Posters

The poster session and reception will take place from 6:30 pm - 7:30 pm on the ground floor of Maxwell Dworkin.

Educational Programs

The GK-12 Program: Involving Graduate Students in K-12 Education

Presenter: Kathryn Hollar

The NSF-funded GK-12 Program is a national outreach program designed to bring current graduate students of local public high schools, as valuable and versatile resources for student, teacher, and curriculum support. The Division of Engineering and Applied Sciences at Harvard University has partnered with the Cambridge Public School System for the past three years to infuse the classroom experience with exposure to cutting edge science and engineering, and to provide the opportunity for students to interact with enthusiastic young scientists. Among the topics overviewed are in-class mentoring and lecturing; classroom activity, laboratory, and demonstration develop- ment; after school enrichment; case study design; science fair project mentoring; teacher professional development; and the overall impact of the graduate student presence at the high school level. The integration of graduate students into physics, chemistry, biology, and computer science classrooms has had a lasting positive impact on all that are involved, and the partnership developed between Harvard and Cambridge Public Schools can serve as a model for building long-term relationships with urban school systems.

Educational Programs of the MRSEC and NSEC Based at Harvard

Presenter: Kathryn Hollar

The NSF-sponsored Materials Research Science and Engineering and Nanoscale Research Science and Engineering Centers based at Harvard University actively promote interdisciplinary education and research in materials and nanoscale science and engineering. Center participants are actively involved in programs that engage the public, teachers, students, and young scientists and engineers in the excitement of scientific discovery and that increase awareness of the impact of scientific research on their daily lives. Our broad goals as Centers are to increase public engagement in and awareness of advances in materials science and engineering, and to promote career advancement for a diverse group of young scientists who represent the future of nanoscale and materials science. Our educational initiatives at the pre-college, undergraduate, graduate, and postdoctoral levels include embedded diversity initiatives and strategic collaborations whenever possible to encourage individuals from underrepresented groups to pursue careers in science and engineering. An overview of our educational activities, including Research Experiences for Teachers, Research Experiences for Undergraduates, K-12 programs, community celebrations of science, and collaborations with partners such as the Museum of Science , Boston , is presented in this poster.

Seeing the Unseen, Presenting Nanotechnology to the Public

Faculty: Carol Lynn Alpert
Contributors: C.L. Alpert, J. Antill, A. Weiss, and G. Murray
Presenter: Daniel Davis

The Current Science & Technology Center at the Museum of Science , Boston performs live multimedia presentations, broadcasts weekly regional cablecasts, produces multimedia web and touch screen stories and news bytes, hosts guest speakers, and conducts student and teacher workshops. Content is conveyed to diverse public audiences varied in age, interest, and education using familiar analogies, dynamic props, and models relating form to function. The center's nanotechnology related work may be reviewed online at www.mos.org/nano, and inquiries are welcomed to Carol Lynn Alpert calpert@mos.org or Daniel Davis danieldavis@mos.org (617) 589-0119.

Division of Engineering and Applied Sciences (DEAS)
Center for Nanoscale Systems (CNS)
Harvard Medical School (HMS)

Center for Nanoscale Systems: Tools for New Discoveries

Faculty: Charles M. Marcus
Contributors: CNS Staff
Presenter: David C. Bell

A brief overview of the Center will be presented.

Mechanical Characterization of Liver for Surgical Simulation

Faculty: Robert D. Howe
Contributors: S.L. Dawson, R.D. Howe, A.E. Kerdok, S. Socrate
Presenter: Amy E. Kerdok

Computer-aided medical technologies such as simulators for surgical training, planning, and assessment, are currently limited by the inability to realistically portray the behavior of the involved tissues. The goal of this work is to accurately characterize the mechanical behavior of liver under large deformations typical of surgical manipulation. Three steps are required for this characterization. First, the effect of testing condition on the sought-after behavior is identified. A study that evaluated the effects of perfusion on the viscoelastic response of liver resulted in the development of an ex vivo perfusion system that nearly approximates the in vivo condition. Second, a mathematical model is derived that is capable of capturing the liver's nonlinear, viscoelastic response based on the physical make-up of the tissue. Third, using the perfusion apparatus, indentation tests are performed to identify and validate the model's parameters by solving the inverse problem using an iterative approach.

The liver is a highly vascular organ. An ex vivo perfusion apparatus was developed that approximated physiologic boundary conditions to study the effects of perfusion on the viscoelastic response of liver. A manually driven indenter was used to study the large deformation (~50% nominal strain) creep response of the whole liver across in vivo , ex vivo perfused, and ex vivo post perfused conditions, and on an ex vivo section. Results indicate that 1) unperfused conditions were stiffer and more viscous than the perfused ones, 2) the ex vivo perfused condition nearly approximates the in vivo condition, and 3 ) two time constants of roughly 1.86 and 51.3 seconds are needed to describe the response. Further investigation of the liver's physiology suggests that the slow time constant is from blood flow through the resistive microvasculature (sinusoids), and that the fast time constant is from extracellular fluid flow and cellular deformation.

The proposed model is a viscoelastic modification of the Arruda-Boyce hyperelastic model whose global response combines contributions from the tissue's microstructure in the form of three parallel networks. These include a hyperelastic 8-chain network to capture the elastic response from collagen and elastin fibrils, a viscoelastic network to establish the time-dependent response from the parenchymal cells and the extracellular fluid, and a network that provides an induced pressure from the vascular flow within the organ using a linear Darcy's law. A two-layered 2-D axisymmetric finite element model has been developed to model the liver and its elastic capsule with indentation boundary conditions. An iterative inverse approach is used to determine the material parameters of the model by minimizing the least-squares error between the models' predicted force-displacement response and data from large strain viscoelastic indentation tests on perfused ex vivo porcine livers. Three indentation tests will be conducted using a linear actuator. Load-unload tests will be used to initially identify the model's parameters using the iterative optimization approach described above. Data from stress-relaxation and creep tests will then be used to validate the identified parameters and provide a measure for their uniqueness.

Guiding Robotic Surgery with 3-D Ultrasound: A Study of Instrument Size on Surgical Performance

Faculty: Robert D. Howe
Contributor: P.M. Novotny
Presenter: Paul M. Novotny

Real-time 3-D ultrasound has become a promising new tool for guiding surgical procedures. In particular, minimally invasive applications within a beating heart are now possible because of ultrasound's high tissue/blood contrast and a temporal resolution capable of capturing fast-moving cardiac structures. Intra-cardiac beating heart procedures present new challenges such as limited workspace and a highly dynamic environment. A surgical robot's abilities are well suited for these conditions, due to increased dexterity, accuracy, and speed. However, the use of ultrasound to guide surgical procedures decreases a surgeon's ability to perceive and manipulate surgical instruments, as ultrasound is not well suited for imaging hard highly reflective objects. We are beginning to explore these new challenges and numerous ways to overcome these shortcomings. In this paper we evaluate the performance of subjects controlling a surgical robot and using real-time 3-D ultrasound for guidance. Specifically we looked at the role of instrument size and how it affects a subject's ability to control a surgical robot.

Signal Transduction Pathways that Regulate Cell Fate in Normal and Cancer Cells

Faculty: Roya Khosravi-Far
Contributors: R. Khosravi-Far, A. Chalah
Presenter: Anas Chalah

Normal development of multicellular organisms is controlled by a delicate balance between signals that regulate cell proliferation, differentiation and programmed cell death. Deregulation of any of these cellular processes leads to a variety of human diseases, including cancer. A major interest of our laboratory is elucidating the mechanism by which oncogenes promote evasion of tumor cells from apoptosis. In addition, we are investigating the signal transduction pathways that regulate cell fate, in particular ones that are involved in mediating apoptosis by members of the TNF family of ligand. Selected examples of our current projects are described briefly:

1) A crucial aspect of tumorigenesis is the evasion of apoptosis. An understanding of how oncogenes cause resistance to apoptosis will help in the design of more suitable anti-cancer therapies, yet, the mechanisms by which some of the most prominent oncogenes, such as BCR-ABL enable protection from apoptosis are far from clear. Recently, we have observed that BCR-ABL induces evasion of transformed cells from apoptosis by down-regulating the transcription of many pro-apoptotic factors. We are currently investigating the molecular mechanism by which BCR-ABL regulates the transcription and expression of these pro-apoptotic genes.

2) Recent discovery of TNF-related apoptosis inducing ligand, TRAIL, that selectively induces apoptosis of transformed cells in vitro , has provided hope for the use of TRAIL as a cancer therapeutic. However, the signaling events that mediate the biological effects of TRAIL and its receptors are not well characterized. Recently, we have identified several proteins that specifically interact with TRAIL receptors. We are characterizing these proteins and have found that one of them specifically interacts with TRAIL-R2 in a ligand-independent manner to prevent spontaneous death signaling. We are currently investigating the mechanism of function of this and other death receptor interacting molecules.

3) Death receptors initiate ubiquitous pathways of cell death in which caspase activation is mediated either directly without mitochondrial amplification or via the release of apoptogenic factors from mitochondria. We have recently observed that death receptor signaling induces alteration in mitochondrial membrane lipids independently of caspase activation, thus facilitating the action of pro-apoptotic proteins in releasing apoptogenic factors through a PI-3K-dependent pathway. We are currently investigating the molecular mechanism for the caspase-independent regulation of the mitochondrial death pathway.

Single Step Biomarker Discovery Using Self-Assembling Protein Microarrays

Contributors: N. Ramachandran, E. Hainsworth, G. Demirkan, D. Schiwek, K. Anderson, J. LaBaer
Presenter: Niroshan Ramachandran

We have developed a novel self-assembling protein microarray called N ucleic A cid P rogrammable P rotein A rray ( NAPPA ). While traditional protein microarrays require individual production and purification of hundreds or thousands of proteins, with NAPPA the proteins are simultaneously expressed directly from DNA on the microarray surface. We have used NAPPA to map protein interactions, build multicomponent complexes and map binding domains of proteins. Currently, we are using this platform technology to present tumor antigens to screen for immune responses. Cancer patients' own immune systems produce humoral responses to cancer antigens released by their tumors due to alterations in protein expression, mutation, degradation, or localization . Antibodies to a number of tumor-associated antigens have been identified in patient sera, including HER-2/neu, NY-ESO-1, p53, MUC1, cyclin B1, ras, myc, and others . For early detection of disease, the responses to these and other antigens must be combined to markedly improve sensitivity and specificity. However, current methods rely on purifying individual proteins for ELISA assays, which is costly and impractical for large numbers of antigens. In comparison to traditional ELISAs, protein microarrays are capable of presenting and assessing hundreds of tumor antigens simultaneously. The responses are rapidly identified because the address of each protein is known in advance and there are no representation issues; all proteins are represented equally (usually in duplicate). The proteins are arrayed on a single microscope slide requiring only a few microliters of serum per assay. Known tumor antigens as well as predicted tumor antigens can be included to generate a comprehensive protein tumor antigen array. An additional advantage of this approach is that the data can be evaluated to look for both informative individual antigens as well as for patterns of antigen responses with good predictive value.

Nanoscale Science and Engineering Center (NSEC)
Harvard University ·MIT·USCB· Museum of Science , Boston

CMOS/Microfluidic Hybrid Microsystem for Manipulation of Biological Cells

Faculty: Donhee Ham, Robert M. Westervelt
Contributors: H. Lee, Y. Liu
Presenter: Yong Liu

We combine microelectronics and microfluidics to precisely manipulate individual biological cells. This CMOS/microfluidic hybrid microsystem is made by fabricating a microfluidic system on top of a CMOS chip. Magnetic-bead-tagged biological cells are suspended in a biocompatible environment. The chip produces patterned magnetic fields using a microcoil array to manipulate individual bead-bound cells. The hybrid prototype manipulated BCE cells with precise spatial control. In the 2nd-generation chip, new features are exploited, such as time-division current sharing to reduce power, current direction control for higher spatial resolution and integrated temperature sensing circuit.

Dielectrophoresis Tweezers

Faculty: Robert M. Westervelt
Contributors: T.P. Hunt
Presenter: Tom P. Hunt

Dielectrophoresis tweezers are a simple means of manipulating individual cells. Two electrodes on either side of a sharp glass tip provide a localized electric field that can be used to trap a single cell. This label-free technique allows us to hold a cell and move it without injury.

Optofluidic Mid-Infrared Laser Platform for Biochemical Applications

Faculty: Federico Capasso
Contributors: F. Capasso, M. Loncar, S. Tang
Presenters: Marko Loncar, Sindy Tang

We are developing an optofluidic laser platform that can be used for on-chip spectroscopy and chemical sensing via intracavity microfluidic delivery of bio/chem materials. Novel "holy" quantum cascade lasers emitting in mid-infrared are used as light sources in this lab-on-a-chip system. Porous nature of our photonic crystal resonators enables introduction of analyte directly into the "heart" of the laser, close to its active region. This results in the strong interaction between light and analyte and increased sensitivity of our system. Microfluidic channels, defined in soft materials (e.g., PDMs rubber) are used for fluid delivery. Changes in emission wavelength or intensity of the light emitted by laser are used to detect the presence of various biochemical reagents. Due to the compact nature of photonic crystal resonators, it is possible to integrate many lasers emitting at different wavelengths on the same chip and therefore interrogate several chemicals in parallel. Another interesting application of our optofluidic platform is reconfigurable laser sources, since emission wavelength and overall light output-current characteristics can be modified by introducing fluids directly into the laser cavity.

Charge and Spin Manipulation in Nanoscale Electronic Devices

Faculty: Charles M. Marcus, Mikhail D. Lukin, Arthur C. Gossard
Contributors: N. Mason, S. Garaj, J.M. Chow, J. Martin, A. Yacoby, D.J. Reilly, T.M. Buehler, V. Chan, R.G. Clark, S. Garaj, and J.R. Petta, J.M. Taylor
Presenter: Michael Biercuk, Alex C. Johnson

We present developments in technology and measurement techniques leading towards the realization of a solid-state quantum computer using quantum dots as the basis for spin qubits. Experimental advances exploiting carbon nanotubes and GaAs 2-D electron systems will be discussed, including recent measurements of electron spin dephasing in GaAs double quantum dots and single charge detection in carbon nanotube quantum dots.

Localization and Visualization of Calcium Sparks in Micropatterned Cardiac Myocytes

Faculty: (Kevin) Kit Parker
Contributors: M-A. Bray, N.A. Geisse, K.K. Parker
Presenter: Mark-Anthony Bray

Elementary calcium release events ("sparks") are believed to underlie the mechanism of mechanoelectrical coupling in the cardiac myocyte. The cytoarchitecture of the myocyte has been determined to be critical in understanding not only mechanical contraction of the cell but also electrical propagation. Knowledge of this mechano-transduction mechanism has implications in the treatment of stretch-activated arrhythmias as well as understanding the role of the extracellular environment on intracellular signaling pathways. Our objective is to micropattern myocytes into various shapes and examine spark occurrence as a function of cell shape.

Haptic Interface for Cardiac Cell Exploration Using AFM

Faculty: (Kevin) Kit Parker
Contributors: D.P. Perrin, C.R. Wagner, N.A. Geisse, R.D. Howe, K.K. Parker
Presenter: Douglas P. Perrin

The atomic force microscope (AFM) is similar in operation to a record player. A micro-fabricated stylus that terminates to tip with an end radius on the order of a few nm is mounted onto a 3-axix piezoelectric material which drags the stylus across the surface of interest. Measuring the amount of light reflected from the AFM tip quantifies the amount of tip deflection. The deflection at a given position can be used to determine a force on the stylus. Combining a haptic display with the AFM allows a user to command the position of the AFM tip while simultaneously sensing the force on the tip. Our system consists of a Asylum Research AFM and a Phantom 1.5 haptic interface. The AFM has a resolution on the order of a few nm in X-Y, and approximately 0.1 nm in Z. The forces encountered by the tip are rendered on the Phantom using a gain of approximately 10,000,000.

Control of Cardiac Myocyte Architecture

Faculty: (Kevin) Kit Parker
Contributors: N. Geisse, S.P. Sheehy, A.P. Ziman, W.J. Lederer
Presenter: Nicholas Geisse

-- The heart uses mechanical stimulus to regulate its physiology via a process known as mechanotransduction.

-- Cell shape and cytoskeletal morphology determine distribution of internal and external cellular stresses, and changes in the failing heart.

-- Controlling cell shape can control cellular stress distribution.

-- Cellular shape and stress determine cytoskeletal architecture and the organ-ization of components of the contraction regulation system.

-- Cardiac cells organize both their contractile machinery and regulatory machinery based on geometrical cues from their environment, which may be optimized for mechanotransduction.

Developing New Approaches to Fabricate Nanoscale Structure

Faculty: George M. Whitesides
Contributors: B.T. Mayers, G.M. Whitesides, Q. Xu
Presenter: Qiaobing Xu

We will present two simple and convenient non-traditional techniques to fabricate nanoscale structures. The first technique uses microtome sectioning of thin films to convert materials that are nanoscale in one dimension, to materials that are nanoscale in two dimensions. The second technique makes use of atomically flat silicon wafers which can be partially cracked to give uniform step gradients from the micron scale to the atomic scale.

Materials Research Science and Engineering Center (MRSEC)
Harvard University

A General Method for Patterning Gradients of Biomolecules on Surfaces Using Microfluidic Networks

Faculty: George M. Whitesides
Contributors: X. Jiang, Q. Xu, S.K.W. Dertinger, A.D. Stroock, T.-M. Fu, G.M. Whitesides
Presenter: Brian Mayers

We will present a general method for the fabrication of immobilized gradients of biomolecules on surfaces. This method utilizes a microfluidic network that generates a gradient of avidin in solution and immobilizes this protein on the surface of glass or PDMS by physical adsorption. The immobilized gradient of avidin is then translated into gradients of biotinylated ligands (e.g., small molecules, oligomers of DNA, polysaccharides) using the specific interaction between biotin and avidin. This method is useful for cell biologists who are interested in phenotypes associated immobilized gradients, such as migration, formation of processes and polarity of cells.

The Phase Chip: High Throughput Screening with Microfluidics

Faculty: David A. Weitz, Seth Fraden
Contributor: J. Shim, D. Link, G.C. Azarkarate
Presenter: Jung uk Shim (Brandeis University)

A high throughput, low volume microfluidic device denoted the Phase Chip has been constructed out of poly(dimethylsiloxane) elastomer. We have demonstrated that sub-nanoliter water-in-oil drops of protein solutions of different composition can be rapidly stored in individual wells, which allows screening of 1000 conditions while consuming a total of only 1 microgram protein on a 20 cm2 chip. This reduction in protein needed for crystal screens allows high-throughput crystallization of mammalian proteins expressed in tissue culture. A significant advance over current microfluidic devices is that each sample well is in contact with a reservoir through a dialysis membrane through which only water and other low molecular weight organic solvents can pass, but not salt, polymer, or amphiphile. This enables the concentration of all solutes in a solution to be reversibly, rapidly, and precisely varied in contrast to current microfluidic methods, which are irreversible. This microfluidic dialysis technology solves a major problem in protein crystallization, the decoupling of nucleation from growth. The Phase Chip will also be useful for general studies of the phase behavior of protein solutions, including diseases associated with protein aggregation and phase separations such as cataracts, gall stone formation, and Alzheimer's Disease.

Discretizing Fluid Streams in Micro-Channels Using Multi-Phase Fluid Flows

Faculty: David A. Weitz, Howard A. Stone
Contributor: A.S. Utada
Presenter: Andrew Shinichi Utada

We manipulate multiple fluid phases within micro-channels, which enables precise control of droplet formation, and fabricate novel encapsulated structures that may have direct applications in fields such as biology and chemistry. The use of immiscible fluid can be used to directly encapsulate other fluids or serve as carriers in compartmentalizing interesting materials. We generate capsules with colloidal, polymer, or fluid shells.

An Integrated Magneto-Optic Microfluidic Device for Biosensing and Sorting

Faculty: Robert M. Westervelt, David A. Weitz
Contributors: C. Kerbage, K. Ahn, T. Hunt, C. Brangwynne
Presenter: Charles Kerbage

We demonstrate an integrated magneto-optic microfluidic device for biosensing and sorting. Optical detection of water drops formed in a continuous oil phase flow is performed using optical fibers which are integrated into the channels of the PDMS (polydimethylsiloxane) based microfluidic device. The size and the velocity of the drops can be determined by measuring the transmission intensity as a function of time. We show that such a device can be used to detect fluorescent materials introduced in the drop or the cell itself. Moreover, introducing nanoscale magnetic particles into the water drops allows for drop or cell sorting by means of a magnetic field gradient, which is generated by thin film permalloy integrated into the device itself and tuned by an external coil.

Mechanical Properties of Semiflexible Biopolymers and Living Cells

Faculty: David A. Weitz
Contributors: C. Brangwynne, K. Kasza, G. Koenderink, D. Vader, Y.-C. Lin, J. Liu
Presenter: Charles Kerbage

We study the mechanical properties of semiflexible biopolymers and living cells. Biopolymer networks form a mechanochemical structural scaffold within living cells that is involved in cell division, migration and contractility. In addition, cells apply forces and move through an extracellular structural scaffolding of collagen and other biopolymers. Understanding the mechanical properties of the intracellular and extracellular environments is critical for elucidating both healthy and cancerous cell behavior, such as the rapid and destructive invasion of brain tumor cells. We use a variety of techniques including fluorescence imaging, bulk and micro-rheology, atomic force microscopy, micromechanical manipulation, and microfluidics to study mechanics of in vitro model systems and living cells.

Colloidal Models of the Plastic Deformation of Crystals and Glasses

Faculty: David A. Weitz, Frans Spaepen
Contributors: P. Schall, I. Cohen
Presenter: Peter Schall

Colloidal suspensions are widely used to study a variety of phenomena in hard condensed matter physics. The particles -- several ten nanometers to micrometers in size -- self-organize into structures similar to atoms in different phases of condensed matter. Crystalline as well as amorphous states can be prepared. Being several orders of magnitude larger than atoms, colloidal particles offer the unique possibility for studies at convenient length and time scales.

We use crystalline and amorphous colloidal suspensions as models to study the behavior of their atomic counterparts under applied stresses. We find that strained colloidal crystals exhibit dislocations, one-dimensional defects in the crystalline lattice, which show remarkable similarities to dislocations in atomic crystals. Using confocal microscopy, we are able to study the nucleation, motion and interaction of dislocations on the particle scale. Furthermore, we are able to follow dislocation propagation on a much larger length scale using a self-made laser diffraction microscope. This apparatus is analogous to the transmission electron microscope used to study dislocations in atomic crystals.

In the amorphous suspension, we are able to follow the motion of the individual particles during deformation and identify local events that are indicative of basic localized shear events of the amorphous state.

Self-Assembled Buckling Channel for Fluidic Network in Compressive Film by Buckle Patterning

Faculty: John W. Hutchinson
Contributors: M.-W. Moon
Presenter: Myoung-Woon Moon

The first attempt to apply the buckling channel structure to fluidic applications is presented with buckle patterning technique in compressive film. Confined on patterned substrate of low adhesion layer surrounded by high adhesion substrate, the self- assembled wavy buckling has been developed on the various pattern layers; straight strip, tapered strip and grid strip.

Openings of buckle channel consists of buckled diamond-like carbon (DLC) film as structure layer and gold film as low adhesion layer giving the buckling channel biological functions. A cross-section profile of buckling channel can be controlled by adjusting film thickness of 8 nm to 260 nm, then the associated opening area less than 0-.01 to 10 mm2, which is comparable to the nanoscale area of hollow hole with less than 50 nm to 1.8 mm in radii, respectively.

To be integrated with outer microfluidic interface, the buckling channel was overlaid with polydimethylsiloxane (PDMS) layer, formed an irreversible seal with DLC buckled film and also protected thin buckled film from collapsing by generated flows. We visualized the fluid inside buckling channel integrated with outer fluidic interface by filling of photoresist(PR)-mixed deionized water and focused ion beam sectioning the cured photoresist-filled buckling channel. The developed nanofluidic network using buckling channel can be used as confined nanofluidic networks connected to world-to-chip microfluidic interface, made of different materials.

Femtosecond Laser Dissection of Neurons in C. elegans

Faculty: Eric Mazur
Contributors: D.A. Clark, C.V. Gabel, A.D.T. Samuel
Presenter: Samuel H. Chung

Tightly-focused femtosecond laser pulses of a few nanojoules sever individual dendrites in the nematode worm C. elegans . Quantification of the resulting behavioral deficits identifies the contribution of the dissected structures. The dissection has submicrometer resolution with no collateral damage, permitting precise studies on live animals. Future work include an examination of the molecular basis of neurode-generation that has application to diseases such as Parkinsons and Alzheimers.

Nanosurgery in Live Cells Using Ultrashort Laser Pulses

Faculty: Donald Ingber, Eric Mazur
Contributors: A. Heisterkamp, S. Kumar
Presenter: Iva Maxwell

We performed sub-cellular ablation in live cells by tightly focusing femtosecond laser pulses. We used this nanosurgery technique to sever individual microtubules and actin fibers in endothelial cells. This method allows real time observation and measurement of the retraction of the severed actin bundles, which can be used to extrapolate the prestress of the actin network.

Combined Microfluidic-Magnetic Separation of Particles and Living Cells in Continuous Flow

Faculty: Donald E. Ingber, Robert M. Westervelt, George M. Whitesides
Contributors: S. Xia, T.P. Hunt, B.T/ Mayers, E. Alsberg
Presenter: Shannon Xia

Methods currently used for separation of living cells and biomolecules from blood and other complex mixtures are critical for basic research, medical diagnostics and clinical care, however, they require expensive and bulky equipment that can only be found in a laboratory. Here we describe a miniaturized integrated device that combines microfluidics and micromagnetics to separate and collect micro- and nanometer-sized magnetic particles under continuous fluid flow. By aligning a high-gradient magnetic concentrator next to one side of a microfluidic channel containing two laminar flow paths, we were able to pull magnetic particles from one flow path to the other by applying magnetic fields, and thus, selectively remove these particles and adherent cells from a complex flowing fluid without any moving parts. Several high-gradient magnetic concentrator designs have been evaluated experimentally for their separation efficiency, and one design that incorporates a microfabricated layer of NiFe has been shown to separate magnetically labeled living bacteria from blood with great efficiency. Due to its simplicity, integrated architecture and low cost, this cell separator microchip technology may potentially allow cell separations to be done outside of hospitals and clinical laboratories.

Aspects of Curved Space Crystallography

Faculty: David R. Nelson
Contributors: J.B. Lucks, D.R. Nelson, V. Vitelli
Presenter: Vincenzo Vitelli

The physics of topological defects in two-dimensional crystals is investigated. Using a continuum effective elastic theory as a starting point, we mapped the problem into curved-space electrostatics with disclinations playing the role of electrostatic charges interacting with a background charge distribution determined by the shape of the surface. Using a Gaussian bump as an analytically tractable model system, the dislocation potential is found to be in strong agreement with numerical computations. Because of the known presence of defects in the protein lattices that make up viral capsids, these results could have direct applicability to common example of viral capsid morphology.

Controlled Assembly and Manipulation of Shells of 2-D Colloidal Crystals

Faculty: Howard A. Stone
Contributors: M. Abkarian, A.B. Subramaniam
Presenter: Manouk Abkarian

Assembly of colloidal particles on fluid interfaces is a promising technique for synthesizing two-dimensional micro-crystalline materials useful in fields as diverse as biomedicine, materials science, mineral flotation and food processing. Current approaches rely on bulk emulsification methods, require further chemical and thermal treatments, and are restrictive with respect to the materials employed. Here we report a microfluidic method that allows direct visualization and understanding of the dynamics of colloidal-crystal growth on curved interfaces. The crystals are periodically ejected to form stable jammed shells, which we refer to as colloidal armour. Our method allows an unprecedented degree of control over armour composition, size and stability.

Microfluidic Manipulation and Measurements Applied to Mechanics of Blood Cells: From the Fahraeus-Lindqvist Effect to Biomedical Applications

Faculty: Howard A. Stone
Contributors: M. Faivre, A.B. Subramaniam
Presenter: Manouk Abkarian

We describe a variety of microfluidic studies of blood cells, including experiments with implications for basic hemodynamic understanding, novel dynamic pressure measurements that are combined with simultaneous visualization, and a device capable of filtering or concentrating suspensions. First, we have shown experimentally that the cell-free layer that accompanies flow in the microcirculation is substantially increased due to a small constriction, which may represent a blockage or a measurement device. We refer to this observation, which we have also quantified and modeled, as the enhanced Fahraeus effect. Second, we have developed a microfluidic technique for measuring the pressure changes over time when small particles block, enter or exit a small channel and we have applied the approach to monitor time evolution of the pressure when cells flow through a channel. Finally, we have fabricated a microfluidic filter or concentrator that can take a suspension of rigid particles or blood cells, and output a particle-free filtrate and a suspension of higher concentration.

Mechanical Behavior of Biomolecules and Biomaterials

Faculty: Mara G. Prentiss, David R. Nelson, George M. Whitesides, Donald E. Ingber
Contributors: V. Krishnamurthy, D. Weibel, M. Narovlyansky, D. Vezenov, E. Alsberg
Presenters: Chiu Hong Lee, Claudia Danilowicz, Efraim D. Feinstein

We have studied the mechanical behavior of biomolecules and biomaterials in several experimental designs. We performed experiments at the single molecule level to evaluate the dissociation of biomolecules in DNA unzipping and ligand-receptor unbinding. Our experimental set-up allows performing m easurements in p arallel which can drastically shorten acquisition times. We used magnetic tweezers where a constant force can be applied between 0.5 and 160 pN. The system is well suited to studying the effect of force on single and double stranded DNA as well as to probing specific ligand-receptor bonds that are crucial in biological recognition. We studied the effect of ionic conditions and temperature in the unzipping of double stranded DNA providing the phase diagram in the temperature-force plane. Also the dissociation of ligand-receptor complexes was studied for (strept)avidin-biotin and carbonic anhidrase-sulfonamide and a comparison of the extrapolated rates of dissociation with values obtained with other methods has been done. Finally, we used polarization microscopy to measure the birefringence and direction of the optical axes of agarose and collagen gels in order to understand the physical properties of the environment in which cells live. We intend to demonstrate that local internal structure and stresses in biologically relevant hydrogels can be detected by measuring their stress birefringence.

Porous Drug Particles of Nanometer Dimensions

Facultly: David A. Edwards
Contributors: J. Fiegel, J. Sung, D.A. Edwards
Presenters: Jennifer Fiegel, Jean Sung

Given the global epidemic of tuberculosis (TB) and emerging public health threat of MDR-TB, there is an unmet medical need requiring the development of new treatment approaches. To help fulfill this need, we have developed a new bioengineered drug therapy for TB treatment by forming TB drugs into large porous particle aerosols (LPPs) or porous nanoparticle-aggregate particles (PNAPs). LPPs provide excellent aerosolization into the lungs, fast drug release and quick sterilization of the lung mucosa. PNAP systems, with aggregate size ranging from 1 micron geometric diameter to 100 microns, were formed by spray drying suspensions of drug-containing nanoparticles to yield drug formulations that 1) were highly dispersible, 2) allowed delivery of large masses of drug to mucosa, and 3) were easily disassembled upon delivery to body fluids to yield nanoparticles with their inherent attractive features for drug delivery (i.e., large surface area to achieve heightened solubility and targeting ability). Several TB drugs, such as capreomycin and rifampicin, were formed into LPPs and PNAPs for delivery by inhalation or ingestion. This approach can be applied more broadly for other infectious diseases such as SARS and smallpox.

Substrate Stiffness Regulates Traction Forces in Parallel with Cellular Phenotype Evaluation Using FRET

Faculty: David J. Mooney
Contributors: H.J. Kong, D.J. Mooney
Presenter: Hyun Joon Kong

Non-viral gene vectors are commonly used in various gene therapy strategies due to safety concerns with viral vectors, but are plagued by low levels of gene transfection and cellular expression. The current emphasis in efforts to increase the efficiency of non-viral gene delivery is manipulating the delivery vector, while the influence of the cellular environment in DNA uptake is often ignored. The mechanical properties (e.g., rigidity) of the substrate to which a cell adheres have been found to mediate many aspects of cell function including proliferation, migration, and differentiation, and this suggests that the mechanics of the adhesion substrate may regulate a cell's ability to transfer intracellulary exogeneous signaling molecules. In this report, we present a critical role for the rigidity of the cell adhesion substrate on the level of gene transfer and expression. The mechanism relates to material control over cell proliferation, and was investigated utilizing a fluorescent resonance energy transfer (FRET) technique. This study provides a new material-based control point for non-viral gene therapy.

Perfusion of VEGF-Induced Neovessel Networks: Dependence on Local Ischemia and Immune Competence

Faculty: David J. Mooney
Contributor: R.R. Chen
Presenter: Ruth R. Chen

Growth factor driven neovascularization may be a useful strategy to engineer microvasculature, and sustained and localized delivery of pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF) greatly increases local vessel density. However, the function of these new vessel networks has not been quantified nor examined in the context of the immune competence of the host nor local ischemia. We have measured perfusion, locally and regionally, resulting from implantation of porous 85:15 poly(lactide-co-glycolide) (PLG) scaffolds releasing VEGF in both subcutaneous tissue and severely ischemic hindlimbs in immune deficient SCID and immune competent C57/Bl6 mice. Sustained VEGF delivery resulted in a ~100% increase in microvessel density in both implant sites and animal models. However, the resulting perfusion varied greatly with the model. The perfusion of tissue within VEGF-releasing scaffolds (local perfusion) placed in subcutaneous tissue was 25%±7% greater than the perfusion resulting from blank scaffold implantation in SCID mice at 2 and 6 weeks. Results were similar in C57/Bl6 mice. However, placement of VEGF-releasing scaffolds in ischemic hindlimbs dramatically increased the local perfusion resulting from the neovasculature. SCID mice exhibited a 93%±35% increase in local perfusion with VEGF compared to blank scaffold implantation at 2 weeks, and a 52%±24% increase at 6 weeks, while C57/Bl6 mice exhibited an increase of 64%±24% at 2 weeks, and 110%±31% at 6 weeks. In the entire ischemic limb (regional perfusion), VEGF delivery resulted in more rapid reconstitution of blood flow. In SCID mice, perfusion of ischemic hindlimbs implanted with blank scaffolds remained at ~10% of the normal value through week 6, while VEGF delivery led to a steady increase in perfusion to 45%±2% of the normal value. C57/Bl6 mice showed a spontaneous partial recovery of limb ischemia, but VEGF delivery still led to a two-fold increase in regional perfusion compared to implantation of blank scaffolds at 2 and 4 weeks. In summary, this system for VEGF delivery leads to a similar, high level of neovascularization in a variety of models. However, the resulting local and regional perfusion is highly dependent on the implantation site and the animal model. This suggests that one must evaluate therapeutic angiogenesis strategies in therapeutically relevant sites and animal models. Most importantly, these results indicate that sustained and localized VEGF delivery can create functional vasculature which amplifies recovery of tissue ischemia, and highlights the potential utility of this strategy to treat ischemic diseases.

Injectable Delivery Vehicle for Angiogenic Molecules

Faculty: David J. Mooney
Contributors: E.A. Silva
Presenter: Eduardo A. Silva

Angiogenesis may provide a new approach to treat ischemic diseases and is a critical element in virtually all tissue-engineering approaches. Angiogenesis results from the sprouting of new vessels from pre-existent vessels, and the temporally distinct signaling of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) represent crucial steps in this process. Biodegradable polymer systems are designed to provide localized and controlled release of factors, and the purpose of this study was to develop an injectable hydrogel-based delivery system that could be utilized with minimally invasive surgical techniques for sequential VEGF and PDGF delivery. Alginate hydrogels (binary molecular weight distribution) and poly (lactide-co-glycolide) (PLG) microspheres were tested as injectable vehicles for the delivery of angiogenic molecules. The growth factor was associated with the alginate using three distinct approaches. The first approach involved simply mixing inductive molecules with the alginate. The second approach consisted of the pre-incorporation of the factors in PLG microspheres and then immobilizing the microspheres within the alginate gel. In the third approach the angiogenic molecules were incorporated into 3-D porous PLG scaffolds that were subsequently ground into small particles that were immobilized in the alginate gel. Both rapidly degradable and non-degradable alginate gels were used in these studies as well. The in vitro release kinetics of VEGF121, VEGF165 and PDGF were assessed using 125I-labeled factors and an ELISA. VEGF release from the microspheres was rapid (74 ± 4% and 81 ± 13% in 24 h; VEGF121 and VEGF165, respectively). However direct immobilization of VEGF and PDGF into gels led to a sustained release (87 ± 3%, 62 ± 5% and 36 ± 6% release in 7 days; VEGF121, VEGF165 and PDGF, respectively). The degradation rate of the alginate gel provided another mean to regulate release from this system. The bioactivity of the factors released from our polymer systems was confirmed by an endothelial cell proliferation assay. These data demonstrate that the factor release rate can be controlled over a wide range by manipulation of the delivery vehicles. Different release profiles obtained with these three distinct approaches may be useful for the putative delivery of multiple factors with distinct kinetics, and should provide a useful tool to study mechanisms related to blood vessel destabilization, regression, and remodeling.

Nanoscale RGD Peptide Organization Regulates Cell Proliferation and Differentiation

Faculty: David J. Mooney
Contributors: S. Hsiong, H.-J. Kong, P. Cooke
Presenter: Susan Hsiong

RGD (arginine-glycine-aspartic acid) containing peptide sequences, common cell attachment sites present in many extracellular matrix (ECM) proteins, mediate many important cellular processes. The role of nanoscale organization of RGD peptides in the regulation in the adhesion, proliferation and differentiation of both preosteoblasts (MC3T3-E1) and multipotential (D1) cell lines in vitro was investigated in this study. Alginate polymer chains with varying RGD peptide degree of substitution were mixed with unmodified polymer chains at different ratios to allow variation of RGD peptide spacing in the nanometer scale, independently of the overall bulk density of peptides presented from the material. Preliminary work to determine the nanoscale RGD peptide distribution in cross-linked alginate hydrogel disks was performed using atomic force microscopy (AFM). Proliferation of both cell types was observed to be closely correlated to RGD island spacing, independently of overall bulk ligand density, following cell adhesion to alginate hydrogels. Differentiation of preosteoblasts was significantly upregulated in response to decreased RGD spacing, whereas differentiation of multipotential cells was modestly regulated by this variable. These results demonstrate that the nanoscale organization of adhesion ligands may be an important variable in controlling cell phenotype and function. In addition, cellular responses to nanoscale ligand organization differ depending on the cell type, and this may be related to the differentiation stage of the cells.

Optimizing and Quantifying Ligand-Cell Receptor Bonds in Hydrogels

Faculty: David J. Mooney
Contributors: T. Boontheekul, H.-J. Kong, Y.-C. Huang, S.X. Hsiong
Presenter: Tanyarut Boontheekul

The cellular adhesion ligands presented in the extracellular matrix (ECM) clearly regulate local cellular activity and many functions of the ECM can be mimicked by small peptide fragments of the entire molecule (e.g., RGD sequence) and have found numerous utilities in tissue engineering when coupled to synthetic polymers so as to present them. However, fundamental of numbers of RGD ligand-receptor bonds per single cell remain unknown. With rheological measurement, we quantify the number of bonds between cell receptors and RGD peptides presented from alginate polymers by obtaining their frequency-dependent storage and loss modulus. Their temperature dependency can subsequently provide activation energy required to dissociate the bonds, and thus the number of bonds per cell is calculated to be about 100-1,000. The nanoscale organization and conformation of RGD ligands are further investigated. Cell receptor-ligand binding is stronger when RGD ligands are presented in a cluster format as compared to uniform distribution or when cyclic RGD ligands are conjugated as compared to linear RGD. This study illustrates the quantification of number of bonds per cells, which may lead to better understanding how RGD presentation (e.g., nanoscale organization, conformation) can control cellular function.

PLG Scaffolds as Cancer Vaccine Delivery Vehicles

Faculty: David J. Mooney
Contributors: D.J. Mooney, O. Ali
Presenter: Omar Ali

Many experimental cancer vaccines utilize the ability of dendritic cells (DC) to initiate an immune response by modifying isolated DCs in vitro to express tumor associated antigens. Results from this approach are promising, but this approach requires the isolation, expansion, and manipulation of DCs in vitro . We hypothesize that in vivo recruitment of DCs to a material that delivers inflammatory adjuvants serially with tumor associated antigens will allow in vivo , local expansion and activation of DC populations in the presence of antigens, leading to an enhanced tumor-specific response without the need for in vitro DC manipulation. To test this hypothesis, three-dimensional porous poly-lactide-co-glycolide (PLG) scaffolds that provide sustained and localized delivery of bioactive GM-CSF and melanoma tumor lysates were implanted into the subcutaneous tissue of rodents. This resulted in the local recruitment and expansion of DC, and GM-CSF delivery increased the number of total DC in a dose dependent manner. The utility of this system as a cancer vaccine was evaluated by implantation into C57/B6 mice that also received B16-F10 melanoma cells. Sustained delivery of melanoma tumor lysates from the material inhibited tumor growth by 90% at day 21, and 72% at day 28. The results of these studies demonstrate that a material system can potentially recruit, activate and target tumor antigens to host DCs in order to induce an anti-tumor response.

Tissue-Engineered Tumors for Investigation of Cancer Progression in a 3-D Microenvironment

Faculty: David J. Mooney
Contributors: C. Fischbach, P. Kumar, P.J. Polverini
Presenter: Claudia Fischbach

Three-dimensional interactions are essential in both initiation and malignant progression of tumors. However, these interactions are not reflected in conventional 2-D cell culture. Although certain approaches mimicking tumor inherent 3-D interactions are increasingly acknowledged for analysis of tumor cell properties appropriate model systems are often still missing. Tissue engineering strategies were applied to generate a 3-D human tumor model consisting of oral squamous cell carcinoma cells (OSCC-3) and porous poly(lactide-co-glycolide) (PLG) scaffolds. The model system was used to examine whether generation of a 3-D environment affects the ability of cultivated tumor cells to promote angiogenesis, as a mimic to 3-D interactions present in vivo . Secretion of angiogenic proteins from the engineered model system was proven to be differentially regulated relative to monolayer culture. Medium conditioned by 3-D cultivated OSCC-3 increased human umbilical vein endothelial cells (HUVEC) proliferation and sprouting to a greater extent than medium collected from monolayer culture. 3-D interactions are proposed to account for these phenomena.

The established model is suggested as a useful tool to address 3-D interactions, which may be critical to tumor angiogenesis and progression.

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Harvard Tech Transfer
Contributor: R. Benson
Presenter: Robert Benson (Licensing Office)

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