Martin L. Yarmush - US grants

Immune complexes (IC) are formed in the circulation or in tissue fluids as a consequence of the interaction between antigens and their corresponding antibodies. Many properties of IC, including clearance from the circulation, complement fixation, and adherence to phagocytes, depend on IC size and composition. The chronic presence of IC has been implicated in the pathology of several diseases including, systemic lupus erythematosus, rheumatoid arthritis, and neoplastic disease. The demonstration that activity which blocks the immune response to tumors can be removed by incubation of serum with protein A has motivated studies of plasma adsorption onto immobilized protein A in a number of tumor systems, with excellent success reported in treatment of lymphosarcoma and leukemia in FeLV-infected cats. Our research objectives are to: (1) determine the parameters that govern size, composition, and structure of IC, (2) Investigate the dependence of complex size and composition on binding and activation of complement, (3) understand the interactions between IC and the cells which bind them, and (4) define the reactions that occur upon contact of IC with immunoadsorbents such as immobilized protein A and conglutinin. Model IC constructed from two or more monoclonal antibodies and bovine serum albumin will be characterized in terms of molecular weight and size distribution, composition, overall structure, and mean hydrodynamic radius using electron microscopy, quasi-elastic light scattering, high performance size exclusion chromatography, radioimmunoassays, and mathematical models. Purified complement components will be incubated with model IC; the dependence of complement fixation and activation on complex size, structure, and composition will be measured. Similarly, IC will be incubated with macrophages and erythrocytes to measure binding and uptake, and with immunoadsorbents to analyze adsorption effects. These fundamental studies will provide the basis for experiments using IC isolated from sera of FeLV-infected cats. The proposed research will enhance our understanding of IC formation and behavior in the presence of complement and specific cells, and will provide a rational basis for future development and design of immunoadsorption techniques which may be applied diagnostically or therapeutically to a variety of disease states.

The PI's research interests are in the fields of applied immunology, bioseparations, artificial organs, and protein science and engineering. In the area of applied immunology, he has an interest in a variety of phenomena with application to important issues in biotechnology and medicine. These include: (1) studies on the structure and dynamics of antigen-antibody complexes and their interaction with various blood-borne molecules and cells, (2) development of novel targeting therapies with light-activatable drugs, and (3) studies of the dynamics of activation of lymphoid cells by lymphokines. In the area of artificial organs, the PI's current research involves development of an artificial liver and an artificial pancreas by encapsulating pancreatic or liver cells with semipermeable membranes. In the field of bioseparations, several; new approaches are under investigation including: (1) affinity chromatographic separations using electric fields and high pressure, (2) development of affinity reversed micelles, and (3) development of electrically controlled polyelectrolyte membrane technology. In the fields of protein science and engineering, the PI's research interests lie in defining the phenomena that contribute to protein stability in different environments.

The proposed studies intend to develop a generalized method for cell activation using immobilized growth or differentiation factors. The experimental studies will initially involve a step by step investigation of methodology for immobilization of active Interleukin-2 (IL-2) on the surface of several supports. A simple proliferation assay will be used to evaluate the effectiveness of the immobilized lymphokine for supporting growth. The overall objective of the proposed research is to demonstrate the feasibility of using lymphokines immobilized on solid surfaces to stimulate and activate lymphokine-sensitive cells. IL-2 activation will be used as the model system for these studies. Successful implementation of such a technique would provide a generalized and more efficient method for stimulating cell growth and/or cell differentiation in general cell cultures.

The overall objective of the studies outlined in the application is to obtain a quantitative and fundamental physicochemical understanding of the interactions of immune complexes (IC) with immunoadsorbents. Special emphasis will be placed upon (1) obtaining a basic understanding of the complex interactions in solution involving antigen, antibody complement, and various other plasma borne molecules; (2) defining and quantifying the reactions in solution between IC both reactive and non-reactive towards complement components; and (3) investigating the reactions occurring during immunosorption of immune complexes with a variety of receptor molecules bound to solid supports and with cells which have receptors for immune complexes. Techniques to be used include classical and dynamic light scattering. HPLC, radioimmumnoassays, and electron microscopy. Experiments will be carried out with model immune complexes and with immune complexes isolated from sera of feline leukemia virus-infected cats. The proposed research will enhance our understanding of IC formation and behavior in the presence of complement and specific blood cells, and may provide a rational basis for future development and design of immunoadsorption techniques which may be applied diagnostically or therapeutically to a variety of disease states.

The equipment requested will be used for several projects, including: Structure and Dynamics of Antigen-Antibody Complexes; Applications of Antibody Engineering; Protein Engineering of Allosteric Antibodies; Protein Separations Utilizing Temperature Sensitive Microemulsions; Engineering Protein Crystallization Processes; Mechanisms of Neurite Outgrowth and Guidance; Development of Hepatocyte Long Term Culture and Storage Techniques; Cellular and Developmental Biology in Plant Cell Cultures; and Immobilized Recombinant Cells. The wide dynamic range offered by the combination of the two instruments, and the simultaneous use of the instruments in certain projects, will allow for an enhanced understanding of underlying biochemical and biophysical phenomena.

This proposal addresses the environmentally important task of removing certain hazardous small organic molecules (e.g. aromatic amines, PCBs and polycyclic aromatics) from water by a unique process utilizing enzyme catalysis in an organic phase. In this process, the aqueous phase is separated from in organic phase by a membrane. The enzyme is dissolved in the organic phase, and the membrane is selected to have a cutoff pore size so that the hazardous small organic molecule, but not the enzyme, can pass through the membrane. The organic molecule diffuses into the organic compartment where the enzyme polymerizes the organic into a high molecular weight species which can subsequently by removed from the organic phase via settling or ultrafiltration. To demonstrate the feasibility of this process, 2 chlorophenol (2CP) will be used as the example organic solvent, and the polymerization will take place in perfluorodecalin with horseradish peroxidase as the enzyme catalyst. This catalysis requires free radicals which will be provided by hydrogen peroxide, itself generated by alcohol oxidation catalyzed by alcohol oxidase.

DESCRIPTION (provided by applicant): Recent animal studies and clinical trials using bioartificial liver devices have shown great promise for the treatment for acute liver failure, and are providing valuable information on the problems and limitations of current ''1st generation" liver assist devices. It is becoming clearer every day, however, that more basic information of the effect of environmental parameters on hepatocellular function, as well as host-bioartificial liver interactions, is necessary before the concept of bioartificial liver becomes a reality available at reasonable cost. Our long-term objective is to help development of 2nd and 3rd generation devices, which are expected to be significantly more effective than currently available devices. In order to reach this goal, we require a better understanding of many critically important questions including: What is the minimum cell mass to support a patient? How long and well do hepatocytes function during plasma exposure? What are the most critical functions for patient survival? What is the impact of bioartificial liver treatment on the immune system and on subsequent liver transplantation? Answers to these questions will often not be obtainable using off-the-shelf tools, and will require the development of new experimental systems. Our main hypothesis is that there is a finite number of hepatic functions which are most critical for patient survival, that it is possible to significantly upregulate them in hepatocyte cultures (both at the single cell level and at the level of tissue), and as a result, reduce the cell mass required in the bioartificial liver. The specific aims are: (1) To use metabolic and genetic engineering approaches to enhance the performance of hepatocyte cultures in plasma; (2) To optimize the oxygenation and geometric configuration of hepatocyte co-cultures for plasma detoxification; (3) To investigate patient-bioartificial liver interactions and characterize the immunological response to extracorporeal perfusion with allo- and xenogeneic cells. These studies will provide the basic knowledge and technologies enabling us to develop the next generation of liver assist devices and speed up the translation of this promising modality to the bedside. The proposed studies will also provide basic tools useful in the development of other engineered tissues and organs.

9409363 Yarmush This American Institute of Medical and Biological Engineering (AIMBE) conference will examine the need for a more quantitative and integrated approach in future developments of biotechnology and the role of engineering in meeting this challenge. Outstanding investigators and prominent policy-makers will present their views on technical and policy issues impacting on the engineering and science base for biotechnology. This overall theme is to be developed over a two-day meeting that is expected to attract 300-500 participants. Leaders in the bioengineering and medical communities are to become aware of: (1) the diversity of contributions that engineers have made in advancing the field of biotechnology; and (2) important areas that require engineering insight and know-how for future developments.

9503227 Yarmush This project is on research to study the use of hydrostatic pressure as a mild and effective means of dissociating antigen-antibody complexes, thus providing a rational basis for the design of strategies which utilize this pressure sensitivity in separation and/or sensing applications. The proposed studies include three aspects: (1) fundamental solution-phase thermodynamic and kinetic characterization of the pressure-induced phenomena for a panel of antibodies using high pressure fluorescence spectroscopy; (2) protein engineering studies directed at understanding the molecular mechanisms mediating pressure effects; and (3) thermodynamic, kinetic and pressure cycling studies with immobilized antibodies to develop operational strategies for practical pressure elution on a large scale. In addition to providing the rational framework for the development of strategies for engineering and utilizing pressure sensitive antibodies, this research could also result in advances in the fundamental understanding of pressure effects on protein structure and function. ***

9800617 Yarmush This SGER proposal deals with increasing the effectiveness often transfer to mammalian cells using recombinant retroviruses. In previous efforts, the PIs have found that glycoproteins produced by virus producer cells inhibit retroviral-mediated gene transfer. Enzymes that degrade these proteoglycans enhance the productivity of the gene transfer to mammalian cells. Since the size of the proteoglycans and the viral particles are similar, physicals separations seem unlikely. The overall goal of this proposal is to better understand the role of the proteoglycans in the inhibition process. This will be accomplished by: Isolating and purifying the proteoglycans and studying their impact, and the impact of their protein and glycan fragments on the transfer process Measuring the binding of these inhibitors to both the target cells and the retrovirus. Identify the inhibitor fragment and the cell receptor responsible for the inhibition. Develop a model of the inhibition process A better understanding of the inhibition mechanism and its scope should facilitate discovery of routes to remedy the problem. One such route already considered involves incorporation of genes for proteoglycan destruction. ***

Profound alterations in the regulation of energy and amino acid metabolism occur after burn injury. Given that the liver plays a major role in regulating whole body amino acid metabolism and is the primary site of nitrogen conversion into urea, we hypothesize that burn injury induces intrinsic metabolic changes in liver, and that these changes are responsible in large part for the aberrant amino acid profile as well as the increased nitrogen loss in the patient after burns. The overall objective of the proposed studies is to investigate, in a well characterized and controlled perfused liver model, the molecular mechanisms which trigger the overall metabolic changes that occur in the liver in response to burn trauma, and how these changes affect the metabolism of conditionally essential amino acids and the conversion of amino acid nitrogen into urea. The specific aims of the proposed work are: 1. To determine the mechanisms of increased amino acid oxidation in burned rat liver. 2. To determine the mechanisms of increased urea production in the burned rat liver and identify critical urea and TCA cycle interactions. The proposed studies will provide basic, quantitative information on the pathways activated by burn injury in liver and on the molecular mechanisms which mediate this activation. This investigation will form a rational basis for (1) the future development of nutritional regimens and therapeutic approaches aimed at reducing the catabolic response to burn injury, and (2) for the investigation of the gene expression component in the modulation of amino acid oxidation.

This project is an unsolicited submission. The goal of this project is to develop strategies to enhance the efficiency and utility of retrovirus-mediated gene transfer. Retroviruses are a widely utilized vector system in current gene therapy clinical trials, which has resulted in the rapid advancement of retroviral vector technology. Despite this, retroviral gene transfer efficiency has remained disappointingly low, hampering the emergence of retroviral gene therapy as an effective clinical tool. It has been found that charged molecules greatly influence the efficiency of the gene transfer process. Specifically, positively charged compounds have been shown to enhance gene transfer, whereas negatively charged compounds inhibit the process. This project seeks to accomplish the following: (1) to investigate in detail the mechanism of charged compound enhancement and inhibition of the retroviral gene transfer process, (2) to develop a mathematical framework for analyzing the effect of charged compounds on virus transport and transduction, and (3) to compare gene transfer efficiency in an animal model using ex vivo and in vivo gene therapy protocols optimized to enhance the electrostatic interaction between the retrovirus vector and target cell. To achieve these goals, the investigators will utilize a complementary set of enzymatic and immunohistochemical assays, molecular imaging techniques, and cytofluorometric analyses to quantitate various steps of the retroviral gene transfer process. Engineering analysis of the resultant biomolecular transport and kinetic reaction data will be used to facilitate the construction of an integrative model of retrovirus transport.

DESCRIPTION (provided by applicant): The tools of modern biology are revolutionizing biomedical research, enabling an exponential growth in the acquisition of data regarding genes, proteins, and their structures and functions in normal and diseased states. Among these advances, the ability to monitor profiles of genes, and to a lesser extent, protein expression accurately, and on a large scale are notable. However, it is often not easy to correlate the trends and relationships observed in normal or abnormal states to the phenotype resulting from the gene expression profile. For physiological states involving metabolic derangements, gene expression profiling does not fully explain the complex molecular mechanisms involved. Thus, in order to develop a comprehensive understanding of metabolic states, it is essential to understand both the gene expression events as well as the cytoplasmic events that control changes in metabolites. The proposed research seeks to accomplish the following: (1) To determine the genes whose expression is altered by molecular mediators of the stress response and to generate green fluorescence protein (GFP)-tagged expression constructs of these genes; (2) To use microfabricated and microfluidic techniques for developing a living cell array systems where differentiated cells can be cultivated and exposed to multiple inputs; (3) To obtain temporal gene expression profiles using the living cell array that has been exposed to combinatorial mixtures of stress mediators that closely mimics the physiological stress response and to use this information to predict the molecular events that determine the cell's progression to recovery or failure during stress. This proposal is an interdisciplinary project that integrates scientific inputs from biology, engineering, and computational methods. Each element of the proposal is integral to the success of the overall project and we anticipate that this will provide excellent training to the postdoctoral fellow and graduate students working on this project. The results should provide valuable new information on the molecular mechanisms governing metabolic states, which will be disseminated via peer-reviewed publications and presentations at national conferences. In summary, the proposed work seeks to provide fundamental research and a base of personnel equipped to solve problems in fields such as metabolic engineering where complex biological phenomena are under investigation.

DESCRIPTION (provided by applicant): Orthotopic liver transplantation (OLT) is a highly successful therapeutic modality for the treatment of both acute and chronic liver failure which is severely limited by the scarcity of donor livers. Among the livers donated after brain death, the most common single predisposing risk factor for postoperative liver failure is steatosis;thus, fatty livers are often considered to be "unacceptable" or "marginally acceptable" for transplantation. The incidence of hepatic steatosis is 10 to 25% based on autopsy studies and donor liver biopsies. It is clear that methods that would salvage discarded donors because of severe steatosis could significantly reduce the number of patient deaths, and help close the gap between supply and demand in liver transplantation. The overall hypothesis is that discarded donor livers, and more specifically steatotic livers (and in the long run donors after cardiac death), can be salvaged by perfusion with artificial solutions under well-controlled conditions and at physiological temperatures in order to promote defatting and cellular repair, and as a result made capable to withstand surgical procedures and reduce the risk of postoperative liver dysfunction to a level similar to that observed in normal livers. In the studies proposed herein, our objective is to apply this approach to fatty livers, and eventually to the more complex case of ischemic livers (i.e. from donors after cardiac death). Our specific aims are: (1) To optimize metabolism for defatting steatotic livers during normothermic or mild hypothermic perfusion;(2) To investigate the combined effects of heat shock and warm perfusion on microvascular function and transplantability in steatotic livers;(3) To develop a normothermic perfusion protocol that restores mitochondrial function and ATP stores in warm ischemic livers. In the short-term, the proposed studies could (a) provide the rationale basis for increasing the donor pool size;(b) improve the outcome of patients which receive marginal donor livers;(c) prolong the useful preservation time of steatotic, defatted, as well as warm ischemic livers. In the long-term, these studies will lead to (a) increased donor pool size and (b) increased organ storage time beyond the limits of current cold storage techniques. These outcomes will significantly alleviate donor shortage and lead the way to donor banking, with the potential to revolutionize donor liver allocation. PUBLIC HEALTH RELEVANCE: The proposed studies will provide basic scientific information and new technologies that will enable the recovery of donor livers that are otherwise rejected from the donor pool. In the long-term, these studies will lead to (a) increased donor pool size and (b) increased organ storage time beyond the limits of current cold storage techniques. These outcomes will significantly alleviate donor shortage and lead the way to donor banking, with the potential to revolutionize donor liver allocation.

This project studies the development of protein-based nano-motors and nano-robots. The goal is to develop novel and revolutionary biomolecular machine components that can be assembled and form multi-degree of freedom nanodevices that will be able to apply forces and manipulate objects in the nanoworld, transfer information from the nano to the macro world and also be able to travel in the nanoenvironment. These
machines are expected to be highly efficient, economical in mass production, work under little supervision and be controllable. The vision is that such ultra-miniature robotic systems and nano-mechanical devices will be the biomolecular electromechanical hardware of future planetary, military or medical missions. Some proteins, due to their structural characteristics and physicochemical properties constitute potential candidates for this role. The specific aims of this project are: a) To identify proteins that can be used as motors in nano / micro machines and mechanisms. We will focus our studies on the mechanical properties of viral proteins to fold or unfold depending on the pH level of environment. Thus, a new, powerful, linear biomolecular actuator type is obtained that we call: Viral Protein Linear (VPL) motor. Various viral proteins will be studied and from them different VPL motors will be produced; b) To develop dynamic models and realistic simulations / animations to accurately predict the performance of the proposed VPL motors; c) To perform a series of biomolecular experiments to demonstrate the validity of the proposed concept of VPL motors; d) To study, both computationally and experimentally, the interface of the proposed protein motors with other biomolecular components such as DNA joints and carbon-nanotube rigid links so that complex, multi-degree of freedom machines and robots are formed.

The broader impact and outreach activities of this project are: a) the initiation of undergraduate students in research; b) the establishment of collaborative projects on nanotechnology with the science and technology high schools of New Jersey with the objective to attract new students in this field; c) the organization of a special session at the annual ASME International Mechanical Engineering Congress and Exposition (IMECE) on nano-robotics; and d) the development and maintenance of a webpage on bio-nano-robotic systems.

This IGERT program at Rutgers University, focused on integratively engineered biointerfaces, will be an intimately collaborative effort of 32 selected faculty from graduate programs in Molecular Biosciences, Physical Sciences (Physics, Chemistry & Chemical Biology), and Engineering (Biomedical Engineering, Ceramics and Materials Engineering, Chemical and Biochemical Engineering, Mechanical and Aerospace Engineering).

Intellectual Merit: The program derives strength from the highly cross-disciplinary nature of over fifteen research project areas identified at the cutting edge of the field of biointerfaces, and programmatic partnerships with five strategic centers of excellence to promote cohesive access for the IGERT community to state-of-the-art research infrastructure. A wide range of thesis project themes is planned for the IGERT trainees, developed around three research and educational thrusts, (1) living cell-based interfaces, (2) microengineered and nanoengineered biointerfaces, (3) biosensing and bioresponsive interfaces. The five major partnering Centers for the IGERT program are: Keck Center for Collaborative Neuroscience, Center for Nanomaterials Research, New Jersey Center for Biomaterials, the Laboratory for Surface Modification, and the Rutgers Center for Computational Design. The educational core of the proposed IGERT program will intimately support the research program, and includes graduate courses in the integrative areas of biointerfacial engineering, as well as course modules on responsible conduct of research, technical communications, entrepreneurship and effective teaching/learning methods.

Broader Impact: The IGERT curriculum is designed to foster a community featuring the next generation of biointerfacial and biomaterials engineers by offering IGERT graduate fellows a range of interactive experiences at multiple levels: multi-disciplinary coursework, lab rotations in two cross-cutting research groups, biannual participation in symposia, and participation in a national/international conference resulting in a white paper. To maximize its impact, the IGERT program will offer varied programmatic pathways to promote diverse modes of professional development of IGERT graduate fellows: (1) Summer research internships at selected international sites for academically inclined students; and (2) Translational research and industrial summer internships for students interested in industrial and entrepreneurial careers. Through a partnership with the Robert Davis Learning Institute of the Rutgers Graduate School of Education Institute, the IGERT program will establish a COLTS (Community of Learners and Thought Shapers) program, inspired by communication-driven cognition models, to encourage IGERT fellows to develop as learners by dynamically communicating their research on integratively engineered biointerfaces.

IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries. In this sixth year of the program, awards are being made to institutions for programs that collectively span the areas of science and engineering supported by NSF.

This project will study the development of protein-based nano-motors and nano-robots. The goal is to develop novel and revolutionary biomolecular machine components that can be assembled and form multi-degree of freedom nanodevices that will be able to apply forces and manipulate objects in the nanoworld, transfer information from the nano to the macro world and also be able to travel in the nanoenvironment. These machines are expected to be highly efficient, economical in mass production, work under little supervision and be controllable. The vision is that such ultra-miniature robotic systems and nano-mechanical devices will be the biomolecular electro-mechanical hardware of future manufacturing, biomedical and planetary applications. Some proteins, due to their structural characteristics and physicochemical properties constitute potential candidates for this role. The specific aims of this project are: (1) To identify proteins that can be used as motors in nano/micro machines and mechanisms. The focus of the study will be on the mechanical properties of viral proteins to open or close depending on the pH level of environment. Thus, a new, powerful, linear biomolecular actuator type is obtained,Viral Protein Linear (VPL) motor. Various viral proteins will be studied and from them different VPL motors will be produced. (2) To develop dynamic models and realistic simulations/animations to accurately predict the performance of the proposed VPL motors. (3) To perform a series of biomolecular experiments to demonstrate the validity of the proposed concept of VPL motors. (4) To study the interface of the proposed protein motors with other biomolecular components such as DNA joints and carbon-nanotube rigid links so that complex, multi-degree of freedom machines and robots powered by the VPL motors are formed.

The educational, broader impact and outreach activities of this project are: (1) The development of a new, inter-departmental course on the design of nano-machines; (2) The initiation of undergraduate students in research; (3) The establishment of collaborative projects on nano-technology with the science and technology high schools of New Jersey with the objective to attract new students in this field; (4) The organization of symposia and journal special issues on bio-nano-robotics; (5) The development and maintenance of a webpage on bio-nano-robotic systems; and (6) The establishment of international collaborative activities in the area of nano-technology.

Ierapetritou
0424968

The objective of this research is to develop systems-based quantitative approaches that consider the whole picture of cell metabolism, including intra and inter compartmental processes within a multi-objective framework. This is needed in order to investigate the characteristics of hepatocyte cells, the role of specific functions in bioartificial liver systems, and the optimization of their performance. Their hypothesis is that there is a finite number of hepatic functions which are most critical for patient survival, that it is possible to significantly upregulate these functions in hepatocyte cultures, and as a result, maximize cell function and reduce the cell mass required in the bioartificial liver. In particular, the specific aims for this research are: (1) to characterize and optimize substrate supplementation to enhance hepatocyte function, (2) to modulate the hepatocyte function through the use of antisense technology with the target to examine different substrates and validate the importance of different pathways as predicted by specific aim 1, and (3) to develop a quantitative analysis to study the effects of uncertainty considerations mainly due to variability of plasma concentration.

[unreadable] DESCRIPTION (provided by applicant): New tools are revolutionizing biomedical research, enabling an exponential growth in the acquisition of data regarding genes, proteins, and their structures and functions in normal and diseased states. Among these advances, the ability to monitor profiles of genes on a large scale is notable. However, it is often difficult to correlate the trends and relationships observed in normal and abnormal states to the phenotype resulting from the gene expression profile. For physiological states involving metabolic derangements, gene expression profiling does not fully explain the complex molecular mechanisms involved. Thus, in order to develop a comprehensive understanding of metabolic states, it is essential to understand both the gene expression events as well as the cytoplasmic events, which control changes in metabolites. The proposed Bioengineering Research Partnership seeks to develop a new functional genomics approach for studying gene expression: the use of intact cells for the simultaneous temporal expression profiling of multiple genes using aequorin-type fluorescent proteins (AFP) in a massively parallel, high throughput format. The specific aims are: (1) to generate and characterize a panel of reporter cell lines that monitors the major events in the inflammatory cytokine signaling cascades; (2) to design a microfluidic system to dynamically control the input stimulus as well as the fluorescent response of an array of primary rat hepatocytes and H35 hepatoma reporter cell lines; (3) to characterize the dynamics of cytokine signal transduction and the impact of steatosis and different metabolic states on the acute phase response of these cells. This project will be carried out by three distinct research groups, which will interact extensively. Dr. Martin Yarmush (MGH), the Principal Investigator, will oversee the Administrative and Technical Core, and lead the Cell Physiology and Imaging group, which will integrate the technologies developed by the other Pl's and carry out the bulk of the cell physiology experiments. Dr. Mehmet Toner, Director of the Microscale Engineering Facility at the MGH, will lead the Microfabrication and Microfluidics group focusing on the development of microfabricated devices. Dr. Jeffrey Morgan (Brown Univ., Providence, RI), a molecular biologist and expert in gene therapy, will lead the Molecular Biology and Cell Analysis group focusing on reporter cell line development. [unreadable] [unreadable]

Roth
0519563

The proposed research involves the development of novel approaches to model regulation of metabolic networks, with a particular focus on gene regulation and cellular signaling. This approach combines gene expression data acquisition and a modeling framework, which utilizes experimental data and biological knowledge in unique ways, and which will have significant impact on the field of systems biology. The effect of stress mediators on hepatocyte metabolism has not been studied extensively and quantitatively from the standpoint of re-directing metabolism from inflammation to differentiated function for tissue engineering applications.

This project will expand on collaborations already developed among the Principal Investigators (PIs) to include a larger and more diverse team, including experts in liver physiology and development of a bioartificial liver, biochemical reaction modeling, optimization, and data mining. Several graduate students will be supported by this grant. Other students are on fellowships from the Rutgers IGERT program on Integratively Engineered Biointerfaces. All students will be co-advised by 2 PIs and receive cross-disciplinary training. This project will be used as a case study in the PI's Molecular and Cellular Bioengineering course. Because of the integrative and interdisciplinary nature of this project, a significant impact on the educational and research infrastructure at Rutgers is expected.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] This proposal for a K18 Career Enhancement Award for Stem Cell Research describes a one year training program which will allow the principal investigator (PI) to appropriately use stem cells in his research on Hepatic Tissue Engineering. The PI, Dr. Martin Yarmush, is an MD PhD bioengineer who for the past 15 years has been developing critical technologies for bioartificial liver support systems. A critical issue in the development of bioartificial liver support systems is the limited availability of large numbers of differentiated hepatocytes necessary to seed these devices. As a means of addressing this problem, the PI has gravitated towards stem cell engineering, specifically focusing on directing embryonic stem cell differentiation into hepatocytes. After some initial work of an empirical nature, the PI seeks more focused training in developmental and stem cell biology. Through a program of didactic coursework, seminars and research, the PI will focus his efforts on embryonic stem cell biology. Dr. George Q. Daly, an internationally recognized stem cell expert will serve as the sponsor. The PI will become intimately familiar with many of the techniques that Dr. Daley's lab has developed related to self-renewal and differentiation of human ES cells, and then apply them to directing ES cells to mature hepatocytes. The research focus in this proposal is to develop techniques to derive adult hepatocytes from embryonic stem (ES) cells in vitro. For the purpose of developing the technology and gaining a better understanding of the underlying basic science, a mouse model will be used. The overall goals of this project are two-fold: (1) to develop an approach to reset the clock of diseased adult hepatocytes to a progenitor stage, and (2) to understand the molecular mechanisms of switching between proliferation and differentiation in progenitor cells, so that higher levels of liver-specific functions can be expressed in the differentiated hepatocytes. Through this work, we hope to produce a clonal source of hepatocytes which could be used in various applications of hepatic tissue engineering, including drug screening, toxicology, and bioartificial liver assist devices. The Childrens Hospital Boston, the Center for Engineering in Medicine at MGH, the Harvard Stem Cell Institute with its distinguished faculty, interdisciplinary environment, well equipped core facilities, and extensive collaborative interactions will provide the PI with everything necessary to pursue his goals. [unreadable] [unreadable]

This Integrative Graduate Education and Research Traineeship program (IGERT) renewal award establishes a training program focused on the science and engineering of stem cells. New cross-cutting thrusts for Ph.D. research, together with a new multidisciplinary graduate curriculum, will integrate stem cell biology with research in biomaterials, process engineering, and computational modeling. Trainees will participate in an IGERT Research Interchange Forum to develop their abilities to communicate across disparate disciplines. Professional development activities encompassing teaching, mentoring, and outreach will enable IGERT trainees to better realize the impact of their technological know-how. Each IGERT trainee will be guided by an advisory constellation of scholars drawn from over 30 faculty members from Engineering, Molecular Biosciences, Physical Sciences, Business, Public Policy, and Management. The IGERT program will leverage Rutgers' active "diversity infrastructure" to help broaden the participation of underrepresented minority students. In addition to providing research opportunities for visiting underrepresented undergraduates, the IGERT will offer two new initiatives: a teacher-student summer institute at Rutgers, and, a bridge-to-IGERT program. New outreach programs at the intersection of stem cell science and engineering with public policy and business include: (1) an initiative with the School of Management and Labor Relations to "bundle" IGERT research and curriculum into portable modules for scientific workforce training; (2) Rutgers Business School-mediated interactions with pharmaceutical management students and industry; (3) public policy workshops with policy makers, facilitated by the Eagleton Institute of Politics. IGERT trainees will acquire global perspectives through internships and workshops with leading stem cell researchers at over 15 sites in Europe and Asia. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

CBET-0828244
M. Yarmush, Rutgers University New Brunswick

The overall goal of the proposed research is to develop effective strategies to increase the stability and titer of retroviral vectors for gene therapy applications. Recently, considerable effort has been dedicated toward utilizing these, and other viral, vectors in a number of clinical trials. Although several publications have described various degrees of success, the clinical success of retroviral vectors is limited by the rapid rate of viral inactivation and limited viral titers. Thus, a retrovirus with enhanced intrinsic stability and high titer would alleviate some of the practical obstacles limiting the success of retroviral vectors in the clinic. It is likely that these principles could also be extended to other viral vectors.

The proposed research seeks to accomplish the following: (1) To determine the mechanisms underlying the loss of retroviral bioactivity and the relative stability of the immature / mature viral particles; (2) To optimize culture conditions and methods, in order to allow the production of high-titer viral vectors; and (3) To modify the viral reverse transcriptase to achieve higher efficiency viral replication. To achieve these goals, physical, chemical, and molecular manipulation techniques will be used with the objective of slowing the rate of retroviral decay and increasing retroviral titer.

The proposed work will provide excellent training for the graduate fellows involved in the project, as well as to students exposed to it through various courses and training programs conducted by the investigators. In addition, the PI will provide an excellent educational environment for various high school and university-wide undergraduate research programs. The scientific approach was designed not only to establish an excellent scientific program, but also to expose students to state of the art technology and methodology through both classroom instruction and direct laboratory interaction. The research itself will provide valuable new information regarding the mechanisms of retroviral decay and methods to address them. The experimental results are likely to have a broad impact on the scientific and clinical venues which utilize or are developing viral vector delivery protocols. In summary, the proposed work seeks to provide fundamental research and a human base of personnel equipped to solve problems related to the implementation of techniques to improve suitability of viral vectors for gene therapy applications.

DESCRIPTION (provided by applicant): About thirty million people in the US undergo a liver disorder for different causes and about 27,000 deaths are registered annually in the US due to liver disease. At this time, the only definitive treatment of hepatic failure is orthotopic transplantation. However, there is a critical shortage of organs, with a deficit of ~3,000 livers per year. Similar numbers affect most organs &tissues, with the total organ waiting list currently at 100,000 requests and the number increasing by 5% every year. Given that only organs in pristine condition are transplantable, orthotopic transplantation will always remain a limited pool. A more elegant, long-term solution is using stem cells to develop tissue-engineered replacements. However, while many in vitro successes have been demonstrated, clinical success has been very limited due to low cell viability and functionality in the long term in vivo. The major gap is the lack of an ideal transplantable scaffold that has all the necessary microstructure and extracellular cues for cell attachment, differentiation, function and vascularization, which has so far proven difficult to manufacture in vitro. Our long-term goal is to engineer transplantable liver grafts for curing or treating liver dysfunction and failure. The objective of the proposed study is to develop functional and transplantable rat liver grafts. The central hypothesis to be tested here is that the natural liver scaffold derived from discarded livers can be extensively repopulated, can provide an adequate maturation environment for stem cell derived hepatocytes, and that these grafts perform the essential hepatic functions in vitro and in vivo. The rationale of the study is that while most research focuses on producing the ideal scaffold from the ground up using synthetic biomaterials, the native ECM is likely to contain the necessary architecture and environmental cues, hence presents a promising, little explored alternative approach for producing organ grafts which can vertically advance the field of tissue engineering. The research team assembled has expertise on perfusion systems, tissue engineering, stem cell engineering, and liver transplantation (see Biographical Sketches), which are necessary to perform the proposed studies. Engineering of functional liver grafts from stem-cell derived hepatocytes and liver's natural matrix is an innovative endeavor, as it has the potential to become a novel platform for hepatic tissue engineering. The work described here is expected to lead to a novel graft engineering approach to provide auxiliary hepatic support. While this work utilizes liver as the model organ, the results of this work will also have a positive impact by establishing the basis of future sophisticated organ engineering techniques that incorporate several different cell types and can be applied to other organs (pancreas, kidney, etc.), and may ultimately lead to development of entire organs in vitro. The ESC maturation protocol developed here is expected to be a significant contribution to the field of stem cell engineering. Hepatocyte culture in the decellularized matrix may also prove to be a new platform for pharmaceutical studies. PUBLIC HEALTH RELEVANCE: About thirty million people in the US undergo a liver disorder for different causes and about 27,000 deaths are registered annually in the US due to liver disease. At this time, the only definitive treatment of hepatic failure is orthotopic transplantation. However, there is a critical shortage of organs, with a deficit of ~3,000 livers per year. Similar numbers affect most organs &tissues, with the total organ waiting list currently at 100,000 requests and the number increasing by 5% every year. Given that only organs in pristine condition are transplantable, orthotopic transplantation will always remain a limited pool. The results of this study are expected to directly improve public health by the developing a novel approach for engineering auxiliary liver grafts using stem cell derived hepatocytes and native matrices from discarded livers, in order to treat patients with liver failure.

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

Proposal Number: 0941313
PI: Juergen Hahn
Institution: Texas Engineering Experiment Station

Proposal Number: 0941287
PI: Martin Yarmush
Institution: Rutgers University


Signal transduction pathways play a key role in many cellular functions as well as intercellular communication. However, elucidating the exact mechanisms involved in signal transduction pathways is non-trivial: crosstalk exists between different pathways, the response within a population of cells can vary significantly, and only limited measurement capabilities are available for observing intracellular signals. One specific example highlighting the importance of signal transduction and how it is affected by cell population is stem cell differentiation. The resulting cell type is affected by the cell population and intercellular communication that activates different signal transduction pathways.

This project is focused on the development of a new computational framework that enable the PIs to investigate the role of cell populations on signal transduction. In order to do so, they will derive techniques that allow them to distinguish between stochastic components and population effects. Unlike their past work, which dealt with average properties only, they will focus on developing techniques that consider information about individual cells within a population and use this information for investigating population effects on signal transduction activity.

Intellectual Merit: This work includes the following portions:
(a) Development of problem formulations and algorithms that can solve inverse problems considering cell populations, rather than just bulk averages, subject to the high level of measurement noise commonly found when studying signal transduction pathways.
(b) Derivation of a new approach for determining the optimal set of parameters to estimate in a nonlinear signal transduction pathway model given the available data for a distribution of cells and considering uncertainty in the model.
(c) Development of a computational technique for large-scale parameter estimation across populations to determine how intercellular communication affects signal transduction in individual cells, leading to a greater understanding of cellular behavior and improved experimental design. This includes determining the number of cells and their spatial location in experiments in order to avoid results that are skewed because cell population effects have not been considered.


In summary, this work will develop and integrate mathematical, computational, and experimental approaches to partition stochastic and population effects with the ultimate goal of developing improved models of signal transduction pathways. These techniques will be applied to the Jak/STAT and the Erk-C/EBPâ signaling pathways which play an important role in many cellular responses, such as stem cell differentiation and the inflammatory response of the liver.

Broader Impact: Synergies can be created by integrating research and teaching efforts in the area of systems biology as well as by establishing long-term collaborations between research groups involved in modeling and in the experimental life sciences. Two of the PIs coteach a senior-level undergraduate/graduate elective class on systems biology which integrates theoretical and experimental aspects required for modeling and analysis of bio-systems. The class aligns with departmental curriculum reform plans and will include several modules which can also be used in other courses and outreach activities. Interactive and web-based learning aids will be developed along with the modules and incorporated throughout the course. Additionally, significant effort will be devoted to disseminating research results in the form of software, case studies, undergraduate student education and training, and outreach programs to underrepresented groups.

ABSTRACT There are currently ~100,000 patients on the organ transplant waiting list in the US, a number that far exceeds the supply of available organs, and that continues to grow ~5% each year. The most promising solutions, bioartificial tissue and organ construction and donor organ reengineering methodologies, are both ultimately limited by biopreservation technologies, as any tissue engineered products prepared in a laboratory will have to be stored for a period of time until utilization. The current gold standard for whole organ preservation is cold storage on ice for up to 72 hours, during which time the organ continuously deteriorates. A superior biopreservation method that extends the tissue storage time beyond current limitations is yet to be developed. Such a method would provide a crucial enabling technology for tissue and organ preservation, tissue and organ transport, and tissue and organ transplantation. The objective of this study is to extend the viable preservation time of hepatic tissues by sub-zero non- freezing (SZNF) storage in a supercooled preservation medium. The central hypothesis of this study relies on two phenomena: 1) that 3-O-methyl-glucose (3OMG) lowers achievable stable SZNF temperature without major toxic side effects, and that 2) rewarming by normothermic perfusion reduces reperfusion damage. Our hypothesis has been formulated based on our preliminary findings establishing 3OMG as a minimally toxic cryoprotectant for hepatocytes, and establishing that normothermic perfusion can significantly reverse the damaging effects of ischemia. The rationale of the study is that if supercooled preservation can be achieved while avoiding antifreeze toxicity, then organ metabolism can be further slowed thereby reducing anoxic/ischemic damage to minimal levels. Establishment of a sub-zero nonfreezing preservation technology will be a welcome innovation to the field. The work described herein will help develop this enabling technology of supercooled storage, and also establish quantitative standards for evaluating the liver and bioartificial organ viability following preservation. While we focus on the liver, we expect that the protocols established here will also serve as the basis for subzero nonfreezing preservation of other tissue engineered products, such as artificial organ substitutes and seeded scaffold constructs.

DESCRIPTION (provided by applicant): The overall goal of this Phase I project is to create technical protocols for the reproducible operation of our proprietary cell-based device for the treatment of acute renal failure. The project specific aims are: (1) To identify active therapeutic secreted factors from MSCs in order to develop quality control assays;and (2) To optimize standard operating procedures for the manufacturing of MSC-devices and perform a dose escalation trial in dogs. The deliverable of this completed project will be a prototype cell-laden dialysis cartridge that will be ready for therapeutic testing in large animal and human trials during Phase II funding. PUBLIC HEALTH RELEVANCE: Sentien Biotechnologies Inc. has developed a cell-based kidney dialysis device that offers unparalleled support to patients undergoing acute renal failure. The device, known as the Sentinel", delivers cell- derived secretions of anti-inflammatory and regenerative molecules directly into the bloodstream in a dynamic and sustained manner. This immunomodulatory and organ sparing approach distinguishes this device from current devices designed to provide artificial kidney support and promises to quickly become a disruptive technology in the realm of renal failure therapy. Sentien now seeks funding to continue to develop a pre-clinical pathway to commercialization of their proprietary technology. This pathway will involve an initial phase of identifying the key molecules secreted by MSCs for quality control purposes and then the creation of standard operating procedures that optimize the viability and secretions of MSCs during bioproccessing of a human scale device. A dose escalation study will be performed in dogs using the manufacturing protocols created for the Sentinel". A second phase of development will involve therapeutic trials in large animals and the clinical preparation and testing of the Sentinel" in a Phase I/II human trial.

DESCRIPTION (provided by applicant): Liver failure is the 7th leading cause of death and is responsible for 50,000 deaths per year in the United States. Orthotopic liver transplantation is the only proven effective treatment of acute liver failure (ALF), but its use is limited due to organ donor shortage, associated high costs, and the requirement of lifelong immunosuppression. The present and expected growth of the population that is affected by liver failure is ever rising and a life-saving alternative to transplantation is needed to support patients. Bioartificial liver devices are a rational approach to support ALF patients as a bridge to transplantation. Five cell- based devices have been tested in humans and pigs and appear safe, but none have shown a survival benefit. The failure of devices to-date suggests an ineffective mechanism of action. End-stage liver failure leads to systemic dysfunction that is occurring simultaneously with an inflammatory response. We hypothesize that a combination approach to therapy that provides hepatocellular support along with cytoprotection, anti-inflammatory, and trophic support will cover the broad spectrum of pathological processes that can stabilize a patient. In proof-of-principle therapeutic trials, we have demonstrated that human mesenchymal stem cell (MSCs) naturally secrete bioactive molecules that have immunomodulatory properties. We have developed MSC-based devices that are operated outside the body and connect to a subject's circulation to provide long-term support, and have shown that when connected to one of these devices for 10 hours, rats undergoing ALF have a 5-fold increase in survival from less than 15% to over 70%. The overall goal of this Phase I project is to develop a composite cellular bioreactor for the treatment of ALF that integrates both hepatocyte and MSC metabolism and secretion in a single unit, and evaluate the added benefit of this two-cell device over and above the effectiveness of the MSC devices. The project specific aims are: (1) To optimize the in vitro coculture of MSCs and hepatocytes and simulate the effect of liver failure serum on the function of the coculture;and (2) To incorporate MSCs and hepatocytes into flat-plate devices and initiate therapeutic testing of bioreactor treatments in rodent models. Upon successful completion of this project, the deliverable will be a prototype cell-laden dialysis cartridge that can be readily scaled up and tested in large animals. PUBLIC HEALTH RELEVANCE: We propose to develop an extracorporeal bioartificial liver device that offers unparalleled support to patients undergoing liver failure. The device will contain hepatocytes and mesenchymal stem cells (MSCs). The addition of MSCs is unique to our technology and is designed to enhance the metabolic functions of hepatocytes exposed to plasma and restore the regulation of the dysfunctional immune system in patients undergoing liver failure by active MSC secretion anti-inflammatory and trophic molecules. This two-pronged approach distinguishes this device from current prototypes. Our objectives are to perform in vitro optimization of the coculture using metabolic engineering of the coculture and in vivo testing of a microfabricated coculture device in two rat models of liver failure. These studies will determine if the combination therapeutic approach can be indicated for a broad range of liver disease etiologies and will motivate testing in large animal models of liver failure, and ultimately in human patients.

DESCRIPTION (provided by applicant): The overall goal of the project is to develop discovery platform based on microfluidics and functional cell assays that is suitable for screening hundreds of proteins or small-molecule compounds simultaneously in a cost-effective and high throughput manner. The projects Specific Aims are: (1) To develop a microfluidic-based platform for high-throughput screening of potential protein and small molecule therapeutics. (2) To develop an insulin stimulated glucose uptake assay in differentiated 3T3-L1 adipocytes using fluorescent 2-deoxy glucose (2-DOG) analog. (3) To integrate and perform a functional 2-DOG uptake assay in the microfluidics based multi-well cell culture biochip. The deliverable from this completed project is a cost-effective microfluidics based platform suitable for high throughput screening of proteins and small molecule therapeutic compounds. PUBLIC HEALTH RELEVANCE: The long term objective of the project is to identify protein and small molecule therapeutics to treat diseases such as diabetes, obesity, cancer and neurological disorders. As a first step towards this objective, a platform based on microfluidics technology and functional cell bioassays is being developed to screen the entire human proteome and small molecule libraries in a cost-effective and time saving manner. Presently, safety and efficacy of most of the drugs in the market is still a primary concern. However, with more therapies becoming available, it is possible that not only safety standards will be met but also the cost of drugs will be lowered.

DESCRIPTION (provided by applicant): Our proposal aims at redefining the essence of team-based Senior Design projects in Biomedical Engineering and is based on the realization that in addition to the basic technical, communication and interpersonal skills acquired in a typical 4-year engineering curriculum, successful careers in the health industry require an understanding of how a business functions (marketing and sales, accounting and finance, and operations) and familiarity with the legal, regulatory, and economic constraints affecting patient care device design and development. While Biomedical Engineers need not become experts in all the above, it is critical for them to understand and appreciate these issues. The goal of this proposal is to implement a new Senior Design experience for BME students focused on the integration of biomedical engineering and clinical disciplines with translation of innovation to industry. There are three specific aims: Aim 1: To incorporate basic aspects of translational medicine and innovation commercialization into the lecture component of the Senior Design course. The lecture component, which is primarily given in the fall semester of the senior year, will be overhauled to include lectures given by instructors from the medical and business schools. Aim 2: To increase the clinical significance of Senior Design projects offered to BME seniors. Students will be able to apply for a clinical immersion program in the summer prior to their senior year and will be encouraged to identify needs that can be addressed through biomedical engineering solutions. Medical and Business School faculty, as well as industrial advisors will be invited to develop and mentor senior design projects. Aim 3: To provide a path for translation and commercialization for the most successful senior design projects. A selection process will be implemented whereby about 20% of the projects will be selected at the end of the fall semester for matching with an entrepreneurship course where they become case studies for marketing and financial analysis. At the end of the spring, one or two projects will be further evaluated and pushed towards actual commercialization. The program participants will be the entire senior class of BME students at Rutgers, and the program will be run through the collaborative efforts of the Rutgers Center for Innovative Ventures of Emerging Technology, the Department of Biomedical Engineering at Rutgers, the Robert Wood Johnson School of Medicine, and the Rutgers Business School. Students will tremendously benefit from the proposed upgrade to the Senior Design course because they will be working on projects that are better tailored towards the needs of patients and physicians. They will also get a better understanding of the down-to-earth practical - yet critical - non-technical issues that must be considered when designing new products, such as the competition, market acceptance, cost, and other related considerations.

DESCRIPTION (provided by applicant): A 3D biomimetic liver sinusoid construct for predicting physiology and toxicity Approximately 90% of drug candidates entering Phase 1 clinical trials fail, and one of the main reasons for drug failure is unexpected toxicity. The liver plays a centra role in the human body, contributing to homeostasis and important functions such as biotransformation and metabolism of drugs. The liver is also the most common target for drug-induced toxicity. Existing in vitro models and in vivo animal models have limited predictive power for human liver toxicity. The goal of this project is to construct a microfluidic liver modul which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This human liver model is expected to be the essential elimination organ for modeling human exposure, provide improved predictions of drug induced liver toxicity, and also serve as a disease model for drug discovery. Our approach will be to develop a 3D microfluidic system with human hepatocyte, kupffer, stellate and endothelial cells, to mimic the liver acinus - the smallest functional unit of the liver. A uniue feature of the model will be the oxygenation of the media, and the establishment of an oxygen gradient, which is believed to account for important metabolic, gene expression and functional heterogeneity of the hepatocytes in the sinusoidal space of normal human liver. Hepatocytes in the oxygen rich zone are efficient in oxidative metabolism, fatty acid oxidation, gluconeogenesis, bile acid extraction, ammonia detoxification to urea and glutathione-conjugation while hepatocytes in the oxygen depleted zone are efficient in glycolysis, liponeogenesis and Cytochrome P-450 biotransformation. Another unique feature of the model will be the incorporation of 'sentinel' biosensor cells, a small fraction of cells with engineered biosensors that indicate changes in cellular functions. When combined with other fluorescent probes, standard biochemical and mass spectroscopy readouts, the model will provide a real-time High Content Analysis (HCA) profile to monitor organ function and response. The selection and validation of readouts and performance of the model will be evaluated based on a panel of reference drugs with available clinical data. To facilitate that comparison, a database of drugs with clinical data, and data from other in vitro and in vivo studies will be constructed. The ultimate goal of this project is to develop a microfluidic model of human liver function that will integrate with a series of other human organ modules, to create a microphysiology platform that reproduces human clinical trial results and provides improved predictivity of exposure, safety and efficacy for drug development. The liver plays a central role in human drug interactions, both within the liver and in other organs, as a result of drug metabolism. The performance of the liver module is central to the performance of the microphysiology platform. We believe the design proposed here will optimally recapitulate human liver function on that platform. PUBLIC HEALTH RELEVANCE: The liver plays a central role in human drug interactions and is also the most common target for drug-induced toxicity, resulting in costly, late stage drug failures. The goal of this project is to construct a microfluidic liver module which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This module will be designed to integrate with other organ models forming a human microphysiology platform to improve drug efficacy and safety testing.

DESCRIPTION (provided by applicant): A 3D biomimetic liver sinusoid construct for predicting physiology and toxicity Approximately 90% of drug candidates entering Phase 1 clinical trials fail, and one of the main reasons for drug failure is unexpected toxicity. The liver plays a centra role in the human body, contributing to homeostasis and important functions such as biotransformation and metabolism of drugs. The liver is also the most common target for drug-induced toxicity. Existing in vitro models and in vivo animal models have limited predictive power for human liver toxicity. The goal of this project is to construct a microfluidic liver modul which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This human liver model is expected to be the essential elimination organ for modeling human exposure, provide improved predictions of drug induced liver toxicity, and also serve as a disease model for drug discovery. Our approach will be to develop a 3D microfluidic system with human hepatocyte, kupffer, stellate and endothelial cells, to mimic the liver acinus - the smallest functional unit of the liver. A uniue feature of the model will be the oxygenation of the media, and the establishment of an oxygen gradient, which is believed to account for important metabolic, gene expression and functional heterogeneity of the hepatocytes in the sinusoidal space of normal human liver. Hepatocytes in the oxygen rich zone are efficient in oxidative metabolism, fatty acid oxidation, gluconeogenesis, bile acid extraction, ammonia detoxification to urea and glutathione-conjugation while hepatocytes in the oxygen depleted zone are efficient in glycolysis, liponeogenesis and Cytochrome P-450 biotransformation. Another unique feature of the model will be the incorporation of 'sentinel' biosensor cells, a small fraction of cells with engineered biosensors that indicate changes in cellular functions. When combined with other fluorescent probes, standard biochemical and mass spectroscopy readouts, the model will provide a real-time High Content Analysis (HCA) profile to monitor organ function and response. The selection and validation of readouts and performance of the model will be evaluated based on a panel of reference drugs with available clinical data. To facilitate that comparison, a database of drugs with clinical data, and data from other in vitro and in vivo studies will be constructed. The ultimate goal of this project is to develop a microfluidic model of human liver function that will integrate with a series of other human organ modules, to create a microphysiology platform that reproduces human clinical trial results and provides improved predictivity of exposure, safety and efficacy for drug development. The liver plays a central role in human drug interactions, both within the liver and in other organs, as a result of drug metabolism. The performance of the liver module is central to the performance of the microphysiology platform. We believe the design proposed here will optimally recapitulate human liver function on that platform. PUBLIC HEALTH RELEVANCE: The liver plays a central role in human drug interactions and is also the most common target for drug-induced toxicity, resulting in costly, late stage drug failures. The goal of this project is to construct a microfluidic liver module which mimics the functions and responses of the human liver, with readouts designed to indicate both normal liver function and toxic responses. This module will be designed to integrate with other organ models forming a human microphysiology platform to improve drug efficacy and safety testing.

? DESCRIPTION (provided by applicant): Biomedical Science and Engineering PhDs are faced with a dwindling number of faculty openings, and therefore, an increasing proportion of trainees conduct research in non-academic venues such as the private sector and the government, and yet another substantial number pursue non-research careers in the broad health and life sciences industry. Predoctoral and postdoctoral training programs provide little formal training for nonacademic jobs, and faculty trainers poorly understand the non-academic paths themselves. Biomedical PhDs are therefore unaware of the opportunities that may exist outside of academia, and lack many professional skills that sought after in these sectors of the job market. To address these gaps, we will establish the Rutgers Interdisciplinary Job Opportunities for Biomedical Scientists (iJOBs) program, open to predoctoral, postdoctoral, and recent Rutgers alumni in all biomedical sciences and engineering disciplines. For trainees, the iJOBS program begins with site visits to partnering companies and monthly networking events with working professionals to learn about various professional options. Participants then receive formal training via courses and shadowing experiences in core professional skills (such as communications, performance management, and team building) and in one of five professional tracks of their choice: i) science and health policy, ii) business management, iii) intellectual property management, iv) clinical and regulatory sciences, and v) health and science data analysis. Each participant is assigned a professional mentorship pod consisting of their research faculty advisor, iJOBS program faculty member, and an external mentor from industry, who assist the trainee through their individual Professional Development Plan. Several months prior to graduation, trainees work jointly with iJOBS staff and Rutgers Career Services for application package development and interview skill support, participation in career fairs and assistance with job placement. Finally, employed alumni of the program are invited to give back by providing mentoring and shadowing opportunities to current trainees. An external assessor will evaluate the program on an ongoing basis using data provided by trainees, faculty, and external mentors to determine its strengths and weaknesses. Simultaneously, the assessor will monitor changes in the supply and demand needs for PhD graduates, so that the program activities and areas of concentration can be adapted to the constantly evolving job market. Workforce data and iJOBS best practices will be disseminated to educate our faculty about the changing needs of our students and postdoctoral fellows. We will also disseminate this information nationally by publishing research articles and opinion pieces, collaborating with other existing BEST programs, and holding annual symposia to encourage dialogue among other universities and the bio-industry. The iJOBs program will enable flexible and informative exploration of careers outside of academia by trainees, and strive to increase cooperation between academia and industry so that a new workforce model for biomedical trainees can emerge.

? DESCRIPTION: Diagnostic blood testing is the most ubiquitous clinical procedure in the US, with over 1 billion tests performed annually. The traditional method of blood testing involves performing a venipuncture, transporting samples to a centralized lab, analyzing the samples using large benchtop instruments, and relaying the results to the clinician. This is a labor intensive, time consuming, and expensive process, and unexpected delays often arise due either to difficulties in performing the venipuncture or the time needed to transport and analyze the sample. Particularly in the hospital emergency department (ED), rapid changes in a patient's condition necessitate immediate response, and thus delays can be life threatening. Point of care (PoC) blood testing has emerged as a way to potentially reduce turnaround times, and several devices based on capillary blood draws have achieved commercial translation. However, in comparison to centralized testing, the quantity of available assays remains limited for existing PoC testing, and the accuracy of results obtained with capillary blood remains controversial. Venous blood draws via a venipuncture allows for the collection of a larger volume of blood, yielding more dependable results as the specimen comes directly from the circulation. However to date, no fully automated venous access devices are available, either as independent units, or coupled with POC testing platforms. To address these current limitations, our group is developing a portable, automated device that performs venipuncture and provides quantitative diagnostic results in 15 to 30 minutes at the point of patient care. The portable device will operate by imaging a patient's veins, autonomously introducing a needle into a selected vein, drawing blood into microfluidics chips, and performing on-board blood analysis via an integrated optical imaging platform. A single device will be able to support the throughput of most emergency departments; in this way, the proposed device would serve as an all-in-one portable STAT lab for rapid, automated emergency diagnostics. Outside of emergency medicine, the device could furthermore have strong impact in areas such as ambulatory and outpatient facilities, pediatric and geriatric care, as well as military use.

? DESCRIPTION (provided by applicant): The overall goal of the Rutgers Ph.D. Training Program in Biotechnology is to provide deserving predoctoral students with an integrated multidisciplinary educational and research training experience in biotechnology. The program provides 2 years of funding to meritorious students, with the contractual understanding that students will participate in all program activities for their entire graduate careers. The training experience involves a unifying and multifaceted curriculum which includes: 1) specialized courses that provide the student with critical perspectives of the field from three different vantage points (academic research, industrial R&D, and new ventures); 2) summer industrial internships; 3) training in the responsible conduct of research; 4) an Annual Symposium; and 5) a Ph.D. dissertation research project in one of the laboratories of our 39 participating faculty mentors who are appointed in many different life science and engineering departments. We are requesting 10 NIH-funded predoctoral positions per year for 5 years. Rutgers has committed significant matching fellowship support, stipend and tuition supplements, and other administrative support. Industrial interaction is exceedingly strong, with industrial investigators participating in the teaching of courses, presenting at the Annual Symposium, hosting summer interns, providing matching support, serving on thesis committees, and occasionally hosting students for a part of their Ph.D. dissertation work. The participating graduate programs and departments, together with several biotechnology related centers provide exceptional facilities for comprehensive biomedical biotechnology research and education. Vigorous and extensive recruitment efforts are expended to attract applicants to the program, and only students of exceptional abilities and motivation are admitted. The program also vigorously recruits students from under-represented minorities into the biotechnology field with excellent success. Student progress is continuously monitored through progress reports (and via individual development plans in the future). These documents, as well as surveys of the students' progress in their careers after they leave the university will be used to evaluate the effectiveness of the program. These efforts will continue unabated through the ongoing program, which will continue to produce professionals who are well-educated within a single discipline and have the cross-disciplinary skills needed to conduct independent research and development at the forefront of the constantly evolving discipline of biotechnology.

PI: Yarmush, Martin L.
Proposal Number: 1512170

Traumatic brain injuries (TBI) are the leading cause of disability each year in the US and are also a major risk factor for epilepsy in both injured civilian and military populations. TBI dramatically reduces quality of life in affected patients and there are significant direct and indirect costs associated with TBI. While some drug TBI treatment protocols are under clinical review, none has been identified which can significantly attenuate the progression of events leading to neurological impairment. Improved in vitro screening methods are critical to expedite drug identification and development. Animal studies are both expensive and time consuming, but most in vitro approaches fail to recapitulate in vivo central nervous system inter-cellular connections and responses. Therefore, the goal of the proposed studies is to develop a novel high content "Brain-on-a-Chip" device, which integrates pairs of brain tissue slices and uses novel microfabrication and optical imaging tools, to identify drug candidates that can be used to treat TBI.

Many recent studies indicate that mitochondrial dysfunction contributes to secondary TBI severity and associated axonal dysfunction. As such, the investigators aim to develop a high-content approach to screen mitochondrial drugs to alleviate post-TBI neuronal decay. An interdisciplinary team of science and engineering investigators will utilize microfabrication techniques to develop a "Brain-on-a-Chip" device which will be used to culture paired brain organotypic tissue slices with individual interconnecting axons that extend over microchannels. Strain injury will be introduced by pressurizing a cavity beneath the microchannels. Integrating a multi-electrode array (MEA) on-chip will enable precise and on-line identification of electrophysiological changes in response to injury. The investigators expect to assess how various strain injuries affect electrophysiological and biochemical responses between two organotypic slices using a novel dynamic optical imaging approach. By using microfabricated "Brain-on-a-Chip" arrays, the investigators will be able to screen, in parallel, drug candidates both individually and in combination, more efficiently than has been previously possible. Establishment of such a novel platform is significant, because it would accelerate the identification of molecular entities which control the injury response and, in concert, the development and screening of drug treatments for complex circuit disorders like TBI and epilepsy. The education plan includes high school, undergraduate, and graduate training components with a focus on underrepresented student education. Furthermore, industrial practitioners will be involved in bioengineering courses, which is an effective approach allowing student exposure to the industrial environment.

With the number of Americans who take more than one drug for their well-being increasing, there is an increasing risk of adverse events due to drug-drug interactions. New tools are necessary to understand biology at the initiation of the drug metabolic process, i.e., transcriptional regulation. Since multiple enzymes may act on a given drug, and multiple transcription factors may activate an enzyme, the problem becomes quite complex. In addition, new in vitro tissue engineered models that recapitulate the human physiology are required to get an accurate approximation for the in vivo response of drugs. We propose to extend out microphysiological in vitro liver model to a liver-on-a-chip array to study in high-throughput the transcriptional activity of enzymes and apply it to the study of drug-drug interactions (DDIs). Our tissue engineered construct will use both primary human cells and induced pluripotent stem cells (iPSC)-derived cells. This project will be carried out by distinct research groups. The PIs have a very strong collaborative record of accomplishment. Dr. Martin Yarmush (MGH) will oversee tissue engineering of the liver-on-a-chip and the drug interaction studies. Dr. Mehmet Toner, Director of the BioMEMs Resource Center at MGH, will lead the microfabrication group focusing on the development of microfabricated array. The iPSC-derived cells will be sourced from Dr. Yoon Y Jang at Johns Hopkins University.

ABSTRACT Although several therapeutic options to treat chronic diabetic wounds exist, ranging from occlusive dressings, vacuum assisted closure, skin grafts, to bioengineered skin substitutes, in many instances the wounds fail to adequately respond to treatment. Chronic wounds are characterized by a failure to progress from the pro- inflammatory to the proliferative phases of wound healing. While it has been proposed to provide exogenous growth factors to the wound to help in this transition, there has been very little success using such an approach in practice. Peptide growth factors are rapidly degraded due to the overabundance of proteases in such wounds. Furthermore, recent evidence suggests that the increased levels of advanced glycation endproducts (AGEs) in the diabetic environment may interfere with signaling pathways thus making target cells poorly responsive to bioactive peptides (such as growth factors and chemokines). We have recently shown that these responses can be restored by blocking the receptor to AGEs (RAGE) using soluble RAGE (sRAGE). We propose to develop a multi-functional nanoparticle system consisting of fusion proteins of elastin-like peptides (ELPs) with relevant bioactive peptides and sRAGE. We hypothesize that these nanoparticles can exclude proteases, protecting the attached biopeptides from degradation, and that the simultaneous release of sRAGE can restore signaling in the diabetic wound. Furthermore, these nanoparticles spontaneously and reversibly self-assemble at physiological temperatures, thus enabling rapid and inexpensive purification of the fusion proteins, and with a size below 1 micrometer, nanoparticles are small enough to be easily incorporated into topical treatment modalities, including advanced methods (e.g. skin substitutes, which typically have pore sizes in excess of 50 micrometers). To test the hypothesis, we will develop a sRAGE-ELP fusion protein and combine it with one of three different bioactive peptides that target different aspects of the wound healing process: KGF-ELP (epidermis), SDF-ELP (dermis), and ARA290-ELP (tissue protective response). Our specific aims are: (1) To develop sRAGE-ELP fusion proteins that reversibly form nanoparticles with themselves and other peptide-ELP fusion proteins. (2) To evaluate the biological activity of ELP-based nanoparticles in a simulated diabetic environment in vitro. (3) To test the effect of sRAGE-ELP nanoparticles in in vivo diabetic wound conditions.

PROJECT SUMMARY With more than five times the number of patients on the wait list than will receive a donor organ in the United States, the field of transplantation is facing a serious donor shortage crisis. Overcoming the organ shortage will require integrated strategies, including a particular focus on overcoming ineffective bio-preservation and stabilization protocols. Longer storage durations will provide the infrastructure required to enable global matching programs, eliminate the need to scramble and conduct unplanned surgeries, and reduce unnecessary waste of quality organs. We believe the method for preserving mammalian organs should employ hibernating and freeze-tolerant strategies in nature that are then further augmented using bioengineering principles. Consequently, we seek to develop a protocol for human organ preservation which will achieve high subzero storage temperatures (ranging from -10 to -20 °C) in the presence of extracellular ice, and storage durations of weeks to months, using inspiration from in nature. Our approach is unique in organ/tissue preservation literature since we aim to actively initiate ice propagation in the vasculature and extracellular spaces, rather than extreme means of inhibiting ice crystallization as is the current standard. The presence of non-injurious ice will be essential in achieving longer storage durations, while also playing an important role in the scale-up to human livers. While this program targets the banking of human liver, our discoveries and solutions will be translatable to other tissues and organ systems. In Specific Aim 1, we will adapt endothelial cell-coated microvascular networks already developed by our group3 in order to model and develop strategies to overcome challenges associated with ice propagation. Since endothelial cells in the vasculature will be the most vulnerable to ice propagation, SA#1 will be an essential proof of concept of our novel strategy and we already have promising data. In Specific Aim 2, we will engineer an ice nucleating agent which will promote non-injurious propagation of ice in extracellular spaces. Ice nucleating agents are essential for restricting ice formation to extracellular spaces and have been identified as critical strategies for freezing survival. In Specific Aim 3, we will reprogram cells to descend into a state of `suspended animation' with enhanced stress tolerance, as inspired by nature. We will achieve this using both passive temperature effects as well as using pharmacological agents. We will perform in-depth characterization of the molecular impact of our cellular reprogramming efforts. In each specific aim, we scale up rapidly to rat whole liver while also validating in human livers in order to maximize impact.

Bioengineering Career Clusters and Frameworks Martin L. Yarmush, M.D, Ph.D., Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA; Susan Engelhardt, Department of Biomedical Engineering, Rutgers University, Piscataway, NJ, USA yarmush@soe.rutgers.edu +1 848 445 6528 Keywords: career framework, career cluster, bioengineering career, career advancement, professional skills, case studies Abstract: A vast majority of biotechnology program graduates seek careers other than those for tenure-track faculty positions. Although current courses in the typical graduate curriculum appropriately deliver strategic discipline-based learning for life science and engineering graduate students, critical is these scientists be made aware of, and prepared for, the full range of careers available within the biomedical science and engineering ecosystem. Employers have defined career frameworks, clarifying the skills and knowledge required for different types of work, defining job families and appropriate career progressions to encourage employee skills-building and retention and enabling managers to have conversations that encourage career development. As each trainee explores his/her career path, it is vitally important for him/her to understand these frameworks and how they should work within them toward career advancement, understanding how to navigate influence of company size, organization structure, performance/associated metrics, work culture, growth/advancement, political/regulatory environment, societal topics/issues, etc., as well as work independence, accountability for impactful results, influence on organizations and (corporate) results, business and societal impacts, and documentation/communication responsibilities. It is based upon this need that we propose the addition of a 3- credit course that guides trainees regarding these career frameworks and discusses specifics relative to overarching biotechnology career paths. The motivation is to enhance our students' competitive skills as, although our curricula produce scientific excellence, technical competence is ?necessary but not sufficient? for carrying out the responsibilities of today's professionals, whether as researchers, or as contributors to business, clinical translation, legal or related areas. The syllabus combines didactic instruction with expository case studies, reinforcing key learnings, as they review and analyze case studies specific to various professional environments and challenges and present recommendations to the class to seed group discussions and further role-play. At the end of the semester, students present a case study based upon their area of professional interest with analysis of the actions and inactions relative to the concepts taught in class. The course will be offered as a permanent component of the Biomedical Engineering graduate program.

7. PROJECT SUMMARY/ABSTRACT The overall goal of the Rutgers Biotechnology Training Program is to provide outstanding predoctoral students with an integrated multidisciplinary educational and research training experience in biotechnology. The specific aims are to: 1) increase the number of well-trained PhDs able to conduct cutting-edge research in biotechnology and related areas; 2) to increase the number of under-represented minorities receiving biotechnology related-degrees; and 3) to enhance the overall training experience by re-envisioning predoctoral education through a diversified set of enrichment activities with appropriate reinforcement throughout the training period. This comprehensive program builds on our nearly 30 years of prior experience and continuous NIGMS support in this area. Students in the program receive a broad understanding across biomedical disciplines and the skills to independently acquire the knowledge needed to advance their field, as well as the ability to think critically and independently, and to identify important research questions and approaches that push forward the boundaries in their areas of study. The program provides 2 years of funding to meritorious students, with the contractual understanding that students will participate in all program activities for their entire graduate careers. The training experience involves a unifying and multifaceted curriculum which includes: 1) specialized courses and experiential activities that provide the student with critical perspectives of the field from multiple vantage points; 2) summer industrial internships; 3) training in responsible conduct of research and rigor/reproducibility; 4) an annual symposium; and 5) a PhD dissertation research project in one of the laboratories of our 43 participating faculty mentors who are appointed in many different life science and engineering departments. We are requesting 13 NIH-funded predoctoral positions per year for 5 years. Rutgers has committed significant matching fellowship support (7 matching fellowships), stipend and tuition supplements, and other salary and administrative support. Industrial interaction is exceedingly strong, with industrial investigators participating in the teaching of courses, presenting at the Annual Symposium, hosting summer interns, providing matching support, serving on thesis committees, and occasionally hosting students for a part of their PhD dissertation work. The participating graduate programs and departments, together with several biotechnology related centers provide exceptional facilities for comprehensive biomedical biotechnology research and education. Vigorous and extensive recruitment efforts are expended to attract applicants to the program, and only students of exceptional abilities and motivation are admitted. The program also vigorously recruits students from under-represented minorities into the biotechnology field with documented excellent long-term success. Student progress is continuously monitored through progress reports and via individual development plans. These documents, as well as surveys of the students? progress in their careers after they leave the university will be used to evaluate the effectiveness of the program.

Abstract Red blood cells (RBCs) are possibly the most transfused blood component and the most widely stored cell type. While cold storage (+4 oC) of RBCs has been vastly improved in the last few decades with a standard storage time of 42 days in clinical settings, recent clinical retrospective as well as laboratory studies indicate that beyond 14 days of storage RBCs might have vastly different biochemical properties and possibly inferior outcomes in patients. Here, we propose to develop an RBC preservation method based on our recent breakthrough in deep supercooling (DSC) of aqueous solutions where we can achieve seemingly stable supercooling for large volumes and at very low temperatures (down to -20 oC) for very long times. This is achieved by surface sealing of aqueous solutions via water-immiscible hydrocarbon-based liquids. Our central hypothesis, based on preliminary studies and prior work, is that DSC of RBCs can provide an immediately practical, high quality, and long-term storage, as an alternative to current clinical standard of cold storage. Our approach is to first establish a robust characterization framework for storage related injuries to a) establish the basic temperature optimization of DSC for RBCs, b) while comparing the cell quality of this basic approach to cold storage and cryopreservation. We will then supplement the DSC approach drawing from our experience in alleviating lipid peroxidation, oxidative stress, and membrane injuries and metabolic suppression along with recent advances in RBC preservation such as anaerobic preservation to achieve long-term storage of RBCs in small volumes. Finally, we aim to extend the range of parameters for DSC to the 150-500 ml range (volume), ~- 25-30 oC (temperature), and 150 days (time) in parallel. Our final goal is to conduct storage with these robust DSC strategies to preserve a clinical unit (~300 ml) of RBCs for 150 days. By completing this project, we expect, to demonstrate a novel method to dramatically extend the storage time for RBCs to 150 days whereby alleviating problems of current approaches. In the long-term the ?large volume DSC? method, is widely applicable to all cell, tissue and organs; especially those that are not amenable to cryopreservation. These advances will positively influence healthcare by enabling storage of living matter for applications in cell/organ transplantation, engineered tissue logistics, and food storage among others