the effect of substrate stiffness on tumor related cells in greater detail, we created hard-soft-hard (HSH) polydimethylsiloxane (PDMS) substrates with alternating regions of different stiffness (200 and 800 kPa). We then cultured WI-38 fibroblasts and A549 epithelial cells to probe their motile response to the substrates. We found that when the 2 cell types were exposed simultaneously to the same substrate, fibroblasts moved at an increased speed over epithelial cells. Furthermore, the HSH substrate allowed us to physically guide and separate the different cell types based on their relative motile speed. We believe that this method and results will be important in a diversity of areas including mechanical microenvironment, cell motility, and cancer biology.

3D In Vitro Neuron on a Chip for Probing Calcium Mechanostimulation.

The evolution of tissue on a chip systems holds promise for mimicking the response of biological functionality of physiological systems. One important direction for tissue on a chip approaches are neuron-based systems that could mimic neurological responses and lessen the need for in vivo experimentation. For neural research, more attention has been devoted recently to understanding mechanics due to issues in areas such as traumatic brain injury (TBI) and pain, among others. To begin to address these areas, a 3D Nerve Integrated Tissue on a Chip (NITC) approach combined with a Mechanical Excitation Testbed (MET) System is developed to impose external mechanical stimulation toward more realistic physiological environments. PC12 cells differentiated with nerve growth factor, which were cultured in a controlled 3D scaffolds, are used. The cells are labeled in a 3D NITC system with Fluo-4-AM to examine their calcium response under mechanical stimulation synchronized with image capture. Understanding the neural responses to mechanical stimulation beyond 2D systems is very important for neurological studies and future personalized strategies. This work will have implications in a diversity of areas including tissue-on-a-chip systems, biomaterials, and neuromechanics.

Decidual Vasculopathy Identification in Whole Slide Images Using Multiresolution Hierarchical Convolutional Neural Networks.

After a child is born, the examination of the placenta by a pathologist for abnormalities, such as infection or maternal vascular malperfusion, can provide important information about the immediate and long-term health of the infant. Detection of the pathologic placental blood vessel lesion decidual vasculopathy (DV) has been shown to predict adverse pregnancy outcomes, such as preeclampsia, which can lead to mother and neonatal morbidity in subsequent pregnancies. However, because of the high volume of deliveries at large hospitals and limited resources, currently a large proportion of delivered placentas are discarded without inspection. Furthermore, the correct diagnosis of DV often requires the expertise of an experienced perinatal pathologist. We introduce a hierarchical machine learning approach for the automated detection and classification of DV lesions in digitized placenta slides, along with a method of coupling learned image features with patient metadata to predict the presence of DV. Ultimately, the approach will allow many more placentas to be screened in a more standardized manner, providing feedback about which cases would benefit most from more in-depth pathologic inspection. Such computer-assisted examination of human placentas will enable real-time adjustment to infant and maternal care and possible chemoprevention (eg, aspirin therapy) to prevent preeclampsia, a disease that affects 2% to 8% of pregnancies worldwide, in women identified to be at risk with future pregnancies.

Chemotactic Responses of Jurkat Cells in Microfluidic Flow-Free Gradient Chambers.

Gradients of soluble molecules coordinate cellular communication in a diverse range of multicellular systems. Chemokine-driven chemotaxis is a key orchestrator of cell movement during organ development, immune response and cancer progression. Chemotaxis assays capable of examining cell responses to different chemokines in the context of various extracellular matrices will be crucial to characterize directed cell motion in conditions which mimic whole tissue conditions. Here, a microfluidic device which can generate different chemokine patterns in flow-free gradient chambers while controlling surface extracellular matrix (ECM) to study chemotaxis either at the population level or at the single cell level with high resolution imaging is presented. The device is produced by combining additive manufacturing (AM) and soft lithography. Generation of concentration gradients in the device were simulated and experimentally validated. Then, stable gradients were applied to modulate chemotaxis and chemokinetic response of Jurkat cells as a model for T lymphocyte motility. Live imaging of the gradient chambers allowed to track and quantify Jurkat cell migration patterns. Using this system, it has been found that the strength of the chemotactic response of Jurkat cells to CXCL12 gradient was reduced by increasing surface fibronectin in a dose-dependent manner. The chemotaxis of the Jurkat cells was also found to be governed not only by the CXCL12 gradient but also by the average CXCL12 concentration. Distinct migratory behaviors in response to chemokine gradients in different contexts may be physiologically relevant for shaping the host immune response and may serve to optimize the targeting and accumulation of immune cells to the inflammation site. Our approach demonstrates the feasibility of using a flow-free gradient chamber for evaluating cross-regulation of cell motility by multiple factors in different biologic processes.

Robust mechanobiological behavior emerges in heterogeneous myosin systems.

Biological complexity presents challenges for understanding natural phenomenon and engineering new technologies, particularly in systems with molecular heterogeneity. Such complexity is present in myosin motor protein systems, and computational modeling is essential for determining how collective myosin interactions produce emergent system behavior. We develop a computational approach for altering myosin isoform parameters and their collective organization, and support predictions with in vitro experiments of motility assays with α-actinins as molecular force sensors. The computational approach models variations in single myosin molecular structure, system organization, and force stimuli to predict system behavior for filament velocity, energy consumption, and robustness. Robustness is the range of forces where a filament is expected to have continuous velocity and depends on used myosin system energy. Myosin systems are shown to have highly nonlinear behavior across force conditions that may be exploited at a systems level by combining slow and fast myosin isoforms heterogeneously. Results suggest some heterogeneous systems have lower energy use near stall conditions and greater energy consumption when unloaded, therefore promoting robustness. These heterogeneous system capabilities are unique in comparison with homogenous systems and potentially advantageous for high performance bionanotechnologies. Findings open doors at the intersections of mechanics and biology, particularly for understanding and treating myosin-related diseases and developing approaches for motor molecule-based technologies.

2D and 3D Mechanobiology in Human and Nonhuman Systems.

Mechanobiology involves the investigation of mechanical forces and their effect on the development, physiology, and pathology of biological systems. The human body has garnered much attention from many groups in the field, as mechanical forces have been shown to influence almost all aspects of human life ranging from breathing to cancer metastasis. Beyond being influential in human systems, mechanical forces have also been shown to impact nonhuman systems such as algae and zebrafish. Studies of nonhuman and human systems at the cellular level have primarily been done in two-dimensional (2D) environments, but most of these systems reside in three-dimensional (3D) environments. Furthermore, outcomes obtained from 3D studies are often quite different than those from 2D studies. We present here an overview of a select group of human and nonhuman systems in 2D and 3D environments. We also highlight mechanobiological approaches and their respective implications for human and nonhuman physiology.

Controlled surface topography regulates collective 3D migration by epithelial-mesenchymal composite embryonic tissues.

Cells in tissues encounter a range of physical cues as they migrate. Probing single cell and collective migratory responses to physically defined three-dimensional (3D) microenvironments and the factors that modulate those responses are critical to understanding how tissue migration is regulated during development, regeneration, and cancer. One key physical factor that regulates cell migration is topography. Most studies on surface topography and cell mechanics have been carried out with single migratory cells, yet little is known about the spreading and motility response of 3D complex multi-cellular tissues to topographical cues. Here, we examine the response to complex topographical cues of microsurgically isolated tissue explants composed of epithelial and mesenchymal cell layers from naturally 3D organized embryos of the aquatic frog Xenopus laevis. We control topography using fabricated micropost arrays (MPAs) and investigate the collective 3D migration of these multi-cellular systems in these MPAs. We find that the topography regulates both collective and individual cell migration and that dense MPAs reduce but do not eliminate tissue spreading. By modulating cell size through the cell cycle inhibitor Mitomycin C or the spacing of the MPAs we uncover how 3D topographical cues disrupt collective cell migration. We find surface topography can direct both single cell motility and tissue spreading, altering tissue-scale processes that enable efficient conversion of single cell motility into collective movement.

Emergent systems energy laws for predicting myosin ensemble processivity.

In complex systems with stochastic components, systems laws often emerge that describe higher level behavior regardless of lower level component configurations. In this paper, emergent laws for describing mechanochemical systems are investigated for processive myosin-actin motility systems. On the basis of prior experimental evidence that longer processive lifetimes are enabled by larger myosin ensembles, it is hypothesized that emergent scaling laws could coincide with myosin-actin contact probability or system energy consumption. Because processivity is difficult to predict analytically and measure experimentally, agent-based computational techniques are developed to simulate processive myosin ensembles and produce novel processive lifetime measurements. It is demonstrated that only systems energy relationships hold regardless of isoform configurations or ensemble size, and a unified expression for predicting processive lifetime is revealed. The finding of such laws provides insight for how patterns emerge in stochastic mechanochemical systems, while also informing understanding and engineering of complex biological systems.

Mechanochemical actuators of embryonic epithelial contractility.

Spatiotemporal regulation of cell contractility coordinates cell shape change to construct tissue architecture and ultimately directs the morphology and function of the organism. Here we show that contractility responses to spatially and temporally controlled chemical stimuli depend much more strongly on intercellular mechanical connections than on biochemical cues in both stimulated tissues and adjacent cells. We investigate how the cell contractility is triggered within an embryonic epithelial sheet by local ligand stimulation and coordinates a long-range contraction response. Our custom microfluidic control system allows spatiotemporally controlled stimulation with extracellular ATP, which results in locally distinct contractility followed by mechanical strain pattern formation. The stimulation-response circuit exposed here provides a better understanding of how morphogenetic processes integrate responses to stimulation and how intercellular responses are transmitted across multiple cells. These findings may enable one to create a biological actuator that actively drives morphogenesis.

Three-dimensional microfiber devices that mimic physiological environments to probe cell mechanics and signaling.

Many physiological systems are regulated by cells that alter their behavior in response to changes in their biochemical and mechanical environment. These cells experience this dynamic environment through an endogenous biomaterial matrix that transmits mechanical force and permits chemical exchange with the surrounding tissue. As a result, in vitro systems that mimic three-dimensional, in vivo cellular environments can enable experiments that reveal the nuanced interplay between biomechanics and physiology. Here we report the development of a minimal-profile, three-dimensional (MP3D) experimental microdevice that confines cells to a single focal plane, while allowing the precise application of mechanical displacement to cells and concomitant access to the cell membrane for perfusion with biochemical agonists. The MP3D device--an ordered microfiber scaffold erected on glass--provides a cellular environment that induces physiological cell morphologies. Small manipulations of the scaffold's microfibers allow attached cells to be mechanically probed. Due to the scaffold's minimal height profile, MP3D devices confine cells to a single focal plane, facilitating observation with conventional epifluorescent microscopy. When examining fibroblasts within MP3D devices, we observed robust cellular calcium responses to both a chemical stimulus as well as mechanical displacement of the cell membrane. The observed response differed significantly from previously reported, mechanically-induced calcium responses in the same cell type. Our findings demonstrate a key link between environment, cell morphology, mechanics, and intracellular signal transduction. We anticipate that this device will broadly impact research in fields including biomaterials, tissue engineering, and biophysics.

Mechanical loading of stem cells for improvement of transplantation outcome in a model of acute myocardial infarction: the role of loading history.

Stem cell therapy for tissue repair is a rapidly evolving field and the factors that dictate the physiological responsiveness of stem cells remain under intense investigation. In this study we hypothesized that the mechanical loading history of muscle-derived stem cells (MDSCs) would significantly impact MDSC survival, host tissue angiogenesis, and myocardial function after MDSC transplantation into acutely infarcted myocardium. Mice with acute myocardial infarction by permanent left coronary artery ligation were injected with either nonstimulated (NS) or mechanically stimulated (MS) MDSCs. Mechanical stimulation consisted of stretching the cells with equibiaxial stretch with a magnitude of 10% and frequency of 0.5 Hz. MS cell-transplanted hearts showed improved cardiac contractility, increased numbers of host CD31+ cells, and decreased fibrosis, in the peri-infarct region, compared to the hearts treated with NS MDSCs. MS MDSCs displayed higher vascular endothelial growth factor expression than NS cells in vitro. These findings highlight an important role for cyclic mechanical loading preconditioning of donor MDSCs in optimizing MDSC transplantation for myocardial repair.

Integrated biomimetic carbon nanotube composites for in vivo systems.

As interest in using carbon nanotubes for developing biologically compatible systems continues to grow, biological inspiration is stimulating new directions for in vivo approaches. The ability to integrate nanotechnology-based systems in the body will provide greater successes if the implanted material is made to mimic elements of the biological milieu especially through tuning physical and chemical characteristics. Here, we demonstrate the highly successful capacity for in vivo implantation of a new carbon nanotube-based composite that is, itself, integrated with a hydroxyapatite-polymethyl methacrylate to create a nanocomposite. The success of this approach is grounded in finely tailoring the physical and chemical properties of this composite for the critical demands of biological integration. This is accomplished through controlling the surface modification scheme, which affects the interactions between carbon nanotubes and the hydroxyapatite-polymethyl methacrylate. Furthermore, we carefully examine cellular response with respect to adhesion and proliferation to examine in vitro compatibility capacity. Our results indicate that this new composite accelerates cell maturation through providing a mechanically competent bone matrix; this likely facilitates osteointegration in vivo. We believe that these results will have applications in a diversity of areas including carbon nanotube, regeneration, chemistry, and engineering research.

Modulation of fluidic resistance and capacitance for long-term, high-speed feedback control of a microfluidic interface.

Existing microfluidic systems can control local chemical environments by directing the interface between laminar flowing streams for applications ranging from subcellular stimulation to fuel cells. However, conventional flow modulation methods have not yet provided a robust and reliable way to dynamically control laminar flow interfaces for very long time periods. Such control is important in biological investigations, since response times for living cells and tissues can be as long as several days. Here, we describe a novel long-term, high-speed approach that employs modulation of fluidic resistance and fluidic capacitance between a fluid reservoir and a microfluidic network with feedback control to enable long-term dynamic control of a microfluidic interface in time and space. Our method involves constricting a narrow tube through a pinching approach to modulate fluidic resistance while also controlling a small variable reservoir in the fluidic network through a squeezing approach to modulate fluidic capacitance. We designed a well-tuned proportional-integral-derivative (PID) controller for the closed-loop control system that resulted in control of pressure for short-term (2 s) and long-term (15 h) experiments. Further, we integrated a pressure-based feedback control approach into this method, which enables both long-term spatiotemporal control of our microfluidic interface at frequencies greater than 1 Hz and a reservoir capacity to enable experiments for longer than 60 days. This long-term and high-speed control is not possible with standard microfluidic laboratory practices. Our system has a diversity of potential applications including long-term cellular studies in cancer metastasis or embryonic development.

Evaluating spatial constraints in cellular assembly processes using a monte carlo approach.

Biomolecular behavior commonly involves complex sets of interacting components that are challenging to understand through solution-based chemical theories. Molecular assembly is especially intriguing in the cellular environment because of its links to cell structure in processes such as chemotaxis. We use a coarse-grained Monte Carlo simulation to elucidate the importance of spatial constraints in molecular assembly. We have performed a study of actin filament polymerization through this space-aware probabilistic lattice-based model. Quantitative results are compared with nonspatial models and show convergence over a wide parameter space, but marked divergence over realistic levels corresponding to macromolecular crowding inside cells and localized actin concentrations found at the leading edge during cell motility. These conclusions have direct implications for cell shape and structure, as well as tumor cell migration.

Sequential involvement of Cdk1, mTOR and p53 in apoptosis induced by the HIV-1 envelope.

Syncytia arising from the fusion of cells expressing the HIV-1-encoded Env gene with cells expressing the CD4/CXCR4 complex undergo apoptosis following the nuclear translocation of mammalian target of rapamycin (mTOR), mTOR-mediated phosphorylation of p53 on Ser15 (p53(S15)), p53-dependent upregulation of Bax and activation of the mitochondrial death pathway. p53(S15) phosphorylation is only detected in syncytia in which nuclear fusion (karyogamy) has occurred. Karyogamy is secondary to a transient upregulation of cyclin B and a mitotic prophase-like dismantling of the nuclear envelope. Inhibition of cyclin-dependent kinase-1 (Cdk1) prevents karyogamy, mTOR activation, p53(S15) phosphorylation and apoptosis. Neutralization of p53 fails to prevent karyogamy, yet suppresses apoptosis. Peripheral blood mononuclear cells from HIV-1-infected patients exhibit an increase in cyclin B and mTOR expression, correlating with p53(S15) phosphorylation and viral load. Cdk1 inhibition prevents the death of syncytia elicited by HIV-1 infection of primary CD4 lymphoblasts. Thus, HIV-1 elicits a pro-apoptotic signal transduction pathway relying on the sequential action of cyclin B-Cdk1, mTOR and p53.