VIIBRE conducts research at the intersection of the physical and biological sciences and medicine, with an emphasis on developing tools, techniques, models, and measurements to understand the dynamic behavior of cellular systems. We use microfabricated devices and state of the art instrumentation and models to explore post-genomic, post-proteomic, multiscale, non-linear, dynamic physiology, i.e., systems biology and bioengineering.

The Challenges of Systems Biology⁺

The scientific goal of Systems Biology is to find the underlying biological explanations for the multitudinous, multiscale observations provided by genomics, proteomics, and physiology. Thousands of genes can be active in a cell at any one time and a gene may encode multiple proteins. Cell-cell interactions are critical to system function, and there are 10⁹ - 10¹¹ interacting cells in some organs, such as the heart and brain. Cell signaling involves highly DYNAMIC biochemical cascades with positive and negative feedback, and there are multiple, overlapping regulatory mechanisms. Many of these interactions are nonlinear.

The complexity of biology, including the multiscale character and inhomogeneities, precludes simple statistical methods for analysis of these multiscale, time-dependent data. Instead the discovery process begins with postulating a biological model from a scientist with a deep understanding of the physical phenomena. From this model, parameters are estimated to fit the data. The degree to which the model explains the data is an indication of the reliability of the model. Several models may be generated and tested, and hence computer models are central to the field. What distinguishes the models of Systems Biology from those of many other disciplines is their multiscale richness in both space and time: these models may eventually have millions of dynamic variables with complex nonlinear interactions. Modeling a single mammalian cell may require >100,000 dynamic variables and equations, maybe > 1,000,000. It is conceivable that the ultimate models for Systems Biology might require a mole of differential equations (called a Leibnitz*) and computations that require a yottaFLOPs (floating point operations per second) computer. However, in general the data don't yet exist to drive these models, and there is a need to develop new classes of instruments to conduct the requisite experiments.

⁺J. P. Wikswo, A. Prokop, F. Baudenbacher, D. Cliffel, B. Csukas, and M. Velkovsky. Engineering challenges of BioNEMS: the integration of microfluidics, and micro- and nanodevices, models, and external control for systems biology. IEE Proc.Nanobiotech. 153 (4):81-101, 2006.

^*S. Huang and J. Wikswo. Dimensions of systems biology. Rev. Physiol., Biochem. Pharmacol. 2007, pp. 81-104.

VIIBRE is developing microfabricated instruments to ask such questions as how does nature pass information from one spatiotemporal scale to another, and how do we simulate this in multiscale mathematical models? How best can we ask a cell what it is thinking during chemotaxis, haptotaxis, and durotaxis? Or during development and differentiation, wound healing, angiogenesis, and cancer growth and metastasis? What can high-speed metabolic measurements tell us about the response of cells to drugs and toxins? Are cellular phenotypes high-dimensioned attractors in metabolic phase space? To what extent are the dynamics of single cells relevant in immune signaling, normal cell migration, cancer metastasis, and wound healing?

The Practical Problems

Our understanding of biological phenomena is often based upon experiments that measure the ensemble averages of populations of 10⁶ - 10⁷ cells, or measurements of a single dependent variable while all independent variables but one are, it is hoped, held constant. It is possible to record one rapid variable on one cell, such as the transmembrane potential or ion channel conductance, but it is not possible to record 100 rapid variables simultaneously. Biochemical measurements often represent averages taken over minutes to hours - a ten-liter bioreactor can measure 50 variables after a one-week reactor equilibration to steady state. It is costly to measure the expression of 12,600 genes, particularly if you need to read mRNA every 30 minutes for seven days from multiple cell cultures. The answering of some biological questions encounters more than one of these limitations. Finally, there is an explosion of qualitative genomic expression data without adequate quantification of expressed protein concentrations.

What Do We Need to Study Cellular Dynamics?

Biologists, chemists, and physicists have been highly successful in developing cell-scale biosensors. With time, the number that can be applied to a single cell will increase, as will their speed. Molecular biology provides us with a plethora of techniques for opening the intracellular feedback and control loops, including mutations, siRNA, and drugs. Synthetic chemists and molecular biologists need to provide new classes of intra- and extracellular actuators for controlled perturbations - reversible devices that can both increase and decrease the concentration of molecular signals within a cell. Finally, control engineers and computer scientists need to develop system algorithms and models that allow us to close and stabilize external feedback loops to provide the same functions of the intracellular loops that we chose to disable. Only then will we be able to understand the subtleties of cellular signaling.

What Microfluidics Provides

Historically, the most widely recognized capabilities of microfluidics are the ability of microscale devices to utilize small volumes of samples and reagents, support massive parallelization, and provide facile control of microfluidic flows at low cost and with rapid turnaround of devices under development. The popular efforts to create Laboratories on a Chip benefit from each of these. VIIBRE is focusing on additional capabilities provided by microfabrication: high cell densities, i.e., a low fluid-to-cell ratio approaching that of tissue, integrated sensors for quantitative measurement, rapid temporal response for diffusion-limited signals, parallel experiments on a single seeding of cultured or primary cells, and, most importantly, the possibility of closed-loop control of biology.

Bioreactors

VIIBRE is developing several types of three-dimensional bioreactors suitable for the study of development, angiogenesis, metastasis, endothelial permeability, and wound healing. Much of the effort is focusing on determining how the extracellular matrix (ECM) and the 3-D microenvironment affect cell mobility and differentiation.

Chemotaxis and Haptotaxis

Microfabricated devices are uniquely well suited to the study of cellular responses to patterned distributions of chemokines and haptokines in development, cancer, and wound healing. Ongoing experiments are addressing questions regarding how external spatiotemporal factors regulate cell migration and differentiation.

Immunology

VIIBRE's program in microfabrication for immunology research involves the development and application of microfluidic devices that can trap arrays of immune cells for sequential observation of cell signaling and cell-cell interactions. Questions being asked include how do dendritic cells stimulate remote T cells, how do calcium dynamics define phenotype, and how are cellular metabolic and signaling activity reflected in the lamellepodial ruffling of non-adherent cells?

Microphysiometers

The Cliffel and Wikswo laboratories are developing microliter-volume microphysiometers to measure metabolic dynamics of cultured cells in response to drugs and toxins, with the goal of distinguishing unknown or maliciously engineered toxins based upon their effect on metabolic dynamics. This approach may allow us to determine whether cellular phenotype is a high-dimensioned attractor in metabolic phase space.

Smaller is Faster

To obtain the requisite bandwidth and sensitivity, we must reduce the physical size of the measuring instrument to match more closely the spatial scale of not only the cell but also the organelles within it and the proteins that serve as the primary sensors, information carriers, and actuators. Small numbers of cells and sub-nanoliter cell-culture/measurement chambers ensure that diffusional mixing can occur in milliseconds and hence will provide faster detector response and require smaller volumes of injected chemicals, allowing faster control. In a larger, slower system, the detailed biochemical dynamics would be averaged out by both diffusion and differing cell states, and biochemical events might not even be detected before they reach saturation and irreversible changes occur. Our small, fast, single-cell system would make possible the high-frequency control of an individual cell.

J. P. Wikswo, A. Prokop, F. Baudenbacher, D. Cliffel, B. Csukas, and M. Velkovsky. Engineering challenges of BioNEMS: the integration of microfluidics, and micro- and nanodevices, models, and external control for systems biology. IEE Proc.Nanobiotech. 153 (4):81-101, 2006.

Nanophysiometers

The Baudenbacher laboratory is developing nanophysiometers whose instrumented nanoliter volumes allow measurement of the signaling and mechanical behavior of single cardiomyocytes contained in cell-sized chambers. Optical and electrochemical readout provides information regarding calcium concentrations, transmembrane potential, mechanical activity, and metabolic rates. These measurements are being used to determine how specific mutations in calcium handling affect excitation and contraction.

I.A. Ges, F.J. Baudenbacher. Microfluidic device to confine single cardiac myocytes in sub-nanoliter volumes for extracellular pH measurements. J. Exp. Nanosci., 3(1) 63-75, 2008.