Shuichi Takayama - US grants

OIA-0116331
PI: Solomon
Abstract

The PI and six colleagues in the Departments of Chemical engineering, Materials Science and Engineering, Biomedical Engineering, and Electrical and Computer Engineering at the University of Michigan are requesting funds to purchase a confocal laser scanning microscope. This will enhance their research and research training in nanoscale engineering of complex fluids and biomaterials. Specifics proposed applications of the instrument are: to quantify defect dynamics during annealing of colloidal crystals, to detect self-assembled proteins, to observe microfluidic flows on cellular development, and to facilitate the efficient design of microfabricated devices.

The objective of this proposed research is to develop micro- and nanotechnologies that will enable precise engineering of cellular environments and allow correlation of subcellular signaling with cellular function. Genomics has greatly advanced our knowledge concerning how biological systems are programmed. This "software", however, is embedded in the "hardware" of a cell, which interacts with its environment to compute its specific outputs such as growth, differentiation, or death. The micro- and nanotechnology developed in this project will enable control over configuration of the cellular hardware such as receptor clustering and cell shape. The technology will then be used to study how different hardware configurations affect subcellular signaling and cell function. Specifically, multiple laminar flow technology will be used to stimulate subcellular microdomains with growth factors and soft lithographic three-dimensional protein nanopatterning technology will be developed to perform nanoscale extracellular matrix engineering. The two technologies will provide, with subcellular resolution, control of cross-talk between growth factor signals and adhesive signals. This type of knowledge is specifically relevant to understanding physiological processes such as embryonic development and wound healing as well as pathological states such as fibrosis and cancer. The research will be tightly coupled with the education and training of a work force that can translate these scientific and engineering discoveries into tangible social and economic improvements. A major component of the educational program is a Biological Micro- and Nanotechnology course that will seamlessly integrate micro- and nanotechnology with cell biology and provide both theoretical training and hands-on laboratory experiences to students.

DESCRIPTION (provided by applicant): Two of the most important problems in cell-based treatments of muscular and neurological diseases and injuries are: i) the regulation of cell proliferation and differentiation, and ii) the control of cell migration. For example, in human myoblast transplant trials for treatment of muscular dystrophies, major problems were the lack of proliferation, migration, and fusion of transplanted cells with existing muscle tissue. In repair of nerve damage, suppressing astrocyte proliferation and promoting neuroblast proliferation, along with guidance of neurite extensions are key issues. Although these cellular behaviors are critically depend on nanoscale adhesive cues in the extracellular matrix (ECM), there is currently a lack of understanding of what these crucial nanostructures are and how dynamic changes in those structures determining cell function. This lack of understanding is due, at least in part, to the lack of efficient, versatile, and convenient methods to engineer the ECM at the nanoscale across biologically relevant areas of micrometers, millimeters, and larger. The specific aims of this proposal are: i) understand and optimize a technology, called nanocrack patterning, to generate nanoengineered substrates with ECM molecule nanolines of defined widths, lengths, spacings, and orientations, ii) test the hypothesis that stretch-induced, nanoscale substrate reconfiguration can contribute to proliferation and lineage determination of myoblasts and neuroblasts, and iii) perform feasibility studies for discovery-driven research on cellular pathfinding where a microarray of criss-crossing nanopatterns of ECM molecules will be fabricated to rapidly profile cellular spreading/migrating preferences. The initial proof-of-concept studies will use C2C12 myoblasts and N27 neuronal precursor cells. Mesenchymal stems cells and primary myoblasts will be studied in the future. Although the biological problem to be addressed in this proposal is limited to myocyte and neuron behavior, the nanobiomaterials developed will be useful for addressing a much broader range of biological questions. The nanocrack patterning technique that will be developed uses nanoscale fracture mechanics and has the advantages of: (i) rapid nanopatterning over large areas (up to square centimeters and larger), (ii) nanopatterning over 3D substrates and inside microfluidic channels, (iii) generation of nanopattems consisting of multiple types of molecules on the same substrate, and (iv) stretch-induced in situ adjustment of the widths of ECM molecule nanolines generated.

The objective of this research is to develop a novel and versatile
nano-manufacturing technology for fabricating defined numbers of nanowire rings on polymer microsphere surfaces. The approach combines compression-induced cracking of polymer-supported thin films, surface chemistry manipulation, and electroless deposition of metals. Detailed relationships between microsphere size, surface film property, degree of compression, electroless deposition procedure, and the type of nanofeatures generated will be determined to enable a cost-effective and well-controlled nanomanufacturing scheme.

This proposal is motivated by the general need for novel nano-manufacturing technologies and concepts. The ability to manufacture micro- and nanospheres with defined sizes, compositions, surface charges, and surface patterns has been critical for advances in many fields such as in the generation of opals, preparation of photonic band gap materials, development of novel electronic devices, manipulation and analysis of biological cells, and production of inks, paints, cosmetics, and other rheological fluids. Most microspheres prepared to date, however, have relatively simple surface structures such as uniform coatings or the "capping" of one half of a sphere. The more elaborate nanopatterned microspheres that will be manufactured through this project will open new possibilities in advanced materials, electronics, and biology. Another important feature of the program is the integration of research and education through the training of students in a technologically significant area. This project is visually striking, experimentally straightforward, and theoretically stimulating. It will provide valuable hands-on laboratory experiences in nanomanufacturing research to graduate and undergraduate students and inspire the next generation of nanotechnology scientists and engineers.

The project will lead to the acquisition of a X-ray photoelectron spectroscopy (XPS) instrument, which represents a major advance in commercially available instrumentation. XPS has become a critical technique in the characterization of the chemical composition, oxidation and bonding states in various materials, including polymers, catalysts, metals, and semiconductors. The XPS instrument will provide real time chemical state and elemental imaging capabilities using the full range of pass energies and multi-point analysis from either real time or scanned images without the need for sample translation. The instrumentation further comes with the ability to obtain data over a large field of view, while maintaining photoelectron and Auger peak positions. The initial research foci of the instrument will include fundamental studies of: designer surfaces for biomedical applications; advanced micro-probes for neural prostheses; chemo-mechanical nanopatterning of polymer substrates for cell culture; surface-modified nano-fibrous scaffolds for tissue engineering applications; surface-modified microfabricated chemical analysis devices; controlled anchoring of DNA molecules at chemically tailored surfaces; coaxial semiconductor nanowires; smart surfaces assembled from molecular switches; directed self assembly of nanostructures by focused ion beam patterning; fuel processors for PEM fuel cells; lean NOx traps for automotive emission control; semiconductor alloy surfaces; surfaces and interfaces for electronic devices; and GaInNAs for high efficiency solar cells. The instrument will also have broad impact upon the research and educational infrastructure at U of M. Most faculty involved in the project have Undergraduate Research Opportunities Program (UROP) and Research Experience for Undergraduates (REU) students in their research groups actively working on characterization of materials. The instrument will be used in summer research projects for minority high school students and young women. For example, a number of students from under-represented groups participating in the NASA Summer High School Apprenticeship Program (SHARP) have worked on projects with instruments at the University of Michigan's Electron Microbeam Analysis Laboratory (EMAL), which will be the home of the new XPS instrument. As soon as the proposed instrument is installed, it will be included in this program.

The project will lead to the acquisition of a X-ray photoelectron spectroscopy (XPS) instrument with advanced capabilities. XPS has become a critical technique in the surface characterization of various materials, including polymers, catalysts, metals, and semiconductors. The XPS instrument will enable the real time chemical state and elemental imaging of surfaces. In essence, it will allow us to determine and image the chemical composition of the outermost layers of a material. Initially, the instrumentation will support research in the areas of biomaterials, surface science, nanoscience, catalysis, and fuel cells. The instrument will also have broad impact upon the research and educational infrastructure at U of M. The new XPS instrument will be used in the undergraduate and graduate curriculum, as well as in summer research projects for minority high school students and young women.

DESCRIPTION (provided by applicant): Two of the most important problems in cell-based treatments of muscular and neurological diseases and injuries are: i) the regulation of cell proliferation and differentiation, and ii) the control of cell migration. For example, in human myoblast transplant trials for treatment of muscular dystrophies, major problems were the lack of proliferation, migration, and fusion of transplanted cells with existing muscle tissue. In repair of nerve damage, suppressing astrocyte proliferation and promoting neuroblast proliferation, along with guidance of neurite extensions are key issues. Although these cellular behaviors are critically depend on nanoscale adhesive cues in the extracellular matrix (ECM), there is currently a lack of understanding of what these crucial nanostructures are and how dynamic changes in those structures determining cell function. This lack of understanding is due, at least in part, to the lack of efficient, versatile, and convenient methods to engineer the ECM at the nanoscale across biologically relevant areas of micrometers, millimeters, and larger. The specific aims of this proposal are: i) understand and optimize a technology, called nanocrack patterning, to generate nanoengineered substrates with ECM molecule nanolines of defined widths, lengths, spacings, and orientations, ii) test the hypothesis that stretch-induced, nanoscale substrate reconfiguration can contribute to proliferation and lineage determination of myoblasts and neuroblasts, and iii) perform feasibility studies for discovery-driven research on cellular pathfinding where a microarray of criss-crossing nanopatterns of ECM molecules will be fabricated to rapidly profile cellular spreading/migrating preferences. The initial proof-of-concept studies will use C2C12 myoblasts and N27 neuronal precursor cells. Mesenchymal stems cells and primary myoblasts will be studied in the future. Although the biological problem to be addressed in this proposal is limited to myocyte and neuron behavior, the nanobiomaterials developed will be useful for addressing a much broader range of biological questions. The nanocrack patterning technique that will be developed uses nanoscale fracture mechanics and has the advantages of: (i) rapid nanopatterning over large areas (up to square centimeters and larger), (ii) nanopatterning over 3D substrates and inside microfluidic channels, (iii) generation of nanopattems consisting of multiple types of molecules on the same substrate, and (iv) stretch-induced in situ adjustment of the widths of ECM molecule nanolines generated.

DESCRIPTION (provided by applicant): This proposal is a design-directed project to develop a novel microfluidic bioreactor that will culture multiple single embryos under simulated physiological conditions while simultaneously performing real-time monitoring of biochemical markers of embryo quality. Reducing the incidence of high-order multiple pregnancies while maintaining the overall IVF success Irate is a holy grail of human IVF and would be greatly assisted by the ability to produce and identify the highest quality embryos. This goal has been elusive to date due, at least in part, to the lack of instrumentation to perform convenient and reliable single embryo manipulation and analysis. Because of the small quantities of biomarkers produced by single embryos, reliable quantification hinges on the ability to culture the embryos in very small volumes of fluid and to directly analyze the culture media for soluble biomarkers secreted by embryos with minimal dilution. The proposed microbioreactor with microfluidic pumps, valves, and sensors will provide an inherently biomimetic milieu for embryo culture as well as enable direct biomarker analysis on chip. The microbioreactor will utilize a computer-controlled integrated microfluidics platform that controls fluid flow inside elastomeric capillaries by deformation of the microchannels with mechanical microactuators. The biomarkers of mouse embryo health that will be monitored in this exploratory grant are embryo metabolites, autocrine factors, and embryo surface biomarker of embryo health. The embryo microbioreactor will be used to specifically test the hypothesis that analysis of select biochemical markers will enable prediction of which 8 cell embryo will proceed to produce healthy blastocysts. This test will mainly serve the purpose of device validation and concept feasibility but it also has clinical relevance. A current trend is to grow embryos to the blastocyst stage and transfer the two morphologically "best" blastocyst. Recently, however, two reports using mouse embryos have independently demonstrated that extended culture to the blastocyst stage causes aberrant genetic imprinting and altered postnatal development, growth, physiology and behavior. Case reports and studies also suggest a general association between in vitro culture to the blastocyst stage and MZ twinning. This proposal will address these issues by developing novel non-invasive means of selecting embryos with the greatest implantation potential, with the least amount of manipulation and culture.

0601237
Kurabayashi

The objective of this research is to develop a novel nano photonic tunable device for high-speed single-detector spectral measurement. The proposed device is integrated in a microfluidic system to achieve high-throughput multi-analyte detection in flow-through microsphere-based fluoroimmunoassay with simple optics and with less computational requirements. The approach is based on the new polymer-silicon hybrid microelectromechanical systems technology. It allows imprinting of nanoscale features on the surface of the three-dimensional polymer microstructure. The spectrum acquisition speed of the system is expected to exceed 100 nm/ms and to allow real-time spectroscopy in a microfluidic system.

Intellectual merit:The innovative nano photonic technology developed in this research will guide future advancements of wavelength-discriminating detection for the identification and quantification of multiple chemical and biological species. Integrated with microfluidic cell culture and immunoassay systems, the device leads to development of a non-existing scientific instrument that permits in-situ monitoring of time variations of cellular parameters in a microfluidic channel.

Broader Impact:The developed microfluidic fluoroimmunoassay system may find new commercial markets because of its cost-effectiveness and utility in life sciences research and development. The proposed project will promote an excellent opportunity to train a new generation of engineers and scientists who will cross the boundaries of traditional research fields and create new avenues of research.

In diseases that involve mucus secretion and movement in the small airways, such as chronic bronchitis, cystic fibrosis or asthma, liquid plugs form occluding bridges that obstruct the airway and disrupt gas exchange. In response to cough, these bridges move and the airway is reopened, with the transmission of mechanical forces to airway epithelial cells. Similarly, in the setting that involve both the airway and alveolar space, such as pneumonia or congestive heart failure, or mechanical ventilation with low tidal volumes, there is cyclic closure and reopening of smaller airways, which may be recognized as crackle sounds heard easily with a stethoscope. The cellular-level effect of the explosive transient pressure waves created by these reopening events, however, has not previously been investigated despite the likelihood that the associated plug rupture produces large stresses and is a major cause of lung injury. This proposal will investigate, experimentally and theoretically, the detrimental effect of fluid mechanical stresses on airway epithelial cells during airway reopening using a micro-engineered airway. The specific hypothesis is that the movement and rupture of liquid plugs in the small airway system during airway reopening will generate large fluid mechanical stresses and damage airway epithelial cells, and that even normally sub-lethal amounts of fluid mechanical stress will become lethal in the presence of other insults such as bacteria or hyperoxia-mediated inflammation, expanding the region and severity of injury. The specific aims of this proposal are: 1. Design and fabrication of a biomimetic microfluidic system to perform in vitro culture of airway epithelial cells under physiological air-liquid interface conditions. 2. Generation of liquid plugs with physiological propagation velocities and rupture frequencies within the engineered microfluidic small airways, and combined computational and experimental assessment of the resulting fluid mechanical stresses and their effect on cell injury. 3. Investigate synergistic cellular damage caused by combination of liquid plug propagation/rupture- mediated fluid mechanical stresses and bacterial infection or hyperoxia-mediated inflammation. Also, evaluate the effect of surfactant as a countermeasure to reduce cellular injuries.

Intellectual Merit: New possibilities would open in many fields if there were straightforward ways to manufacture microspheres with complex surface features. The challenge in accomplishing this goal is the lack of methods for manufacturing micro/nano features on three-dimensional micro-objects in a controlled, yet rapid, robust, and parallel process. This proposal takes inspiration from nature, where fracture of thin films is utilized to produce patterns such as features on cantaloupe skin that form as the surface is strained in response to growth of its interior, to develop a novel fracture-based manufacturing technology. Specifically, a combination of strain-induced fracturing of polymer-supported thin films, surface chemistry manipulations, microfluidics, colloidal assembly, and electroless deposition of metals is used to form micro/nano patterned microspheres. Manufacture of the desired features is guided by computational analysis of the mechanics of thin film fracture on microspheres.

Broader Impact: Efficient manufacture of micro/nano-patterned microspheres will impact many fields such as preparation of photonic materials, study of rheology, development of novel electronic devices, and in the study and manipulation of biological cells. The technology will also impact micro/nano-manufacturing education because the visually striking, experimentally straightforward, and theoretically stimulating nature of the process makes it an inherently useful system to provide introductory hands-on laboratory experiences to students of all levels. This project will particularly focus on providing research opportunities for minority female engineering students and undergraduate students. The theories and experiments developed will also be incorporated into graduate courses in biomedical, mechanical, and materials engineering.

DESCRIPTION (provided by applicant): Active Nanofluidics for Analysis of Chromatin and Genomic DNA Structures A. Specific Aims This project will develop nanotechnology to fill an unmet need in genome-wide analysis of DNA and chromatin structures. This capability will greatly enhance our understanding of how genetics and epigenetics translate the DNA-encoded information of the nucleus into cellular functions and phenotypes. The approach will use parallel nanochannels whose cross-sectional profiles can be reversibly regulated to be narrow (nanometers) or wide (micrometers). The tunable channels will be widened to enable efficient loading of the relatively large chromatin or genomic DNA molecules in their folded states. Then the channels will be gradually narrowed, under the precise control of the operator. Simultaneous application of an electric field within the nanochannel will allow controlled linearization of the chromatin or DNA inside the channels. The stretched out chromatin or DNA will be analyzed optically to map and observe genomic structures, such as replication forks, and epigenetic structures, as well as the distribution of nucleosomes, and organized chromatin regions. These capabilities will be used for comparative genomics and epigenomics of healthy and diseased/stressed cells. Aim 1. Construction of Tunable Nanochannel Arrays: Material properties and processing methods will be tested and optimized to construct parallel arrays of nanochannels. The nanochannels provide reproducible control of channel cross-sectional profile, microfluidic flow, and surface chemistry. Aim 2. DNA Linearization and Stabilization: Mechanisms and software will be developed to coordinate and control channel cross-sectional shape adjustments with electrical field application. Both direct current and pulsed-field current regimes will be tested. The nanochannel profile and electric fields will be optimized to allow linearization and stable molecular control using lambda bacteriophage DNA (48 kb) as an initial test. Aim 3. Image-based Analysis of Linearized DNA: Computerized image capture and analysis programs will be developed. As an initial biological test, we will examine replication forks on linearized genomic DNA samples from cultured mammalian cells exposed or not exposed to pharmacologic replication stress. Aim 4. Analysis of Histone-Associated DNA: Procedures for the gentle dissociation of live cells within the devices will be developed. Dynamic changes in chromatin structures, including nucleosomes, will be observed within the channels using controlled currents, temperatures, and channel morphologies. Public Health Relevance Statement: This project will develop broadly useful nanotechnology to fill an important unmet need in genome-wide analysis of DNA and chromatin structures. The specific initial biological application of the nanotechnology in this proposal will be to analyze genomic and epigenomic structures related to DNA replication. Despite intense efforts, the orderly activation of replication sites in genomes of higher organisms remains largely unexplained. This is due, at least in part, to the complexity of the process which orchestrates activation of an estimated 10,000 to million replication sites, where the sites are determined not only by sequence but by epigenetic factors as well. This type of analysis is important clinically because faulty replication is involved in a variety of diseases such as Werner syndrome, Seckel syndrome, Fanconi anemia and cancer.

"This award is funded under the American Recovery and Reinvestment Act of 2009
(Public Law 111-5)."

In this project new technologies based on the emerging sciences of microfluidics and microelectro-poration have been developed in a partnership between neuroscientists and engineers to address fundamental questions of nervous system and sensory system development. Using a model organism, the tiny zebrafish, newly recognized factors "immune system cytokines" are being studied for their roles in the earliest stages of development of the nervous system and sensory systems (ear and eye). A zebrafish microfluidic bioreactor was developed to expose specific circumscribed parts of the growing embryo's nervous system or sensory systems to streams of morphogens (fundamental growth promoting molecules), in controlled concentrations. In the bioreactor, small areas of the developing nervous system, the ear or the eye can be bathed in such morphogens, including MIF (macrophage migration inhibitory factor), which is an "inflammatory" cytokine. Cytokines are vital for immune system development as well as triggers of damaging inflammatory reactions after the immune system forms. The primary investigator's laboratory has made the surprising discovery that immune system cytokines, and especially MIF, are also vital for the very earliest development of nervous system neurons (and pathfinding of neurites) and sensory cells in the eye and ear. MIF could also be involved in repair processes at concentrations orders of magnitude below those that cause inflammation. MIF and other cytokines therefore act as "neurotrophins" or directional nerve growth factors in these systems, a surprising outcome to scientists in a field that had long thought only classical (already identified) neurotrophins could play such a role. The use of microfluidic technologies as well as electroporation of molecules directly into the developing zebrafish inner ear were key to this new line of research. This project investigates the mechanism of action by which MIF exposure to circumscribed parts of the nervous system and developing sensory systems promotes their development and how blocking it alters development. Fundamental links between the developing nervous and sensory systems and the immune system can be studied in this model. Zebrafish auditory system development is, in many ways, the same as that in the human ear. The same molecules that are active in zebrafish are active in humans, but zebrafish are easier to study. Development takes place outside the mother and is extremely rapid (a scale of hours to a few days rather than weeks or months). This project will provide training opportunities for interdisciplinary teams of neuroscientists and engineers at all levels from undergraduate and graduate students (who developed the zebrafish bioreactor) to the postdoctoral and faculty level, who supervised the work.

Trace elements (those present at the parts-per-million level) play a critical role in virtually all biological processes; they are essential to life but can also be toxic if present at the wrong concentration. Consequently, the trace element levels within cells tend to be tightly regulated, with a complex set of machinery to control the "inorganic physiology" of cells, that is, the regulation, uptake, storage, and efflux of trace elements. Although there have been many studies of trace elements in biology, virtually all have relied on bulk analyses (e.g., how much metal is present on average in an ensemble of cells; and how does this change as a function of chemical and/or biological treatment?). The new instrument that will be developed will permit the direct determination of the concentrations of trace elements in intact individual cells. The instrument will allow, for the first time, facile determination of the inorganic composition of individual cells, thereby providing direct insight into the distribution of compositions within the population, and permitting studies aimed at correlating other cellular properties with chemical composition. By permitting determination of not only average concentrations but also the cell-to-cell variations in individual concentrations, and by allowing these data to be correlated with conventional measures of cell status, this will provide a transformative capability, opening the possibility of dissecting the cellular roles of both essential and toxic trace elements with an unprecedented level

DESCRIPTION (provided by applicant): Oscillatory signals regulate a wide variety of integral physiological and cellular processes, from G- protein coupled receptor (GPCR) signaling to circadian rhythms. Although an actively studied area, even the most well-known and commonly studied pathways can have controversy and lack of clarity on circuit architecture. This is because pathway perturbation studies using conventional molecular or genetic tools only provide limited information resulting in multiple plausible mechanisms. This proposal will develop tools and methods based on non-linear frequency and waveform response analysis to dissect such oscillatory pathways in ways that are not possible with conventional molecular or genetic perturbations alone. Specifically, we will use microfluidics to apply a periodic chemical input to cells and observed phase-locked cellular responses using real-time fluorescent readouts of intracellular signaling. The observed frequency response characteristics will be evaluated using computer models of the signaling pathway. Signaling circuit architecture as well as modes of action and mechanisms of inhibitors, agonists, and modulators will be dissected. Although the method should be applicable to any oscillatory signaling pathway, we will first focus on two GPCR signaling pathways (M3 muscarinic acetylcholine receptor and type 5 metabotropic glutamate receptor) that have very different proposed mechanisms of oscillation and that are physiologically and pharmacologically important (diabetes and schizophrenia). Aim 1. Analyze Phase Locking Response of Cells Under Base Conditions: Perform microfluidic pulsed stimulation of live cells with receptor ligands. Obtain high time resolution real-time imaging of intracellular signals using genetically encoded fluorescent indicators of calcium and IP3. Aim 2. Construct Mathematical Models of Signaling Circuitry: First construct plausible mathematical models based on published data. Then refine the circuit architecture and parameters to match observations in Aim 1, guided by results of uncertainty and sensitivity analyses. Aim 3. Delineate Mechanisms of Action of Modulators Through Phase Locking Analysis: Study how phase locking responses of cells change in the presence of inhibitors, agonists, and modulators. Use the experimental observations with mathematical models to delineate mechanisms of action. Aim 4. Disseminate self-regulating chips that make microfluidic phase-locking studies accessible to anyone.

DESCRIPTION (provided by applicant): The field of microfluidics is uniquely poised to make a broad impact in biomedical sciences through the miniaturization and mass parallelization of biological experimentation. For example, future advances in microfluidics could revolutionize disease diagnosis, drug discovery, and pathogen detection. For these impacts to be realized we need individuals conversant in engineering, chemistry, biology, and medical sciences. Currently however, such cross-trained people are in short supply. To address this shortage, we propose a five-year program to train the next generation of biomedical microfluidics experts. This program combines the expertise of faculty from engineering, chemistry, physics, and the medical school plus the world-class Solid State Electronics Laboratory (SSEL) for state-of-the-art micro- and nanofabrication. The premise of the program is that students should have significant training in both the methods of microfluidics and in the biomedical applications of this technology. Such training enhances the communication between disciplines, identification of solvable biomedical and clinical problems, and selection of appropriate tools to solve these problems. The training of the students in this program will involve a combination of course work, seminar series, hands-on workshop, annual symposium, journal club, and cross-disciplinary lab rotation or collaboration. All of these activities are beyond the requirements of the trainee's home department. The core course for this program Microfluidic Science and Engineering has been taught for several years with success. The seminar and workshop were originally developed by graduate students independent of the training program showing the enthusiasm of the graduate students for this topic. We feel that this combination of practical teaching, cross- disciplinary exposure, and intensive microfluidic study will produce students well positioned to answer the growing need for highly educated and trained microfluidics experts.

DESCRIPTION (provided by applicant): As determined by the cancer research community and NCI, inefficiencies and inaccuracies of existing methods for drug testing are critical obstacles preventing development and clinical translation of new drugs to dramatically improve cancer therapy (Provocative Question 17). To overcome these obstacles, we will develop a new 3D cell culture model of disseminated breast cancer cells in the bone marrow microenvironment. Our focus on the bone marrow microenvironment is driven by the high frequency of disseminated cancer cells even in patients with seemingly localized primary tumors, limited activity of drugs against metastases relative to primary tumors, and > 90% of cancer mortality caused by metastatic disease. Our model will incorporate multiple types of human bone marrow stromal cells, including mesenchymal stem cells, endothelium, and osteoblasts. One or more of these cell types form protective niches that may confer drug resistance to metastatic breast cancer cells through intercellular signaling pathways. We will optimize culture conditions to reproduce hypoxia normally present in human bone marrow, using an innovative imaging technique to quantify oxygenation within 3D spheroids. We will test activity of standard chemotherapeutic drugs in breast cancer and promising molecularly-targeted compounds against human cell lines representative of intrinsic molecular subtypes of breast cancer integrated into 3D bone marrow spheroids. We also will test compounds against primary human breast cancer cells passaged only as mouse xenografts and correlate responses in 3D culture with patient outcomes. For both cell lines and primary tumor specimens, we will use advanced optical imaging methods to measure drug targeting, potential mechanisms of drug resistance, and heterogeneous responses of breast cancer cells to treatment. We will answer Provocative Question 17 by accomplishing the following specific aims: 1) develop an advanced 3D culture system to analyze treatment of disseminated human breast cancer cells in bone marrow; 2) quantify effects of compounds on breast cancer cell lines representative of molecular subclasses of human breast cancer and tumor-initiating cells; 3) determine activities of compounds against primary patient tumor samples. Collectively, this research will establish a facile, inexpensive, reproducible model to test potential cancer drugs and accurately match compounds with patient subpopulations highly likely to respond to treatment. The strategy will accelerate clinical translation of new, more effective cancer drugs while reducing costs of drug development.

No one biomarker gives all the information needed to diagnose or treat a patient reliably. Thus a major goal of clinical diagnostics is to efficiently analyze a panel of diagnostic biomarkers. Despite this well-known need, cost-effective, sensitive, and quantitative multi-protein (multiplexed) diagnostic immunoassays hardly exist. While hundreds of research articles have described multiplexed immunoassays, they cannot be validated for clinical applications due to increased background and or cross-talk that occur when multiple detection antibodies are mixed together into one solution. The team utilizes a proprietary microfluidic technology to provide an innovative solution to this problem by creating multiplexed immunoassay microarrays where no mixing of detection antibodies occurs. The project will advance microfluidic science and technology as well as provide new tools for use in basic science studies that utilize proteomics. In addition to science and technology advancement, this project will advance concepts and methods in translating laboratory research results into commercial products and services. The project will increase understanding of customer needs in the area of multiplexed biomarker assays and proteomics. The project will also propose the most efficient and commercially-viable methods to bring the technology to users.

The team envisions the project to benefit society through development of robust, user-friendly, multiplexed immunoassays that will enhance clinical diagnostics as well as basic biology studies through providing robust and user-friendly protein assays. As the project goal is technology commercialization, success of this project will directly provide a large benefit to society through economic as well as technological means. All three personnel involved (entrepreneurial lead, PI, and mentor) will also learn new ideas and methodology as well as actual hands on experience that is part of this project. These experiences will surely impact not only this particular technology commercialization effort but other commercialization projects in the future. Additionally, the technology transfer lessons learned will also be incorporated into the classroom through courses taught by the PI as well as through one-time lectures.

? DESCRIPTION OF THE OVERALL U19 APPLICATION (provided by applicant): Enteric infectious diseases continue to represent a major cause of morbidity and mortality worldwide. In addition to gastrointestinal pathogens that have been known for centuries, there continues to be an emergence of enteric disease agents as a product of manmade and natural changes in the environment. The appearance of antibiotic resistance and the lateral transfer of virulence factors have also impacted our ability to deal with well known pathogens. To counter these infectious disease threats, novel methods of studying these pathogens are needed. We have assembled an interdisciplinary team to address the need for novel alternative model systems for enteric diseases research. With expertise in viral and bacterial pathogenesis, immunology, tissue engineering, stem cell biology, infectious diseases and bioengineering, this team will utilize human intestinal or- ganoids (HIOs) generated from human pluripotent stem cells (hPSCs) as a model gut epithelium. Three integrated projects will address the common specific aim of utilizing HIOs as a system to investigate the interaction between the intestinal epithelium, immune cells, microbiota and enteric pathogens. The first project will focus on the interaction of the HIO epithelium with normal members of the gut microbiota and specific enteric pathogens. Changes in the function of both the microbes and the HIO epithelium will be investigated. The second project will focus on interactions between the model epithelium found in the organoids and cellular elements of the immune system. Human immune cells will be allowed to interact with HIOs in both the presence and absence of microbes. The final project will employ a bioengineering approach to create a system that both facilitates the use of HIOs as a platform for scientific discovery and serves as a flexible platform for drug discovery and testing. These three projects will form an integrated cooperative research center that will involve investigators with a wide range of complementary expertise. Successful completion of the three projects will generate a powerful new system to study the biology of enteric disease agents and a platform for the development of novel therapeutics for their control.

PROJECT SUMMARY/ABSTRACT This project is a combined design-driven and hypothesis-driven project to bioengineer microscale models of enteric disease. Starting with the Spence lab's in vitro intestine system that accurately reflects both the complex cellular makeup and the appropriate layered organization of the human intestine, this project will provide these 3-Dimensional (3D) Human Intestinal Organoids (HIOs) with physiologicaly soft but confining mechanical cues as well as microscale fluid perfusion capabilities that will mimic luminal flow, to further induce physiological structures such as crypts and villi. Both of these properties (constraint, flow) have a significant impact on intestine development, differentiation and function. Our hypothesis is that by providing a mechanically confined culture condition and fluid perfusion, as opposed to the free expanding culture with a static, enclosed lumen as is currently used for HIO formation, that the epithelial layer will self-organize additional levels of physiological complexity, including as crypts and villi, along with associated spatial organization of intestinal stem cells (ISCs) in crypts and differentiated cells on the villi. Incorporation of microscale fluid perfusion capabilities in HIO culture devices will also allow precise regulation of intraluminal flow of nutrients, and long-term colonization with bacteria, and pathogens. Technologically, this project will be innovative in developing a method (?supersoft lithography?) for reproducibly creating supersoft PDMS structures with physiological moduli of 1-100 kPa. To enable closed-loop control for maintenance of tissue homeostasis as well as to provide readouts of tissue function, this project will also integrate miniature oxygen sensors and electrodes for trans-epithelial electrical resistance (TEER) measurements. Additionally, sampling capabilities from the interior and exterior of the HIO will be incorporated to enable off-line measures of fluid and drug absorption/secretion. HIO microscale culture devices will also facilitate measurement of cytokine production in integrated HIO-immune co-cultures. Finally, we will demonstrate modularity and utility of the bioengineered and instrumented HIO system by integrating NAMSED Projects 1, 2 and 3. Specifically, instrumented-HIOs with luminal flow will be generated, co-cultured with immune cells and colonized by probiotic microbes (Lactobacillus GG, LGG) and/or pathogens (S.typhimurium). In each co-culture, (probiotic/HIO/immune vs. probiotic/pathogen/HIO/immune), we will test the ability of the system to generate real-time physiological data by measuring epithelial barrier function (TEER, FITC-Dextran), oxygen concentration, cytokine production, and finally by examining epithelial invasion by S.typhimurium. We will also test the utility of this system to screen drugs/compounds by generating instrumented LGG/S.typhimurium/HIO/immune co-cultures and adding Cefoperazone, an antibiotic that will selectively target the pathogen S.typhimurium, but not the probiotic LGG. The ability of Cefoperazone to kill S.typhimurium will be examined by culturing the luminal effluent to determine S.typhimurium colony forming units before, during and after antibiotic treatment. Finally, when live cultures are terminated, we will harvest the system and examine cellular and molecular difference between the different groups using immunofluorescence or qRT-PCR on purified immune cells and epithelium.

? DESCRIPTION (provided by applicant): Directional migration of malignant cells toward a gradient of one or more signaling molecules underlies fundamental steps in metastasis, including local invasion of cancer cells, vascular intravasation, and extravasation of cancer cells at secondary sites. Understanding formation of gradients in complex environments with multiple cells and extracellular matrix molecules remains a central challenge in cell migration not only in cancer but also in normal physiology and other diseases. The challenge of understanding gradient formation and cell migration becomes even more difficult in the disordered cellular and extracellular matrix architecture of a tumor. We will meet this challenge through an integrated systems bioengineering approach combining microscale technologies for cell migration, in vitro and in vivo cellular and molecular imaging, and sophisticated multi-scale computational models. This approach will enable us to investigate gradient formation and cell migration in increasingly complex environments, ranging from a 2D system with defined positions of three different cell types to the disorganized structure of a tumor. Using computational modeling to identify key parameters controlling gradient formation and cell migration, we also will experimentally test and validate interventions to block cell migration, which will provide new targets for anti-metastatic therapies. Our research will focus on gradient formation and cell migration controlled by chemokine CXCL12, a signaling molecule that drives metastasis in more than 20 human cancers. CXCL12 exists as six alternatively-spliced isoforms, four of which are expressed in human breast cancers. We recently have shown CXCL12-isoform specific differences in cell migration, resistance to targeted inhibitors, and correlations with disease recurrence and survival in breast cancer. We propose that CXCL12 molecules bound to the extracellular environment drive cell migration, a process referred to as haptotaxis, and differences in binding to the extracellular matrix underlie isoform-specific differences in gradient formation and cell migration. To investigate CXCL12 isoforms in cell migration, we will complete the following specific aims: 1) derive basic cell migration response parameters under simple, defined gradients; 2) using tissue-like geometries, test effects of extracellular matrix composition on migration potency of CXCL12 isoforms; and 3) Quantify in vivo migration in tumor environments with different CXCL12 isoforms. Collectively, this research will advance knowledge of gradient formation in cell migration and point to new treatment strategies for targeting CXCL12 in cancer.

Project Description This proposal aims to linearize, immobilize and perform super-resolution imaging of native chromatin fibers using a platform of elastomeric nanochannels. The immediate biological goal is to track the transmission of histones during DNA replication and the redistribution of histone modifications during transcription activation, revealing how epigenetic information is coordinated on individual chromatin fibers. Conventional nanochannels, while capable of linearizing DNA and chromatin, do not allow super-resolution imaging due to mobility of the biopolymers within the channels. This proposal will overcome this key limitation by using completely collapsible elastomeric nanochannels to not only linearize chromatin but also immobilize it. The required nanochannels will be fabricated by guided fracture of elastomer-sandwiched brittle thin films. The ?tunable? channels are opened to enable efficient loading of the relatively large chromatin in their coiled states. Then the channels are gradually closed in several steps. The combined hydrodynamic flow and confinement within the nanochannel linearize and immobilize individual chromatin fibers, which will be imaged and mapped with super-resolution optical microscopy. The process can be reversed and repeated, allowing the same chromatin fiber to be examined multiple times. This will not only improve confidence of the chromatin status revealed, but also enable us to study various epigenetic marks, particularly histone modifications, on the same chromatin fiber. For biology, we will focus on Tetrahymena rDNA mini-chromosome, which can also be engineered as a high copy number expression vector. Its size (~20 kb) is optimal for linearization in nanochannels. Labelling with fluorescent proteins or antibodies coupled with fluorescent dyes enables optical differentiation of old and new histones, as well as various histone modifications. This will allow direct visualization of how histones are transmitted during DNA replication. We will also examine redistribution of various histone modifications accompanying transcription activation. Importantly, we will be able to directly examine the status of histones and histone modifications in nucleosomes discretely positioned in individual chromatin fibers. This will reveal the connectivity or coordination between different epigenetic marks. The aims of this project are: Aim 1. Computational analysis of fabrication and operation of normally-closed tunable nanochannels Aim 2. Construction of straining system, and optimization of chromatin linearization and imaging Aim 3. Analysis of histones and histone modifications on individual chromatin fibers We will examine the inheritance of old histones during DNA replication, in normal as well as replicative stress conditions. We will also analyse the redistribution of histone variants and histone modifications, upon induction of transcription activation or silencing. These studies will be extended to mutants deficient in epigenetic pathways, as well as in pharmacologically perturbed cells.

ABSTRACT: 3D High Throughput Model to Predict Drug Efficacy in Fibrosis Progression vs Reversal Idiopathic pulmonary fibrosis (IPF) is the most relentlessly progressive and fatal fibrotic lung disorder, which disproportionately affects the elderly. Although two drugs have recently gained FDA-approval for IPF, these drugs only moderately slow the progression of lung decline and do not improve quality of life for patients. There are no available therapies that can `reverse' fibrosis. Despite efforts by numerous groups to develop IPF treatments, progress has been aggravatingly slow. This proposal focuses on two possible reasons for these difficulties: (1) Current pre-clinical screening models fail to reliably predict the success of drug candidates in humans, and (2) Although IPF is widely regarded as an age-related disease, drug treatments have not targeted age-associated pathologic mechanisms. The existing paradigm, that pathologic fibrosis is a ?fibro-proliferative? process, has not led to effective IPF treatments. This proposal integrates expertise in fibroblast aging and novel IPF therapeutics in development (Hecker lab) with cutting edge technologies for microscale bioprinting and 3D cell assays (Takayama lab) to develop a high throughput phenotypic cellular screening assay to determine efficacy for fibrosis reversal. The proposed studies will utilize normal ?control?, aged ?senescent?, and IPF human lung fibroblasts in small numbers to bioengineer a high-throughput phenotypic assay that will evaluate fibrosis over a 21 day period. An aqueous two phase system (ATPS) bioprinting of these cells will be used to create microscale contraction assays that are several order of magnitude smaller in volume compared to conventional assays. Importantly, the project will repeatedly micro-print fresh collagen around already contracted cell-laden gels to enable repeated contractions over 21 days. The proposed model will enable the first high-throughput phenotypic screening assay with the capability to determine a drug candidate's efficacy for fibrosis progression and reversal. The new cellular assay will be validated for its ability to identify fibrosis reversal drugs using ?Noxindoline? a highly selective Nox4 inhibitor that is currently in preclinical development by the Hecker lab. Noxindoline was identified by the Hecker lab through studies of age-dependent alterations in Nox4 that results in a sustained redox imbalance, and promotes senescence and apoptosis-resistance of myofibroblasts. The proposal hypothesizes that current therapies (Nintedanib and Pirfenidone) will inhibit the progression of pro-fibrotic phenotypes (but not reversal), whereas treatment with Noxindoline will promote the reversal of established pro-fibrotic phenotypes. The aims are: Aim1: Develop high throughput bioprinted cellular assay for fibrosis progression using non-senescent cells Aim2: Monitor fibrosis progression and reversal of senescent cells and IPF patient cells

The broader impact/commercial potential of this I-Corps project is to improve gene and cell therapies which hold potential to make life-altering improvements in healthcare for a broad spectrum of diseases, including cancers, blood disorders, and genetic diseases. If successful, this project can help to expand the population that has access to these treatments, both by reducing the cost of production and by enabling new therapies that are currently infeasible due to the low efficiency of existing cell engineering processes. The team will seek to learn new ideas and methodologies for engaging participants and stakeholders in the cell therapy community. This includes contract manufacturing organizations, clinicians/physicians, regulatory representatives, pharmaceutical companies, and patient advocates, among others. In addition to cell and gene therapies, the team will connect with researchers using gene modification in basic biology and drug discovery applications. If successful on all fronts, this project has potential to aid the discovery and delivery of many new therapies for untreatable diseases.

This I-Corps project addresses an inefficiency in the cell engineering process. In cell therapies, patient cells are reprogrammed with a genetic sequence to alter the cell behavior. This can involve teaching immune cells to clear cancer, or reprogramming bone marrow cells to produce missing clotting factor proteins to cure congenital disease. Although these treatments have demonstrated tremendous potential, many of the treatments suffer from the low efficiency of gene transfer. In some cases, the low efficiency may prevent the treatment from being effective. The state of the art uses lentivirus to introduce genes into cells, typically using 20-50 viral particles per cell. However, it has been clearly shown that a great portion of the virus vector is wasted, due largely to mass transport limitations. This project proposes to provide a reagent to decrease the quantity of lentivirus used in cell transductions by 10-fold and improve transduction efficiency with minimal disruption of current transduction protocols. This process co-localizes cells with virus to reduce diffusion distances, while maintaining a mild, aqueous environment and retaining a large reservoir of cell culture nutrients.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.