To prevent the loss of blood following a break in blood vessels, components in blood and the vessel wall interact rapidly to form a thrombus (clot) to limit hemorrhage. In this talk we will describe a multiscale model of thrombus formation consisting of components for modeling viscous, incompressible blood plasma; coagulation pathway; quiescent and activated platelets; blood cells; activating chemicals; fibrinogen; the vessel walls and their interactions. At macro scale blood flow field is described by the incompressible Navier-Stokes equations and is numerically solved using the projection method. At micro scale, cell movement, cell-cell adhesion, cell-flow and cell-vessel wall interactions are described through an extended stochastic discrete Cellular Potts Model (CPM). Model is tested for robustness with respect to fluctuations of basic parameters. Simulation results demonstrate the development of an inhomogeneous internal structure of the thrombus which is conformed by the preliminary experimental data. We also make predictions about different stages in thrombus development which can be tested experimentally and suggest specific experiments. Lastly, we demonstrate that dependence of the thrombus size on the blood flow rate in simulations is close to the one observed experimentally.
Directing objects across functional streamlines at low Reynolds number is difficult but important since this motion can be used to label, lyse, and analyze complex biological objects on-chip without cross-contamination. Here we use an asymmeteric post array to move cells across coflowing reagents and show on-chip, immunofluorescent labeling of platelets with washing and E.Coli cell lysis with simultaneous separation of bacterial chromosome from the cell contents. Furthermore, we develop the concept of a microfluidic metamaterial by using the basic asymmetric post array as a building block for complex particle handling modes. These modular array elements could be of great use for developing robust techniques for on-chip, continuous flow manipulation and analysis of cells, large bio-particles, and functional beads.
As cells navigate the in vivo microenvironment, they encounter spatiotemporally encoded diffusible and contact cues from growth factors, the extracellular matrix and surrounding cells. To reduce this complexity and permit molecular mechanistic investigations, many current biological studies manipulate cells in very simple culture platforms. However, the simplicity of the microenvironment in these cases can limit the relevance of resulting discoveries. Furthermore, many in vitro microenvironments are treated as static, whereas results must often be interpreted in the context of spatial and temporal dynamics. Our lab aims to define cellular microenvironments that are dynamically complex compared with conventional in vitro culture platforms, yet still permit the manipulations and data collection necessary to gain insight into biologic processes. Here, we will present methods to incorporate both diffusible and contact dynamics into culture platforms using microfabrication-based approaches. Our studies suggest that spatiotemporal dynamics of soluble (growth factors) and insoluble (adhesion) factors have important consequences for cell behaviors in vitro. We are currently studying how cells transduce such complex dynamics via biochemical signaling into behavioral responses such as proliferation, differentiation or migration.
Cell culture technology is falling behind in the pace of progress. As animal and bacterial genomes and proteomes are being fully probed with DNA chips and a wide array of analytical techniques, a picture of cells with dauntingly complex inner workings is emerging. Yet cell culture methodology has remained basically unchanged for almost a century: it consists essentially of the immersion of a large population of cells in a homogeneous fluid medium. This approach is becoming increasingly expensive to scale up and cannot mimic the rich biochemical and biophysical complexity of the cellular microenvironment.
Microtechnology offers the attractive possibility of modulating the microenvironment of single cells and, for the same price, obtain data at high throughput for a small cost. Microfluidic or "Lab on a Chip" devices, in particular, promise to play a key role for several reasons: 1) the dimensions of microchannels can be comparable to or smaller than a single cell; 2) the unique physicochemical behavior of liquids confined to microenvironments enables new strategies for delivering compounds to cells on a subcellular level; 3) the devices consume small quantities of precious/hazardous reagents (thus reducing cost of operation/disposal); and 4) they can be mass-produced in low-cost, portable units. Not surprisingly, in recent years there has been an eruption of microfluidic implementations of a variety of traditional bioanalysis techniques. I will review the latest efforts of our laboratory in the development of cell-based microdevices for neurobiology studies, such as neuromuscular synaptogenesis, axon guidance, and olfaction.
The current state of automation in biological instrumentation revolves around the use of liquid handling robots or systems built with discrete fluidic components. This has enabled many exciting breakthroughs, such as the sequencing of the human genome. But these systems are large and expensive to build and maintain, and there is a limit to the scale and complexity of automation that can be achieved with them. Microfluidic Large Scale Integration may enable much higher degrees of biological automation than conventional technologies while also making automation more ubiquitous. In this talk I will present a highly automated platform for culturing and studying mammalian cells, built around a microfluidic device that contains hundreds of micromechanical valves. One of the most important benefits of this platform's automation is the ability to stimulate cells with mixtures of reagents than can be changed over time in arbitrary ways and read the cells' responses optically, at single-cell resolution, with minimal human supervision. Automation not only reduces the amount of labor involved, but also prevents human errors associated with repetitive pipetting. It also achieves excellent fluid dispensing accuracy and performs semi-continuous (time-lapse) optical imaging of the cells during the complete duration of an experiment. Using appropriate optical reporters the response of single pathways, or networks of pathways to complex, time-changing chemical stimuli can be studied as a function of time and chemical composition. I will present results on the study of the motility and differentiation of human Mesenchymal Stem Cells, and will describe preliminary results on the culturing of human Colon Cancer Stem Cells and on the study of NFkB dynamics in mouse fibroblasts.
Micro-Electro-Mechanical-Systems (MEMS) technology enables us to design and fabricate miniature sensors and actuators which can be used to directly manipulate microscale subjects. Many recently developed transducers further extend the range of applications to nano scales. With these micro/nano modalities, we can interrogate and manipulate cells and bio-molecules with unprecedented capabilities.
Moving, stopping, mixing and separation of fluids and particle are the basic processes in medical diagnoses and drug developments. For those applications, macro complex molecules commonly found in the bio-fluid flow present many challenges which do not exist in simple fluid flows. In bio-medical applications, target collection, sample preparation and bio-marker detection involve flows from the macro to nano scales. Handling the multi-scale problem with different governing forces is not commonly encountered in traditional fluid dynamics. The surface effects play a major role in the fluid molecules and also on the surface functionalities. In this presentation, we will review the present capabilities and discuss the challenges.
Micro- and nanoscale technologies are emerging as powerful tools to control the interaction between cells and their surroundings for biological studies, tissue engineering, diagnostics and cell-based screening. In our lab we have developed various approaches at the interface between materials science, engineering and biology to control and study the cellular microenvironment with emphasis on controlling stem cell differentiation and generating 3D tissues. In this talk I will present our work in controlling the cell-microenvironment interactions in 2D and 3D using variety of microscale technologies. Specifically I will talk about the use of microfluidics for generating tissue-like structures with biomimetic microvasculature and complexity by using microengineered cell-laden hydrogels with controllable biochemical and architectural features. Also, I will describe our efforts to immobilize cells within microfluidic devices and their emerging biomedical applications.
The creation of a DNA double-strand-break constitutes the most dangerous type of DNA damage. Inefficient response to DNA damage may lead to hypersensitivity to cellular stressors, susceptibility to genomic defects and resistance to apoptosis, which can lead to cancer. Current research on DNA repair has enabled numerous breakthroughs in our understanding of the DNA repair mechanisms at the population level. However, similar understanding at the level of single cells has been lacking mainly because of two reasons: 1) population level measurements do not visualize the repair process and therefore the exact mechanism by which the donor and recipient sequences are brought together is not well understood. 2) without using a microfluidic device, the control of the microenvironment of the single cell is difficult.
In my lab we utilize a multidisciplinary approach, which combines the sciences of microbiology and physics with microfluidic-engineering, to address these specific aspects of the DNA repair at the single cell level. By tagging several locations on DNA, the dynamics of a single molecule of DNA and the exact timing of the repair process will be visualized for individual cells in a genetically identical cell population. These quantitative measurements will be used to formulate a predictive mathematical model of the repair process. In all of these experiments, individual cells will be followed over long periods of time and many cellular generations in a microfluidic device, in which a precise control of the microenvironment of the cells is possible.
Microscale technologies have provided cell biologists with two key opportunities: 1) To manipulate the cellular microenvironment with unprecedented control and 2) to parallelize environments to permit simultaneous interrogation. We have developed a device to monitor dynamic single cellular responses to numerous perturbations within a single chip. This simple medium-throughput platform, without flow valves or specialized equipment, provides for the near-simultaneous timelapse imaging of living cells at high numerical aperture in many isolated environments. The single-layer modular design incorporates a conserved 96-well, ~6 x 4 mm2 imaging area and a variable input/output channel design selected for the number of cell types and microenvironments under investigation. We tested the system by loading cells harboring fluorescent protein fusions to monitor mitotic spindle or chromosome dynamics. For each cell line we monitored cell division at high resolution in an array of anti-mitotic agents similar to those used in cancer chemotherapy. Timelapse protocols of over five days indicate that cells survive, divide, and respond to exogenous agents within the device. These studies demonstrate the ability to perturb and observe multiple elements of a signaling pathway in a single, simple experimental platform that yields dynamic information in time and space.
Many biological studies, drug screening methods, and cellular therapies require culture and manipulation of living cells outside of their natural environment in the body. The gap between the cellular microenvironment in vivo and in vitro, however, poses challenges for obtaining physiologically relevant responses from cells used in basic biological studies or drug screens and for drawing out the maximum functional potential from cells used therapeutically. One of the reasons for this gap is because the fluidic environment of mammalian cells in vivo is microscale and dynamic whereas typical in vitro cultures are macroscopic and static. This presentation will give an overview of efforts in our laboratory to develop programmable microfluidic systems that enable spatio-temporal control of both the chemical and fluid mechanical environment of cells. The technologies and methods close the physiology gap to provide biological information otherwise unobtainable and to enhance cellular performance in therapeutic applications. Specific biomedical topics that will be discussed include subcellular signalling in normal and cancer cells, in vitro fertilization on a chip, studies of the effect of physiological and pathological fluid mechanical stresses on airway epithelial cells. Time permitting, use of tunable nanofluidic systems for single molecule genetic analysis will also be discussed.
The environment afforded to cells in tissue is remarkably different from that typical of in vitro cell culture. In vivo, cells live in complex, biologically and chemically heterogeneous microenvironments with highly restricted extracellular spaces, and are subject to a large variety of simultaneous, time-dependent signals: soluble ones secreted by the cell itself (autocrine) and by other cells (paracrine), by physical contact to membrane-bound molecules on adjacent cells or extracellular matrix (juxtacrine), or by mechanical stresses and strains from neighboring cells or the substrate. This is in stark contrast to typical in vitro cultures, where monocultures of cells are grown either adhered to plastic or suspended in stirred media. As a result the cells and processes studied are often those that are self-selected for this dilute and artificial environment. Hence many important biological processes are difficult to study and control in vitro, such as stem cell differentiation, cell migration, angiogenesis, cancer metastasis, and viral infection. The observation of signaling in single, unattached cells is particularly challenging, since single-pass cytometry or imaging of suspended cells does not allow tracking of the time course of multiple signals in individual cells in their native microenvironment.
VIIBRE is developing and applying BioMEMS devices that address the limitations of conventional in vitro cell culture, and enable, for example, the study of paracrine signaling dynamics in T cells; cellular haptotaxis in response to gradients of surface-bound proteins; cell migration and differentiation in wound healing, tissue remodeling, angiogenesis and metastasis; the role of various genes in cardiac electromechanical activity; and cellular metabolic responses to toxins and drugs. Our ultimate goal is to use recorded signals as feedback to control the microenvironment and the cells within it.