April Kloxin, University of Delaware
Danielle Benoit, University of Rochester
Chien-Chi Lin, Indiana University-Purdue University at Indianapolis
Violaine See, University of Liverpool
Symposium Support 3M Co
Journal of Visual Experiments
National Science Foundation
Purdue School of Engineering and Technology
Royal Society of Chemistry
Society for Biomaterials
University of Delaware
University of Rochester BME Department
University of Rochester Chemistry Department
University of Rochester School of Engineering and Applied Science
F2: Engineered Materials to Understand and Control Cell Response to Biophysical and Biochemical Signals
Monday PM, December 02, 2013
Sheraton, 2nd Floor, Republic A
2:30 AM - *F2.01
A Materials Approach to Understanding Forces, Organization, and Function of Multicellular Tissue
Christopher Chen 1 2
1Boston University Boston USA2The Harvard Wyss Institute Boston USAShow Abstract
A long recognized tenet of biological systems is that structure gives rise to function. Mechanical force in contrast has emerged only recently as a critical dimension that links form and function, providing the central effector to sculpt the body plan during morphogenesis, as well as a mechanism for cells to sense and respond to local changes in tissue structure and mechanics. Despite the realization that forces, form, and function permeate all living systems, we as a research community sorely lack methods to control the mechanics of the environment, the spatial organization of cells, or the architecture of cell-matrix and cell-cell interfaces, which collectively define the boundary conditions for how forces are transmitted into cells. Here, I will describe our efforts to design and build physical microenvironments that explicitly manipulate and monitor the structure and mechanics of cellular interactions with their surroundings, and how we have used these approaches to gain insights into their role in regulating cell and tissue structure, signaling, and function. I will use our studies to illustrate 1) the multiple means by which cell-material interactions can control cell signaling and function; 2) the importance of novel engineering and materials approaches to understanding cellular decision making; and 3) opportunities and challenges for how to connect these insights to the ultimate translational objectives set by regenerative medicine.
3:00 AM - F2.02
Two Dimensional Crosslinked Polymeric Mats for Peptide Immobilization and Stem Cell Adhesion
Padma Gopalan 1 Samantha K Schmitt 1 William Murphy 1
1University of Wisconsin-Madison Madison USAShow Abstract
We have designed a lightly crosslinked PEG based copolymer coating with compositional flexibility as well as extended stability for studying human mesenchymal stem cells (hMSC). The copolymer contains a majority of poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) as a cytophobic background with poly(ethylene glycol) methacrylate (PEGMA) for peptide coupling, and less than 10% glycidyl methacrylate (GMA) for crosslinking. Copolymer thin films were crosslinked into 30 nm thick mats by either thermal treatment or ultraviolet light and were stable for 35 days in water at 37 °C. The amount of PEGMA in the copolymer was optimized to ~ 11% to minimize non-specific cell-protein interactions while maximizing the amount of total bound peptides. Following the binding of RGDSP to the mat, hMSC were seeded. The hMSC adhesion, spreading and focal adhesion formation was promoted in a concentration dependent manner. Mats coupled with a non-adhesive scramble (RDGSP) maintained their cytophobicity. Competitive detachment experiments further demonstrated that cell adhesion was mediated by receptor binding to the RGDSP peptide. Due to the very thin nature of the mats, XPS was successfully used to quantify the total average peptide. XPS was effectively used to quantify average total peptide concentration as 12.6 ± 6.14 pmol cm-2. A square 2.2 mm N (1s) element map shows an average value of 17.9 pmol cm-2 of RGDSP, which correlates well with the multipoint high resolution data. The stability, compositional flexibility, ease of application and the ability to precisely quantify bound peptides on the mats, make these materials ideal for the study of cellular processes, where stability, functionality and topography of the biointerface are relevant. The large compositional flexibility provided by the copolymer chemistry and the crosslinking make it feasible to present multiple peptide sequences and to investigate proliferation and differentiation in future studies.
3:15 AM - F2.03
Probing Cellular Response to Heterogeneous Rigidity at the Nanoscale
Jinyu Liao 1 Manus Biggs 2 Ryan Cooper 3 Carl Dahlberg 3 Jeffrey Kysar 3 Shalom J Wind 2
1Columbia University New York USA2Columbia University New York USA3Columbia University New York USAShow Abstract
Physical factors in the environment of a cell regulate cell function and behavior and are involved in the formation and maintenance of tissue. There is evidence that substrate rigidity plays a key role in determining cell response in culture. Previous studies have demonstrated the importance of rigidity in numerous cellular processes, including migration and adhesion and stem cell differentiation. Atypical response to rigidity is also a characteristic of transformed (cancerous) cells. In vivo, a cell encounters features of different size and stiffness in its environment. Thus, it is important to understand how cells sense rigidity, particularly in the context of tactile cell sensing of discrete localized areas of increased or decreased rigidity.
We have developed a new technique for the creation of biomimetic surfaces comprising regions of heterogeneous rigidity on the micro- and nanoscale. The surfaces are formed by exposing an elastomeric film of polydimethylsiloxane (PDMS) to a focused electron beam to form patterned regions of micro- and nanoscale spots. Finite element analysis of nanoindentation measurements performed on irradiated PDMS films show that in a thin layer near the film surface, where approximately 90% of the electron energy is absorbed, the Young&’s modulus of the elastomer undergoes a significant increase as a function of electron beam dose.
Human skeletal stem cells plated upon electron beam-exposed PDMS in a pattern of spots with diameters ranging from 2 µm to 100 nm display differential co-localization of focal adhesions to the exposed features, with the degree of co-localization depending on both rigidity and feature size. This behavior persists as the area of the exposed regions is reduced below ~1 µm. On spots with diameters of ~ 250 nm and below, co-localization is lost, and cells appear unable to identify the rigid spots. This implies that there exists a length scale for cellular rigidity sensing, with the critical length in the range of a few hundred nanometers.
Understanding the fundamental underpinnings of cellular rigidity sensing can shed light on a variety of cell functions and behaviors. Surfaces that are designed to elicit different cellular responses can be used to guide the design of new types of tissue scaffolds and may be useful in the development of adoptive immunotherapies and treatments for a broad variety of diseases.
3:30 AM - F2.04
Cavitation Microrheology: A Tool to Probe Microscale Mechanical Properties of Natural Biomaterials
Whitney L Stoppel 1 Alfred J Crosby 2 Susan C Roberts 1
1University of Massachusetts Amherst Amherst USA2University of Massachusetts Amherst Amherst USAShow Abstract
A thorough understanding of cell-material interactions is critical for controlled manipulation of these interactions for applications in tissue engineering. Literature shows that changes in mechanical properties within a three-dimensional (3D) scaffold can elicit changes in encapsulated cell function through mechanotransduction. Furthermore, microstructure and mechanical flexibility of a biomaterial scaffold significantly influence cell behavior. For improved scaffold design, it is paramount that researchers are able to fully characterize the mechanical environment surrounding a cell or cluster of cells over time and position within the construct. Bulk rheology and current micro- and nano-scale rheological techniques do not provide sufficient spatiotemporal correlations between microscale mechanical properties and cell function within 3D scaffolds. Thus, there is a critical need for new technologies capable of spatiotemporal quantification of mechanical properties within a 3D cell-seeded scaffold.
To address this need, we employed cavitation microrheology (CMR) with the goal of increasing spatial resolution of mechanical properties (e.g., Young&’s Modulus) within 3D biomaterials. CMR uses mathematical correlations to relate the pressure at the point of elastic instability (by employing bubble formation at the tip of a needle inserted into the hydrogel) to the Young&’s modulus. The length scale probed is determined by the inner radius of the needle applied, which allows CMR to access mechanical properties at very specific locations within a material over a wide range of length scales by simply varying the needle gauge. Our results demonstrate that CMR measures spatiotemporal changes in mechanical properties on smaller size scales than traditional rheology (100-300 µm used here) and with greater precision and accuracy than what is afforded by other techniques.
We use ionically crosslinked alginate hydrogels which supply a flexible scaffold architecture, allowing seeded fibroblasts to reorganize their external environment. Gradients in crosslinking density, and therefore elastic moduli, can be created by modulating calcium diffusion within the construct. Using CMR, we were able to quantify alginate hydrogel degradation in cell culture media, demonstrating radial variation in crosslinking density over 7 days when gels were uniformly crosslinked on day 0. CMR was also used to quantify the mechanical contribution of cell-secreted extracellular matrix within fibroblast seeded alginate hydrogels. Overall, this work demonstrates the power of CMR in quantifying mechanical properties within 3D biomaterials with precise control over measurement position within the construct. Current and future work focuses on the implication of varying microscale mechanical properties on encapsulated cell function, with a long-term goal of improving fundamental and translational knowledge on cellular response to mechanical cues in 3D biomaterials.
3:45 AM - F2.05
A Computational Platform Integrating Molecular and Systems Level Understandings of Mechanotransduction
John Kang 1 Kathleen Puskar 2 Russell Schwartz 1 Philip LeDuc 1 3
1Carnegie Mellon University Pittsburgh USA2Point Park University Pittsburgh USA3Carnegie Mellon University Pittsburgh USAShow Abstract
Biological phenomena that are difficult to investigate experimentally can often be better understood by developing computational models. Mechanical forces on cells are increasingly becoming understood as playing a crucial role in biological phenomenon in fields ranging from stem cell differentiation to cancer metastases to vasculogenesis. Mechanotransduction has been divided into the three phases of mechanotransmission, mechanosensing, and mechanoresponse yet how the cell performs all three functions using a shared set of components is still poorly understood and a challenge to tackle experimentally. Here, we present a structurally-driven mechanotransduction model that integrates actin network changes under stretch with a mixture model of molecular release to further elucidate the spatiotemporal interplay between network morphology and resultant biochemical signaling.
As stretch is applied to our model of the discrete actin filament network, the distribution of individual bond angles in the network transitions from a more peaked to a flatter distribution. We used our approach to explore various angle thresholding models of how actin filament network deformations might influence rates of release of bound signaling molecules. These thresholding models allow us to project how the cell may interpret an applied mechanical stimpulus into a biochemical response. We train and validate these simulations using published experimental data and use our parameterized model to then test different predictive capabilities of how mechanotransduction may function. Our integrated mechanotransduction model represents a potentially versatile mechanistic platform for examining biophysical systems that link mechanical stimulus at the cellular level to response at the protein level.
4:30 AM - *F2.06
Bioinspired Platform Technologies for Next Generation Regenerative Therapeutics
Jeffrey Karp 1 2
1Brigham and Women's Hospital Cambridge USA2Harvard Medical School Boston USAShow Abstract
This talk will explore platform technologies that are currently being developed
in the KarpLab to tackle some of the most challenging medical problems.
Namely, sealing tissues/closing wounds, achieving long term local drug delivery
for treatment of diseases such as rheumatoid arthritis and glioblastoma,
development of surfaces to separate cells for disposable point of care diagnostics
and for cell therapy, engineered cell therapy, and needles that sense different
levels of tissue for the delivery of cells and drugs. Many of these platforms were
inspired by solutions observed in nature.
5:00 AM - F2.07
Protection of Pancreatic Islet Cells with Immunomodulatory Ultrathin Polymer Coatings
Veronika Kozlovskaya 1 Hubert Tse 2 Eugenia Kharlampieva 1
1University of Alabama at Birmingham Birmingham USA2University of Alabama at Birmingham Birmingham USAShow Abstract
Despite transplantation of pancreatic islet cells is a promising treatment for Type 1 diabetes, its clinical application remains limited due to adverse effects of immunosuppression, declining allograft function, and recurrence of autoimmunity. We report on a novel ultrathin cytocompatible coating which displays an inherent anti-inflammatory property to protect living pancreatic islets. The coating consists of a hydrogen-bonded multilayer assembly of a natural polyphenol (tannic acid; TA) and poly(N-vinylpyrrolidone) (PVPON) to allow for conformal coating of individual islets with an ultrathin film of controllable thickness and composition. The coating is conformal over the surfaces of rat, non-human primate, and human islets. The coated islets maintain their viability and β-cell functionality. Coated islets displayed an increased insulin stimulation index after 96 hours in vitro as compared to uncoated islets. Also, the (TA/PVPON) coating material demonstrated immunomodulatory properties showing significant suppression of innate immune pro-inflammatory cytokine and adaptive immune Th1 cytokine responses. The developed material combines high chemical stability under physiologically relevant conditions with capability of scavenging free-radicals, two crucial parameters for prolonged islet integrity, viability, and function in vitro/in vivo. Our study offers new opportunities in the area of advanced transplant materials to be used in Type 1 diabetes treatment as well as in various areas of cell-based therapy.
5:15 AM - F2.08
Supramolecular Hydrogel of Chemoattractant as a Depot for Neutrophils Attraction In Vivo
Fan Zhao 1 2 Jingyu Li 3 4 5 Jiro Sakai 3 4 5 Ning Zhou 1 Hongbo R. Luo 3 4 5 Bing Xu 1
1Brandeis University Waltham USA2Brandeis University Waltham USA3Harvard Medical School Boston USA4Childrenamp;#8217;s Hospital Boston Boston USA5Dana-Farber/Harvard Cancer Center Boston USAShow Abstract
Most of immunomodulatory materials (e.g., vaccine adjuvants such as alum) modulate adaptive immunity, and yet little effort has focused on developing materials to regulate in-nate immunity (e.g., short-lived effector cells such as neutrophils). Here we show that the incorporation of unnatural amino acids into a well-known chemoattractant—N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF) offers a simple and efficient approach to create a de novo, multifunctional chemoattractant that self-assembles to form supramolecular nano-fibrils and hydrogels. This de novo chemoattractant not only maintains the potent chemotactic activity to human neutrophils as well as mouse neutrophils, but also exhibits dramatically increased biostability against proteolysis. Thus, its hydrogel, in vivo, sustainably releases the chemoattractant and attracts neutrophils to the desired location in a sustainable manner. This work illustrates a novel approach to generate a new class of biomaterials for modulating innate immunity and offers an unprecedented prolonged acute inflammation model for developing various new applications.
5:30 AM - F2.09
Antifreeze Glycoprotein Mimetic Polymers for Improving Biological Cryopreservation; Enhanced Cell Recovery, Function and Application to Regenerative Medicine
Robert Deller 1 Thomas Congdon 1 Matthew Gibson 1
1University of Warwick Coventry United KingdomShow Abstract
With an ever-ageing population in the Western world, the need for regenerative medicine, especially transplantation is increasing, but the number of donors remains static. Therefore improved storage methods for cells/tissues/organs is urgently required to overcome their limited shelf-life and also for improved storage of progenitor cells. Antifreeze glycoproteins (AFGPs) from polar fish slow ice crystal growth and may find application in cryopreservation, but are limited by their extremely high costs, challenging synthesis and potential toxicity. To address this, my team is developing novel synthetic materials which can reproduce the desirable properties of AFGPs (and other glycoproteins), without the side effects, at lower cost. We have recently demonstrated that synthetic polymers designed to inhibit ice recrystallisation (growth) improve cryopreservation, without the need for any toxic organic solvents (traditional method), using 400 x less material.[3,4] A thorough biophysical analysis on the underlying mechanisms of action of these polymers is underway[5,6] alongside biocompatibility and cellular preservation studies. This research is truly interdisciplinary, crossing the boundaries of materials, polymers, chemistry, and the life sciences.
 Gibson, M.I., Polymer Chemistry, 2010, 1, 1141-1152,, Richards, S-J., Jones, MW, Hunaban, M., Haddleton, DM., Gibson, M.I., Angewandte Chemie,.2012, 51, 7812 - 7816;  Gibson, M. I., Barker, C. A., Spain, S. G, Albertin, L., Cameron, N. R. Biomacromolecules, 2009, 10, 328-333;  Deller, R.C., Vatish, M., Mitchell, D.A., Gibson, M.I. 2012, Submitted;  Congdon, TC, Notman, R., Gibson, MI, Biomacromolecules, 2013, 14, 1578 - 1586;  Deller, R.C., Congdon, T., Sahid, M., Morgan, M., Vatish, M., Mitchell, D.A., Notman, R., Gibson, M.I., Biomaterials Science, 2013, 1, 478 - 485
5:45 AM - F2.10
Time-Dependent Extracellular Matrix Organization and Secretion from Vascular Endothelial Cells Due to Macromolecular Crowding
Frances D Liu 1 2 Adam S Zeiger 2 3 Krystyn J Van Vliet 1 2 3
1Massachusetts Institute of Technology Cambridge USA2Singapore-MIT Alliance in Research amp; Technology (SMART) Singapore Singapore3Massachusetts Institute of Technology Cambridge USAShow Abstract
The extracellular matrix (ECM) is composed primarily of proteins secreted from support cells such as fibroblasts in connective tissue. The ECM not only acts to physically support cells, but also has been shown to affect cell migration and proliferation. Epithelial cells such as vascular endothelial cells (VECs), however, are also known to secrete and remodel their own extracellular matrix. We mimicked the crowded in vivo physiological microenvironment surrounding VECs by using macromolecular crowding nanoparticles (MMCs). We tracked ECM deposition and organization of confluent VECs over the course of four weeks using immunocytochemistry with and without the presence of MMCs. We stained for collagen type IV, the extracellular collagen found primarily in the basal lamina, and used average angular standard deviation to quantify the orientation and organization of the ECM fibers with and without MMCs. Detectable differences in ECM architecture were identified within one week in vitro . Our results suggest that the ECM remodeling may play a role in quiescence and angiogenesis in vivo. Moreover, our results suggest we can control endothelial cell behavior and the organization of native ECM in vitro through biophysical rather than biochemical tools.
F3: Poster Session
Monday PM, December 02, 2013
Hynes, Level 1, Hall B
9:00 AM - F3.01
Engineering Transparent UV-Absorbing ZnO Nanorods with Reduced DNA Damage
Georgios Sotiriou 1 Christa Watson 1 Kim Murdaugh 1 Alison Elder 2 Philip Demokritou 1
1Harvard University Boston USA2University of Rochester Medical Center Rochester USAShow Abstract
Zinc oxide (ZnO) is a wide-bandgap metal oxide semiconductor that can be excited at room temperature by UV irradiation, and finds applications in a variety of fields and products including paints, pigments, batteries, photocatalysis, foods, to name a few. When in nanometer size range, it may be used in polymer nanocomposites and sunscreens as an efficient UV-filter with high transparency in the visible wavelength range. However, the photocatalytic activity of ZnO causes the degradation of the surrounding polymer rendering it unsuitable for long-term employment. Furthermore, ZnO nanoparticles are highly toxic and may pose risks to the public and the environment. Here, ZnO nanorods are made by scalable flame synthesis and are in-situ encapsulated by an amorphous nanothin SiO2 layer [1,2,3]. The as-prepared nanoparticles are characterized by electron microscopy (EM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and N2 adsorption. The hermetic nature of the SiO2 coating is evaluated by a detailed dissolution study and the photocatalytic activity is monitored by the decomposition of methylene blue dye. The presence of SiO2 facilitates the dispersion of these nanorods in relevant solutions. The core ZnO nanorods exhibit the characteristic optical properties as determined by UV/vis and diffuse reflectanse spectrometry, while the SiO2 coating eliminates the photocatalytic activity  and minimizes the DNA damage to human cells, as illustrated in cellular studies using multiple cell lines. Therefore, these flame-made ZnO nanorods may be safely used as fillers in polymer UV-filter nanocomposites and in sunscreens exhibiting superior performance while mitigating their impact on environmental health.
 Sotiriou, G. A., Sannomiya, T., Teleki, A., Krumeich, F., Vörös, J. and Pratsinis, S. E. Non-toxic dry-coated nanosilver for plasmonic biosensors. Adv. Funct. Mater.20, 4250-4257, (2010).
 Sotiriou, G. A., Hirt, A. M., Lozach, P. Y., Teleki, A., Krumeich, F. and Pratsinis, S. E. Hybrid, silica-coated, Janus-like plasmonic-magnetic nanoparticles. Chem. Mater.23, 1985-1992, (2011).
 Sotiriou, G. A., Franco, D., Poulikakos, D. and Ferrari, A. Optically Stable Biocompatible Flame-Made SiO2-Coated Y2O3:Tb3+ Nanophosphors for Cell Imaging. ACS Nano6, 3888-3897, (2012).
 Gass, S., Cohen, J. M., Pyrgiotakis, G., Sotiriou, G. A., Pratsinis, S. E. and Demokritou, P. Safer Formulation Concept for Flame-Generated Engineered Nanomaterials. ACS Sustainable Chem. Eng.in press, DOI: 10.1021/sc300152f, (2013).
9:00 AM - F3.03
Effects of Dynamic Rotation on Human Umbilical Vein Endothelial Cells in Circular PDMS Microchannels
Kevin Mercurio 1 William Turner 1 Kara McCloskey 1 Valerie Leppert 1
1University of California at Merced Merced USAShow Abstract
In the past decade, researchers have sought to develop circular microchannels for cell culture studies that closely mimic in vivo vasculature. 3D cell culture techniques in tubular structures typically involve the physical rotation of the channels to achieve a uniform distribution of cells on the channel circumference. Depending on the rotation frequency, cells may not distribute uniformly and can clog the tube passage. Blockage in the microchannel may potentially prevent fluid flow through the structure, which could impact oxygen and nutrition flow in vascular networks. To address this issue, a low-cost, motorized, hands-free device was built to produce consistent, dynamic rotation of the channeled structures at low speeds. Circular microchannels having an internal diameter of 1mm were fabricated in polydimethylsiloxane (PDMS) using a wire-sacrificed technique, and were dynamically rotated at a speed of 0.7 rotations per second. Optical microscopy showed human umbilical vein endothelial cells (HUVECs) adhering to the surface of the tubular PDMS channel at 2 days in dynamic culture. This device demonstrates potential benefits for use in applications for vascular network fabrication.
9:00 AM - F3.05
Optimization of Thermal Drawing of High Aspect Ratio Polymer Nanoprobes for Insertion into Chlamydomonas reinhardtii
Dasom Yang 1 Hyeonaug Hong 1 WonHyoung Ryu 1
1Yonsei University Seoul Republic of KoreaShow Abstract
For monitoring of biological events within cells, the importance of cell insertion technique is on the rise in various bioengineering fields. To maintain cell viability during such cell insertion by nanoprobe structures, seamless and damage-free insertion of nanoprobe structures through cell membranes is required. Although silicon or metal nanowires were often used as cellular nanoprobes, their fabrication processes are expensive and time-consuming. As a rapid and low cost fabrication method, we introduce a thermal drawing process for vertically-aligned polymer nanoprobes optimized for cell insertion. This thermal drawing allows for rapid fabrication of high aspect-ratio polymer nanoprobes with about 500 nm diameter and 20 mu;m heights. The whole process takes only a few minutes. In this study, we optimized processing parameters for thermal drawing, such as temperatures of substrates and micro pillars, drawing speed, drawing steps, contact depth of micro pillars, and contact time. First, SU-8 2150 was spin-coated on a glass substrate and soft baked for solvent removal. Afterwards, a tungsten pillar was heated above the glass transition temperature (Tg) of SU-8 2150, and polymer substrates were heated just below the Tg. The heated micro pillar was dipped in the heated SU-8 layer for a pre-determined time. Vertical drawing of the pillar created a liquid bridge between the pillar and substrate. Variation of the processing parameters mentioned above indicated that fast drawing after short contact time produced the nanoprobes with smaller diameters. Dipping depth also affected the profile of the nanoprobes. Photosynthetic algal cells, Chlamydomonas reinhardtii, were used to test cell insertion and the inserted algae maintained the integrity of the cell membrane for 2 hours without any apparent damage or leakage.
9:00 AM - F3.06
Deciphering the Molecular Basis for Alpha-Synuclein Toxicity in Yeast Using Microfluidics Integrating Single Cell Traps and a Chemical Gradient Generator
Joao Tiago S. Fernandes 1 2 Andreia Gameiro 1 Sandra Tenreiro 2 Virginia Chu 1 Tiago F. Outeiro 2 3 Joao Pedro Conde 1 4
1INESC Microsistemas e Nanotecnologias and IN - Institute of Nanoscience and Nanotechnology Lisbon Portugal2Faculdade de Medicina da Universidade de Lisboa Lisbon Portugal3University Medical Center Goettingen Goettingen Germany4Instituto Superior Tamp;#233;cnico Lisbon PortugalShow Abstract
Alpha-synuclein (aSyn) is a central player in Parkinson&’s disease (PD) and other neurodegenerative disorders collectively known as synucleinopathies. It misfolds and aggregates in the typical pathological hallmarks of these disorders, the Lewy bodies, and is associated with familial and sporadic cases when mutated. However, the mechanisms underlying aSyn aggregation and toxicity are not well understood. One way to gain further insight into the biological effects of normal and mutant aSyn is to use yeast cell models. S. cerevisiae (Sc) has been widely used in biology since it shares basic cellular mechanisms of animal cells and can be genetically modified to produce and correctly fold some human proteins. To study aSyn effects in PD, yeast cells were genetically engineered to produce aSyn fused with GFP. The production of this aSyn-GFP fusion protein is induced by exposing cells to galactose (GAL), and can provide insight into what happens when overexpression occurs.
To study how these cells react when exposed to different concentrations of promoter inducer and known stressors, as well as to follow and compare the behaviour of individual and cell populations over time in different controlled and sustained environments, we have developed a PDMS-based microfluidic device that combines a chemical gradient generator with hydrodynamic single cell traps. The chemical gradient generator is a series of serpentine channels that allow laminar flowing liquids to mix by diffusion. By increasing the number of channels through several mixing stages, we created a 9 step gradient from just 3 initial solutions of different concentrations. This gradient was used to expose 9 chambers to fluid flow with precise chemical compositions. Each chamber has an array of hydrodynamic cell traps designed to capture a single Sc cell. The combination of these two modules allows to expose the genetically modified yeast cells to different chemical environments and, since they are trapped, to follow the accumulation and effects of aSyn on a large set (several hundred) of single Sc cells over time.
The first tests done with this device focused on the calibration and optimization of the gradient generator using FITC. Also, the geometry and spacing of traps was optimized in terms of overall trap occupation and single cell trapping. Experiments performed over 3 h with a gradient of GAL showed that the yeast cells exposed to higher concentrations responded with increased production of aSyn. We will show how individual and a population of yeast cells respond in time to different concentrations of the promoter inducer and then report on the study of the effect of different concentrations of chemical stressors such as hydrogen peroxide and iron chloride on the expression and intracellular distribution of aSyn. Ultimately, this approach will enable us to gain novel insight into the molecular basis of aSyn dysfunction in synucleinopathies.
9:00 AM - F3.07
High-Throughput Ion-Beam Modified Micronozzles to Probe Live Cells
Eva Campo 1 Maria Jose Lopez 2 Elizabet Fernandez 3 Jose Antonio Plaza 4
1Bangor University Bangor United Kingdom2University of Hull Hull United Kingdom3Universitat Autonoma Bellaterra Spain4IMB-CNM Bellaterra SpainShow Abstract
Novel medical and biological applications drive increasing interest towards development of materials and processes instrumental to probing- and affecting- biological phenomena. We have combined microfabrication technologies with ion beam milling techniques to successfully produce cantilever-type polysilicon and silicon oxide micro-dispensers with 3D etched microchanels. Ion beam polishing successfully produced sharp cantilevers with pre-determined taper angles and smooth edges. The main purpose of this work was to test the piercing efficiency of custom-fabricated, sharp, polished micronozzles on mouse embryos in absence of piezoelectric-assisted drills.
Systematic study of perforation with different angles and in absence of drills was performed over mouse oocytes and embryos. Namely, geometries involved in sharper angles and narrower pipettes were more successful in perforation. Despite nanometer thin tips, no structural damage to the structures was observed upon piercing. Micronozzles were intact after perforation, suggesting this technology could be useful in cell handling. Significantly, pierced embryos are seen to continue their division to blastocyst, hinting at biocompatibility. These results suggest that residual chemical agents or Ga+ ions injected during milling are not arresting cell development.
This innovative technology could prove invaluable in answering fundamental biophysical questions such as intrinsic mechanic properties of bio-layers, since perforation was conducted in absence of vibrational piercing aids. Moreover, focus ion beam made-to-order pipettes could be highly instrumental towards versatile platforms. These platforms are amenable to sensing and actuating capabilities at cellular and subcellular levels, inclusive of targeted drug delivery, owing to their biological-relevant architecture and hierarchical structures: macro (chip-reservoir) to micro (channels) and nano (beam-sharpened tips).
9:00 AM - F3.08
Intravital Microscopy for Smart Transdermal Vaccine Delivery Systems
Ki Su Kim 1 Hyemin Kim 2 Jeonga Yang 2 Seok-Hyun Yun 1 Sei Kwang Hahn 1 2
1Massachusetts General Hospital, Harvard Medical School Boston USA2Pohang University of Science and Technology (POSTECH) Pohang Republic of KoreaShow Abstract
A variety of vaccine delivery systems have been investigated for facile and efficient immunization with patients&’ compliance. Among them, transdermal vaccine delivery might be a strong alternative candidate due to well-designed immune systems in the skin tissues containing Langerhans and dendritic cells. In this work, we developed smart transdermal vaccine delivery systems using hyaluronic acid - ovalbumin (HA-OVA) conjugates as a model system. HA-OVA conjugates were synthesized by site-specific reaction between HA-aldehyde (HA-ALD) and N-terminal amine group of OVA. The successful synthesis of HA-OVA conjugate was confirmed by 1H NMR, GPC and ELISA. Two-photon microscopy clearly visualized the effective transdermal delivery of HA-OVA conjugates labeled with red fluorescence dye (Rhodamine B and Texas Red). Remarkably, the transdermal delivery of HA-OVA conjugate via the ear skin of Balb/c mice resulted in statistically significant immunization according to the ELISA for the concentration of anti-OVA IgG in cardiac blood samples. Taken together, we could confirm the feasibility of HA-based transdermal vaccine delivery systems for further development.
9:00 AM - F3.09
Why the Dish Makes a Difference: Quantitative Comparison of Polystyrene Surfaces for Cell Culture
Adam S Zeiger 1 Ben Hinton 2 Krystyn J Van Vliet 1 3
1Massachusetts Institute of Technology Cambridge USA2University of Minnesota Minneapolis USA3Singapore-MIT Alliance in Research amp; Technology (SMART) Singapore SingaporeShow Abstract
There is wide anecdotal recognition that biological cell viability and behavior can vary significantly as a function of the source of commercial tissue culture polystyrene (TCPS) culture vessels to which those cells adhere. However, this marked material dependency is typically resolved by selecting and then consistently using the same manufacturer&’s product - following protocol - rather than by investigating the material properties that may be responsible for such experimental variation. Here, we quantified several physical properties of TCPS surfaces obtained from a wide range of commercial sources and processing steps, through the use of atomic force microscopy (AFM)-based imaging and analysis, goniometry, and protein adsorption quantification. We identify qualitative differences in nano- and micro-scale surface features, as well as quantitative differences in surface roughness and wettability that cannot be attributed solely to differences in surface chemistry. We also find significant differences in cell morphology and proliferation among cells cultured on different TCPS surfaces, and resolve a correlation between nanoscale surface roughness and cell proliferation rate for both cell types considered. Interestingly, AFM images of living adherent cells on these nanotextured surfaces demonstrate direct interactions between cellular protrusions and topographically distinct features. These results illustrate and quantify the significant differences in material surface properties among these ubiquitous materials, allowing us to better understand why the dish can make a difference in biological experiments.
9:00 AM - F3.10
3D Bioprinting of Multi-Material, Cell-Laden Tissue Constructs
David B. Kolesky 1 2 Kimberly A. Homan 2 1 A. Sydney Gladman 1 2 Ryan L. Truby 1 2 Travis A. Busbee 1 2 Michael A. Bell 1 2 Jennifer A. Lewis 1 2
1Harvard University Cambridge USA2Harvard University Cambridge USAShow Abstract
Natural tissues are composed of multiple cell types, structural extracellular matrix (ECM), and blood vessels. 3D bioprinted constructs are beginning to emerge as viable options in regenerative medicine for situations where the tissue is simple and does not require complex vasculature to function properly, e.g., bladder, cartilage, and the trachea. However, to vastly expand the application of these types of tissues, one must be able to integrate cells, structural, and vascular components concurrently and with precision. Using a custom-designed bioprinter, we are creating 3D multi-material, cell-laden architectures with embedded vascular networks. We will describe the printing platform and enabling ink designs as well as the structure and viability of the printed 3D tissues.
9:00 AM - F3.11
Covalent Attachment of Polyelectrolyte Multilayers to Cells
Rosanna M. Lim 1 Robert E. Cohen 1 Michael F. Rubner 2
1Massachusetts Institute of Technology Cambridge USA2Massachusetts Institute of Technology Cambridge USAShow Abstract
Biohybrid materials integrate synthetic materials with biological systems in order to confer unique properties to existing biological functions. This class of materials can be used in a variety of applications, such as sensing, drug delivery, or biological imaging. One type of biohybrid material is the cell backpack, which incorporates polyelectrolyte multilayer (PEM) patches with cells. Various cargoes, such as small molecules, nanoparticles, or labeling moieties, can be incorporated into the backpacks for the cells to carry, making cellular backpacks a versatile platform for drug delivery. These PEM backpacks are fabricated through a combination of photolithography and layer-by-layer (LbL) assembly. The backpacks are hundreds of nanometers thick, a few microns wide, and attach to part of the cell surface.
The original cell adhesive PEMs produced in our group contained hyaluronic acid paired with chitosan, since CD44 receptors on lymphocyte surfaces bind to hyaluronic acid. However, this receptor-ligand binding interaction is not strong and is specific to certain types of cells, so more robust and versatile techniques for cell attachment need to be developed, such as covalent attachment. This would not only create an irreversible chemical bond between the backpack and the cell, but it would also allow the backpacks to attach to a greater variety of cell types.
Covalent attachment can be achieved by introducing functional groups into the polymers of the cell adhesive multilayers, which would react with proteins on the cell surface. One target functional group includes the maleimide group, which reacts with thiols present on cysteine residues of proteins. Maleimide groups are grafted onto poly(allylamine hydrochloride) using a heterobifunctional cross-linker, sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC). The functionalized polymer is then paired with poly(acrylic acid) for LbL assembly. The cellular backpacks are then reacted with lymphocytes for the covalent attachment reaction. The cells are incubated with both an array of backpacks attached to a glass substrate as well as releasable backpacks that react with cells in solution. In addition to incorporation into cell backpacks, polymers that covalently attach to cell surfaces can also be used to create other types of biohybrid materials for a variety of applications. The development of polymers that covalently attach to cells could thus open a broad area of new ideas to explore.
9:00 AM - F3.12
Carbon Dot-Gold Nanoparticle-DNA Molecular Assembly for Real-Time Intracellular Trafficking and Enhanced Transfection
Jinhwan Kim 1 2 Juhee Park 1 2 Hyunwoo Kim 1 2 Kaushik Singha 1 2 Won Jong Kim 1 2
1Institute for Basic Science (IBS) Pohang Republic of Korea2Postech Pohang Republic of KoreaShow Abstract
The effort to develop polymeric vectors for gene delivery has been drifted from viral vectors mainly due to the safety and less viable production concerns associated with viral vectors. However, polymeric vectors are impaired significantly with low delivery efficiency compared to viral vectors, and thus to overcome diverse impediments plethora of research activities have been undertaken. Nevertheless, for practical clinical realization it is essential to find out rational design method for polymeric gene delivery system with high efficiency. To develop biologically viable and efficient delivery constructs, it is of paramount importance to decipher the underlying mechanisms of polymer-mediated delivery which comprises several extra- or intracellular events such as circulation in blood stream, cellular uptake, endosomal escape, and dissociation of cationic polymer/gene complex (polyplex). Especially, the dissociation of polyplex is considered as the rate-limiting step for efficient trafficking of gene to nucleus. A study about prompt and opportune dissociation of polyplex bears an immense importance in achieving high gene transfection efficiency, however, only a few work has highlighted this aspect. Therefore, there is a pressing need to develop tools which can monitor the dissociation of polyplex in real-time without compromising the delivery efficiency and the safety issues.
Herein, we employ carbon dot (CD), which has been emerging as a fluorescent nanomaterial with excellent biocompatibility, to monitor the association/dissociation of polymeric carrier/plasmid DNA (pDNA) complex during transfection. To shed light on the underlying post-endosomal events, the adopted strategy exploited the quenching of the fluorescence of CD by Au nanoparticles. The surface of CD and Au was modified with polyethylenimine (PEI) to make cationic surface. Simple subsequent treatment of negative charged pDNA triggered the quenching of the CD fluorescence by reducing distance between CD and Au. The dissociation of pDNA from the complex induced fluorescence recovery due to the increase in distance between CD and Au by charge repulsion. The fluorescence intensity changes during transfection, especially post-endosomal step, were monitored by fluorescence measurement using fluorescence microscope. This nano-assembly system was found to be very effective at monitoring the carrier/pDNA dissociation in a non-labeled manner, providing efficient strategy to study the mechanistic aspect of polymer mediated pDNA delivery.
9:00 AM - F3.13
Ag+ Ion Release from Nanosilver in Aqueous Solutions: Effect of Size, pH and O2 Concentration
Kakeru Fujiwara 1 Georgios A Sotiriou 1 2 Pratsinis E Sotiris 1
1ETH Zurich Zurich Swaziland2Harvard University Boston USAShow Abstract
Silver nanoparticles (nanosilver) have attracted strong interest in biomedical applications  for their plasmonic and superior antibacterial properties. The mechanism for the latter depends on bacterial contact with either the nanosilver surface or the released (or leached) Ag+ ions from it . Here, nanosilver with closely controlled particle size (7-30 nm) and without any surface functionalization (that could interfere with its ion release or leaching) is prepared, and characterized by X-ray diffraction, nitrogen adsorption and transmission electron microscopy. The release or leaching of Ag+ ions in de-ionized (DI) water (pH 6.8) and in low pH (4.65) buffer solution is monitored electrochemically. The presence of Ag2O on nanosilver surface is detected by optical (UV-vis) spectroscopy and quantified by thermogravimetric analysis and mass spectroscopy. The Ag+ ion release in DI water depends on nanosilver size and is traced to the dissolution of one Ag2O surface layer.  On the other hand, higher Ag+ ion release occurs in a low pH (4.65) buffer solution stemming from metallic Ag dissolution  beyond that of the surface Ag2O layer. This dissolution of the smallest nanosilver (< 10 nm) occurs almost instantly (within 3 min) while that of the larger ones (> 10 nm) continuously occurs for seven days. Furthermore, the release of Ag+ ions from metallic nanosilver is examined by continuous bubbling of O2 or N2 though such suspensions. Higher Ag+ ion release occurs from metallic Ag at high O2 concentrations in water, which depends also on nanosilver size, indicating that even metallic nanosilver is oxidized and eventually release Ag+ ions. During N2 bubbling, the smallest nanosilver appear to release some ions but this is attributed to its high surface availability by O2 that is gradually removed by the bubbling N2.
1. Chen, X. and H.J. Schluesener, Toxicol. Lett., 2008. 176(1): p. 1-12.
2. Sotiriou, G.A. and S.E. Pratsinis, Environ. Sci. Technol., 2010. 44(14): p. 5649-5654.
3. Sotiriou, G.A., A. Meyer, J.T. Knijnenburg, S. Panke, and S.E. Pratsinis, Langmuir, 2012. 28(45): p. 15929-15936.
4. Liu, J.Y. and R.H. Hurt, Environ. Sci. Technol., 2010. 44(6): p. 2169-2175.
9:00 AM - F3.14
Internalization of Carbohydrate-Conjugated Nanoparticles by Mycobacterium Smegmatis
Kalana Wijesinghe Jayawardana 1 H. Surangi Nelusha Jayawardena 1 Thareendra De Zoysa 1 Mingdi Yan 1
1Umass Lowell Lowell USAShow Abstract
ABSTRACT: Mycobacterium smegmatis is a non-pathogenic microorganism and has been widely used as a model organism to study infections caused by M. tuberculosis and other mycobacterial pathogens. We report that nanoparticles conjugated with selected carbohydrate show a striking increase in the surface adherence and internalization by M. smegmatis. This applies to silica nanoparticles and magnetic nanoparticles ranging from 100 nm to 5 nm. Under the same experimental conditions, minimum adhesion or internalization was observed for unfunctionalized nanoparticles. The synthesis and characterization of the glyconanoparticles will be presented. The finding is applied to imaging M. smegmatis-infected lung epithelial cells, and the results will be discussed.
9:00 AM - F3.15
Electrochemical Evaluation of Reactive Oxygen Species (ROS) Scavenging Capacity of Naturally-Derived Antioxidant Substances under the Environment of Lamellar Structure
Yu Aoki 1 Chihiro Kaise 2 1 Teruhisa Kaneko 2 1 Tatsuo Aikawa 1 Takeshi Kondo 1 3 Makoto Yuasa 1 3
1Tokyo University of Science Noda Japan2L.V.M.C. INC. Toshima-ku Japan3Tokyo University of Science Noda JapanShow Abstract
Continuous ultraviolet (UV) radiation to the skin generates reactive oxygen species (ROS) including singlet oxygen (1O2), superoxide anion radical (O2-), etc. The ROS can be a factor of oxidative damage such as rough skin, lipid peroxidation and skin irritation. Therefore, in the field of prevention and treatment such as cosmetics and pharmaceuticals, development of superior ROS scavenging system is desired. In this study, we focus on quercetin and gallic acid, which have been reported to have a high ROS scavenging ability. Experiments were performed under the environmental of lamellar structure model imitating the skin stratum corneum intercellular lipid. The ROS scavenging capacity was measured with high accuracy by monitoring the concentration of ROS with a short lifetime by electrochemical technique.
Environment of lamellar structure model was prepared by mixing of ultrapure water, arginine, ethylene glycol, glycerol, oleic acid and linoleic acid. Antioxidants (20mM quercetin, 20mM gallic acid and 10mM quercetin + 10mM gallic acid) were added to the model for the test. A needle-type electrochemical sensor with a platinum working electrode and a stainless steel counter electrode was fabricated for hydrogen peroxide (H2O2) detection, and with an iron porphyrin complex-modified carbon working electrode and a stainless steel counter electrode for O2- detection. H2O2 and O2- were generated in the model systems.
Result & Discussion
We confirmed that the model was prepared successfully by observing a lamellar structure consisting of the lipid and the water layers in the model by a polarizing microscope. Calibration curves for ROS detection were obtained with response current at constant potential electrolysis. From the result of the ROS scavenging capasity test, smaller response current for both H2O2 and O2- was observed in the presence of the antioxidants than in the absence of the antioxidants. From the electrochemical evaluation, the ROS scavenging capacity was higher at the mixed system (quercetin + gallic acid) than at the system with a single antioxidant. This should be due to a synergistic effect of the combination of hydrophobic (quercetin) and hydrophilic (gallic acid) antioxidants.
9:00 AM - F3.16
Surface-Initiated Living Radical Polymerization for the Synthesis of Stealth Nanoparticles
Kohji Ohno 1
1Kyoto University Kyoto JapanShow Abstract
The physiological properties of polymer brush-afforded silica particles prepared by surface-initiated living radical polymerization were investigated in terms of the circulation lifetime in the blood and distribution in tissues. Hydrophilic polymers consisting mainly of poly(poly(ethylene glycol) methyl ether methacrylate) (PPEGMA) were grafted onto silica particles by surface-initiated atom transfer radical polymerization that was mediated by a copper complex to produce hairy hybrid particles. The hybrid particles were injected intravenously into mice to systematically investigate their blood clearance and body distribution. It was revealed that the structural features of the hybrid particles significantly affected their in vivo pharmacokinetics. Some particles exhibited excellent stealth properties, for instance, the hybrid particles with SiP cores with a diameter of 15 nm and a shell of PPEGMA brush with an number-average molecular weight of 68000 have an extremely long circulation period with a halflife of about 20 h in the blood, and were found to preferentially accumulate into tumor tissue with the aid of the EPR effect.
9:00 AM - F3.17
Fluorescence Resonance Energy Transfer-Based Mesoporous Silica Nanoparticles for Real Time Monitoring of Drug Release
Birju Shah 1 Jinping Lai 1 Ki-Bum Lee 1
1Rutgers University Piscataway USAShow Abstract
Diseased microenvironments, such as tumors release different biological cues as compared to normal physiological tissues. Hence, it is important to develop effective delivery vehicles which can deliver multiple payloads and modulate their payload release in response to these biological cues for enhancing their efficacy. In this regard, mesoporous silica nanoparticles (MSNs) have shown excellent potential owing to their unique porous structure, tunable dimensions, ease of surface functionalization and overall versatility. Futhermore, by aptly modifying their pores with molecular valves, it is also possible to trigger the release of the entrapped drugs in response to various chemical and physical stimuli such as light, pH, redox potential, enzyme and temperature. While there have been numerous reports on the development of stimuli-sensitive MSNs for drug/gene delivery and subsequent intracellular release, monitoring the release of drugs from these MSNs in real-time is still in its nascent stage. Hence, it is essential to develop and integrate a real-time monitoring system within the stimuli-responsive MSNs to investigate real-time monitoring of drug release in complex cellular microenvironments. To address the afore-mentioned challenges, we have developed a versatile fluorescence resonance energy transfer (FRET)-based real time monitoring system consisting of (a) coumarin-cysteine-tethered MSNs as drug carriers, (b) FITC-conjugated β-cyclodextrin (FITC-β-CD) as redox-responsive molecular gate and (c) FRET donor-acceptor pair of coumarin and FITC integrated within the pore-unlocking event to allow real-time monitoring of drug release. Under non-reducing conditions, the intact disulfide bond brings coumarin and FITC in close proximity, thereby turning FRET ON. However, in the presence of reducing environment, such as that encountered by the FMSNs in the cytoplasm of cancer cells, the disulfide bond is cleaved which results in the release of FITC-β-CD, thereby unlocking the pores and release of encapsulated drug. Since the modulation of FRET signal was incorporated into the pore-unlocking event, the triggered release of the drug in target location can be monitored in real-time by simultaneous monitoring the change in FRET signal. Thus, the proposed drug delivery system provides a versatile strategy for real-time monitoring of drug delivery which can be extended to a wide array of biological applications.
9:00 AM - F3.18
MTS Assay for Evaluating the Antimicrobial Efficacy of Ag/BSA Nanoparticles
Almaz Gebregeorgis 1 John Stubbs 2 Dharmaraj Raghavan 1
1Howard University Washington USA2Howard University College of Medicine Washington USAShow Abstract
The primary objective of this study is to develop a quantitative analytical tool to evaluate the antimicrobial efficacy of Ag/BSA Nanoparticles. Ag/BSA Nanoparticles were prepared by chemical reduction and capping of silver nanoparticles with Bovine Serum Albumin (BSA). The synthesized nanoparticles were characterized by a sleuth of microanalytical techniques to obtain morphological information of nanoparticles. The antibacterial efficacy of silver nanoparticles against E. coli was measured by performing the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay (used to measure the viability of cells). Minimum Lethal Concentration (MLC) and the Minimum Inhibitory Concentration (MIC) of the nanoparticles against E. coli were established. Colony forming unit and optical density measurement were used to validate the results from MTS results. Future studies will investigate the optimum Ag/BSA nanoparticle concentrations needed to exhibit MIC and MLC behavior on biodegradable PHBV for potential osteomyelitis application.
Acknowledgment : NSF-DMR, NIH S06 GM 08016 -32
9:00 AM - F3.20
Inflammation-Triggered Degradation and Cargo Release from Biocompatible Polymeric Nanoparticles
Caroline de Gracia Lux 1 Shivanjali Joshi-Barr 1 Trung Nguyen 1 Enas Mahmoud 1 Eric Schopf 1 Nadezda Fomina 1 Adah Almutairi 1
1UC San Diego La Jolla USAShow Abstract
Inflammation, characterized by increased levels of reactive oxygen species, such as hydrogen peroxide (H2O2), is involved in various chronic diseases, including atherosclerosis, rheumatoid arthritis, neurodegeneration, and cancer. Targeting drug delivery specifically to these conditions may be possible with a new biocompatible, nontoxic polymeric nanoparticle capable of degrading and releasing its contents upon exposure to biologically relevant levels of H2O2. While delivery vehicles designed to respond to inflammatory conditions have been previously developed, none have yet been demonstrated to release cargo in response to sufficiently low concentrations of H2O2 to promise likely specificity of delivery in vivo. Our nanoparticles, consisting of a newly designed poly-cresol bearing benzyl ether-linked boronic ester triggering groups in each monomer, release their cargo upon exposure to 50 µM H2O2 or activated neutrophils and remain stable in aqueous solution. Release was indicated by quenching of fluorescence of Nile red, LCMS for paclitaxel of supernatants from centrifuged particles (both in vitro), and fluorescence of fluorescein diacetate (in cells; intracellular esterases cleave the dye to a fluorescent form). Nanoparticle degradation was confirmed by transmission electron microscopy; exposure to H2O2 induces significant ripping or crumpling in most particles. 1H NMR and GPC confirm polymer degradation into easily cleared small molecules upon H2O2 exposure and stability in its absence. These particles have no significant effects on cell viability, cytotoxicity, or apoptosis at concentrations up to 100 µg/mL. We are currently translating this material into an MRI-based ROS sensor and exploring its ability to enhance the efficacy of antioxidants in models of inflammatory disease.
9:00 AM - F3.21
Direct Translocation of and Cell-Penetrating Peptides and Cationic Nanoparticles across Lipid Bilayers under Transmembrane Potentials
Jiaqi Lin 1 Alfredo Alexander-Katz 1
1MIT Cambridge USAShow Abstract
Cell membranes regulate the transport of large molecules through endocytosis, which is a pathway for cells to bring substances from the extracellular medium into cytosol. Very recently, however, it was discovered that cationic nanoparticles (NPs) can enter cells in an energy-independent fashion, escaping the traditional endocytosis route. This unconventional translocation happens on the timescale of seconds, significantly faster than endocytosis, and can be somewhat toxic to cells. This has led the field to believe that cationic nanoparticles enter cells by generating holes on membranes, yet direct evidence in cells has not been observed. Meanwhile, cationic cell-penetrating peptides (CPPs) have demonstrated the ability of effectively translocating cargos into cells. Although there have been several different mechanisms proposed for the cell entry pathway of CPPs, there is still much debate and thus no consensus has been reached on the issue. Here, using coarse-grained simulations we show that cationic gold nano-particles, as well as HIV-1 Tat peptide, spontaneously translocate a model cell membrane by generating nanoscale pores in the presence of a transmembrane (TM) potential. The presence of cationic particles alters the electric field across the membrane, forcing the formation of holes, which in turn allow the translocation of particles. We demonstrate that the threshold of TM potential for nanoparticle translocation is concentration dependent, and is of the order of that appearing under physiological conditions. Furthermore, by comparing a linear chain of CPPs and a compact cluster of CPPs, we show that the shape of the cationic object is crucial in determining if it can translocate or not across. After translocation, the TM potential is strongly diminished due to the transport of ions in the local area and the membrane reseals (self-heals) very rapidly (e.g. within a microsecond). The mechanism put forward here provides fundamental information on the uptake kinetics of the cationic nanoparticles/biomolecules as well as their potential cytotoxicity, which is critical for understanding common biological processes such as viral invasion, or the design principles of drug/gene delivery system utilizing cationic agents.
F1: Advanced Techniques, High-Throughput Assessment, and Microfluidics to Probe Cellular Systems
Monday AM, December 02, 2013
Sheraton, 2nd Floor, Republic A
9:30 AM - *F1.01
Microfabricated Hydrogels: From Tissue Engineering to Stem Cell Bioengineering
Ali Khademhosseini 1 2 3
1Brigham and Womenamp;#8217;s Hospital, Harvard Medical School Cambridge USA2Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology Cambridge USA3Harvard University Cambridge USAShow Abstract
Micro and nanoscale technologies have been increasingly applied in various scientific disciplines from physics and chemistry to biology and medicine. In particular, these technologies have immensely benefited the fields of experimental biology and medicine through designing of complex biomaterials for cell-based studies. In addition, these technologies have allowed for an unprecedented ability to control cell-cell, cell-microenvironment and cell-soluble factors interactions through miniaturized assays for high-throughput cell-based studies. The focus of our research group has been on merging mico- and nanofabrication techniques with innovative biomaterials for tissue engineering and stem cell bioengineering applications. Our research encompasses a wide range of scientific areas from biomaterials design to microfabrication and biology. We have developed novel polymeric and hydrogels based biomaterials and have used ‘bottom-up&’ and ‘top-down&’ fabrication techniques to create three dimensional (3D) vascularized tissue constructs. In addition, we have shown successful incorporation of various nanoparticles and carbon nanotubes within the hydrogel constructs in order to enhance their mechanical and electrical properties for potential applications in bone and cardiovascular tissue engineering. On the stem cell bioengineering aspect, we have created suitable microenvironments, such as high density microwell topographies, to module stem cell behavior and direct their differentiation toward the desired lineages. In this talk, I will present the recent findings in our lab covering biomaterials, tissue engineering and stem cell bioengineering aspects. I will also discuss our major biological findings related to tissue engineering and stem cell differentiation using miniaturized systems.
10:00 AM - F1.02
Chemically Anchored Micro-Fabricated Landscapes for Single-Cell Based Biophysical Studies
Prasana Kumar Sahoo 1 Richard Janissen 1 Joao H. Clerici 1 Monica A. Cotta 1
1Universidade Estadual de Campinas Campinas BrazilShow Abstract
Spatially defined tethering of individual cells are of great interest in biomedical topics, applied microbiology, environmental research up to forensics and toxicology applications. The methodological possibility to study spatially separated individual cells facilitates simultaneous observation of cell motility behavior and division processes, and allows further to perform in-vivo studies within the cellular environment. In this context, we developed a universal and rapid fabrication of arrays of micro-patches with different shape and size for controlled and localized tethering of living prokaryotic and eukaryotic cells. Human cervical cancer cells (HeLa) and phytopathogen bacteria (Xylella Fastidiosa) have been used for the present study.
Initially, arrays of micro-patches using Ag 5214 photoresist with sizes ranging from 1 µm to 80 µm were fabricated on covalently amino-coated glass substrates which facilitate the cellular adhesion. The substrates were further selectively functionalized with poly(ethylene glycol) (PEG) at the surroundings of the predefined areas which were developed using mask-free laser-lithographic approach. The covalent ligation of the cell-repellant PEG polymer around the tethering areas has been successfully demonstrated via sensitive fluorescence experiments. Within our study we analyze how the cells can be tethered selectively on the predefined areas as a response to different chemical surface compositions and changes in pattern shape and size. While monitoring a single cell, we can able to disentangle the parameters which influence the cell motility behavior, degree of spatial organization and complexity while tethered in a micro environment. Our results unravel the potential use of present methodology for single cell studies as well as developing future biomedical devices in a more versatile and reliable way.
10:15 AM - F1.03
A Microfluidic Platform for the Study of Cell-Cell Communication in the Context of Parkinsonrsquo;s Disease
Joao Tiago S. Fernandes 1 2 Oldriska Marques 2 Joana Branco-Santos 2 Federico Herrera 2 Virginia Chu 1 Tiago F. Outeiro 2 3 Joao Pedro Conde 1 4
1INESC Microsistemas e Nanotecnologias and IN - Institute of Nanoscience and Nanotechnology Lisbon Portugal2Faculdade de Medicina da Universidade de Lisboa Lisbon Portugal3University Medical Center Goettingen Goettingen Germany4Instituto Superior Tamp;#233;cnico Lisbon PortugalShow Abstract
Alpha-synuclein (aSyn) is an abundant protein in the brain that is deeply implicated in Parkinson&’s disease (PD) and other synucleinopathies. PD is characterized by the loss of dopaminergic cells and by the presence of intraneural Lewy bodies, which are mostly composed of lipids and aggregated aSyn. Although the mechanisms behind neural cell death are not yet fully understood it is known that aSyn plays a double role: it is toxic to cells and, in certain aggregated states, activates microglia, the immune cells in the brain. To find long-term therapies for PD, the mechanisms underlying the relation between aSyn, microglia activation, and cell death are currently the subject of intense investigation.
Microfluidic technology can provide new ways to explore these cellular mechanisms and interactions, its key advantage being the fine control that can be achieved over the cellular microenvironment.
In order to further explore the mechanisms of PD, a microfluidic device was built for the co-culture of neural and glial cells and to monitor their interaction through soluble factors. This device has two chambers, one for each cell population, connected by microfluidic channels allowing the exchange of soluble materials while preventing direct cell-cell interaction. The incorporation of micro-pneumatic valves allows to 1) populate the chambers individually; 2) isolate chambers so that both cell populations are exposed to different chemical environments; and 3) control fluid flow to individual chambers or between chambers.
Preliminary tests on the microfluidic device were carried out with HEK cells. In the first series of experiments, cell viability and proliferation tests were performed. By coating the device with fibronectin, an extracellular matrix protein, and supplying cell culture medium with a pH buffer, cell mortality was kept at about 20% and cell proliferation reached 150% after 4 days, which is comparable to typical macroscopic cell culture protocols. In the second series of experiments, HEK cells producing aSyn fused with GFP were seeded on one chamber and HEK cells producing aSyn fused with DsRED were seeded on the other. Confocal microscopy imaging revealed the presence of green fluorescent signal in the chamber containing red fluorescent cells, strongly suggesting the traffic of aSyn between chambers and that cell-cell communication through soluble factors is possible in this device.
We will show that the environment of each cell population can be independently controlled, by testing whether inducing apoptosis in one cell population increases aSyn transmission to the other. We will also show the results of viability and proliferation assays on the co-culture of neural cells and microglia.
Overall, this novel device will contribute to a better understanding of the importance of the traffic of soluble factors between cell populations in normal and pathological conditions, such as PD or other neurodegenerative disorders.
10:30 AM - F1.04
Trap and Release of Single Motile Cells in Sub-Microfluidics; Methods and Applications
Andreas E. Vasdekis 1 G. Stephanopoulos 2
1Pacific Northwest National Laboratory Richland USA2Massachusetts Institute of Technology Cambridge USAShow Abstract
Single cell analysis unmasks information inaccessible in population level data, such as intracellular dynamics and cell-to-cell heterogeneity . In this presentation, the microfluidic manipulation of single motile cells will be discussed, including both trapping and release . The focus will particularly be on motile cells of reduced size, namely bacteria and fungi. The present approach was based on the integration of micron-scale features with sub-micron ones. The reali